Columbia ®nit)em'tp .
College of ^fjpsiitians! anlJ ^urseonjs
Xibrarp
(iH Presented by
, DR. WILLIAM J. OIES^J^
;o enrich the library resources ' |f
avciiUble to holders
OlES FELLOWSHIP
t>j Biologic&l Chemistry
PRACTICAL
PHYSIOLOGICAL CHEMISTRY
HAWK
Absorption Spectra.
Oxyhaemoglobin.
Haemoglobin.
Carboxy-
haemoglobin.
Neutral Met-
haemoglobin.
Alkaline Met-
haemoglobin.
Alkali
Haematin.
Absorption Spectra.
Reduced Alkali
Haematin or
HaemochromoKen.
Acid Haematin in
ethereal solution.
Acid Haemato-
porphyrln.
Alkaline
Haematopor-
phyrln.
Urobilin or Hydro-
bilirubin In acid
solution.
Urobilin or Hydro-
bllirublivJn alkaline
solution after the
addition of zinc
chloride solution.
Blllcyanin or
Cholecyanin in
alkaline solution.
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PRACTICAL
PHYSIOLOGICAL CHEMISTRY
A BOOK DESIGNED FOR USE IN COURSES IN PRACTIC
PHYSIOLOGICAL CHEMISTRY IN SCHOOLS
OF MEDICINE AND OF SCIENCE
BY
PHILIP B. HAWK, M. S., Ph. D.
PROFESSOR OF PHYSIOLOGICAL CHEMISTRY AND TOXICOLOGY IN THE
JEFFERSON MEDICAL COLLEGE OF PHILADELPHIA
FOURTH EDITION, REVISED AND ENLARGED
WITH TWO FULL-PAGE PLATES OF ABSORPTION SPECTRA IN COLORS
FOUR ADDITIONAL FULL-PAGE COLOR PLATES AND ONE
HUNDRED AND THIRTY-SEVEN FIGURES OF WHICH
TWELVE ARE IN COLORS
PHILADELPHIA
P. BLAKISTON'S SON & CO.
1012 WALNUT STREET
1912
First Edition, Copyright, 1907, by P. Blakiston's Son & Co.
Second Edition, Copyright, 1909, by P. Blakiston's Son & Co.
.Third Edition, Copyright, iqio, by P. Blakiston's Son & Co.
Fourth Edition, Copyright, 1912, by P. Blakiston's Son Sz Co.
P
THE. MAPLE. PRESS. TORK- PA
THESE PAGES ARE
AFFECTIONATELY DEDICATED
TO
MY MOTHER
PREFACE TO FOURTH EDITION.
The continued rapid development of the many phases of biochemistry,
which has taken, place in the interval of two years since the last edition
of this volume appeared has necessitated a rather comprehensive revision
and the consequent inclusion of considerable new matter. The main
bulk of the material will be found in the chapters on Enzymes, Carbo-
hydrates, Proteins, Blood and Lymph, Feces, Putrefaction and Quanti-
tative Analysis of Urine. The publishers have wisely reduced the
marginal space of the page thus necessitating but slight increase in the
size of the volume.
The author wishes to express his gratitude to Professor William J.
Gies, Professor Lafayette B. Mendel and Dr. Thomas B. Osborne for
many valuable suggestions for the betterment of this revised volume. He
is also under obligations to Dr. Martha Tracy and Professors Marshall
P. Cram, Paul E. Howe, E. C. L. Miller, Charles J. Robinson and
A. P. Sy for similar offices and to Messrs. Olaf Bergeim, Lawrence T.
Fairhall, Edwin F. Hirsch, Melvin A. Saylor and Theodore F. Zucker
for assistance in the verification of tests and methods, the translation of
papers, the sketching of crystals and the reading of proof.
The author would be grateful if those using the volume in their
classes would make suggestions regarding insertions, omissions or
corrections.
Philip B. Hawk.
Philadelphia.
IIV
PREFACE TO THIRD EDITION.
The increasing approval with which this volume is being received has
rendered necessary the preparation of a new edition, although the period
elapsing since the last edition appeared is little more than one year.
The present edition has been brought up to date by the insertion of
various additions and corrections as well as by the inclusion of a number
of qualitative tests and quantitative methods. Because of the very short
intervening period since the last edition of the volume, the new material
inserted is rather small in quantity when compared with that incorporated
at the previous revision.
The author wishes to thank Dr. W. H. Welker and Dr. Croll for
permission to insert unpublished material.
Philip B. Hawk.
Urbana, Illinois.
UC
PREFACE TO SECOND EDITION.
The kind fCccption accorded this volume by the instructors in
physiological chemistry in the United States and Great Britain has
made the preparation of a new edition imperative, notwithstanding
the fact that less than two years have elapsed since the former edition
appeared. The advance and development made in the field of physio-
logical chemistry during this period have been both rapid and impor-
tant; conditions which would of themselves have necessitated the revision
of the volume at an early date.
The book has been thoroughly revised in all department and in
part rewritten, the system of spelling officially adopted by the American
Chemical Society having been followed throughout the volume. Besides
introducing many new qualitative tests and quantitative methods, the
author has added a chapter on "Enzymes and Their Action" and has
rewritten the two chapters on Proteins. The term "protein" has been
substituted for "proteid" and the classification of proteins as recently
adopted by the American Physiological Society and the American Society
of Biological Chemists has been introduced and is followed throughout
the text; the classification adopted by the Chemical and Physiological
Societies of England is also included.
The original plan of the book has been adhered to with the excep-
tion that the chapter on "Enzymes and Their Action" has been made
Chapter I and the practical work upon the proteins is preceded by a
chapter giving a brief discussion of protein substances from the stand-
point of their decomposition and synthesis. We believe that the student
will be able to pursue his practical work more intelligently and will
derive greater benefit therefrom if the plan of instruction as suggested
in Chapters IV and V be followed in the presentation of the subject
of "Proteins."
The author wishes to express his thanks to all those who so kindly
offered suggestions for the betterment of the book. He is particularly
desirous of expressing his gratitude to Professor Lafayette B. Mendel
and Dr. Thomas B. Osborne for the many helpful suggestions they
have so kindly given him. His thanks are also due Professor C. A.
Herter, Dr. H. D. Dakin, Dr. S. R. Benedict, and Mr. S. C. Clark
for permission to insert unpublished material, to Mr. Paul E. Howe
xi
XU PREFACE TO SECOND EDITION.
for valuable assistance rendered in the reading of proof and in the verifi-
cation of tests and methods, and to Dr. M. E. Rehfuss for assistance in
proof reading.
The author takes this opportunity of making an acknowledgment
which was inadvertently omitted from the first edition. He wishes to
express his obligation to the laboratories of physiological chemistry at
Yale University and at Columbia University (College of Physicians and
Surgeons) in the latter of which he was Assistant to Professor W. J.
Gies for two years. The courses given in these laboratories formed the
basis of many of the experiments included in this volume, and it is with
feelings of deepest gratitude that he records this acknowledgment of the
assistance thus rendered by those in charge of these courses.
Philip B. Hawk.
Uebana, Illinois.
PREFACE TO FIRST EDITION.
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, however, he feels to be a logical
one and has followed it with satisfactory 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
proposed 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 physiological chemistry. For the
specimens of the decomposition products, the crystalHne forms of which
are reproduced by original drawings or by microphotographs, the author
is indebted to Dr. Thomas B. Osborne of New Haven, Conn.
Because of the increasing importance attached to the examination
of feces for the purposes of diagnosis, the author has devoted a chapter
to this subject. He feels that a careful study of this topic deserves
to be included in the courses in practical physiological chemistry, of
medical schools in particular. 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 grati-
tude 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 assistance in
proof reading.
For the micro-photographs of oxyhaemoglobin and haemin 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.
xiii
XIV PREFACE TO FIRST EDITION.
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.
CONTENTS
chapti:r I.
Page
Enzymes and Their Action i
CHAPTER II.
Carbohydrates 25
CHAPTER III.
Salivary Digestion 59
CHAPTER IV.
Proteins: Their Decomposition AND Synthesis 68
CHAPTER V.
Proteins: Their Classification and Properties 92
CHAPTER VI.
Gastric Digestion 124
CHAPTER VII.
Fats 139
CHAPTER VIII.
Pancreatic Digestion , . 148
CHAPTER IX.
Bile 158
CHAPTER X.
Putrefaction Products 169
CHAPTER XI.
Feces 178
CHAPTER.XII.
Blood and Lymph 194
XV
XVI CONTENTS.
CHAPTER XIII.
Pagb
^JlLK 235
CHAPTER XIV.
Epithelial and Connective Tissues 245
CHAPTER XV.
Muscular Tissue 254
CHAPTER X\T.
Nervous Tissue 268
CHAPTER XVII.
Urine: Geneila.l ChlVracteristics of Normal and Pathological Urine 274
CH.APTER XVIII.
Urine: Physiological Constituents 283
CHAPTER XIX.
Urine: Pathological Constituents 323
CHAPTER XX.
Urine: ORGAN^ZED and Unorganized Sediments 361
CHAPTER XXI.
Urine: Calculi 379
CHAPTER XXII.
Urine: Quantitative Analysis 383
CHAPTER XXIII.
Quantitative Analysis of Milk, Gastric Juice and Blood 435
Appendix 443
Index 455
LIST OF ILLUSTRATIONS
Plate
I. Absorption Spectra "1 c • •
II. Absorption Spectra / ^
III. Osazones Opposite page 28
IV. Normal Erythrocytes and Leucocytes Opposite page 196
V. Uric Acid Crystals Opposite page 291
VI. Ammonium Urate Opposite page 365
Figure Page
1. Apparatus for Quantitative Determination <jr Catalase 24
2. Dialyzing Apparatus for Students' Use 30
3. Einhorn Saccharometer o6
4. One Form of Laurent Polariscope 37
5. Diagrammatic Representation of the Course of the Light through the
Laurent Polariscope ■jg
6. Polariscope (Schmidt and Hansch Model) 38
7. Iodoform 4y
8. Potato Starch 4p
9. Bean Starch ^p
10. Arrowroot Starch ^g
11. Rye Starch ^g
12. Barley Starch ^g
13. Oat Starch ^p
14. Buckwheat Starch 40
15. Maize Starch ^g
16. Rice Starch 4^
17. Pea Starch 4g
18. Wheat Starch 4q
19. Microscopical Constituents of Saliva 63
20. Glycocoll Ester Hydrochloride y-j
21. Serine 78
22. Phenylalanine jg
23. Fischer Apparatus 80
24. Tyrosine 81
25. Cystine 81
26. Histidine Dichloride 8?
27. Leucine 85
28. Lysine Picrate 86
29. Aspartic .\cid 86
xvii
XVlll LIST OF ILLUSTEATICNS.
Figure Page
30. Glutamic Acid 87
31. Laevo- a-Proline 88
32. Copper Salt of Proline 89
33. Coagulation Temperature Apparatus 107
34. Edestin no
35. Excelsin, the Protein of the Brazil Nut no
36. Beef Fat 139
37. Mutton Fat 142
38. Pork Fat • 144
39. Palmitic Acid 145
40. Melting-Point Apparatus 146
41. Bile Salts 160
42. Bilirubin (Hsematoidin) i6r
43. Cholesterol 166
44. Taurine 167
'45. Glycocoll 167
46. Ammonium Chloride 175
47. Microscopical Constituents of Feces 178
48. Hsematoidin Crystals from Acholic Stools 179
49. Charcot-Leyden Crystals ■ . 181
50. Boas' Sieve 184
51. Oxyhsemoglobin Crystals from Blood of the Guinea Pig 198
52. Oxyhsemoglobin Crystals from Blood of the Rat 198
53. Oxy haemoglobin Crystals from Blood of the Horse 199
54. Oxyhaemoglobin Crystals from Blood of the Squirrel 199
55. Oxy haemoglobin Crystals from Blood of the Dog 200
56. Oxyhaemoglobin Crystals from Blood of the Cat 200
57. Oxyhaemoglobin Crystals from Blood of the Necturus 201
58. Effect of Water on Erythrocytes 208
59. Haemin Crystals from Human Blood 211
60. H^min Crystals from Sheep Blood .211
61. Sodium Chloride 213
62. Direct-vision Spectroscope 216
63. Angular- vi.sion Spectroscope Arranged for Absorption Analysis . .217
64. Diagram of Angular-\dsion Spectroscope 217
65. Fleischl's Haemometer 220
66. Pipette of Fleischl's Haemometer 221
67. Colored Glass Wedge of Fleischl's Haemometer 221
68. Dare's Haemoglobinometer 222
69. Horizontal Section of Dare's Haemoglobinometer 223
70. Method of Filling the Capillary Observation Cell of Dare's Haemo-
globinometer 223
71. Thoma-Zeiss Counting Chamber 224
72. Thoma-Zeiss Capillary Pipettes 225
LIST Ol ILLUSTRATIONS. xix
FicLRE Page
73. Ordinary Ruling of Thoma-Zeiss Counting Chamber 226
74. Zappert's Modified Ruling of Thoma-Zeiss Counting Chamber. . . 227
75. Biirker's Pipettes, Mixing Flasks, and Counting Chamber 229
76. Ruling of Biirkcr Counting Chamber 232
77. Schema 232
78. Burker Counting Chamber 233
79. Normal Milk and Colostrum 236
80. Lactose 238
8r. Calcium Phosphate 242
82. Creatine 257
83. Xanthine 258
84. Hypoxanthine Silver Nitrate 265
85. Xanthine Silver Nitrate 266
86. Deposit in Ammoniacal Fermentation 277
87. Deposit in Acid Fermentation 277
88. Urinometer and Cylinder 278
89. Beckmann-Heidenhain Freezing-point .Apparatus 280
90. Urea 285
91. Urea Nitrate 287
92. Melting-point Tubes Fastened to Bulb of Thermometer 288
93. Urea Oxalate 289
94. Pure Uric Acid 293
95. Creatinine 295
96. Creatinine-Zinc Chloride 296
97. Hippuric Acid 300
98. AUantoin from Cat's Urine 304
99. Benzoic Acid 308
100. Calcium Sulphate 316
loi. "Triple Phosphate" 310
102. The Purdy Electric Centrifuge 361
103. Sediment Tube for the Purdy Electric Centrifuge 361
104. Calcium Oxalate 363
105. Calcium Carbonate 363
106. Various Forms of Uric Acid 365
107. Acid Sodium Urate 366
108. Cystine 366
109. Crystals of Impure Leucine 367
no. Epithelium from Different Areas of the Urinary Tract 370
111. Pus Corpuscles 3yi
112. Hyaline Casts 3-2
113. Granular Casts ^-jj
114. Granular Casts 374
115. Epithelial Casts 374
116. Blood, Pus, Hyaline and Epithelial Casts 374
XX LIST OF ILLUSTRATIONS.
FifiuRE Page
117. Fatty Casts 375
118. Fatty and Waxy Casts 375
119. Cylindroids 376
120. Crenated Erythrocytes 377
121. Human Spermatozoa 377
122. Esbach's Albuminometer 384
123. Marshall's Urea Apparatus 392
124. Hiifner's Urea Apparatus 394
125. Doremus-Hinds Ureometer 395
126. Folin's Urea Apparatus 396
127. Folin's Ammonia Apparatus 399
128. Folin Improved Absorption Tube 400
129. 130. Forms of Apparatus used in Methods of Folin and Associates for
Determination of Total Nitrogen, Urea and Ammonia 403
'131. Van Slyke's Amino-nitrogen Apparatus 405
132. Berthelot-Atwater Bomb Calorimeter 411
133. Hall's Purinometer 431
134. Centrifuge Tube used in Babcock Fat Method 435
135. Croll's Fat Apparatus 436
136. Soxhlet Apparatus 437
137. Feser's Lactoscope 438
PHYSIOLOGICAL CHEMISTRY.
CHAPTER I.
- ENZYMES AND THEIR ACTION.
According to the old classification ferments were divided into two
classes, the organized ferments and the unorganized ferments. As organized
ferments or true ferments there w^ere grouped such substances as yeast
and certain bacteria which were supposed to act by virtue of vital processes,
whereas the unorganized ferments included salivary amylase (ptyalin),
gastric protease (pepsin), pancreatic protease (trypsin), etc., which were
described as "non-li^ing unorganized substances of a chemical nature."
Kiihne designated this latter class of substances as enzymes {iv ^vfirj-m yeast).
This division into organized ferments (true ferments) and unorganized
ferments (enzymes) was generally accepted and was practically unc^ues-
tioned until Buchner overthrew it in the year 1897 by his epoch-making
investigations on zymase. Previous to this time many writers had ex-
pressed the opinion that the action of the ferment organisms was similar
to that of the unorganized ferments or enzymes and that therefore the
activity of the former was possibly due to the production of a substance
in the cell, which was in nature similar to an enzyme. Investigation
after investigation, however, failed to isolate any such principle from an
active cell and the exponents of the "vital" theory became strengthened
in their belief that certain fermentative processes brought about by living
cells could not occur apart from the biological acti\-ity of such cells. How-
ever, as early as 1858, Traube had enunciated, in substance, the principles
which were destined to be fundamental in our modern theory of fermenta-
tion. He expressed the belief that the yeast cell produced a product in
its metabolic actiA-ities which had the property of reacting with sugar with
the production of carbon dioxide and alcohol, and further that this
reaction between the product of the metabolism of the yeast cell and the
sugar occurred without aid from the original cell. It was not until 1897,
however, that this theory was placed upon a firm experimental basis.
This was brought about through the efforts of Buchner who succeeded in
isolating from the living yeast cells a substance (zymase) which, when
freed from the last trace of organized cellular material, was able to bring
about the identical fermentative processes, which, up to this time, had
been deemed possible only in the presence of the active, living yeast cell.
Buchner's manipulation of the yeast cells consisted in first grind-
2 PHYSIOLOGICAL CHEMISTRY.
ing them with sand and infusorial earth, after which the finely divided
material was subjected to great pressure (300 atmospheres) and yielded
a liquid which possessed the fermentative activity of the unchanged
yeast cell/ This liquid contained zymase, the principal enzyme of
the yeast cell. Later the lactic-acid- and acetic-acid-producing bac-
teria were subjected by Buchner to treatment similar to that- accorded
the yeast cells, and the active intracellular enzymes were obtained. Many
other instances are on record in which a soluble, active agent has been
isolated from ferment cells, with the result that it is pretty well estab-
lished that all the so-called organized ferments elaborate substances of
this character. Therefore, basing our definition on the work of Buchner
and others we may define an enzyme as an unorganized, soluble ferment,
which is elaborated by an animal or vegetable cell and whose activity is
entirely independent of any of the life processes of such a cell. According
to this definition the enzyme zymase elaborated by the yeast cell is entirely
comparable to the enzyme pepsin elaborated by the cells of the stomach
mucosa. One is derived from a vegetable cell, the other from an animal
cell, yet the activity of neither is dependent upon the integrity of the cell.
Enzymes act by catalysis and hence may be termed catalyzers or
catalysts. A simple rough definition of a catalyzer is "a substance
which alters the velocity of a chemical reaction mthout undergoing
any apparent physical or chemical change itself and without becoming
a part of the product formed." It is a well-known fact that the velocity
of the greater number of chemical reactions may be changed through
the presence of some catalyzer. For example, take the case of hydro-
gen peroxide. It spontaneously decomposes slowly into the water and
oxygen. In the presence of colloidal platinum,^ however, the decom-
position is much accelerated and ceases only when the destruction of
the hydrogen peroxide is complete. Without multiplying instances,
suffice it to say that there is an analogy between inorganic catalyzers
and enzymes, the main point of difference between the enzymes and
most of the inorganic cata yzers being that the enzymes are colloids.^
Inasmuch as each of the enzymes has an action which is more or less
specific in character, and since it is a fairly simple matter, ordinarily, to
determine the character of that action, the classification of the enzymes
is not attended with very great difficulties. They are ordinarily classi-
fied according to the nature of the substrate* or according to the type
' In later investigations the process was improved by freezing the ground cells with liquid
air and finely fiulverizing them before applying the pressure.
^ Produced by the passage of electric sparks between two platinum terminals immersed
in distilled water, thus liberating ultra-microscopic particles.
' Bredig has been able to obtain certain inorganic catalyzers in colloidal solution. These
he calls " inorganic enzymes."
* Substance acted upon. See Lippmann: Ber. d. Deulsch. Chem. Ces., 36, 331, 1903.
ENZYMES AND THEIR ACTION. 3
of reaction they bring about. Thus we have various classes of enzymes,
such as amylolytic,^ proteolytic, lipolytic, glycolytic, uricolytic, autolytic,
oxidizing, reducing, inverting, protein-coagulating, deamidizing, etc. In
every instance the class name indicates the individual type of enzy-
matic activity which the enzymes included in that class are capable of
accomplishing. For example, amylolytic enzymes facilitate the hydro-
lysis of starch (amylum) and related substances, lipolytic enzymes
facilitate the hydrolysis of fats (Aittos), whereas through the agency of
uricolytic enzymes uric acid is broken down. There is a tendency,
at the present time, to harmonize the nomenclature of the enzymes by
the use of the termination, -ase. According to this system of nomen-
clature, all starch-transforming enzymes, or so-called amylolytic en-
zymes, are called amylases, all fat-splitting enzymes are called lipases,
etc. Thus ptyalin the amylolytic enzyme of the saliva, would be
termed salivary amylase in order to distinguish it from pancreatic amy-
lase (amylopsin) and vegetable amylases (diastase, etc.). According
to the same system, the fat-splitting enzyme of the gastric juice would
be termed gastric lipase to differentiate it from pancreatic lipase (steap-
sin), the fat-splitting enzyme of the pancreatic juice.
Euler^ claims that enzymatic cleavage and synthesis are often brought
about by two different components of an enzyme preparation. He
would indicate this fact by giving the termination -ese to those enzymes
exerting a synthetic function. For example, the enzyme which catalyzes
the formation of nitriles Euler would call miriXese in distinction from nitril-
ase which splits nitriles. He would further designate as phosphatase
the enzyme which builds up phosphoric acid esters of carbohydrates in
distinction from phosphatase which causes their cleavage. In the same
way he would differentiate the lipolytic enzymes into lipases and lipeses.
Our knowledge regarding the distribution of enzymes has been
wonderfully broadened in recent years. Up to within a few years,
the real scientific information as to the enzymes of the animal organism,
for example, was limited, in the main, to a rather crude understanding
of the enzymes intimately connected with the main digestive func-
tions of the organism. We now have occasion to believe that enzymes
arc doubtless present in every animal cell and are actively associated
with all vital phenomena. As a preeminent example of such cellular
activity may be cited the liver cell with its reputed complement of 15-
20 or more enzymes.
'Armstrong suggests the use of the termination "clastic" instead of "lytic." He calls
attention to the fact that amylolytic, in analogy with electrolytic, means "decomposition by
means of starch" and is therefore a misnomer. He suggests the use of amyloclastic, proteo-
clastic, etc.
* Euler: Zeitschrift fiir physiologische chemie, 74, 13, 1911.
PHYSIOLOGICAL CHEMISTRY.
A list of the more important enzymes together with their class, dis-
tribution, substrate and end-products is given below.
Name.
CLASSIFICATION OF ENZYMES.
Class. i Distribution. Substrate.
End-products.
Adenase Deamidizing \ Animal tissues
Adenine Hypoxanthine.
Atnylases
(a) Pancreatic . . .
(amylopsin)
(b) Salivary
(ptyalin)
(c) Vegetable. . .
(diastase)
Amylolytic.
Pancreatic juice.. . . Starch, dextrine, Malsose.
etc. _ I
Saliva . .■ ' Starch, dex trine, , Maltose.
etc.
Plant tissues ' Starch, dex trine, i Maltose.
etc. !
Arginase .
Argenine-split-
ting.
Mucosa of intes-
tine and in paren-
chyma of liver,
kidney, spleen,
etc.
Arginine .
Ornithine and urea.
Bromelin
Proteolytic
Pineapple
.... Proteins
Proteoses, peptones,
etc.
Carboxylase
Decarboxylizing.
Yeast
COOH group of ah-
phatic acids.
Carbon dioxide.
Catalase
Oxidizing
Tissues
..... Peroxides
Oxygen, water.
Emulsin (synap-
tase).
Glucoside-split-
ting.
Plants
. . . . i Amygdalin, etc ... .
Glucose, etc.
Enterokinase Activating.
Intestinal epithe- Trypsinogen.
lium.
Trypsin.
Erepsin (protease) . ! Proteoljrtic .
Glycogenase .
Glycogen-s p 1 i t-
ting.
Intestinal mucosa Proteoses, peptones,
of man and dogs. peptides, and
Animal and vege- casein,
table tissues, and
pancreatic juice.
Simple cleavage
products, such as
amino acids.
Liver, intestinal Glycogen,
mucosa (?), mus-
cles (?).
Maltose and dextrin
(dextrose ?) .
Glycolytic enzymes . Glycolytic.
Blood and various . . Sugar.
organs.
Lactic acid, alcohol,
carbon dioxide and
water.
Guanase Deamidizing.
Animal tissues Guanine Xanthine.
Laccase Oxidizing.
Sap of lac tree and
other saps and •
juices; fungi; gum
arable, etc.
Polyhydric p-phe-
nols such as hy-
droquinone and
pyrogallol.
Oxidation products.
Lactase .
Lactose-splitting. Intestinal juice and
mucosa.
Lactose.
Dextrose and galac-
tose.
Lipases .
(a) Pancreatic.
(steapsin)
(b) Gastric. . .
(c) Vegetable .
Lipolytic .
Pancreatic juice.
Gastric juice. . . .
Plant tissues ....
Neutral fats.
Neutral fats.
Neutral fats.
(d) Animal.
Animal tissues Neutral fats.
Fatty acid and
alcohol.
Fatty acid and
alcohol.
Fatty acid and
alcohol.
Fatty acid and
alcohol.
Maltose ' Maltose-splitting.
Blood serum, liver. Maltose,
saliva, pancreatic
and intestinal |
juice and lymph. '
Dextrose.
Nuclease.
Nucleoprotein Tissues Nucleoprotein.
hydrolyzing.
Purine bases.
Oxidases.
Oxidizing .
Plant and animal Various tissue con- Oxidation products,
tissues. stituents.
Pancreatic rennin. . Coagulating Pancreatic juice.
Caseinogen I Casein.
ENZYMES AND THEIR ACTION.
CLASSIFICATION OF ENZYMES.— Contmued.
Name. | Class. j Distribution. [ Substrate. End-products.
Papain (papayotin) , Proteolytic [ Pawpaw ' Proteins Proteoses, peptones,
I etc.
^ 1 j ^
Pepsin (pepsase or j Proteolytic Gastric juice Proteins Proteoses, peptones
acid -proteinase). ; and peptides.
Peroxidases Oxidizing Plant and animal Peroxides, or hy- Oxidation products.
^ tissues. droperoxides and
] carries oxygen to
tissue constitu-
ents.
Phytase
Phytin-splitting. .
Rice bran
Phytin
. . . . Inosite and phos-
phoric acid.
Protease (erepsin) . .
Proteolytic
Kachree gourd
Proteins
. . . . Proteoses, peptones,
peptides, etc.
Rennin (rennase
or caseinase.)
Coagulating
Gastric and pan-
creatic juices.
Caseinogen ....
. . . . Casein.
Sucrose (invertase
or invertin).
Inverting
Mucosa and juice
of the intestine.
Sucrose
. . . . Dextrose and Lse-
vulose (invert-
sugar) .
Thrombin
Coagulating
Blood
Fibrinogen
. . . . Fibrin.
Trypsin (trypsase
or alkali-protein-
ase.)
Proteolytic
Pancreatic juice.. . .
Proteins
, . . . Proteoses, peptones,
peptides and ami-
no acids.
Tyrosinase Oxidizing Plant and animal Tyrosine , Homogentisic acid,
tissues. etc.
Urease Urea-splitting. . . . Micrococcus ureae. . Urea Carbon dioxide and
1 ammonia.
Uncase (uricolytic , Uric acid-split- j Tissues Uric acid AUantoin, urea, gly-
enzyme). ting. cocoU and glyoxy-
lic acid (?}.
Xantho-oxidase. . .
. Oxidizing
Tissues
. Xanthine and
poxanthine.
hy-
Uric acid.
Zymase
. S u g a r-ferment-
ing.
Yeast
. Sugar
Alcohol and carbon
dioxide.
Inulase
. Hydrolytic
Plants and fungi . .
Inulin
Laevulose.
Rhamnase
. Hydrolytic
Fungi
. Rhamnose.
Trehalase
. Hydrolytic
Fungi
. Trehalose.
1
In text-book discussions of the enzymes it is customary to say that
very little is known regarding the chemical characteristics of these sub-
stances since no member of the enzyme group has, up to the present
time, been prepared in an absolutely pure condition. Apparently, how-
ever, from the nature of the facts in the case, it would be much more
accurate to say that we absolutely do not know whether a specific enzyme
has, or has not, been prepared in a pure state. (Some authors, like
Arthus, have assumed that enzymes are not chemical individuals, but
properties conferred upon bodies.) The enzymes are very difficult to
prepare in anything like a condition approximating purity, since they
are very prone to change their nature during the process by which the
investigator is attempting to isolate them. For this reason we have
absolutely no proof that the final product obtained is, or is not, in the
t) PHYSIOLOGICAL CHEMISTRY.
same state of purity it possessed in the original cell. Some of the en-
zymes are more or less closely associated with the proteins from the fact
that they are both formed in every cell as the result of the cellular ac-
tiWty, both may be removed from solution by "salting-out," both are
for the most part non-diffusible and are probably very similar as re-
gards elementary composition. Hence in the preparation of some
enzymes it is extremely difficult to make an absolute separation from
the protein.* Under certain conditions enzymes are readily adsorbed
by shredded protein material, such as fibrin, and may successfully
resist the most prolonged attempts at washing them free. We may
summarize some of the properties of the great body of enzymes as fol-
lows: Enzymes are soluble in dilute glycerol, sodium chloride solu-
tion, dilute alcohol and water, and precipitable by ammonium sulphate
and strong alcohol. Their presence may be proven from the nature
of the end-products of their action and not through the agency of any
chemical test. They are colloidal and non- diffusible, and occur closely
associated with protein material with which they possess many proper-
ties in common. Each enzyme shows the greatest activity at a certain
temperature called the optimum temperature; there is also a minimum
and a maximum temperature for each specific enzyme. Their action
is inhibited by sufficiently lowering the temperature, and the enzyme, if
in solution, is entirely destroyed by subjecting it to a temperature of
ioo° C. The best known enzymes, whether derived from warm-blooded
or cold-blooded animals, are most active between 35°-45° C. The
nature of the surrounding media alters the velocity of the enzymatic
action, some enzymes being more active in acid solution whereas others
require an alkaline fluid.
Many of the more important enzymes do not occur preformed
within the cell, but are present in the form of a zymogen or mother-
substance. In order to yield the active enzyme this zymogen must be
transformed in a certain specific manner and by a certain specific sub-
stance. This transformation of the inactive zym.ogen into the active
enzyme is termed activation. For instance, the zymogen of the enzyme
pepsin of the gastric juice, termed pepsinogen, is activated by the hydro-
chloric acid secreted by the gastric cells (see p. 127), whereas the acti-
vation of the trypsinogen of the pancreatic juice is brought about by a
substance termed enterokinase^ (see p. 15). These are examples of
many well-known activation processes going on continually within the
animal organism. The agency which is instrumental in activating a
zymogen is generally termed a zymo-exciter or a kinase. In the cases
' Others seem to be like the substrate on which they act, e. g., carbohydrate.
^ According to Delezenne, trypsinogen may be rapidly activated by soluble calcium salts
ENZYMES AND THEIR ACTION. 7
cited hydrochloric acid would be termed a zymo-exciter and entero-
kinase would be termed a kinase.
After filterintT yeast juice, prej)ared by the Buchner process (see p.
i), through a Martin gelatin filter, Harden and Young showed that
the colloids left behind and the filtrate were both inactive fermenta-
tively. Upon treating the colloid material (enzyme) with some of the
filtrate, however, the mixture was shown to be able to bring about pro
nounced fermentation. It is believed that a co-enzyme present in the
filtrate was the eflScient agent in the transformation of the inactive
enzyme. It is necessary to make frequent renewals of the co-enzyme
in order to maintain continuous fermentation. It was further shown
that this co-enzyme, in addition to being diffusible, was not destroyed
by boiling and that it disappeared from yeast juice when this latter
w^as fermented or allowed to undergo autolysis. The exact nature of
this co-enzyme of zymase is unknown. The co-enzyme action, in this
case, is probably dependent upon the presence of two individual agencies,
one of which is phosphates.
It has been shown by Loevenhart that the property of acting as a
pancreatic lipase co-enzyme is vested in bile salts, and Magnus has
further shown that the synthetic salts are as efficient in this regard as
the natural ones. A few other instances of co-enzyme demonstrations
have been reported.
Electrolytes are very important factors in facilitating, or inhibiting
enzyme action. ^ For example, magnesium hydroxide inhibits the action
of salivary amylase" whereas the CI ion facilitates the action of this and
other amylases.^ In fact Bierry^ has very recently gone so far as to assert
that the presence of the CI or Br ion is ''absolutely essential to the activity
of pancreatic amylase."
The so-called "specificity" of enzyme action is an interesting and
important fact. That enzymes are very specific as to the character of
the substrate, or substance acted upon, is well known. Emil Fischer
investigated this problem of specificity extensively in connection with
the fermentation of sugars and reached the conclusion that enzymes,
with the possible exception of certain oxidases, can act only upon such
substances as have a specific stereo-isomeric relationship to themselves.
He considers that the enzyme and its substrate must have an inter-
relation, such as the key has to the lock, or the reaction does not occur.
Fischer was able to predict, in certain definite cases, from a knowledge
of the constitution and stereo-chemical relationships of a substance,
'For literature, see Kendall and Sherman: Jour, Am. Client. Soc, 32, 10S7, 1910.
'^ Bergeim and Hawk: Unpublished data.
'Wohlgemuth: Biochemische Zeitschrift, 9, 10, 1908.
*Bierry: Ibid., 40, 357, 1912.
8 PHYISOLOGICAL CHEMISTRY.
whether or not it would be acted upon by a certain enzyme. An appli-
cation of this specificity of enzyme action may be seen in the well-known
facts that certain enzymes act on carbohydrates, others on fats, and others
on protein; and, moreover, that the group of those which transform car-
bohydrates, for example, is further subdivided into specific enzymes each
of which has the power of acting alone upon some one sugar.
It has been conclusively shown, in the case of certain enzymes,^
at least, that their action is a reversible one and is, in all its main fea-
tures, directly analogous to the reversible reactions produced by chem-
ical means. For instance, in the saponification of ethyl-butyrate by
means of pancreatic lipase, it has been shown that upon the formation
of the end-products of the reaction, i. e., butyric acid and ethyl alcohol,
there is reversion^ and the reaction is stationary. This does not mean
CjH^COO.CjHj + H^O^i^CgH.COOH-FCsHsOH.
Ethyl butyrate. Butyric acid. Ethyl alcohol.
there are no chemical changes going on, but simply indicates that chem-
ical equilibrium has been established, and that the change in one direction
is counterbalanced by the change in the opposite direction. Pancreatic
lipase was one of the first enzymes to have the reversibility of its reaction
clearly demonstrated.^ A knowledge of the fact that lipase possesses
this reversibility of action is of extreme physiological importance and
aids us materially in the explanation of the processes involved in the diges-
tion, absorption, and deposition of fats in the animal organism (see
p. 141).
In respect to many enzymes it has been found that the law govern-
ing the action of inorganic catalyzers is directly applicable, i. e., that
the intensity is almost directly proportional to the concentration of the enzyme.
In the case of enzymes, however, there is a difference in that a maximum
intensity is soon reached and that subsequent concentration of the en-
zyme is productive of no further increase in intensity. The enzymes which
have been shown to obey this linear law are lipase, invertase, rennin, and
trypsin. In certain instances, where this law of direct proportionality
between the intensity of action and the concentration of enzyme does not
hold, it has been found that the Schiltz-Borissow law, first experimentally
demonstrated by E. Schutz, was applicable. This is to the effect that
the intensity is directly proportional to the square root of the concentra-
tion, or conversely, that the relative concentrations of enzymes are directly
proportional to the squares of the intensities}
' This is probably a general condition.
*The re-synthesis of ethyl-butyrate from its hydrolysis products. This may be indicated
thus:
'The principle was first demonstrated in connection with the enzyme maltase (see p. 62).
* This SchUtz-Borissow law is not generally applicable.
ENZYMES AND THEIR ACTION. 9
It has been shown that there are certain substances which possess
the property of directly inhibiting or preventing the action of a catalyzer.
These are called anti-catalyzers or paralyzers and have been compared
to the anti-toxins. Related to this class of anti-catalytic agents stand
the anti-enzymes. The first anti-enzyme to be reported was the anti-
rennin of Morgenroth. This was produced by injecting into an animal
increasing doses of rennet solution, whereupon an "anti" substance
was subsequently found both in the serum and in the milk, which
prevented the enzyme rennin from exerting its normal activity in the
presence of caseinogen. In other words, anti-rennin had been formed
in the serum of the animal,^ through the repeated injections of rennet
solution. Since the discovery of this anti-enzyme, anti-bodies have
been demonstrated for pepsin, trypsin, lipase, urease, amylase, laccase,
tyrosinase, emulsin, papain, and thrombin. According to Weinland,
the reason why the stomach does not digest itself is, that during life
there is present in the mucous membrane of the stomach an anti-enzyme
{anti-pepsin) which has the property of inhibiting the action of pepsin.
A similar substance (anti-trypsin) is present in the intestinal mucosa
as well as in the tissues of various intestinal worms. Some investigators
are not inclined to accept the enzyme nature of these inhibitory agents as
proven.
The very recent investigations of Ehrlich^ and of Neuberg^ have
served to cause a complete revision of our ideas regarding yeast fermenta-
tion. Ehrlich, for example, has shown that yeast will liberate ammonia
from amino acids and leave behind a non-nitrogenous complex. Among
these complexes amyl alcohol, succinic acid and others may be mentioned.
Thus, amyl alcohol results from the fermentation of leucine, whereas ethyl
alcohol results from the fermentation of sugar. Neuberg has demon-
strated the presence in the yeast of an enzyme termed carboxylase which has
the property of splitting off carbon dioxide from the carboxyl group of
amino and other aliphatic acids. The findings mentioned above constitute
the basis for much important work on so-called "sugar- free fermentation."
For a more extended consideration of enzymes the student is referred to
the following sources: —
Bayliss. — The Nature of Enzyme Action, Second Edition, Longmans,
Green and Co., New York and London.
DuCLAUX. — Traite de Microbiologie, Masson & Co., Paris.
Effront. — Enzymes and their Applications, Translated by Prescott,
Wiley and Sons, New York.
' Serum is normally anti-tryptic.
^Ehrlich: Biochemische Zeitschrift, 36, 477, 191 1.
^Neuberg and Collaborators: Biochemische Zeitschrift, 31, 170; 32, 323; 36 (60, 68, and
76), 1911.
lO PHYSIOLOGICAL CHEMISTRY.
EuLER. — (a) Allgemeine Chemie der Enzyme, Bergmann, Wiesbaden,
1910. (b) Ergebnisse der Physiologie, 1909-10.
Oppenheimer. — Die Fermente und Ihre Wirkungen, Dritte Auflage,
Vogel, Leipzig.
Samuely. — Handbuch der Biochemie des Menschen und der Thiere
(Oppenheimer), Gustav Fischer, Jena.
Vernon. — Intracellular Enzymes, Murray, London.
EXPERIMENTS ON ENZYMES AND ANTI-ENZYMES.
A. Experiments on Enzymes.^
I. AMYLASES.
1. Demonstration of Salivary Amylase.^ — To 25 c.c. of a one
per cent 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 of a test-tablet and test by the iodine test.^
If the blue color with iodine still forms after five minutes, add another
five drops of saliva. The opalescence of the starch solution should
soon disappear, indicating the formation of soluble starch {amidulin)
which gives a blue color with iodine. This body should soon be trans-
formed into erythro dextrin which gives a red color with iodine, and this,
in turn, should pass into achroodextrin which gives no color with iodine.
This point is called the achromic point. When this point is reached
test by Fehling's test* to show the production of a reducing substance
(maltose). A positive Fehling's test may be obtained whib the solution
still reacts red with iodine inasmuch as some sugar is formed from the
soluble starch coincidently with the formation of the erythrodextrin. For
further discussion of the transformation of starch see p. 61.
2. Demonstration of Pancreatic Amylase/ — ^Proceed exactly
as indicated above in the Demonstration of Salivary Amylase except
that the saliva is replaced by 5 c.c. of pancreatic extract prepared as
described on p. 153. Pancreatic amylase transforms the starch in a
manner entirely analogous to the transformation resulting from the
action of salivary amylase.
3. Preparation of Vegetable Amylase. — Extract finely ground
malt with water, filter and subject the filtrate to alcoholic fermentation
by means of yeast. When fermentation is complete filter off the yeast
' If it is deemed advisable Ijy the instructor to give all the practical work upon enzymes at
this point in the course, additional experiments will be found in Chapters III, VI and VIII.
^ For a discussion of this enzyme see p. 60.
^See p. 50.
^See p. 32.
' For a discussion of this enzyme see p. 1 50.
ENZYMES AND THEIR ACTION. II
and precipitate the amylase from the filtrate by the addition of alcohol.
The precipitate may be filtered off and obtained in the form of a fmc white
powder.
4. Demonstration of Vegetable Amylase. — This enzyme may
be demonstrated according to the directions given under Demonstra-
tion of Salivary Amylase, p. 10, with the exception that the saliva used
in that experiment is replaced by an aqueous solution of the vegetable
amylase powder prepared as described above. ^
II. PROTEASES.
1. Preparation of Gastric Protease.- — Treat the finely com-
minuted mucosa of a pig's stomach with 0.4 per cent hydrochloric
acid and extract at 38° C. for 24-48 hours. The filtrate from this mix-
ture constitutes a very satisfactory acid extract of gastric protease (see
p. 130).
2. Demonstration of Gastric Protease. — Introduce some pro-
tein material (fibrin, coagulated egg-white, or washed lean beef) into
the acid extract of gastric protease prepared as above described,-^ add
an equal volume of 0.4 per cent hydrochloric acid and place the mix-
ture at 38° C. for 2-3 days. Identify the products of digestion according
to directions given on p. 130.
3. Preparation of Pancreatic Protease.^ — A satisfactory ex-
tract of this enzyme may be made from the pancreas of a pig or sheep
according to the directions given on p. 153.
4. Demonstration of Pancreatic Protease. — Into an alkaline
extract of pancreatic protease,^ prepared as directed on p. 153, introduce
some fibrin, coagulated egg-white or lean beef and place the mixture
at 38° C. for 2-5 days." At the end of that period separate and identify
the end-products of the action of pancreatic protease according to the
directions given on p. 153.
5. Demonstration of a Vegetable Protease. — A commercial
preparation of papain {papayotin, carase or papase), the protease of the
fruit of the pawpaw {carica papaya), may be used in this connection.
Follow the same procedure as that described under Gastric Protease
(see above).
* If desired the first aqueous extract of the original malt may be used in this demonstration.
Commercial taka-diastase may also be employed.
-Also called pepsin, pepsase, gastric proteinase, and acid protease. For a discussion of this
enzyme see p. 127.
^ If so desired, a solution of commercial pepsin powder in 0.2 per cent, hydrochloric acid
may be substituted.
* .\lso called trypsin, trypsase, pancreatic proteinase and alkali proteinase. For a discussion
of this enzyme see p. 149.
W 0.25 per cent sodium carbonate solution of commercial trypsin may be substituted.
*.\ few c.c. of toluol or an alcoholic solution of thymol should be added to prevent
putrefaction.
12 PHYSIOLOGICAL CHEMISTRY.
It has been demonstrated by Mendel and Blood ^ that the presence of
HCN will accelerate the proteolytic activity of papain. It is suggested
that the HCN acts as a so-called co-enzyme (see p. 7).
. Vines- believes that "papain" consists of a mixture of two enzymes,
a pepsin and an erepsin. Mendel and Blood do not consider the evidence
on this point as conclusive.
III. LIPASES.
I. Preparation of Pancreatic Lipase.^ — An extract of this en-
zyme may be prepared from the pancreas of the pig or sheep accord-
ing to the directions given on p. 153.*
.2. Demonstration of Pancreatic Lipase. — Into each of two test-
tubes introduce 10 c.c. of milk and a small amount of litmus powder.
To the contents of one tube add 3 c.c. of a neutral extract of pancreatic
lipase and to the contents of the other tube add 3 c.c. of a boiled neutral ex-
tract of pancreatic lipase. Keep the tubes at 38° C. and watch for color
changes. The blue color of the litmus powder will gradually give place
to a red. This change in color of the litmus from blue to red has been
brought about by the fatty acid which has been produced through the
lipolytic action exercised by the lipase upon the milk fats.
3. Preparation of Vegetable Lipase. — This enzyme may be
readily prepared from castor beans, several months old, by the following
procedure:^ Grind the shelled beans very fine® and extract for twenty-
four-hour periods with alcohol-ether and ether, in turn. Reduce the semi-
fat-free material to the finest possible consistency by means of mortar
and pestle and pass this material through a sieve of very fine mesh. Place
this material in a Soxhlet extractor and extract for one week. This
fat-free powder may then be used to demonstrate the action of vegetable
lipase. Powder prepared as described may be used in quantitative tests.
For ordinary qualitative tests it is not necessary to remove the last traces
of fat and therefore the extraction period in the Soxhlet apparatus may
be much shortened.
4. Demonstration of Vegetable Lipase. — The lipolytic action
of the lipase prepared from the castor bean, as just described, may
be demonstrated in a manner entirely analogous to that used in the
Demonstration of Pancreatic Lipase, see above. Proceed as indicated
' Mendel and Blood: Journal of Biological Chemistry, 8, 177, 1910.
'Vines: Annals of Botany, 19, 174, 1905.
'Also called steapsin. For a discussion of this enzyme see p. 151. A very active lipolytic
extract may also be prepared from the liver.
*If preferred, a glycerol extract may be prepared according to the directions given by
Kanitz; Zeitschriftfur physiologische Chemie, 1906, 46, p. 4cS2.
* A. E. Taylor: On Fermetitalion; University of California Publications, 1907.
* The shells should be removed without the use of water. These beans are poisonous, due
to their content of ricin.
ENZYMES AND THEIR ACTION, 1 3
in that experiment and substitute the vegetable lipase powder for the
neutral extract of pancreatic lipase. The type of action is entirely
analogous in the two instances.
An experiment similar to Experiment 2, p. 157, may also be tried
if desired. In this experiment 0.2 c.c. of cither ethyl butyrate or amyl
acetate may be employed.
IV. INVERTASES.'
1. Preparation of an Extract of Sucrase.- — Treat the finely
divided epithelium of the small intestine of a dog, pig, rat, rabbit, or hen
with about three volumes of a two per cent solution of sodium fluoride
and permit the mixture to stand at room temperature for twenty-four
hours. Strain the extract through cloth or absorbent cotton and use the
strained material in the following demonstration.
2. Demonstration of Sucrase. — To about 5 c.c. of a one per cent
solution of sucrose, in a test-tube, add about one cubic centimeter of a
two per cent sodium fluoride intestinal extract, prepared as described
above. Prepare a control tube in which the intestinal extract is boiled
before being added to the sugar solution. Place the two tubes at 2,^° C.
for two hours.^ Heat the mixture to boiling to coagulate the protein
material, filter, and test the filtrate by Fehling's test (see p. 32). The
tube containing the boiled extract should give no response to Fehling's
test, whereas the tube containing the imhoiled extract should reduce the
Fehling's solution. This reduction is due to the formation of invert
sugar (see p. 46) from the sucrose through the action of the enzyme su-
crase which is present in the intestinal epithelium.
3. Preparation of Vegetable Sucrase. — Thoroughly grind about
100 grams of brewer's yeast in a mortar with sand. Spread the ground
yeast in thin layers on glass or porous plates and dry it rapidly in a current
of dry, warm air. Powder this dry yeast, extract it with distilled water
and filter. Pour the filtrate into acetone, stir and after permitting the
acetone mixture to stand for a few minutes filter on a Buchner funnel.
The resulting precipitate, after drying and pulverizing, may be used to
demonstrate vegetable sucrase.
4. Demonstration of Vegetable Sucrase. — To about 5 c.c. of a one
per cent solution of sucrose in a test-tube add a small amount of the
sucrase powder prepared as directed above. Place the tube at 2,^° C.
for 24-72 hours and at the end of that period test the solution by Fehling's
test. Reduction indicates that the active sucrase powder has transformed
' The invertirig enzymes of the alimentary tract; Mendel and Mitchell: American Journal of
Physiology, 20, 81, 1907-08.
* For a discussion of this enzyme see p. 152.
' If a positive result is not obtained in this time permit the digestion to proceed for a longer
period.
14 PHYSIOLOGICAL CHEMISTRY.
the non-reducing sucrose into dextrose and Isevulose, and these sugars,
in turn, have reduced the Fehling solution.
5. Preparation of an Extract of Lactase.^ — Treat the finely
divided epithelium of the small intestine of a kitten, puppy, or pig embryo
with about three volumes of a two per cent solution of sodium fluoride
and permit the mixture to stand at room temperature for twenty-four
hours. Strain the extract through cloth or absorbent cotton and use
the strained material in the following demonstration.
6. Demonstration of Lactase.^ — To about 5 c.c, of a one per
cent solution of lactose in a test-tube add about one cubic centimeter
of a toluol-water or a two per cent sodium fluoride extract of the first
part of the small intestine^ of a kitten, puppy, or pig embryo prepared as
described above. Prepare a control tube in which the intestinal
extract is boiled before being added to the sugar solution. Place the two
tubes at 38° C. for 24 hours. At the end of this period add one cubic
centimeter of the digestion mixture to 5 c.c. of Barfoed's* reagent and
place the tubes in a boiling water-bath.^ Examine the tubes at the end of
three minutes against a black background in a good light. If no cuprous
oxide is visible replace the tubes and repeat the examination at the end
of the fo^irth and fifth minutes. If no reduction is then observed permit
the tubes to stand at room temperature for 5-10 minutes and examine
again.®
It has been determined that disaccharide solutions will not reduce
Barfoed's reagent until after they have been heated for 9-10 minutes on a
boiling water-bath in contact with the reagent.^ Therefore in the above
test, if the tube containing the unboiled extract exhibits any reduction
after being heated as indicated, for a period of five minutes or less, and
the control tube containing boiled extract shows no reduction, it may be
concluded that lactase was present in the intestinal extract.^
7. Preparation of an Extract of Maltase.^^ — Treat the finely
divided epithelium of the small intestine of a cat, kitten, or pig {embryo or
adult) with about three volumes of a two per cent solution of sodium
fluoride and permit the mixture to stand at room temperature for twenty-
1 For a discussion of this enzyme see p. 152.
^Roaf; Bio-Chemical Journal, 3, 182, 1908.
' Duodenum and first part of jejunum.
* To 4.5 grams of neutral crystallized copper acetate in 900 c.c. of water, add 0.6 c.c. of
glacial acetic acid and make the total volume of the solution one liter.
* Care should be taken to see that the water in the bath reaches at least to the upper level
of the contents of the tubes.
" Sometimes the drawing of conclusions is facilitated by pouring the mixture from the tube
and examining the bottom of the tube for adherent cuprous oxide.
'The heating for 9-10 minutes is sufficient to transform the disaccharide into mono-
saccharide.
* The reduction would, of course, be due to the action of the dextrose and galactose which
had been formed from the lactose through the action of the enzyme lactase.
" For a discussion of this enzyme see p. 62.
ENZYMES AND THEIR ACTION. 1 5
four hours. Strain the extract through cloth and use the strained material
in the followinfi; demonstration.
8. Demonstration of Maltase. — Proceed exactly as indicated in the
demonstration of lactase, above, except that a one per cent solution of
maltose is substituted for the lactose solution. The extract used may be
prepared from the upper part of the intestine of a cat, kitten, or pig {embryo
or adult). In the case of lactase, as indicated, the intestine used should
be that of a kitten;* puppy, or pig {embryo).
V. EREPSm.i
1. Preparation of Erepsin. — Grind the mucous membrane of the
small intestine of a cat, dog, or pig with sand in a mortar. Treat the
mortared membrane with toluol- or chloroform-water and permit the
mixture to stand, with occasional shaking, for 24-72 hours. ^ Filter the
extract thus prepared through cotton and use the filtrate in the following
experiment.
2. Demonstration of Erepsin. — To about 5 c.c. of a one per cent
solution of Witte's peptone in a test-tube add about i c.c. of the erepsin
extract prepared as described above and make the mixture slightly
alkaline (o.i per cent) with sodium carbonate. Prepare a second tube
containing a like amount of peptone solution but boil the erepsin extract
before introducing it. Place the two tubes at 38° C. for 2-3 days. At
the end of that period heat the contents of each tube to boiling, filter and try
the biuret test on each filtrate. In making these tests care should be taken
to use like amounts of filtrate, potassium hydroxide and copper sulphate
in each test in order that the drawing of correct conclusions may be
facilitated. The contents of the tube which contained the boiled extract
should show a deep pink color with the biuret test, due to the peptone still
present. On the other hand, the biuret test upon the contents of the tube
containing the unboiled extract should be negative or exhibit, at the most, a
faint pink or bhie color, signifying that the peptone, through the influence
of the erepsin, has been transformed, in great part at least, into amino
acids which do not respond to the biuret test.^
3. The Glycyl-Tryptophane Reaction. — The dipeptide glycyl-
tryptophane* may be used in place of the peptone solution for the
demonstration of erepsin. It is used widely in the diagnosis of gastric
cancer. It has been found that a peptide-splitting enzyme (erepsin) is
' For a discussion of this enzyme see p 152.
^ The enzyme may also be e.xtracted by means of glycerol or alkaline " physiological" salt
solution if desired.
^Strictly speaking, this erepsin demonstration is not adequate unless a control test is made
with native protein (except caseinogen, histones and protamines) to show that the extract is
trypsin-free and digests peptone but not native protein.
^ This dipeptide is sold commercially under the name "Ferment Diagnosticon."
l6 PHYSIOLOGICAL CHEMISTRY,
present in the stomach contents of individuals suffering from cancer
of the stomach, whereas the stomach contents of normal individuals
contains no such enzyme. The glycyl-tryptophane test, therefore, furn-
ishes a means of aiding in the diagnosis of this disorder. As applied to
stomach contents, the test is as follows:^ Introduce about lo c.c. of the
filtrate from the stomach contents into a test-tube, add a little glycyl-
tryptophane, and a layer of toluol and place the tube in an incubator
at 38° C. for 24 hours. At the end of this time by means of a pipette
transfer 2-3 c.c. of the fluid from beneath the toluol to a test-tube,
add a few drops of 3 per cent acetic acid and carefully introduce bromine
vapors. Shake the tube and note the production of a red color if
tryptophane is present. The tryptophane has, of course, been liberated
from the peptide through the action of the peptide-splitting enzyme
(erepsin) elaborated by the cancer tissue.
If an excess of bromine is added the color will vanish. If no rose
color is noted, add more bromine vapors carefully with shaking until
further addition of the vapors causes the production of a yellowish color.
This indicates an excess of bromine and constitutes a negative test.
Occasionally the rose color indicating a positive test is so transitory as to
escape detection unless the test be very carefully performed.
VI. URICOLYTIC ENZYME.2
1. Preparation of Uricase (Uricolytic Enz3rme). — Extract pulped
liver tissue with toluol- or chloroform-water at 2)^° C. for 24 hours, with
occasional shaking. Filter the extract and use the filtrate in the following
experiment.
2. Demonstration of Uricase (Uricolytic Enzyme). — Add about
0.1 gram of uric acid to 10 c.c. of water and bring the uric acid into solu-
tion by the addition of the minimal quantity of potassium hydroxide.
To 5 c.c. of this uric acid solution, in a test-tube, add 5 c.c. of the uricolytic
enzyme extract prepared as described above. Prepare a second tube
containing a like amount of uric acid solution, but boil the extract before
it is introduced. Place the two tubes at T)d)° C. for 3-4 days and titrate
the two digestive mixtures with a solution of potassium permanganate
according to directions given under Folin-Schaffer Method, Chapter
XXII. It will be found that the mixture containing the boiled extract
requires a much larger volume of the permanganate to complete the
titration than the other tube. This indicates that a uricolytic enzyme
has destroyed at least a portion of the uric acid which was originally
present in the tube containing the unboiled extract.
* Neubauer and Fischer; Deutsches Archiv f. klinische Medizin, 97, 499, 1909.
* Mendel and Mitchell; American Journal of Physiology, 20, 97, 1908.
ENZYMES AND THEIR ACTION. 1 7
VII. CATALASE.
Demonstration of Catalase. — The various animal tissues, such
as liver, kidney, blood, lung, muscle and brain, contain an enzyme called
catalase which possesses the property of decomposing hydrogen peroxide.
The presence of this enzyme may be demonstrated as follows: Introduce
into a low, broad, wide-mouthed bottle some pulped liver tissue and a
porcelain crucible containing neutral hydrogen peroxide.^ Connect the
bottle with a eudiometer filled with water, upset the crucible of
hydrogen peroxide upon the liver pulp and note the collection of gas
in the eudiometer. This gas is oxgyen which has been liberated from
the hydrogen peroxide through the action of the catalase of the liver
tissue.
See p. 23 for a method for the quantitative determination of catalase
based on the above principle.
B. Experiments on Anti-Enzymes.
1. Preparation of an Extract of Anti-Pepsin.^ — Grind up a
number of intestinal worms (ascaris)^ with quartz sand in a mortar.
Subject this mass to high pressure, filter the resultant juice and treat
it with alcohol until a concentration of sixty per cent is reached. If
any precipitate forms it should be filtered oflf* and alcohol added to the
filtrate until the concentration of alcohol is 85 per cent, or over. The
anti-enzyme is precipitated by this concentration. Permit this precipitate
to stand for twenty-four hours, then filter it off, wash it with 95 per cent
alcohol, absolute alcohol, and ether, in turn, and finally dry the substance
over sulphuric acid. The sticky powder which results may be used in
this form or may be dissolved in water as desired and the aqueous
solution used.^
2. Demonstration of Anti-Pepsin.® — Introduce into a test-tube
a few fibrin shreds and equal volumes of pepsin-hydrochloric acid''
and ascaris extract made as indicated above. Prepare a control tube
in which the ascaris extract is replaced by water. Place the tubes at
^S° C. Ordinarily in one hour the fibrin in the control tube will be com-
pletely digested. The fibrin in the tube containing the ascaris extract
* Mendel and Leavenworth; American Journal of Physiology, 21, 85, 1908.
^ Anti-gastric-protease or anti-acid-protease.
' These may be readil\- obtained from pigs at a slaughter house.
* This precipitate consists of impurities, the anti-enzyme not being precipitated until a
higher concentration of alcohol is reached.
* The original ascaris extract possesses much greater activity' than either the powder or
the aqueous solution.
* Martin H. Fischer; Physiology of Alimentation, 1907, p. 134.
' Made by bringing 0.015 gram of pepsin into solution in 7 c c. of water and o. 23 gram of
concentrated hydrochloric acid.
16 PHYSIOLOGICAL CHEMISTRY.
may, however, remain unchanged for days, thus indicating the inhibitory
influence exerted by the anti-enzyme present in this extract.
3. Preparation of an Extract- of Anti-Trypsin/ — The extract
may be prepared from the intestinal worm, ascaris, according to the
directions given on page 17.
4. Demonstration of Anti-Trypsin. — Introduce into a test-tube
a few shreds of iibrin and equal volumes of an artificial tryptic solution^
and the ascaris extract made as described on page 17. Prepare a control
tube in which the ascaris extract is replaced by water. Place the two
tubes at 38° C. Ordinarily the fibrin in the control tube will be com-
pletely digested in two hours. The fibrin in the tube containing the
ascaris extract may, however, remain unchanged for days, thus indicating
the inhibitory influence of the anti-enzyme.
Blood serum also contains anti-trypsin. This may be demonstrated
as follows: Introduce equal volumes of serum and artificial tryptic solu-
tion (prepared as described above) into a test-tube and add a few shreds of
fibrin. Prepare a control tube containing boiled serum. Place the two
tubes at 7,8° C. It will be observed that the fibrin in the tube containing
the boiled serum digests, whereas that in the other tube does not digest.
The anti-trypsin present in the unboiled serum has exerted an inhibitory
influence upon the action of the trypsin.
C. Quantitative Applications.
I. Quantitative Determination of Amylolytic Activity. — Wohl-
gemuth's Method.^ Arrange a series of test-tubes with diminishing
quantities of the enzyme solution under examination, introduce into each
tube 5 c.c. of I per cent solution of soluble starch^ and place each tube at
once in a bath of ice-wafer.^ When all the tubes have been prepared in
this way and placed in the ice-water bath they are transferred to a water-
bath or incubator and kept at 38° C. for from thirty minutes to an hour.**
' Anti-pancreatic-protease or anli-alkali-protease.
^ Made by dissolving 0.04 gram of sodium carbonate and 0.015 gram of trypsin in 8 c.c.
of water.
^ Wohlgemuth; Biochemische Zeitschrift, 9, i, igo8.
* Kahlbaum's soluble starch is satisfactory. In preparing the i per cent, solution, the
weighed starch powder should be dissolved in cold distilled water in a casserole and stirred
until a homogeneous suspension is obtained. The mixture should then be heated, with con-
stant stirring, until it is clear. This ordinarily takes about 8-10 minutes. .\ slightly opaque
solution is thus obtained which should be cooled and made up to the proper volume before
using.
* Ordinarily a series of six tubes is satisfactory, the volumes of the enzyme solution used
ranging from i c.c. to o.i c.c. and the measurements being made by means of a i c.c. gradu-
ated pipette. Each tube should be placed in the ice- water bath as soon as the starch solution is
introduced. It will be found convenient to use a small wire basket to hold the tubes.
" Longer digestion periods may be used where it is deemed advisable. If exceedingly
weak solutions are being investigated, it may be most satisfactory to permit the digestion to
extend over a period of 24 hours.
ENZYMES AND THEIR ACTION. I9
At the end of this digestion period the tubes are again removed to
the bath of ice-water in order that the action of the enzyme may be
stopped.
Dilute the contents of each tube, to within about one-half inch of the
top, with water, add one drop of a N/io solution of iodine and shake the
tube and contents thoroughly. A series of colors ranging from dark blue
through bluish-violet and reddish-yellow to yellow, will be formed.' The
dark blue color shows the presence of unchanged starch, the bluish-violet
indicates a mixture of starch and erythrodextrin, whereas the reddish-
yellow signifies that erythrodextrin and maltose are present and the
yellow solution denotes the complete transformation of starch into maltose.
Examine the tubes carefully before a white background and select the
last tube in the series which shows the entire absence of all blue color, thus
indicating that the starch has been completely transformed into dcxtrins
and sugar. In case of indecision between two tubes, add an extra drop of
the iodine solution, and observe them again, after shaking.
Calculation. — The amylolytic activity' of a given solution is expressed
in terms of the acti\ity of i c.c. of such a solution. For example, if it is
found that 0.02 c.c. of an amylolytic solution, acting at 38° C, completely
transformed the starch in 5 c.c. of a i percent starch solution in 30 minutes,
the amylolytic activity of such a solution would be expressed as follows:
Dll' = 2So.
This indicates that i c.c. of the solution under examination possesses the
power of completely digesting 250 c.c. of i per cent starch solution in 30
minutes at t,8° C.
Wohlgemuth has suggested a slight alteration in the above procedure
for use in the determination of the amylase content of the feces. ^ A mod-
ification of the Wohlgemuth procedure* for this purpose is given in
the latter part of the chapter on Feces.
2. Quantitative Determination of Peptic Activity. — (a) Mett's
Method. — The determination of the actual rate of peptic actix-ity is a most
important procedure under certain conditions. Several methods of
making this determination are in use. The method of Sprigg^ is probably
the most accurate method yet devised for this purpose. It is, however,
too complicated and time-consuming for clinical purposes. The method
of Mett, given below, is very simple although not strictly accurate. The
procedure is as follows: To about 5 c.c. of the gastric juice under exami-
' See p. 61.
- Designated by "D" the first letter of "diastatic."
^ Wohlgemuth; Berliner klinische Wochenschrift, 47, 92, 1910.
* Hawk; .Archives of Internal Medicine, 8, 552, 191 1.
*Sprigg: Zeitschrift fiir physiologische Chemie, 35, 465, 1902.
20 PHYSIOLOGICAL CHEMISTRY.
nation in a test-tube add 1-3 sections of a Mett tube^ and place the mixture
at 38° C. for ten hours. At the end of this period, the tube should be re-
moved from the gastric juice and the length of the column of coagulated
albumin which has been digested carefully determined by means of a low-
power microscope and a millimeter scale. In normal human gastric
juice the upper limit is 4 mm. However, control tests should always be
made to determine the digestibility of the coagulated albumin in artificial
gastric juice, inasmuch as this factor will vary with different albumin
preparations. This fact of the variation in the digestibility has been
emphasized by the recent work of Frank. ^ This investigator demon-
strated that the digestibility of the egg albumin in the Mett tube would
vary according to the temperature at which the albumin was coagulated.
Therefore in making a series of comparative tests the albumin in the
Mett tubes should be coagulated under uniform conditions in order to
insure accuracy.
In connection with this test the Schiitz-Borissow law should be borne
in mind. This principle is to the effect that the amount of proteolytic
enzyme present in a digestion mixture is proportional to the square of the
number of millimeters of albumin digested. Therefore a gastric juice which
digests 2 mm. of albumin contains /owr times as much pepsin as a gastric
juice which digests only i mm. of albumin. And further, if the quantities
of albumin digested are 2 mm. and 3 mm., respectively, the ratio between
the pepsin values will be as 4 : 9.
It is claimed by Nirenstein and Schiff^ that the principle of Schiitz does
not apply to gastric juice unless this fluid be diluted with fifteen volumes
of N/20 hydrochloric acid.
(6) Fuld and Levison's Method. — This test is founded upon the fact,
shown by Osborne, that edestin when brought into solution in dilute acid
will change in its solubility, due to the contact with the acid, and that a
protean called edestan, which is insoluble in neutral fluid, will be formed.
The procedure is as follows: Dilute the gastric juice under examination
with 20 volumes of water and introduce gradually decreasing volumes of the
diluted juice into a series* of narrow test-tubes about i cm. in diameter.
' In the preparation of these tubes, egg-white is diluted with an equal volume of water,
the precipitated globulin filtered off and the filtrate collected in a tall, narrow beaker or a
large test-tube. A bundle of capillary tubes about lo cm. in length and 2 mm. in diameter
are now placed in this vessel in such a manner that they are completely submerged in the
albumin solution. After an examination has sh^nvn that the tubes are completely filled with
the albumin solution and that there are no interfering air-bubbles, the vessel and its contained
tubes is heated for 5-15 minutes in a boiling water-bath, in order to coagulate the albumin.
When this coagulation is complete, the tubes are removed, all albumin adhering to them is
carefully cleaned off, and the tubes rendered air-tight by the application of sealing wax at
either end. When needed for use, these tubes are cut into sections about 2 cm. in length.
^ Frank: Journal 0/ Biological Chemistry, g, 463, 191 1.
* Nirenstein and Schiff; Archiv. fur Verdauungekrankheiten, 8, 559, 1902.
* The longer the series, the more accurate the deductions which may be drawn.
ENZYMES AND THEIR ACTION. 21
The measurements of gastric juice may conveniently be made with a i
c.c. pipette which is accurately graduated in i/ioo c.c. Into the first
tube in the scries may be introduced i c.c. of gastric juice, and the tubes
which follow in the scries may receive volumes which differ, in each
instance, from the volume introduced into the preceding tube by i/ioo,
1/50, 1/20, or i/io of a cubic centimeter. Now rapidly introduce into
each tube the same volume {e. g., 2 c.c.) of a i : 1000 solution of edestin^
and place the tGbes at 40° C. for one-half hour. At the end of this time
stratify ammonium hydroxide upon the contents of each tube,^ place
the tubes in position before a black background and examine them
carefully. The ammonium hydroxide, by diffusing into the acid fluid,
forms a neutral zone and in this zone will be precipitated any undigested
edestan which is present. Select the tube in the series which contains the
least amount of gastric juice and which exhibits no ring, signifying that the
the edestan has been completely digested, and calculate the peptic activity
of the gastric juice under examination on the basis of the volume of
gastric juice used in this particular tube.
Ca/cw/a//(W.^Multiply the number of c.c. of cdestin solution used by
the dilution to which the gastric juice was originally subjected and divide
the volume of gastric juice necessary to completely digest the edestan by
this product. For example, if 2 c.c. of the edestin solution was com-
pletely digested by 0.25 c.c. of a 1:20 gastric juice we would have the
following expression: 0.25^20X2 or 1:160. This peptic activity may
be expressed in several ways, e. g., (a) 1:160 pepsin; (b) 160 pepsin con-
tent; (c) 160 parts.
(c) Rose's Modification^ of the Jacoby-Solms Method/— Dissolve
0.25 gram of the globulin of the ordinary garden pea,® Pisum sativum, in
* This edestin should be prepared in the usual way (see p. 109), and brought into solution
in a dilute hydrochloric acid of approximately the same strength as that which occurs normally
in the human stomach. This may be conveniently made by adding 30 c.c. of N/io hydro-
chloric acid to 70 c.c. of water. Ordinarily it should not take longer than one minute to
introduce the edestin solution into the entire series of tubes. However, if the edestin is added
to the tubes in the same order as the ammonium hydroxide is afterward stratified, no appreci-
able error is introduced
^ Making the stratification in the same order as the edestin solution was added.
^ Rose: Archives of Internal Medicine, 5, 459, 1910.
* Sohns: Zeitschrift fiir klinische Medizin, 64, 159, 1907.
* The globulin may be prepared as follows: "The finely ground peas, freed as much as
possible from the outer coating, are repeatedly extracted with large quantities of 10 per cent
sodium chloride solution, the extracts combined, strained through fine bolting-cloth, and
allowed to stand over night in large cylinders to deposit insoluble matter. The supernatant
fluid is siphoned off and saturated with ammonium sulphate. The precipitate of albumin and
globulin is filtered off, suspended in a litUe water, and dialyzed in running water for three days,
until the salt has been removed, and the albumins have been dissolved. The globulins are
filtered off and washed two or three times to remove the last trace of albumins. To purify
further, the precipitate is extracted with 10 per cent sodium chloride solution, and filtered
until perfectly clear. The resulting solution is neutralized to litmus paper by the cautious
addition of dilute sodium hydroxide, and again dialyzed in running water for three days to
remove the salts completely. The precipitated globulins are then filtered off and dried on a
water-bath at 40° C. During the entire process of separation the proteins should be preserved
with a mixture of alcoholic thymol and toluol." This dried globulin is used in the clinical
procedure.
22 PHYSIOLOGICAL CHEMISTRY.
loo c.c. of lo per cent sodium chloride solution, warming slightly if
necessary/ Filter and introduce i c.c. of the clear filtrate into each of a
scries of six^ test-tubes about i cm. in diameter. Introduce into each
tube I c.c. of 0.6 per cent hydrochloric acid and permit a period of about
five minutes to elapse for the development of the turbidity. Make a
known volume of the gastric juice (5-10 c.c. is sufl&cient) exactly neutral
to litmus paper with dilute alkali; and record the volume of the alkali so
used. If acid metaprotein precipitates, filter it off; if there is no precipi-
tate proceed without filtration. Dilute the clear neutral solution with a
known quantity of distilled water (usually five volumes) making proper
allowance for the volume of alkali used in the neutralization. Boil 5-10
c.c. of the diluted juice, filter and add the following decreasing volumes
(c.c.) to the series of six tubes: i.o, 0.9, 0.7, 0.5, 0.2, 0.0. Make the
measurements by means of a i c.c. pipette graduated in o.oi c.c. Now
rapidly introduce the imboiled, diluted juice in the following increasing
volumes (c.c.) in order: 0.0, o.i, 0.3, 0.5, 0.8, i.o. Each tube now contains
a total volume of 3 c.c. and a total acidity of 0.2 per cent hydrochloric
acid. Shake each tube thoroughly and place them at 50-52° C. for fifteen
minutes or at 35-36° C. for one hour. Examine the series of tubes at the
end of the digestion period and select that tube which contains the smallest
quantity of gastric juice and which shows no turbidity. The volume of
the juice used in this tube is taken as the basis for the calculation of the
peptic activity.
Calculation. — The peptic activity is expressed in terms of i c.c. of the
undiluted juice. For example, if it requires 0.5 c.c. of the diluted juice
(five-fold dilution) to clear up the turbidity in i c.c. of the globulin solu-
tion in the proper experimental time interval (15 minutes or one hour
according to temperature) the peptic activity would be expressed as
follows :
(I-^o.5)X5 = Io (peptic activity) .
According to this scale of pepsin units 10 may be considered as
"normal" peptic activity. These units are about i/io as large as those
expressed by the Jacoby-Solms scale.
Inasmuch as it has been shown^ that blood serum contains an anti-
pepsin it is advisable to test the gastric juice for blood before determining
its proteolytic power.
3. Quantitative Determination of Tryptic Activity. — Gross'
Method. — This method is based upon the principle that faintly alkaline
' This solution may be preserved at least two months under toluol.
^ A longer series of tubes may be used if desired. However, experience has shown that a
series of six ordinarily affords sufTicient range for all diagnostic purposes.
^ Oguro: Biochemische Zeitschrifl, 22, 266, 1909.
ENZYMES AND THEIR ACTION. 23
solutions of casein are precipitated upon the addition of dilute (i per cent)
acetic acid whereas its digestion products are not so precipitated. The
method follows: Prepare a series of tubes each containing lo c.c. of a
0.1 per cent solution of pure, fat-free casein/ which has been heated to a
temperature of 40° C. Add to the contents of the series of tubes increas-
ing amounts of the trypsin solution under examination,^ and place them
at 40° C. iov fifteen minutes. At the end of this time remove the tubes and
acidify the conteftts of each with a few drops of dilute (i per cent) acetic
acid. The tubes in which the casein is completely digested will remain
clear when acidified, while those tubes which contain undigested casein
will become more or less turbid under these conditions. Select the first
tube in the series which exhibits no turbidity upon acidification, thus
indicating complete digestion of the casein, and calculate the tryptic
acti\ity of the enzyme solution under examination.
Calculatian. — The unit of tryptic activity is an expression of the power
of I c.c. of the fluid under examination exerted for a period of fifteen
minutes on 10 c.c. of a o.i per cent casein solution. For example, if 0.5
c.c. of a trypsin solution completely digests 10 c.c. of a o.i per cent solu-
tion of casein in fifteen minutes the acti\'ity of that solution would be
expressed as follows:
Tryptic acti\'ity== i-=-o.5 = 2.
Such a trypsin solution would be said to possess an activity of 2. If
0.3 c.c. of the trypsin solution had been required the solution would be
said to possess an activity of 2,-y, i- ^•, i -=-o-3 ==3-3-
4. Quantitative Determination of Catalase.^^In the determination
of the catalase values of tissues weighed portions of the tissue under
examination should be ground with sand in a mortar then treated with
four volumes of chloroform water and permitted to extract for 24 hours at
room temperature. An apparatus such as that shown in Fig. i may be
employed in determining the catalase values. The main features of the
apparatus are based upon those of a delivery funnel for introducing liquids
under increased or diminished pressure.
In making a determination introduce a measured volume (1-4 c.c.)
of the filtered extract* into the small fliask and insert the modified Johnson
burette graduated to 5 c.c. and containing 50 c.c. of hydrogen peroxide
(Oakland dioxygen neutral^ to congo red) into the neck of the flask.
* Made by dissolving one gram of Griibler's casein in a liter of o.i per cent sodium car-
bonate. A little chloroform may be added to prevent bacterial action.
^ The amount of solution used may var}' from o.i-i c.c. The measurements may con-
veniently be made by means of a i c.c. graduated pipette.
'Hawk: Journal of American Chemical Society, ^;j, 425, 191 1.
* If less than 4 c.c. of extract are used the volume should be made up to 4 c.c. by the addi-
tion of distilled water.
* An acid reaction modifies the rate of the oxygen evolution. (See Mendel and Leaven-
worth, American Journal of Physiology, 21, 85, 1908.)
24
PHYSIOLOGICAL CHEMISTRY,
Shake the contents of the flask briskly^ and record the volume of oxygen
evolved in a two-minute period taking readings at intervals of fifteen
seconds.
Calculation. — When a series of comparative tests are made on different
tissues or on the same tissue under different conditions it is considered
satisfactory to make a comparison of the catalase values upon the basis
Fig. I. — ^Apparatus for Quantitative Determination of Catalase.
of the volume of oxygen evolved in a period of two minutes from 5 c.c. of
neutral hydrogen peroxide by means of i c.c. of a 1:4 chloroform-water
extract of the tissue.
' In making a series of comparative tests it is essential that tlie shaking process should be
uniform from determination to determination.
CHAPTER II.
CARBOHYDRATES.
The 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 proteins. They
are called carbohydrates because 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 inosite, 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^H^fi^.
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 oxygen atoms present in the molecule, e. g.,
trioses, pentoses, and hexoses.
The more common carbohydrates may be classified as follows:
I. Monosaccharides.
1. Hexoses, CgHjjOg.
(a) Dextrose.
(b) Laevulose.
(c) Galactose.
2. Pentoses, CgHj^O..
(a) Arabinose.
(b) Xylose.
(c) Rhamnose (Methyl-pentose), CgHj^Og.
II. Disaccharides, C^^li^^O^^.
1. Maltose.
2. Lactose.
3. Iso-Maltose.
4. Sucrose.
III. Trisaccharides, C^^H^fi^^.
I. Raffinose.
25
26 PHYSIOLOGICAL CHEMISTRY.
IV. Polysaccharides, (CeH^gOg)^.
1. Starch Group.
(a) Starch.
(b) Inulin.
(c) Glycogen.
(d) Lichenin.
2. Gum and Vegetable Mucilage Group.
(a) Dextrin.
(b) Vegetable Gums.
3. Cellulose Group.
(a) Cellulose.
(b) Hemicelluloses.
(i) Pentosans.
Gum Arabic.
(2) Hexosans.
Galactans.
Agar-agar.
Each member of the above carbohydrate classes, except the members
of the pentose group, may be supposed to contain the group CgH^^Og,
called the saccharide group. The polysaccharides consist of this group
alone taken a large number 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, (CgHjQ05)^ = polysaccharide, (CqHjo05)2 + H20— 'disaccharide,
CgHj^Og + HgO-^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 soluble. This means simply that, as a class, the monosaccharides
(hexoses) are the most soluble and the polysaccharides (starches and
cellulose) are the least soluble.
MONOSACCHARIDES.
Hexoses, CgH^^Og.
The hexoses are monosaccharides containing six oxygen atoms in a
molecule. They are the most important of the simple sugars, and two of
the principal hexoses, dextrose and Isevulose, occur widely distributed in
plants and fruits. Of these two hexoses, dextrose results from the
hydrolysis of starch whereas both dextrose and laevulose are formed in
the hydrolysis of sucrose. Galactose, which with dextrose results from
the hydrolysis of lactose, is also an important hexose. These three hexoses
CARBOHYDRATES. 27
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 phenylhydrazine they
form characteristic osazones.
CH3OH
i
DEXTROSE, (CHOH),.
I
CHO
Dextrose, also called glucose or grape 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 sucrose, lactose or dextrose, causing the
assimilation limit to be exceeded, an alimentary glycosuria may arise.
The assimilation limit for dextrose has been shown ^ to be between loo
and 150 grams. In diabetes mellitus very large amounts of dextrose arc
excreted in the urine. The following structural formula has been
suggested by Victor Meyer for (f-dextrose:
COH
I
H— C— OH
I
HO— C— H
H— C— OH
H— C— OH
I
CH.OH
(For further discussion of dextrose see section on Hexoses, page 26.)
Experiments on Dextrose.
1. Solubility. — Test the solubility of dextrose in the "ordinary
solvents" and in alcohol. (In the solubility tests throughout the book we
shall designate the following solvents as the "ordinary solvents": H^O;
10 per cent NaCl; 0.5 per cent NajCOg; 0.2 per cent HCl; concentrated
KOH; concentrated HCl.)
2. Molisch's Reaction. — Place approximately 5 c.c. of concentrated
HjSO^ 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
Molisch's reagent (a 15 per cent alcoholic solution of a-naphthol) has been
added, so that the sugar solution will not mix with the acid. A reddish-
' Brasch: Zeitschrift fur Biologic, 50, 113, 1907.
28 PHYSIOLOGICAL CHEMISTRY.
violet zone is produced at the point of contact. The reaction is due to the
formation of furfurol,
HC— CH
HC C-CHO,
• \/
O
by the acid. The test is given by all bodies containing a carbohydrate
group and is therefore not specific and, in consequence, of very little
practical importance.
3, Phenylhydrazine Reaction. — Test according to one of the follow-
ing methods: (a) To a small amount of phenylhydrazine mixture,
furnished by the instructor,^ add 5 c.c. of the sugar solution, 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). 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 osazones are formed from certain
sugars under these conditions, in general each individual sugar giving
rise to an osazone of a definite crystalline form which is typical for that
sugar. It is important to remember in this connection that of the simple
sugars of interest in physiological chemistry, dextrose and laevulose yield
the same osazone. Each osazone has a definite melting-point and as a
further and more accurate means of identification it may be recrystallized
and identified by the determination of its melting-point and nitrogen
content. The reaction taking place in the formation of phenyldextrosazone
is as follows:
Dextrose. Phenylhydrazine. Phenyldextrosazone.
(6) Place 5 c.c. of the sugar solution in a test-tube, add i c.c. of the
phenylhydrazine-acetate solution furnished by the instructor,^ 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 phenylhydrazine test has been so modified by Cipollina as to be of
use as a rapid clinical lest. The directions for this test are given in the
next experiment.
* This mixture is prepared by combining one part of phenylhydrazine hydrochloride and
two parts of sodium acetate, by weight. These are thoroughly mixed in a mortar.
^ 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 phenylhydrazine (the base).
PLATF. III.
OSAZONES.
Upper form, de.xtrosazone; tentral form, maltosazonc; lower form, lactosazone.
CARBOHYDRATES. 29
4. CipoUina's Test. — Thoroughly mix 4 c.c. of dextrose solu-
tion, 5 drops of phenylhydrazine (the base) and 1/2 c.c. of glacial acetic
acid in a test-tube. Heat the mixture for about one minute over a low
flame, shaking the tube continually 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. Ordinarily the crystals form
at once, especially if the sugar solution 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 28.
5. Riegler's Reaction.^ — Introduce o.i gram of phenylhydra-
zine-hydrochloride and 0.25 gram of sodium acetate into a test-tube,
add 20 drops of the solution under examination and heat the mixture
to boiling. Now introduce 10 c.c. of a 3 per cent solution of potassium
hydroxide and gently shake the tube and contents. If the solution
under examination contains dextrose the liquid in the tube will assume
a red color. One per cent dextrose yields an immediate color whereas
0.05 per cent yields the color only after the lapse of a period of one-
half hour from the time the alkali is added. In urinary examination
if the color appears after the thirty-minute interval the color change is
without significance inasmuch as sugar-free urine will respond thus.
The reaction is given by all aldehydes and therefore the test cannot
be safely employed in testing urines preserved by formaldehyde. Al-
bumin does not interfere with the test.
6. Bottu's Test.^ — To 8 c.c. of Bottu's reagent' in a test-tube add
I c.c. of the solution under examination and mix the liquids by gentle
shaking. Now heat the upper portion of the mixture to boiling, add
an additional i c.c. of the solution and heat the mixture again imme-
diately. The appearance of a blue color accompanied by the precipi-
tation of small particles of indigo blue indicates the present of dextrose
in the solution under examination. The test will serve to detect the
presence of o.i per cent of dextrose.
7. 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 53.
8. Iodine Test. — Make the regular iodine test as given under Starch,
'Riegler; Compt. rend. soc. biol., 66, p. 795.
-Bottu; Compt. rend. soc. bid., 66, p. 972.
'This reagent contains 3.5 grams of o-nitrophenylpropiolic acid and 5 c.c. of a freshly
prepared 10 per cent solution of sodium hydro.xide per liter.
30 PHYSIOLOGICAL CHEMISTRY.
5, page 50, and keep this result in mind for comparison with the results
obtained later with starch and with dextrin.
9. Diffusibility of Dextrose. — Test the diffusibility of dextrose
solution through animal membrane, or parchment paper, making a
dialyzer like one of the models shown in Fig. 2.
A most satisfactory dialyzing bag may be made of collodion as follows:
Pour a solution of collodion into a clean dry Erlenmeyer flask or test-
tube. While rotating the vessel on its longitudinal axis, gradually pour
out the collodion, at the same time being careful that the interior surface
of the flask is completely coated with the solution. Continue the rotation
in the inverted position until the collodion ceases to flow. After the
solution has evaporated such that the collodion skin on the rim is dry
Fig. 2. — -Dialyzing Apparatus for Students' Use.
and stiff, cut or loosen it around the edge of the rim. With a pipette
or wash bottle run in a few cubic centimeters of water between the mem-
brane and the wall of the flask or test-tube. Shake the inclined vessel
while rotating on its longitudinal axis, thus detaching the membrane.
Now withdraw the detached bag and fill with water, to determine whether
or not it contains defects.^
TO. Moore's Test. — To 2-3 c.c. of sugar solution in a test-tube
add an equal volume of concentrated KOH or NaOH, and boil. The
solution darkens and finally assumes a brown color. At this point the
odor of caramel may be detected. This is an exceedingly crude test
and is of little practical value. The brown color is due to the oxida-
tion of the dextrose and the resulting formation of the potassium or
sodium salts of certain organic acids which are formed as oxidation
products.
II. Reduction Tests.— To their aldehyde or ketone structure
many sugars owe the property of readily reducing alkaline solutions
of the oxides of metals like copper, bismuth and mercury; they also
' Gies: Quoted by Clark. Bioch. Bull., i, 198, 1911
CARBOHYDRATES. 3 1
possess the property of reducing ammoniacal silver solutions with the
separation of metallic silver. Upon this property of reduction the
most widely used tests for sugars are based. When whitish-blue cu-
pric hydroxide in suspension in an alkaline liquid is heated it is con-
verted 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 blownish-red or red cuprous oxide. These changes
are indicated as follows:
OH
/
Cu -^Cu^O + H^O.
\ Cupric oxide
\ (black).
OH
Cupric hydroxide
(whitish- blue).
OH
/
Cu
\
OH
— 2Cu-0H + H,0 + 0.
OH
Cuprous hydroxide
(yellow>.
Cu
\
OH
Cu— OH
Cu— OH
Cu
\
O + H.,0.
/
Cu
Cuprous hydro.vide C'uprou.s oxide
(yellow). (brownish-red).
The chemical equations here discussed are exemplified in Trom-
mer'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 copper sulphate. Con-
tinue 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 copper sulphate used
is too strong a small brownish-red precipitate produced in a weak sugar
32 PBT^SIOLOGICAL CHEMISTRY.
solution may be entirely masked. On the other hand, particularly
in testing for sugar in the urine, if too little copper sulphate is used a
light-colored precipitate formed by uric acid and purine 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.
Salkowski ^ has very recently proposed a modification of the Trommer
procedure which he claims is a very accurate sugar test.
{b) Feliling's Test. — To about i c.c. of FehHng's solution^ in a
test-tube add about 4 c.c. of water, and boil. This is done to deter-
mine whether the solution will of itself cause the formation of a pre-
cipitate of brownish-red cuprous oxide. If such a precipitate forms,
the Fehling's solution must not be 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 glycuronates, uric acid, nucleoprotein and homogen-
tisic acid when present in sufficient amount may produce a result sim-
ilar 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 cuprous hydroxide. Cupric hydroxide may also
be reduced to cuprous oxide and this in turn be dissolved by creatinine,
a normal urinary constituent. This will give the urine under examina-
tion a greenish tinge and may obscure the sugar reaction even when a
considerable amount of sugar is present.
According to Laird^ even small amounts of creatinine will retard
the reaction velocity of reducing sugars with Fehling's solution.
* Salkowski; Zeil. physiol. Chem., 79, 164, 1912.
* Fehling's solution is composed of two definite solutions — a copper sulphate solution and
an alkaline tartrate solution, which may be prepared as follows:
Copper sulphate solution =34.65 grams of copper 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 bottles and mixed
in equal volumes when needed for use. This is done to prevent deterioration.
' Laird: Journal of Pathology and Bacteriology, 16, 398, 1912.
CARBOHYDRATES. 33
(c) Benedict's Modifications of Fehling's Test. — First Modification. —
To 2 c.c. of Benedict's solution^ in a test-tube add 6 c.c. of distilled
water and 7-9 drops (not more) of the solution under examination.
Boil the mixture vigorously for about 15-30 seconds and permit it to
cool to room temperature spontaneously. (If desired this process
may be repeated, although it is ordinarily unnecessary.) If sugar
is present in the solution a precipitate will form which is often bluish-
green or green at Tirst, especially if the percentage of sugar is low, and
which usually becomes yellowish upon standing. If the sugar present
exceeds 0.06 per cent this precipitate generally forms at or below the
boiling-point, whereas if less than 0.06 per cent of sugar is present the
precipitate forms more slowly and generally only after the solution has
cooled.
Benedict claims, whereas the original Fehling test will not serve
to detect sugar when present in a concentration of less than o.i per
cent, that the above modification will serve to detect sugar when
present in as small cjuantity as 0.015-0.02 per cent. Corroboration
of this claim of increased delicacy has recently been submitted by
Harrison.^
The modified Fehling solution used in the above test differs from
the original Fehling solution in that 100 grams of sodium carbonate
is substituted for the 125 grams of potassium hydroxide ordinarily used,
thus forming a Fehling solution which is considerably less alkaline
than the original. This alteration in the composition of the Fehling
solution is of advantage in the detection of sugar in the urine inasmuch
as the strong alkalinity of the ordinary Fehling solution has a tendency,
when the reagent is boiled with a urine containing a small amount of
dextrose, to decompose sufficient of the sugar to render the detection of
the remaining portion exceedingly difficult by the usual technic. Bene-
dict claims that for this reason the use of his modified solution permits
the detection of much smaller amounts of sugar than does the use of the
ordinary Fehling solution. He has further modified his solution for
use in the quantitative determination of sugar (see Chapter XXII).
Second Modification.^ — Very recently Benedict has further modi-
fied his solution and has succeeded in obtaining one which does not
' Benedict's modified Fehling solution consists of two definite solutions — a copper sulphate
solution and an alkaline tartrate solution, which may be prepared as follows:
Copper sulphate solution =34.65 grams of copper sulphate dissolved in water and made
up to 500 c.c.
Alkaline tartrate solution =100 grams of anhydrous sodium carbonate and 173 grams of
Rorhelle 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.
* Harrison: Pharnt. Jour., 87, 746, igii.
* Benedict: Jour. Am. Med. Ass'n., 57, 1193, 1911.
3
34 PHYSIOLOGICAL CHEMISTRY.
deteriorate upon long standing.^ The following is the procedure for
the detection of dextrose in solution : To five cubic centimeters of the
reagent in a test-tube add eight (not more) drops of the solution under
examination. Boil the mixture vigorously for from one to two minutes
and then allow the fluid to cool spontaneously. In the presence of dex-
trose the entire body of the solution will he filled with a precipitate, which
may be red, yellow or green in color, depending upon the amount of sugar
present. If no dextrose is present, the solution will remain perfectly
clear. (If urine is being tested, it may show a very faint turbidity, due
to precipitated urates.) Even very small quantities of dextrose (o.i
per cent) yield precipitates of surprising bulk with this reagent, and the
positive reaction for dextrose is the filling of the entire body of the solu-
tion with a precipitate, so that the solution becomes opaque. Since
amount rather than color of the precipitate is made the basis of this test,
it may be applied even for the detection of small quantities of dextrose,
as readily in artificial light as in daylight.
{d) Boettger^s Test.- — To 5 c.c. of sugar solution in a test-tube add
I c.c. of KOH or NaOH and a very small amount of bismuth subni-
trate, and boil. The solution will gradually darken and finally as-
sume a black color due to reduced bismuth. 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).
{e) Nylander^s Test {Almens' Test). — To 5 c.c. of sugar solution
in a test-tube add one-tenth its volume of Nylander's reagent^ and heat
for five minutes in a boiling water-bath.^ 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, since with albumin a
similar change of color is produced. Dextrose when present to the
' Benedict's new solution has the following composition:
Copper sulphate 17.3 grams.
Sodium citrate 1730 grams.
Sodium carbonate (anhydrous) 100. o grams.
Distilled water to make i liter.
With the aid of heat dissolve the sodium citrate and carbonate in about 600 c.c. of water.
Pour (through a folded filter paper if necessary) into-a glass graduate and make up to 850 c.c.
Dissolve the copper sulphate in about 100 c.c. of water and make up to 150 c.c. Pour the
carbonate-citrate solution into a large beaker or casserole and add the copper sulphate solu-
tion slowly, with constant stirring. The mixed solution is ready for use and does not deterio-
rate upon long standing.
- Nylander's reagent is prepared by digesting 2 grams of bismuth subnitrate 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.
' Hammarsten suggests that the mixture should be boiled 2-5 minutes (according to the
sugar content) over a free flame and the tube then permitted to stand 5 minutes before
drawing conclusions.
CARBOHYDRATES.
35
extent of 0.08 per cent may be detected by this reaction. It is claimed
by Bech'old that Nylander's and Boettger's tests give a negative reaction
with solutions containing sugar when mercuric chloride or chloroform
is present. Other observers^ have failed to verify the inhibitory action
of mercuric chloride and have shown that the inhibitory influence of
chloroform may be overcome by raising the temperature of the urine
to the boiling-point for a period of five minutes previous to making the
test. Urines rich in indican, urochrome, nroerythrin or hcEmatopor phyrin,
as well as urines excreted after the ingestion of large amounts of certain
medicinal substances, may give a darkening of Nylander's reagent similar
to that of a true sugar reaction. It is a disputed point whether the urine
after the administration of urotropin will reduce Nylander's reagent.^
Strausz' has recently shown that the urine of diabetics to whom '' lothion "
(diiodohydroxypropane) has been administered will give a negative
Nylander's reaction and respond positively to the Fehling and polari-
scopic tests. "lothion" also interferes with the Nylander test w ^'//ro
whereas KI and I do not.
According to Rustin and Otto, the addition of PtCl^ increases the
delicacy of Nylander's reaction. They claim that this procedure causes
the sugar to be converted quantitatively. No quantitative method has
yet been devised, however, based upon this principle.
Bohmansson* before testing the urine under examination treats
it (10 c.c.) with 1/5 volume of 25 per cent hydrochloric acid and about
1/2 volume of bone black. This mixture is shaken one minute, then
filtered and the neutralized filtrate tested by Nylander's reaction. Boh-
mansson claims that this procedure removes certain interfering substances,
in particular urochrome.
A positive Nylander or Boettger test is probably due to the following
reactions:
(a) Bi (OH) 2NO3 + KOH— Bi(OH) 3 + KNO3.
{b) 2Bi(OH)3-30--Bi3 + 3H30.
12. 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. 3, p. 36) 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 fermenta-
^ Kehixxss a.nd}i&\\\!i; Journal of Biological Chemistry, -J, 26-], 1910; also Zeidlitz; Upsala
Lakdreforen Fork., N. F., 11, igo6.
^ Abt; Archives oj Pediatrics, 24, 275, 1907; also Weitbrecht; Schweiz. Wochschr., 47,
577> 1909-
' Strausz; Miinch. med. Woch., 59, 85, 1912.
* Bohmansson: Biochem. Zeit., 19, p. 281.
36
PHYSIOLOGICAL CHEMISTRY.
tion introduce a little potassium hydroxide solution into the graduated
portion by means of a bent pipette, place the thumb tightly over the open-
ing in the apparatus and invert the saccharometer. Explain the result.
The important findings of Neuberg and associates^ recently re-
ported indicate very clearly that the liberation of carbon dioxide by yeast
is not necessarily a criterion of the presence of sugar. The presence
of a new enzyme called carboxylase has been
demonstrated in yeast which has the power of
splitting off CO ^ from the carhoxyl group of amino
and other aliphatic acids.
13. Barfoed's Test. — Place about 5 c.c. of
Barfoed's solution^ in a test-tube and heat to
boiling. Add dextrose solution slowly, a few
drops at a time, heating after each addition.
Reduction is indicated by the formation of a
red precipitate. If the precipitate does not form
upon continued boiling allow the tube to stand a
few minutes and examine. Sodium chloride
interferes with the reaction (Welker).
Barfoed's test is not a specific test for dextrose
as is frequently stated, but simply serves to
detect monosaccharides. Disaccharides will also
respond to the test, under proper conditions of
acidity.^ Also if the sugar solution is boiled
sufl&ciently long, in contact with the reagent, to
hydrolyze the disaccharide through the action of the acetic acid present
in the Barfoed's solution a positive test results.*
14. Formation of Caramel. — Gently heat a small amount of pul-
verized 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.
15. Demonstration of Optical Activity. — A demonstration of the
use of the polariscope, by the instructor, each student being required
to take readings and compute the "specific rotation."
Fig. 3. — EixHORN Sac-
charometer.
Use of the Polariscope.
For a detailed description of the different forms of polariscopes, the
method of manipulation and the principles involved, the student is
'Neuberg and Associates: Biochem. Zeitsch., 31, 170; 32, 323; 36, (60, 68, 76), 1911.
' Barfoed's solution is prepared as follows: Dissolve 4.5 grams of neutral crystallized
copper acetate in 100 c.c. of water and add 1.2 c.c. of 50 per cent acetic acid.
* Mathews and McGuigan: Am. Jour. Physiol., 19, 175, 1907.
* Hinkle and Sherman: Jour. Am. Chem.Soc, 29, 1744, 1907.
CARBOHYDRATES.
37
referred to any standard text-book of physics. A brief description fol-
lows: 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
two 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 polar-
ized. Many organic substances (sugars, proteins, 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
Fig. 4. — 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.
the polarized light passes. Substances which possess this power are
said to be "optically active." The specific rotalmi of a substance is the
rotation expressed in degrees which is afforded by one gram of substance
dissolved in i c.c. of water in a tube one decimeter in length. The spe-
cific rotation, (a)^, may be calculated by means of the following formula,
p.l
in which
£>=;= sodium light.
a = observed rotation in degrees.
/» = 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.
P =
(«)d I
38
PHYSIOLOGICAL CHEMISTRY.
The value of p multiplied by loo will be the percentage of the sub-
stance in solution.
An instrument by means of which the extent of the rotation may
be determined is called a polariscope or polarimeter. Such an instru-
Kini
Fig. V — D1AGRAMM.A.TIC Representatiox of the Course of the Light through the
Laurent Polariscope. (The direction is reversed from that of Fig. 4, p. 37.)
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; /and g, ocular lenses.
ment designed especially for the examination of sugar solutions is termed
a saccharimeter or polarizing saccharimeter. The form of polariscope
in Fig. 4, p. 37, consists essentially of a long barrel provided with a
Fig. 6. — Polariscope (Schmidt and Haensch Model).
Nicol prism at either end (Fig. 5 , above) . 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 eyepiece for focusing and
CARBOHYDRATES. 39
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 ])ohirizcd
by passing through a Nicol ])rism. This polari^^cd ray then traverses the
column of liquid within the tube mentioned 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 I cevo -rotatory.
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. e., ''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 per-
centage of sugar in solution is determined by making an observation
and multiplying the value of each division on a horizontal sliding scale
by the value of the division expressed in terms of dextrose. This factor
may vary according to the instrument.
"Optical" methods embracing the determination of the optical
activity are being utilized in recent years in many "quantitative"
connections.^
CH^OH
L^EVULOSE, (CH0H)3.
CO
1
CH,OH
As already stated, laevulose, sometimes called fructose or fruit sugar,
occurs widely disseminated throughout the plant kingdom in company
with dextrose. Its reducing power is somewhat weaker than that of
dextrose. Laevulose does not ordinarily occur in the urine in diabetes
mellitus, but has been found in exceptional cases. With phenylhydrazine
it forms the same osazone as dextrose. With methylphenylhydrazine,
laevulose forms a characteristic methylphenyllaev^losazone.
(For a further discussion of laevulose see the section on Hexoses,
p. 26.)
I Abderhalden and Schmidt: "Determination of blood content by means of the optica
method," Zeit. physiol. Chem. 66, 120, 1910; also C. Neuberg; "Determination of nucleic acid
cleavage by polarization," Biodiemische Zeitschrijt, 30, 505, 191 1
40 physiological chemistry.
Experiments on L.evulose.
1-13. Repeat these experiments as given under Dextrose, pages
27-36.
14. Seliwanoff's Reaction. — To 5 c.c' of Seliwanoff's reagent^
in a test-tube add a few drops of a Isvulose solution and heat the mix-
ture to boiling. A positive reaction is indicated by the production
of a red color and the separation of a red precipitate. The latter may
be dissolved in alcohol to which it will impart a striking red color.
If the boiling be prolonged a similar reaction may be obtained with
solutions of dextrose or maltose. This has been explained^ in the case
of dextrose as due to the transformation of the dextrose into laevulose by the
catalytic action of the hydrochloric acid. The precautions necessary for a
positive test for laevulose are as follows: The concentration of the
hydrochloric acid must not be more than 12 per cent. The reaction
(red color), and the precipitate must be observed after not more than
20-30 seconds boiling. Dextrose must not be present in amounts ex-
ceeding 2 per cent. The precipitate must be soluble in alcohol with a
bright red color.
15. Borchardt's Reaction. — To about 5 c.c. of a solution of laevulose
in a test-tube add an equal volume of 25 per cent hydrochloric acid and a
few crystals of resorcinol. Heat to boiling and after the production of a
red color, cool the tube under running water and transfer to an evapo-
rating dish or beaker. Make the mixture slightly alkaline with solid
potassium hydroxide, return it to a test-tube, add 2-3 c.c. of acetic ether
and shake the tube vigorously. In the presence of lae\Talose, the
acetic ether is colored yellow. (For further discussion of the test see
Chapter XIX.)
16. Formation of Methylphenyllaevulosazone. — To a solution
of 1.8 grams of laevulose in 10 c.c. of water add 4 grams^ of methyl-
phenylhydrazine and enough alcohol to clarify the solution. Intro-
duce 4 c.c. of 50 per cent acetic acid and heat the mixture for 5-10 min-
utes on a boiling water-bath.^ On standing 15 minutes at room tem-
perature, crystallization begins and is complete in two hours. By scratch-
ing the sides of the flask or by inoculation, the solution quickly con-
geals to form a thick paste of reddish-yellow silky needles. These are
the crystals of inethylphenyllcBVulosazone. They may be recrystallized
from hot 95 per cent alcohol and melt at 153° C.
* Seliwanoff's reagent may be prepared by dissolving 0.05 gram of resorcinol in 100 c.c.
of dilute (1:2) hydrochloric acid.
^Koenigsfeld: Bioch.'Zeit., 38, 311, 19 12
'3.66 grams if absolutely pure.
* Longer heating is to be avoided.
CARBOHYDRATES. 4I
CH.OH
GALACTOSE, (CHOH),.
CHO
Galactose occurs with dextrose as one of the products of ihe hydro-
lysis of lactose. It is dextro-rotatory, forms an osazone with j)hcnyl-
hydrazine and ■ferments slowly with yeast. Upon oxidation with nitric
acid galactose yields mucic acid, thus differentiating this monosac-
charide from dextrose and laevulose. Lactose also yields mucic acid
under these conditions. The mucic acid test may be used in urine
examination to differentiate lactose and galactose from other reducing
sugars. The assimilation limit for galactose is 30-40 grams. ^
Experiments on Galactose.
1. Tollens' Reaction. — To equal volumes of galactose solution and
hydrochloric acid (sp. gr. 1.09) add a little phloroglucinol, and heat the
mixture on a boiling water-bath. Galactose, pentose and glycuronic
acid will be indicated by the appearance of a red color. Galactose
may be differentiated from the two latter substances in that its solutions
exhibit no absorption bands upon spectroscopical examination.
2. Mucic Acid Test. — Treat 100 c.c. of the solution containing
galactose with 20 c.c. of concentrated nitric acid (sp. gr. 1.4) and evapo-
rate the mixture in a broad, shallow glass vessel on a boiling water-
bath until the volume of the mixture has been reduced to about 20 c.c.
At this point the fluid should be clear, and a fine white precipitate of
mucic acid should form. If the percentage of galactose present is low
it may be necessary to cool the solution and permit it to stand for some
time before the precipitate will form. It is impossible to differentiate
between galactose and lactose by this test, but the reaction serves to
differentiate these two sugars from all other reducing sugars. Differ-
entiate lactose from galactose by means of Barfoed's test (p. 36).
3. Phenylhydrazine Reaction. — Make the test according to direc-
tions given under Dextrose, 3 or 4, pages 28 and 29.
Pentoses, C.HjoOg.
In plants and more particularly in certain gums, very complex car-
bohydrates, called pentosans (see p. 55), 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
"Brasch: Zeitschrift fur Biologic, 50, 113, 1907.
42 PHYSIOLOGICAL CHEMISTRY.
urine of morphine habitues and others, their occurrence sometimes
being a persistent condition without known cause. They may be ob-
tained from the hydrolysis of nucleoproteins being present in the nucleic
acid complex of the molecule. Pentoses are non-fermentable, have
strong reducing power and form osazones with phenylhydrazine. Pen-
toses are an important constituent of the dietary of herbivorous animals.
Glycogen is said to be formed after the ingestion of these sugars containing
five oxygen atoms. This, however, has not been conclusively proven.
On distillation with strong hydrochloric acid pentoses and pentosans
yield furfurol, which can be detected by its characteristic red reaction
with aniline-acetate paper.
CH^OH
ARABINOSE, (CHOHjg.
CHO
Arabinose is one of the most important of the pentoses. The l-
arabinose may be obtained from gum arabic, plum or cherry gum. by
boiling for lo minutes with concentrated hydrochloric acid. This pentose
is dextro-rotatory, forms an osazone and has reducing power, but does not
ferment. The ^'-arabinose has been isolated from the urine and yields
an osazone which melts at i66°-i68° C.
Experiments on Arabinose.
1. Bial's Reaction/— To 5 c.c. of Bial's reagent^ in a test-tube
add 2-3 c.c. of the arabinose solution and heat the mixture gently until
the first bubbles rise to the surface. Immediately or upon cooling the
solution becomes green and a flocculent precipitate of the same color
may form. (For further discussion see Chapter XIX) . The test may also
be performed by adding the pentose to the hot reagent.
2. ToUens' Reaction.— To equal volumes of arabinose solution
and hydrochloric acid (sp. gr. 1.09) add a little phloroglucinol and heat
the mixture on a boiling water-bath. Galactose, 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 glycuronic
acid. Differentiate between the two latter bodies by the melting-points
of their osazones.
' Bial: Deut. med. Woch., 28, 252, 1902.
^ Orcinol 1.5 gram.
Fuming HCl 500 grams.
Ferric chloride (10 per cent) . .20-30 drops.
CARBOHYDRATES. 43
Compare the reaction with that obtained with galactose (page 41).
3. Orcinol Test. — Repeat i, using orcinol instead of phloroglucinol.
A succession of colors from red through reddish-blue to green is pro-
duced. A green precipitate is formed which is soluble in amyl alcohol
and has absorption Ijands between C and D.
4. Phenylhydrazine Reaction. — Make this test on the arabinose
solution according to directions given under Dextrose, 3 or 4, pages
28 and 29.
CH2OH
XYLOSE, (CHOH)3.
CHO
Xylose, or wood sugar, is obtained by boiling wood gums with di-
lute acids as explained under Arabinose, page 43. It is dextro-rota-
tory, forms an osazone and has reducing power, but does not ferment.
Experiments on Xylose.
1-4. Same as for arabinose (see above).
RHAMNOSE, C«H,,0..
' 6 12 5
Rhamnose or methyl-pentose is an example of a true carbohydrate
which does not have the H and O atoms present in the proportion to
form water. Its formula is CpH, ,0.. It has been found that rham-
C 12 o
nose when ingested by rabbits or hens has a positive influence upon the
formation of glycogen in those organisms.
DISACCHARIDES, C.^H^^O^^.
The disaccharides as a class may be divided into two rather dis-
tinct groups. The first group would include those disaccharides which
are found in nature as such, e. g., sucrose and lactose and the second
group would include those disaccharides formed in the hydrolysis of more
complex carbohydrates, e. g., maltose, and iso-maltose.
The disaccharides have the general formula C^^'B.^Jd^^, 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. The products of the hydrolysis of the more common dis-
accharides are as follows:
Maltose = dextrose -1- dextrose.
Lactose = dextrose + galactose.
Sucrose = dextrose -|- laevulose.
44 PHYSIOLOGICAL CHEMISTRY.
All of the more common disaccharides except sucrose 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, C^^H^^O,,.
Maltose or malt sugar is formed in the hydrolysis of starch through
the action of an enzyme, vegetable amylase {diastase), contained in sprout-
ing 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, reduces metallic oxides in alkaline solution and is fer-
mentable by yeast after being inverted (see Polysaccharides, page 47)
by the enzyme maltase of the yeast. In common with the other disac-
charides, maltose may be hydrolyzed with the formation of two molecules
of monosaccharide. In this instance the products are two molecules of
dextrose. With phenylhydrazine maltose forms an osazone, mallo-
sazone. The following formula represents the probable structure of
maltose:
CH2OH CHO
I I
CHOH CHOH
CHO — CHOH
I I
CHOH CHOH
CHOH CHOH
C-^^ O CH2
\
H
Maltose.
Expp:riments on Maltose.
1-13. Repeat these experiments as given under Dextrose, pages 27-36.
ISO-MALTOSE, C^^^fi^^.
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 hydro-
chloric acid upon dextrose. It also occurs with maltose as one of the
products of salivary digestion. It is dextro-rotatory and with phenylhy-
drazine gives an osazone which is characteristic. Iso-maltose is very
CARBOHYDRATES. 45
soluble and reduces the oxides of bismuth and copper in alkaline solution.
Pure iso-maltose is probably only slightly fermentable.
LACTOSE, Cj,H,,0,,.
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 persons after the ingestion of
unusually large amounts of lactose in the food. It has a strong reducing
power, is dextro-rotatory and forms an osazone with phenylhydrazine.
Upon hydrolysis lactose yields one molecule of dextrose and one molecule
of galactose.
In the souring of milk the bacterium laclis and certain other micro-
organisms bring about lactic acid fermentation by transforming the lac-
tose of the milk into lactic acid,
H OH
H— C — C— COOH,
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 fermenta-
tion, through the action of ferments other than yeast, and at the same
time lactic acid is produced. Lactose and galactose yield mucic acid on
oxidation with nitric acid. This fact is made use of in urine analysis to
facilitate the differentiation of these sugars from other reducing sugars.
Lactose is not fermentable by pure yeast.
Experiments on Lactose.
1-13. Repeat these experiments as given under Dextrose, pages 27-36.
14. Mucic Acid Test. — Treat 100 c.c. of the solution containing
lactose with 20 c.c. of concentrated nitric acid (sp. gr. 1.4) and evaporate
the mixture in a broad, shallow glass vessel on a boiling water-bath, until
the volume of the mixture has been reduced to about 20 c.c. At this
point the fluid should be clear, and a fine white precipitate of mucic acid
should form. If the percentage of lactose present is low it may be neces-
sary to cool the solution and permit it to stand for some time before the pre-
cipitate will appear. It is impossible to differentiate between lactose
and galactose by this test, but the reaction serves to differentiate these
two sugars from all other reducing sugars.
46
PHYSIOLOGICAL CHEMISTRY.
Differentiate lactose from galactose by means of Barfoed's test,
page 36.
SUCROSE, C,,H,20,,.
Sucrose, also called saccharose or cane sugar, is one of the most
important of the sugars and occurs very extensively distributed in plants,
particularly in the sugar cane, sugar beet, sugar millet and in certain
palms and maples, ^
Sucrose is dextro-rotatory and upon hydrolysis, as before mentioned,
the molecule of sucrose takes on a molecule of water and breaks down
into two molecules of monosaccharide. The monosaccharides formed in
this instance are dextrose and laevulose. This is the reaction:
C12H22O11 + H2O— CeH^20g+CgH^20(
Sucrose.
Dextrose.
Laevulose.
This process is called inversion and may be produced by bacteria, enzymes,
and certain weak acids. After this inversion the previously strongly
dextro-rotatory solution becomes laevo-rotatory. This is due to the fact
that the laevulose molecule is more strongly laevo-rotatory than the dex-
trose molecule is dextro-rotatory. The product of this inversion is called
invert sugar.
Sucrose does not reduce metallic oxides in alkaline solution and forms
no osazone with phenylhydrazine. It is not fermentable directly by yeast,
but must first be inverted by the enzyme sucrase {invertase or invertin)
contained in the yeast. The probable structure of sucrose may be repre-
sented by the following formula. Note the absence of any free ketone
or aldehyde group.
CH2OH
CHOH
CH2OH
CHO
Sucrose.
Experiments on Sucrose.
1-13, Repeat these experiments according to the directions given
under Dextrose, pages 27-36.
CARBOHYDRiVTES.
47
14. Inversion of Sucrose. — To 25 c.c. of sucrose solution in a
beaker add 5 drops of concentrated HCl and boil one minute. Cool
the solution, render alkaline with solid KOH and upon the resulting fluid
repeat experiments 3 (or 4) and 11 as given under Dextrose, pages 28-30.
Explain the results.
15. Production of Alcohol by Fermentation. — Prepare a strong
(io-2o per cent) solution of sucrose, add a small amount of egg albumin
or commercial peptone and introduce the mixture into a bottle of appro-
priate 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 gas may be
collected in a eudiometer tube and examined.
When the activity of the yeast has 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
-,-,,. . Fig. 7. — Iodoform. {Autenneth.)
for alcohol by one of the following reactions :
(a) Iodoform Test. — Render 2-3 c.c. of the distillate alkaline with
potassium hydroxide solution and add a few drops of iodine solution.
Heat gently and note the formation of iodoform crystals. Examine these
crystals under the microscope and compare them with those in Fig. 7.
(b) Aldehyde Test. — Place 5 c.c. of the distillate in a test-tube, add a
few drops of potassium dichromate solution, K^CrjO^, and render it acid
with dilute sulphuric acid. Boil the acid solution and note the odor of
aldehyde.
TRISACCHARIDES, CjgHg.Oie.
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 partly fermentable by yeast.
Raffinose may be hydrolyzed by weak acids the same as the poly-
saccharides are hydrolyzed, the products being lasvulose and melibiose;
further hydrolysis of the melibiose yields dextrose and galactose.
POLYSACCHARIDES, (C,'ii,,0,)^.
In general the polysaccharides are amorphous bodies, a few, how-
ever, are crystallizable. Through the action of certain enzvmes or
48 PHYSIOLOGICAL CHEMISTRY.
weak acids the polysaccharides may be hydrolyzed with the formation of
monosaccharides. As a class the polysaccharides are quite insoluble and
are non-fermentable until inverted. By inversion is meant the hydrolysis
of disaccharide or polysaccharide sugars to form monosaccharides, as
indicated in the following equations:
(a) C,3H,30,,H-H30-2(C,H,,Oe).
(b) C«H,,0, + H,0-CeH,30,.
STARCH, (CeH^.OJ,.
Starch is widely distributed throughout the vegetable kingdom,
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 concentric rings of granulose and cellulose. Ordinary
starch is insoluble in cold water, but if boiled with water the cell walls
are ruptured and starch paste results. In general starch gives a blue
color with iodine.
Starch is acted upon by amylases, e. g., salivary amylase {ptyalin)
and pancreatic amylase (amylopsin) , with the formation of soluble starch,
erythro-dextrin, achroo-dextrins, maltose, iso-maltose and dextrose (see
Salivary Digestion, page 6i). Maltose is the principal end-product of
this enzyme action. Upon boiling 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
^dgorously 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 repeated decantation. Allow the compact mass of starch to
drain thoroughly and spread it out on a watch glass to dry in the air.
If so desired this preparation may be used in the experiments which follow.
2. Microscopical Examination. — Examine microscopically the gran-
ules of the various starches submitted and compare them with those
shown in Figs. 8-18, page 49. The suspension of the granules in a drop
of water will facilitate the microscopical examination.
3. Solubility. — Try the solubility of one form of starch in each of
the ordinary solvents (see page 27). If uncertain regarding the solubility
in any reagent, filter and test the filtrate with iodine solution as given
CARBOHYDRATES.
49
Fig. 8. — Potato.
Fig. g. — Bean.
Fig. io. — .'\rrowroot.
Fig. II. — Rye.
Fig. 12. — Barley.
Fig. 13. — Oat.
Fig. 14. — Buckwheat.
Fig. 15. — Maize.
Fig 16. — KicE.
Fig. 17.— Pea. Fig. 18.— Wheat.
Starch Granules krom \'arious Sources. (Leffmai.n and Beam )
so PHYSIOLOGICAL CHEMISTRY.
under 5 below. The production 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 depres-
sions of a porcelain test-tablet and treat with a drop of a dilute solution of
iodine in potassium iodide. The granules 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" 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 32).
7. Hydrolysis of Starch. — ^Place about 25 c.c. of starch paste in a
small beaker, add 10 drops of concentrated HCl, 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 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, Fehling's test (see page 32)
should give a positive result due to the formation of a reducing sugar
from the starch. Make the phenylhydrazine test upon some of the
hydrolyzed starch. 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 yellowish-white precipitate is produced.
Compare this result with the result of the similar experiment on dextrin
(page 53).
9. Diffusibility of Starch Paste. — Test the diffusibility of starch
paste through animal membrane, parchment paper or collodion, making
a dialyzer like one of the models shown in Fig. 2, page 30.
mULIN, (CeH,,0,),.
Inulin is a polysaccharide which may be obtained as a white, odorless,
tasteless powder from the tubers of the artichoke, elecampane, or dahlia.
' Preparation of Si arch 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.
' for this particular test a starch paste of very satisfactory strength may be made by
mixing i c.c. of a i per cent starch paste with 100 c.c. of water.
CARBOHYDRATES. 51
It has also been prepared from the roots of chicory, dandelion, and ];ur-
dock. 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 nurely
the normal color of the iodine solution. It is very difficult to prepare inu-
lin which does not reduce Fehling's solution slightly. This reducing
power may be due to an impurity. Practically all commercial prepara-
tions of inulin possess considerable reducing power.
Inulin is laevo-rotatory and upon hydrolysis by acids or by the enzyme
inulase it yields the monosaccharide Itevulose which readily reduces
Fehling's solution. The ordinary amylolytic enzymes occurring in the
animal body do not digest inulin. A small part of the ingested inulin
may be hydrolized by the acid gastric juice, but Lewis ^ has recently
shown that " the value of inulin as a significant source of energy in human
dietaries must be questioned."
Experiments on Inulin.
1. Solubility. — Try the solubility of inulin powder in each of the
ordinary solvents. If uncertain regarding the solubility 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 potassium
hydroxide and try Fehling's test. What 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 directions
given under Dextrose, 2, page 27.
4. Fehling's Test. — Make this test on the inulin solution according
to the instructions given under Dextrose, page 32. Is there any
reduction ?^
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.
Now cool the solution, neutralize it with concentrated KOH and test the
' Lewis: Journal American Medical Ass'n., 58, 1176, 1912.
- See the discussion of the properties of inulin, above.
52 PHYSIOLOGICAL CHEMISTRY.
reducing action of i c.c. of the solution upon i c.c. of diluted (i 14) Feh-
ling's solution. Explain the result. ^
GLYCOGEN, {C,U,,0,),.
(For discussion and experiments see Muscular Tissue, Chapter XV.)
LICHENIN, (CgH^^OJ,.
Lichenin may be obtained from Cetraria islandica (Iceland moss). It
forms a diflBicultly 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, (CeH^^OJ,.
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 insol-
uble in alcohol or ether. Dextrins are dextro-rotatory and are not fer-
mentable by yeast.
The dextrins may be hydrolyzed by dilute acids to form dextrose.
With iodine one form of dextrin (erythro-dextrin) gives a red color. Their
power to reduce Fehling's solution is questioned.
Experiments on Dextrin.
1. Solubility. — Test the solubility of pulverized dextrin in the
ordinary solvents (see page 27).
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 due to the formation of the red
iodide of dextrin. If the reaction is not sufficiently pronounced make a
stronger solution from pulverized dextrin and repeat the test. The
solution should be slightly acid to secure the best results.
Make proper tests to show that the red iodide of dextrin is influenced by
heat, alkali, and alcohol in a similar manner to the blue iodide of starch
(see page 50).
' If the inulin solution gave a positive Fehling test in the last experiment 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 i c.c. of the resulting solution upon i c.c.
of diluted (i :4) Fehling's solution. This will show the normal reducing power of the inulin
solution. In case the inulin was hydrolyzed, the Fehling's test in the hydrolysis experiment
should show a more pronounced reduction than that observed in the check experiment.
CARBOHYDRATES. 53
3. Fehling's Test. — Sec if the dextrin solution will reduce Fehling's
solution.
4. Hydrolysis of Dextrin. — Take 25 c.c. of dextrin solution in a
small beaker, add 5 drops of dilute hydrochloric acid, and boil. 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 concentrated potassium hydroxide-
Try Fehling's test (see page 32). This reaction is now strongly positive,
due to the formation of a reducing sugar. Determine the nature of the
sugar by means of the phenylhydrazine test (see pages 28 and 29).
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 50).
6. Diffusibility of Dextrin. — (See Starch, 9, page 50.)
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 concentrated dextrin
solution. Dextrin is thrown out of solution as a gummy white precipitate.
Compare the result with that obtained under Dextrose, 5, page 50.
CELLULOSE, (C^H^^OJ,
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 ordi-
nary absorbent cotton are good types of cellulose.
At one time there was but a single known solvent for cellulose. Recent
investigation has, however, revealed a long list of cellulose solvents. (See
Experiment 7.)
Cellulose is not hydrolyzed by boiling with dilute mineral acids. It
may be hydrolyzed, however, by treating with concentrated sulphuric
acid then subsequently diluting the solution with water and boiling.
There is some difference of opinion as to the exact extent to which
cellulose is utilized in the animal organism. It is no doubt, more effi-
ciently utilized by herbivora than by carnivora or by man. It is claimed
that about 25 per cent may be utilized by herbivora, less than 5 per cent by
dogs whereas the quantity utilized by man is "too small for it to play a
r6le of importance in the diet of a normal individual."* In neither man
nor the lower animals has there been demonstrated any formation of
' Swartz: Transactions of the Connecticut Academy of Arts and Sciences, i6, 247, 191 1.
54 PHYSIOLOGICAL CHEMISTRY.
sugar or glycogen from cellulose. ^ It is probable that the cellulose which
disappears from the intestine is transformed for .the most part into fatty
acids. ~
Experiments on Cellulose.
1. Solubility. — Test the solubility of cellulose in the ordinary solvents
(see page 27).
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.^ — Add 10 c.c. of dilute and 5 c.c. of
concentrated H2SO4 to some absorbent cotton in a test-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, neutralize with solid KOH and test with Fehling's
solution. Dextrose has been formed from the cellulose by the action of
the acid.
4. Schweitzer's Solubility Test. — ^Place a little absorbent cotton in a
test-tube, add Schweitzer's reagent,^ and stir the cellulose with a glass
rod. When completely dissolved acidify the solution with acetic acid.
An amorphous precipitate of cellulose is produced.
5. Cross and Bevan's Solubility Test.^ — ^Place a little absorbent
cotton in a test-tube, add Cross and Bevan's reagent," and stir the cellulose
with a glass rod. When solution is complete reprecipitate the cellulose
with 95 per cent alcohol.
6. Iodine-Zinc Chloride Reaction. — Place a little absorbent cotton
or quantitative filter paper in a test-tube and treat it with the iodine-zinc
chloride reagent.^ A blue color forms on standing. Amyloid has been
' Lusk: American Journal of Physiulogy, 27, 467, 1911; also Hoffmann, Inaugural dis-
sertation, Halle-Wittenberg, igio.
^Tappeiner: ZeUschrift fiir Biologie, 24, 105, 1888.
^ This bofly derives its name from amylum (starch) and is not to be confounded with
amyloid, the glycoprotein.
* Schweitzer's reagent is made by adding potassium hydroxide to a solution of copper
sulphate which contains some ammonium chloride. A precipitate of cupric hydroxide
forms and this is filtered off, washed, and 3 grams of the moist cupric hydroxide brought
into solution in a liter of 20 per cent ammonium hydroxide.
^ Cross and Bevan: Chemical News, 63, p. 66.
® Cross and Bevan's reagent may be prepared by combining two parts of concentrated
hydrochloric acid and one part of zinc chloride, by weight.
' The iodine-zinc chloride reagent as suggested by Nowopokrowsky (Beihefte Bolan.
Centr., 28, 90, 1912) may be made by dissolving 20 grams ZnCl_, in 8.5 c.c. water and when
cool introducing the iodine solution (3 grams KI-I- 1.5 gram I in 60 c.c. water) drop by drop
until iodine begins to precipitate.
CARBOHYDRATES. 55
formed from the cellulose through the action of the ZnCl, and the iodine
solution has stained the amyloid blue.
7. New Cellulose Solvents. — It has recently been demonstrated by
Deming^ that there are many excellent solvents for cellulose (filter paper).
For example, the concentrated aqueous solutions of certain salts such as
antimony trichloride, stannous chloride and zinc bromide. In hydro-
chloric acid solution the solvent action of the abo\'e salts is increased.
The following'salts are also good solvents in hydrochloric acid solution:
mercuric chloride, bismuth chloride, antimony pentachloride, tin tetrachloride
and titanium tetrachloride. In the case of the last-mentioned salt the
swollen, transparent character of the cellulose libers preliminary to solution
can be seen very nicely.
Try selected solvents suggested by the instructor.
HEMICELLULOSES.
The hemicelluloses differ from cellulose in that they may be hydrolysed
upon boiling with dilute mineral acids. They differ from other poly-
saccharides in not being readily digested by amylases. Hemicellulose
may yield pentosans, or hexosans upon hydrolysis.
Pentosans. — ^Pentosans yield pentoses upon hydrolysis. So far as is
known they do not occur in the animal kingdom. They have, however,
a very wide distribution in the vegetable kingdom, being present in leaves,
roots, seeds and stems of all forms of plants, many times in intimate
association or even chemical combination with galactans. In herbivora,
pentosans are 40-80 per cc^nt utilized.' The few tests on record as to the
pentosan utilization by man^ indicate that 80-95 P^r cent disappear from
the intestine. According to Cramer,* bacteria are efficient hemicellulose
transformers. It has not yet been demonstrated that pentosans form
glycogen in man, and for this reason they must be considered as playing an
unimportant part in human nutrition. Gum arable an important pento-
san may be hydrolyzed by concentrated hydrochloric acid if boiled for a
short time. The pentose arabinose results from such hydrolysis.
Galactans. — In common with the pentosans the galactans have a very
wide distribution in the vegetable kingdom. The pure galactans yield
galactose upon hydrolysis. One of the most important members of the
galactan group is agar-agar, a product prepared from certain types of
Asiatic sea-weed. This galactan is about 50 per cent utilizable by
herbivora^ and 8-27 per cent utilizable by man." Agar ingestion has
I Deming: Journal American Chemical Society, t,t„ 1515, iqii.
-Swartz: Transactions of the Connecticut Academy of Arts and Sciences, 16, 247, 1911.
' Konig and Reinhardt: Zeit. f. Uutersuchung der Xalirungs u. Centissmitlel, 5, 110,1902.
* Cramer: Inaug. Diss., Halle, 1910.
* Lohrisch: Zeit.f. exper. Path. u. Phartn., 5, 478, 1908.
* Saiki: /our. Biol. Cheni., 2, 251, 1906.
56 PHYSIOLOGICAL CHEMISTRY.
been shown to be a very efficient therapeutic aid in cases of chronic
constipation.^' ^ This is particularly true when the constipation is due
to the formation of dry, hard, fecal masses (scybala), a type of fecal forma-
tion which frequently follows the ingestion of a diet which is very thoroughly
digested and absorbed. The agar, because of its relative indigestibility
and its property of absorbing water yields a bulky fecal mass which is
sufficiently soft to permit of easy evacuation. Agar has been used with
good results in the treatment of constipation in children.^ The function
of agar is not limited to its use in connection with constipation, it may
serve in other capacities as an aid to intestinal therapeutics.^
Experiments on a Pentosan.
1. Solubility. — Test the solubility of gum arable in the ordinary
solvents (see page 27).
2. Iodine Test. — Add a drop of dilute iodine solution to a little gum
arable on a test-tablet. It resembles cellulose in giving no color with
iodine.
3. Hydrolysis of Gum Arabic. — Introduce a little gum arable into a
test-tube, add 5-10 c.c. of strong hydrochloric acid (cone. HCl and water
1:1) and heat to boiling for 5-10 minutes. Cool, neutralize with
potassium hydroxide and test by the Fehling or some other reduction test.
A positive reaction should be obtained indicating that the gum arable
has been hydrolyzed by the acid with the production of a reducing sub-
stance. What is this reducing substance ? How would you identify it ?
Experiments on a Galactan.
1. Solubility. — Test the solubility of agar-agar in the ordinary sol-
vents (see page 27). Observe its marked property of imbibing water
(see page 255).
2. Iodine Test. — Add a drop of dilute iodine solution to a little
agar-agar on a test- tablet. It resembles cellulose in giving no color
with iodine.
3. Hydrolysis of Agar-agar. — Introduce a few pieces of agar-agar
into a test-tube, add 5-10 c.c. of strong hydrochloric acid (cone. HCl
and water I :i) and heat to boiling for 5-10 minutes. Cool, neutralize
with potassium hydroxide and test by the Fehling or some other re-
duction test. A positive reaction should be obtained indicating that
' Mendel: Zentralblat, f. d. ^esammle Phys. u. Path, des Sloffw., No. 17, i, 1908.
^Schmidt: Munch, med. Woch., 52, 1970, 1905.
' Morse: Journal American Medical Ass'?i., 55, 934, 1910.
* Einhorn: Berl. klin. Woch., 49, 113, 1912.
CARBOHYDRATES.
57
the agar-agar has been hydrolyzed by the acid with the production of a
reducing substance. What is this reducing substance? How would
you identify it?
REVIEW OF CARBOHYDRATES.
In order to facihtate the student's ^e^'ie^v of the carhobydrates, the
preparation of a chart similar to the appended model is recommended.
MODEL CHART FOR REVIEW PURPOSES.
Carbo-
hydrate.
Dextrose.
1
"o
Iodine Test.
Moore's Test.
Trommer's Test.
Fehling's Test.
Boettger's Test.
m
'u
4)
•a
c
ea
■>.
is
Barfoed's Test.
Seliwanoff's
Reaction.
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1
<
'3
0
S
0Q«
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ll
.£■<
4)
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q
rt
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i
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—
—
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The signs + and — may be conveniently used to indicate positive
and negative reaction. Only those carbohydrates which are of greatest
importance from the standpoint of physiological chemistry have been
included in the chart.
"Unknown" Solutions of Carbohydrates.
At this point the student will be given several "unknow^n" solutions,
each solution containing one or more of the carbohydrates 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 instructor, a written report of his findings, on slips
furnished by the laboratory.
The scheme given on page 58 may be of use in this connection.
58
PHYSIOLOGICAL CHEMISTRY.
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CHAPTER III.
SALIVARY DIGESTION.
The saliva i? secreted by three pairs of glands, the submaxillary,
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 combined product of all the
glands mentioned. The fact that there are pronounced variations in the
composition of dift'erent fractions of saliva secreted by the same normal
individual on a uniform diet has recently been emphasized by Lothrop
and Gies.^
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 par-
ticular class of stimuli employed as well as upon the character of the
indindual 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 thrown into 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 having 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, results were obtained similar to those
secured when the substances were actually placed in the animal's mouth.
For example, when a pretense was made of throwing sand into the dog's
mouth, a watery saliva was secreted, whereas food under the same con-
ditions excited a thicker and more slimy secretion. The exhibition of dry
food, in which the dog had no particular interest (dry bread) caused the
' Lothrop and Gies: Journal of the Allied {Dental) Societies, 6, 65, 1911.
59
6o PHYSIOLOGICAL CHEMISTRY,
secretion of a large amount of watery saliva, while the presentation of
moist food, which was eagerly desired by the animal, called forth a much
smaller secretion, slimy in character. These experiments show it to be
rather difficult to differentiate between the influence of physiological and
psychical stimuli.
The amount of saliva secreted by an adult in 24 hours has been vari-
ously placed, as the result of experiment and observation, between 1000
and 1500 c.c, the exact amount depending, among other conditions, upon
the character of the food.
The saliva of adults ordinarily has a weak, alkaline reaction to litmus,
but becomes acid, in some instances, 2-3 hours after a meal or during fast-
ing. The saliva of the newborn is generally neutral to litmus, whereas that
of infants, especially those breast-fed, is generally acid. ^ The alkalinity of
saliva is due principally to di-sodium hydrogen phosphate (NajHPOJ
and its average alkalinity may be said to be equivalent to 0.08-0.1 per
cent sodium carbonate. The saliva is the most dilute of all the digestive
secretions, having an average specific gravity of 1.005 ^^^ containing only
o. 5 per cent of solid matter. Among the solids are found albumin, globulin,
mucin, urea, the enzymes salivary amylase (ptyalin), maltase, and peptide
splitting enzymes; phosphates, and other inorganic constituents. Potas-
sium thiocyanate, 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 thiocyanate in the saliva is not
known; it probably comes from the ingested thiocyanates and from the
breaking down of protein material. The attempts to show some relation-
ship between tooth decay and the thiocyanate content of the saliva
secreted into the mouth cavity have met with failure. The most recent
experiments^ indicate a virtual absence of such relationship.
The so-called tartar formation on the teeth is composed almost
entirely of calcium phosphate with some calcium carbonate, mucin,
epithelial cells, and organic debris derived from the food. The calcium
salts are held in solution as acid salts, and are probably precipitated by
the ammonia of the breath. The various organic substances just men-
tioned are carried down in the precipitation of the calcium salts.
The suggestion has been made that mucin is the salivary constituent
" which is particularly influential in the development of local conditions
favoring the onset of dental decay. "^
The principal enzyme of the saliva is known as salivary amylase or
ptyalin. This is an amylolytic enzyme (see page 4), so called because it
possesses the property of transforming complex carbohydrates such as
' Allaria: Monalsschr. fiir Kinderheilkunde, lo, 179, 191 1.
^ Lothrop and Gies: Journal of the Allied {Dental) Societies, 6, 65, 1911.
*Id.; Ibid., 5, No. 4, 1910.
SALIVARY DIGESTION. 6l
starch and dextrin into simpler bodies. The action of salivary amylase
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 drylliro-dcxtrin, a-achroo-dextrin, ^-achroo-dextrin, and
y-achroo-dextrin, the erythro-dextrm being formed directly from soluble
starch and later being itself transformed into a-achroo-dextrin from which
in turn are produced ^-achroo-dextrin, y-achroo-dextrin and perhaps other
dextrins. Accompanying each dextrin a small amount of iso-maltose is
formed, the quantity of iso-maltose growing gradually larger as the process
of transformation progresses. (Erythro-dextrin gives a red color with
iodine, the other dextrins give no color.) The next stage is the transforma-
tion of the final dextrin into f50-wa//05e and subsequently the transforma-
tion of the iso-maltose into maltose, the latter being the principal end-
product of the salivary digestion of starch. At this point a small amount
of dextrose is formed from the maltose, through the action of the enzyme
maltase.
Salivary amylase acts in alkaline, neutral, or combined acid solu-
tions. It will act in the presence of relatively strong combined HCl (see
page 126), whereas a trace (0.003 P^i" cent to 0.006 per cent) of ordinary
free hydrochloric acid will not only prevent the action but will destroy the
enzyme. By sufficiently increasing the alkalinity of the saliva to litmus,
the action of the salivary amylase is inhibited.
It has been claimed by Roger ^ that the activation of human saliva
inactivated by the action of heat or hydrochloric acid could be brought
about by the addition of traces of fresh human saliva. Very recent at-
tempts- to verify this claim have met with failure.
It has recently been shown by Cannon 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.
Food in the pyloric end of the stomach is soon mixed with the gastric
secretion, but food in the cardiac end is not mixed with the acid gastric
juice for a considerable period of time, and in this region during that time
salivary digestion may proceed undisturbed.
It has very recently been found that salivary amylase acts more
eflSciently when the saliva is diluted.^ The optimum dilution for sodium
' Roger: Rev. Gen. des. ScL, i8, 544, IQ07.
- Bergeim and Hawk: Unpublished data.
*Bergeim and Hawk: Unpublished data.
62 PHYSIOLOGICAL CHEMISTRY.
chloride solution (0.3 per cent) was found to be about four volumes,
whereas that for tap water ^ and distilled water was about seven volumes.
These findings are of interest in connection with the more efficient utili-
zation of ingested carbohydrate which has been found to accompany the
drinking of large volumes of water at meal time.^ Lipase also acts better ,
in dilution.^
It has further been demonstrated very recently that the action of sali-
vary amylase is inhibited in the presence of softened water.'' The
inhibitory factor was found to be magnesium hydroxide.^ Electrolytes
have an important influence upon the action of amylases. The CI ion
has a pronounced facilitating action (see Pancreatic Amylase).
The question of the adaptation of the salivary secretion to diet is one
which has received considerable attention in recent years. It has been
claimed, on the basis of experimental evidence,^ that the continued feeding
of a carbohydrate diet causes the secretion of a saliva which contains a
higher concentration of salivary amylase and one which is therefore able to
more efficiently digest the carbohydrate fed. On the other hand strong
evidence^ has been submitted that the amylase content of the saliva is not
increased through the continued feeding of a carbohydrate diet. The
balance of evidence is however opposed to adaptation. In general
the concensus of opinion is opposed to the adaptation of digestive secretions
to diet.
Maltase, sometimes called glucase, is the second enzyme of the saliva.
The principal function of maltase is the splitting of maltose into dextrose.
Besides occurring in the saliva it is also present in the pancreatic and
intestinal juices. For experimental purposes the enzyme is ordinarily
prepared from corn. The principles of the "reversibility" of enzyme
action were first demonstrated in connection with maltase by Croft Hill.
The presence in the saliva of dipeptide- and tripeptide-splitting
enzymes has recently been demonstrated.* Leucyl-glycyl-alanine was
the tripeptide split whereas the cleavage of several dipeptids was
brought about. The action is similar to that of intestinal erepsin (see
Chapter VIII).
Microscopical examination of the saliva reveals salivary corpuscles,
' University of Illinois Water Supply.
^ Mattill and Hawk: Jour. Am. Chem. Soc, 33, 2019, 1911.
^Bradley: Jour. Biol. Chem., 8, 251, 1910.
* Prepared by treating top water with one-sixth its volume of saturated lime water, allowing
to stand 24 hours and filtering.
' Bergeim and Hawk: Unpublished data.
" Neilson and Terry: American Journal of Physiology, 15, 406, 1905; Neilson and Lewis:
Journal of Biological Chemistry, 4, 501, 1908.
^Mendel: American Journal of the Medical Sciences, Oct., 1909; Mendel and Underbill:
Journal of Biological Chemistry, 3, 135, 1907. Mendel, Chapman and Blood: Medical
Record, Aug. 27, 1910.
* Koelker: Zeitschrift fiir physiol. Chem., 76, 27, 191 1.
SALIVARY DIGESTION. 63
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 experi-
ments which follow is to chew a small piece of pure paralBn 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 examination.
I. Microscopical Examination. — Examine a drop of unfiltered
saliva microscopically and compare with Fig. 19 below.
Fig. 19. — Microscopical Constituents of Saliva.
a, Epithelial cells; b, salivary corpuscles; c fat drops; d, leucocytes; e, / and g. bacteria;
h, i and k, fission-fungi.
2. Reaction. — Test the reaction to litmus, phenolphthalein and
Congo red.
3. Specific Gravity. — Partially fill a urinometer cylinder with saliva,
introduce the urinometer, 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.^ — 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) copper sulphate solution. The formation of a purplish-violet
color is due to mucin.
6. Millon's Reaction.- — Add a few drops of Millon's reagent to a
little saliva. A light yellow precipitate formed by the mucin gradually
turns red upon being gently heated.
7. Preparation of Mucin. — Pour 25 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
' The significance of this reaction is pointed out on page 98.
- The significance of this reaction is pointed out on page 97.
64 PHYSIOLOGICAL CHEMISTRY,
ether. Finally dry the precipitate, remove it from the paper and make
the following tests on the mucin: (a) Test its solubility in the ordinary
solvents (see page 27); (b) 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 HCl 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 conjugated protein or glycoprotein (see
p. 94) and upon boiling wdth the acid the carbohydrate group in the mole-
cule has been split off from the protein 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 HNO3 and add AgXOg. For
phosphates, acidify with HNO3, ^^^^ ^^^ ^.dd molybdic solution.^ For
sulphates, acidify with HCl and add BaCl, and warm. For calcium,
acidify with acetic acid, CH3COOH, and add ammonium oxalate,
(NHJX.O,.
9. Viscosity Test. — Place filter papers in two funnels, 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. Xote
the progress of filtration in each case. ^^Tiy does one solution filter more
rapidly than the other ?
10. Test for Nitrites.— Add 1-2 drops of dilute HjSO^ 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. Thiocyanate 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 HCl. Red ferric thiocyanate forms.
To show that the red coloration is not due to iron phosphate add a drop
of HgClj when colorless mercuric thiocyanate forms.
(6) Solera's Reaction. — This test depends upon the liberation of iodine
through the action of thiocyanate upon iodic acid. Moisten a strip of
starch paste-iodic acid test paper^ with a little saliva. If thiocyanate be
present the test paper will assume a blue color, due to the liberation of
iodine and the subsequent formation of the so-called iodide of starch.
* Molybdic solution is prepared as follows, the parts being by weight:
I part molybdic acid.
4 parts ammonium hydroxide (sp. gr. 0.96).
15 parts nitric acid (sp. gr. i. 2).
^ This test paper is prepared as follows: Saturate a good quality of filter paper with 0.5
per cent starch paste to which has been added sufficient iodic acii to make a i per cent
solution of iodic acid and allow the paper to dry in the air. Cut it in strips of suitable size and
preserve for use.
SALIVARY DIGESTION 65
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
solution should soon disappear, indicating the formation of soluble starch
which gives a blue color with iodine. This body should soon be trans-
formed 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 positive
Fehling's test may be obtained while the solution still reacts red with
iodine inasmuch as some iso-maltose is formed from the soluble starch
coincidently with the formation of the erythro-dextrin. 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 ID drops of saliva and place the tube in the incubator or water-bath
at 40° C. After one-half hour test the solution by Fehling's test. ^ Is any
reducing substance present? What do you conclude regarding the
salivary digestion of inulin ?
15. Influence of Temperature. — In each of four tubes place about
5 c.c. of starch paste. Immerse one tube in cold water from the faucet,
keep a second at room temperature, and place a third in the incubator or
the water-bath at 40° C. Now add to the contents 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 test,
using the test-tablet, and note in which tube the most rapid digestion
occurs. Explain the results.
16. Influence of Dilution."— Take a series of six test-tubes each
containing 9 c.c. of water. Add i c.c. of saliva to tube i and shake
thoroughly. Remove i c.c. of the solution from tube i to tube 2 and
after mixing thoroughly remove i 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
* If the inulin solution gives a reduction before being acted upon b)' the saliva it wrill be
necessary to determine the extent of the original reduction by means of a "check" test (see
page 52).
^ The technic of Wohlgemuth's method (see page i8) may be employed in this test if
so desired.
66 PHYSIOLOGICAL CHEMISTRY.
very thoroughly, and place in the incubator or 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 six tubes in each of which is placed 4 c.c. of one of the
follo\nng strengths oifree HCl: 0.2 per cent, o.i per cent, 0.05 per cent,
0.025 per cent, 0.0125 per cent and 0.006 per cent. Now 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: o.i per cent, 0.05 per cent,
0.025 per cent, 0.0125 per cent, 0.006 per cent and 0.003 P^^ 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 find ?
(&) Influence of Combined Acid {Protein Salt). — Repeat the first three
experiments of the above series using combined hydrochloric acid (see
page 126) instead of the/ree acid. How does the action of the combined
acid differ from that of \htfree acid? (For a discussion of combined acid
see page 126.J
(c) Influence of Alkali. — Repeat the first four experiments under (a)
replacing the HCl by 2 per cent, i per cent, 0.5 per cent and 0.25 per cent
NajCOg. Neutralize the alkalinity before trying the iodine test (see
Starch, 5, page 50).
(d) Nature of the Action of Acid and Alkali. — ^Place 2 c.c. of saliva and
2 c.c. of 0.2 per cent HCl in a test-tube and leave for 15 minutes. Neu-
tralize the solution, add 4 c.c. of starch paste and place the tube in the
incubator or water-bath at 40° C. In 10 minutes test by the iodine and
Fehling's tests and explain the result. Repeat the experiment, replacing
the 0.2 per cent HCl by 2 per cent Na2C03. Whd.i 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 1/2 c.c. of one of the solutions named below.
Shake well, add 1/2 c.c. of saliva to each tube, thoroughly mix, and place
in the incubator or 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 lead acetate, 2 per
cent copper sulphate, 5 per cent ferric chloride, 8 per cent mercuric
chloride; Neutral salts, 10 per cent sodium chloride, 10 per cent mag-
nesium sulphate, 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 conclusions ?
SALIVARY DIGESTION 67
19. Excretion of Potassium Iodide. — Ingest a small dose of potas-
sium iodide (0.2 gram) contained in a gelatin capsule, 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 i c.c. of NaNOz
and I c.c. of dilute H.SO/ in a test-tube, add a little saliva directly
from the mouth, and a small amount of starch paste. 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 inges-
tion of the potassium iodide and the appearance of the first traces of the
substance in the saliva. If convenient, the urine may also be tested.
The chemical reactions taking place in this experiment are indicated in
the following equations :
(a) 2NaN03 + H^SO,— 2HNO3 + Na^SO,.
(b) 2KI + H2SO, — 2HI + K2SO,.
(c) 2HN02-h 2HI— I2 + 2H2O-I-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 (p. 32) and the phenylhydrazine reaction (see Dextrose, 3,
page 28). On the dextrin precipitate try the iodine test (page 50). Also
hydrolyze the dextrin as given under Dextrin, 4, page 53.
' Instead of this mixture a few drops of HNO3 possessing a yellowish or brownish color
due to the presence of HNO2 may be employed.
CH.\PTER IV.
PROTEINS:^ THEIR DECOMPOSITION AND SYNTHESIS.
The proteins are a class of substances, which in the light of our present
knowledge, consist, in the main, of combinations of a-amino-acids or
their derivatives. These protein substances form the chief constituents
of many of the fluids of the body, constitute the organic basis of animal
tissue, and at the same time occupy a decidedly preeminent position among
our organic food-stuffs. They are absolutely necessary to the uses of the
animal organism for the continuance of life and they cannot be satis-
factorily replaced in the diet of such an organism by any other dietary
constituent either organic or inorganic. Such an organism may exist
mthout protein food for a period of time, the length of the period varying
according to the specific organism and the nature of the substitution
offered for the protein portion of the diet. Such a period is, however,
distinctly one of existence rather than one of normal life and one which is
consequently not accompanied by such a full and free exercise of the
various functions of the organism as would be possible upon an evenly
balanced ration, i. e., one containing the requisite amount of protein food.
These protein substances are, furthermore, essential constituents of all
living cells and therefore without them vegetable life as well as animal life
is impossible.
The proteins, which constitute such an important group of substances,
differ from the carbohydrates and fats very decidedly in elementary com-
position. In addition to containing carbon, hydrogen, and oxygen, which
are present in fats and carbohydrates, the proteins invariably contain
nitrogen in their molecule and generally sulphur also. Proteins have also
been described which contain phosphorus, iron, copper, iodine, manganese,
and zinc. The percentage composition of the more important members
of the group of protein substances would fall within the following limits :
0 = 50-55 per cent, H = 6-7.3 P^^ cent, 0=19-24 per cent, N= 15-19 per
cent, 5=0.3-2.5 per cent, P=o.4-o.8 per cent when present. When iron,
copper, iodine, manganese, or zinc are present in the protein molecule they
are practically without exception present only in traces and with the
exception of iodine are probably not constituents of the protein molecule.^
* The term proteid has been very widely used by English-speaking scientists to signify
the class of substances we have called proteins.
* Some investigators regard these elements as contaminations, or constituents of some
non-protein substance combined with the protein.
68
PROTEINS. 69
Of all the various elements of the protein molecule, nitrogen is by far
the most important. The human body needs nitrogen for the continua-
tion of life, but it cannot use the nitrogen of the air or that in various other
combinations as we lind it in nitrates, nitrites, etc. However, in the pro-
tein molecule the nitrogen is present in a form which is utilizable by the
body. The nitrogen in the protein molecule occurs in at least four
different forms as follows:
I. Monamino acid nitrogen.
II. Diamino acid nitrogen or banc nitrogen.
III. Amide nitrogen.
IV. A guanidine residue.
The actual structure of the protein molecule is stilf unknown, and we
have as yet no means by which its molecular weight can be even approxi-
mately established. The many attempts which have been made to deter-
mine this have led to very different results, some of which are given in the
following table:
Globin =15000 — 16086
Oxyhaemoglobin = 14800 — 15000— 16655 — 16730
Of these figures, those given for oxyhaemoglobin deserve the most
consideration, for these are based on the atomic ratios of the sulphur
and iron contained in this substance. The simplest formula that can be
calculated from analyses of oxyhaemoglobin, namely, CesgHugiNjoySj-
FeOjjo, serves to show the great complexity of this substance.
The decomposition^ of protein substances may be brought about by
oxidation or hydrolysis, but inasmuch as the hydrolytic procedure has
been productive of the more satisfactory results, that type of decomposition
procedure alone is used at present. This hydrolysis of the protein mole-
cule may be accomplished by acids, alkalis, or superheated steam, and in
digestion by the action of the proteolytic enzymes. The character of the
decomposition products varies according to the method utilized in tearing
the molecule apart'. Bearing this in mind, we may say that the decom-
position products of proteins include proteoses, peptones, peptides, carbon
dioxide, ammonia, hydrogen sulphide, and amino acids. These amino acids
constitute a long list of important substances which contain nuclei belong-
ing either to the aliphatic, carbocyclic, or heterocyclic series. The list
includes glycocoll, alanine, serine, phenylalanine, tyrosine, cystine, trypto-
phane, histidine, valine, arginine, leucine, isoleucine, lysine, asparlic acid,
glutamic acid, proline, oxyproline, and diaminotrihydroxydodecanoic acid.
'The terms "degradation," "dissociation," and "cleavage," are often used in this
connection.
70 PHYSIOLOGICAL CHEMISTRY.
Of these amino acids, tyrosine and phenylalanine contain carbocyclic
nuclei: histidine, proline, and tryptophane contain heterocyclic nuclei:
and the remaining members of the list, as given, contain aliphatic nuclei.
The amino acids are preeminently the most important class of protein
decomposition products. These amino acids are all a-amino acids, and,
^A^ith the exception of glycocoll, are all optically active. Furthermore,
they are amphoteric substances and consequently are able to form salts
with both bases and acids. These properties are inherent in the NHj and
COOH groups of the amino acids.
The decomposition products of protein may be grouped as primary
and secondary decomposition products. By primary products are meant
those which exist as radicals within the protein molecule and which are
liberated, upon cleavage of this molecule, with their carbon chains intact
and the position of their nitrogen unaltered. The secondary products are
those which result from the disintegration of the primary cleaA^age prod-
ucts. No matter what method is used to decompose a given protein
molecule, the primary products are largely the same under all conditions.^
In the process of hydrolysis the protein molecule is gradually broken
down and less complicated aggregates than the original molecule are
formed, which are known as proteoses, peptones, and peptides, and which
still possess true protein characteristics. Further hydrolysis causes the
ultimate transformation of these substances, of a protein nature, into the
amino acids of known chemical structure. In this decomposition the
protein molecule is not broken down in a regular manner into 1/2, 1/4,
1/8 portions and the amino acids formed in a group at the termination of
the hydrolysis. On the contrary, certain amino acids are formed very
early in the process, in fact while the main hydrolytic action has pro-
ceeded no further than the proteose stage. Gradually the complexity
of the protein portion undergoing decomposition is simplified by the split-
ting off of the amino acids and finally it is so far decomposed through pre-
vious cleavages that it yields only amino acids at the succeeding cleavage.
In short, the general plan of the hydrolysis of the protein molecule is
similar to the hydrolysis of starch. In the case of starch there is
formed a series of dextrins of gradually decreasing complexity and coin-
cidently with the formation of each dextrin a small amount of sugar is
split off and finally nothing but sugar remains. In the case of protein
hydrolysis there is a series of proteins of gradually decreasing complexity
produced and coincidently with the formation of each new protein sub-
stance amino acids are split off and finally the sole products remaining
are amino acids.
' Alkaline hydrolysis yields urea and ornithine which result from arginine, the product of
acid hydrolysis.
PROTEINS. 71
Inasmuch as diversity in the method of decomposing a given protein
does not result in an cc[ually diversified line of decomposition products,
but, on the other hand, yields products which are quite comparable in
character, it may be argued that there are probably well-defined lines of
cleavage in the indi\'idual protein molecule and that no matter what the
force brought to bear to tear such a molecule apart, the disintegration,
when it comes, will yield in every case certain definite fragments. These
fragments may be called the "building stones" of the protein molecule, a
term used by some of the German investigators. Take, for example, the
decomposition of protein which may be brought about through the action
of the enzyme trypsin of the pancreatic juice. When this enzyme is allowed
to act upon a given protein, the latter is disintegrated in a series of definite
cleavages, resulting in the formation of proteoses, peptones, and peptides
in regular order, the peptides being the last of the decomposition products
which possess protein characteristics. They are all built up from
amino acids and are therefore closely related to these acids on the one
side and to peptones on the other. We have di-, tri-, tetra-, penta-, deca-,
and poly-peptides which are named according to the number of amino
acids included in the peptide molecule. Following the peptides there are
a diverse assortment of monamino and diamino acids which constitute the
final products of the protein decomposition. These acids are devoid of
any protein characteristics and are therefore decidedly different from the
original substance from which they were derived. From a protein of huge
molecular weight, a typical colloid, perhaps but slightly soluble, and
entirely non-diffusible, we have passed by way of proteoses, peptones, and
peptides to a class of simpler crystalline substances which are, for the most
part, readily soluble and diffusible.
These amino acids after their production in the process of digestion,
as just indicated, are synthesized within the organism to form protein
material which goes to build up the tissues of the body. It is thus seen
that the amino acids are of prime importance in the animal economy. It
was formerly believed that these essential factors in metabolism and
nutrition could not be produced within the animal organism from their ele-
ments, but were only yielded upon the hydrolysis of ingested protein of
animal or vegetable origin. Recent experiments, however, by Abderhol-
den and by Grafe and Schlapfcr and others indicate that the nitrogen
of food protein may in part be replaced by ammonium salts. Experi-
ments by Osborne and others also indicate amino acid synthesis by
animals.
There are formed, by life processes in both the animal and the
vegetable kingdom certain transformation products of amino acids.
Our knowledge regarding these has been advanced principally through the
72
PHYSIOLOGICAL CHEMISTRY.
efforts of Kutscher and his colleagues. This class of substances has
been given the name aporrhegmas.^ Among the aporrhegmas are
included acids and bases formed in putrefaction as well as a number of
similar compounds which have not been isolated from putrefaction mix-
tures but are formed normally in the plant or animal body. (For further
discussion of aporrhegmas, see chapter on Putrefaction.)
Important data regarding the decomposition products of the protein
molecule are given in the tables which follow.
COMPARISON OF THE DECOINIPOSITION PRODUCTS OF PROTAMINES, AND
OTHER PROTEINS.
I Protamines. -
(Per cent of total nitrogen of
amino acid).
Other Proteins.
(Per cent of amino acids in
proteins.)
4- .... + + ....
2 . 00
3.6
i-S
0.8 4.2
9.79
Valine
+ + 4- T.65
3-34
6.2
7.2
I.O '
1.88
+
6.62
14-5
9.4
2.1 Izg.o
19-55
Proline
.... 3 .8' 1+43
13.22
4.1
6.7
5-2 1 2.3
9.04
Phenylalanine
2-3 5
31
3-2
0.4 : 4-2
6.55
Aspartic acid
0.58
4-5
1-4
0.56 4.4
I. 71
Glutamic acid . . . .
43.66
18.74
II .0
i.88i 1.7
26.17
+325
O.I3
0.33
o.S
0.4 1 0.6
I . 20
2 . 1
4-5
0! 1.3
3.55
Arginine
. . . . 88.8 67.7 63. 5 8.7 28.0 88.0 89.2
3.16
14.2
4.84
7.62[ 5.4
i.SS
0
1-7
S.9S
2-75 4-3
Histidine
II. 8
0.61
2 . 2
2.50
1
0.4011 .0
0.82
Tryptophane
+ +
I .0
4-
I -5
0 4-
0
_
1 .00 0.065 °
0.3
J
Oxyproline .... i .... i . . ' 1 '
2.0 0.23 6.4
I .0
f
Diaminotrihydroxydo- 1 . . . .
0.7S
1
?
decanoic acid. [ ; ,
.... 1
5.22 ]
2.3
1. 61
3.64
1
' Ackermann and Kutscher: Zeit. physiol. Chent., 69, 263, 1910; Ackermann: Ibid., 273: Engeland
and Kutscher: Ibid., 282.
^ Kossel: Zeit. physiol. Chem., 44, 347, 1905.
' Osborne and Guest: Jour. Biol. Chem., 9, 425, 1911.
< Abderhalden, Kcsseland others.
' Abderhalden, Fischer, Momer and others.
•Fischer, Levene and Aders: Zeit. physiol. Chem., 35, 70, 1902; also Levene and Beatty: Ibid, 49,
252, 1906.
' Abderhalden : Zei/. physiol. Chem., 37^ 484, 1903.
' Osborne and Liddle, Am. Jour. Physiol., 26, 295, 1910.
* This unique and important protein has probably been more carefully analyzed than any other.
FK(JTi;iN.S.
73
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74
PHYSIOLOGICAL CHEMISTRY.
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§ i:2
PROTEINS. 75
When we examine the formulas of the principal members of the
crystalline end-products of protein decomposition we note that they are
invariably acids, as has already been mentioned, and contain an NH2
group in the a position. This relation of the NH, group to the acid radi-
cal is constant, no matter what other groups or radicals are present. We
may have straight chains as in alanine and glutamic acid, the benzene ring
as in phenylalanine, or we may have sulphurized bodies as in cystine and
still the formula is always of the same type, i. e.,
NH,
I
R— CH— COOH
It is seen that this characteristic grouping in the amino acid provides
each one of these ultimate fragments of the protein molecule with both a
strong acid and a strong basic group. For this reason it is theoretically
possible for a large number of these amino acids to combine and the result-
ing combinations may be very great in number, since there is such a varied
assortment of the acids. The protein molecule, which is of such mam-
moth proportions, is probably constructed on a foundation of this sort.
Of late much valuable data have been collected regarding the synthetic
production of protein substances, the leaders in this line of investigation
being Fischer and Abderhalden. After ha\dng gathered a mass of data
regarding the final products of the protein decomposition and demonstrat-
ing that amino acids were the ultimate results of the various forms of
decomposition, these investigators, and notably Fischer, set about in an
effort to form, from these amino acids, by synthetic means, substances
which should possess protein characteristics. The simplest of these
bodies formed in this way was synthesized from two molecules of glyco-
coll with the liberation of water, thus:
CH,-(NH,)-CO OH H HN-CH^-COOH.
The body thus formed is a dipeptide, called glycyl- glycine. In an analo-
gous manner may be produced leucyl-leucine, through the synthesis of
two molecules of leucine or leucyl-alanyl- glycine through the union of one
molecule of leucine, one of alanine, and one of glycocoll. By this pro-
cedure Fischer and his pupils have been able to make a large number of
peptides containing varied numbers of amino-acid radicals, the name
polypeptides being given to the whole group of synthetic substances thus
formed. The most complex poplypeptide yet produced is one containing
fifteen glycocoll and three leucine residues.
Notwithstanding the fact that most synthetic polypeptides are pro-
duced through a union of amino acids bv means of their imide bonds, it
76 PHYSIOLOGICAL CHEMISTRY.
must not be imagined that the protein molecule is constructed from amino
acids linked together in straight chains in a manner analogous to the
formation of simple peptides, such as glycyl-glycine. The molecular
structure of the proteins is much too complex to be explained upon any
such simple formation as that. There must be a variety of linkings, since
there is a varied assortment of decomposition products of totally different
structure.
Many of these synthetic bodies respond to the biuret test, are precipi-
tated by phosphotungstic acid, and behave, in other ways, as to leave no
doubt as to their protein characteristics. For instance, a number of
amino acids each possessing a sweet taste have been synthesized in such a
manner as to yield a polypeptide of hitter taste, a well-known characteristic
of peptones. From the fact that the polypeptides formed in the manner
indicated have free acidic and basic radicals we gather the explanation of
the amphoteric character of true proteins.
For the benefit of those especially interested in such matters a photo-
graph of the Fischer apparatus (Fig. 23, page 80) used in the fractional
distillation, in vacuo, of the esters of the decomposition products of the
proteins, as well as micro-photographs and drawings of preparations of
several of these decomposition products (Figs. 20 to 32, pages 77 to 89)
are introduced. For the preparations and the photograph of the appa-
ratus the author is indebted to Dr. T. B. Osborne, of New Haven, Conn.,
who has made many important observations upon the hydrolysis of
proteins. The reproduction of the crystalline form of some of the more
recent of the products may be of interest to those viewing the field of
physiological chemistry from other than the student's aspect.
An extended discussion of the various decomposition products being
out of place in a book of this character, we will simply make a few general
statements in connection with the primary decomposition products.
DISCUSSION OF THE PRODUCTS.
Ammonia, NH3. — Ammonia is an important decomposition product
of all proteins and probably arises from an amide group combined
with a carboxyl group of some of the amino acids. It is possible that the
dibasic acids, aspartic and glutamic, furnish most of these carboxyl
groups. This is indicated by the more or less close relationship which
exists between the amount of ammonia and that of the dibasic acids which
the several proteins yield upon decomposition. The elimination of the
ammonia from proteins under the action of acids and alkalis is very
similar to that from amides like asparagine.
PROTEINS.
77
Glycocoll, C2H5NO2. — Glycocoll, or amino acetic acid, is the simplest
of the amino acids and has the following formula:
NH,
I
H— C— COOH.
1
H
Glycocoll, as the* formula shows, contains no asymmetric carbon atom,
and is the only amino acid yielded by protein decomposition which is
optically inactive. Glycocoll and leucine were among the first decom-
position products of proteins to be discovered. Upon administering benzoic
acid to animals the output of hippuric acid in the urine is greatly increased,
Fig. 20. — Glycocoll Ester Hydrochloride.
thus showing a synthesis of benzoic acid and glycocoll in the organism
(see page 168, Chapter IX). Glycocoll, ingested in small amount, is
excreted in the urine as urea, whereas if administered in excess it appears
in part unchanged in the urine. It is usually separated from the mixture
of protein decomposition products as the hydrochloride of the ester. The
crystalline form of this compound is shown in Fig. 20.
Alanine, CjH^NOg. — Alanine is a-amino-propionic acid, and as such
it may be represented structurally as follows :
H NH2
I i
H— C— C— COOH.
1 I
H H
Obtained from protein substances, alanine is dextro-rotatory, is very
soluble in water, and possesses a sweet taste. Tyrosine, phenylalanine,
78
PHYSIOLOGICAL CHEMISTRY.
cystine, and serine are derivatives of alanine. This amino acid has been
obtained from nearly all proteins examined. Its absence from those
proteins from which it has not been obtained has not been proven. Most
proteins yield relatively small amounts of alanine.
Serine, C3H7NO3. — Serine is a-amino-^-hydroxy-propionic acid and
possesses the following structural formula:
OH NH,
I I "
H — C — C— COOH
H H
Serine obtained from proteins is laevo-rotatory, possesses a sweet taste,
and is quite soluble in water. Serine is not obtained in quantity from
most proteins, but is yielded abundantly by silk glue. Owing to the
difficulty of separating serine it has not been found in a ' number of
proteins in which it probably occurs. Serine_crystals are shown in Fig.
2T, below.
Fig. 21. — Serine.
Phenylalanine, CgHjjNO,. — This product is ^-phenyl- a-amino-
propionic acid, and may be represented graphically as follows:
H NH,
I I "
C-C-COOH.
I I
H H
The Isevo-rotatory form is obtained from proteins. Phenylalanine has
been obtained from all the proteins examined except from the protamines
and some of the albuminoids. The yield of this body from the decom-
PROTEINS,
79
The
position of proteins is frequently greater than the yield of tyrosine,
crystalline form of phenylalanine is shown in Fig. 22.
Tyrosine, CgHjjNOg. — Tyrosine, one of the first discovered end-
products of protein decomposition, is the amino acid, p-ox y- .3 -phenyl- a-
amino-propianic acid. It has the following formula:
H NH2
I I
C-C-COOH.
H H
OH
The tyrosine which results from protein decomposition is usually Icevo-
rotatory. Tyrosine is one of the end-products of tryptic digestion and
usually separates in conspicuous amount early in the process of digestion.
Fig. 22. — Phexyl.\laxixe.
It does not occur, however, as an end-product of the decomposition of
gelatin.
Tyrosine is found in old cheese, and derives its name from this fact.
It crystallizes in tufts, sheaves, or balls of fine needles, which decompose
at 295° C. and are sparingly soluble in cold (1-2454) water, but much
more so in boiling (1-154) water. Tyrosine forms soluble salts with
alkalis, ammonia, or mineral acids, and is soluble, with difficulty, in
acetic acid. It responds to Millon's reaction, thus showing the presence
of the hydroxyphenyl group, but gives no other protein test. The aro-
matic groups present in tyrosine, phenylalanine, and tryptophane cause
proteins to yield a positive xanthoproteic reaction. In severe cases of
typhoid fever and smallpox, in acute yellow atrophy of the liver, and in
8o
PHYSIOLOGICAL CHEMISTRY.
acute phosphorus poisoning, tyrosine has been found in the urine. Tyro-
sine crystals are shown in Fig. 24, page 81.
Fig. 23. — Fischer Apparatus.
Reproduced from a phoiograph made by Prof. E. T. Reichert, of the University of Penn-
sylvania. The negative was furnished by Dr. T. B. Osborne, of New Haven, Conn.
A, Tank into which freezing mixture is pumped and from whicla it flows through the
condenser, B; C, fiask from which the esters are distilled, the distillate being collected in D;
E, a Dewar flask containing liquid air serving as a cooler for condensing tube F; G and G',
tubes leading to the Geryck pump by which the vacuum is maintained; /, tube leading to a
McLeod gauge (not shown in figure) ; J, a bath containing freezing mixture in which the
receiver D is immersed; K, a bath of water during the first part of the distillation and of
oil during the last part of the process; 1-5, stop cocks which permit the cutting out of different
parts of the apparatus as the procedure demands.
Cystine, CgHj204N2S2. — Friedmann has recently shown cystine to
be a-diamino-[i-dithiolactyl acid and to possess the following structural
formula :
CH^S-SCH,
I I
CHNH, CHNH,
COOH COOH
PROTEINS. 8 1
Cystine is the principal sulphur-containing body obtained from the
decomposition of protein substances. It is obtained in greatest amount
as a decomposition product of such keratin-containing tissues as horn,
Fig. 24. — Tyrosine.
hoof, and hair. Cystine occurs in small amount in normal urine and is
greatly increased in quantity under certain pathological conditions. It
crystallizes in thin, colorless, hexagonal plates which are shown in Fig. 25.
Cystine is very slightly soluble in water but its salts, with both bases and
acids, are readily soluble in water. It is laevo-rotatory.
Fig. 25. — Cystixe.
It was formerly claimed that cystine occurred in two forms, i. e.,
stone-cystine and protein-cystine and that these two forms are distinct in
their properties. This view is incorrect.
6
82 PHYSIOLOGICAL CHEMISTRY.
For a discussion of cystine sediments in urine see Chapter XX.
Tryptophane, CiiHj2N202. — Recently Ellinger and Flamand have
shown that tryptophane possesses the following formula :
/\ C-CH2-CH(NH,)-C00H
CH
NH
It is therefore indol-a-amino-propionic acid. Tryptophane is the
mother-substance of indole, skatole, skatole acetic acid and skatole carhoxylic
acid, all of which are formed as secondary decomposition products of
proteins. Its presence in protein substances may be shown by means of
the Adamkiewicz reaction or the Hopkins-Cole reaction (see page 98).
It may be detected in a tryptic digestion mixture through its property of
giving a violet color-reaction with bromine water. Tryptophane is
yielded by nearly all proteins, but has been shown to be entirely absent from
zein, the prolamin (alcohol-soluble protein) of maize and also from gelatin.
Upon being heated to 285° C. tryptophane decomposes with the evolu-
tion of gas.
Histidine, CgHgNgO,. — Histidine is a-amino-^-imidazol-propionic
acid with the following structural formula :
H NH,
I I
HC - C-C-C-COOH.
I I
H H
HN. ^N
CH
The histidine obtained from proteins is laevo-rotatory. It has been
obtained from all the proteins thus far examined, the majority of them
yielding about 2.5 per cent of the amino acid. However, about 11 per
cent was obtained by Abderhalden from globin, the protein constituent of
oxy haemoglobin and about 13 per cent by Kossel and Kutscher from the
protamine sturine.
Crystals of histidine dichloride are shown in Fig. 26, page 83.
Knoop^s Color Reaction for Histidine. — To an aqueous solution of
histidine or a histidine salt in a test-tube add a little bromine water. A
yellow coloration develops in the cold and upon further addition of bro-
mine water becomes permanent. If the tube be heated,^ the color will
disappear and will shortly be replaced by a faint red coloration which
• The same reaction will take place in the cold more slowly.
PROTEINS. 83
gradually passes into a deep wine red. Usually black, amorphous par-
ticles separate out and the solution becomes turbid.
The reaction cannot be obtained in solutions containing free alkali. It
is best to use such an amount of bromine as will produce a permanent
yellow color in the cold. The use of a less amount of bromine than this
produces a weak coloration whereas an excess of bromine prevents the
reaction. The test is not v^ry delicate, but a characteristic reaction may
always be obtained in 1:1000 solutions. The only histidine derivative
which yields a similar coloration is imidazolethylamine, and the reaction
\C£
Fig. 26. — Histidine Bichloride.
in this case is rather weak as compared with the color obtained with
histidine or histidine salts.
Valine, C-Hj^NOj. — The amino- valerianic acid obtained from
proteins is a-amino-isovalerianic acid, and as such bears the following
formula :
CH3 NH2
H-C C-COOH.
1 I
CH3 H
It closely resembles leucine in many of its properties, but is more soluble
in water. It is a difficult matter to identify valine in the presence of
leucine and isoleucine inasmuch as these amino acids crystallize together
in such a way that the combination persists even after repeated recrystal- .
lizations. Valine is dextro-rotatory.
Arginine, CgHj^N^Oj. — Arginine is guanidine-a-amino-valerianic
acid and possesses the following structural formula:
84 PHYSIOLOGICAL CHEMISTRY.
H H H NH^
NH-C-C-C-C-COOH.
I 1 I I I
NH=C H H H H
It has been obtained from every protein so far subjected to decomposition.
The arginine obtained from proteins is dextro-iotatory, and has pro-
nounced basic properties, reacts strongly alkaline to litmus, and forms
stable carbonates. Because of these facts, Kossel considers arginine to be
the nucleus of the protein molecule. It is obtained in widely different
amounts from different proteins, over 85 per cent of certain protamines
having been obtained in the form of this amino acid. It is claimed that in
the ordinary metabolic activities of the animal body arginine gives rise
to urea. While this claim is probably true, it should, at the same time,
be borne in mind that the greater part of the protein nitrogen is eliminated
as urea and that, therefore, but a very small part can arise from arginine.
Leucine, CgH^gNOg. — Leucine is an abundant end-product of the
decomposition of protein material, and was one of the first of these
products to be discovered. It is a-amino-isohutyl-acetic acid, and
therefore has the following formula:
CH3 NH2
H-C-CH^-C-COOH.
CH3 H
The leucine which results from protein decomposition is /-leucine.
Leucine is present normally in the pancreas, thymus, thyroid, spleen, brain,
liver, kidneys, and salivary glands. It has been found pathologically in
the urine (in acute yellow atrophy of the liver, in acute phosphorus
poisoning, and in severe cases of typhoid fever and smallpox), and in
the liver, blood, and pus.
Pure leucine crystallizes in thin, white, hexagonal plates. Crystals of
pure leucine are reproduced in Fig. 27. It is rather easily soluble in water
(46 parts), alkalis, ammonia, and acids. On rapid heating to 295° C,
leucine decomposes with the formation of carbon dioxide, ammonia, and
amylamine. Aqueous solutions of leucine obtained from proteins are laevo-
rotatory, but its acid or alkaline solutions are dextro-rotatory. So-called
impure leucine^ is a slightly refractive substance, which generally crystal-
* These balls of so-called impure leucine do contain considerable leucine, but inasmuch
as they may contain many other things it is a bad practice to allude to them as leucine.
PROTEINS.
85
lizes in balls having a radial structure, or in aggregations of spherical
bodies, Fig. 109, Chapter XX.
Isoleucine, CgHjgNOj. — Isoleucine is a-amino-^-methyl-^-elhyl-pro-
pionic acid, and possesses the following structural formula:
CIL
H-C
C2H5
NH,
C-COOH.
I
H
This amino acid was discovered by Ehrlich in 1903. Its presence has
been established among the decomposition products of only a few proteins
Fig. 27. — Leucine.
although it probably occurs among those of many or most of them. Ehr-
lich has shown that the d-o^myl alcohol which is produced by yeast fermen-
tation originates from isoleucine and the isoamylalcohol originates from
leucine. Isoleucine is dextro-rotatory.
Lysine, CgHi^NjOg. — The three bodies, lysine, arginine, and his-
tidine, are frequently classed together as the hexone bases. Lysine was
the first of the bases discovered. It is a-e-diainino-caproic acid and hence
possesses the following structure:
NH^H H H NH,
I ! I I I
H-C-C-C-C-C-COOH
i I I I I
H H H H H
It is dextro-rotatory and is found in relatively large amount in casein and
gelatin. Lysine is obtained from nearly all proteins, but is absent from
the vegetable proteins which are soluble in strong alcohol. It is the
mother-substance of cadaverin and has never been obtained in crystalline
PHYSIOLOGICAL CHEMISTRY.
form. Lysine is usually obtained as the picrate which is sparingly soluble
in water and crystallizes readily. These crystals are shown in Fig. 28.
Fig. 28. — Lysine Picrate.
Fig. 29. — AsPARTic Acid.
Aspartic Acid, C^H^NO^. — Aspartic acid is amino-succinic acid and
has the following structural formula:
H-CCOOH
H-CCOOH.
H
The amide of aspartic acid, asparagine, is very widely distributed in
the vegetable kingdom. The crystalline form of aspartic acid is exhibited
in Fig. 29.
PROTEINS.
87
Aspartic acid has been found among the decomposition products of
all the proteins examined, except the protamines. It has not been obtained,
however, in very large proportion from any of them. The aspartic acid
obtained from protein is laevo-rotatory.
Glutamic Acid, C^HgNO^. — This acid is a-amino-normal-glutaric
acid and as such bears the following graphic formula :
NH,
I
H-CCOOH
I
H-C-H
I
H-CCOOH.
H
Glutamic acid is yielded by all the proteins thus far examined, except
the protamines, and by most of these in larger amount than any other of
Fig. 30. — Glutamic Acid.
Reproduced from a micro-photograph made by Prof. E. T. Reichert, of the University of
Pennsylvania.
their decomposition products. It is yielded in especially large proportion
by most of the proteins of seeds, 43.66 per cent ha^•ing been obtained
very recently by Osborne and Guest ^ by the hydrolysis of gliadin, the
prolamin of wheat. This is the largest amount of any single decompo-
sition product yet obtained from any protein except the protamines.
Glutamic acid and aspartic acid are the only dibasic acids which have
thus far been obtained as decomposition products of proteins. As there
is an apparent relation between the proportion of these acids and that of
' Osborne and Guest; Jour. Biol. Ghent., 9, 425, igii
88
PHYSIOLOGICAL CHEMISTRY.
ammonia which the different proteins yield it is possible that one of the
carbox}'! groups of these acids is united with NH2 as an amide, the other
carboxyl group being united in polypeptide union (see page 75) with some
other amino acid. This might be represented by the following formula:
R-CHNH-COOH
/
CO- CHXH3- CH.- CH,- CONH2.
It has not been definitely proven, however, that this form of linking
actually occurs.
The glutamic acid, }delded by proteins upon hydrolysis, is dextro-
rotatory. Crystals of glutamic acid are reproduced in Fig. 30, p. 87.
Fig. 31.— L^vo-a-PROLENT;.
Proline, CgHgNOg. — ^Proline is a-pyrroUdine-carhoxylic acid and
possesses the following graphic structure:
-CH,
H,C-
H^C^^/CHCOOH.
NH
Proline was first obtained as a decomposition product of casein. Proline
obtained from proteins is laevo-rotatory and is the only protein decomposi-
tion product which is readily soluble in alcohol. It is also one of the few
heterocyclic compounds obtained from proteins. Prohne has been found
among the decomposition products of all proteins except the protamines.
The maximum yield reported is 13.73 per cent obtained by Osborne and
Clapp from the hydrolysis of hordein. More recently Fischer and
PROTEINS.
89
Boehner* reported having obtained 7.7 per cent from the hydrolysis of
gelatin. The crystalline form of Icrvo- a- proline is shown in Fig. 31, and
the copper salt of proline is represented by a micro-photograph in Fig. 32,
below. The crystals of the copper salt have a deep blue color, but when
they lose their water of crystallization they assume a characteristic violet
color.
-\^^
Fig. 32. — Copper Salt of Proline.
Reproduced from a micro- photograph made by Prof. E. T. Reichert, of the Universit}- of
Pennsylvania.
Oxyproline, C5H9XO3. — Oxyproline was discovered by Fischer.
It has as yet been obtained from only a few proteins, but this may be due
to the fact that only a few have been examined for its presence. The
position of the hydroxyl group has not yet been established.
Diaminotrihydroxydodecanoic Acid, Ci2H2gN,05. — This amino
acid was discovered by Fischer and Abderhalden as a product of the
hydrolysis of casein. It has thus far been obtained from no other source.
It is laevo-rotatorv and its constitution has not been determined.
Experiments.
While the ordinary courses in physiological chemistry preclude any
extended study of the decomposition products of proteins, the manipula-
tion of a simple decomposition and the subsequent isolation and study of a
few of the products most easily and quickly obtained will not be without
interest.^ To this end the student may use the following decomposition
* Fischer and Boehner: Zeit. phys. client., 65, p. 118, 1910.
* The procedure here set forth has nothing in common with the procedure by means
of which the long line of decomposition products just enumerated are obtained. This latter
process is an exceedingly compUcated one which is entirely outside the province of any course
in physiological chemistry.
90 PHYSIOLOGICAL CHEMISTRY.
procedure : Treat the protein in a large flask with water containing 3-5
per cent of H^SO^ and place it on a water-bath until the protein material
has been decomposed and there remains a fine, fluffy, insoluble residue.
Filter off this residue and neutralize the filtrate with Ba(0H)2 and BaCOg.
Filter off the precipitate of BaSO^ which forms and when certain that
the fluid is neutral or faintly acid, ^ concentrate (first on a wire gauze and
later on a water-bath) to a syrup. This syrup contains the end-products
of the decomposition of the protein, among which are proteoses, peptones,
tyrosine, leucine, etc. Add 95 per cent alcohol slowly to the warm syrup
until no more precipitate forms, stirring continuously with a glass rod.
This precipitate consists of proteoses and peptones. Gather the sticky
precipitate on the rod or the sides of the dish, and, after warming the
solution gently for a few moments, filter it through a filter paper which has
not been previously moistened. After dissolving the precipitate of pro-
teoses and peptones in water^ the solution may be treated according to
the method of separation given on page 120.
The leucine and tyrosine, etc., are in solution in the warm alcoholic
filtrate. Concentrate this filtrate on the water-bath to a thin syrup,
transfer it to a beaker, and allow it to stand over night in a cool place for
crystallization. The tyrosine first crystallizes (Fig. 24, page 81), followed
later by the formation of characteristic crystals of impure leucine (see Fig.
109, Chapter XX). After examining these crystals under the microscope,
strain off the crystalline material through fine muslin, heat it gently in a
little water to dissolve the leucine (the tyrosine will be practically insoluble)
and filter. Concentrate the filtrate and allow it to stand in a cool place
over night for the crude leucine to crystallize. Filter off the crystals and
use them in the tests for leucine given on page-9i. The crystals of tyrosine
remaining on the paper from the first filtration may be used in the tests for
tyrosine as given below. If desired, the tyrosine and leucine may be
purified by recry stallizing in the usual manner. Habermann has suggested
a method of separating leucine and tyrosine by means of glacial acetic acid.
Experiments on Tyrosine. .
Make the following tests with the tyrosine crystals already prepared
or upon some pure tyrosine furnished by the instructor.
I. Microscopical Examination. — ^Place a minute crystal of tyrosine
on a slide, add a drop of water, cover with a coverglass, and examine
' If the solution is alkaline in reaction at this point, the amino acids will be broken down
and ammonia will be evolved.
^ At this point the aqueous solution of the proteoses and peptones may be filtered to remove
any BaSO^ which may still remain. Tyrosine crystals will also be found here, since it is
less soluble than the leucine and may adhere to the proteose-peptone precipitate. Add the
cr}'stals of tyrosine to the warm alcohol filtrate.
PROTEINS. 91
microscopically. Now run more water under the cover-glass and warm
in a bunsen flame until the tyrosine has dissolved. Allow the solution to
cool slowly, then examine again microscopically, and compare the crystals
with those shown in Fig. 24, page 81.
2. Solubility. — Try the solubility of very small amounts of tyrosine in
cold and hot water, cold and hot 95 per cent alcohol, dilute NH^OH,
dilute KOH and dilute HCl.
3. Sublimation. — Place a little tyrosine in a dry test-tube, heat
gently and notice that the material does not sublime. How does this
compare with the result of Experiment 3 under Leucine ?
4. Hoffman's Reaction. — This is the name given to Millon's reaction
when employed to detect tyrosine. Add about 3 c.c. of water and a few
drops of Millon's reagent to a little tyrosine in a test-tube. Upon dissolv-
ing the tyrosine by heat the solution gradually darkens and may assume a
dark red color. What group does this test show to be present in tyrosine ?
5. Piria's Test. — Warm a little tyrosine on a watch glass on a boiling
water-bath for 20 minutes w'ith 3-5 drops of cone. HjSO^. Tyrosine-
sulphuric acid is formed in the process. Cool the solution and wash it
into a small beaker with water. Now add CaCOg in substance slowly
v\ath stirring, until the reaction of the solution is no longer acid. Filter,
concentrate the filtrate, and add to it a few drops (avoid an excess) of
very dilute neutral ferric chloride. A purple or violet color, due to the
formation of the ferric salt of tyrosine-sulphuric acid, is produced. This
is one of the most satisfactory tests for the identification of tyrosine.
6. Morner's Test. — Add about 3 c.c. of Morner's reagent^ to a little
tyrosine in a test-tube, and gently raise the temperature to the boiling-
point. A green color results.
Experiments ox Leucine.
Make the following test upon the leucine crystals already prepared or
upon some pure leucine furnished by the instructor.
I, 2 and 3. Repeat these experiments according to the directions
given under Tyrosine (pages 90 and 91).
' Morner's reagent is prepared by thoroughly mixing i volume of formalin, 45 volumes
of distilled water, and 55 volumes of concentrated sulphuric acid.
CHAPTER V.
PROTEINS : THEIR CLASSIFICATION AND
PROPERTIES.
From what has already been said in Chapter IV regarding the protein
substances it will be recognized that the grouping of the diverse forms of
this class of substances in a logical manner is not an easy task. The fats
and carbohydrates may be classified upon the fundamental principles of
their stereo-chemical relationships, whereas such a system of classification
in the case of the proteins is absolutely impossible since, as we have
already stated, the molecular structure of these complex substances is
unknown. Because of the diversity of standpoint from which the proteins
may be viewed, relative to their grouping in the form of a logically classi-
fied series, it is obvious that there is an opportunity for the presentation
of classifications of a widely divergent character. The fact that there
were until recently at least a dozen different classifications which were
recognized by various groups of English-speaking investigators emphasizes
the difficulties in the way of the individual or individuals who would
offer a classification which should merit universal adoption. Realizing
the great handicap and disadvantage which the great diversity of the
protein classifications was forcing upon the workers in this field, the
Chemical and Physiological Societies of England recently drafted a classi-
fication which appealed to these groups of scientists as fulfilling all require-
ments and presented it for the consideration of the American Physiological
Society and the American Society of Biological Chemists. The outcome
of this has been that there are now only two protein classifications which
are recognized by English-speaking scientists, one the British Classification,
the other the American Classification. These classifications are very
similar and doubtless will ultimately be merged into a single classification.
In our consideration of the proteins we shall conform in all details to the
American Classification. In this connection we will say, however, that
we feel that the English Societies have strong grounds for preferring the
use of the term scleroproteins for albuminoids and chromoproteins for
heemoglobins. The two classifications are as follows:
92
PROTEINS. 93
CLASSIFICATION OF PROTEINS ADOPTED BY THE AMERI-
CAN PHYSIOLOGICAL SOCIETY AND THE AMERICAN
SOCIETY OF BIOLOGICAL CHEMISTS.
I. SIMPLE PROTEINS.
Protein substances which yield only a-amino acids or their derivatives
on hydrolysis.
(a) Albumins. — Soluble in pure water and coagulable by heat,
e. g., ovalbumin, serum albumin, lactalbumin, vegetable albumins.
(b) Globulins. — Insoluble in pure water but soluble in neutral
solutions of salts of strong bases with strong acids/ e. g., serum globulin,
ovo globulin, edeslin, amandin, and other vegetable globulins.
(c) Glutelins. — Simple proteins insoluble in all neutral solvents, but
readily soluble in very dilute acids and alkalis/ e. g., glutenin.
id) Alcohol-soluble Proteins (Prolamins).^ — Simple proteins sol-
uble in 70-So per cent alcohol, insoluble in water, absolute alcohol, and
other neutral solvents,* e.g., zein, gliadin, hordein, and bynin.
ie) Albuminoids. — Simple proteins possessing a similar structure to
those already mentioned, but characterized by a pronounced insolubility
in all neutral solvents,^ e.g., elastin, collagen, keratin.
(J) Histones. — Soluble in water and insoluble in very dilute ammo-
nia, and, in the absence of ammonium salts, insoluble even in excess of
ammonia; yield precipitates with solutions of other proteins and a coagu-
lum on heating which is easily soluble in very dilute acids. On hydrolysis
they yield a large number of amino acids among which the basic ones pre-
dominate. In short, histones are basic proteins which stand between
protamines and true proteins, e.g., globin, thymus histone, scombrone.
(g) Protamines. — Simpler polypeptides than the proteins included
in the preceding groups. They are soluble in water, uncoagulable by
heat, have the property of precipitating aqueous solutions of other pro-
teins, possess strong basic properties and form stable salts with strong
mineral acids. They yield comparatively few amino acids, among which
the basic ones predominate. They are the simplest natural proteins, e. g.,
salmine, sturine, clupeine, scombrine.
' The precipitation limits with ammonium sulphate should not be made a basis for dis-
tinguishing the albumins from the globulins.
-' Such substances occur in abundance in the seeds of cereals and doubtless represent a
well-defined natural group of simple proteins.
' The name prolamins has been suggested for these alcohol-soluble proteins by Dr. Thomas
B. Osborne (Science, iqoS, XX\'III, p. 417). It is a very fitting term inasmuch as upon
hydrolysis they yield particularly large amounts of proline and ammonia.
* The subclasses defined (a, b, c, d,) are exemplified by proteins obtained from both plants
and animals. The use of appropriate prefixes will suffice to indicate the origin of the com-
pounds, e. g., tnoglobuiin, laclalhumin, etc.
^ These form the principal organic constituents of the skeletal structure of animals and also
their external covering and its appendages. This definition does not provide for gelatin
which is, however, an artificial derivative of collagen.
94 PHYSIOLOGICAL CHEMISTRY.
II. CONJUGATED PROTEINS.
Substances which contain the protein molecule united to some other
molecule or molecules otherwise than as a salt.
{a) Nucleoproteins. — Compounds of one or more protein molecules
with nucleic acid, e. g., cy to globulin, nucleohistone.
(b) Glycoproteins. — Compounds of the protein molecule with a
substance or substances containing a carbohydrate group other than a
nucleic acid. e. g., mucins and mucoids {osseomucoid, iendomucoid, sero-
mucoid, ichthulin, helicoprotein).
(c) Phosphoproteins. — Compounds of the protein molecule with
some, as yet undefined, phosphorus-containing substances other than a
nucleic acid or lecithin,^ e. g., caseinogen, vitellin.
(d) Haemoglobins. — Compounds of the protein molecule with
haematin, or some similar substance, e.g., hcemoglobin, hcEmocyanin.
{e) Lecithoproteins. — Compounds of the protein molecule with
lecithins, e. g., lecithans, phosphatides.
III. DERIVED PROTEINS.
I. Primary Protein Derivatives.
Derivatives of the protein molecule apparently formed through
hydrolytic changes which involve only slight alteration of the protein
molecule.
{a) Proteans. — Insoluble products which apparently result from
the incipient action of water, very dilute acids or enzymes, e. g., myosan,
edesian.
(b) Metaproteins. — ^Products of the further action of acids and alkalis
whereby the molecule is so far altered as to form products soluble in very
weak acids and alkalis but insoluble in neutral fluids, e.g., acid meta-
protein {acid albuminate) , alkali metaprotein {alkali albuminate).
ic) Coagulated Proteins.— Insoluble products which result from
(i) the action of heat on their solutions, or (2) the action of alcohol on the
protein.
2. Secondary Protein Derivatives.^
Products of the further hydrolytic cleavage of the protein molecule.
{a) Proteoses. — Soluble in water, non-coagulable by heat, and
precipitated by saturating their solutions with ammonium — or zinc
sulphate,' e. g., protoproteose, deuteroproteose.
* The accumulated chemical evidence distinctly points to the propriety of classifying
the phosphoproteins as conjugated compounds, i. e., they are possibly esters of some phosphoric
acid or acids and protein.
^ The term secondary protein derivatives is used because the formation of the primary
derivatives usually precedes the formation of the secondary derivatives.
' As thus defined, this term does not strictly cover all the protein derivatives commonly
called proteoses, e. g., heteroproteose and dysproteose.
PROTEINS. 95
{b) Peptones. — Soluble in water, non-coagulable by heat, but not
precipitated by saturating their solutions with ammonium sulphate/ e. g.,
antipeptaue, a mpho peptone.
(c) Peptides. — Defmitely characterized combinations of two or more
amino acids, the carboxyl group of one being united with the amino group
of the other with the elimination of a molecule of water, ^ e. g., dipeptides,
tripeptides, tetrapeptides, pentapeptides.
CLASSIFICATION OF PROTEINS ADOPTED BY THE CHEM-
ICAL AND PHYSIOLOGICAL SOCIETIES
OF ENGLAND.
I. Simple Proteins.
1. Protamines, e.g., salmine, clupeine.
2. Histones, e. g., globin, scombrone.
3. Albumins, e.g., ovalbumin, serum albumin, vegetable albumins.
4. Globulins, e.g., serum globulin, ovoglobulin, vegetable globulins.
5. Glutelins, e. g., glutenin.
6. Alcohol-soluble proteins, e. g., zein, gliadin.
7. Scleroproteins, e. g., elastin, keratin.
8. Phosphoproteins, e.g., caseinogen, vitellin.
II. Conjugated Proteins.
1. Glucoproteins, e.g., mucins, mucoids.
2. Nucleoproteins, e. g., nucleohistone, cyto globulin.
3. Chromoproteins, e. g., hamo globin, hcemocyanin.
III. Products of Protein Hydrolysis.
1. Infraproteins, e. g., acid infraprotein {acid albuminate), alkali
infraprotein {alkali albuminate) .
2. Proteoses, e.g., protoproteose, heteroproteose, deuteroproteose.
3. Peptones, e.g., amphopeptone, antipeptone.
4. Polypeptides, e.g.. dipeptides, tripeptides, tetrapeptides.
CONSIDERATIONS OF THE VARIOUS CLASSES
OF PROTEINS.
SIMPLE PROTEINS.
The simple proteins are true protein substances which, upon hy-
drolysis, yield only a-amino acids or their derivatives. "Although
' In this group the kjTines may be included. For the present it is believed that it will
be helpful to retain this term as defined, reserving the expression peptide for the simpler
compounds of definite structure, such as dipeptides, etc.
^ The peptones are undoubtedly peptides or mixtures of peptides, the latter term being
at present used to designate those of definite structure.
96 PHYSIOLOGICAL CHEMISTRY.
no means are at present available whereby the chemical individuality of
any protein can be established, a number of simple proteins have been
isolated from animal and vegetable tissues which have been so well
characterized by constancy of ultimate composition and uniformity of
physical properties that they may be treated as chemical individuals
until further knowledge makes it possible to characterize them more
definitely." Under simple proteins we may class albumins, globulins,
glutelins, prolamins, albuminoids, histones and protamines.
ALBUMINS.
Albumins constitute the first class of simple proteins and may be
defined as simple proteins which are coagulable by heat and soluble
in pure (salt-free) water. Those of animal origin are not precipitated
upon saturating their neutral solutions at 30° C. with sodium chloride
or magnesium sulphate, but if a saturated solution of this character
be acidified with acetic acid the albumin precipitates. All albumins
of animal origin may be precipitated by saturating their solutions with
ammonium sulphate.^ They may be thrown out of solution by the
addition of a sufficient quantity of a mineral acid, whereas a weak
acidity produces a slight precipitate which dissolves 'upon agitating the
solution. Metallic salts also possess the property of precipitating al-
bumins, some of the precipitates being soluble in excess of the reagent,
whereas others are insoluble in such an excess. Of those proteins
which occur native the albumins contain the highest percentage of sul-
phur, ranging from 1.6 to 2.5 per cent. Some albumins have been
obtained in crystalline form, notably egg albumin, serum albumin, and
lactalbumin, but the fact that they may be obtained in crystalline form
does not necessarily prove them to be chemical individuals.
GENERAL COLOR REACTIONS OF PROTEINS.
These color reactions are due to a reaction between some one or
more of the constituent radicals or groups of the complex protein molecule
and the chemical reagent or reagents used in any given test. Not all
proteins contain the same groups and for this reason the various color
tests will yield reactions varying in intensity of color according to the
nature of the groups contained in the particular protein under examina-
tion. Various substances not proteins respond to certain of these color
reactions, and it is therefore essential to submit the material under ex-
amination to several tests before concluding defmitely regarding its
nature.
' In this connection, Osborne's observation that there are certain vegetable albumins
which are precipitated by saturating their solutions with sodium chloride or magnesium
sulphate or by half-saturating with ammonium sulphate, is of interest.
PROTEINS. 97
TECHNIC OF THE COLOR REACTIONS.
1. Millon's Reaction. — To 5 c.c. of a dilute solution of egg aK
bumin in a test-tube add a few drops of Millon's reagent. A white
precipitate forms which turns red when heated. This test is a partic-
ularly satisfactory one for use on solid proteins, in which case the reagent
is added directly to the solid substance and heat applied, which causes
the substance to assume a red color. Such proteins as are not precipitated
by mineral acids, for example certain of the proteoses and peptones,
yield a red solution instead of a red precipitate.
The reaction is due to the presence of the hydroxy-phenyl group,
— CgH^OH, in the protein molecule and certain non-proteins such as
tyrosine, phenol (carbolic acid) and thymol also respond to the reaction.
Inasmuch as the tyrosine grouping is the only hydroxyphenyl grouping
which has definitely been proven to be present in the protein molecule it
is e^ident that protein substances respond to Millon's reaction because
of the presence of this tyrosine complex. The test is not a very satis-
factory one for use in solutions containing inorganic salts in large amount,
since the mercury of the Millon's reagent^ is thus precipitated and the
reagent rendered inert. This reagent is therefore never used for the
detection of protein material in the urine.
2. Xanthoproteic Reaction. — To 2-3 c.c. of egg albumin solu-
tion in a test-tube add concentrated nitric acid. A white precipitate
forms, which upon heating turns yellow and finally dissolves, imparting
to the solution a yellow color. Cool the solution and carefully add
ammonium hydroxide, potassium hydroxide, or sodium hydroxide in
excess. Note that the yellow color deepens into an orange. This
reaction is due to the presence in the protein molecule of the phenyl
group, with which the nitric acid forms certain nitro modifications.
The particular complexes of the protein molecule which are of especial
importance in this connection are those of tyrosine, phenylalanine, and
tryptophane. The test is not a satisfactory one for use in urinary ex-
amination because of the color of the end-reaction.
3. Adamkiewicz Reaction. — Thoroughly mix i volume of con-
centrated sulphuric acid and 2 volumes of acetic acid in a test-tube,
add a few drops of egg albumin solution and heat gently. A reddish-
violet color is produced. Gelatin does not respond to this test. This
reaction shows the presence of the tryptophane group (see next experi-
ment). The test depends upon the presence of glyoxylic acid, CHO.-
C00H4-H,0 or CH(OH)XOOH, in the reagents. This is shown
' Millon's reagent consists of mercury dissolved in nitric acid containing some nitrous
acid. It is prepared by digesting one part (by weight) of mercury with two parts (by weight)
of HNOj (sp. gr. 1.42) and diluting the resulting solution with two volumes of water.
98 PHYSIOLOGICAL CHEMISTRY.
by the failure to secure a positive reaction when acetic acid free from
glyoxyhc acid is used.
Rosenheim has recently advanced the view that the reaction may
be due to the presence of oxidizing agents such as nitrous acid and
ferric salts in the sulphuric acid.
4. Hopkins-Cole Reaction.^ — Place 1-2 c.c. of egg albumin solu-
tion and 3 c.c. of glyoxylic acid, CHO.COOH + H^O or CH(OH)2-
COOH, solution (Hopkins-Cole reagent^) in a test-tube and mix thor-
oughly. In a second tube place 5 c.c. of concentrated sulphuric acid.
Incline the tube containing the sulphuric acid and by means of a pipette
allow the albumin-glyoxylic acid solution to flow carefully down the side.
When stratified in this manner a reddish-violet color forms at the zone of
contact of the two fluids. This color is due to the presence of the trypto-
phane group. Gelatin does not respond to this test. For formula for
tryptophane see page 82.
Benedict^ has recently suggested a new reagent for use in carrying out
the Hopkins-Cole reaction.*
5. Biuret Test. — To 2-3 c.c. of egg albumin solution in a test-tube
add an equal volume of concentrated potassium hydroxide solution, mix
thoroughly, and add slowly a very dilute (2-5 drops in a test-tube of
water) copper sulphate solution until a purplish-violet or pinkish-violet
color is produced. The depth of the color depends upon the nature of the
protein; proteoses, and peptones giving a decided pink, while the color
produced with gelatin is not far removed from a blue. This reaction is
given by those substances which contain two amino groups in their molecule,
these groups either being joined directly together or through a single
atom of nitrogen or carbon. The amino groups mentioned must either
be two CONH2 groups or one CONH, group and one CSNH2, C(NH)-
NHj or CH2NH2 group. It follows from this fact that substances which
are non-protein in character but which contain the necessary groups will
respond to the biuret test. As examples of such substances may be cited
oxamide,
' Hopkins and Cole: Journal of Physiology, 27, 418, 1902.
^ Hopkins-Cole reagent is prepared as follows: To one liter of a saturated solution
of oxalic acid add 60 grams of sodium amalgam and allow the mixture to stand until the
evolution of gas ceases. Filter and dilute with 2-3 volumes of water.
^ Beneriict: Journal uf Biological Chemistry, 6, 51, 1909.
* Benedict's modified Hopkins-Cole reagent is prepared as follows: Ten grams of
powdered magnesium are placed in a large Erlerimeyer flask and shaken up with enough dis-
tilled water to liberally cover the magnesium. Two hundred and fifty c.c. of a cold, saturated
solution of oxalic acid is now added slowly. The reaction proceeds verj' rapidly and with the
liberation of much heat, so that the flask should be cooled under running water during the
addition of the acid. The contents of the flask are shaken after the addition of the last
jxjrtion of the acid and then poured upon a Alter, to remove the insoluble magnesium oxalate.
A little wash water is pfjured through the Alter, the filtrate a( idified with acetic acid to prevent
the partial precipitation of the magnesium on long standing, and made up to a liter with
distilled water. This solution contains only the magnesium salt of glyoxylic acid.
PROTEINS, 99
CONH,
I
CONH^
and biuret,
CONH2
\
NH.
CONH,
The test derives its name from the fact that this latter substance which
is formed on heating urea to 180° C. (see page 286), will respond to the
test. Protein material responds positively since there are two CONH,
groups in the protein molecule.
According to Schiff the end-reaction of the biuret test is dependent
upon the formation of a copper-potassium-biuret compound (cupri-potas-
sium biuret or biuret potassium cupric hydroxide). This substance was
obtained by Schiflf in the form of long red needles. It has the following
formula :
OH OH
I ■ !
CO -NH, Cu NH,CO
\ /
NH HN
/ \
CO NH,— K K— NH,CO
I " I "
OH OH
6. Gies's Biuret Reagent.^ — Gies has recently devised a reagent for
use in the biuret test. This reagent consists of 10 per cent KOH solution,
to which enough 3 per cent CuSO^ solution has been added to impart a
slight though distinct blue color to the clear liquid. The CuSO^ should
be added drop by drop with thorough shaking after each addition. This
reagent is of material assistance in performing the biuret test.
7. Biuret Paper of Kantor and Gies. — According to Kantorand Gies^
when filter paper is immersed in the above reagent and subsequently
dried it forms a very satisfactory "biuret paper" which may be used in a
manner analogous to indicator papers. Moist papers may be used in the
examination of powders which are neutral or alkaline in reaction. In
preparing the "biuret paper" if the filter paper is left for a suflficient length
of time in the reagent all traces of the copper sulphate will be removed
from the solution.
' Gies: Proceedings of Society of Biological Chemists, Journal of Biological Chemistry,
7, 60, 1910.
- Kantor and Gies: Proc. Soc. Biol. Chem., p. ii, 1910.
lOO PHYSIOLOGICAL CHEMISTRY.
8. Posner's Modification of the Biuret Test. — This test is par-
ticularly satisfactory for use on dilute protein solutions, and is carried out
as follows: To some dilute egg albumin in a test-tube add one-half its
volume of potassium hydroxide solution. Now hold the tube in an
inclined position and allow some very dilute copper sulphate solution,
made as suggested on page 98 (5), to flow down the side, being especially
careful to prevent the fluids from mixing. At the juncture of the two
solutions the typical end-reaction of the biuret test should appear as a
colored zone (see Biuret Test, page 98).
9. Testing Colored Solutions by Biuret Test. — If the color of the
solution is such as to interfere with the end-reaction of the biuret test
proceed as follows: Make the solution strongly alkaline with potassium
hydroxide and add a solution of copper sulphate. Shake up the mixture
with alcohol and if protein is present the alcohol vdW assume the typical
biuret coloration. This procedure is not applicable in case the pigment
of the original solution is soluble in alcohol. Excess of the copper salt
need not be avoided in this test.
ID. Liebermann's Reaction. — Add about 10 drops of concentrated
egg albumin solution (or a little dry egg albumin) to about 5 c.c. of con-
centrated HCl in a test-tube. Boil the mixture until a pinkish-violet
color results. This color was originally supposed to indicate the presence
of a carbohydrate group in the protein molecule, the furfurol formed
through the action of the acid upon the protein reacting with the hydroxy-
phenyl group of the protein producing the pinkish-\'iolet color. It is now
considered uncertain whether the carbohydrate group enters into the
reaction. Cole has called attention to the fact that a blue color results if
protein material which has been boiled ^ith alcohol and subsequently
washed with ether be used in making the test. He believes the blue color
to be due to an interaction between the glyoxylic acid, which was present
as an impurity in the ether used in washing the protein, and the trypto-
phane group of the protein molecule which was split off through the action
of the acid.
II. Acree-Rosenheim Formaldehyde Reaction. — Add a few drops
of a dilute (1:5000) solution of formaldehyde to 2-3 c.c. of egg albumin
solution in a test-tube. Mix thoroughly and after 2-3 minutes carefully
introduce a little concentrated sulphuric acid into the tube rn such a
manner that the two solutions do not mix. A \'iolet zone will be observed
at the point of juncture of the two solutions especially if the mixture is
slightly agitated. This color probably results through the union of the
protein and the formaldehyde. If the sulphuric acid is added to the
protein before the formaldehyde is added the typical end-reaction is not
obtained. So far as is known this is a specific test for proteins.
PROTEINS, lOI
The reaction cannot be applied satisfactorily with concentrated formal-
dehyde.
Rosenheim claims the reaction is due to the presence of oxidizing
material in the sulphuric acid and that when pure sulphuric acid is used
no reaction is obtained. He advises the use of a slight amount of an
oxidizing agent, e. g., ferric chloride or potassium nitrate (0.005 K^am per
100 c.c. of sulphuric acid) in order to facilitate the reaction. Rosenheim
further states tTiat proteins respond to the formaldehyde reaction because
of the presence of the tryptophane group, a statement which Acree does not
accept as proven.
12. Bardach's Reaction/ — This is one of the most recent tests which
have been described for the detection of protein material. The test
depends upon the property possessed by protein substances of preventing
the formation of typical iodoform crystals through the interaction of an
alkaline acetone solution with iodopotassium iodide. Instead of the
typical hexagonal plates or stellar formations of iodoform there are pro-
duced, under the conditions of the test, fine yellow needles which are
apparently some iodine compound other than iodoform. The technic
of the test is as follows: Place about 5 c.c. of the protein solution^ under
examination in a test-tube, add 2-3 drops of a 0.5 per cent solution of
acetone and sufficient Lugol's solution^ to supply a moderate excess of
iodine and produce a red-brown coloration. (The amount of Lugol's
solution necessary will depend upon the content of protein, sugar, and
other iodine-reacting substances in the solution under examination and
may vary from one drop to several cubic centimeters.) Add an excess
(ordinarily about 3 c.c.) of concentrated ammonium hydroxide and
thoroughly mix the solution. Place the tube in the test-tube rack, ex-
amine the contents at intervals of five minutes, and when it is evident that
crystals have formed, place a drop of the mixture upon a microscopic
slide, put a coverglass in position, and examine the mixture under the
microscope. The formation of canary yellow crystals indicates the pres-
ence of protein material in the solution examined. The crystals are
ordinarily needle-like in appearance and show a tendency to assume
rosette or bundle-like formations, but under certain conditions they may
show knobbed (nail-like) and branching variations.
If a moderate excess of iodine is used in making the test, a black pre-
cipitate of iodonitro compounds is at once formed upon the addition of
the ammonium hydroxide, and yellow needles are subsequently deposited
upon it. In case just the proper amount of iodine is used, the solution
* Bardach: Zeitschrift fur Physiologische Chemie, 54, 355, iqoS; also Seaman and
Gies: Proceedings of the Society for Experimental Biology and Medicine, 5, 125, 1908.
* The solution should not contain more than 5 per cent of protein material.
' Dissolve 4 grams of iodine and 6 grams of potassium iodide in 100 c.c. of distilled water.
I02 PHYSIOLOGICAL CHEMISTRY.
soon assumes a yellow color and the black precipitate formed upon the
addition of the ammonium hydroxide is gradually transformed more or
less completely into theyellow crystals. In either case the needles ordinarily
form within an hour, and frequently in a much shorter time. If too great
an excess of iodine is employed the heavy black precipitate may obscure
or even prevent the reaction. The presence of insufficient iodine or
excess protein may likewise prevent the reaction. In tests in which a
concentrated protein solution and an excess of iodine are used, the addi-
tion of ammonium hydroxide immediately produces a grayish-green
precipitate. In such instances, if the proportions are favorable, and the
mixture be stirred with a glass rod for a few minutes, the precipitate is
gradually transformed into the crystals before mentioned.
It is probable that all soluble proteins will respond to Bardach's
reaction, but the relative delicacy of the reaction as well as the value of the
test as compared with other protein tests remain to be determined. The
only disturbing factor noted thus far is the presence of earthy phosphates
in the solution under examination.
PRECIPITATION REACTIONS AND OTHER PROTEIN TESTS.
There are three forms in which proteins may be precipitated, i. e.,
unaltered, as an albitminate, and as an insoluble salt. An instance of the
precipitation in a native or unaltered condition is seen in the so-called
salting-out experiments. Various salts, notably (NHJ2SO4, ZnSO^,
MgSO^, NajSO^ and NaCl possess the power when added in solid form to
certain definite protein solutions, of rendering the menstruum incapable of
holding the protein in solution, thereby causing the protein to be precipi-
tated or salted-out to use the common term. Mineral acids and alcohol
also precipitate proteins unaltered. In the case of concentrated acids
the protein is dissolved in the presence of an excess of acid with the
formation of a protein salt. Proteins are precipitated as albuminates
(protein salts) when treated with certain metallic salts, and precipitated as
insoluble salts when weak organic acids such as certain of the alkaloidal
reagents are added to their solutions.
If certain acids (picric, tungstic, phosphomolybdic, tannic, or chromic)
be added to a neutral albumin solution a precipitate of a protein salt
occurs. If, however, the salts of these acids be added no precipitate
occurs. The addition of a small amount of acid, as acetic acid, to such a
solution will cause a precipitate to form.'
The effect of the addition of the salts of the heavy metals is in the first
instance to cause a precipitation of the protein. In many cases, however,
' Mathews: Amer. Jour, of Physiology, 1, 445, 1898.
PROTEINS. 103
the addition of an excess of such saUs causes the solution of the precipitate
while a further excess may cause a reprecipitation. The precipitate which
is first formed in a protein solution by the addition of the salts of the heavy
metals may be redissolved not only by an excess of such salts but by an
excess of protein as well/
It is generally stated that globulins are ])recij)itated from their solutions
upon half saturation with ammonium suli)hate and that albumins are
precipitated upon complete saturation by this salt. Comparatively few
exceptions were found to this rule until proteins of vegetable origin came
to be more extensively studied. These studies, furthered especially by
Osborne and associates, have demonstrated very clearly that the char-
acterization of a globulin as a protein which is precipitated by half
saturation with ammonium sulphate, can no longer hold. Certain vege-
table globulins have been isolated which are not precipitated by this salt
until a concentration is reached greater than that secured by half-saturation.
As an example of an albumin which does not conform to the definition of
an albumin as regards its precipitation by ammonium sulphate, may be
mentioned the leucosin of the wheat germ which is precipitated from its
solution upon /;tz//-saturation with ammonium sulphate. The limits of
precipitation by ammonium sulphate, therefore, do not furnish a suffi-
ciently accurate basis for the differentiation of globulins from albumins.
It has further been determined that a given protein which is precipitable
by ammonium sulphate cannot be " salted-out " by the same concentration
of the salt under all conditions.
Experiments.
1. Influence of Concentrated Mineral Acids, Alkalis and Organic
Acids. — Prepare five test-tubes each containing 5 c.c. of concentrated egg
albumin solution. To the first add concentrated HjSO^, drop by drop,
until an excess of the acid has been added. Note any changes which may
occur in the solution. Allow the tube to stand for 24 hours and at the
end of that period observe any alteration which may have taken place.
Heat the tube and note any further change which may occur. Repeat the
experiment in the four remaining tubes with concentrated hydrochloric
acid, concentrated nitric acid, concentrated potassium hydroxide and
acetic acid. How do strong mineral acids, strong alkalis, and strong
organic acids differ in their action toward protein solutions?
2. Precipitation by Metallic Salts. — Prepare four tubes each con-
taining 2-3 c.c. of dilute egg albumin solution. To the first add mercuric
* Pauli: Hofmeister's Beitrage, 6, 233, 1904-5. Robertson: Ergebnisse tier Physiologic,
10, 290, 19T0.
I04 PHYSIOLOGICAL CHEMISTRY.
chloride, drop by drop, until an excess of the reagent has been added, noting
any changes which may occur. Repeat the experiment with lead acetate,
silver nitrate, copper sulphate, ferric chloride, and barium chloride.
Egg albumin is used as an antidote for lead or mercury poisoning.
Why?
3. Precipitation by Alkaloidal Reagents. — ^Prepare six tubes each
containing 2-3 c.c. of dilute egg albumin solution. To the first add
picric acid drop by drop until an excess of the reagent has been added,
noting any changes which may occur. Repeat the experiment with
trichloracetic acid, tannic acid, phosphotungstic acid, phosphomolybdic acid,
and potassio-mercuric iodide. Acidify with hydrochloric acid before testing
with the three last reagents.
4. Heller's Ring Test. — ^Place 5 c.c. of concentrated nitric acid in a
test-tube, incline the tube, and by means of a pipette allow the dilute
albumin solution 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 is a very delicate test and is further discussed on p. 333.
An apparatus called the albumoscope or horismascope has been devised
for use in the tests of this character and has met with considerable favor.
The method of using the albumoscope is described below.
Use of the Albumoscope. — This instrument is intended to facilitate
the making of "ring" tests such as Heller's and Roberts'. In making a
test about 5 c.c. of the solution under examination is first introduced into
the apparatus through the larger arm and the reagent used in the particu-
lar test is then introduced through the capillary arm and allowed to flow
down underneath the solution under examination. If a reasonable
amount of care is taken there is no possibility of mixing the two solutions
and a definitely defined white "ring" is easily obtained at the zone of
contact.
5. Roberts' Ring Test. — Place 5 c.c. of Roberts' reagent^ in a test-
tube, incline the tube, and by means of a pipette allow the albumin solu-
tion 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 satis-
factory. The albumoscope may also be used in making this test. (See
page 334. j
6. Spiegler's Ring Test. — Place 5 c.c. of Spiegler's reagent^ in a test-
* Roberts' reagent is composed of i volume of concentrated HNO3 and 5 volumes of a
saturated solution of MgSO^.
* Spiegler's reagent has the following composition:
Tartaric acid 20 grams.
Mercuric chloride 40 grams.
Glycerol 100 grams.
Distilled water 1000 grams.
PROTEINS. TO
tube, incline the tube, and by means of a pipette allow 5 c.c. of albumin
solution, 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 delicate for ordinary clinical purposes, since it
serves to detect albumin when present in the merest trace (i : 250,000).
This test is further discussed on page 335.
7. Jolles' Reaction. — Shake 5 c.c. of albumin solution with i c.c. of
30 per cent acetic acid and 4 c.c. of Jolles' reagent^ in a test-tube. A
white precipitate of albumin should form. Care should be taken to use
the correct amount of acetic acid. For further discussion of the test see
page 335-
8. Tanret's Test. — To 5 c.c. of albumin solution in a test-tube add
Tanret's reagent,^ drop by drop, until a turbidity or precipitate forms.
This is an exceedingly deHcate test. Sometimes the albumin solution is
stratified upon the reagent as in Heller's or Roberts' ring tests. In urine
examination it is claimed by Repiton that the presence of urates lowers
the delicacy of the test. Tanret has, however, very recently made a state-
ment to the effect that the removal of urates is not necessary inasmuch as
the urate precipitate will disappear on warming and the albumin precipi-
tate will not. He says, however, that mucin interferes with the delicacy
of his test and should be removed by acidification with acetic acid and
filtration before testing for albumin.
9. Sodium Chloride and Acetic Acid Test. — Mix 2 volumes of
albumin solution and i volume of a saturated solution of sodium chloride
in a test-tube, acidify with acetic acid, and heat to boiling. The pro-
duction of a cloudiness or the formation of a precipitate indicates the
presence of albumin.
10. Potassium Iodide Test. — Stratify a dilute albumin solution
upon a solution of potassium iodide made slightly acid with acetic acid.
In the presence of 0.01-0.02 per cent of albumin a white ring forms
immediately. If the test be allowed to stand two minutes after the
stratification it will serve to detect 0.005 P^r cent of albumin.
11. Acetic Acid and Potassium Ferrocyanide Test. — To 5 c.c.
of dilute egg albumin solution in a test-tube add 5-10 drops of acetic
acid. Mix well, and add potassium ferrocyanide, drop by drop, until
a precipitate forms. This test is very delicate.
' Jolles' reagent has the following composition:
Succinic acid 40 grams.
Mercuric chloride 20 grams.
Sodium chloride 20 grams.
Distilled water 1000 grams.
^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 acetic acid to
the combined solutions.
Io6 PHYSIOLOGICAL CHEMISTRY.
Schmiedl claims that a precipitate of Fe(Cn)gK2Zn or Fe(Cn)Q-
Zn^, is formed when solutions containing zinc are subjected to this test,
and that this precipitate resembles the precipitate secured with protein
solutions. In the case of human urine a reaction was obtained when
0.000022 gram of zinc per cubic centimeter was present. Schmiedl
further found that the urine collected from rabbits housed in zinc-lined
cages possessed a zinc content which was sufl&cient to yield a ready re-
sponse to the test. Zinc is the only interfering substance so far reported.
12. Salting-out Experiments. — (a) To 25 c.c. of egg albumin
solution in a small beaker add solid ammonium sulphate to the point
of saturation, keeping the temperature of the solution below 40° C.
Filter, test the precipitate by Millon's reaction and the filtrate by the biu-
ret test. What are your conclusions? (&) Repeat the above experi-
ment making the saturation with solid sodium chloride. How does
this result differ from the result of the saturation with ammonium sul-
phate ? Add 2-3 drops of acetic acid. . What occurs ? All proteins
except peptones are precipitated by saturating their solutions with ammo-
nium sulphate. Glohulins are the only proteins precipitated by satu-
rating with sodium chloride (see Globulins, page 109), unless the satu-
rated solution is subsequently acidified, in which event all proteins except
peptones are precipitated.
Soaps may be salted-out in a similar manner (see p. 145).
13. Coagulation or Boiling Test. — Heat 25 c.c. of dilute egg
albumin solution to the boiling-point in a small evaporating dish. The
albumin coagulates. Complete coagulation may be obtained by acidify-
ing the solution with 3-5 drops of acetic acid^ at the boiling-point. Test
the coagulum by Millon's reaction. The acid is added to neutralize any
possible alkalinity of the solution, to dissolve any substances which are
not albumin and to facilitate coagulation (see further discussion on pages
117 and 335).
14. Coagulation Temperature. — Prepare 4 test-tubes each con-
taining 5 c.c. of neutral egg albumin solution. To the first add i drop of
0.2 per cent hydrochloric acid, to the second add i drop of o. 5 per cent
sodium carbonate solution, to the third add i drop of 10 per cent sodium
chloride solution and leave the fourth neutral in reaction. Partly fill a
beaker of medium size with water and place it within a second larger
beaker which also contains water, the two vessels being separated by
pieces of cork. Fasten the four test-tubes compactly together by means
of a rubber band, lower them into the water of the inner beaker and sus-
pend them, by means of a clamp attached to one of the tubes, in such a
' Nitric acid is often used in place of acetic acid in this test. In case nitric acid is used,
ordinarily^i-2 drops is sufTu ient.
PROTEINS.
Jo:
0& P
Q--^
manner that the albumin solutions shall be midway between the upper
and lower surfaces of the water. In one of the tubes place a thermometer
with its bulb entirely beneath the surface of the albumin solution (Fig. 33).
Gently heat the water in the beakers, noting carefully any changes which
may occur in the albumin solutions and record the exact tem-
perature at which these changes occur. The first appearance of an
opacity in an albumin solution indicates the
commencement of coagulation and the
temperature at which this occurs should
be recorded as the coagulation temperature
for that particular albumin solution.
What is the order in which the four
solutions coagulate?
Repeat the experiment, adding to the
first tube i drop of acetic acid, to the second
I drop of concentrated potassium hydroxide
solution, to the third 2 drops of a 10 per
cent sodium chloride solution and leave
the fourth neutral as before.
What is the order of coagulation here ?
Why ?
15. Precipitation by Alcohol. — Pre-
pare 3 test-tubes each containing about 10
c.c. of 95 per cent alcohol. To the first
add one drop of 0.2 per cent hydrochloric
acid, to the second one drop of potassium
hydroxide solution and leave the third
neutral in reaction. Add to each tube a
few drops of egg albumin solution and
note the results. What do you conclude
from this experiment ? Alcohol precipitates
proteins unaltered, but if allowed to re-
main under alcohol the protein is trans-
formed. The "fixing" of tissues for histological examination by means
of alcohol is an illustration of the application of this transformation pro-
duced by alcohol. It apparently is a process of dehydration.
16. Preparation of Powdered Egg Albumin. — This may be pre-
pared as follows : Ordinary egg-white finely divided by means of scissors
or a beater is treated with four volumes of water and filtered. The
filtrate is evaporated on a water-bath at about 50° C. and the residue
powdered in a mortar.
17. Tests on Powdered Egg Albumin. — With powdered albumin
Fig.
5. — Co.-VGULATiox Temper-
ature Apparatus.
Io8 PHYSIOLOGICAL CHEMISTRY.
prepared as described above (by yourself or furnished by the instructor) ^
try the following tests:
(a) Solubility.
{b) Millon's Reaction.
(c) Hopkins-Cole Reaction. — When used to detect the presence of
protein in solid form this reaction should be conducted as follows: Place
5 c.c. of concentrated sulphuric acid in a test-tube and add carefully,
by means of a pipette, 3-5 c.c. of Hopkins-Cole reagent. Introduce a
small amount of the solid substance to be tested, agitate the tube slightly,
and note that the suspended pieces assume a reddish-violet color, which
is the characteristic end-reaction of the Hopkins-Cole test; later the
solution will also assume the reddish-violet color.
(d) Composition Test. — Heat some of the powder in a test-tube in
which is suspended a strip of moistened red litmus paper and across the
mouth of which is placed a piece of filter paper moistened with lead
acetate solution. As the powder is heated it chars, indicating the presence
of carbon; the fumes of ammonia are evolved, turning the red litmus
paper blue and indicating the presence of nitrogen and hydrogen; the
lead acetate paper is blackened, indicating the presence of sulphur^
and the deposition of moisture on the side of the tube indicates the
presence of hydrogen.
(e) Immerse a dry test-tube containing a little powdered egg albumin
in boiling water for a few moments. Remove and test the solubility of the
albumin according to the directions given under (a) above. It is still
soluble. Why has it not been coagulated? Repeat the above experi-
ments with powdered serum albumin and see how the results compare
with those just obtained.
SULPHUR IN PROTEIN.
Sulphur is believed to be present in two different forms in the pro-
tein molecule. The first form, which is present in greatest amount^
is that loosely combined with carbon and hydrogen. Sulphur in this
form is variously termed unoxidized, loosely combined, mercaptan, and
lead-blackening sulphur. The second form is combined in a more stable
manner with carbon and oxygen and is known as oxidized or acid sulphur.
The protamines are the only class of sulphur-free proteins.
Tests for Sulphur.
I. Tests for Loosely Combined Sulphur. — (a) To equal volumes of
KOH and egg albumin solutions in a test-tube add 1-2 drops of lead
acetate solution and boil the mixture. Loosely combined sulphur is
PROIKINS. 109
indicated by a darkening of the solution, the color deepening into a
black if sufl'icicnt sulphur is present. Add hydrochloric acid and note
the characteristic odor evolved from the solution. Write the reactions
for this test, (b) Place equal volumes of KOH and egg albumin solu-
tions in a test-tube and boil the mixture vigorously. Cool, make acid
with glacial acetic acid. and add 1-2 drops of lead acetate. A darken-
ing indicates the presence of loosely combined sulphur.
2. Test for Total Sulphur (Loosely Combined and Oxidized). —
Place the substance to be examined (powdered egg albumin) in a small
porcelain crucible, add a suitable amount of solid fusion mixture (potas-
sium hydroxide and potassium nitrate mixed in the proportion 5:1) and
heat carefully until a colorless mixture results. (Sodium peroxide may
be used in place of this fusion mixture if desired.) Cool, dissolve the cake
in a httle warm water and filter. Acidify the filtrate with hydrochloric
acid, heat it to the boiling-point and add a small amount of barium
chloride solution. A white precipitate forms if sulphur is present.
What is this precipitate ?
GLOBULINS.
Globulins are simple proteins especially predominant in the vege-
table kingdom. They are closely related to the albumins and in com-
mon with them give all the ordinary protein tests. Globulins differ
from the albumins in being insoluble in pure (salt-free) water. They
are, however, soluble in neutral solutions of salts of strong bases with
strong acids. Most globulins are precipitated from their solutions by
saturation 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 protein 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 proteins, 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 sodium chloride for
one-half hour at 60° C. Filter while hot through a paper moistened
with 5 per cent sodium chloride solution. Place the filtrate in the water-
bath at 60° C. and allow it to stand for 24 hours in order that the globulin
no PHYSIOLOGICAL CHEMISTRY.
may crystallize slowly. In case the filtrate is cloudy it should be warmed
to 60° C. in order to produce a clear solution. The globulin is soluble in
hot 5 per cent sodium chloride solution and is thus extracted from the
Fig. 34. — Edestin.
hemp seed, but upon cooling this solution much of the globulin separates in
crystalline form. This particular globulin is called edestin. It crystal-
lizes in several different forms, chiefly octahedra (see Fig. 34, above).
(The crystalline form of excelsin, a protein obtained from the Brazil nut,
is shown in Fig. 35, below. This vegetable protein crystallizes in the
Fig. 35. — Excelsin, the Protein or the Brazil Nut.
(Drawn from crystals furnished by Dr. Thomas B. Osborne, New Haven, Conn.)
form of hexagonal plates.) Filter oflf the edestin and make the following
tests on the crystalline body and on the filtrate which still contains some
of the extracted globulin.
PROTEIN'S. 1 1 1
Tests on Crystallized Edestin. — (i) Microscopical examination
(see Fig. 34, p. no).
(2) Solubility. — Try the solubility in the ordinary solvents (see page
27). Keep these solubilities in mind for comparison with those of
edestan, to be made later (see page 115).
(3) Mil Ion's Reaction.
(4) Coagulation Test. — Place a small amount of the globulin in a
test-tube, add "a little .water and boil. Now add dilute hydrochloric
acid and note that the protein no longer dissolves. It has been coagulated.
(5) Dissolve the remainder of the edestin in 0.2 per cent hydro-
chloric acid and preserve this acid solution for use in the experiments
on proteans (see page 115).
Tests on Edestin Filtrate. — (i) Influence of Protein Precipi-
tants. — Try a few protein precipitants such as nitric acid, tannic acid,
picric acid, and mercuric chloride.
(2) Biuret Test.
(3) Coagulatian Test. — Boil some of the filtrate in a test-tube. What
happens ?
(4) Saturation with Sodium Chloride. — Saturate some of the filtrate
with solid sodium chloride. How does this result differ from that ob-
tained upon saturating egg albumin solution with solid sodium chloride ':!
(5) Precipitation by Dilution. — Dilute some of the filtrate with ia-15
volumes of water. Why does the globulin precipitate ?
Glutelins.
It has been repeatedly shown, particularly by Osborne, that after
extracting the seeds of cereals with water, neutral salt solution, and
strong alcohol, there still remains a residue which contains protein
material which may be extracted by very dilute acid or alkali. These
proteins which are insoluble in all neutral solvents, but readily soluble
in very dilute acids and alkalis are called glutelins. The only member
of the group which has yet received a name, is the glutenin of wheat,
a protein which constitutes nearly 50 per cent of the gluten. It is not
definitely known whether glutelins occur as constituents of all seeds.
Prolamins (Alcohol-soluble Proteins).
The term prolamin has been proposed by Osborne, for the group of
proteins formerly termed "alcohol-soluble proteins." The name is
very appropriate inasmuch as these proteins yield, upon hydrolysis,
especially large amounts of proline and ammania. The prolamins are
simple proteins which are insoluble in water, absolute alcohol and other
112 PHYSIOLOGICAL CHEMISTRY.
neutral solvents, but are soluble in 70 to 80 per cent alcohol and in dilute
acids and alkalis. They occur widely distributed, particularly in the
vegetable kingdom. The only prolamins yet described are the zein of
maize, the hordein of barley, the gliadin of wheat and rye, and the hynin
of malt. They yield relatively large amounts of glutamic acid on hydroly-
sis but no lysin. The largest percentage of glutamic acid (43.66 per cent)
ever obtained as a decomposition product of a protein substance has
very recently been obtained by Osborne & Guest from the hydrolysis of
the prolamin gliadin.'^ This yield of glutamic acid is also the largest
amount of any single decomposition product yet obtained from any
protein except protamines.
Albuminoids. (Scleroproteins.)
The albuminoids yield similar hydrolytic products to those obtained
from the other simple proteins already considered, thus indicating that
they possess essentially the same chemical structure. They differ from
all other proteins, whether simple, conjugated, or derived, in that they
are insoluble in all neutral solvents. The albuminoids include "the
principal organic constituents of the skeletal structure of animals as
well as their external covering and its appendages. Some of the principal
albuminoids are keratin, elastin, collagen, reticulin, spongin, Sind fibroin.
Gelatin cannot be classed as an albuminoid although it is a transformation
product of collagen. The various albuminoids differ from each other in
certain fundamental characteristics which will be considered in detail
under Epithelial and Connective Tissue (see Chapter XIV, p. 245).
CONJUGATED PROTEINS.
Conjugated proteins consist of a protein molecule united to some
other molecule or molecules otherwise than as a salt. We have glyco-
proteins, nucleo proteins, hcBmoglobins (chromoproteins) , phospho proteins
and lecitho proteins as the five classes of conjugated proteins.
Glycoproteins may be considered as compounds of the protein mole-
cule with a substance or substances containing a carbohydrate group
other than a nucleic acid. The glycoproteins yield, upon decomposition,
protein and carbohydrate derivatives, notably glycosamine, CHgOH.-
(CHOH)3.CH(NH3).CHO,and galactosamine, GHCH^. (CHOH)3.CH-
(NHJ.CHO. The principal glycoproteins are mucoids, mucins, and chon-
droproteins. By the term mucoid we may in general designate those glyco-
proteins which occur in tissues, such as tendomucoid from tendinous
' Up to this time the yield of 41.32 per cent obtained by Kleinschmitt from hordein was
the maximum yield.
s.
C.
H.
0.
2-33
48.76
6.53
30.60
2.32
47-43
6.63
31-40
PROTEINS. 113
issue and osseomucoid from bone. The elementary composition of these
ypical mucoids is as follows:
N.
Tendomucoid • 1 1 -7 S
Osseomucoid' 12.22
The term mucins may be said in general to include those forms of glyco-
proteins which occur in the secretions and fluids of the body. Seroww-
coid^ is, however, the term applied to the glycoprotein of blood serum.
Chondroproteins are so named because chondromiicoid, the principal
member of the group, is derived from cartilage (chondrigen). Amyloid,*
which appears pathologically in the spleen, liver, and kidneys, is also a
chondroprotein.
The tmdeoproteins occur principally in animal and vegetable cells,
and following the destruction of these cells they are found in the fluids
of the body. These proteins are discharged into the tissue fluids by
the acti\-ity or disintegration of cells. Combined with the simple pro-
tein in the ncuclcoprotein molecule we find nucleic acid, a body which
contains phosphorus and which yields purine bases and pyrimidine bases
{thymine, cytosine, and uracil) upon decomposition. The so-called
nucleins are formed in the gastric digestion of nucleoproteins.
Wheeler-Johnson Reaction for Uracil and Cytosine. — To about
5 c.c. of the solution under examination add bromine water until the color
is permanent.^ In case the solution contains only small quantities of
cytosine or uracil, it is advisable to remove the excess of bromine by
passing a stream of air through the solution. Now add an excess of an
aqueous solution of barium hydroxide and note the appearance of a
purple color.
Very dilute solutions do not give the test. Under these conditions the
solution should be evaporated to dryness, the residue dissolved in a
little bromine water and the excess of bromine removed. Then upon
adding an excess of barium hydroxide a decided bluish-pink or lavender
color will appear in the presence of as small an amount as o.ooi gram of
uracil.
In testing solutions for cytosine, it is preferable to warm or boil the
solution with bromine water, and after cooling the solution to apply the
test as suggested above, being careful to have a slight excess of bromine
present before adding barium hydroxide.
' Chittenden and Gies: Jour. Exp. Med, i, 186, 1896.
- Hawk and Gies: Amer. Jour. Physiol., 5, 387, 1901.
' Bywaters: Biochemische Zeitschrfft, 15, 322, 1909.
* Not to be confused with the substance amyloid which may be formed from cellulose (see
page 54)-
^ Avoid the addition of a large excess of bromine inasmuch as this will interfere with the
test.
8
114 PHYSIOLOGICAL CHEMISTRY.
The phosphoproteins are called nucleo albumins in many classifications
and are grouped among the simple proteins. They are considered to be
''compounds of the protein molecule and some, as yet undefined, phos-
phorus-containing substances other than a nucleic acid or lecithin."
The percentage of phosphorus in phosphoproteins is very similar to that
in nucleoproteins but they dift'er from this latter class of proteins in that
they do not yield any purine bases upon hydrolytic cleavage. Two of the
common phosphoproteins are the caseinogen of milk and the ovovitellin of
the egg-yolk.
The hemoglobins (chromoproteins) are compounds of the protein
molecule with haematin or some similar substance. The principal member
of the group is the haemoglobin of the blood. Upon hydrolytic cleavage
this haemoglobin yields a protein termed globin and a coloring matter
termed hmnochromogen. The latter substance contains iron and upon
coming in contact with oxygen is oxidized to form hcematin. Hamocyanin,
another member of the class of haemoglobins, occurs in the blood of certain
invertebrates, notably cephalopods, gasteropods, and Crustacea. Haemo-
cyanin generally contains either copper, manganese, or zinc in place of the
iron of the haemoglobin molecule.
The lecithoproteins include such substances as lecithans and phospha-
tides which consist of a protein molecule joined to lecithin. They have
been comparatively little studied until recently, and in much of the older
research they were undoubtedly considered as lecithins.
For experiments on conjugated proteins see pages 63, 162, 247, 251,
271, and 308.
DERIVED PROTEINS.
These substances are derivatives which are formed through hydrolytic
changes of the original protein molecule. They may be divided into two
groups, the primary protein derivatives and the secondary protein deriva-
tives. The term secondary derivatives is made use of in this connection
since the formation of the primary derivatives generally precedes the
formation of these secondary derivatives. These derived proteins are
obtained from native simple proteins by hydrolyses of various kinds, e. g.,
through the action of acids, alkalis, heat, or enzymes. The particular
class of derived protein desired regulates the method of treatment to which
the native protein is subjected.
Primary Protein Derivatives.
The primary protein derivatives are "apparently formed through
hydrolytic changes which involve only slight alterations of the protein
PROTEINS.
molecule." This class includes proteans, melaproteins, and coagulated
proteins.
PROTEANS.
Proteans arc those insoluble protein substances which are produced
from proteins originally soluble through the incipient action of water,
enzymes, or very dilute acids. It is well known that globulins become
insoluble upon repeated reprecipitation and it may possibly be found that
the greater number of the proteans are transformed globulins. Osborne,
however, believes that nearly all proteins may give rise to proteans. This
investigator who has so very thoroughly investigated many of the vege-
table proteins claims that the hydrogen ion is the active agent in the trans-
formation. The protein produced from the transformation of edestin is
called edestan, that produced from myosin is called myosan, etc. The
name protean was first given to this class of proteins by Osborne in 1900
in connection with his studies of edestin.
Experiments on Proteans.
Preparation and Study of Edestan. — Prepare edestin according to
the directions given on page log. Bring the edestin into solution in
0.2 per cent hydrochloric acid and permit the acid solution to stand for
about one-half hour.^ Neutralize, with a 0.5 per cent solution of sodium
carbonate, filter off the precipitate of edestan and make the following
tests:
1. Solubility. — Try the solubility in the ordinary solvents (see page
27). Note the altered solubility of the edestan as compared with that of
edestin (see page no).
2. Millons Reaction.
3. Coagulation Test. — Place a small amount of the protean in a
test-tube, add a little water and boil. Now add dilute hydrochloric acid
and note that the protein no longer dissolves. It has been coagulated.
4. Tests on Edestan Solution. — Dissolve the remainder of the
edestan precipitate in 0.2 per cent hydrochloric acid and make the follow-
ing tests:
(a) Biuret Test.
(b) Influence of Protein Precipitants. — Try a few protein precipitants
such as picric acid and mercuric chloride.
METAPROTEINS.
The metaproteins are formed from the native simple proteins through
an action similar to that by which proteans are formed. In the case of
' The edestan solution preserved from experiment (5), page iii, may be used.
Il6 PHYSIOLOGICAL CHEMISTRY.
•
the metaproteins, however, the changes in the original protein molecule
are more profound. These derived proteins are characterized by being
soluble in very weak acids and alkalis, but insoluble in neutral fluids.
The metaproteins have generally been termed albuminates, but inasmuch
as the termination ate signifies a salt it has always been somewhat of a
misnomer.
Two of the principal metaproteins are the acid metaprotein or so-
called acid albuminate and the alkali metaprotein or so-called alkali
albuminate. They differ from the native simple proteins principally
in being insoluble in sodium chloride solution and in not being coagula-
ted except when suspended in neutral fluids. Both forms of metaprotein
are precipitated upon the approximate neutralization of their solutions.
They are precipitated by saturating their solutions with ammonium sul-
phate, and by sodium chloride, also, provided they are dissolved in
an acid solution. Acid metaprotein contains a higher percentage of
nitrogen and sulphur than the alkali metaprotein from the same source,
since some of the nitrogen and sulphur of the original protein is
liberated in the formation of the latter. Because of this fact, it is impos-
sible to transform an alkali metaprotein into an acid metaprotein, while it
is possible to reverse the process and transform the acid metaprotein into
the alkali modification.
Experiments on Metaproteins.
ACID METAPROTEm (ACID ALBUMINATE).
Preparation and Study. — 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 HCl. Place it on a
boihng water-bath for one-half hour, filter, cool, and divide the filtrate
into two parts. Neutralize the first part with dilute KOH solution,
filter oflf the precipitate of acid metaprotein and make the following tests:
(i) Solubility. — Solubility in the ordinary solvents (see page 27).
(2) Milton's Reaction.
(3) Coagulation Test. — Suspend a little of the metaprotein 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 metaprotein is still
soluble in dilute alkali. What is the result and why ?
(4) Test for Loosely Combined Sulphur (seepage 108).
Subject the second part of the original solution to the following tests:
(i) Coagulation Test. — Heat some of the solution to boiling in a test-
tube. Does it coagulate ?
(2) Biuret Test.
PROTEINS. 117
(3) Influence of Protein Precipitants. — Try a few protein prccipitants
such as picric acid and mercuric chloride. How do the results obtained
compare with those from the experiments on egg albumin? (See
page 102.)
ALKALI METAPROTEIN (ALKALI ALBUMINATE).
Preparation and Study. — Carefully separate the white from the
yolk of a hcn.'s egg and place the former in an evaporating dish. Add
concentrated potassium hydroxide solution, drop by drop, stirring con-
tinuously. The mass gradually thickens and finally assumes the con-
sistency of jelly. This is solid alkali metaprotein or "Lieberkiihn's
jelly." Do not add an excess of potassium hydroxide 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 hydrochloric acid, noting the
odor of the liberated hydrogen sulphide as the alkali metaprotein precipi-
tates. Filter off the precipitate and test as for acid metaprotein, page
116, noting particularly the sulphur test. How does this test compare with
that given by the acid metaprotein? Make tests on the second part of
the solution the same as for acid metaprotein, page 116.
Coagulated Proteins.
These derived proteins are produced from unaltered protein mate-
rials by heat, by long standing under alcohol, or by the continuous move-
ment 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 195), the coagulation may be produced by enzyme action.
Ordinary soluble proteins after having been transformed into the coagu-
lated modification are no longer soluble in the ordinary solvents. Upon
being heated in the presence of strong acids or alkalis, coagulated proteins
are converted into metaproteins.
Many proteins coagulate at an approximately fixed temperature under
definite conditions (see pp. 106 and 254). This characteristic may be
applied to separate different coagulable proteins from the same solution
by fractional coagulation. The coagulation temperature frequently may
serve in a measure to identify proteins in a manner similar to the melting-
point or boiling-point of many other organic substances. The separation
of proteins by fractional coagulation is thus analogous to the separation
of volatile substances by means oi fractional distillation. This method of
Il8 PHYSIOLOGICAL CHEMISTRY.
separating proteins is not a satisfactory one, however, inasmuch as proteins
in solution have different effects upon one another and also because of the
fact that the nature of the solvent causes a variation in the temperature at
which a given protein coagulates. The nature of the process involved in
the coagulation of proteins 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 libera-
tion of minute amounts of hydrogen, nitrogen, and sulphur. The pres-
ence of a neutral salt or a trace of a mineral acid may facilitate the coagu-
lation of a protein solution (see page io6), whereas any appreciable
amount of acid or alkali will retard or entirely prevent such coagulation.
It has recently been shown that the coagulation of proteins by heat
proceeds in two stages,^ first, a reaction between the protein and the hot
water (denaturation) and second, an agglutination or separation of the
altered protein in particulate form. The concentration of acid, or hydro-
gen ion, in the solution influences the coagulation of proteins, such that
the original protein is acted upon less readily by hot water alone than in the
presence of acid. The formation of the coagulum is accompanied by the
disappearance of the free acid from the solution, indicating the formation
of a protein salt. A disturbance of the equilibrium between the hydro-
lyzed and unhydrolyzed portions of the protein salt, due to the greater
rapidity with which the unhydrolyzed portion is precipitated, results in
the gradual removal of both protein and acid from the solution. This
has been offered as an explanation of the decreasing acidity.
According to Chick and Martin, the addition of neutral salts to the
acid solution of the salt-free protein to be coagulated results in a decreased
rate of coagulation. This is due in part to the decrease in the concen-
tration of the free acid, which results from the disturbance of the equilib-
rium between the protein and acid and also in part to the direct influence
which the salts exert upon the protein. The presence of neutral salts
may under certain circumstances facilitate the coagulation of proteins by
heat.
The temperature at which egg white is coagulated causes a difference
in the appearance of the coagulum.^ Coagulated egg white which has
been immersed in water at a low temperature and then gradually heated
to the coagulating temperature is more translucent and has a bluish color,
whereas, egg white which has been immersed in water heated to a tem-
perature above the coagulating temperature is creamy-white in color.
The varying digestibitily, as the result of the different methods of heating,
has been discussed in the chapter on Enzymes.
' Chick and Martin: Journal of Physiology, 43, i, 1911.
'■'P'rank: Journal of Biological Chemistry, 9, 463, 191 1
proteins. 119
Experiments on Coagulated Protein.
Ordinary coagulated egg-white may be used in the following tests:
1. Solubility. — Try the solubility of small pieces of the coagulated
protein in each of the ordinary sclvents (see page 27).
2. Millon's Reaction.
3. Xanthoproteic Reaction. — Partly dissolve a medium-sized piece
of the protein in concentrated nitric acid. Cool the solution and add an
excess of ammonium hydroxide. Both the protein solution and the
undissolved protein will be colored orange.
4. Biuret Test. — Partly dissolve a medium-sized piece of the protein
in concentrated potassium hydroxide solution. If the proper dilution of
copper sulphate solution is now added the white coagulated protein, as
well as the protein solution, will assume the characteristic purplish-violet
color.
5. Hopkin's-Cole Reaction.— Conduct this test according to the
moditication given on page 98.
Secondary Protein Derivatives.
These derivatives result from a more profound cleavage of the protein
molecule than that which occurs in the formation of the primary deriva-
tives. The class includes proteoses, peptanes, and peptides.
PROTEOSES AND PEPTONES.
Proteoses are intermediate products in the digestion of proteins by
proteolytic enzymes, as well as in the decomposition of proteins by hydrol-
ysis and the putrefaction of proteins through the action of bacteria.
Proteoses are called albiimoses by some writers, but it seems more logical
to reserve the term ablumose for the proteose of albumin.
Peptones are formed after the proteoses and it has been customary to
consider them as the last product of the processes before mentioned which
still possess true protein characteristics. In other words it has been
considered that the protein nature of the end-products of the cleavage of
the protein molecule ceased with the peptones, and that the simpler
bodies formed from peptones were substances of a different nature (see
page 70). However, as the end-products have been more carefully
studied, it has been found to be no easy matter to designate the exact
character of a peptone or to indicate the exact point at which the peptone
characteristic ends and the peptide characteristic begins. The situation
regarding the proteoses, peptones and peptides, is at present a most
unsatisfactory one because of the unsettled state of our knowledge regard-
ing them. The exact differences between certain members of the peptone
I20 PHYSIOLOGICAL CHEMISTRY.
and peptide groups remain to be more accurately established. It has been
quite well established that the peptones are peptides or mixtures of peptides
but the term peptide is used at present to designate only those possessing a
definite structure.
There are several proteoses (protoproteose, heteroproteose and deutero-
proteose), and at least two peptones (amphopeptone and antipeptone),
which result from proteolysis. The differentiation of the various proteoses
and peptones at present in use is rather unsatisfactory. These compounds
are classified according to their varying solubihties, especially in ammo-
nium sulphate solutions of different strengths. The exact differences in
composition between the various members of the group remain 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, diffusible bodies
which are non-coagulable by heat. Peptones differ from proteoses in being
more diffusible, non-precipitable by (NH4)2SO^, and by their failure to give
any reaction with potassium ferrocyanide and acetic acid, potassio-mer curie
iodide and HCl, picric acid, and trichloracetic acid. The so-called primary
proteoses are precipitated by HNO3 and are the only members of the pro-
teose-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:
(i) Solubility. — Solubihty in the ordinary solvents (see page 27).
(2) Millon^s Reaction.
Dissolve a little of the powder in water and test the solution as
follows:
(i) Precipitation by Picric Acid. — To 5 c.c. of proteose-peptone
solution in a test-tube add picric acid until a permanent precipitate forms.
The precipitate disappears on heating and returns on cooling.
(2) Precipitation by a Mineral Acid.- — Try the precipitation by nitric
acid.
(3) Coagulation Test. — Heat a little proteose-peptone solution to boil-
ing. Does it coagulate like the other simple proteins studied?
SEPARATION OF PROTEOSES AND PEPTONES."
Place 50 c.c. of proteose-peptone solution in an evaporating dish or
casserole, and half-saturate it with ammonium sulphate solution, which
' The separation of proteoses and peptones by means of fractional precipitation with
ammonium sulphate does not possess the significance it was once supiposed to possess inas-
much as the boundary between these substances and peptides is not well defined (see p. 119).
PROTEINS. 121
may be accomplished by adding an equal volume of saturated ammonium
sulphate solution. At this point note the appearance of a precipitate of
the primary proteoses (protoproteose and hctero-proteose). Now heat the
half-saturated solution and its suspended precipitate to boiling and
saturate the solution with solid ammonium sulphate. At full saturation the
secandary proteoses (deuteroproteoses) are precipitated. The peptones
remain in solution.
Proceed as fallows with the precipitate of proteoses: Collect 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 ammonium sulphate, which adhered to the precipitate
and is now in solution, add barium carbonate, boil, and filter off the
precipitate of barium sulphate. Concentrate the proteose solution to a
small volume^ and make the following tests:
(i) Biuret Test.
(2) Precipitatian by Nitric Acid. — A\Tiat would a precipitate at this
point indicate ?
(3) Precipitatian by Trichloracetic Acid. — This precipitate dissolves
on heating and returns on cooling.
(4) Precipitatian by Picric Acid. — This precipitate also disappears on
heating and returns on cooling.
(5) Precipitation by Potassio-mer curie Iodide and Hydrochloric Acid.
(6) Coagulatian 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 filtered,
and the ammonium sulphate in solution removed by boiling with barium
carbonate as described above. After filtering off the barium sulphate
precipitate, concentrate the peptone filtrate to a small volume and repeat
the test as given under the proteose solution, above. In the biuret test
the solution should be made very strongly alkaline with solid potassium
hydroxide.
PEPTIDES.
The peptides are "definitely characterized combinations of two or
more amino acids, the carboxyl (COOH) group of one being united
with the amino (NHj) group of the other with the elimination of a mole-
cule of water." These peptides are more fully discussed on pages 71
and 119,
* 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.
122 PHYSIOLOGICAL CHEMISTRY.
REVIEW OF PROTEINS.
In order to facilitate the student's review of the proteins, the prepara
tion of a chart similar to the model given is recommended. The signs + ,
and — may be conveniently used to indicate positive and negative reactions.
MODEL CHART FOR REVIEW PURPOSES.
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At this point the student's knowledge of the characteristics of the
various proteins studied will be tested by requiring him to examine several
'•'unknown" protein mixtures or solutions and make full report upon the
same. The scheme given on page 123 may be used in this examination.
PROTEINS.
123
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CHAPTER VI.
GASTRIC DIGESTION.
Gastric digestion takes place in the stomach and is promoted by
the gastric juice, which is secreted by the glands of the stomach mucosa.
These glands are of two kinds, fundus glands and pyloric glands which
are situated, as their names imply, in the regions of the fundus and
pylorus. The principal foods acted upon in gastric digestion are the
proteins which are so changed by its processes as to become better pre-
pared for further digestion in the intestine and for their final absorption.
From reliable experiments made upon lower animals it is evident
that the gastric juice is secreted as the result of stimuli of two forms,
i. e., psychical stimuli and chemical stimuli. The psychical form of
stimuli may be produced by the sight, thought, or taste of food, and the
chemical stimuli may be produced by certain substances, such as water,
milk, the extractives of meat, etc., when coming in contact with the
stomach mucosa. The stimulatory power of water has been very strik-
ingly demonstrated/ Experiments have been made which indicate
clearly that the outpouring of gastric juice increases in direct proportion
to the volume of water which comes into contact with the gastric mucosa.^
The claim that the drinking of water with meals is harmful because such
a procedure causes a dilution of the gastric juice, has no basis in fact.
The drinking of water with meals by normal individuals has been found
to be accompanied by a more economical utilization of the ingested pro-
teins, fats and carbohydrates. Various other desirable and no undesirable
features have been demonstrated as accompanying or following such a
dietary procedure.^ No experimental evidence has been submitted which
can justly be interpreted as showing any harmful influence to accompany
or follow the drinking, by normal persons, of large quantities of water at
meal time.
* Foster and Lambert: Journ Exper. Med., lo, 820, 1908.
2 Wills and Hawk: Jour. Biol. Chem., 9, xxx, 191 1. (Proceedings).
'Hawk: University 0/ Pennsylvania Medical Bulletin, 18, i, 1905.
Fowler and Hawk: Jour. Exper. Med., 12, 388, 1910.
Hattrem and Hawk: Arch. Int. Med., 7, 610, 1911.
Mattill and Hawk: Jour. Am. Chem. Soc, 33, pp. 1978, 1999, and 2019, 1911.
Hawk: Arch. Int. Med., 8, 382, 1911.
Hawk: Proceedings Soc. Exp. Biol, and Med., 8, 36, 1910.
Fairhall and Hawk: Jour. Am. Chem. Soc, 34, 546, 1912.
Howe and Hawk: Jour. Biol. Chem., 11, 129, 1912.
124
GASTRIC DIGESTION. 1 25
The volume of gastric juice secreted during any given period of
digestion, \aries with the quantity and kind of the food. These con-
clusions were deduced principally from a series of so-called delusive
feeding experiments. A dog was prepared with two oesophageal openings
and a gastric fistula. When thus prepared and fed foods of various kinds
such as meat and bread, the material instead of passing to the stomach,
would invariably find its way out of the animal's body at the upper
oesophageal opening. Through the medium of the gastric fistula the
course of the secretion of gastric juice could be carefully followed. It
was found that when the dog ate meat, for example, there was a large
secretion of gastric juice notwithstanding no portion of the food eaten
had reached the stomach. Further experiments made through the
medium of a cul-de-sac formed from the stomach wall have given us
many valuable conclusions, among others those regarding the influence
of the chemical stimuli. The method followed was to feed the animal
certain substances and note the secretion of gastric juice in the miniature
stomach while the real process of digestion was taking place in the
stomach proper.
Normal gastric juice is a thin, light colored fluid which is acid in
reaction and has a specific graA^ity varying between i.ooi and i.oio.
It contains only 2-3 per cent of solid matter which is made up prin-
cipally of hydrochloric acid, sodium chloride, potassium chloride, earthy
phosphates, mucin and the enzymes pepsin, gastric rennin, and gastric
lipase; the hydrochloric acid and the enzymes are of the greatest im-
portance. The acidity of the gastric juice is due to free hydrochloric.
It was formerly believed that this acid was secreted by the parietal cells
of the fundus as well as by the chief cells of both the fundus and pyloric
glands. It has recently been claimed^ however, that the parietal cell is
the seat of the formation of the hydrochloric acid. This conclusion is based
upon the formation of Prussian blue after the subcutaneous injection of
potassium ferrocyanide and ammonium ferric citrate (rabbits and
guinea-pigs) and the subsequent (3 to 30 hours) microscopical examina-
tion of the gastric mucosa. The acid was shown to be present in the
lumina of the gland tubules and in the canaliculi of the parietal cells;
traces were also apparently present in the cytoplasm. Still more recently
Bensley and Harvey' have shown by means of dyes which act as vital
stains and as indicators very sensitive to alkali that the secretion in the
parietal cells is slightly alkaline whereas that in the lumen of the gland
proper is very nearly neutral. Therefore, the acid is formed entirely above
the level of the gland proper i. e. in the foveolae and on the surface. It is
' Fitzgerald: Proceedings Royal Society (B), 83, 56, igio.
- Bensley and Harvey: Unpublished data furnished by Dr. Bensley.
126 PHYSIOLOGICAL CHEMISTRY.
apparent from the work of Fitzgerald, and Bensley and Harvey that
the question as to the seat of formation of the hydrochloric acid must be
considered as undecided.
Hydrochloric acid is generally present in the gastric juice of man to
the extent of o . 2-0 . 3 per cent. When the amount of hydrochloric acid
varies to any considerable degree from these values a condition of
hypoacidity or hyperacidity is established. Hydrochloric acid has the
power of combining with protein substances taken in the food-, thus
forming so-called combined hydrochloric acid. This combined acid is a
less potent germicide than/ree hydrochloric acid and has less power to
destroy the amylolytic enzyme salivary amylase (ptyalin) of the saliva.
This last fact explains to a degree the possibility of the continuance of
salivary digestion in the stomach.
The term combined hydrochloric acid is really a misnomer. When
free hydrochloric acid is treated with a protein the latter functions as a
base metal and a salt is formed. Therefore, instead of having, "com-
bined hydrochloric acid" we have a protein salt of hydrochloric acid.
This salt ionizes differently from the free acid. This fact explains the
variation in the gemicidal properties of the two solutions as well as their
different action toward enzymes, such, for example, as salivary amylase
(see page 66).
The hydrochloric acid of the gastric juice forms a medium in which
the pepsin can most satisfactorily digest the protein food, and at the
same time it acts as an antiseptic or germicide which prevents putre-
factive processes in the stomach. It also possesses the power of inverting
cane sugar, this property being due to the hydrogen ion. When the
hydrochloric acid of the gastric juice is diminished in quantity (hypoacid-
ity) or absent, as it may be in many cases of functional or organic dis-
ease, there is no check to the growth of micro-organisms in the stomach.
There are, however, certain of the more resistant spores which even the
normal acidity of the gastric juice will not destroy. A condition of
hypoacidity may also give rise to fermentation with the formation of
comparatively large amounts of such substances as lactic acid and butyric
acid.
The f|uestion of the origin of the hydrochloric acid of the gastric
juice is a problem to whose solution many investigators have given
much attention. Many theories have been proposed, among them
being Bunge's mass action theory, Koppe's electrolytic dissociation theory.
and the more recent theory based upon the interaction of sodium chloride
and lactic acid. We cannot go into a discussion of these various theories.
Each of them has met with objection and we have, as yet, no generally
accepted theory as to the origin of the hydrochloric acid of the gastric
GASTRIC DIGESTION. 1 27
juice. That this hydrochloric acid originates from the chlorides of the
blood is apparently a well established fact, but farther than this no positive
statement can be made.
The most important of the enzymes of the f^astric juice is the pro-
teolytic enzyme pepsin. The pepsin does not originate as such in the
gastric cells but is formed from its precursor, the zymogen or mother-
substance pepsinogen, which is produced by the parietal cells of the
fundus as well as by the chief cells of the fundus and pyloric glands.
Pepsinogen may be differentiated from pepsin from the fact that it is
more resistant to alkali.^ Upon coming in contact with the hydrochloric
acid of the secretion this pepsinogen is immediately transformed into
pepsin. Pepsin is not active in alkaline or neutral solutions but requires
at least a faint acidity before it can exert its power to dissolve and digest
proteins. The percentage of hydrochloric acid facilitating the most
rapid peptic action varies with the character of the protein acted upon,
e. g., 0.08 per cent to o.i per cent for the digestion of fibrin and 0.25
per cent for the digestion of coagulated egg-white. While hydrochloric
acid is the acid usually employed to promote artificial peptic proteolysis,
other acids, organic and inorganic, will serve the same purpose. Acidity
of the liquid is necessary to promote the activity of the pepsin, but the
acidity need not necessarily be confined to hydrochloric acid.
In common with many other enzymes pepsin acts best at about
38^-40° C. and its digestive power decreases as the temperature is lowered,
the enzyme being only slightly active at o° C. Its power is only tempo-
rarily inhibited by the appHcation of such low temperatures, however,
and the enzyme regains its full proteolytic power upon raising the tem-
perature to 40° C. As the temperature of a digestive mixture is raised
above 40° C. the pepsin gradually loses its activity until at about 80°-
100° C. its proteolytic power is permanently destroyed.
Our ideas regarding the nature of the products formed in the course
of peptic proteolysis have undergone considerable revision in recent
years. The former view that these products included only acid albu-
minate (acid metaprotein), proteoses and peptones is no longer tenable.
From the investigations of numerous observers we have learned that
artificial gastric digestion if permitted to proceed for a sufjiciently long
period will yield, in addition to proteoses and peptones, a long list of
protein cleavage products which are crystalline in character, including
leucine, tyrosine, alanine, phenylalanine, aspartic acid, glutamic acid,
proline, leucininiide, valine, and lysine. A similar group of substances
may result from the action of the enzyme trypsin (see p. 149). The
relative amounts of proteoses, peptones, and crystalline substances
'Langley: Jour, of Physiol., 3. p. 246.
128 PHYSIOLOGICAL CHEMISTRY.
formed depends to a great extent upon the character of the protein under-
going digestion, e. g., a, greater proportion of proteoses results from the
digestion of fibrin than from the digestion of coagulated egg-white. We
must not be led into the error of thinking that the large number of protein
cleavage products just mentioned are formed in the course of normal
gastric digestion within the animal organism. They are formed only
after comparatively long-continued hydrolysis. In pancreatic digestion,
however, there are formed even under normal conditions, the .large
number of cleavage products to which reference has been made. Peptic
proteolysis, therefore, within the animal organism differs from tryptic
proteolysis (see page 149) in that the former yields larger amounts of
proteoses, smaller amounts of peptones and no considerable quantity
of crystalline bodies as end-products in the brief period during which
proteins are ordinarily subjected to gastric digestion. Prolonged hydrol-
ysis with gastric juice does, however, yield considerable quantities of
the non-protein end-products. In cases of cancer of the stomach a
peptide-splitting enzyme (erepsin) is present in the stomach contents.
This enzyme is believed to be elaborated by the cancer tissue and its
identification is of importance in connection with the diagnosis of gastric
cancer. The glycyl-tryptophane test^ is used for this purpose
(see p. 15).
Abderhalden and Meyer^ have very recently shown active pepsin to
be present in the contents of all parts of the small intestine. It is suggested
that pepsin may be adsorbed in the stomach by such protein substances
as pass into the intestine in solid form and that the pepsin thus protected
may bring about gastric digestion whenever the reaction of the surrounding
intestinal contents is favorable. This fact may be of importance in
connection with the profound proteolysis taking place in the intestine.
Heretofore, this process was believed to be furthered alone by trypsin
and erepsin. The passage of adsorbed pepsin into the intestine may be
an efficient aid to the proper digestion of solid proteins which are ingested
without sufficient mastication ("bolted")^ and which consequently, at
times, pass into the intestine in rather large pieces (see chapter on Feces).
Gastric rennin, the second enzyme of the gastric juice, is what is
known as a milk curdling or protein coagulating enzyme. Rennin acts
upon the caseinogen of the milk, splitting it into a proteose-like body
and soluble casein. This soluble body, in the presence of calcium
salts, combines with calcium, forming calcium casein or true casein
which is insoluble and precipitates. There is some uncertainty re-
' Neubauer and Fischer: Deut. Arch./, klin. Med., 97, 499, 1909.
* Abderhalden and Meyer: Zeit.fur physiol. Chem., 74, 67, 191 1.
' Foster and Hawk: Proceedings of the Eighth International Congress of Applied Chemistry,
New York, September, 1912.
GASTRIC DIGESTION. 1 29
garding the reaction to litmus in which gastric rcnnin shows the greatest
activity. It is, however, said to be active in neutral, alkaline, or acid
solution. However, it probal^ly possesses its greatest activity in the
presence of a slight acid reaction, as would naturally be expected. It
is especially abundant in the gastric mucosa of the calf, and is used
to curdle the milk used in cheese-making. Gastric rennin is always
present normally in the gastric juice but in certain pathological con-
ditions such as atrophy of the mucosa, chronic catarrh of the stomach,
or in carcinoma it may be absent.
The theory that the proteolytic activity and the milk curdling property
of the gastric juice reside in a single substance is causing much con-
troversy at the present time. The theory was originally advanced by the
Pawlow school.^ According to Nencki and Sieber^ the milk curdling
and protein hydrolyzing activities reside in defmite and distinct side chains
of a single mammoth molecule. The view which has rather the strongest
support, however, is to the effect that there are two entirely distinct
enzymes. Important evidence has been advanced in favor of this view
by Hammarsten,^ Taylor,^ and Hemmeter.^ Very recently Burge" has
reported experiments upon the influence of a direct electric current upon
solutions possessing typical rennin and peptic activities. By this means
he was able to prepare a solution possessing strong rennin activity but
entirely void of peptic activity. This furnishes strong evidence against
the identity of the two enzymes but does not necessarily deny the accuracy
of the side-chain theory.
Gastric lipase, the third enzyme of the gastric juice, is a fat-splitting
enzyme. It possesses but slight activity when the gastric juice is of
normal acidity, but evinces its action principally at such times as a
gastric juice of low acidity is secreted either from physiological or patho-
logical cause. The digestion of fat in the stomach is, however, at most,
of but slight importance as compared with the digestion of fat in the
intestine through the action of the lipase of the pancreatic juice (see
page 151).
PREPARATION OF AN ARTIFICIAL GASTRIC JUICE.
Dissect the mucous membrane of a pig's stomach from the mus-
cular portion and discard the latter. Divide the mucous membrane
into two parts (4/5 and 1/5). Cut up the larger portion, place it in
' Tawlow and Parastschuk: Zeitschrift fiir Physiologische Chemie, 42, 415, 1904.
- Xencki and Sieber: Zeitschrift fiir Physiologische Chemie, 23, 2gi, 1901.
' Ilammarsten: Zeitschrift fiir Physiologische C hemic, 56, 18, 1908.
* Taylor: Journal of Biological C hemistry, 5, 399, 1909.
^ Hemmeter: Berliner klinische Wochenschrift, Ewald Festnummer, 44, 1905.
^ Burge: American Journal of Physiology, 29, 1912.
130 PHYSIOLOGICAL CHEMISTRY.
a large-sized beaker with 0.4 per cent hydrochloric acid and keep at
38°-4o° C. for at least 24 hours. Filter off the residue, consisting prin-
cipally of nuclein and anti-albumid, and use the filtrate as an artificial
gastric juice. This filtrate contains pepsin, rennin, and the products
of the digestion of the stomach tissuue, i. e., acid metaprotein (acid
albuminate), proteoses, peptones, etc.
Preparation of a Glycerol Extract of Pig's Stomach.
Take the one-fifth portion of the mucous membrane of the pig's
stomach not used in the preparation of the artificial gastric juice, cut
it up very finely, place it in a small-sized beaker and cover the mem-
brane with glycerol. Stir frequently and allow to stand at room tem-
perature for at least 24 hours. The glycerol will extract the pepsinogen.
Separate, with a pipette or by other means, the glycerol from the pieces
of mucous membrane and use the glycerol extract as required in the
later experiments.
Products of Gastric Digestion.
Into the artificial gastric juice, prepared as above described, place
the protein material (fibrin, coagulated egg-white, or lean beef) pro-
vided for you by the instructor, add 0.4 per cent hydrochloric acid as
suggested by the instructor and keep the digestion mixture at 40° C,
for 2 to 3 days. Stir frequently and keep free hydrochloric acid present
in the solution (for tests for free hydrochloric acid see p. 131).
The original protein has been digested and the solution now con-
tains the products of peptic proteolysis, i. e., acid metaprotein (acid
albuminate), proteoses, peptones, etc. The insoluble residue may
include nuclein and anti-albumid. Filter the digestive mixture and
after testing ior free hydrochloric acid neutralize the filtrate with potas-
sium hydroxide solution. If any of the acid metaprotein (acid albu-
minate) is still untransformed into proteoses it will precipitate upon neutral-
ization. If any precipitate forms heat the mixture to boiling, and filter.
If no precipitate forms proceed without filtering.
We now have a solution containing a mixture consisting princi-
pally of proteoses and peptones. Separate and identify the proteoses
and peptones according to the directions given on pages 120 and 121.
Tests for Free and Combined HCl.
These tests are made with a class of reagents known as indicators,
so-called because they show changes of color according to the degree of
acidity (or alkalinity) of the solution. They behave as though they
were weak acids or bases whose ions and unionized molecules have
GASTRIC DIGESTION. I31
different colors. Modern theories of color in organic compounds how-
ever class them as tautomeric substances.
A neutral solution is one in which there are equal numbers of hydro-
gen and hydroxyl ions. An acid solution has a preponderance of hydro-
gen ion and an alkaline solution an excess of hydroxyl ion. All indi-
cators do not show changes of color at the true neutral point, but at
some fixed decree of acidity (or alkalinity), i. e., at a definite hydrogen or
hydroxyl ion concentration. Those indicators which change color at the
approximate true neutral point are litmus and rosolic acid, while phenol-
phthalein changes color in a slightly alkaline solution. Congo red,
sodium alizarin sulphonate and tropaeolin OO are examples of indicators
which change color in an acid solution.
Organic acids are not sufficiently strong, i. e., do not produce enough
hydrogen ion, to cause color changes with the last-mentioned class of
indicators; litmus, rosolic acid, and phenolphthalein however indicate
the hydrogen ion concentration of organic acids or their solutions. Even
very dilute solutions of mineral acids are sufficiently acid to produce
color changes with congo red, etc. Phenolphthalein, which changes
color in a weakly alkaline solution, is used to indicate the presence of
acid combined with weakly alkaline substances (as protein), as well as
the other types of acid and, hence, is used to indicate the total acidity.
The differentiation between the various forms of acidity depends upon
the above facts.
The hydrogen ion concentrations at which some common indicators
show the most characteristic change of color are given below. Concen-
trations are expressed in approximate moles of hydrogen ion per liter.
True nature
Indicator. Hydrogen ion of solution
concentration. when the
color changes.
Rosolic acid i X io~"' Neutral.
Litmus Between i X lo"*^ and i X io"~' . . Neutral.
Tropa-olin OO i X io~^ Acid.
Dimethyl-amino-azobenzene .. Between i X io~' and iXio~*.. .\cid.
Sodium alizarin sulphonate. . . . Between i X lO""* and i X io~*. . Acid.
Congo red Between i X io~° and i X 10—^ . . Acid.
Phenolphthalein Between i X lO""' and i X lo""'. . Alkaline.
Examine each of the following solutions by means of the tests given
below and report the results in a form similar to the chart given on
page 133: (i) 0.2 per cent free hydrochloric acid. (2) 0.05 per cent
free hydrochloric acid. (3) 0.0 1 per cent free hydrochloric acid. (4)
0.05 per cent combined hydrochloric acid (see p. 126). (5) i per cent
lactic acid. (6) Equal volumes of 0.2 per cent free hydrochloric acid and
I per cent lactic acid. (7) i per cent potassium hydroxide.
132 PHYSIOLOGICAL CHEMISTRY.
1. Dimethyl-amino-azobenzene (or Topfer's Reagent),^
N(CH3),-C,H,-N^N-C,H,.
Place 1-2 drops of the reagent in the solution to be tested. Free min-
eral acid (hydrochloric acid) is indicated by the production of a pinkish-
red color. If free acid is absent a yellow color ordinarily results.
2. Giinzberg's Reagent.^ — ^Place 1-2 drops of the reagent in a
small porcelain evaporating dish and carefully evaporate to dryness
over a low flame. Insert a glass stirring rod into the mixture to be
tested and draw the moist end of the rod through the dried reagent.
Warm again gently and note the production of a purplish-red color
in the presence oifree hydrochloric acid.
3. Boas' Reagent.^ — Perform this test in the same manner as
2, above. Free hydrochloric acid is indicated by the production of
a rose-red color which becomes less pronounced on cooling.
4. Congo Red,*
NH2 • SOgNa
SOgNa NH2
Conduct this test according to the directions given under i or 2, above
A blue color indicates free hydrochloric acid, a violet color indi-
cates an organic acid and a brown color indicates combined hydro-
chloric acid. Congo-red paper, made by immersing ordinary fiilter
paper in the indicator and subsequently drying, may be used in this
test.
5. Tropaeolin 00/
NH(CeH5) -CgH, -N =N -C,H, -SOgNa.
Place 2 drops of the solution to be tested and i drop of the indicator
in an evaporating dish and evaporate to dryness over a low fiame. The
formation of a reddish-violet color indicates /r^e hydrochloric acid.
This test may also be conducted in the same manner as 2,
above.
' To prepare Topfer's reagent dissolve 0.5 gram of climethyl-amino-azobenzene in 100 c.c.
of 95 per cent alcohol.
* Giinzberg's reagent is prepared by dissolving 2 grams of phloroglucinol and i gram of
vanillin in 100 c.c. of 95 per cent alcohol.
■^ Boas' reagent is prepared by dissolving 5 grams of resorcinol and 3 grams of sucrose in
100 c.c. of 50 per cent alcohol.
' This indicator is prepared by dissolving 0.5 gram of congo red in 90 c.c. of water and
adding 10 c.c. of 95 per cent alcohol.
^Prepared by dissolving 0.05 gram of tropa;olin OO in 100 c.c. of 50 per cent alcohol.
GASTRIC DIGESTION.
133
6. Phenolphthalein/
C„H,OH
C— C«H,OH
C„H,
O
\
o
Add the indicator directly to the solution, or apply the test according to
the directions given under 2 on page 134. This indicator serves to
denote the total acidity since it is acted upon by free mineral acids,
combined acids, organic acids, and acid salts. A red color indicates the
presence of an alkali and the indicator is colorless in the presence of a
neutral or acid reaction. This indicator is unsatisfactory in the pres-
ence of ammonia.
7. Sodium Alizarin Sulphonate,^
CO
(OH),
C„H
C„H
CO S03Na
This indicator may be used directly in the solution to be tested, or the
test may be applied as 2, page 134. It serves to indicate all acid re-
actions except those due to combined acids. A reddish-violet color
indicates an alkaline reaction, while a yellow color indicates an acid
reaction due to a free mineral acid, an organic acid, or an acid salt.
Report the results of your tests tabulated in the form given below:
Solutions Examined.
Name of Indicator.
0.2 ■o
HCl.
0.05 %
HCl.
O.OI .0
HCl.
0.0s % I 7c
Combined Lactic
HCl. Acid.
Equal Vols.
0.2% HCl 1^0
and I % ' KOH.
Lactic Acid, i
Topfer's Reagent.
1
Giinzberg's Reagent. j
Boas' Reagent. |
Congo Red.
Tropaeolin GO. ' j
Phenolphthalein.
Ali7.arin. [
' This indicator is prepared by dissolving i gram of phenolphthalein in 100 c.c. of 95
per cent alcohol.
- Prepare this indicator by dissolving i gram of sodium alizarin sulphonate in 100 c.c.
of water.
134 PHYSIOLOGICAL CHEMISTRY.
GENERAL EXPERIMENTS ON GASTRIC DIGESTION.
1. Conditions Essential for the Action of Pepsin. — ^Prepare four
test-tubes as follows:
(a) Five c.c. of pepsion solution.
(b) Five c.c. of 0.4 per cent hydrochloric acid.
(c) Five c.c. of pepsin-hydrochloric acid solution.
{d) Two or three c.c. of pepsin solution and 2-3 c.c. of 0.5 per cent
sodium carbonate solution.
Introduce into each tube a small piece of fibrin and place them in the
incubator or water-bath at 40° C. for one-half hour, carefully noting any
changes which occur. ^ Now combine the contents of tubes (a) and
(b) and see if any further change occurs after standing at 40° C. for
15-20 minutes. Explain the results obtained from these five experi-
ments.
2. Influence of Different Temperatures. — In each of four test-
tubes place 5 c.c. of pepsin-hydrochloric acid solution. Immerse one
tube in cold water from the faucet, keep a second tube at room tem-
perature and place a third in the incubator or water-bath at 40° C. Boil
the contents of the fourth tube 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 ? Explain this.
3. The Most Favorable Acidity. — Prepare three tubes as follows:
(a) Five c.c. of 0.2 per cent pepsin-hydrochloric acid solution.
{b) Two or three c.c. of 0.2 per cent hydrochloric acid + i c.c. of
concentrated hydrochloric acid + 5 c.c. of pepsin solution.
(c) One c.c. of 0.2 per cent pepsin-hydrochloric acid solution -f 5 c.c.
of water.
Introduce a small piece of fibrin into each tube, keep them at 40°
C, and note the progress of digestion. In which degree of acidity
does the fibrin digest the most rapidly ?
4. Differentiation Between Pepsin and Pepsinogen. — Prepare
five tubes as follows:
(a) Few drops of glycerol extract of pepsinogen -\- 2-3 c.c. of water.
(b) Few drops of glycerol extract of pepsinogen -1- 5 c.c. of 0.2 per
cent hydrochloric acid.
' Digestion of fibrin in a pepsin-hydrochloric acid solution is indicated first by a swelling
of the protein due to the action of the acid, and later by a disintegration and dissolving of
the fibrin due to the action of the pepsin-hydrochloric acid. If uncertain at any time whether
digestion has taken place, the solution under examination may be filtered and the biuret
test ajjj>lied to the filtrate. A positive reaction will signify the presence of acid metaprotein
(acid albuminate;, proteoses (albumoses), or peptones, the presence of any one of which
would indicate that digestion has taken place.
GASTRIC DIGESTION. I35
(c) Few drops of glycerol extract of pepsinogen + 5 c.c. of 0.5 per
cent sodium carbonate.
(d) Two or three c.c. of pepsin solution + 2-3 c.c. of i per cent
sodium carbonate.
(e) Few drops of glycerol extract of pepsinogen + 5 c.c. of i per
cent sodium carbonate.
Add a small piece of fibrin to the contents of each tube, keep the
live tubes at 46° C. for one-half hour and observe any changes which
may have occurred. To (a) add an ec[ual volume of 0.4 per cent hydro-
chloric acid, neutralize (c), (d) and (e) with hydrochloric acid and add
an equal volume of 0.4 per cent hydrochloric acid. Place these tubes
at 40° C. again and note any further changes which may occur. What
contrast do we find in the results from the last three tubes T-* Why is
this so ?
5. Comparative Digestive Power of Pepsin with Different
Acids. — Prepare a series of tubes each containing one of the following
acids: 0.5 per cent acetic, lactic, oxalic, salicylic, tannic, and butyric,
and 0.2 per cent hydrochloric, sulphuric, nitric, arscnious, and com-
bined hydrochloric. To each acid add a few drops of the glycerol extract
of pig's stomach and a small piece of fibrin. Shake well, place at 40°
C, and note the progress of digestion. In which tubes does the most
rapid digestion occur ?
6. Influence of Metallic Salts, etc. — ^Prepare a series of tubes
and into each tube introduce 4 c.c. of pepsin-hydrochloric acid solu-
tion and 1/2 c.c. of one of the chemicals listed in Experiment 18 under
Salivary Digestion, page 66. Introduce a small piece of fibrin into
each of the tubes and keep them at 40° C. for one-half hour. Note
the variations in the progress of digestion. Where has the least rapid
digestion occurred ?
7. Sahli's Desmoid Reaction. — This is a method for testing gastric
function without using the stomach tube. The underlying principle
of the test is the fact that raw catgut may be digested in gastric juice
but is entirely indigestible in pancreatic juice. The test is made as
follows: A methylene-blue pill is introduced into a small rubber bag
and the mouth of the bag subsequently tied with catgut.^ The small
bag is then ingested immediately after the mid-day meal and the urine
examined 5, 7, 9 and 18-20 hours later for methylene blue. If methylene
' About 0.05 gram of methylene blue is mixed with sufficient ext. glycyrrhiza to form
a pill about 3-4 mm. in diameter. The pill is then placed in the center of a square piece of
thin rubber dam and a little bag-like receptacle constructed by a twisting movement. The
neck of the bag is then closed by wrapping three turns of catgut about it. The most satis-
factory catgut to use is number 00 raw catgut which has previously been soaked in water
until soft. When ready for use the bag should sink instantly when placed in water and
be water-tight.
136 PHYSIOLOGICAL CHEMISTRY.
blue is present in appreciable qaantity, it will impart to the urine a
greenish-blue color. If not present in sufficient amount to impart this
color the urine should be boiled with 1/5 its volume of glacial acetic
acid, w^hereupona gieenish-bluecolor results if thechromogenof methylene
blue is present. This contingency seldom arises, however, inasmuch
as in most cases of uncolored urine it will be found that the rubber bag
has passed through the stomach unopened. If the methylene blue is
found in the urine inside of 18-20 hours a satisfactory gastric function
is indicated.
For Einhorn's bead method for the study of digestive function, see
chapter on Feces.
8. Testing the Motor and Functional Activities of the Stomach.
— This test is performed the same as Experiment 19 under Salivary
Digestion, page 67. If the experiment was carried out under salivary
digestion it will not be necessary to repeat it here.
9. Influence of Bile. — ^Prepare five tubes as follows:
(a) Five c.c. of pepsin-hydrochloric acid solution + 1/2-1 c.c. of
bile.
(b) Five c.c. of. pepsin-hydrochloric acid solution + 1-2 c.c. of
bile.
(c) Five c.c. of pepsin-hydrochloric acid solution + 2-3 c.c. of bile.
(d) Five c.c. of pepsin-hydrochloric acid solution + 5 c.c. of bile.
(e) 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 ?
10. Influence of Gastric Rennin on Milk. — Prepare a series of
five tubes as follows:
(a) Five c.c. of fresh milk + 0.2 per cent hydrochloric acid (add
slowly until precipitate forms).
{b) Five c.c. of fresh milk -|- 5 drops of rennin solution.
(c) Five c.c. of fresh milk + 10 drops of 0.5 per cent sodium car-
bonate solution.
(d) Five c.c. of fresh milk + 10 drops of a saturated solution of
ammonium oxalate.
(e) Five c.c. of fresh milk -h 5 drops of 0.2 per cent hydrochloric acid.
Now 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 diflerent tubes. Give a reason for each
particular result.
11. Tests for Lactic Acid. — (a) Ufelmann's Reaction. — To a small
GASTRIC DIGESTION. 1 37
quantity of UlTelmann's reagent' 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 gi\'e a similar reaction.
Mineral acids such as hydrochloric acid discharge the blue coloration
lea%ing a colorless solution. In other words, the color of the reagent
is weakened in the presence of an acid reaction.
(b) Ferric Chloride Test. — Place lo c.c. of very dilute ferric chloride
in each of five tubes. To the first add 2 c.c. of 0.2 per cent hydro-
chloric acid, to the second 2 c.c. of 10 per cent alcohol, to the third
2 c.c. of 2 per cent sucrose, 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, evapora-
ting the ether extract to dryness, and dissolving the residue in water.
This residue will not contain any of the contaminations which interfere
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.
(c) Hopkins^ Thiophenc Reaction.- — Place about 5 c.c. of concen-
trated sulphuric acid in a test-tube and add one drop of a saturated solu-
tion of copper sulphate." Introduce a few drops of the solution to be
tested, shake the tube well, and immerse it in the boiling water of a
beaker-water-bath for one or two minutes. Now remove the tube, cool
it under running water, add 2-3 drops of a dilute alcoholic solution^ of
thiophene, C^H^S, from a pipette, replace the tube in the beaker and
carefully observe any color change which may occur. Lactic acid is
indicated by the appearance of a bright cherry-red color which forms
rapidly. This color may be made more or less permanent by cooling
the tube as soon as the color is produced. Excess of thiophene produces
a deep yellow or brown color with sulphuric acid. The test is not
wholly specific though the author claims it to be more so than Uffelmann's
reaction.
12. Qualitative Analysis of Stomach Contents. — Take 100 c.c.
of stomach contents and analyze it according to the following scheme:
■ Uffelmann's reagent is prepared by adding ferric chloride solution to a i per cent
solution of carbolic acid until an amethyst-blue color is obtained, due to the formation of a
ferric salt of carbolic acid.
- This is added to catalyze the oxidation which follows.
' About 10-20 drops in loo c.c. of 95 per cent alcohol.
138 PHYSIOLOGICAL CHEMISTRY.
Stomach Contents.
Filter and test the filtrate for free hydrochloric acid.
I I
Filtrate I. Residue.
Divide into two parts. Discard after making a microscopical exami-
I nation.
I I
Filtrate II. Filtrate III.
One-fifth portion. Four-fifths portion.
Test for: Neutralize carefully; any precipitate is acid meta-
(a) Pepsin. protein (acid albuminate). If a precipitate forms
(b) Bile (see page 162). filter and divide the filtrate into two parts. If no
(c) Starch. precipitate forms divide the solution into two parts
(d) Dextrin. without filtering.
Filtrate IV. Filtrate V.
Two-thirds portion. One-third portion.
Heat to boiling to remove coagulable
proteins. If any precipitate forms filter
it off; if there is no precipitate proceed Test for:
directly with the tests. (a) Lactic acid.
Test for: (b) Gastric rennin.
(a) Sugar. (Differentiate between the various (c) Salivary amylase
sugars by the use of the scheme on page
5S.)
(b) Proteoses.
(c) Peptones.
CHAPTER Vn.
FATS.
Fats occur very widely distributed in the plant and animal king-
doms, 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.
Fig. 36. — Beef Fat. {Long.)
Chemically considered the fats are esters* of the tri-atomic alcohol,
glycerol, and the mono-basic fatty acids. In the formation of these
fats three molecules of water result. This water may arise in either
of two ways. First, by the replacement of the H of each of the OH
groups of glycerol by a fatty acid radical, giving the following formula
in which R, R' and R" represent fatty acid radicals,
CH3OR
CHOR'
I
CH2OR"
' .\n ester is an oxyacid, one of whose acid hydrogens is replaced by an organic radical
I40 PHYSIOLOGICAL CHEMISTRY.
Second, by the replacement of the H's of the carboxyl groups of the
three fatty acid molecules by the glycerol radical, thus yielding the
following type of formula in which R represents the glycerol radical,
OOCH3,C,,
R-00CH3,C,
\
OOCH3,C,,
Of these two processes the second is the more logical procedure from
the standpoint of the ionic theory. The three fatty acid radicals entering
into the structure of a neutral fat may be the radicals of the same fatty
acid or they may consist of the radicals of three different fatty acids.
By hydrolysis of a neutral fat, i. e., by the addition to the molecule of
those elements which are eliminated in the formation of the fat from
glycerol and fatty acid, it may be resolved into its component parts, /'. e.,
glycerol and fatty acid. In the case of palmitin the following would
be the reaction:
C3H,(0-C„H3,CO)3 + 3H,0-^C3H,(OH)3 + 3(C,,H3,COOH).
Palmitin. Glycerol- 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, e. g., pancreatic lipase (see page 151). The
cells forming the walls of the intestines evidently possess the peculiar
property of synthesizing the glycerol and fatty acid thus formed so that
after absorption these bodies appear in the blood not in their individual
form but as neutral fats. This synthesis is similar to that enacted in
the absorption of protein 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, palm-
itin, olein, and butyrin. Such less important forms as laurin and myristin
may occur abundantly in plant organisms. The older system of nomencla-
ture for these fats was to apply the prefix "tri" in each case {e. g., tri-
palmitin) since there 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 olein, palmitin
and 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 stearin and less
olein than the pork fat. Human fat contains from 67 per cent to 85 per
FATS. 141
cent of olcin and according to Hcncdict and Osterbcrg, upon analysis
yields 76.08 per cent of carbon and 11.78 per cent of hydrogen. Butter
consists in large part of olein and palmitin. Stearin, butyrin, caproin
and traces of other fats are also present.
Pure neutral fats are odorless, tasteless, and generally colorless.
Thev arc insoluble in the ordinary protein solvents such as water, salt
solutions, and dilute acids and alkalis, but are very readily soluble in ether,
benzene, chloroform, and boiling alcohol. The neutral fats are non-
volatile substances possessing a neutral reaction. If allowed to remain
in contact with the air for a sufficient length of time they become yellow
in color, assume an acid reaction and are said to be rancid. The neutral
fats may be crystallized, some of them with great facility. The crystalline
forms of some of the more common fats arc reproduced in Figs. 36, 37
and 38 on pages 139, 142 and 144. 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 coag-
ulation temperature (see page 117) is used for the dillferentiation of coag-
ulable proteins. When shaken with water, or a solution of albumin,
soap, or acacia, the liquid fats are finely divided and assume a condition
known as an emulsion. The emulsion with water is transitory, while the
emulsions with soap, acacia, or albumin, are permanent.
The fat ingested continues essentially unaltered until it reaches the in-
testine where it is acted upon by pancreatic lipase (steapsin) the fat-split-
ting enzyme of the pancreatic juice (see page 151), and glycerol 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 absorp-
tion of fat is indicated by the increase of fat in the feces when for any
reason bile does not pass into the intestine. That fat is not absorbed
unsplit in the form of an emulsion has recently been redemonstrated by
Whitehead * in a histological study of the absorption in the cat's intestine of
fat stained with Sudan III. Whitehead considers that fat was not
absorbed unsplit because no dye was jound in the lacteals. Mendel"
has pointed out that Sudan III is soluble in fatty acids as well as fats,
and therefore its presence in the lacteals furnishes no evidence " for or
against the possibility of the absorption of fats prior to their digestion."
The failure to find Sudan III in the lacteals may have been due to the
* Whitehead: American Journal of Physiology, 24, 294, 1909.
* Mendel: Ibid., p. 493,
142 PHYSIOLOGICAL CHEMISTRY.
fact that in postmortem examinations these vessels are often found
collapsed and empty.
The fat distributed throughout the animal body is formed partly
from the ingested fat and partly from carbohydrates and the "carbon
mioety" of protein material. The formation of adipocere and the
occurrence oi fatty degeneration are sometimes given as proofs of the
formation of fat from protein. This is questioned by many investigators.
Rather more satisfactory and direct proof of the formation of fat from
protein material has been obtained by Hofmann in experimentation
Fig. 37. — Mutton Fat. {Long.)
with fly-maggots. The normal content of fat in a number of maggots
was determined and later the fat content of others which haddeveloped in
blood (84 per cent of the solid matter of blood plasma is protein material)
was determined. The fat content was found to have increased 700 to
1 100 per cent as a result of the diet of blood proteins. The celebrated
experiments of Pettenkofer and Voit, however, have furnished what is,
perhaps, the most substantial positive evidence of the formation of
fat from protein. These investigators fed dogs large amounts of lean
meat, daily, and through subsequent urinary and fecal examinations
were enabled to account for only part of the ingested carbon, although
obtaining a satisfactory nitrogen balance. The discrepancy in the carbon
balance was explained upon the theory that the protein of the ingested
meat had been split into a nitrogenous and a non-nitrogenous portion in
the organism, and that the non-nitrogenous portion, the so-called "carbon
moiety" of the protein, had been subsequently transformed into fat and
deposited as such in the tissues of the organism. Some investigators are
not inclined to accept these data regarding the formation of fat from
protein as conclusive.
FATS. 143
Later evidence in favor of the formation of fat from protein has
been furnished by the experiments of Weinland. This investigator
worked with the larvae of Calliphora,^ these larvae being rubbed up
in a mortar^ with Witte's peptone and water to form a homogeneous
mixture. After placing these mixtures at 38° C. for 24 hours the fat
content was found to have increased, as much as 140 per cent in some
instances. The active agency in this transformation of fat is the larval
tissue since the? tissues of both the dead and li\'ing larvae possess the
property. Data are given from control tests which show that the action
of bacteria in this transformation of protein was excluded.
Experiments on Fats.
1. Solubility. — Test the solubility of olive oil in each of the ordi-
nary solvents (see page 27) and in cold alcohol, hot alcohol, chloroform,
ether, and carbon tetrachloride.
2. Formation of a Transparent Spot on Paper. — Place a drop
of olive oil upon a piece of ordinary writing paper. Note the trans-
parent appearance of the paper at the point of contract with the fat.
3. Reaction. — Try the reaction oi fresh olive oil to litmus, congo red
and phenolphthalein. Repeat the test with rancid olive oil. WTiat 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 dry potassium bisulphate, KHSO^, and rub up thoroughly. Trans-
fer to a dry test-tube and cautiously heat. Note the irritating odor of
acrolein. The glycerol of the fat has been dehydrolyzed and acrylic
aldehyde or acrolein has been produced. This is the reaction which
takes place:
CH^OH CHO
I I
CHOH -^ CH+2H2O.
I II
CH^OH CH2
Glycerol. Acrolein.
5. Emulsification. — (a) Shake up a drop of neutral^ olive oil with
a little water in a test-tube. The fat becomes finely divided, forming
an emulsion. This is not a permanent emulsion since the fat separates
and rises to the top upon standing.
' The ordinary "blow-fly."
^ Intact larva; were used in some experiments.
' Neutral olive oil may be prepared by shaking ordinary olive oil with a lo per cent solution
of sodium carbonate. This mixture should then be extracted with ether and the ether
removed by evaporation. The residue is neutral olive oil.
144
PHYSIOLOGICAL CHEMISTRY.
(b) To 5 c.c. of water in a test-tube add 2 or 3 drops of 0.5 per cent
NaoCOg. Introduce into this faintly alkaline solution a drop of neutral
olive oil and shake. The emulsion while not permanent is not so transi-
tory as in the case of water free from sodium carbonate.
(c) Repeat {b) using rancid olive oil. What sort .of an emulsion
do you get and why ?
{d) Shake a drop of neutral olive oil with dilute albumin solution.
\^^lat is the nature of this emulsion ? Examine it under the microscope.
6. Fat Crystals. — Dissolve a small piece of lard in ether in a test-
tube, add an equal volume of alcohol and allow the alcohol-ether mixture
Fig. t,S. — Pork Fat.
to evaporate spontaneously. Examine the crystals under the microscope
and compare them with those reproduced in Figs. 36, 37 and 38, on pages
39, 142 and 144.
7. Saponification of Bayberry Tallow.^ — ^Fill a large casserole
two-thirds full of water rendered strongly alkaline with solid potassium
hydroxide (a stick one inch in length). Add about 10 grams of bay-
berry tallow and boil, keeping the volume constant by adding water as
needed. When saponification is complete" remove 25 c.c. of the soap
solution for use in Experiment 8 and add concentrated hydrochloric
acid slowly to the remainder until no further precipitate is produced.^
Cool the solution and the precipitate of free fatty acid will rise to the sur-
face and form a cake. In this instance the fatty acid is principally pal-
' Baybern- tallow is flerivcd from the fatty covering of the berries of the wax myrtle. It
s therefore frequently (ailed "myrtle wax" or " Ijayberry wax,"
^ Place 2 or 3 drops in a test-tube full of water. If saponification is complete the prod-
ucts will remain in solution and no oil will separate.
^ Under some conditions a purer product is obtained if the soap solution is cooled before
precipitating the fatty acid.
FATS.
145
mflic acid. Remove the cake, break it iato small pieces, wash it with
water by decantation and transfer to a small beaker by means of 95 per
cent alcohol. Heat on a water-bath until the palmitic acid is dissolved,
then filter through a dry filter paper and allow the filtrate to cool slowly in
order to obtain satisfactory crystals. Write the reactions which have
taken place in this experiment.
When the palmitic acid has completely crystallized filter off the
alcohol, dry the crystals between the filter papers and try the tests
given in E.xperiment 9, below.
Fig. 39 — Palmitic Acid.
8. Salting-out Experiments. — To 25 c.c. of soap solution, pre-
pared as described above, add solid sodium chloride to the point of
saturation, with continual stirring. A menstruum is thus formed in
which the soap is insoluble. This salting-out process is entirely anal-
ogous to the salting-out of proteins (see page 106).
9. Palmitic Acid. — (a) Examine the crystals under the microscope
and compare them with those shown in Fig. 39, above.
(b) Solubility. — Try the solubility of palmitic acid in the same sol-
vents as used on fats (see page 143).
(c) Melting-point. — Determine the melting-point of palmitic acid
by one of the methods given on page 146.
(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 writing paper.
How does this compare with the action of a fat under similar circum-
stances ?
{e) Acrolein Test. — Apply the test as given under 4, page 143. Explain
the result.
146
PHYSIOLOGICAL CHEMISTRY.
10. Saponification of Lard. — To 25 grams of lard in a flask a'dd
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 solution from the flask to an evaporating dish con-
taining about 100 c.c. of water and heat on a water-bath until all the
alcohol has been driven off. Precipitate
the fatty acid with hydrochloric acid and
cool the solution. Remove the fatty acid
which rises to the surface, neutralize the
solution with sodium carbonate and evap-
orate to dryness. Extract the residue with
alcohol, remove the alcohol by evaporation
upon a water-bath and on the residue of
glycerol thus obtained make the tests as
given below.
II. Glycerol, (a) Taste. — What is the
taste of glycerol ?
(h) Solubility. — Try the solubility of
glycerol in water, alcohol and ether.
(c) Acrolein Test. — Repeat the test as
given under 4, page 143.
(rf) Borax Fusion Test. — Fuse a little
glycerol on a platinum wire with some
powdered borax and note the character
istic green flame. This color is due to the
glycerol ester of boric acid.
(e) Fehling's Test. — How does this re-
sult compare with the results on the sugars ?
(/) Solution of Cu (OH),. —Form a httle
cupric hydroxide by mixing copper sulphate
and potassium hydroxide. Add a httle
glycerol to this suspended precipitate and note what occurs.
12. Melting-Point of fat. First Method.— Insert one of the melt-
ing-point tubes, furnished by the instructor, into the hquid 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 fas-
ten 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 ther-
mometer (Fig. 40, above). 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.
Fig. 40. — Meltixg-Point
Apparatus.
FATS. 147
Immerse the bulb of the thermometer and the attached tube in such a
way that the bulb is al^out 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 surrounding 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 determine the congealing-point remove the flame and
note the temperature at which the fat begins to solidify. Record the
melting- 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 coverglass 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 VIII.
PANCREATIC DIGESTION.
As soon as the food mixture leaves the stomach it comes into inti-
mate 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 previously 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.
Therefore, any factor which produces an increased flow of gastric juice
such, for example, as water ^ will cause a stimulation of the pancreatic
secretion. The secretion of pancreatic juice is probably not due to a nerv-
ous reflex as was believed by Pawlow but rather, as Bayliss and Starling
have shown, is dependent upon the presence, in the epithelial cells of
the duodenum and jejunum 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 absorbed by the blood, passes to the
pancreas and stimulates the pancreatic cells, causing a flow of pancreatic
juice. The quantity of juice secreted under these conditions is propor-
tional to the amount of secretin present. The activity of secretin solutions
is not diminished by boihng, hence the body does not react like an
enzyme. Further study of the body may show it to be a definite chem-
ical individual of relatively low molecular weight. It has not been
possible thus far to obtain secretin from any tissues except the mucous
membrane of the duodenum and jejunum.
This secretin mentioned above belongs to the class of substances
called hormones or chemical messengers. These hormones play a very
important part in the coordination of the activities of certain functions
and glands. Other important hormones are those elaborated by the
thyroids, the adrenals, the pituitary body (hypophysis), the embryo and
the reproductive glands. It is claimed that all active organs of the body
produce hormones.
The juice as obtained from a permanent fistula differs greatly in
' .See chapter on Gastric Digestion.
148
PANCREATIC DIGESTION. 1 49
its properties from the juice as obtained from a temporary fistula, and
neither form of fluid possesses the properties of the normal fluid. Pan-
creatic juice collected by Glaessner from a natural fistula has been found
to be a colorless, clear, strongly alkaline fluid which foams readily. It is
further characterized by containing albumin, globulin, proteose, and pep-
tone; nucleoprotein is also present in traces.^ The average daily secre-
tion of pancreatic juice is 650 c.c. and its specific gravity is 1.008. The
fluid contains 1.3 per cent of solid matter and the freezing-point is — 0.47°
C. The normal pancreatic secretion contains at least four distinct
enzymes. They arc trypsin, a proteolytic enzyme; pancreatic amylase
(amylopsin), an amylolytic enzyme; pancreatic lipase (steapsin), a fat-
splitting enzyme; and pancreatic rennin, a milk-coagulating enzyme.
Lactase, the lactose-splitting enzyme, is also present at certain times.
The most important of the four enzymes of the pancreatic juice is
the proteolytic enzyme trypsin. This enzyme resembles pepsin in so
far as each has the power of breaking down protein material, but the
trypsin has much greater digestive power and is able to cause a more
complete decomposition of the complex protein molecule. In the
process of normal digestion the protein constituents of the diet are for
the most part transformed into proteoses (albumoses) and peptones
before coming in contact with the enzyme trypsin. This is not abso-
lutely essential however, since trypsin possesses digestive activity suffi-
cient to transform unaltered native proteins and to produce from their
complex molecules comparatively simple fragments. Among the prod-
ucts of tryptic digestion are proteoses, peptones, peptides, leucine, tyrosine,
aspartic acid, glutamic acid, alanine, phenylalanine, glycocoll, cystine,
serine, valine, proline, oxyproline, isoleucine, arginine, lysine, histidine, and
tryptophane. (The crystalline forms of many of these products are repro-
duced in Chapter IV.) Trypsin does not occur preformed 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 destroyed by comparatively
weak alkali (2 per cent sodium carbonate) if left in contact for a suflS-
ciently long time. Trypsinogen, on the other hand, is more resistant to
the action of alkalis. In pancreatic digestion the protein does not swell
as is the case in gastric digestion, but becomes more or less "honey-
combed" and it finally disintegrates.
The presence of active pepsin in the contents of the intestine has been
* Glaessner: Zeitschri/t/ur physiologische Chemie, 40, 476, 1904.
150 PHYSIOLOGICAL CHEMISTRY. :
demonstrated very recently by Abderhalden and Meyer. ^ It may
possibly be that pepsin may play a part in the profound intestinal pro-
teolysis which has up to this time been assigned to trypsin and erepsin
(see chapter on Gastric Digestion).
The pancreatic juice which is collected by means of a fistula pos-
sesses practically no power to digest protein matter. A body called
enter okinase occurs in the intestinal juice and has the power of converting
trypsinogen into trypsin. This process is known as the "activation" of
trypsinogen and through it a juice which is incapable of digesting protein
may be made active. Enterokinase is not always present in the intestinal
juice since it is secreted only after the pancreatic juice reaches the intes-
tine. 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
is capable of activating only a definite quantity of trypsinogen. It is,
however, generally classified as an enzyme. Enterokinase 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. Mendel and Rettger^ and others have demon-
strated that activation of trypsinogen into trypsin may be brought about
in the gland as well as in the intestine of the living organism. The manner
of the activation in the gland and the nature of the body causing it are
unknown at present. Prym^ denies that such an activation occurs.
Delezenne claims that trypsinogen may be activated by soluble
calctMrn salts. He reports experiments which indicate that proteolytic-
ally inactive pancreatic juice, obtained directly from the duct, when
treated with salts of this character, assumes the property of digesting
protein material. This process by which the trypsinogen is activated
through the instrumentality of calcium salts is very rapid and is desig-
nated by Delezenne as an "explosion." The recent suggestion of
Mays that there may possibly be several precursors of trypsin one of
which is activated by enterokinase and the others by other agents, is
of interest in this connection.
Pancreatic amylase (amylopsin), the second of the pancreatic en-
zymes, is an amylolytic enzyme which possesses somewhat greater diges-
tive power than the salivary amylase (ptyalin) of the saliva. As its
name implies, its activity is confined to the starches, and the products
of its amylolytic action are dtxtrins and sugars. The sugars are prin-
cipally iso-maltose and maltose and these by the further action of an
inverting enzyme are partly transformed into dextrose.
.* Abderhalden and Meyer: Zeit. physiol. Chem., 74, 67, 191 1.
* Mendel and Rettger: American Journal of Physiology, 7.
'Prym: Pfliiger's Archiv., 104 and 107.
PANCREATIC DIGESTION. I5I
It is possible that the saliva as a digestive fluid is not absolutely
essential. The salivary amylase (ptyalin) is destroyed by the hydro-
chloric 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 by the active pancreatic amy-
lase. 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.
The pronounced influence of electrolytes upon the action of pancreatic
amylase and other amylases has been demonstrated many times. ^ In
this connection Bierry^ has very recently shown that the removal of
electrolytes from pancreatic juice by dialysis yields a juice which possesses
no power to split starch. He further claims that the CI or Br ion is ''abso-
lutely essential to the activity of animal amylases." It is generally rec-
ognized that the presence of the CI ion facilitates amylolytic action.'
It has been claimed that pancreatic amylase has a slight digestive
action upon unboiled starch.
The extent to which amylase is present in the feces has been taken as
the index of pancreatic activity.
The third enzyme of the pancreatic juice is called pancreatic lipase
(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 glycerol. A
typical reaction would be as follows:
C3H,(0-C,,H3,CO)3 + 3H30-3(C,,H3,COOH) + C3H,(OH)3.
Palmitin. Palmitic acid. Glycerol.
Recent researches make it probable that fats undergo saponifica-
tion to a certain extent prior to their absorption. The fatty acids formed,
in part unite with the alkalis of the pancreatic juice and intestinal secre-
tion to form soluble soaps; in part they are doubtless absorbed dissolved
in the bile. Some obsen'ers believe that the fats may also be absorbed
in emulsion — a condition promoted by the presence of the soluble soaps.
After absorption the fatty acids are re-synthesized to form neutral fats
with glycerol.
It has been demonstrated that lipase acts best in dilution.* This
fact is of importance when considered in connection ^\-ith the fact that in-
gested fat is better utilized in the human organism when large volumes
of water (looo c.c.) are taken with meals. ^
' For the literature see Kendall and Sherman: Jour. Am. ChemSuc, 32, 10S7, 1910.
- Bierr)-: Biochem. Zeit., 40, 357, 1912.
^Wohlgemuth: Biochem Zeit., 9, 10, 1908; and Kendall and Sherman: Jow. Am. Cliem.
Sac., 32, 10S7, 1910.
* Bradley: Jour. Biol. Chem., 8, 251, 1910.
* Mattill and Hawk: Jour. Am. Chem.Soc, ^2,^ 1978, 1911.
152 PHYSIOLOGICAL CHEMISTRY.
Pancreatic lipase is very unstable and is easily rendered inert by the
action of acid. For this reason it is not possible to prepare an extract
ha\dng 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 pancreatic rennin.
It is a milk-coagulating enzyme whose action is very similar to that
of the enzyme gastric rennin found in the gastric juice. It is supposed
to show its greatest activity at a temperature varying from 60° to 65° C.
The enzymes of the intestinal juice {succus entericus) are of great
importance to the animal organism. These enzymes include erepsin,
sucrase, maltase, lactase, and enterokinase. According to Boldyreff lipase
is also present.
Erepsin is a proteolytic enzyme which has the property of acting
upon the proteoses, peptones and peptides which are formed through the
action of trypsin and further splitting them into amino acids. Erepsin
has no power of digesting any native proteins except caseinogen, histones,
and protamines. It possesses its greatest activity in an alkaline solution
although it is slightly active in acid solution. An extract of the intestinal
erepsin may be prepared by treating the finely divided intestine of a
cat, dog, or pig with toluol- or chloroform-water and permitting the
mixture to stand with occasional shaking for 24-72 hours. ^ Enzymes
similar to erepsin occur in various tissues of the organism.
In cases of gastric cancer a peptide-splitting enzyme is present in
the stomach contents. The glycyl-tryptophane test is used for its
detection (see chapters on Enzymes and Gastric Digestion).
The three invertases sucrase, maltase, and lactase are also important
enzymes of the intestinal mucosa. The sucrase acts upon sucrose
and inverts it with the formation of ifivert sugar (dextrose and laevulose).
Some investigators claim that sucrase is also present in saliva and gastric
juice. It probably does not exist normally in either of these digestive
juices, however, and if found owes its presence to the excretory processes
of certain bacteria. Sucrases may also be obtained from several vegetable
sources. For investigational purposes it is ordinarily obtained from
yeast (see p. 13 j. It exhibits its greatest activity in the presence of a
slight acidity but if the acidity be increased to any extent the reaction is
inhibited.
Lactase is an enzyme which inverts lactose with the consequent
formation of dextrose and galactose. Its action is entirely analogous,
in type, to that of sucrase. It has apparently been proven that lactase
occurs in the intestinal mucosa of the young of all animals which suckle
* See page 15.
PANCREATIC DIGESTION. 1 53
their offspring.' It may also occur in the intestinal mucosa of certain
adult animals if such animals be maintained u])on a ration containing
more or less lactose. Fischer and Armstrong have demonstrated the
reversible action" of lactase.
For discussions of maltase and cnterokinase sec pages 62 and 150
respectively.
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 tv^^enty-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 potassium hydroxide solution and then exactly neutralize it with
0.5 per cent sodium carbonate.
Products of Tryptic 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. Introduce
equal volumes of the pancreatic extract prepared as above and 0.5
per cent sodium carbonate, 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 undis-
solved residue through cheese cloth, nearly neutralize the solution with
dilute hydrochloric acid and then exactly neutralize it with 0.2 per
cent hydrochloric acid. A precipitate at this point would indicate
alkali metaprotein (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 120.
Place about 5 c.c. of the three-fourth portion in a test-tube and
* Mendel and Mitchell: American Journal of Physiology, 20, 81, 1907.
- See p. 8.
'For other methods of preparation see Karl Mays: Zeitschrift fUr physiologische Chemie,
38, 428, 1903.
154 PHYSIOLOGICAL CHEMISTRY.
add about i c.c. of bromine water. A violet coloration indicates the
presence of tryptophane (see page 82. Also see glycyl-tryptophane
reaction in chapter on Enzymes.) Concentrate^ the remainder of the
three-fourth portion to a thin syrup and make the separation of leucine
and tyrosine according to the directions given on page 82.
GENERAL EXPERIMENTS ON PANCREATIC DIGESTION.
Experiments on Trypsin.
. I. The Most Favorable Reaction for Tryptic Digestion. —
Prepare seven tubes as follows:
{a) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of water.
{b) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of i per cent
sodium carbonate.
(c) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.5 per cent
sodium carbonate.
{d) 2-3 c.c. of neutrfil pancreatic extract + 2-3 c.c. of 0.2 per cent
hydrochloric acid.
{e) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.2 per cent
combined hydrochloric acid.
(/) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.4 per cent
boric acid.
is) 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 indi-
cations 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 fol-
lowing series of experiments under tryptic digestion use the neutral
extract plus an equal volume of 0.5 per cent sodium carbonate.) In
each of four tubes place 5 c.c. of alkaline pancreatic extract. Immerse
one tube in cold water from the faucet, keep a second at room tempera-
ture and place a third in the incubator or 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 pancre-
' If the solution is alkaline in reaction, while it is being concentrated, the amino acids
will be broken down and ammonia will be liberated.
PANCREATIC DIGESTION. 1 55
atic extract and i volume of one of the chemicals listed in Experiment
1 8 under Salivary Digestion, page 66.
Introduce a small piece of fibrin into each of the tubes and keep
them at 40° C. for one-half hour. Shake the tubes frequently. In
which tubes do we get the least digestion ?
4. Influence of Bile. — Prepare live tubes as follows:
(a) Five c.c. of pancreatic extract + 1/2-1 c.c. of bile.
(b) Five c.c. of pancreatic extract + 1-2 c.c. of bile.
(c) Five c.c. of pancreatic extract + 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 progress of digestion.
Does the presence of bile retard tryptic digestion? How do these
results agree with those obtained under gastric digestion ?
Experiments on Pancreatic Amylase.
1. The Most Favorable Reaction. — Prepare seven tubes as follows:
(a) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of water.
(b) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of I per cent sodium carbonate.
(c) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of 0.5 per cent sodium carbonate.
(d) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of 0.2 per cent hydrochloric acid.
(e) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of 0.2 per cent combined hydrochloric acid.
(/) One c.c. oi neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of 0.4 per cent boric acid.
(g) One c.c. of neutral pancreatic extract + i c.c. of starch paste +
2 c.c. of 0.4 per cent acetic acid.
Shake each tube thoroughly and place them in the incubator or 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 bv
Fehling's test. Where do you find the most satisfactory digestion ?
How do the results compare with those obtained from the similar series
under Trypsin, page 154?
2. The Most Favorable Temperature. — (For this and the fol-
lowing series of experiments upon pancreatic amylase use the neutral
extract plus an equal volume of 0.5 per cent sodium carbonate.) In
156 PHYSIOLOGICAL CHEMISTRY.
each of four tubes place 2-3 c.c. of alkaline pancreatic extract. Immerse
one tube in cold water from the faucet, keep a second at room tempera-
ture, 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 bv 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 experiments under Trypsin (see page 154) ?
3. Influence of Metallic Salts, etc. — Prepare a series of tubes
and into each place 3 volumes of water, 3 volumes of alkaline pancreatic
extract, i volume of one of the chemicals listed in Experiment 18 under
Salivary Digestion, page 66, and 3 volumes of starch paste. Be sure
to introduce the starch paste into the tube last. Why? Shake the
tubes well and place them in the incubator or 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 -f 1/2-1
c.c. of bile.
(b) 2-3 c.c. of pancreatic extract -h 2-3 c.c. of starch paste -j- 1-2
c.c. of bile.
(c) 2-3 c.c. of pancreatic extract + 2-3 c.c. of starch paste 4- 2-3
c.c. of bile.
(d) 2-3 c.c. of pancreatic extract -|- 2-3 c.c. of starch paste -f 5 c.c.
of bile.
(e) 2-3 c.c. of pancreatic extract -|- 2-3 c.c. of starch paste.
Shake the tubes thoroughly and place them in the incubator or 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
pancreatic amylase ?
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 in the incubator
or water-bath at 40° C. At the end of a half-hour filter and test separate
portions of the filtrate by the iodine and Fehling tests. What do you con-
clude regarding the action of pancreatic amylase upon dry starch?
Compare this result with that obtained in the similar experiment under
Salivary Digestion (page 65).
PANCREATIC DIGESTION. 1 57
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 in the incubator or
water-bath at 40° C. After one-half hour test the solution by Fehling's
test/ Is any reducing substance present? What do you conclude
regarding the digestion of inulin by pancreatic amylase?
Experiments on Pancreatic Lipase.
1. "Litmus-milk" Test. — Into each of two test-tubes introduce
10 c.c. of milk and a small amount of litmus powder. To^ the con-
tents of one tube add 3 c.c. of neutral pancreatic extract and to the con-
tents of the other tube add 3 c.c. of water or of boiled neutral pancre-
atic 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 introduce
4 c.c. of water, 2 c.c. of ethyl butyrate, CgH^COO.CjH^, 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 of boiled neutral pancreatic 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 four test-tubes as follows :
(a) Five c.c. of milk -t- 10 drops of neutral pancreatic extract.
(b) Five c.c. of milk -|- 20 drops of neutral pancreatic extract.
(c) Five c.c. of milk + 10 drops of a/)^a//;;f pancreatic extract.
{d) Five c.c. of milk + 20 drops of a/^a/m^ pancreatic extract.
Place the tubes at 6o°-65° C. for a half hour ivithout shaking. Note
the formation of a clot.^ How does the action of pancreatic rennin
compare with the action of the gastric rennin ?
' 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 52).
^ This reaction will not always succeed, owing to conditions which are not well
understood.
CHAPTER IX.
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 intestine. 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 charac-
teristics of the secretion depending upon the nature of the food ingested.
Fats, the extractives of meat and the protein end-products of gastric
digestion (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 protein diet is supposed to increase the
amount of bile secreted, whereas a carbohydrate diet would cause a
much less decided increase and might even tend to decrease the amount.
It has been demonstrated by Bayliss and Starling that the secretion of
bile is under the control of the same mechanism that regulates the flow
of pancreatic juice (see p. 148). In other words, the hydrochloric
acid of the chyme, as it enters the duodenum transforms prosecretin
into secretin and this in turn enters the circulation, is carried to the
liver, and stimulates the bile-forming mechanism to increased activity.
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, cholesterol, and the decomposition products of
haemoglobin 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 ropy, 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 from the living organism ordi-
narily has a green or golden-yellow color. Postmortem bile is variable
in color. It is very difficult to determine accurately the amount of
normal bile secreted during any given period. For an adult man it
has been variously estimated at from 500 c.c. to iioo c.c. for twenty-
' It does not contain any/^ee hydroxyl ions, however.
158
BILE. J 59
four hours. The specific gravity of the bile varies between i.oio 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 relatively low
content of solid matter. Such bile would hav^e a specific gravity of
approximately i.oio. After it reaches the gall-bladder, however, it
becomes mixed with mucous material from the walls of the gall-bladder,
and this process coupled 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-bladder, ordinarily possesses a higher spe-
cific gravity than that of the freshly secreted fluid. The specific grav-
vity under these conditions may run as high a 1.040.
The principal constituents of the bile are the salts of the bile acids,
bile pigments, neutral fats, lecithin, phosphatides, and cholesterol, besides
the salts of iran, copper, calcium, and magnesium. Zinc has also fre-
quently 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 predomi-
nates, while taurocholic acid is the more abundant in the bile of car-
nivora. The bile acids are conjugate amino-acids, the glycocholic
acid yielding glycocoll,
CH^NH,
COOH,
and cholic acid upon decomposition, whereas taurocholic acid gives
rise to taurine,
CH,HN,
CH.-SO^-OH,
and cholic acid under like conditions. Glycocholic acid contains some
nitrogen but no sulphur, whereas taurocholic acid contains both these
elements. The sulphur of the taurocholic acid is present in the taurine
(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 constitution
depending upon the nature of the cholic acid which enters into the com-
bination. The bile acids are present in the bile as salts of one of the
alkalis, generally sodium. The sodium glycocholate and sodium tau-
rocholate 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. 41, p. 160). The salts of the bile acids are dextro-rotatory.
l6o PHYSIOLOGICAL CHEMISTRY.
Among other properties these salts have the power of holding the choles-
terol and lecithin of the bile in solution.
Hammarsten has demonstrated a third group of bile acids in the
bile of the shark. This same group very probably occurs in certain
other animals also. These acids are very rich in sulphur and resemble
ethereal sulphuric acids inasmuch as upon treatment with boiling hydro-
chloric acid they yield sulphuric acid.
Fig 41. — Bile Salts.
The bile pigments are important and interesting biliary constit-
uents. The following have been isolated: bilirubin, biliverdin, bili-
fiiscin, biliprasin, bilihumin, bilicyanin, choleprasin, and choletelin. Of
these, bilirubin and biliverdin are the most important and predominate in
normal bile. The colors possessed by the various varieties of normal
bile are due almost 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
calculi or in altered bile obtained as 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 com-
position 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 isolated in the form of a reddish-
yellow powder or may be obtained in part, in the form of reddish-yellow
rhombic plates (Fig. 42, p. 161) upon the spontaneous evaporation of
its chloroform solution. The crystalline form of bilirubin is practically
the same as that of haematoidin. It is easily soluble in chloroform,
BILE, l6l
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 \nth the formation
of the green biliverdin. Bilirubin occurs in animal fluids as soluble
bilirubin-alkali._
I
^ I
Fig. 42. — Bilirubin (H^vl^toidix). (Ogden.)
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 (Absorp-
tion 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, chloro-
form, and ether. Biliverdin is formed from bihrubin upon oxidation. It
is an amorphous substance, and in this differs from bilirubin which may
be at least partly crystallized under proper conditions. Biliverdin may
be obtained in the form of a green powder. In common with bilirubin,
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 alkaline solution is green
with no appreciable flourescence 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 be-
tween 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
1 62 PHYSIOLOGICAL CHEMISTRY.
be divided into three classes, cholesterol calculi, pigment calculi, and
calculi made up almost entirely of inorganic material. This last class
of calculus is formed principally of the carbonate and phosphate of calciurfi
and is rarely found in man although quite common to cattle. The pigmii^iit
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 cholesterol calculus is the one found most fre-
quently in man. These may be formed almost entirely of cholesterol,
in which event the color of the calculus is very light, or they may contain
more or less pigment and inorganic matter mixed with the cholesterol,
which tends to give us calculi of various colors.
For discussion of cholesterol see page 270.
Experiments on Bile.
1. Reaction. — Test the reaction of fresh ox bile to litmus, phenol-
phthalein and congo red.
2. Nucleoprotein. — Acidify a small amount of bile with dilute
acetic acid. A precipitate of nucleoprotein forms. Bile acids will also
precipitate here under proper conditions of acidity.
3. Inorganic Constituents. — Test for chlorides, sulphates, and
phosphates (see page 64).
4. Tests for Bile Pigments, {a) Gmelin'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
bile 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) Nakayama's Reaction. — -To 5 c.c. of diluted bile in a test-tube
add an equal volume of a 10 per cent solution of barium chloride, centrifu-
gate the mixture, pour off the supernatant fluid, and heat the precipitate
with 2 c.c. of Nakayama's reagent.^ In the presence of bile pigments
the solution assumes a blue or green color.
{d) Huppert^s Reaction. — Thoroughly shake equal volumes of undiluted
bile and milk of lime in a test-tube. The pigments unite with the calcium
and are precipitated. Filter off the precipitate, wash it with water, and
' Prepared by combining 99 c.c. of alcohol and i c.c. of fuming hydrochloric acid con-
taining 4 grams of ferric chloride per liter.
BILE. 163
transfer to a small beaker. Add alcohol acidified slightly with hydro-
chloric acid and warm upon a water-bath until the soluticm becomes
colored an emerald green.
In examining urine for bile pigments, according to Steensma, this
procedure may give negative results even in the presence of the pigments,
owing to the fact that the acid-alcohol is not a sufficiently strong oxidizing
agent. He therefore suggests the addition of a drop of a 0.5 per cent
solution of sodium nitrite to the acid-alcohol mixture before warming on
the water-bath. Try this modification also.
(e) Hammarsten's Reaction. — To about 5 c.c. of Hammarsten's
reagent^ in a small evaporating dish add a few drops of diluted bile. A
green color is produced. If more of the reagent is now added the play
of colors as observed in Gmelin's test may be obtained.
(/) 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.
(g) Salkowski-S chipper s Reaction. — To 10 c.c. of diluted bile in a
test-tube add 5 drops of a 20 per cent solution of sodium carbonate and
10 drops of a 20 per cent solution of calcium chloride. Filter off the re-
sultant precipitate upon a hardened filter-paper and wash it with water.
Remove the precipitate to a small porcelain dish, add 3 c.c. of an acid-
alcohol mixture- and a few drops of a dilute solution of sodium nitrite and
heat. The production of a green color indicates the presence of bile
pigments.
Qi) Bonanno's Reaction.^ — Place 5-10 c.c. of diluted bile in a small
porcelain evaporating dish and add a few drops of Bonanno's reagent.'*
An emerald-green color will develop.
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 sucrose.
Now run about 2-3 c.c. of concentrated 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 gradually
assumes a reddish color. As the tube becomes warm, it should be cooled
in running water in order that the temperature of the solution may not
rise above 70° C.
' Hammarsten's reagent is made by mixing i volume of 25 per cent nitric acid and 19
volumes of 25 per cent hydrochloric acid and then adding i volume of this acid mixture
to 4 volumes of 95 per cent alcohol.
^ Made by adding 5 c.c. of concentrated hydrochloric acid to 95 c.c. of 96 per cent alcohol.
' // Tommasi, 2, No. 21.
* This reagent may be prepared by dissolving 2 grams of sodium nitrite in 100 c.c. of
concentrated hydrochloric acid.
164 PHYSIOLOGICAL CHEMISTRY.
(b) Mylius's Modification of. Pettenkofer^s Test. — To approximately
5 c.c. of diluted bile in a test-tube add 3 drops of a very dilute (1:1000)
aqueous solution of furfurol,
HC — CH
II II
HC CCHO.
O
Now run about 2-3 c.c. of concentrated sulphuric acid carefully 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) Neukomm's Modification of Pettenkofer^s Test. — To a few drops
of diluted bile in an evaporating dish add a trace of a dilute sucrose
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 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 (1:1,000) 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 of a small pipette add 2-3 drops of con-
centrated sulphuric acid to the foam and note the dark pink coloration
produced.
(e) Guerin's Reaction. — To equal volumes of diluted bile and alcohol
in a test-tube add 5-6 drops of a saturated aqueous solution of furfurol
and 5-6 drops of concentrated sulphuric acid. A blue color indicates
bile acids.
if) Hay's Test. — This test is based upon the principle that bile acids
have the property of reducing the surface tension of fluids in which they
are contained. The test is performed as follows: Cool about 10 c.c. of
diluted bile in a test-tube to 17° C. or lower and sprinkle a Httle finely
pulverized 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. Remove the residue, grind it in
BILE.
i6s
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. 41, page 160. Try one of the tests for bile acids upon some of the
crystals.
7. Analysis of Biliary Calculi. — Grind the calculus in a mortar
with 10 c.c. of ether. Filter.
Filtrate I.
Add an equal volume of 95 per cent
alcohol' to the ether extract, allow the
mixture to evaporate and examine for
cholesterol crj-stals (Fig. 43, p. 166).
(For further tests see Experiment 8,
below.)
Residue I.
(On paper and in mortar.)
Treat with dilute hvdrochloric acid and
filter.
Filtrate II. Residue II.
Test for calcium, phosphates, and (On paper and in mortar.)
iron. Evaporate remainder of filtrate Wash with a little water. Dr\' the filter paper.
to dryness in porcelain crucible and I
ignite. Dissolve residue in dilute |
hydrochloric acid and make alkaline Treat with 5 c.c. chloroform and filter,
with ammonium hydroxide. Blue I
color inilicates copper. . ■ ■
Filtrate III. Residue III.
Bilirubin. (On paper and in mortar.)
(Apply test for j
bile pigments.) |
Treat with 5 c.c. of hot
alcohol I
Biliverdin.
8. Tests for Cholesterol.
{a) Microscopical Examination. — Examine the crystals under the
microscope and compare them with those shown in Fig. 43, p. 166.
(6) Iodine-sulphuric Acid Test. — Place a few crystals of cholesterol
in one of the depressions of a test-tablet and treat with a drop of con-
centrated 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 Liehermann-Burchard Test. — Dissolve a few crystals of
cholesterol in 2 c.c. of chloroform in a dry test-tube. Now add 10
drops of acetic anhydride and 1-3 drops of concentrated sulphuric acid.
The solution becomes red, then blue, and finally bluish-green in color.
' The alcohol is added because of the fact that it is often found that crystallization from
pure ether does not yield typical cholesterol crystals.
i66
PHYSIOLOGICAL CHEMISTRY.
(d) Salkou'ski^s Test. — Dissolve a few crystals of cholesterol in a
little chloroform and add an equal volume of concentrated 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 cholesterol in an evaporating
dish add a few drops of Schiff's reagent.^ Evaporate to dryness over
a low flame and observe the reddish-violet residue which changes to a
bluish-violet.
Fig. 43. — Cholesterol.
9. Preparation of Taurine. — To 300 c.c. of bile in a casserole
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 glycocholic 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 dryness. Dis-
solve the residue in 5 per cent hydrochloric acid and precipitate with
ten volumes of 95 per cent alcohol. Filter off the taurine and recrystallize
it from hot water. (Save the alcoholic filtrate for the preparation of
glycocoll, below.) Make the following tests upon the taurine crystals.
(a) Examine them under the microscope and compare with Fig. 44.
(b) Heat a crystal upon platinum foil. The taurine at first melts,
then turns brown, and finally carbonizes as the temperature is raised.
Note the suffocating odor. What is it ?
* Schiff's reagent consists of a mixture of three xolumes of concentrated sulphuric acid
and one volume of lo per cent ferric chloride.
BILK
iby
(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 liltle dilute sulphuric acid
Fig. 44.. — Taurine.
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 fead sulphide.
C
/S?^^^'-
<^^^>%^
Fig. 45. — Glycocoll.
10. Preparation of Glycocoll. — Concentrate the alcoholic filtrate
from the last experiment (9) until no more alcohol remains. The
glycocoll is present here in the form of an hydrochloride and may be
liberated from this combination by the addition of freshly precipitated
1 68 PHYSIOLOGICAL CHEMISTRY.
lead hydroxide or by lead hydroxide solution. Remove the lead by
hydrogen sulphide. Filter and decolorize the filtrate by animal charcoal.
Filter again, concentrate the filtrate, and set it aside for crystallization.
Glycocoll separates as colorless crystals (Fig. 45.)
II. Synthesis of Hippuric Acid. — To some of the glycocoll pre-
pared in the last experiment or furnished by the instructor, add a
little water, about i 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 hydrochloric acid, add an equal
volume of petroleum ether, and shake thoroughly to remove the benzoic
acid. (Evaporate this solution and note the crystals of benzoic acid.
Compare them with those shown in Fig. 99, page 308.) Decant the
ethereal solution into a porcelain 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. 97, page 300.
The chemistry of the synthesis is represented thus:
CH,NH2 COCl • OC-NH-CH^-COOH.
/\ /\
+ "^ i I +^^^-
COOH \/ \/
Glycocoll. Benzoyl chloride. Hippuric acta.
CHAPTER X.
PUTREFACTION PRODUCTS.
The putrefactive processes in the intestine are the result of the
action of bacteria upon the protein 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 proteins are identical vi^ith those formed
in tryptic digestion, although the decomposition of the protein material
is much more extensive when subjected to putrefaction. Some of the
more important of the putrefaction products are the following: Indole,
skatole, paracresol, phenol, para-oxyphenylpropionic acid, para-oxyphenyl-
acetic acid, volatile fatty acids, hydrogen sulphide, methane, methyl
mercaptan, hydrogen, and carbon dioxide, beside proteoses, peptones,
ammonia, and amino acids. Of these the indole, skatole, phenol, and
paracresol appear in part in the urine as ethereal sulphuric acids,
whereas the oxyacids mentioned pass unchanged into the urine. The
potassium indoxyl sulphate (page 298) content of the urine is a rough
indicator of the extent of the putrefaction within the intestine.
The portion of the indole which is excreted in the urine is first sub-
jected to a series of changes within the organism and is subsequently
eliminated as indican. These changes may be represented thus :
CH /\ C(OH)
+ 0 -^
CH \/\/CH
NH NH
Indole- Indoxyl-
C(OH) /\ CCO-SOgH)
+ H3SO, -^ +H,0
CH \/\/CH
NH NH
Indoxyl. Indoxyl sulphuric acid.
In the presence of potassium salts the indoxyl sulphuric acid is then
transformed into indoxyl potassium sulphate (or indican),
/\_ C(0S03K),
\/\/CH
NH
and eliminated as such in the urine.
169
170 PHYSIOLOGICAL CHEMISTRY.
Indican may be decomposed by treatment with concentrated hydro-
chloric acid (see tests on page 298) into sulphuric acid and indoxyl.
The latter body may then be oxidized to form indigo-blue thus:
/\__C(OH) /\ COOC _/\
2 +2O-- 4-2H20
\ / ^v/ CH -xy \/c=. c\/\/
NH NH NH
Indoxyl. Indigo-blue.
This same reaction may also occur under pathological conditions
within the organism, thus giving rise to the appearance of crystals of
indigo-blue in the urine.
Skatole is hkewise changed within the organism and eliminated in
the form of a chromogenic substance. Skatole is, however, of less impor-
tance as a putrefaction product than indole and ordinarily occurs in
much smaller amount. The tryptophane group of the protein molecule
yields the indole and skatole formed in intestinal putrefaction, but the
reasons for the transformation of the major portion of this tryptophane
into indole and the minor portion into skatole are not well understood.
Indole is more toxic than skatole.
Phenol occurs in fairly large amount in certain abnormal conditions
of the organism, but ordinarily the amount is very small. It is probably
derived from the tyrosine group of the protein molecule. Phenol is
conjugated in the liver to form phenyl potassium sulphate and appears
in the urine in this form (Baumann and Herter). Para-cresol occurs in
the urine as cresyl potassium sulphate.
Regarding the claim of Nencki that methyl mercaptan is formed
as a gas during intestinal putrefaction it is an important fact that Herter^
has been unable to detect the mercaptan in fresh feces. He is, therefore,
not inclined to accept the theory that methyl mercaptan is formed in
ordinary intestinal putrefaction but beheves that it may be formed in
exceptional cases. Hydrogen sulphide is, however, formed in all cases of
intestinal putrefaction.
It has been shown by Kutscher and his associates^ that many acids
and bases formed in putrefaction and which have been considered as
originating alone from bacterial action, may also be formed in certain
phases of metabolism in both the plant and animal kingdom. These
transformation products of amino acids have been termed "aporrhegmas."
The following aporrhegmas may result from putrefaction processes:
* Herter: Bacterial Infections of the Digestive Tract, p. 227.
^ Ackermann and Kutscher; Zeit. physiol. Ckem., 69, 265, 1910.
Ackermann: Ibid., 273.
Engelanrl anrl Kutscher; Ibid., 282.
PUTREFACTION PRODUCTS. I71
Aporrhegma. Amino Acid Source.
Iminazolethylamine 1 Histidine.
Iminazolpropionic acid J
Ornithine 1
Tetramethylendiamine 1- Arginine.
Aminovalerianic acid J
Pentamethylendiamine Lysine.
Amjnobutyric acid Glutamic acid.
^'^"!"^- • 1 Aspartic acid.
Succmic acid J ^
Isovalerianic acid Leucine.
Phenylethylamine 1
Phenylaretic acid [ Phenylalanine.
Phenylpropionic acid J
/.-Oxyphenylacetic acid. 1 Tyrosine.
/'-O.xyphenylpropionic acid J ^
Indole
Skatole. .••■•:, \ Tryptophane.
Indolacetic acid -^^ ^
Indolpropionic acid J
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 conditions the scheme here
submitted may be used profitably in the way of 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 individual
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 protein used.
Sterilize the vessel and contents, inoculate with the colon bacillus, and
keep at 40° C. for two or three weeks. If cultures of the colon bacillus
are not available, add 60 c.c. of a cold saturated solution of sodium
carbonate for every liter of water previously added and inoculate with
some putrescent material (pancreas or feces). ^ Mix the putrefaction
mixture ver\' thoroughly by shaking and insert a cork furnished with
a glass tube to which is attached a wash bottle containing a 3 per cent
solution of mercuric cyanide. ^ This device is for the purpose of collecting
the methyl mercaptan, a gas formed during the process of putrefaction.
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
' Putrefying protein may be prepared by treating lo grams of finely ground lean meat
with 100 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.
- 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 e.xclude moisture from the isatin solution.
172 PHYSIOLOGICAL CHEMISTRY.
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.
PART I.
MANIPULATION OF THE DISTILLATE.
Acidify with hydrochloric acid and extract with ether.
Ether Extract No. i. Residue No. i.
Add an equal volume of water, make Allow the ether to volatilize. Evapo
alkaline with potassium hydroxide, and rate and detect ammonium chloride
shake thoroughly. I crystals (Fig. 46, p. 173).
Ether Extract No. 2. Alkaline Solution No. i.
Evaporate spontaneously. Indole and Acidify with hydrochloric acid, add
skatole remain. Try proper reactions sodium carbonate, and extract with
(see pages 175 and 176). ether.
Ether Extract No. 3. Alkaline Solution No. 2,
Evaporate. Detect phenol and cresol Acidify with hydrochloric acid, and
(paracresol). See p. 177. extract with ether.
Ether Extract No. 4. Final Residue.
Evaporate. Volatile fatty acids re- (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 portion of the distillate. Repeat the process until the
entire distillate has been extracted. Add a small amount of fresh 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. Evaporate until crystal-
lization begins. Examine the crystals under the microscope. Ammonium
chloride predominates. Explain its presence.
Ether Extract No. i. — Add an equal volume of water, render the
mixture alkaline with potassium hydroxide, and shake thoroughly by
PUTREFACTION PRODUCTS.
173
means of a separatory funnel as before. The volatile fatty acids, con-
tained among the putrefaction products, would be dissolved by the alka-
line solution (No. i) whereas any indole or skatole would remain in the
ethereal solution (No. 2).
Alkaline Solution No. i. — 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. E.xtract the whole mixture with ether
in the usual way, using care in the manipulation of the stop cock to
Fig. 46. — .\mmoxium Chloride.
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 solution (No. 2)
as alkali salts.
Ether Extract Xo. 2. — Drive off the major portion of the ether at a
low temperature on a water-bath and allow the residue to evaporate
spontaneously. Indole and skatole should be present here. Prove the
presence of these bodies. For tests for indole and skatole see pp. 175
and 176.
Alkaline Solution Xo. 2. — Make strongly acid w^th hydrochloric
acid and extract with a small amount of ether, using a separator}'
funnel. As carbon dioxide is liberated here, care must be used in the
manipulation of the stop cock of the funnel in relie\-ing the pressure within
the vessel. The volatile fatty acids are dissolved by the ether (Ether
Extract No. 4).
Ether Extract Xo. 3. — Evaporate this ethereal solution on a water-
bath. The oily residue contains phenol and cresol. The cresol is
174 PHYSIOLOGICAL CHEMISTRY.
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 177.
Ether Extract No. 4. — Evaporate on a water-bath. The volatile
fatty acids remain in the residue.
PART II.
MANIPULATION OF THE RESIDUE.
Evaporate, filter, and extract with ether.
Ether Extract. Aqueous Solution.
Evaporate, extract the residue with Evaporate until crystals begin to
warm water, and filter. form. Stand in a cold place until
crystallization is complete. Filter,
Crystalline Deposit. Filtrate No. i.
Consists of a mixture of Contains proteose^ peptone,
leucine and tyrosine crystals aromatic acids, and tryptophane.
(Figs. 24, 27 and 109, pages
81, 85 and 367.)
Filtrate No. 2. Residue.
Contains oxyacids and Contains non-volatile
skatole-carbonic acid. 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 satisfactory extraction
the mixture should be shaken very thoroughly. Separate the ethereal
solution from the ac{ueous portion and treat them according to the
directions given on p. 172.
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 crystallization
begins. Stand the solution in a cold place until no more crystals form.
This crystalline mass consists of impure leucine and tyrosine. Filter
off the crystals.
Crystalline Deposit. — Examine the crystals under the microscope
and compare them with those reproduced in Figs. 24, 27, and 109, pages
PUTREFACTION PRODUCTS. 1 75
8i, 85 and 367. Do the forms of the crystals of leucine and tyrosine
resemble those previously examined ? Make a separation of the leucine
and tyrosine and apply typical tests according to directions given on
pages 90 and 91.
Filtrate No. i. — Make a test for tryptophane with bromine water
(see page 153), and also with the Hopkins-Cole reagent (see page 98),
Use the remainder of the liltratc for the separation of proteoses and pep-
tones. Make The separation according to the directions given on
page 120.
Filtrate No. 2. — This solution contains para-oxyphenylacetic acid,
para-oxyphenylpropionic acid and skatole-carbonic acid. Prove the
presence of these bodies by appropriate tests. Tests for oxyacids and
skatole-carbonic acid are given on page 177.
TESTS FOR VARIOUS PUTREFACTION PRODUCTS.
Tests for Indole.
I. Herter's ;?-Naphthaquinone Reaction. — (a) To a dilute aque-
ous solution of indole (1:500,000) add one drop of a 2 per cent solution
of /3-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 fa-
cilitates the development of the color reaction.
One part of indole in one million parts of water may be detected by
means of this test if carefully performed.
{h) If the alkali be added to a more concentrated indole solution
before the introduction of the naphthaquinone the course of the re-
action is different, particularly if the indole solution is somewhat more
concentrated than that mentioned above and if heat is used. Under
these conditions the blue indole compound ultimately forms as fine
acicular crystals which rise to the surface.
If we do not wait for the production of the crystalline body but as
soon as the blue color forms, shake the aqueous solution with chloro-
form, the blue color disappears from the solution and the chloroform
assumes a pinkish-red hue. This is a distinguishing feature of the indole
reaction and facilitates the differentiation of indole from other bodies
which yield a similar blue color. A very satisfactory method for the
quantitative determination of indole is based upon the principle under-
lying this test.
176 PHYSIOLOGICAL CHEMISTRY.
2. Konto's Reaction. — Distil the solution to be tested until only
one-third of the original solution remains. Make the distillate alkaline
with sodium hydroxide and distil again in order to separate the indole from
the phenol, the latter remaining in the residue. Inasmuch as this second
distillate generally contains a large amount of ammonia it should be
acidified with dilute sulphuric acid and again distilled. To i c.c. of this
ammonia-free distillate in a test-tube add 3 drops of a 40 per cent solution
of formaldehyde and i c.c. of concentrated sulphuric acid. Now agitate
the mixture and note the appearance of a violet red color if a trace
of indole is present. The test is said to serve for the detection of indole
when present in a dilution of i : 700,000.
Skatole gives a yellow or brown color under the above conditions.
3. 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 concentrated sulphuric
acid down the side of the tube so that it forms a layer at the bottom.
Note the purple color. Neutralize with potassium hydroxide and
observe the production of a bluish-green color.
\^i 4. 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 nitro-
prusside, Na2Fe(CN)5NO-f2H20. Render alkaline with potassium
hydroxide and note the production of a violet color. If the solution is
now acidified with glacial acetic acid the violet is transformed into a
blue.
5. Pine "Wood Test. — Moisten a pine splinter with concentrated
hydrochloric acid and insert it into the residue. The wood assumes
a cherry-red color.
6. Nitroso-indole Nitrate Test. — Acidify some of the residue
with nitric acid, add a few drops of a potassium nitrite solution and
note the production of a red precipitate of nitroso-indole nitrate. If
the residue contains but little indole simply a red coloration will result.
Compare this result with the result of the similar test on skatole.
Tests for Skatole.
I. Herter's Para-dimethylaminobenzaldehyde Reaction.^ — To
5 c.c. of the distillate or aqueous solution under examination add
I c.c. of an acid solution of para-dimethylaminobenzaldehyde^ and
heat the mixture to boiling. A purplish-blue coloration is produced^
' Herter: Bacterial hifeclions of the Digestive Tract, 1907, p. 141.
^ Made by dissolving 5 grams of para-dimethylaminobenzaldehyde in 100 c.c. of 10
per cent sulphuric acid.
* If the color does not appear add more of the aldehyde solution.
PUTREFACTION PRODUCTS. 177
which may be intensified through the addition of a few drops of concen-
trated hydrochloric acid. If the solution be cooled under running water
it loses its purplish tinge of color and becomes a definite blue. The
solution at this point may be somewhat opalescent through the separation
of uncombined para-dimethlaminobenzaldehyde. Care should be taken
not to add an excess of hydrochloric acid inasmuch as the end-reaction
has a tendency fo fade under the influence of a high acidity.
A rough idea regarding the actual quantity of skatole in a mixture
may be obtained by extracting this blue solution with chloroform and
subsequently comparing this chloroform solution, by means of a color-
imeter (Duboscq), with the maximal reaction, obtained with a skatole
solution of known strength.
2. Color Reaction with Hydrochloric Acid. — Acidify some of
the residue with concentrated hydrochloric acid. Note the production
of a \iolet 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 indole.
Tests for Phenol and Cresol.
1. Color Test. — Test a little of the solution Avith Millon's reagent.
A red color results. Compare this test with the similar one under Tyro-
sine (see page 91).
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 bromine water
to a little of the fluid under examination. Note the crystalline precipi-
tate 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 Skatole-carbonic Acid.
Ferric Chloride Test. — Acidify some of the filtrate with hydro-
chloric acid, add a few drops of ferric chloride solution, and heat. Com-
pare the end-reaction with that given by phenol.
CHAPTER XI.
FECES.
The feces is the residual mass of material remaining in the intes-
tine 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 various authorities claim that 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 vegetable diet will be much greater and may
Fig. 47. — Microscopical Constituents 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.
even be as great as 350 grams and possess a solid content of 75 grams.
In the author's own experience the average daily output of moist feces,
calculated on the basis of data secured from the examination of over 1,000
stools, was about 100 grams. The variation in the normal daily output
being so great renders this factor of very little value for diagnostic pur-
poses, except where the composition of the diet is accurately known.
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. This
178
FECES. 179
pigment originates from the bilirubin which is secreted into the intes-
tine in the bile, the transformation from bilirubin to hydrobilirubin
being brought about through the activity of certain bacteria. Hydro-
bilirubin is sometimes called stercobilin and bears a close resemblance
to urobilin or may even be identical with that pigment. 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 brownish-black stool, whereas
the stool resulting from a milk diet is
invariably light colored. Certain pig-
mented foods such as the chlorophyllic
vegetables, and various varieties of ber-
ries, each afford stools having a charac- ^^
teristic color. Certain drugs act in a ! ^^^ 1 .^"*'*' ^^J^\ /
similar way to color the fecal discharge. v^ J «" \-^ '
This is well illustrated bv the occurrence "^ _ ^'^
. I1G.48. — H^MATOiDLv Crystals FROM
of green stools following the use ot Acholic Stools, {v. Jaksch.)
calomel and of black stools after bismuth Color of crystals same as the color of
those in Fig. 42, p. 161.
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 incorrect 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 skatole and
indole, two bodies formed in the course of putrefactive processes occurring
within the intestine (see page 169). Such bodies as methane, methyl
mercaptan, and hydrogen sulphide 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 pathological source.
A neutral reaction ordinarily predominates in normal stools although
sHghtly 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.
l8o PHYSIOLOGICAL CHEMISTRY.
Recent experiments^ in which the actual hydrogen ion concentration
of the feces was determined indicated that the reaction of the excreta
was uniformly slightly alkaline. Pronounced dietary changes e. g., low
protein diet, high protein diet, fasting, water drinking with meals, produced
at most only minor changes in the reaction of the feces.
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 normal conditions the consistency may vary from a
thin, pasty discharge to a firmly formed stool. Stools which are ex-
ceedingly thin and watery ordinarily have a pathological significance.
In general the feces of the carnivorous animals is of a firmer consistency
than that of the herbivora.
The continued ingestion of a diet which is very thoroughly digested
and absorbed is frequently accompanied by the formation of dry, hard
fecal masses (scybala). Constipation generally results, due to the small
bulk of the feces and its lack of moisture. To counteract this tendency
toward constipation the ingestion of agar-agar^ has been suggested.^ This
agar is relatively indigestible and readily absorbs water thus forming a
bulky fecal mass which is sufficiently soft to permit of easy evacuation.
The function of agar is not limited to its use in connection with consti-
pation; it may serve in other capacities as an aid to intestinal therapeutics.'*
It is frequently desirable for clinical or experimental purposes to
make an examination of the fecal output which constitutes the residual
mass from a certain definite diet. Under such conditions, it is customary
to cause the person under observation to ingest some substance, at the
beginning and end of the period in question, which shall sufficiently
differ in color and consistency from the surrounding feces as to render
comparatively easy the differentiation of the feces of that period from
the feces of the immediately preceding and succeeding periods. One
of the most satisfactory methods of making this "separation" is by means
of the ingestion of a gelatin capsule containing about 0.2 gram of powdered
charcoal at the beginning and end of the period under observation. This
procedure causes the appearance of Iwo black zones of charcoal in the
fecal mass and thus renders comparatively simple the differentiation of
the feces of the intermediate period. Carmine (0.3 gram) may be used in
a similar manner and forms two dark red zones. Some similar method
for the "separation of feces" is universally practised in connection with
the scientifically accurate type of nutrition or metabolism experiment
' Howe and Hawk; Jour. Biol. Ghent., ii, 129, 1912.
^ Agar-agar is a product prepared from certain types of Asiatic sea-weed. It is a carbo-
hydrate and is classified as a galactan in tiie polysaccharide group.
'Mendel: Zent. f. ges. Physiol, u. Path, des Stoffw., No. 17, p. i, 1908; Schmidt: Milnch.
med. Woch., 52, 1970, 1905.
^Einhorn: Berl. klin. Woch., 49, 113, 1912.
FECES. l8l
which embraces the collection of useful data regarding the income and
outgo of nitrogen, and other elements.
Among the macroscopical constituents of the feces may be men-
tioned the following: Intestinal parasites, undigested food particles,
gall stones, pathological products of the intestinal wall, enteroliths,
intestinal 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 microscope are as follows:
Constituents derived from the food, such as muscles 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 con-
stituents named the following crystalline deposits may
be detected : cholesterol, soaps, fatty acid, fat, bismuth
sulphide, hcematoidin, "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 recog- Fig. 49.— Charcot-
... , . • r • 1 • 1 Leyden Crystals.
mzed 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 microscopical identification through the detection of erythrocytes
are both unsatisfactory in their results. Of the tests given for the
detection of "occult blood" the benzidine reaction and the phenol-
phthalein and aloin-turpentine tests (page 185) are 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.
It has been quite clearly shown that the intestine of the newly born
is sterile. However, this condition is quickly altered and bacteria may
be present in the feces before or after the first ingestion of food. There
are three possible means of infecting the intestine, i. e., by way of the
mouth or anus or through the blood. The infection by means of the
blood seldom occurs except under pathological conditions, thus limiting
the general infection to the mouth and anus.
In infants with pronounced constipation two-thirds of the dry sub-
stance of the stools has been found to consist of bacteria. In the stools
of normal adults probably about one-third of the dry substance is bacteria. *
' Schittenhelm and ToUens found bacteria to comprise 42 per cent of the dry matter.
This value is, however, undoubtedly too high.
1 82 PHYSIOLOGICAL CHEMISTRY.
The average excretion of dry bacteria in twenty-four hours for an adult
is about 8 grams. The output of fecal bacteria has been found to undergo
a decrease under the influence of water drinking with meals. ^ There was
also a decrease in intestinal putrefaction,^ a fact which indicates that at
least a part of the bacterial deficit was made up of putrefactive organisms.
Over 50 per cent of the total nitrogen of feces has been shown to be
bacterial nitrogen.^
Various enzymes have been detected in the feces. The first one so
demonstrated w^as pancreatic amylase. "* The amylase content of the
feces is believed to be an index of the activity of the pancreatic function.^
The excretion of this enzyme has been found to increase under the
influence of water drinking with meals. ^ Other enzymes which have
been found in the feces under various conditions are trypsin, rennin,
maltase, sucrase, lactase, nuclease and lipase. '^
Some of the more important organisms met with in the feces are the
following:^ B. coli, B. lactis aero genes ^ Bad. Welchii, B. bijidus, and
coccal forms. Of these the first three types mentioned are gas-forming
organisms. The production of gas by the fecal flora in dextrose-bouillon
is subject to great variations under pathological conditions: alterations
in the diet of normal persons will also cause wide fluctuations. In this
connection Herter has observed a marked reduction or even complete
cessation of gas production by the mixed fecal bacteria while considerable
doses of benzoate were being given. A return to the former plane of gas
production followed the discontinuation of the benzoate.® Data as to the
production of gas are of considerable importance in a diagnostic way
although the exact cause of the variations is not yet established. It should
be borne in mind in this connection that gas volumes are frequently vari-
able with the same individual. For this reason it is necessary in every
instance to follow the gas production for a considerable period of time
before drawing conclusions. ^**
The nitrogen present in the feces consists principally of bacteria,
unabsorhed intestinal secretions, epithelial cells, mucus material and food
residues. In the early days of nutrition study the fecal nitrogen was
' Mattill and Hawk: Jour. Am. Chem. Soc, 2iZ, 1999, 1911; B'latherwick, Shenvin and
Hawk: Jour. Biol. Chem., 11, viii, 1912 (Proceedings).
^ Hattrem and Hawk: Arch. Int. Med., 7, 610, 191 1; Blatherwick, Sherwin and Hawk:
loc. cit.
' MacNeal, Latzer and Kerr: Jour. Inf. Dis., 6, 123, 190Q; Mattill and Hawk: Jour
JLxp. Med., 14, 433, 1911; Blatherwick and Hawk: Unpublished data.
* Wcgscheider: Inaug. Diss., Strassburg, 1875.
^ Wohlgemuth: Berl. klin. Woch., 47, 3, 92, 1910.
"Hawk: Arch. Int. Med., 8, 382, 1911.
' Ury: Biochem. Zeit., 23, 152, 1909.
' Herter and Kendall: Journal of Biological Chemistry, 5, 283, 1908.
" Private communication from Professor C. A. Herter.
'" Herter and Kendall: loc. cit.
FECES. 183
believed to consist principally of food residues. We now know that such
residues ordinarily make up but a small part of the nitrogen quota of the
stools of normal individuals who exercise normal mastication.^ When
meat has been "bolted," however, from 1/2 gram to 16 grams of macro-
scopical meat residues has been found in a single stool. ^ The phrase
"metabolic product nitrogen" is frequently used as a designation for
all fecal nitrogen except that present as food residues and bacteria.
Bacteria cannot logically be classed under "metabolic" nitrogen since
they doubtless develop at the expense of food nitrogen as well as at the
expense of that in the form of intestinal secretions. In the accurate
study of " protein utilization"^ a correction should be made for " metabolic
nitrogen." Data regarding the output of metabolic nitrogen may be
secured by determining the fecal nitrogen excretion on a diet of proper
energy value but amtaining no nitrogen.* Agar-agar may be utilized
advantageously in connection with such a nitrogen-free diet.
Feces is still excreted from the intestine even when no food is ingested.
Carefully conducted fasting experiments have demonstrated this. A dog
nourished on an ordinary diet to which bone ash has been added will
excrete a grey feces. W^hen fasted such an animal will, after a few days,
excrete a small amount of a greenish-brown mass, containing no bone ash.
This is fasting feces. It is of a pitch-like consistency and turns black
on contact with the air.* Adult fasting men have been found to excrete
7-8 grams of feces per day, the daily nitrogen value being about o.i gram.®
No separating medium such as charcoal or carmine (p. 180) should be used
in differentiating fasting feces.
In recent years the examination of feces for evidences of parasitism
(detection of parasites and their ova) has taken on an added importance.
The investigation of the hookworm has been particularly developed. (For
methods and discussion see Bulletin 135, Bureau of Animal Industry,
U. S. Department of Agriculture, 191 1 (M. C. Hall.)
For diagnostic purposes the macroscopical and microscopical exami-
nations of the feces ordinarily yield much more satisfactory data than
are secured from its chemical examination.
' Kerraauner: Zeit.fiir Biol., 35, 316, 1897.
- Foster and Hawk: Proceedings of Eighth International Congress of .Applied Chem., New
York, September, 1912.
^ The percentage of the ingested protein which is absorbed from the intestine. This
may be calculated by subtracting the metabolic nitrogen from the total fecal nitrogen and
dividing this value by the food nitrogen.
* Tsuboi: Zeit.fiir Biol., 35, 68, 1897; Mendel and Fine: Jour. Biol. Chem., ii, 5, 191 2.
^ Howe and Hawk: Jour. Am. Chem.Soc, ^^, 215, 1911.
*Howe, Mattill and Hawk: Ibid., ^3, 56S, 191 1.
1 84
PHYSIOLOGICAL CHEMISTRY.
Experiments on Feces.
I. Macroscopical Examination. — If the stool is watery pour it
into a shallow dish and examine directly. 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 satisfactorily
by means of a Boas sieve (Fig. 50). 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 contained 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
stirred by means of a glass rod during the washing
process. After 15-30 minutes' washing nothing but
the coarse fecal constitutents remain upon the sieve.
2. Microscopical Examination. — Watery stools
should be placed in a shallow dish, thoroughly mixed,
and a small amount removed to a slide for examina-
tion. Stools of a firm or pasty consistency should be
rubbed up in a mortar with physiological salt solution
and a small portion of the resulting mixture trans-
ferred to a slide for examination. In normal feces
look for food particles, bacteria and crystalline bodies. In pathological
stools, in addition to these substances, look for animal parasites and
pathological products of the intestinal wall. See Fig. 47, page 178.
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 reaction may change
very rapidly. The reaction of the normal stools of adult man is ordi-
narily neutral or faintly alkaline to litmus, but seldom acid. Infants'
stools are generally acid in reaction. Try the reaction to Congo red paper.
Also test tte reaction of fecal extract to phenolphthalein.
4. Starch. — If any imperfectly cooked starch-containing food has
been ingested it will be possible to detect starch granules by a micro-
scopical examination of the feces. If the granules are not detected
by a microscopical examination, the feces should be placed in an evaporat-
ing 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 50).
Fig.
-Boas'
Sieve.
FECES. 185
5. Cholesterol and Fat. — Extract the dry feces with ether in a
Soxhlet apparatus (see Fig. 136). If this apparatus is not available
transfer the dry feces to a flask, add ether, and shake frequently for
a few hours. Filter and remove the ether by evaporation. The residue
contains cholesterol and the mixed fats of the feces. For every gram
of fat add about i 1/2 gram 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 cholesterol. Add sodium chloride, in substance, to the mixture
and extract with ether to dissolve out the cholesterol. Remove the ether
by evaporation and examine the residue microscopically for cholesterol
crystals. Try any of the other tests for cholesterol as given on page 272.
6. Blood. — Undecomposed blood may be detected macroscopically.
If uncertain, look for erythrocytes under the microscope, and spectro-
scopically for the spectrum of oxyhaemoglobin (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 just mentioned,
the following tests may be tried :
{a) Benzidine Reaction. — Make a thin fecal suspension using about
5 c.c. of distilled water, and heat it to boiling to render oxidizing enzymes
inactive. To 2 c.c. of a saturated solution of benzidine in glacial acetic
acid add 3 c.c. of 3 per cent hydrogen peroxide and 2-3 drops of the cooled
fecal suspension. A clear ^reg« or blue color appears within 1-2 minutes
in the presence of blood. If the mixture is 7iot shaken a ring of color will
form at the top. Minute traces of blood are more easily detected by the
latter procedure.
{h) Phenol phthalein Tesl.^ — Make a thin fecal suspension using
about 5 c.c. of distilled water. Heat to boiling,' cool and add 2 c.c. of
the suspension to i c.c. of the phenolphthalein reagent^ and a few drops
of hydrogen peroxide. A pink or red color promptly forms in the presence
of blood.
(c) Aloin-turpenline 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
' Boas: Deut. Med. Woch., 37, 62, igii.
^ Boas suggests using an ether extract of the fecal suspension thus eliminating the necessity
of boiling. However, oxidizing enzymes are the main sources of error here and the action
is easily and effectively eliminated by boiling. (See White: Boston Medical and Surgical
Journal, 164, 876, iqii.)
^Prepared by dissolving 1-2 grams of phenolphthalein and 25 grams of KOH in 100 c.c.
of distilled water. .A.dd 10 grams of powdered zinc and heat gently until the solution is de-
colorized. Prepared in this way the solution will not deteriorate on standing.
1 86 PHYSIOLOGICAL CHEMISTRY.
with an equal volume of ether to remove any fat which may be present.
Now treat the extracted feces with one-third its volume of glacial acetic
acid and lo 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 instances 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 negative test. Hydrogen peroxide is not a
satisfactory substitute for turpentine in the test.
(d) 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 solu-
tion of guaiac (strength about i : 60) , ^ drop by drop, until the fluid becomes
turbid. Now add hydrogen peroxide or old turpentine. In the presence
of blood a blue color is produced (see page 209).
(e) Cowie's Guaiac Test. — To i gram of moist feces add 4-5 c.c. of
glacial acetic acid and extract the mixture with 30 c.c. of ether. To 1-2
c.c. of the extract add an equal volume of water, agitate the mixture, intro-
duce a few granules of powdered guaiac resin, and after bringing the resin
into solution, gradually add 30 drops of old turpentine or hydrogen
peroxide. A blue color indicates the presence of blood. Cowie claims
that by means of this test an intestinal hemorrhage of i gram can easily
be detected by an examination of the feces.
(/) Acid-h(zmatin. — Examine some of the ethereal extract from
Experiment {d) spectroscopically. Note the typical spectrum of acid-
haematin (see Absorption Spectra, Plate II).
7. Hydrobilirubin. Schmidt's Test. — 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 6-24 hours.
The presence of hydrobilirubin will be indicated by a deep red color being
imparted to the particles of feces containing this pigment. This red color
is due to the formation of hydrobilirubin-mercury. If unaltered bilirubin
is present in any portion of the feces that portion will be green in color due
to the oxidation of bilirubin to biliverdin.
' Buckmaster advises the use of an alcoholic solution of guaiaronic acid instead of an
alcoholic solution of guaiac resin.
FECES. 187
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 alcohol extracts the pigment and
assumes a reddish color. Examine a little of this fluid spectroscopically
and note the typical spectrum of hydrobilirubin (Absorption Spectra,
Plate 11).
S. Bilirubin/ (a) Gmelin's Test. — ^Place a few drops of concentrated
nitric acid in an evaporating dish or on a porcelain test-tablet and allow a
few drops of the feces and water to mix with it. The usual play of colors
of Gmelin's test is produced, i. e., green, blue, violet, red, and yellow.
If so desired, this test may be executed on a slide and observed under a
microscope.
(6) HupperCs 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.
Steensma advises the addition of a drop of a 0.5 per cent solution of
sodium nitrite to the acid-alcohol mixture before warming on the water-
bath. Try this modification also.
9. Bile Acids. — Extract a small amount of feces with alcohol 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 solution try any of the tests for bile acids
given on page 163.
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 proteins. 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 caseinogen would dissolve. Filter off the caseinogen and
test it according to directions given on page 241. 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 could you make on the newly precipitated body to differentiate
between mucin and caseinogen ?
11. Nucleoprotein. — Mix the stool thoroughly with water, transfer
to a flask, and add an equal amount of saturated lime water. Shake
' The detection of bilirubin in the feces is comparatively simple provided it is not accom-
panied by other pigments. When other pigments are present, however, it is difficult to
detect the bilirubin and, at times, may be found impossible.
l88 PHYSIOLOGICAL CHEMISTRY.
frequently for a few hours, filter, and precipitate the nucleo-protein with
acetic acid. Filter off this precipitate and test it as follows:
{a) Phosphorus. — Test for phosphorus by fusion (see page 271).
ih) Solubility, — Try the solubility in the ordinary solvents.
(c) Protein Color Test. — Try any of the protein color tests.
What proof have you that the above body was not mucin? What
other test can you use to differentiate between nucleoprotein and mucin?
12. Albumin and Globulin. — Extract the fresh feces with a dilute
solution of sodium chloride. (The preliminary extract from the prepa-
ration of caseinogen (10), above, 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 signi-
fies albumin. Make a protein 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
120. 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.
(h) Phosphates. — Acidify with nitric acid, add molybdic solution,
and warm gently.
(c) Sulphates. — Acidify with hydrochloric acid, add barium chloride,
and warm.
15. Konto's Reaction for Indole. — Rub up the stool with water
to form a thin paste. From this point the test is the same as for the
detection of indole in putrefaction mixtures (see page 176).
. 16. Schmidt's Nuclei Test. — This test serves as an aid to the diag-
nosis of pancreatic insufficiency. The test is founded upon the theory
that cell nuclei are digestible only in pancreatic juice, and therefore
that the appearance in the feces of such nuclei indicates insufficiency of
pancreatic secretion. The procedure is as follows: Cubes of fresh beef
about one-half centimeter square are enclosed in small gauze bags and
ingested with a test meal. Subsequently the fecal mass resulting from
this test-meal is examined, the bag opened, and the condition of the
enclosed residue determined. Under normal conditions the nuclei would
be digested. Therefore if the nuclei are found to be for the most part
undigested, and the intervening period has been sufficient to permit of
FECES. 189
the full activity of the pancreatic function (at least six hours), it may be
considered a sign of pancreatic insufficiency.
It has been claimed by Steele that under certain conditions the non-
digestion of the nuclei may indicate a general lowering of the digestive
power rather than a true pancreatic insufficiency.
Kashiwado^ has recently suggested the use of stained cell nuclei in
this test.
17. Einhorn's Bead Test.^ — This is a method for testing the digestive
function. In some respects it is similar to Sahli's desmoid reaction.
The procedure consists in wrapping the material under examination
(catgut, fish-bone, raw beef, cooked potatoes, thymus gland or mutton
fat, etc.) in gauze to which glass beads of various colors are attached and
enclosing gauze and beads in a gelatine capsule.^ The gelatine capsule
is swallowed and the beads serve to facilitate the separation of the gauze
from the feces. The residue within the gauze is then examined. If
beads appear in much less than 24 hours an accelerated motility is indi-
cated, whereas an interval of 48 hours or over elapsing indicates retarded
motility. If gastric function alone is to be studied silk threads are
attached to the beads and the latter are withdrawn and examined before
they have passed into the intestine.
18. "Separation" of Feces. — In order to become familiar with the
method ordinarily utilized in metabolism experiments to differentiate the
feces which corresponds to the food ingested during any given interval,
and at the same time to secure data as to the length of time necessary
for ingested substances to pass through the alimentary tract proceed as
follows: Just before one of the three meals of the day ingest a gelatine
capsule (No. 00) containing 0.2-0.3 ^^ ^ gram of carmine or charcoal.
Make an inspection of all stools subsequently dropped and note the time
interval elapsing between the ingestion of the capsule and the appearance
of its contents in the feces. Under normal conditions this period is ordi-
narily 24 hours.
19. Quantitative Determination of Fecal Amylase (The Author's*
Modification of Wohlgemuth's^ Method). — Weigh accurately about 2
grams of fresh feces into a mortar," add 8 c.c. of a phosphate-chloride
solution (o.i mol dihydrogen sodium phosphate and 0.2 mol disodium
hydrogen phosphate per liter of i per cent sodium chloride), 2 c.c. at a
time, rubbing the feces mixture to a homogeneous consistency after each ad-
* Kashiwado: Dent. Arch. Klin. Med., 104, 584, 1911.
''Einhorn: The Post-Graduate, May 1912: Boas' Arch., 12, 26, 1906; 13, 35, 1907; Ibid,
475; 15. part 2, 1909.
' Ordinarily two substances are attached to each bead, three beads tied together and
enclosed in one capsule. Test capsules may be obtained from Eimei and Amend, New York.
* Hawk: .Arch. Int. Med., 8, 552, 1911.
' Wohlgemuth: Berl. klin. Woch.. 47, 3, 92, 1910; also see chapter on Enzymes, this book.
" Duplicate determinations should be made.
190
PHYSIOLOGICAL CHEMISTRY.
dition of the extraction medium. Permit the mixture to stand at room tem-
perature for a half-hour with frequent stirring. We now have a neutral
fecal suspension. Transfer this suspension to a 15 c.c. graduated centrifuge
tube, being sure to wash the mortar and pestle carefully with the phosphate-
chloride solution and add all washings to the suspension in the centrifuge
tube. The suspension is now made up to the 15 c.c. mark with the
phosphate-chloride solution and centrifugated for a fifteen-minute period,
or longer if necessary, to secure satisfactory sedimentation. At this point,
read and record the height of the sediment column. Remove the super-
natant Hquid by means of a bent pipette, transfer it to a 50 c.c. volumetric
flask and dilute it to the 50 c.c. mark with the phosphate-chloride solution.
Mix the fecal extract thoroughly by shaking and determine its amylolytic
activity. For this purpose a series of six graduated tubes is prepared, con-
taining volumes of the extract ranging from 2.5 c.c. to 0.078 c.c. Each of
the intermediate tubes in this series will thus contain one-half as much
fluid as the preceding tube. Now make the contents of each tube 2. 5
c.c. by means of the phosphate-chloride solution in order to secure a uni-
form electrolyte concentration. Introduce 5 c.c. of a i per cent soluble
starch solution^ and three drops of toluol into each tube, thoroughly mix
the contents by shaking, close the tubes by means of stoppers and place
them in an incubator at 38° C. for twenty-four hours. At the end of this
time remove the tubes, fill each to within half an inch of the top with ice-
water, add one drop of tenth-normal iodin solution, thoroughly mix the
contents and examine the tubes carefully with the aid of a strong light.
Select the last tube in the series which shows entire absence of blue color,
thus indicating that the starch has been completely transformed into dex-
trin and sugar, and calculate the amylolytic activity on the basis of this
dilution. In case of indecision between two tubes, add an extra drop of
the iodin solution and observe them again. ^
* In preparing the i per cent solution, the weighed starch powder should be dissolved
in cold distilled water in a casserole and stirred until a homogeneous suspension is obtained.
The mixture should then be heated with constant stirring, until it is clear. This ordinarily
takes from eight to ten minutes. A slightly opaque solution is thus obtained, which should
be cooled and made up to the proper volume before using.
''■ Theoretically we would expect the colors to range from a light yellow to a dark blue,
with red tubes holding an intermediate position in the series. This color sequence does often
occur, but its occurrence is far from universal. Many times the first tubes in the series, i. e.,
those containing the largest quantities of the fecal extract, will exhibit a bluish cast of color
which should not be confused with the starch color reaction. When these blue tubes are
present, they are generally followed by yellow, red and blue tubes in order, the final blue tube,
of course, being the regulation starch reaction. Occasionally greenish colors will be obtained
to the left of the red color. It also sometimes happens that it is somewhat difficult to determine
in which tube to the right of the red color the starch blue color is first detected, unless the
tube be examined carefully before a strong light. In every instance, however, when these blue
anri green colors are observed, it is noted that tubes possessing the true dextrin red color are
always present between these tubes and the tubes possessing the true starch blue color. It
is evident, therefore, that these bluish tints in the tubes to the left of the dextrin color cannot
be due to the presence of starch. The cause of the blue color reaction in the first tubes of
the series has not been ascertained as yet.
FECES. 191
The amylolytic value, Df, of a given stool, may be expressed in temrs
of I c.c. of the sediment obtained by centrifugation as above described.
For example, if it is found that 0.31 c.c. of the phosphate-chloride extract
of the stool acting at 38° C. for twenty-four hours completely transformed
the starch in 5 c.c. of a i per cent starch solution, then we would have the
following proportion:
0.31 : 5 (c.c. starch) : : i (c.c. extract): X
The value of X in this case is 16. i, which means that i c.c. of the fecal
extract possesses the power of completely digesting 16. r c.c. of a i per cent
starch solution in twenty-four hours at 38° C.
Inasmuch as stools vary so greatly as to water content, it is essential
to an accurate comparison of stools that such comparison be made on the
basis of the solid matter. Supposing, for example, that in the above
determination we had 6.2 c.c. of sediment. Since the supernatant fluid
was removed and made up to 50 c.c. before testing its amylolytic value,
it is evident that i c.c. of this sediment is equivalent to 8.1 c.c. of extract.
Therefore, in order to derive the amylolytic value of i c.c. of sediment,
we must multiply the value (16. i) as obtained above for the extract, by 8.1.
This yields 130.4 and enables us to express the activity as follows:
^r38 c
Df^ =130.4
24 h
The above method of calculation is that suggested by Wohlgemuth. In
case time and facilities permit of the determination of the moisture con-
tent of the feces, it is much more accurate and satisfactory to place the
amylolytic values of the stools on a "gram of dry matter" basis. The
amylolytic values of the stools are expressed as the number of cubic centi-
meters of I per cent starch solution which the amylase content of i gram
of dry feces is capable of digesting.
20. Quantitative Determination of Fecal Bacteria. ' — The method
is a simplification of MacNeal's adaptation of the Strasburger procedure.'
"About two grams of feces are accurately weighed and placed in a 50 c.c.
centrifuge tube. To the feces in the tube a few drops of 0.2 per cent
hydrochloric acid are added, and the material is mixed to a smooth paste
by means of a glass rod. Further amounts of the acid are added with con-
tinued crushing and stirring until the material is thoroughly suspended.
The tube is then whirled in the centrifuge at high speed for one half to one
minute. The suspension is found sedimented into more or less definite
layers, the uppermost of which is fairly free from the larger particles.
The upper and more liquid portion of the suspension is now drawn off by
'Mattill and Hawk: Jour. Exp. Med., 14, 433, 1911.
' MacNeal, Latzer and Kerr, Jour. Inf. Dis., 6, 123, 1909.
192 PHYSIOLOGICAL CHEMISTRY,
means of a pipette and transferred to a beaker, ^ The sediment remaining
in the tube is again rubbed up with the glass rod with the addition of
further amounts of dilute acid, and again centrifugalized for one half to
one minute. The supernatant liquid is pipetted off and added to the
first, the same pipette being used for the one determination throughout.^
A third portion of the dilute acid is then added to the sediment, which is
again mixed by stirring and again centrifugalized. All the washings are
added to the first one, and during the process care is taken to wash the
material from the walls and mouth of the centrifuge tube down into it.
Finally, when the sediment is sufficiently free from bacteria, the various
remaining particles are visibly clean, and the supernatant liquid after
centrifugalization remains almost clear. This is removed to the beaker in
which are now practically all the bacteria present in the original portion
of feces, together with some solid matter not yet separated. In the centri-
fuge tubes there is a considerable amount of bacteria-free solid matter.
The suspension is now transferred to the same centrifuge tube, centrif-
ugalized for a minute, and the supernatant hquid transferred to a clean
beaker by means of the same pipette. The tube is then refilled from the
first beaker and thus all the suspension centrifugalized a second time.
The beaker is finally carefully washed with the aid of a rubber-tipped
glass rod, the second sediment in the centrifuge tube is washed free of
bacteria by means of this wash water and by successive portions of the
dilute acid, and the supernatant liquid after centrifugalization is added to
the contents of the second beaker. The second clean sediment is added
to the first. The bacterial suspension now in the second beaker is again
centrifugalized in the same way and a third portion of bacteria-free
sediment is separated. Frequently a fourth serial centrifugalization is
performed — always if the third sediment is of appreciable quantity.
At all stages of the separation, small portions of the dilute hydro-
chloric acid are used, so that the final suspension shall not be too vo-
luminous. Ordinarily it amounts to 125 to 200 c.c. At the same time,
the final amount of fluid should not be too small, as shown by
Ehrenpfordt,^ because the viscosity accompanying increased concentration
prevents proper arid complete sedimentation.
To the final bacterial suspension an equal volume of alcohol is added
and the beaker set aside to concentrate. A water bath at 50° to 60° C. is
very satisfactory. After two or three days, when the liquid is concen-
trated to about 50 c.c, the beaker is removed and about 200 c.c. of alcohol
' A 25 c.c. pipette is the most satisfactory size; to facilitate observation, the delivery tube
is bent near the bulb to an angle of about 120 degrees.
^ A convenient support for the pipettes is a wire spring on a glass base, such as is used on a
desk, for pen-holders. The delivery tube, just where it is bent, is inserted between the wires,
and any liquid not delivered collects in the bend of the tube.
' Ehrenpfordt: Zeil. exp. Path. Ther., 7, 455, iqoq.
FECES. 193
are added. The beaker is covered and allowed to stand at room tempera-
ture for twenty-four hours. At the end of this time the bacterial sub-
stance is generally settled, so that most of the clear supernatant liquid, of
dark, brown color, can be directly siphoned off without loss of solid matter.
The remainder is then transferred to centrifuge tubes, centrifugalized,
and the remaining clear liquid pipetted off. ^ The sediment consists of the
bodies of the bacteria, and is transferred to a Kjeldahl flask for nitrogen
determination. "This is the bacterial nitrogen. Where a determination
of bacterial dry substance is desired, the sediment of bacteria is extracted
by absolute alcohol and ether in succession, transferred to a weighed
porcelain crucible, and dried at 102° C. to constant weight. This dried
sample is then used in the nitrogen determination. Our procedure differs
from that of MacNeal in that the bacterial dry matter is not determined.
A saving of about seven days' time and of considerable labor is accom-
plished by this omission.
Inasmuch as it has been shown by various investigators that such
bacteria as are present in the feces contain on the average about i r per
cent of nitrogen, the values for bacterial nitrogen as determined by our
method may conveniently serve as a basis for the calculation of the actual
output of bacterial substance.
' In more recent work (see Blatherwick and Hawk: unpublished) it has been found
advantageous to centrifugalize with alcohol and ether in succession before transferring the
bacterial cells to Kjeldahl flasks.
13
CHAPTER XII.
BLOOD AND LYMPH.
Blood is composed of four types of form-elements (erythrocytes or
red blood corpuscles, leucocytes or white blood corpuscles, blood plates
or plaques and blood dust or hasmoconien) 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 trans-
parent. Blood so altered was formerly said to be laked. The term
hemolysis is now used in this connection and substances which cause such
action are spoken of as hcEmolytic agents. The haemolytic process is
simply a liberation of the haemoglobin from the stroma of the red blood
corpuscle. Normal blood is alkaline in reaction^ to litmus, the alkalinity
being due principally to sodium carbonate and phosphate. The specific
gravity of the blood of adults ordinarily varies between 1.045 ^^^ '^•°1S-
It varies somewhat with the sex, the blood of males having a rather higher
specific gravity than that of females of the same species. Under patholog-
ical conditions also the density of the blood may be very greatly altered.
The freezing-point {A) of normal blood is about — 0.56° C. Variations
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. Abderhal-
den and Schmidt^ have recently suggested a unique method for the
determination of this value. It is based upon the change in the optical
activity of the blood upon injection of a body of known optical activity,
such, for example, as dextrin.
Among the most important constituents of blood plasma are the four
protein bodies, fibrinogen, nudeo protein, serum globulin (euglobulin and
pseudo-globulin) and serum albumin. Plasma contains about 8.2 per cent
of solids of which the protein constituents named above constitute approxi-
mately 84 per cent and the inorganic constituents (mainly chlorides,
phosphates and carbonates) approximately 10 per cent. Among the
inorganic constituents sodium chloride predominates. To prevent coagu-
' Recently it has been shown by physico-chemical methods that the blood is in reality
neutral in reaction.
* Abderhalden and Schmidt: Zeit. physiul. chem., 66, 120, 1910.
194
BLOOD AND LYMPH. I95
lation, blood plasma is ordinarily studied in the form of an oxalated or
salted plasma. The former may be obtained by allowing the blood to
flow from an opened artery into an equal volume of 0.2 per cent ammo-
nium oxalate solution, whereas in the preparation of a salted plasma 10
per cent sodium chloride solution may be used as the diluting fluid.
Fibrinogen is perhaps the most important of the protein 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 nucleoprotein of blood possesses many of the characteristics of
serum globulin. In common with this body it is easily soluble in sodium
chloride, and is completely precipitated from its solutions upon saturation
with magnesium sulphate. It is much less soluble in dilute acetic acid
than serum globuHn, and its solutions coagulate at 65°-69° C.
The body formerly called serum globulin is probably not an indi-
vidual substance. Recent investigations seem to indicate that it
may be resolved into two individual bodies called euglohulin and pseudo-
globidm. 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 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 egg albumin in being more laevorotatory, in being rendered less
insoluble by alcohol, and in the fact that when precipitated by hvdro-
chloric acid it is more easily soluble in an excess of the reagent.
When blood coagulates and the usual clot forms, a light yellow fluid
exudes. This is blood serum. It differs from blood plasma in containing
a large amount oi fibrin ferment, a body of great importance in the coagu-
lation of the blood, and also in possessing a lower protein content. The
protein material present in plasma and not found in serum is the fibrin-
ogen which is transformed into fibrin in the process of coagulation and
196 PHYSIOLOGICAL CHEMISTRY.
removed. The specific gravity of the serum of human blood varies
between 1.026 and 1.032. If blood be drawn into a vessel and allowed
to remain \^dthout stirring or agitation of any sort the major portion of the
red corpuscles will sink away from the upper surface, causing this portion
of the clot to assume a lighter color due to the predominance of leuco-
cytes. This light colored portion of the clot is called the "buffy coat."
Beside the protein constituents already mentioned, other bodies which
are found in both the plasma and serum are the following: Sugar (dex-
trose), fat, enzymes, lecithin, cholesterol and its esters, gases, coloring-
matter (lutein or lipochrome) and mineral substances. In addition to
these bodies the following substances have been detected in normal human
blood: Creatine, carhamic acid, hippuric acid, paralactic acid, urea and
uric acid (urates). Some of the pathological constituents of blood are
proteoses, leucine, tyrosine and other amino acids, biliary constituents and
purine bodies.
By waters^ reports the presence of a glycoprotein in blood serum.
This he has termed seromucoid.
There has 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 normally in the circulation, however, they are prob-
ably thin, non-nucleated, biconcave discs. When examined singly under
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 birds, fishes, amphibians and
reptiles the erythrocytes 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 ^^as of an inch.
Guinea-pig x-h % o^ an inch.
Man V . . ^3^5 u of an inch.
Monkey s-h-x of an inch.
Dog -irhi of an inch.
Rat aoS i of an inch.
Rabbit ^/r, s of an inch.
Mouse ^-V;i- of an inch.
Lion ii-i ;f of an inch.
Ox .f .jS 9 of an inch.
Horse 52^4;:^ of an inch.
Pig j-jV, „ of an inch.
Cat ^/f a of an inch.
Sheep -4 oVa of an inch.
Goat Tj-ji^ ,, of an inch.
Musk-deer Tslsr, of an inch.
' Bywaters: Biochemische Zeilschrift, 15, 322, 1909.
* Wormley's Micro-Chemistry of Poisons, second edition, p. 733.
I'LATK IV.
Normal Erythrocytes and Leucocytes.
BLOOD AND LYMPH. 197
The erythrocytes from whatever source obtained, consist essentially
of two parts, the stroma or protoplasmic tissue and its enclosed pigment,
hcemoglohin. 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. ^ The normal content of the blood
of adult females is from 4,000,000 to 4,500,000 per cubic millimeter.
The number oi erythrocytes varies greatly 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 yellow 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 poly-
cythaemia and increases nearly as great in cyanosis. The number has
been known to decrease to 500,000 per cubic millimeter or lower in per-
nicious anaemia.
Erythrocytes possess the property, when properly treated, of "clump-
ing" together in masses and precipitating, producing so-called aggluti-
nation. Cells other than erythrocytes {e. g., bacteria) possess this property.
When spoken of in connection with the blood such action is termed
hcemagglutination. A substance w'hich vAW bring about haemagglutination
is said to contain hcEmagglutinins. These haemagglutinins are particu-
larly abundant in the vegetable kingdom.^ For a demonstration of
haemagglutination see page 208.
Oxyhsemoglobin, the coloring matter of the blood, is a conjugated
protein. Through treatment with hydrochloric acid it may be split into
a protein body called globin, and hcemochromogen, an iron-containing
pigment. The latter body is rapidly transformed into hcEniatin in the
presence of oxygen, and this in turn gives place to haematin- hydrochloride
or Immin (Figs. 59 and 60, page 211). The pigment of arterial blood is
for the most part loosely combined with oxygen and is termed oxyhsercvo-
globin, whereas the pigment of venous blood is principally haemoglobin
(so-called reduced haemoglobin). Oxyhaemoglobin is the oxygen-carrier
of the body and belongs to the class of bodies known as respiratory pig-
* This statement is based upon observations made upon the blood of athletes in training.
See Hawk: Anter. Jottr. Physiol 1904. It is generally stated in text-books that the blood
of males contains about 5,000,000 per cubic millimeter.
* Mendel: Archivio di fisiologia, 7, 168, 1909; also Schneider: Journal 0/ Biological Chem.,
II, 47. 1912.
198
PHYSIOLOGICAL CHEMISTRY.
Fig. 51. — Oxyhemoglobin Crystals from Blood of the Guinea-pig.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
V'
>1^
Fig. 52. — Oxyhemoglobin Crystals from Blood of the Rat.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
BLOOD AND LYMPH.
199
Fig. 53. — OxYH>EMOGLOBiN Crystals from'Blood of the Horse.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
i>
Fig. 54. — Oxyhemoglobin Crystals from Blood of the Squirrel.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
200
PHYSIOLOGICAL CHEMISTRY.
di=«^"
Fig. 55. — Oxyhemoglobin Crystals from Blood of the Dog.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
Fig. 56. — OXYHiEMOGLOBIN CRYSTALS FROM BlOOD OF THE CaT.
Ref)roduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania.
BLOOD AND LYMPH.
20I
Fig. 57. — OxYH,«MOGLOBiN Crystals from Blood of the Necturus.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University
of Pennsylvania. '
ments. It is held within the stroma of the erythrocyte. The reduction
of oxyhaemoglobin to form haemoglobin (so-called reduced haemoglobin)
occurs in the capillaries. O.xyhaemoglobin may be crystallized and a
specific form of crystal obtained from the blood of each individual species
(see Figs. 51 to 57, pages 198 to 201). This fact seems to indicate that
there are many varieties of oxyhaemoglobin. The interesting findings
of Reichert and Brown are of great value in this connection. These
investigators prepared oxyhaemoglobin crystals from the blood of over
mic hundred species of animal and subsequently studied the character-
istics of the crystals very minutely from the standpoint of crystallography.
Their findings may prove of importance from the standpoint of heredity
and the origin of species. They emphasize the following facts:
1. Crystals from all species of a certain genus have certain charac-
teristics in general. Crystals from different genera, however, exhibit
marked differences in system, axial ratios, etc.
2. Crystals of different species of a genus may generally be differen-
tiated by difference in the angles.
3. The oxyhaemoglobin of some species crystallizes in several types
of crystals in the same preparation. Generally the crystals first formed
belong to a system of a lower grade of symmetry than those formed
later. When such dift'erent types of crystals occur they may be arranged
in isomorphous series.
4. Certain definite angles recur in the crystals from the blood of
^ The micro- photographs of oxyhaemoglobin (see pages 108-201) and h.nemin (see page
211) are reproduced through the courtesy of Professors E. T. Reichert and Amos P. Brown,
of the Universitv of Pennsylvania, who are investigating the crystalline forms of biochemic
substances.
202 PHYSIOLOGICAL CHEMISTRY.
various species of animal, although the zoological connection may be
remote and the crystals belong to different systems.
5. The constant recurrence of certain types of "twinning" in all
the crystalline forms was observed.
6. Differences have been observed in the crystalline form of oxy-
haemoglobin and haemoglobin from the blood of the same species in
certain cases.
The follo\\dng bodies may be derived from haemoglobin, and each
possesses a specific spectrum which serves as an aid in its detection
and identification: Oxyhaemoglobin, methaemoglobin, carbon-monoxide
haemoglobin, nitric-oxide haemoglobin, haemochromogen, haematin,
acid-hasmatin, alkah-hsematin 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 many particulars, such as being
somewhat larger in size, in containing at least a single nucleus and in
possessing amoeboid movement (see Plate IV, opposite page 196).
They are typical animal cells and therefore contain the following bodies
which are customarily present in such cells: Proteins, fats, glycogen,
purine bodies, enzymes, phosphatides, lecithin, cholesterol, inorganic salts
and water. Compound proteins make up the chief part of the protein
quota of leucocytes, the nucleo-proteins predominating. Of the enzymes
present the proteolytic are the most important. It is claimed^ that
there are two proteolytic enzymes in leucocytes, one active in alkaline
solution and present in the polynuclear cells ^ and the other active in acid
medium and present in mononuclear cells. It is claimed that the granu-
lar leucocytes originate in the bone marrow, whereas the non-granular
leucocytes (lymphocytes) have a lymphatic origin (lymph glands or
lymphoid tissue); this matter of origin is uncertain. The normal number
of leucocytes in human blood varies between 5000 and 10,000 per cubic
milhmeter. The ratio between the leucocytes and erythrocytes is about
1 : 3 50-500. A leucocytosis is said to exist when the number of leucocyte s
is increased for any reason. Leucocytoses may 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 maHgnant
disease.
* Opie: Jour, of Experimental Med., 8; Opie and Barker: Ibid., g.
^For discussion of different types of leucocytes, see "Da Costa's Clinical Hematology"
or some similar volume.
BLOOD AND LYMPH. 203
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. 3^., 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 haemoconein or so-called ''blood dust" is made up of round
granules which usuallv have a diameter somewhat less than one micron.
The serum of normal as well as of pathological blood contains these
granules. They were first described by Miiller to whom they appeared
as highly refractile granules possessed of Brownian movement. The
"blood dust" is apparently not concerned with the coagulation of the
blood. The granules are insoluble in alcohol, ether and acetic acid
and are not blackened by osmic acid. According to Miiller, the gran-
ules making up the so-called "blood dust" constitute a new organized
constituent of the blood, whereas other investigators believe them to be
merely free granules from certain of the forms of leucocytes. In common
with blood plates the "blood dust" possesses no clinical significance.
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 throughout the blood mass but instead will
cling to the device used in beating. In this way the fibrin may be re-
moved and the remaining fluid is termed dejibrinated 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) -f Fibrinogen =Fibrin.
HowelP has very recently suggested an ingenious modification of the
' Howell: .American Journal of Physiology, 29, 187, 191 1.
204 PHYSIOLOGICAL CHEMISTRY,
above theory. He says "In the circulating blood we find as constant
constituents, fibrinogen, prothrombin, calcium salts and antithrombin.
The last-named substance holds the prothrombin in combination and
thus prevents its conversion or activation to thrombin. When the blood
is shed, the disintegration of the corpuscles (platelets) furnishes material
(thromboplastin) which combines with the antithrombin and liberates
the prothrombin; the latter is then activated by the calcium and acts on
the fibrinogen. According to this view the actual process of coagulation
involves only three factors, fibrinogen, prothrombin and calcium. These
three factors exist normally in the circulating blood, but are prevented
from reacting by the presence of antithrombin."
Among the medico-legal tests for blood are the following: (i)
Microscopical identification of the erythrocytes, (2) spectroscopic
identification of blood solutions, (3) the guaiac test, (4) the benzidine
reaction, (5) preparation of haemin crystals. Of these five tests the
two last named are generally considered to be the most satisfactory.
They give 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
technic of the tests is simple and the formation of the dark brown
or chocolate colored crystals of haemin or the production of the green or
blue color with benzidine is indisputable proof of the presence of blood
in the fluid, clot or stain examined. The weak point of the tests, medico
legally, lies in the fact that they do not differentiate between human blood
and that of certain other species of animal.
The guaiac test (see page 209), although generally considered less
accurate than the haemin test, is really a more delicate test than the haemin
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 con-
centrated and which, when brought into contact with the aqueous blood
solution, 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 i 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 be considered a
specific test for blood and is of value only in a negative sense. We have
demonstrated to our own satisfaction, 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 advocated the use of an alcoholic solution of guaia-
BLOOD AND LYMPH. 205
conic acid instead of an alcoholic solution of guaiac resin. He claims
that he was able to produce the blue color ujK)n 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 applica-
tion of the guaiac test to the detection of blood, he states that he was able
"to detect laked blood when present in the ratio i : 5,000,000 and unlaked
blood when present in the ratio i : 1,000.000. This author considers the
guaiac test to be far more trustworthy than is generally believed.
Up to within recent times it has been impossible to make an absolute
difTercntiation 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 scrum of an animal into which has been injected the blood of
another animal of different species develops the property of agglutinating
and dissolving erythrocytes similar to those injected, but exerts this influence
■upon the 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 necefsary
to combine the antiserum and the solution under examination in the
proportion of i : 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
within three hours to a distinctly flocculent precipitate. This antiserum
will react thus with no other known substance.
Lymph may be considered as the "middle man" in the transactions
between blood and tissues. It is the medium by which the nutritive
material and oxygen transported by the blood for the tissues is brought into
intimate contact with those tissues and thus utilized. In the further ful-
fillment of its function, the lymph bears from the tissues \vater, salts and
the products of the activity and catabolism of the tissues and passes these
into the blood. Lymph, therefore, exercises the function of a "go-between"
for blood and tissues. It bathes every active tissue of the animal body,
and is believed to have its origin partly in the blood and partly in the
tissues.
In chemical characteristics, lymph resembles blood plasma. In fact,
it has been termed "blood without its red corpuscles." Lymph from the
thoracic duct of a fasting animal or from a large lymphatic vessel of a well-
nourished animal is of a variable color (colorless, yellowish or slightly
reddish) and alkaline in reaction to litmus. It contains fibrinogen, fibrin
2o6 PHYSIOLOGICAL CHEMISTRY.
ferment and leucocytes and coagulates slowly, the clot being less firm and
bulky than the blood clot. Serum albumin and serum globulin are both
present in lymph, the albumin predominating in a ratio of about 3 or 4 : i.
The principle inorganic salts are sodium salts (chloride and carbonate),
whereas the phosphates of potassium, calcium, magnesium and iron are
present in smaller amount.
Substances which stimulate the flow of lymph are termed lympha-
gogues. Such substances, as sugar, urea, certain salts (especially sodium
chloride) peptone, egg albumin, extracts of dogs' liver and intestine, crab
muscles and blood leeches are included in this class.
In a fasting animal, the lymph coming from the intestine is a clear,
transparent fluid possessing the characteristics already outlined. After
a meal containing fat has been ingested, this intestinal lymph is white
or "milky." This is termed chyle and is essentially lymph possessing an
abnormally high (5-15 per cent) content of emulsified fat. This chyle
is absorbed by the lacteals of the intestine and transported to the lower
portion of the thoracic duct. Apart from the fat value, the composition of
lymph and chyle are similar.
Experiments ox 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.
Test by congo-red paper also.
2. Microscopical Examination. — Examine a drop of defibrinated
blood under the microscope. Compare the objects you observ^e ^ith
Plate IV, opposite page 196. Repeat the test with a drop of your own
blood.
3. Specific Gravity. — Determine the specific gra\'ity of defibrinated
blood by means of an ordinary specific gravity spindle. Compare this
result with the specific gravity as determined 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 showing it to be of higher specific gravity
than the surrounding fluid, add chloroform until the blood drop remains
BLOOD AND LYMPH. 207
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 specific gravity accurately
determined. What is the specific graWty of the blood under examination ?
5. Tests for Various Constituents. — Place lo c.c. of defibrinated
blood in an evaporating dish, dilute with loo c.c. of water and heat to
boiling. Is there any coagulation, and if so what bodies form the coag-
ulum? At the boiling-point acidulate slightly with dilute acetic acid.
Filter. The filtrate should be clear and the coagulum dark brown. Re-
serve this coagulum. What body gives the coagulum this color ? Evap-
orate 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) Fehlings Test. — Test for sugar according to directions given on
page 32.
{h) Chlorides. — To a small amount of the filtrate in a test-tube add
a few drops of nitric acid and a little silver nitrate. In the presence
of chloride, a white precipitate of silver chloride will form.
(c) Phosphates. — Test for phosphates by nitric acid and molybdic
solution according to directions given on page 64.
{d) Proteose and Peptone. — Test a small amount of the solution for
proteose and peptone by saturating with ammonium sulphate according
to directions given on page 120.
{e) Crystallizatmi of Sodium Chloride. — Place the remainder 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. 61, page 213.
6. Test for Iron.- — Incinerate a small portion of the coagulum 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 thiocyanate. Which of the constituents of the blood
contains the iron ?
7. Haemolysis ("Laky Blood)." — Note the opacity of ordinary defibri-
nated 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 rendered transparent.
Hcemolysis has taken place. How does the water act in causing this
transparency? Examine a drop of haemolyzed blood under the micro-
scope. How does its microscopical appearance differ from that of
2o8 PHYSIOLOGICAL CHEMISTRY.
unaltered blood? What other agents may be used to bring about
haemolysis ?
8. Osmotic Pressure. — ^Place a few cubic centimeters of blood in
each of three test-tubes. Haemolyze 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 chloride to the blood in the second tbue,
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 exam-
ine a drop from each of the three tubes under the microscope (see Figs.
58 and 120, below and p. 377 ). What do you find and what is your
explanation from the standpoint of osmotic pressure ?
Fig. 58. — Efpect of Water on Erythrocytes.
9. Haemagglutination. — The common garden bean, such as the
Scarlet Runner/ contains a protein substance which exhibits the inter-
esting property of causing a clumping or agglutination of red blood
corpuscles.^
Dilute defebrinated blood^ ten times with physiological sodium
chloride solution (0.9 per cent) and place i c.c. in each of three small test-
tubes.
Grind three beans in a coffee mill, or with mortar and pestle to a fine
meal and extract for a few minutes with 0.9 per cent sodium chloride
solution. Filter and add 0.05 c.c. (about 2-3 drops) of the filtered
' The Scarlet Runner is a familar variety purchasable in every seed store. Ricin a protein
constituent of the castor bean also possesses pronounced agglutinating properties. Because
of its poisonous nature it is, however, not suitable for use in class experiments.
^Mendel: Archivio di fisiologia, 7, 168, 1909; Schneider: Journal Biol. Chem., 11, 47,
1912.
' Rabbit's blood is especially desirable (Mendel: Loc. cit.) and may be obtained for the
purpose by bleeding from a small cut on the animal's ear and defibrinating.
BLOOD AND LYMPH. 209
extract to the first of the blood tubes; o.oi c.c. to the second; and 0.05
of 0.9 per cent sodium chloride solution to the third.
Invert each tube to mix the contents thoroughly, and note the rapid
agglutination, and precipitation of the blood corpuscles in the first
tube, a less rapid agglutination in the second, while the third or
control tube remains unaltered. In one-half hour the corpuscles in
the first tube ^often are packed solid and one is able to pour off perfectly
clear serum.
If the remainder of the bean extract is boiled for a few minutes, the
coagulum filtered out and 0.05 c.c. of the filtrate added to the control
tube, still no agglutination occurs, indicating that the hasmagglutinin
has been destroyed or removed by the boiling.
10. Diffusion of Haemoglobin.— Prepare some hcBtnolyzed ("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. 2, page 30. How does haemoglobin differ from other well-
known crystallizable bodies ?
11. Guaiac Test. — To 5 c.c. of water in a test-tube add two drops
of blood. By means of a pipette drop an alcoholic solution of guaiac
(strength about 1:60)^ into the resulting mixture until a turbidity is
observed and add old turpentine or hydrogen peroxide, drop by drop,
until a blue color is obtained. Do any other substances respond in a
similar manner to this test ? Is a positive guaiac test a sure indication of
the presence of blood ?
12. Schiunm's Modification of the Guaiac Test. — To about 5 c.c.
of the solution under examination^ in a test-tube add about ten drops
of freshly prepared alcoholic solution of guaiac. Agitate the tube gently,
add about 20 drops of old turpentine, subject the tube to a thorough
shaking and permit it to stand for about 2-3 minutes. A blue color
indicates the presence of blood in the solution under examination. In
case there is insufficient blood to yield a blue color under these
conditions, a few c.c. of alcohol should be added and the tube gently
shaken, where-upon a blue coloration will appear in the upper alcohol-
turpentine layer.
A control test should always be made, using water in place of the
solution under examination. In the detection of very minute traces of
blood only 3-5 drops of the guaiac solution should be employed.
13. Adler's Benzidine Reaction. — This is one of the most delicate
of the reactions for the detection of blood. Different benzidine prepara-
' Buckmaster advises the use of an alcoholic solution of guaiaconic acid instead of an
alcoholic solution of guaiac resin.
- Alkaline solutions should be made slightly acid with acetic acid, as the blue end-reaction
is very sensitive to alkali.
14
210 PHYSIOLOGICAL CHEMISTRY.
tions vary greatly in their sensitiveness, however. Inasmuch as benzidine
solutions change readily upon contact with light it is essential that they
be kept in a dark place. The test is performed as follows : To a saturated
solution of benzidine in alcohol or glacial acetic acid add an equal volume
of 3 per cent hydrogen peroxide and one c.c. of the solution under exami-
nation. If the mixture is not already acid render it so with acetic acid,
and note the appearance of a green or blue color. A control test should
be made substituting water for the solution under examination. The
sensitiveness of the benzidine reaction is greater when applied to aqueous
solutions than when applied to the urine. According to Ascarelli^ the
benzidine reaction serves to detect blood when present in a dilution of
I : 300,000. Walter^ has also recently shown the test to be very delicate
and claims it to be more satisfactory than the guaiac test.
14. Haemin Test. — (a) Teichmann''s Method. — ^Place a very small
drop of blood on a microscopic slide, add a minute grain of sodium
chloride^ 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 formation of gas bubbles is noted. Add another
drop of glacial acetic acid, cool the preparation, examine under the
microscope and compare the crystals with those shown in Figs. 59 and 60,
page 211. 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 between human
blood and the blood of other species by means of the haemin test ? .
{h) Atkinson and Kendall's Method. — Introduce a small amount of
the solution under examination into a tube closed at one end, add sodium
chloride and glacial acetic acid as in Teichmann's method,* fuse or
tightly plug the open end of the tube and heat for fifteen minutes in a
boiling water-bath.^ Remove the tube and permit it to cool to room
temperature spontaneously. When the tube has cooled, break it open,
transfer the contents to a watch glass or small evaporating dish and
concentrate on a water-bath until the volume of the fluid in the watch
glass or dish has been reduced to a few drops. Transfer a drop of this
fluid to a slide, cover with a cover slip, allow the slide to stand for a few
minutes and examine it under a microscope. Compare the crystals
with those shown in Figs. 59 and 60, page 211. In case crystals of sodium
chloride (see Fig. 61, page 213) obstruct the view of the haemin crystals,
* Ascarelli: II policlin sez. prat., igog.
^ Walter: Deul. med. Woch., 36 p. 3og.
^ Buckmaster considers the use of potassium chloride preferable.
* Care should be taken not to add too great an excess of these reagents.
' This process insues constancy of temperature and strength of reagents.
BLOOD AND LYMPH.
211
>f
^ W^<^ WrA '<>^
Fig. 59. — H^MiN Crystals from Human Blood.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University of
Pennsylvania.
^"^Z
Fig. 60. — H.EMIN Crystals from Sheep Blood.
Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University of
Pennsylvania.
212 PHYSIOLOGICAL CHEMISTRY.
dissolve the sodium chloride crystals by running a drop of water under
the cover slip.
(c) V. Zeynek and Nencki^s Method. — To lo c.c. of defibrinated
blood add acetone until no more precipitate forms. Filter off the pre-
cipitated protein and extract it with lo 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 microscope. 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 stoppered vessel
and allowing it to remain overnight.
(d) Schalfijew^s Method. — ^Place 20 c.c. of glacial acetic acid in a
small beaker and heat to 80° 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. 59 and 60, page 211.
15. 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?
16. Preparation of Haematin. — ^Place 100 c.c. of hcemolyzed {laked)
blood in a beaker and add 95 per cent alcohol until precipitation ceases.
What bodies are precipitated? Transfer the precipitate 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
protein 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
spectroscopic examination later after becoming familiar with the use of
the spectroscope. How does the spectrum of oxy haemoglobin differ from
that of the derived alkali hcematin?
17. Variation in Size of Erythrocytes. — Prepare two small funnels
with filter papers such as are used in quantitative analysis. Moisten each
paper with physiological (isotonic) salt solution. Into one funnel intro-
duce a small amount of defibrinated 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 ?
BLOOD AND LYMPH.
II. Blood Serum.
213
1. Coagulation Temperature. — ^Placc 5 c.c. of undiluted serum in a
test-tube and determine its temperature of coagulation according to the
method described on page 106. Note the temperature at which a cloudi-
ness occurs as well as the temperature 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 protein. Explain the result.
3. Proteins of Blood Serum.— Place about 10 c.c. of serum in a
small evaporating dish, dilute with 5 c.c. of water and heat to boiling.
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) Millori's Reactian. — Make the test according to directions given
on page 97,
{h) Hopkins-Cole Reaction. — Make the test according to directions
given on page 98.
tJ^ /'^.
i^ J '
Fig. 61. — Sodium Chloride.
4. Sugar in Serum. — Test a little of the filtrate from Experiment 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 silver
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. 61, above.
* For directions as to preparation of serum, see Appendix.
214 PHYSIOLOGICAL CHEMISTRY.
6. Separation of Serum Globulin and Serum Albtmiin. — Place
lo 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 con-
clude ?
III. Blood Plasma.
1. 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 solution. Note the pre-
cipitation of fibrinogen. Filter off the precipitate (reserve the filtrate)
and test it by a protein color test (see page 97).
3. Effect of Calcium Salts. — 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 cool 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 Oxyhaemoglobin. Reicheri's Method. — Add
to 5 c.c. of the blood of the dog, horse, guinea-pig, or rat, before or after
laking, or defibrinating, from i to 5 per cent of ammonium oxalate in
substance. Plcice a drop of this oxalated blood on a slide and examine
under the microscope. The crystals of oxyhsemoglobin will be seen to
form at once near the margin of the drop, and in a few minutes the entire
drop may be a solid mass of crystals. Compare the crystals with those
shown in Figs. 51 to 57, pages 198 to 201.
IV. Fibrin.
I. 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 constructed 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 further traces of
blood. The pure product should be very light in color. It may be pre-
served under glycerol, dilute alcohol, or chloroform water.
BLOOD AND LYMPH. 215
2. Solubility. Try the solubility of small shreds of freshly prepared
li])rin in the usual solvents.
3. Millon's Reaction. — Make the test according to directions given
on page 97.
4. Hopkins-Cole Reaction. — Make the test according to directions
given on page g8.
5. Biuret Test. — Make the test according to directions given on page
98.
V. Detection of Blood in Stains on Cloth, Etc.
1, Identification of Corpuscles. — If the stain under examination is
on cloth a i)ortion should be extracted with a few drops of glycerol or
physiological (0.9 per cent) sodium chloride solution. A drop of this
solution should then be examined under the microscope to determine if
corpuscles arc 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:
{a) HcBmochromogen. — Make a small amount of the extract 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 216) and make a spectroscopic examination.
Compare the spectrum with that of haemochromogen (see Absorption
Spectra, Plate II).
{h) HcBmin Test. — Make this test upon a small drop of the aqueous
extract according to the directions given on page 210.
(c) Guaiac Test. — Make this test on the aqueous extract according to
the directions given on page 209. The guaiac solution may also be
applied directly to the stain without previous 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) Benzidine Reaction. — Make this test according to directions given
on p. 209.
(e) Acid Hamatin. — If the stain fails to dissolve in water extract with
acid alcohol and examine the spectrum for absorption bands of acid
haematin (see Absorption Spectra, Plate II).
* VI. Spectroscopic Examination of Blood.
(For Absorption Spectra see Plates I. and II.)
Either the a7igiilar-\ision spectroscope (Figs. 63 and 64, page 217) or
the direct-\\%\on spectroscope (Fig. 62, page 216) may be used in making
2l6 PHYSIOLOGICAL CHEMISTRY.
the spectroscopic examination of the blood. For a complete description
of these instruments the student is referred to any standard text-book of
physics.
I. Oxyhaemoglobin. — Examine dilute (1:50) defibrinated blood
spectroscopically. Note the broad absorption-band between 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 oxyhaemoglobin obtained from dilute
solutions of 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. 62." — Direct- VISION Spectroscope.
2. Haemoglobin (so-called Reduced Haemoglobin). — To blood which
has been diluted sufficiently to show well-defined oxyhaemoglobin absorp-
tion-bands add a small amount of Stokes' reagent. ^ The blood immedi-
ately changes in color from a bright red to violet-red. The oxyhaemo-
globin has been reduced through the action of Stokes' reagent and haemo-
globin (so-called reduced haemoglobin) has been formed. This has been
brought about by the removal of some of the loosely combined oxygen
from the oxyhaemoglobin. Examine this haemoglobin spectroscopically.
Note that in place of the two absorption bands of oxyhaemoglobin we now
have a single broad band lying almost entirely between D and E. This
is the typical spectrum of haemoglobin. If the solution showing this
spectrum be shaken in the air for a few moments it will again assume the
bright red color of oxyhaemoglobin and show the characteristic spectrum
of that pigment.
3. Carbon Monoxide Haemoglobin.— The peparation of this pig-
ment may be easily accomplished by passing ordinary illuminating gas^
through defibrinated ox-blood. Blood thus treated assumes a brighter
tint (carmine) than that imparted by oxyhaemoglobin. In very dilute
solution oxyhaemoglobin appears yellowish-red whereas carbon monoxide
haemoglobin under the same conditions appears bluish-red. Examine the
' 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 ferrolartrale which is a reducing agent.
* The so-called water gas with which ordinary illuminating gas is diluted contains usually
as much as 20 per cent of carbon monoxide (CO).
BLOOD AND LYMPH.
217
carbon monoxide haemoglobin solution spectroscopically. Observe that
the spectrum of this body resembles the spectrum of oxyhaemoglobin in
showing two absorption-bands between D and E. The bands of carbon
Fig. 63. — Angular-vision Spectroscope Arranged for Absorption Analysis.
monoxide haemoglobin, however, are somewhat nearer the violet end of
the spectrum. Add some Stokes' reagent to the solution and again
examine spectroscopically. Note that the position and intensity of the
absorption-bands remain unaltered.
Fig. 64. — Diagram of Angular- vision Spectroscope. iLong.)
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 eye-piece 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 following is a delicate chemical test for the detection of carbon
monoxide haemoglobin :
Tannin Test. — Divide the blood to be tested into two portions and
dilute each with four volumes of distilled water. Place the diluted blood
2i8 PHYSIOLOGICAL CHEMISTRY.
mixtures in two small flasks or large test-tubes and add 20 drops of a 10
per cent solution of potassium ferricyanide/ Allow both solutions to
stand for a few minutes, then stopper the vessels and shake one vigorously
for 10-15 minutes, occasionally removing the stopper to permit air to
enter the vessel.^ Add 5-10 drops of ammonium sulphide (yellow) and
10 c.c. of a 10 per cent solution of tannin to each flask. The contents of
the shaken flask will soon exhibit the formation of a dirty olive green
precipitate, whereas the flask which was not shaken and which, therefore,
still contains carbon monoxide haemoglobin, will exhibit a bright red
precipitate, characteristic of carbon monoxide haemoglobin. This
test is more delicate than the spectroscopic test and serves to detect
the presence of as low a content as 5 per cent of carbon monoxide
haemoglobin.
4. Neutral Methaemoglobin. — Dflute a little defibrinated blood
(i : 10) and add a few drops of a freshly prepared 10 per cent solution of
potassium ferricyanide. Shake this mixture and observe that the bright
red color of the blood is displaced 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 occupied by the absorp-
tion bands of oxy haemoglobin. Add a few drops of Stokes' reagent to the
methaemoglobin solution while it is in position before the spectroscope
and note the immediate appearance of the oxyhaemoglobin spectrum
which is quickly followed by that of haemoglobin.
5. Alkaline Methaemoglobin. — Render a neutral solution of met-
haemoglobin, such as that used in the last experiment (4), slightly alkaline
with a few drops of ammonia. The solution 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 mentioned, lies
between D and E somewhat nearer E.
6. Alkali Haematin. — Observe the spectrum of the alkali haematin
prepared in Experiment 16 on page 212. Also make a spectroscopic
examination of a freshly prepared alkali haematin.^ The typical spec-
trum of alkali haematin shows a single absorption-band lying across D
and mainly toward the red end of the spectrum.
' This transforms the oxyha.-moglobin into mc-tha-moglobin.
^ This is done to free the blood from carbon monoxide haemoglobin.
" Alkali htematin may be prepared by mixing one volume of a concentrated potassium
hydroxide or sodium hydroxide solution and two volumes 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.
HLOOD AM) l.VMFH. 219
7. Reduced Alkali Haematin or Haemochromogen. — Dilute the
alkali haematin solution used in the last exj)eriment (6) to such an extent
that it shows no absorption band. Now add a few drops of Stokes'
reagent 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 hsemochromogen or reduced alkali hcTmatin. Examine this solution
spectroscopically and observe the narrow, dark absorption-ljand lying
midway betweCVi D and E. If the dilution is not too great a faint band
may be observed in the green extending across E and I).
8. Acid Haematin. — To some delibrinated blood add half its vol-
ume of glacial acetic acid and an equal volume of ether. Mix thor-
oughly. The acidified ethereal solution of haematin rises to the top and
may be poured off and used for the spectroscopic 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 methaemoglobin 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 thoroughly by agitation
after the addition of each drop. A wine-red solution is produced. Ex-
amine this solution spectroscopically. 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 haemato-
porphyrin solution just examined into an excess of distilled water. Cool
the solution and add potassium hydroxide 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
facilitates the formation of this precipitate. Filter off the precipitate 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
220
PHYSIOLOGICAL CHEMISTRY.
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. 65, below). — This is an instrument
used quite extensively clinically, for the quantitative determination of
hemoglobin. The instrument consists of a small cylinder which is pro-
vided 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
mechanism which resembles the base and
stage of an ordinary microscope. Under-
neath the stage is placed a colored glass
wedge (see Fig. 67, p. 221), so arranged
as to run immediately beneath the glass
bottom of one of the compartments of the
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 of this frame indicates the percentage of the normal
amount of haemoglobin which each particular variation in the depth of
color of the ground wedge represents, taking the normal haemoglobin
content as 100.'^ In a position corresponding to the position of the
mirror on the ordinary microscope is attached a light-colored opaque
plate which serves to reflect the light upward through the colored wedge
and the cylinder to the eye of the observer.
In making a determination of the percentage of haemoglobin by
this instrument the procedure is as follows: Fill each compartment
about three-fourths full of distilled water. Puncture 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. 66), 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 determination inaccurate, it is
customary to apply a very thin coating of mutton fat to the outer surface
before using or to 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
* The scale of the ordinary instrument is usually too high.
Fig. 65.
-Fleischl's H^mometer.
{Da Costa.)
BLOOD AND LYMPH.
221
Fig. 66. — Pipette
OF Fleischl's
H^MOMETER.
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 distributed
throughout the compartment and docs not form a homogeneous solution
the contents of the compartment should be mixed thoroughly by means
of the metal handle of the capillary measuring pipette. When this has
been done eacli, 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
containing 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 cor-
responding to this particular color should be read and the actual per-
centage of hcxmoglobin computed. For instance, if the scale reading is
90 it means that the blood under examination contains 90 per cent of the
normal quantity of haemoglobin, 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 precautions are taken,
the margin of error in the absolute quantity 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: (i) The scale of each instrument is supplied with
a caliber table of absolute haemo-
globin values, expressed in milli-
grams: the scale of Fleischl's haemo-
meter shows the percentage of
haemoglobin in relation to an
average selected somewhat arbi-
trarily. Thus many errors arising
from the irregular coloring of the
glass wedge of the older apparatus are avoided in the instrument as
modified. (2) Each instrument is accompanied by a measuring pipette
(melangeur) which allows of a more accurate measurement 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 concentration may be compared. In this way the individual
examinations are controlled and a check upon the accuracy of the gradu-
FiG. 67. — Colored Glass Wedge of
Fleischl's H^mometer. {Da Cosla.)
222
PHYSIOLOGICAL CHEMISTRY.
ation 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 (see page 225).
The capillary portion, however, is
graduated, i, 2/3 and 1/2 which
enables the observer to dilute the
blood sample in the proportion of
1:200, 1:300 or 1:400 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 gradu-
ation may be determined by means
of certain delicate cross-lines which
are placed directly above and below
the graduation. Each cross-line
corresponds to i/ioo of the volume
of the capillary tube from the tip
to the I graduation.
A o. I per cent solution of sodium
carbonate is used to dissolve 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 carbonate 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. 68). — This instrument, as
the name signifies, is used for the determination of haemoglobin. In
using either Fleischj's haemometer or the instrument as modified by
Miescher the blood is diluted 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
apparatus.
-Dare's H^moglobinometer.
{Da Costa.)
R, Milled wheel acting by a friction bear-
ing on the rim of the color disc; S, case in-
closing color disc, and provided with a stage
to which the blood chamber is fitted ; T, mov-
able wing which is swung outward during the
observation, to serve as a screen for the ob-
server'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
position for examination; V, aperture admit-
ting 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 indicated on the
rim of the color disc is read.
BLOOD AND LYMPH.
223
The instrument consists essentially of the following parts: (i) A
capillary observation cell, (2) a semicircular colored glass wedge, (3)
a milled wheel for manipulating the wedge, (4) a candle used to illuminate
portions of the capillary 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 percentages 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, polished rectangular plates of glass,
one being transparent and the other opaque.
When held in position on the instrument, 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-
FiG. 69. — Horizontal Sec-
tion OF Dare's H^emoglo-
BiNOMETER. {Da Cosia.)
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 par-
ticular 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 be 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
Fig. 70.— Method of Filling the Capillary Observation Cell of Dare's Hj:mo-
GLOBINOMETER. (Da Cosla.)
blood specimen is viewed. For a further description of the instrument
see Figs. 68, 69, and 70.
In using the Dare haemoglobinometer proceed as follows: Puncture
the tmger-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
224
PHYSIOLOGICAL CHEMISTRY.
the flat capillary observation cell in contact with the drop and allow it to
fill by capillary attraction (Fig. 70) . 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 semicircular colored glass wedge is visible. These
two circles are 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 haemometer. The scale
reading gives the percentage of the normal quantity of haemoglobin which
the blood sample under examination contains. Compute the actual
haemoglobin contfent in the same manner as from the scale reading of the
Fleischl haemometer (see page 221).
4. Tallquist's Haemoglobin Scale. — This consists essentially of
a series of ten colors corresponding to stains produced by blood con-
taining 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 ac-
companying haemoglobin value is read off directly. This is a very con-
venient method for determining haemoglobin at the bedside. There
is a possibility of the colors being inaccurately printed, however, and
even if originally correct in tint, under the continued influence of air and
light they must eventually alter somewhat.
Fig. 71. — Thoma-Zeiss Counting Chamber. (Da Costa.)
5. Thoma-Zeiss Haemocytometer. — This is an instrument used
in "blood counting," i. e., in determining the number of erythrocytes
and leucocytes. The instrument consists of a microscopic slide con-
structed of heavy glass and provided with a central counting cell (see
Fig. 71, 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
BLOOD AND LYMPH.
22i
U
squares each of which is i 400 of a square millimeter in area (see Fig.
73, page 22O). The volume of blood therefore between any particular
square and the cover glass above must be 1/4000 cubic millimeter. Ac-
companying each instrument are two capillary i)ipetles (Fig. 72. below),
each constructed with a mixing bulb in its upper por-
tion. 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 graduation at the upper end of the bulb is 10 1
on the pipette used in mixing the blood sample in
which the erythrocytes are counted (erythrocytom-
eter, see Fig. 72, page 225), and 11 on the pipette
used in mixing the blood sample for the leucocyte
count (leucocytometer, see Fig. 72, page 225). In
making ''blood counts" with the haemocytometer it is
necessary to use some diluting fluid. Two very satis-
factory forms of fluid for this purpose are Toison's
and Sherrington's solutions.^ When either of these
solutions is used as the diluting fluid it is possible to
make a very satisfactory count of both the erythro-
cytes 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 haemo-
cytometer, proceed as follows: Thoroughly cleanse the
tip of the finger or lobe of the car of the subject by
the use of soap and water, alcohol and ether applied
in the sequence just given. Puncture 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 10 1 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
V
B
Fig. 72. — Thoma-
Zeiss Capillary
Pipettes.
A,Erythrocytometer;
B, Leucocytometer.
' Toison"< solution has the following
formula:
Methyl-violet 0.025 gram.
Sodium chloride i gram.
Sodium sulphate 8 grams.
Glycerol 30 grams.
Distilled water i6o grams.
Sherrington's solution has the follo\Aing
formula :
Methylene-blue o . i gram.
Sodium chloride 1.2 gram.
Neutral potassium oxalate.... i .2 gram.
Distilled water 300.0 grams.
226
PHYSIOLOGICAL CHEMISTRY.
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 contained 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 examination. Transfer a drop from
the pipette to the ruled floor of the counting chamber and, after placing
the cover glass firmly in position/ allow an 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 number of squares, counting the corpuscles which are in contact
Fig. 7.-
-Ordinary Ruling of Thoma-Zeiss Counting Chamber. {Da Costa.)
with the upper and the right-hand boundaries of the square as belonging
to that square. Take the squares in some definite sequence in order that
the recounting of the same corpuscles may be avoided. A satisfactory
procedure is to begin in the upper right-hand corner and proceed from
left to right counting the cells in each individual square. Take the next
lower row of squares and count from left to right and so on (see Fig. 77,
p. 232). Of course, all things being equal, the greater the number of
squares examined the more accurate the count. It is considered essen-
tial 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. Under no conditions should less than 200 squares
be examined.
To calculate the number of erythrocytes per cubic millimeter of
undiluted blood proceed as follows: Determine the number of corpuscles
' If the cover glass is in accurate apposition to the counting cell Newton's rings may be
plainly observed.
BLOOD AND LYMPH.
227
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 cubic millimeter of diluted blood, and multiply this product by 100
or 200, according to the dilution, to obtain the number of erythrocytes
per cubic millimeter of undiluted blood. Thus:
Average number .,of erythrocytes
per square
^, , N Xumher of erythrocytes per
X 4,000 X 200 (or 100)= ,. .,,. .- ■' '
^' ^ cubic milhmeler.
Great care should be taken to see that the capillary pipette is prop-
erly cleaned. After using, it should be immediately rinsed out with the
Fig. 74. — Zappert's Modified Ruxing of Thoma-Zeiss Cox.'xting Chamber. {Da Costa.)
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 haemocytometer proceed as
follows: As mentioned above, if the diluting fluid is either Toison's or
Sherrington's solution the leucocytes may be counted in the same specimen
of blood in which the erythrocytes are counted. When this is done it
is customary to use a slide provided with Zappert's modified ruling
(Fig. 74, above). This method is rather more accurate than the older
one of counting the leucocytes in a separate specimen of blood. Further-
more, it is obviously preferable to count both the erythrocytes and the
leucocytes from the same blood 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 eigh-
teen times as great as the old style Thoma-Zeiss central ruling. This
228 PHYSIOLOGICAL CHEMISTRY.
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 7,200 ^. ^. Number of leucocytes per cubic
' X200X4, 000-^7,200= .,,. , ' ^
squares ^ ' millimeter.
If a Zappert slide is not available, a good plan to follow is to place a
diaphragm in the tube of the ocular of the microscope consisting of a circle
of black cardboard or metaP having a square hole in the center 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 wdthin or without the
ruled area. In counting by means of this device it is, of course, helpful
if the microscope is provided with a mechanical stage, but even without
this arrangement, if the observer is careful to see that the leucocytes at
the extreme boundary of one field move to the opposite boundary when the
position of the slide is changed, the device may be very satisfactorily em-
ployed. 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 leuco-
cytes alone remain visible. Under these conditions the dilution is
customarily made in the pipette having 11 as the final graduation. The
capillary portion is of larger caliber and so rec^uires a greater amount of
blood to fill it to the 0.5 or i mark than is recjuired in the use of the other
form of pipette. In counting the leucocytes according to this method it
is customary to draw blood into the pipette up to the i mark and immedi-
ately fill the remaining portion of the apparatus to the 1 1 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 Soo . . ^, „ Number of leucocytes per cubic
■' X 4,000 X 10-^-800= .,,. . - '
squares millimeter.
6. Biirker's Haemocytometer.- — This is an improved apparatus^ for
the more accurate counting of erythrocytes than is possible by the
Thoma-Zeiss apparatus. The principles involved arc somewhat dif-
ferent from those in force with the latter apparatus. For example, the
blood is diluted in a separate vessel, not in the pipette with which the
sample is drawn, and furthermore the cover glass is applied to the counting
' Ehrlich's mechanical eye-piece with iris diaphragm is also yery satisfactory for this
purpose.
^ Biirker: Pfliiger's Archiv., 142, 337, 191 1; Miinch. med. Woch., 59, pp. 14 and 89, 1912.
' Manufactured by C. Zeiss, Jena.
BLOOD AM) I.V.MPH.
229
chamber and clamped in place before the diluted blood is applied to the
ruled area. Hayem's solution' is used as the diluting fluid. Toison'r-
solution is not satisfactory for use with the Hiirker counting chamber as
its viscosity is too great. The corpuscles settle rapidly in Hayem's fluid
as the specific gravity of the fluid is 1015 whereas that of the erythrocytes
is iO()o.
The pipet^te for measuring the cjuantily of blood (F'ig. 75, uj)per
pipette) has a point which is not ground dull but is polished. This
Fig. 75. — Burker's 1'ipkttks, Mixing Flasks and C )inting Chamber.
allows of better judgment in deciding whether the column of blood ex-
tends to the very tip. The volume of the pipette between tip and mark
is 25 cubic millimeters. The mark extends all the way around the tube
so that errors of parallax may be avoided.
The pipette for measuring the diluting fluid (Fig. 75, middle pipette)
also has a polished point and circular mark and delivers 4975 cubic
millimeters. This volume of diluting fluid with 25 cubic millimeters of
blood gives a dilution of i :20c. Both pipettes are provided with a piece
of rubber tubing and mouth-piece.
' Hayem's solution has the following formula:
Mercuric chloride 0.25 gram.
Sodium chloride 0.5 gram.
Sodium sulphate 2.5 grams.
Distilled water 100 c yrams.
230 PHYSIOLOGICAL CHEMISTRY.
For transferring the diluted blood from the diluting flask to the
chamber a plain pipette provided with a rubber cap is used (Fig. 75,
lower pipette). It is filled by pressing the cap slowly with the index
finger, inserting the tip into the lic|uid and then releasing the pressure.
The diluting is done in a small round-bottomed flask as shown in
Fig. 75. Several of these flasks should be kept on hand in a wooden
rack which will hold them in an upright position. Each flask is provided
with a parafi&ned, or smooth cork stopper.
In the older counting chambers the floor of the chamber is circular and
the counting is done in the center of this space. The corpuscles are
therefore counted in the center of a capillary, circular film where on
account of surface tension their number is slightly greater than elsewhere.
This source of error is avoided in the new counting chamber (Fig. 78) in
which the floor is represented by the upper surface of a piece of glass 25
mm. long and 5 mm. wide which is rounded off at both ends and divided
into two portions by a groove 1.5 mm. wide through the center. At each
side of this floor piece, separated from it by a groove is a glass plate
(7.5 mm. X21 mm.) of such height that the space between the floor of
the cell and a cover glass placed across the plates is o.ioo mm. A cover
glass 23 mm. long and 21 mm. wide with rounded polished edges is
used so that the rounded ends of the floor piece project beyond it. The
chamber is provided with clamps to press the cover glass firmly upon
both plates (Fig. 75).
The ruling on each portion of the floor piece is that shown in Fig. 76,
which will be explained below.
Measuring the Diluting Fluid. — Four thousand nine hundred and
seventy-five cubic millimeters of diluting fluid (Hayem's) are measured
out into the diluting flask. To do this the pipette is filled by suction to
slightly above the mark and the rubber tube is carefully clamped off.
Then with a soit piece of linen the tip is wiped dry. The meniscus is
then accurately adjusted to the mark by lightly touching the point of the
pipette to the cleaned tip of the finger. The pipette is then inserted into
the diluting flask and with the tip nearly touching the bottom of the flask
the fluid is allowed to run out. The time of the flow should be about
forty seconds and is controlled by placing the tip of the index finger
loosely upon the mouth piece. The pipette is emptied completely by
alternately blowing through it and touching it to the wall of the flask
slightly above the level of the liquid. The drops clinging to the wall are
united with the bulk of the Hquid by a suitable motion of the flask. The
flask is then stoppered, care being taken from now on that none of the
liquid ever touches the neck of the flask or the stopper.
Taking the Blood Sample. — Usually the best time to draw the blood is
BLOOD AND LYMPH. 23 1
before breakfast. For a single determination the author prefers to draw-
it from the tip of the fourth finger of the left hand. For repeated deter-
minations it is well to change off between third, fourth and fifth fingers of
left hand. The temperature of the room should not be below 17° C.
to prevent an undue contraction of the cutaneous vessels. The instru-
ment used to puncture the finger should have a chisel-shaped point which
is preferable to the ordinary lancet-shaped point. The first drop of blood
is wiped off. Into the second one the tip of the pipette is inserted and
blood is drawn in until the meniscus is even with or a little beyond the
mark. The tip is then wiped off without touching the capillary opening
and the observer assures himself that the column of blood extends to the
very end of the capillary. The meniscus is then accurately adjusted to
the mark.
Mixing of the Blood and Diluting Fluids. — The tip of the pipette is now
dipped into the diluting fluid which has been measured into the llask and
the blood is slowly blown out. The blood having a much higher specific
gra%-ity than the Hayem's fluid sinks to the bottom. The pipette is then
filled with the pure supernatant diluting fluid and emptied again, care
being taken to avoid air bubbles. This is repeated until the blood is
removed as completely as possible. To mix the blood and diluting fluid
the flask is rotated for two minutes in spiral curves of continually decreas-
ing radius. The motion should be alternately clockwise and counter-
clockwise. After complete mixing the pipette is rinsed out several times
with the diluted blood.
Transferral of the Diluted Blood to the Chamber. — The counting cham-
ber which has been cleaned with distilled water and alcohol-ether and
then wiped dry with a soft cloth as free from lint as possible is placed upon
a black surface and carefully brushed with a camel's hair brush. The
cover glass is now placed over the chamber by sliding it over the two
glass plates with both thumbs while the index fingers are pressing it
down. By means of the clamps it is held in place firmly so that Newton's
rings (if possible of the first order: brown and black) may be seen over
the entire area of the plates. The chamber is placed upon the stage of the
microscope and is brought into a horizontal position.
Before transferring the diluted blood to the chamber the flask must be
shaken for two minutes as described before. The liquid shows a cloudy
appearance and must be allowed to stand until the turbidity has become
uniform.
One of the plain pipettes described above is now inserted into the
diluted blood while slight pressure is being exerted on the rubber cap.
The pressure is released slowly and the liquid rises into the pipette. The
point of the pipette is now immediately placed upon one of the projecting
232
PHYSIOLOGICAL CHEMISTRY.
ends of the floor plate and very slight pressure is exerted on the rubber
cap until the liquid coming from the pipette just reaches the cover glass
when the pressure is released. An instantaneous filling of the capillary
Fig. 76. — Ruling of Bdrker Counting Chamber.
\5 C
5
1
3
13
^1
1
-^
—
^
^5
5
-^
^
-»
«-9
9
"
-^
^
^
<^13
13
1
5
t
J
>
I.
J
Fig. 77. — Schema.
space results. The pipette should be emptied immediately, rinsed with
distilled water and placed in an upright position in a beaker of water.
The other portion of the counting chamber is now filled in the same way
HI.OOI) AM) LYMPH.
^2>3
with a second pipette and about one minute is allowed for the settling of
the corpuscles. During this time the jjipettes may be washed with dis-
tilled water and ether-alcohol and dried In- suction. Occasionally, the
pipettes should l)e cleaned with a horse hair and with concentrated II.^SO^
containing a little KXr.O^.
To see whether the distribution of the corpuscles has been uniform
the chamber is Hluminated with a wide-oj)en diaj)hragm and viewed at an
angle. If the opacity is not uniform in either of the portions of the
chamber, that one should not be used for counting. If the counting must
be interrupted or requires a longtime a moist chamber' should be used to
prevent evaporation of the diluting fluid. The diluted blood may be
retained in the mixing flasks and duplicate countings obtained after the
lapse of twenty-four hours or niore according to Biirker.
Tiefe
O.lOOmni.
C.Zeiss
Jerta
kJ
©
O.Olmm.
I I
Fig. 78. — BuRKER Counting Ch.\mber.
Counting and Calculation. — A mechanical stage movable in two direc-
tions is indispensable. With a magnification of 320 diameters the
counting is begun in the left upper corner of the ruling. Proceed from
left to right along one row then move from right to left along the next
lower row, and so on. Only the small sc|uares are used for counting (see
Fig. 76), and the figures are recorded in the schema'- (see Fig. 77) in which
the squares crossed by horizontal or vertical lines correspond to the small
squares used for counting. Usually 80 squares are counted and by
recording the figures in the schema the count may be verified and an idea
' Biirker: Pfliiger's Archiv, iiS, 465, IQ07.
- The firm of II. Laupp in Tubingen has put this schema on the market (in pac ks of 100)
234 PHYSIOLOGICAL CHEMISTRY.
of the uniformity of the distribution may be formed. Half of the counted
squares should be in the one, half in the other portion of the counting
chamber. For more accurate measurements more squares may be
counted.
The observer will do well not to attempt counting each individual
corpuscle in a square. After some practice each typical group of cor-
puscles will immediately suggest a number. A very common form of
grouping is one corpuscle surrounded by four others. This should
immediately suggest the number five. In this way the counting will
become more rapid and also more reliable.
The calculation is very simple. The number of corpuscles in 80
squares di\dded by 100 will give the number of millions per cubic milli-
meter. If, for example, 536 corpuscles have been counted in 80 squares
then with a dilution of i : 200 the number of corpuscles per cubic milli-
meter is 5,360,000. Thus, ■^^^— X4,oooX2oo =5,360,000 erythrocytes per
80
cubic millimeter. More than two decimal places are without significance.
CHAPTER XIII.
MILK.
Milk is the most satisfactory individual food material elaborated by
nature. It contains the three nutrients, protein, 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, olein, palmitin,
stearin, butyrin, caseinogen, lact-alhumin, laclo- globulin, lactose, and calcium
phosphate. It also contains at least traces of lecithin, cholesterol, urea,
creatine, creatinine, and the tri-glycerides of caproic, lauric, and myristic
acids. Citric acid is also said to be present in milk in minute quantity.
Considered from the standpoint of colloid chemistry we may classify the
main constituents of milk as follows:^
In suspension Fat (olein, palmitin, etc.).
r Caseinogen — an unstable or irreversible
In colloidal solution colloid.
[ Lact-alhumin — a stable or reversible colloid.
^ ^ 11 -1 1 X- [ Salts (calcium phosphate, etc.).
In crystalloid solution I ^ ;, t- t > j
[ Sugar (lactose).
Fresh milk is amphoteric in reaction to litmus,' 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 consisting
of a combination of caseinogen forms on the surface. If the film be
' Alexander and Bullowa: Jour. Am. Med. Ass'n., 55, 1196, igio.
- Human milk as well as cow's milk. It is, however, acid to phenolphthalein.
235
236 PHYSIOLOGICAL CHEMISTRY.
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 of fat facilitate the formation
of the film, but are not essential (Rettger\). As Jamison and Hertz-
have shown, a similar film will form on heating any protein solution con-
taining fat or parafiin. If the milk is acid in reaction, through the inception
of lactic acid fermentation, or from any other cause, no film will form when
heat is applied, but instead a true coagulation will occur. When milk is
boiled certain changes occur in its odor and taste. These changes,
according to Rettger,^ are due to a partial decomposition of the milk
proteins and are accompanied by the liberation of a volatile sulphide,
probably hydrogen sulphide.
Fig. 7g. — Normal Milk and Colostrum.
a, Normal milk; h, Colostrum.
The milk-curdhng 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 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 sur-
rounding the curd is known as whey.
There is still considerable confusion of terms when different authorities
discuss milk proteins and the actibn of milk curdling enzymes upon them.
The English-speaking scientists ^uite uniformly accept the classification
of Halliburton'' as given above. On the other hand, the Germans in
particular give the name casein to the milk protein and paracasein to the
' Rettger: American Journal of Physiolof^y, 7, 325, 1902.
^ JamLson and Hertz: Journal of Fhysiology, 27, 26, igo2.
' Rettger: American Journal oj Physiology, 6, 450, 1902.
'Halliburton: Journal of Physiology, 11, 448, 1900.
MILK. 237
product of the action of rennin upon this protein. The confusion of
terms may be represented thus:
English. German.
Caseinogen. = Casein.
Casein. • = Paracasein.
The most ])ronounced difference between human milk and cow's milk
is in the proteintontent, although there are also differences in the fats and
likewise striking biological differences difficult to define chemically. It
has 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 gastric 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 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
investigations by Proscher^ and by Abderhalden- in Bunge's laboratory.
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.
Period in which
„ . Weight of the
Species. New-born is
100 Parts of Milk Contain
Doubled (Days). Proteins. Salts. Calcium. ^^'Jj;^''"''
Man ' 180 1.6 0.2 0033 0047
Horse 60 2.0 0.4 0.124 0131
Cow 47 3.5 0.7 0.160 0.197
Goat 22 3.7 0.8 o 197 0.284
Sheep 15 4.9 0.8 0.245 0.293
Pig 14 5.2 0.8 0.249 0.308
Cat 9.5 7.0 i.o
"Dog.. 9 7.4 1.3 0.455 0.508
Rabbit 6 10.4 2.5 0.891 0.997
The secretion of the mammary glands of the newborn of both sexes
is called '"witches' milk." The name is centuries old and evidently
refers to the mystery of the useless secretion. Basch^ has recently sug-
gested that this secretion of "wtches' milk" is brought about by the
passage of hormones (see chapter on Pancreatic Digestion) from the blood
of the mother to the fetus.
Lactose, the principal carbohydrate constituent of milk, is an impor-
' Proscher: Zeit.f. physiol. Chemie, 24, 285, 1898.
^ .\bderhalden: Ibid.. 26, 487, 1899; ^"^1 27, pp. 408 and 457, 1899.
* Basch: Miinch. med. Woch., 58, 2266, 191 1.
238 PHYSIOLOGICAL CHEMISTRY.
tant member of the disaccharide group. It occurs only in milk, except as
it is found in the urine of women during pregnancy, during the nursing
period, and soon after weaning; it also occurs in the urine of normal per-
sons 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 phenylhydrazine. 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. It was recently claimed that lactosin, a new carbohy-
drate, had been isolated from milk.
Fig. 80. — Lactose.
Caseinogen, the principal protein constituent of milk, belongs to the
group of phosphoproteins. It has acidic properties and combines with
bases to produce salts. It is not coagulable upon boihng and is precipi-
tated from its neutral solution by certain metallic salts as well as upon
saturation with sodium chloride or magnesium sulphate. Its acid solu-
tion is precipitated by an excess of mineral acid.
Lactalbumin and lacto-globulin, the protein constituents of milk,
next in importance to caseinogen, closely resemble serum albumin and
serum globulin in their general properties. According to Wroblewski,
a protein called opalisin is also present in milk.
Butter (milk fat) consists in large part of olein and palmitin. Stearin,
butyrin, caproin and traces of other fats arc also present. When butter
becomes rancid through the cleavage of certain of its constituent fats by
bacteria the odors of caproic and butyric acids are in evidence.
Colostrum is the name given to the product of the mammary gland
MILK. 239
secreted for a short time before parturition and during the early period
of lactation (see Fig. 79, p. 236). It is yellowish in color, contains more
solid matter than ordinary milk, and has a higher specific granty (1.040-
1.080). The most striking dilTerence between colostrum and ordinary
milk is the high percentage of lactalbumin and lacto-globulin in the
former. This abnormality in the protein content is responsible for the
coagulation of colostrum upon boiling.
Such enzymes as Hpase, amylase, galactase, catalase, oxidases, peroxi-
dases, and reductases have been identified in milk, but not all of them
in milk of the same species of animal.
Among the principal preservatives used in connection with milk are
formaldehyde, hydrogen peroxide, boric acid, borates, salicylic acid,
and salicylates.
Experiments on Milk.
1. Reaction. — Test the reaction of fresh cow's milk to litmus,
phenol phtJialcin and co7igo red.
2. Biuret Test. — Make the biuret test according to directions given
on page 98.
3. Microscopical Examination. — Examine fresh whole milk,
skimmed or centrifugated milk, and colostrum under the microscope.
Compare the microscopical appearance with Fig. 79, page 236.
4. Specific Gravity. — Determine the specific gravity of both whole
and skimmed milk (see p. 278). 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 coagu-
lation 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 boiling. Do you get
any coagulation ? Why ?
7. Action of Hot Alkali.— To a little milk in a test-tube add a few
drops of potassium hydroxide 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-tube add
a few drops of very dilute nitric acid to form a precipitate. Filter off this
precipitate and test the filtrate for chlorides. Does milk contain any
chlorides ?
240 PHYSIOLOGICAL CHEMISTRY.
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 1:60)^
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 60° 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 perox-
ide 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 guaiac without the addition of hydrogen peroxide or old tur-
pentine. See discussion on page 204.
10. Tests to Differentiate Between Raw Milk and Heated Milk.—
(a) Kastle's Peroxidase Reaction. — The peroxidase reaction of milk is
founded upon the fact that small amounts of raw milk will induce the
oxidation of various leuco compounds by hydrogen peroxide. This
reaction has been used in a practical way as the most convenient means
of differentiating between raw milk and heated milk. Many substances
have been employed for this purpose, e. g., guaiac, paraphenylenediamine,
ortol, amidol, etc. Kastle has found that a dilute solution of "trikresol"^
acts as a sensitizing agent in the peroxidase reaction and offers the follow-
ing test which is based upon this fact: To 2-5 c.c. of raw milk in a test-
tube add 0.1-0.3 c-c. of M/io hydrogen peroxide and i c.c. of a i per cent
solution of "trikresol." A slight though unmistakable yellow color will
be observed to develop throughout the solution.
Repeat the test using milk which has been boiled or heated to 80° C.
for 10-20 minutes, and cooled, and note that no yellow color is produced.
The color reaction in the case of the raw milk probably results from
the oxidation of the cresols by the hydrogen peroxide. The first product
of this oxidation^ then oxidizes the leuco compound, when such is present,
and causes the color observed.
(b) Wilkinson and Peters' Test.* — To 10 c.c. of the milk to be tested
add 2 c.c. of a 4 per cent alcoholic solution of benzidine, sufficient acetic
acid to coagulate the milk (usually 2-3 drops) and finally 2 c.c. of a 3 per
cent solution of hydrogen peroxide. Raw milk yields an immediate blue
color. In adding the peroxide it is best to permit it to flow slowly down
the wall of the vessel containing the mixture instead of allowing it to mix
with the milk. Milk which has been heated to 78° C. or above remains
unchanged.
' Buckmaster advises the use of an akoholic solution of guaiaconic acid instead of an
alcoholic solution of guaiac resin. Guaiaconic acid is a constituent of guaiac resin.
^ "Trikresol" is the trade name of an antiseptic which contains the three cresols in ap-
proximately equal proportions.
" Probably some organic peroxide or quinoid compound.
* Wilkinson and Peters: Z. Nahr-Cenussm., 16, No. 3, p. 172.
MILK. 241
11. Saturation with Magnesium Sulphate. — Place about 5 c.c.
of milk in a test-tube and saturate with solid magnesium sulphate.
WTiat is this ])recipitate ?
12. Influence of Gastric Rennin on Milk. — Prepare a series of five
tubes as follows:
(a) 5 c.c. of fresh milk+0.2 per cent HCl (add drop by drop until a
precipitate forms).
(b) 5 c.c. of fresh milk+ 5 drops of rennin solution.
(f) 5 c.c. of fresh milk+ 10 drops of 0.5 per cent XajCOg.
(d) 5 c.c. of fresh milk-i- 10 drops of ammonium oxalate.
(e) 5 c.c. of fresh milk -f 5 drops of 0.2 per cent HCl.
Now 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 different tubes. Give a reason for each particular
result.
13. Preparation of Caseinogen. — Fill a large beaker one-third
full of skimmed (or ccntrifugated) milk and dilute it with an equal volume
of water. Add dilute hydrochloric acid until a flocculent precipitate
forms. Stir after each acidification 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 (14-16) experiments.
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 re-
move 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 precipi-
tate as dry as possible between 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:
(a) Solubility. — Try the solubility in the ordinary solvents.
(6) Millans Reaction. — Make the test according to the directions
given on page 97.
(c) Biuret Test. — Make the test according to directions given on
page 98.
((/) Hopkins-Cole Reaction. — Make the test according to the directions
given on page 98.
{e) Loosely Combined Sulphur. — Test for loosely combined sulphur
according to the directions given on page 108.
16
242 PHYSIOLOGICAL CHEMISTRY.
(/) Fusion Test for Phosphorus. — Test for phosphorus by fusion
according to directions given on page 271.
14. Coagulable Proteins 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
lactalhimin and lactoglobidin will form. Continue to concentrate the solu-
tion until the volume is about one-half that of the original solution.
Filter off the coagulable proteins (reserve the filtrate) and test them as
follows :
(a) Millon^s Reaction.— Make the test according to the directions given
on page 97.
(&) Biuret Test. — Make the test according to the directions given on
page 98.
(c) Hopkins-Cole Reaction.' — Make the test according to the directions
given on page 98.
15. Detection of Calcium Phosphate. — Evaporate the filtrate from
the coagulable proteins, on a water-bath, until crystals begin to form.
It may be necessary to concentrate to 15 c.c.
before any crystallization will be observed.
Cool the solution, filter off the crystals (reserve
the filtrate), and test them as follows:
(a) Microscopical Examination.- — Examine
the crystals and compare them with those in
Fig. 81.
Fig. 81. — Calcium Phosphate. ,,, ,-.. , . .... . , ^r^ ,
[b) Dissolve the crystals m nitric acid. 1 est
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. 104, p. 363.
16. Detection of Lactose. — Concentrate the filtrate from the cal-
cium 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 com-
pare them with those in Fig. 80, page 238.
(b) Fehling's Test. — Try Fehling's test upon the mother liquor.
(c) Phenylhydrazine Test. — Apply the phenlhydrazine test to some of
the mother liquor according to the directions given on page 28.
17. Milk Fat. — (a) Evaporate the ether filtrate from the caseinogen
(Experiment 13) and observe the fatty residue. The milk fat was
carried down with the precipitate of caseinogen and was removed when
the latter was treated with ether. If ccntrifugated milk was used in the
MILK. 243
preparation of the caseinogen the amount of fat in the ether filtrate may
be very small. To secure a larger yield of fat proceed according to direc-
tions 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 ?
18. Saponification of Butter. — Dissolve a small amount of butter in
alcohol made strongly alkaline with potassium hydroxide. 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 casserole in a hood and neutralize the solution with sulphuric
acid. Note the odor of volatile fatty acids, particularly butyric acid.
Under certain conditions the odor of ethyl butyrate may also be detected.
19. 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 0.2-0.3 c.c. of a saturated alcoholic solu-
tion 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 down the side of the tube. A green ring, which fmally
changes to blue, is formed at the juncture of the fluids. This is claimed,
by Sherman, to be twice as delicate as either the sulphuric acid or the
hydrochloric acid test for formaldehyde.
II. Leaches 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, on a water-bath, nearly to the
boiling-point, w^ith 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 i : 250,000.
(b) Salicylic and Salicylates. — Remont's Method.' 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 alcoholic
solution. Make the volume 10 c.c, filter through a dry paper if necessary
' Sherman's Organic Analysis, First Edition, p. 232.
244 PHYSIOLOGICAL CHEMISTRY.
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 indicates the presence of salicylic acid.
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 comparisons maybe made in a Duboscq
colorimeter.
(f) Hydrogen Peroxide. — Add 2-3 drops of a 2 per cent aqueous
solution of para-phenylenediamine 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 according to the
directions given in Experiment 4, page 438, add 2 drops of dilute hydro-
chloric acid and i 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 presence of boric acid is indicated by the pro-
duction 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 XIV.
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 protein 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 con-
taining 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.
The percentage composition of some typical keratins is given in the
following table:
Percentage Composition.
S
N C H
1
0
Nails'
2.80
17.51 ' 5100 6.94
1
21.75
. Horn-
1
3.20
';o 86
6.94
S
Indian
4.82
15.40 44.06 6.53
29.19
Japanese. . . .
Negro
4.96
14.64 42.99 5-91
31-50
4.84
14.90 43.85 1 6.37
30.04
Caucasian
(adults).
5.22
1 15.79 44-49 6.44
28.66
Caucasian
(children) .
' 4-93
1 1458 43 23
6.46
30.80
The composition of human hair is influenced by its color and by the
race, sex, age and purity of breeding of the individual.^
' Mulder: Versuch einer allgent. physiol. Chem., Braunschweig, 1844-51.
- Horhaczewski: Ladenburg^ s Handworterbuch d. Chem., 3.
' Rutherford and Hawk: Jour. Biol. Chem., 3, 459, 1907.
245
246 physiological chemistry.
Experiments on Epithelial Tissue.
Keratin.
Horn shavings or nail parings may be used in the experiments which
follow:
1. Solubility. — Test the solubility of keratin in the ordinary solvents
(see page 27).
2. Afillon^s Reaction.
3. Xanthoproteic Reaction.
4. Adamkiewicz^ s Reaction.
5. Hopkins-Cole Reaction.
6. Test for Loosely Combined Sulphur.
CONNECTIVE TISSUE.
I. WHITE FIBROUS TISSUE.
The principal solid constituent of white fibrous connective tissue is the
albuminoid collagen. This body is also found in smaller percentage in
cartilage, bone, and ligament, but the callogen from the various sources
is not identical in composition. In common with the keratins, collagen
is insoluble in the usual protein solvents. It differs from keratin in con-
taining less sulphur. One of the chief characteristics of collagen is,
according to Hofmeister, the property of being hydrolyzed by boiling acid
or water with the formation of gelatin. Emmett and Gies^ claim that
under these conditions there is an intramolecular rearrangement of
collagen and the resultant gelatin is consequently not the product of
hydrolysis. The liberation of ammonia from the collagen during the
process apparently confirms this view. Collagen gives Millon's reaction
as well as the xanthoproteic and biuret tests.
The form of white fibrous tissue most satisfactory for general experi-
ments is the tendo Achillis of the ox. According to Buerger and GieSj
the fresh tissue has the following composition:
Water; 62 .87%
Solids 37-13
Inorganic matter o . 47
Organic matter 36.66
Fatty substance (ether-soluble) i . 04
Coagulable protein 0.22
Mucoid 1-28
Elastin i .63
Collagen 31-59
Extractives, etc o. 90
' Emmett and Geis: Jour. Biol, chem., 3, xxxiii (Proceedings), 1907.
-' Buerger and Gies: Am. Jour. Physiol., 6, 219, 1901.
EPITHELIAL AND CONNECTIVE TISSUES. 247
The mucoid munlioncd above is called tendomucoid^ and is a glyco
protein. It possesses properties similar to those of other connective-tissue
mucoids, e. g., osseomucoid and chondromucoid.
Gelatin, the body which results from the hydrolysis of collagen (see
statement of Emmett and Gies above) , is also an albuminoid. It responds
to nearly all the protein tests. It dififers from the keratins and collagen in
being easily digested and absorbed. Gelatin is not a satisfactory sub-
stitute for the protein constituents of a normal diet, however, since a
certain portion of its nitrogen is not available for the uses of the organism.
Gelatin from cartilage differs from gelatin from other sources in containing
a lower percentage of nitrogen. Tyrosine and tryptophane are not
numbered among the decomposition products of gelatin, hence it does not
respond to Millon's reaction or the Hopkins-Cole reaction.
Experiments on White Fibrous Tissue.
The tendo Achillis of the ox may be taken as a satisfactory type of the
white fibrous connective tissue.
1. Preparation of Tendomucoid. — Dissect away the fascia from
about the tendon and cut the clean tendon into small pieces. Wash the
pieces in running w^ater, subjecting them to pressure in order to remove
as much as possible of the soluble protein and inorganic salts. This
washing is very important. Transfer the washed pieces of tendon to a
flask and add 300 c.c. of half- saturated lime water.^ Shake the flask at
intervals for twenty-four hours. Filter off the pieces of tendon and pre-
cipitate 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 page 27).
(6) Biuret Test. — First dissolve the mucoid in potassium hydroxide
solution and then add a dilute solution of copper sulphate.
(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 hydro-
chloric acid and boil until the solution becomes dark brown. Cool the
solution, neutralize it with concentrated potassium hydroxide, 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 following tests upon the
'Cutter and Gies: Am. Jour. Physiol., 6,155, iQoi.
- Made by mixing equal volumes of saturated lime water and water from the faucet.
248 PHYSIOLOGICAL CHEMISTRY.
pieces of tendon from which the mucoid, soluble protein, and inorganic
salts were removed in the last experiment, we may consider the tests as
being made upon collagen.
(a) Sohibility. — Cut the collagen into very fine pieces and try its
solubility in the ordinary solvents (see page 27).
{b) Millon's Reaction.
(c) Biuret Test.
(d) Xanthoproteic Reaction,
{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 potassium hydroxide solution.
Heat until the collagen is partly decomposed, then add 1-2 drops of lead
acetate and again heat to boiling.
(g) Formation of Gelatin from 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 transformed and a body known as gelatin is
produced (see p. 247).
3. Gelatin. — On the gelatin formed from the transformation of colla-
gen in the above experiment (g), or on gelatin furnished by the instructor
make the following tests:
(a) Solubility. — Try the solubility in the ordinary solvents (see page
27) and in hot water.
(b) Millon's Reaction.
ic) Hopkins-Cole Reaction. — Conduct this test according to the modi-
fication given on page 98.
id) Test for Loosely Combined Sulphur.
Make the following tests upon a solution of gelatin in hot water:
{a) Precipitation by Mineral Acids. — Is it precipitaed by strong
mineral acids such as concentrated hydrochloric acid ?
(b) Salting-out Experiment. — Saturate a little of the solution with
solid ammonium sulphate. Is the gelatin precipitated ? Repeat the
experiment with sodium chloride. What is the result?
(c) Precipitation by Metallic Salts. —Is it precipitated by metallic ^alts
such as copper sulphate, mercuric chloride, and lead acetate?
{d) Coagulation Test. — Does it coagulate upon boiling?
{e) Precipitation by Alkaloidal Reagents. — Is it precipitated by such
reagents as picric acid, tannic acid, and trichloracetic acid?
(J) Biuret Test. — Does it respond to the biuret test ?
ig) Bardach's Reaction. — Does it yield the typical crystals of this
reaction? (See page 10 1.) ^
EPITHELIAL AND CONNECTIVE TISSUES. 249
{li) Precipitatian by Alcohol. — Fill a test-tube one-half full of 95 per
cent alcohol and pour in a small amount of concentrated gelatin solution.
Do you get a precipitate ? How would you prepare pure gelatin from
the tcndo A chill is of the ox?
11. YELLOW ELASTIC TISSUE (ELASTIN).
The ligameniitm niichce of the ox may be taken as a satisfactory type of
the vellow elastic connective tissue. The principal solid constituent of
this tissue is elaslin, a member of the albuminoid group. In common with
the keratins and collagen, elastin is an insoluble body and gives the pro-
tein 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.
It has recently been demonstrated that elastin has the property of
adsorbing pepsin from the gastric juice and thus protecting it so the
enzyme can function later in the intestine^ (see chapter on Gastric
Digestion).
Yellow elastic tissue also contains mucoid and collagen but these arc
present in much smaller amount than in white fibrous tissue, as may be
seen from the following percentage composition of the fresh Ugamentiim
nuchcB of the ox as determined by Vandegrift and Gies."
Water 57-57%
Solids 42 -43
Inorganic matter o • 47
Organic matter 41 • 96
Fatty substance (ether-soluble) 1.12
Coagulable protein 0.62
Mucoid ^Si
Elastin 31-67
Collagen 7-23
Extractives, etc o . 80
Experiments on Elastin.
I. Preparation of Elastin (Richards and Gies).^ — Cut the liga-
ment 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 the bottom of p. 247) and
allow the hashed ligament 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 protein 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
' Abderhalden and Meyer: Zeit. physiol. Chem., 74, 67, 191 1.
-Vandegrift and Gies: Am. Jour. Physiol., 5, 287, iqoi.
' Richards and Gies: Am. Jour. Physiol., 7, 93, 1902.
250 PHYSIOLOGICAL CHEMISTRY.
hydrochloric acid at room temperature 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, pre-
pared by yourself or furnished by the instructor, in the ordinary solvents
(see page 27). How does its solubility 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 98.
7. Test for Loosely Combined Sulphur.
III. CARTILAGE.
The principal solid constituents of the matrix of cartilaginous tissue
are chondromucoid, 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
decomposition. 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 than
dextrose. Sulphuric acid is a by-product in the formation of chondroitin,
and acetic acid is a by-product in the formation of chondrosin.
Chondroalbumoid is similar in some respects to elastin and keratin.
It dijGfers 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 protein 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 from the sur-
rounding tissue. Use the cartilage so obtained in the following experi-
ments:
2. Solubility. — Cut one of the rings into very small pieces and try the
solubility of the cartilage in the ordinary solvents (see page 27).
EPITHELIAL AND CONNECTIVE TISSUES. 25 1
3. Millon's Reaction.
4. Xanthoproteic Reaction.
5. Hopkins-Cole Reaction. — Conduct this test according to the
modification given on page 98.
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 w^hile 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) Bardach's Reaction.
(c) Test for Loosely Combined Sulphur.
(d) 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 pre-
cipitate due ?
(e) 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 precipitate any larger than
that obtained in the preceding experiment ? Why ?
(J) To the remainder of the solution add a little dilute hydrochloric
acid and boil for a few moments. Cool the solution, neutralize with
solid potassium hydroxide, and try Fehling's test. Explain the result.
IV. OSSEOUS TISSUE.
Of the solids of bone about equal parts are organic and inorganic
matter. The organic portion, called ossein, may be obtained by removing
the inorganic salts through the medium of dilute acid. Ossein is practi-
cally the same body which is termed collagen in the other connective
tissues, and in common with collagen yields gelatin upon being boiled
with dilute mineral acid.
In common with the other connective tissues bone contains a mucoid
and an albumoid. Because of their origin these bodies are called osseo-
mucoid and osseoalbumoid. Osseomucoid, when boiled with hydro-
chloric 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 113).
The inorganic basis of the dry, fat-free bone is a chemical substance,
not a mixtur/s. This fact is indicated by the uniform composition of
the bones of fasting animals as well as by the definite relationship existing
252 PHYSIOLOGICAL CHEMISTRY.
between the elements present. Bones of normal and fasting animals
of the same species present no profound differences in percentage compo-
sition. The percentage composition of the dry, fat-free femurs of two
dogs^ after the animals had fasted for 104 and 14 days respectively was
as follows:
Dog. No.
Length of fast.
Ash.
N.
CaO.
MgO.
I
p.o^.
I.
104 days.
61 .50
4.6
33-3
o.S
12.80
2.
14 days.
61.56
41
33-^
O.Q
12 .90
The marked uniformity in composition notwithstanding the wide
variation in the fasting periods is significant. The tensile strength of
the femur of the dog has been found to be at least 25,000 pounds to the
square inch^ whereas that of oak is 10,000 and that of cast iron 20,000
pounds to the square inch.
Experiment on Osseous Tissue.
Qualitative Analysis of Bone Ash. — Take i gram of bone ash in
a small beaker and add a little dilute nitric acid. What does thf efferves-
cence indicate ? Stir throughly 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 pre-
cipitate of phosphates results. (What phosphates are precipitated
here by the ammonia ?) Filter and test the filtrate for chlorides, sulphates,
phosphates, and calcium. Add dilute acetic acid to <he precipitate on
the paper and test this filtrate for calcium and phosphates. To the
precipitate remaining undissolved on the paper add a little dilute hydro-
chloric acid and test this last filtrate for phosphates and iron.
Reference to the following scheme may facilitate the analysis.
* Johnston and Hawk: Unpublished data. For data on a 117-day fast by dog No. i, see
Howe, Mattill and Hawk: Jour. Biol. Chem., 11, 103, 1912.
EPITHELIAL AND CONNECTIVE TISSUES.
BONE ASH.
253
Add dilute nitric acid, stir thoroughly and after the major portion of the ash has been
brought into solution add a little distilled water and filter.
Residue I. Filtrate I.
(disiard) Add ammonium hydroxide to
alkaline reaction and filter.
Residue II.
Treat oti paper with acetic acid.
Residue III.
Treat i»i paper with hydro-
chloric acid.
Filtrate III.
Test for:
1. Phosphates.
2. Calcium.
Filtrate II.
Test for:
1. Chlorides.
2. Sulphates.
3. Phosphates.
4. Calcium.
Filtrate IV.
Test for:
1. Iron.
2. Phosphates.
y. ADIPOSE TISSUE.
For discussion and experiments see chapter on Fats, page 139.
CHAPTER XV.
MUSCULAR TISSUE.
The muscular tissues are divided physiologically into the voluntary
(striated) and the involuntary (non-striated or smooth). 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 protein material and the remaining one-fifth extractives and
inorganic salts.
The proteins are the most important of the constituents of muscular
tissue. In the living muscle we find two proteins, myosinogen and para-
myosinogen. These may be shown to be present in muscle plasma ex-
pressed 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. According to Halliburton^ and others in
the onset of rigor mortis we have an indication of the formation of this
myosin clot within the body. The relation between the proteins of
living and dead muscle is represented graphically by Halliburton as
follows:
Proteins of the living muscle.
Para-myosinogen (25%). Myosinogen (75%).
Soluble myosin.
Myosin.
(The protein of the muscle clot.)
Of the total protein content of li\TLng muscle about 75 per cent is made
up by the myosinogen and the remaining 25 per cent is para-myosinogen.
These proteins may be separated by subjecting the muscle plasma to
fractional coagulation in the usual way. Under these conditions the
para-myosinogen is found to coagulate at 47° C. and the myosinogen to
coagulate at 56° C. It is also claimed by some investigators that it is
possible to separate these two proteins 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 protein precip-
itation tests and is insoluble in water. Myosinogen, on the contrary,
' Halliburton: Biochemistry of Muscle and Nerve, 1904, p. 4.
254
MUSCULAR TISSUE. 255
is not a typical globulin since it is soluble in water. It has been called a
pseudo-globulin. Myosin possesses the globulin characteristics. It is
insoluble in water but soluble in the other protein solvents and is precipi-
tated from its solution upon saturation with sodium shloride.
Mellanby has recently reported observations which he claims indicate
that there is only one. protein in muscle and that rigor mortis is due to the
coagulation of this protein under the combined inlluences of the salt
present in the muscle and the lactic acid developed upon the death of the
muscle. He further states that the disappearance of rigor is due to the
fact that the lactic acid which is continually formed brings this protein
into solution. There is a difference of opinion as to whether true rigor
ever occurs in conection with non-striated (smooth) ^ muscle.
Our ideas concerning the cause of rigor have undergone an impor-
tant re\dsion quite recently. A very attractive theory has been advanced
by Meigs^ and experimental confirmation has been accorded it by von
Fiirth and Lenk.' According to this theory, rigor has no connection
with the coagulation of the muscle proteins and may even be hindered
or prevented by such coagulation. The cause of rigor, from this new
view point, lies in the imbibition of water by the muscle colloids. It is well
known that colloids possess the property of absorbing whatever fluid
may be in contact with them. Moreover, the capacity of the colloid for
water is increased if the fluid is slightly acid in reaction. Therefore the
postmortem production of lactic acid facilitates the imbibition of muscle
fluid by the muscle colloids. Under such conditions, the fibers swell,
become rigid and the condition known as rigor mortis results. The
disappearance of rigor is believed to be due to the coagulation of the
muscle protein through the agency of the accumulated lactic acid.
This change is accompanied by a release of the imbibed water by the
colloids, inasmuch as the capacity of a colloid for retaining fluid is lowered
by coagulation.
Under the name extractives w^e 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, inosite,
C5Hg(OH)g, and fat. In the class of nitrogenous extractives we have
creatine, creatinine, xanthine, hypoxanthine, uric acid, urea, carnine, guanine,
phosphocarnic acid, inosinic acid, carnosine, taurine, carnitine, novaine,
ignotine, neosine, oblitine, carnomiiscarine and methylguanidine (see for-
mulas on page 260). Not all of these extractives are present in the
' Saxl: Beitrdge zur chcmischeti Physiologic und Pathologic, g, i, IQ07.
^ Meigs: American Journal of Physiology, 26, 191, igio.
' von Fiirth and Lenk: Wiener klinische Wochenschrift, 24, 1079, igri.
25O PHYSIOLOGICAL CHEMISTRY.
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
would, however, be unprofitable in this place.
Glycogen is an important constituent of muscle. The content
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 w^hich 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.
Glycogen is a polysaccharide and has the same percentage com-
position as starch and dextrin. It resembles starch in forming an opal-
escent 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 glycogen from the aqueous solution by alcohol: dilute
or concentrated potassium hydroxide may also be used to extract the
glycogen. Glycogen may be prepared in the form of a white, tasteless,
amorphous powder. It is completely precipitated from its solution by
saturation with solid ammonium sulphate, but is not precipitated 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.
Mendel and Leavenworth have recently drawn the conclusion,
from the examination of embryo pigs, that embryonic structures do not
contain exceptionally large amounts of glycogen. The distribution of
the glycogen was not observed to differ from that in the adult animal
except that the liver of the embryo does not assume its glycogen-storing
function early. They further draw the conclusion that the metabolic
transformations of glycogen in the embryo and the adult are entirely
analogous.
The lactic acid occurring in the muscular tissue of vertebrates is
paralactic or sarcolactic acid,
H OH
I i
H-C-C-COOH.
I I
H H
MUSCULAR TISSUE.
257
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 protein origin.
Among the nitrogenous extratives of muscle, those which are of the
most interest in this connection are creatine and the purine bases, xanthine
and hypoxanthine. Creatine is found in varying amounts in the muscles
of "different species, the muscles of birds having shown the largest amount.
It-has also been found in the blood, the brain, in transudates and in the
thvroid gland. Creatine may be crystalHzed and forms colorless rhombic
prisms (Fig. 82, below) which are soluble in warm water and practically
Fig. 82. — Creatine.
insoluble in alcohol and ether. Upon boiling a solution of creatine with
dilute hydrochloric acid it is dehydrolyzed and its anhydride creatinine is
formed. The theory that the creatine of ingested meat is transformed
into creatinine and excreted in the urine has been proven untenable
through the researches of Folin, KJercker, and Wolf and Shaffer. It is
now known that under normal conditions the ingestion of creatine in no
way influences the e.xcretion of creatinine. In the case of Eck fistula dogs,
however, London and Bolyarskii^ found ingested creatine to increase the
output of creatinine in the urine. This finding is of importance as throw-
ing light upon the role of the liver in creatine and creatinine metabolism.
In this connection it is important to note that there is no normal excretion
of endogenous (see p. 291) creatine, a statement proven by the fact that
' London and Bolyarskii: Zeit. phys. chem., 62, 465, 1909.
17
258
PHYSIOLOGICAL CHEMISTRY.
if no creatine be ingested none will be excreted. Folin^ has shown that the
main bulk of ingested creatine is retained in the body, unless the diet
contains a large amount of protein material. Under certain pathological
conditions the urine may contain endogenous creatine which is probably
derived from the catabohsm of muscular tissue, as Benedict, Mellanby, and
Shaffer have suggested.
Amberg and Morrill,- Sedgwick,^ Rose^ and Folin^ have shown that
creatine is a normal constitutent of the urine of infants and children.
Folin explains this phenomenon on the basis of the relatively high protein
intake, whereas Rose believes it is due to a peculiar carbohydrate
metabolism.
Fig. 83. — Xanthine.
After the drawings of^Horbaczewski, as represented in Neubauer and Vogel. {Ogden.)
Besides being a normal constituent of muscle, xanthine has been
found in the brain, spleen, pancreas, thymus, kidneys, testicles, liver,
and in the urine. It may be obtained in crystalline form (Fig. ^7,, above),
but ordinarily it is amorphous. Xanthine is easily soluble in alkalis, less
soluble in water and dilute acids, and entirely insoluble in alcohol and
ether.
Hypoxanthine occurs ordinarily in those tissues and fluids which
contain xanthine. It has been found, unaccompanied by xanthine, in
bone marrow and in milk. Unlike xanthine it may be easily crystallized
in the form of small, colorless needles. It 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.
' Folin: HammarslenFetschriJt, p. 15.
- Amberg and Morrill: Jour. Biol, client., 3, 311, 1907.
^Sedgwick: Jour. Am. Med. Ass'n, 55, 1178, 1910.
* Rose: Jour. Biol, chem., 10, 265, igii.
* Folin: Ibid, 11, 253, 1912.
MUSCULAR TISSUE.
59
Besides this salt we have present chlorides and sahs of sodium, calcium,
magnesium, and iron. Sulphates are also present in traces.
Mendel and Saiki have made some interesting observations upon the
chemical composition of nan-striated or smooth (involuntary) mammalian
muscle, such as the urinary bladder and the muscular coat of the stomach
of the pig. Ilypoxanthine was found to be the y)redominant purine base
present. Creatine and paralactic acid were also isolated. These investi-
gators were iinable to demonstrate, definitely, the presence of glycogen
in the non-striated muscles studied, but state that "the tissues possess the
property of transforming glycogen in the characteristic enzymatic way."
The most important part of their investigation consists in a rather complete
analysis of the inorganic constituents of these muscles. A notable differ-
ence in the relative distribution of the various inorganic constituents was
observed, a difference which, according to the authors, "can be accounted
for in part only by an admixture of lymph. ' ' The comparative composition
of the inorganic portion of striated and non-striated muscle and of blood
serum for comparison is shown in the appended table:
Per loo parts of fresh muscle.
K,0
Na,0
Fe,0.
CaO MgO
CI
p,o,
H,0
Non-striated muscle (Mendel and
Saiki) 0.081 0.328 o.oii 0.044 0.007 0.171! 0-184 80.6
Skeletal muscle (Katz) 0.306, 0.210
Blood serum (Abderhalden) 0.0271 0.425
0.008^0.0110.0470.048 0.487 72.9
io.oi2 0.004 0.363 0.020 91.8
An interesting comparative study of the ash of the smooth muscle of
the stomach of the frog and the striated muscle from the same animal was
very recently reported by Meigs and Ryan.^ Their data indicate "that
smooth muscle contains somewhat less potassium and phosphorus and
somewhat more sodium and chlorine than the striated muscle of the same
animal, but that the differences in these respects between the two tissues
are not by any means so marked as has sometimes been supposed."
Their average figures for each type of muscle follow:
Muscle.
Per 100 parts of fresh muscle.
Na Fe I Ca
Mg
CI I S
Solids H,0
Striated 0.350 0.054 o.oio 0.028 0.030 0.155 0.066 0.141 20.13 79.87
Smooth '0.325 0.073 0.0007 0.004 0.013 0.137 0.1200.1611 17 .70:82 .30
' Meigs and Ryan: Journal of Biological Chemistry, 11, 401, 1912.
26o
PHYSIOLOGICAL CHEMISTRY,
The preparation from which the above data for smooth muscle were
obtained were shown by histological examination to consist in large
part of smooth muscle fibers.
Muscular tissue is said to contain a reddish pigment called myo-
hcBmatin, which is a derivative of haemoglobin.
The so-called "fatigue substances" of muscle are carbon dioxide,
paralactic acid, and potassium dihydrogen phosphate.
The ordinar)^ commercial "meat extract" is composed principally
of the water-soluble constituents of muscle and contains practically nothing
of nutritive value. The protein material to which meat owes its value as an
article of diet is ordinarily practically all removed in the preparation of
the extract. Occasionally some preparations are found to contain pro-
teose, which is formed from the meat proteins in the process of preparation.
The structural formulas of some of the nitrogenous extractives of
muscle are as follows:
NH, HN CO
HX= C
HN=C
N.CCHJ.CH^.COOH.
Creatine, C4H9N3O2.
M ethyl- guanidine acetic acid.
N.CCHJ.CH,
Creatinine, C4H7N3O.
Creatine anhydride.
NH,
1
C=0
I
NH,
Urea^ CON.H4.
CH,.NH,
CH2.SO3OH
Taurine, C2H7NSO3.
Amino-ethyl-sulphonic acid.
O
CO
(CH3)3.N
CH,— CH . OH— CH2
Carnitine," C-HisNOs.
/■-trimethyloxybutyrobetaine.
Carnosine, CgHj^N^Oj.
Neosine, CgH^NOj.
Novaine, C^Hj^NO,.
Ignotine, CgHi.N.Og.
Phosphocarnic acid, C,oHj7N305 or C10H1-N3O5.
Inosinic acid, (HO)2.PO.O.CH2(CHOH)3.CH:(C5H3N,0).
The following extractives as a group are called purine bodies. Their
formulas, together with that of purine from which they are derived and
the hypothetical "purine nucleus" follow:
MUSCULAR TISSUE.
N=CH *N— C"
I ! II
HC C— NH ,C C'— N^
261
— C — N
Purine, C6H4N4.
HN— CO
I I
HC C— NH
/
CH
.,N-C-N„
Purine Nucleus.
HN— CO
I I
OC C— NH
N— C— N
HVPOXANTHINE, C5H4N4O.
t-oxypurine.
HN— CO
OC C— NH
CH
HN— C— N
Xanthine, CSH4N4OC
2-t-dioxy purine.
N=C.NH,
I I
HC C— NH
>C0
.CH
HN— C— NH
Uric Acid, C5H4N4O3.
2-i>-i-trioxy purine.
N— C— N^
Adenine, CsHjNs.
6-atnitiopurine.
HN— CO
I I
H^N.C C— NH
II 11 \
/
CH
N— C— H
Guanine, C5H.iN.iO.
2-amino-6-oxy purine.
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 remove 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 sulphate. Filter
off the salted muscle plasma and make the following tests:
{a) Reaction. — Test the reaction to litmus phenolphthalein and congo
red. What is the reaction of this fresh muscle plasma ?
(b) Fractional Coagulation. — Place a Httle muscle plasma in a test-
tube and arrange the apparatus for fractional coagulation as explained
262 PHYSIOLOGICAL CHEMISTRY.
on page 106. 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 protein (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 proteins ? Which protein
was present in greater amount ?
(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 above. 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 solu-
tion of Fuchsin "S" into the lymph spaces of a frog two or three times
daily for one or two days, in this way thoroughly saturating the tissues
with the dye. Pith the animal (insert a heavy wire or blunt needle through
the occipito atlantoid 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 prevent 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 stimulation 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 action of the alkalinity
of the blood. Upon stimulating the muscles, however, as above explained,
MUSCULAR TISSUE. 263
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.
1. Preparation of Myosin. — Take 25 grams of finely divided lean
beef which has been carefully washed to remove blood and lymph constit-
uents and place it in a beaker with 10 per cent sodium chloride. Stir
occasionally for several hours. Strain oflf the meat pieces by 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. If this precipitate remains
for any length of time on the paper in contact with the air it will become
transformed into the protean myosan. 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 97.
(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 sus-
pended material and try its solubility in 10 per cent sodium chloride.
What property does this experiment 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 120) Do you find any
peptone? From your experiments on "living" and ''dead" muscle what
are your ideas regarding the proteins of muscle ?
2. Preparation of Glycogen. — Grind a few oysters or scallops^ in a
mortar with sand. Transfer to an evaporating dish, add water, and boil
for 20 minutes. Note the opalescence of the solution. At the boiling-
point faintly acidify with acetic acid. Why is this acid added ? Filter,
and divide the filtrate into two parts. Test one part of the filtrate as
follows :
(a) Iodine Test. — To 50 c.c. of the solution in a test-tube add 5-10
drops of iodine solution and 2-3 drops of 10 per cent sodium chloride.
* Glycogen may also be prepared from the liver of an animal which has been fed a high
carbohydrate diet for 1-2 days previously. The best yield of glycogen can, however, generally
be obtained from scallops.
264 PHYSIOLOGICAL CHEMISTRY.
What do you observe ? Is this similar to the iodine test upon any other
body with which we have had to deal ?
If difl&culty is experienced in securing a satisfactory iodine test proceed
as follows: Make equal volumes of glycogen solution acid in reaction
with hydrochloric acid. Boil one solution to hydrolyze the glycogen.
Add equal volumes of iodine solution to each and note the more pro-
nounced iodine reaction in the unhydrolyzed solution.
(b) Reduction Test. — Does the solution reduce Fehling's solution ?
(c) Hydrolysis oj Glycogen. — Add 10 drops of concentrated hydro-
chloric 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 oj Saliva. — ^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 super-
natant fluid, and filter the remainder. Heat the glycogen on a water-
bath to remove the alcohol, then subject it to the following tests:
{a) Solubility. — Try its solubility in the ordinary solvents.
(h) Iodine Test. — Place a small amount of the glycogen in a depression
of a test-tablet and add 2-3 drops 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.
I. Creatine. — Dissolve about 10 grams of a commercial extract
of meat in 200 c.c. of warm water. Precipitate the inorganic con-
stituents 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 hydrogen sulphide. 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 creatine should form
at this point. Examine under the microscope (Fig. 82, page 257). 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 purine bases
have been dissolved and are in the filtrate, whereas the creatine 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
MUSCULAR TISSUE.
26q
them into solution in a little hot water. Decolorize the solution by animal
charcoal and concentrate it to a small volume. Allow the solution to
cool and note the separation of colorless crystals of creatine. Examine
these crystals under the microscope and compare them with those rejjro-
duced in Fig. 82, page 257.
2. Hypoxanthine. — Evaporate the alcoholic filtrate from the creatine
to remove the alcohol. Make the solution ammoniacal and add am-
moniacal silver nitrate until precipitation ceases. The precipitate con-
sists principally of hypoxanthine silver and xanthine silver. Collect these
silver salts on a filter paper and wash them with water. Place the pre-
cipitate and paper in an evaporating dish and boil for one minute with
nitric acid having a specific gravity of i.i. Filter while hot through a
Fig. 84. — Hypoxanthine Silver Nitr.\te.
(Drawn from a student preparation by Mr. E. F. Hirsch).
double paper, wash with the same strength of nitric acid and allow the
solution to cool. By this treatment with nitric acid hypoxanthine silver
nitrate and xanthine silver nitrate have been formed. The former is
insoluble in the cold solution and separates on standing. After standing
several hours filter off the hypoxanthine silver nitrate and wash \\\\.\\
water until the wash-water is only slightly acid in reaction. Examine
the crystals of hypoxanthine silver nitrate under the microscope and
compare them with those in Fig. 84, above. Now wash the crystals
from the paper into a beaker with a little water and warm the liquid.
Remove the silver by hydrogen sulphide and filter. By this means
hypoxanthine nitrate has been formed and is present in the filtrate. Con-
centrate on a water-bath to drive off hydrogen sulphide and render the
solution slightly alkaline with ammonia. Warm for a time, to remove
266
PHYSIOLOGICAL CHEMISTRY.
the free ammonia, filter, concentrate the filtrate to a small volume and
allow it to stand in a cool place. Hypoxanthine should crystallize in
small colorless needles. Examine the crystals under the microscope.
3. Xanthine. — To the filtrate from the above experiment containing
the xanthine silver ?iitrate add ammonia in excess. (The crystalline form
of xanthine silver nitrate is shown in Fig. 85, below.) A brownish-red
precipitate of xanthine silver forms. Treat this suspended precipitate
with hydrogen sulphide (do not use an excess of hydrogen sulphide),
warm the mixture for a few moments and filter while hot. Concentrate
Fig. 85. — Xanthixe Silver Nitrate.
the filtrate to a small volume and put away in a cool place for crystalli-
zation (Fig. 83, p. 258). To obtain xanthine in crystalline form special
precautions are generally necessary. Evaporate the solution to dryness.
Make the following tests on the crystals or residue:
(a) Xanthine Test. — Place about one-half of the crystalline or amor-
phous material in a small evaporating dish, add a few drops of concen-
trated 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 results
which yields a red residue upon evaporation. How does this differ from
the Murexide test upon uric acid ?
{b) WeideVs Reaction.— By gently heating bring the remainder of the
xanthine 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 xanthine is shown by the residue
MUSCULAR TISSUE. 267
assuming a red color. A somewhat brighter color may be obtained by
using a trace of nitric acid with the bromine-water. By the use of this
modification, however, we may get a positive reaction with bodies other
than xanthine.
Hurthle's Experiment.
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 ammonium magnesium phosphate,
NH,-0
\
Mg-0-P = 0
\/
O
distributed everywhere throughout the muscle fiber, thus demonstrating
the abundance of phosphates and magnesium in the muscle (Fig. loi,
page 319.)
CHAPTER XVI.
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 proteins, cho-
lesterol, cerebrosides (cerebrin, etc.), lecithin, kephalin, protagon {?), para-
nucleoprotagon, nuclein, neurokeratin, collagen, extractives, and inorganic
salts. The proteins are present in the greatest amount and comprise
about 50 per cent of the total solids. Three distinct proteins, two globu-
lins, and a nucleoprotein, have been isolated from nervous tissue. The
globulins coagulate at 47° C. and 70-75° C, respectively, while the
nucleoprotein coagulates at 56-60° C. This nucleoprotein contains
about 0.5 per cent of phosphorus (Halliburton, Levene). Nervous
tissue is composed of a relatively large quantity of a variety of com-
pounds which collectively 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 choles-
terol, the cerebrosides and the phosphorized fats as lipoids.
The consideration of lipoids (or lipins^) is assuming added importance.
These substances constitute one of the two great groups of tissue col-
loids, the proteins being the remaining group. So far as structure and
chemical properties are concerned the various classes of lipoids are entirely
unlike.
The group of phosphorized fats are very important constituents 'of
nervous tissue. The best known members of this group are 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 decom-
position lecithin yields fatty acid, glycero-phosphoric acid, and choline.
* Rosenbloom and Gies: Biochemical Bulletin, i, 51, 191 1. The term lipoid was intro-
duced by Overton (Studien iiber die Narkose, Jena, 1901, Gustav Fischer.)
268
NERVOUS TISSUE. 269
Each lecithin molecule contains two fatty acid radicals which may be
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:
CHO -C^HgjCO
I
CH^O-PO-O-C^H,
\
I /
OH HO
This lecithin would be called distearyl-lecithin or choline-distearyl-
glycero-phosphoric acid. Upon decomposition the molecule splits
according to the following reaction:
C,,H,„NPO«+3H,0->2(C,,H3„0,) + C3H„PO«+C,H,,N03.
Lecithin. Stearic acid. Glycero-phosphoric Choline.
acid.
The lecithins are not confined to the nervous tissues but are found in
nearly all animal and vegetable tissues. Lecithin is a primary con-
stituent of the cell. It is soluble in chloroform, ether, alcohol, benzene,
and carbon disulphide. The chloroform or alcohol-ether solution
may be precipitated by acetone. Lecithin may be caused to crystal-
lize in the form of small plates by cooling the alcoholic solution to a
low temperature. It has the power of combining with acids and bases,
and the hydrochloric acid combination has the power of forming a
double salt with platinic chloride.
Choline, as was indicated above, is one of the decomposition pro-
ducts of lecithin. It is trimethyl-oxy ethyl-ammonium hydroxide and
has the following formula :
CH,.CH,(OH)
N = (CH3)3
\
OH
Recent researches have shown that great importance is to be attached
to the detection of choHne in the cerebro-spinal fluid and the blood in
certain cases of degenerative disease of the nervous system. In this
connection tests for choline (see p. 273) are of interest and value.
Protagon, another nitrogenous phosphorized substance is a body
over which there has been much discussion. Upon decomposition it
270 PHYSIOLOGICAL CHEMISTRY.
is said by some investigators to yield cerebrin and the decomposition
products of lecithin. It has been shown by Posner and Gies^ as well
as by Rosenheim and Tebb" that protagon is a mixture and has no exist-
ence as a chemical individual. Koch^ very recently reported data
obtained from purified preparations which indicate that protagon con-
tains at least three substances: "a phosphatide containing cholin, a
cerebroside containing sugar, a complex combination of a chohn-free
phosphatide with a cerebroside to which an ethereal sulphuric acid
group is attached." On the basis of his data, he beheved the term
protagon to have no chemical significance. He proposed the term sul-
phatide. Koch's preparation analyzed as follows (per cent) :
Choline Sugar Nitrogen Phosphorus Sulphur
i.o 12.0 2.3 1.7 1.9
He suggested the following structure:
O
11
Phosphatide — O — S — O — Cerebroside
grouping jj grouping
O
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^^B.^^-
NPOj3. Kephalin may be a stage in lecithin metabohsm.
The cerebrosides are substances containing nitrogen but no phos-
phorus, and are important constituents of the white matter of nerA^ous
tissue. Certain ones have also been found in the spleen, pus, and in egg
yolk. They may be extracted from the tissue by boiling alcohol and are
insoluble in cold alcohol, cold and hot ether, and in water and dilute
alkalis. The cerebroside termed cerebrin is a mixture containing phre-
nosin (pseudo-cerebrin or cerebron), a body yielding the carbohydrate
galactose on decomposition.
Cholesterol, one of the primary cell constituents, is present in fairly
large amount in nervous tissue. It is a mon-atomic alcohol containing
at least one double bond and possesses the formula C27H^50H or C27-
H^gOH. There is still some uncertainty as to the exact structure of
cholesterol. It may possess a terpene structure. It was formerly called
a "non-saponifiable fat" but since it is not changed in any way by
boiHng alkalis it is not a fat. It is soluble in ether, chloroform, benzene,
and hot alcohol. It crystallizes in the form of thin, colorless, trans-
* Posner and Gies: Journal 0/ Biological Chemistry, i, 59, 1905-6.
^ Rosenheim and Tebb: Journal of Physiology, 36 and 37, 1907-8.
^ Koch: Journal 0/ Biological Chemistry, 11, March, 1912, Proceedings.
NERVOUS TISSUE. 27 1
parent plates (Fig. 43, p. 166). Cholesterol occurs abundantly in
one form of biliary calculus. It has also been found in feces, wool
fat, egg yolk, and milk, frequently in the form of its esters of higher
fatty acids. It is generally believed that the cholesterol present in the
animal body has its origin in the vegetable kingdom. However evidence
has recently been submitted' indicating a synthesis of cholesterol under
certain conditions in the animal body.
Paranucleoprotagon is a phosphorized substance originally isolated
from brain tissue by Ulpiani and Lelli and recently reinvestigated by
Steel and Gies. It is said to possess lecithoprotein characteristics.
Nervous tissue yields about i per cent of ash which is made up in
great part of alkaline phosphates and chlorides.
Experiments on the Lipoids of Nervous Tissue.'
1. Preparation of Lecithin. — Treat the macerated brain of a
sheep with ether and allow it to stand in the cold for 48-72 hours. The
cold ether will extract lecithin and cholesterol. Filter and add 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 sHde and examine under the microscope.
{h) Osmic Acid Test. — Treat a small portion with osmic acid. What
happens ?
(c) Acrolein Test. — Make the acrolein test according to directions
on page 143.
{d) "Fusiau" 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 potassium hydroxide and potassium
nitrate (5 : i) and heat carefully until the resulting mixture is colorless.
Cool, dissolve the mass in a little warm water, acidify with nitric acid,
heat to boiling, and add a few cubic centimeters of molybdic solution.
In the presence of phosphorus a yellow precipitate forms. What is it?
2. Preparation of Cholesterol. — Place a small amount of macer-
ated brain tissue under ether and stir occasionally for one hour. Filter,
evaporate the filtrate to dryness on a water-bath, and test the cholesterol
according to directions given below. (If it is desired, the ether extract
* Klein: Biochem. Zeil., 30, 465, 1910.
- 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 cholesterol are caused to precipitate. Filter the cold solution rapidly and
treat the precipitate on the paper with ice cold ether to dissolve out the cholesterol. The pro-
tagon may now be redissolved in warm 85 per cent alcohol from which solution it will precipi-
tate upon cooling.
272 PHYSIOLOGICAL CHEMISTRY.
from the so-called protagon, or the ether-acetone filtrate from the lecithin
may be used for the isolation of cholesterol. In these cases it is simply-
necessary to evaporate the solution to dryness on a water-bath.) Upon
the cholesterol prepared by either of the above methods make the fol-
lowing tests:
(a) Microscopical Examination. — Examine the crystals under the
microscope and compare them with those in Fig. 43, page 166.
{b) Iodine-sulphuric Acid Test.- — ^Place a few crystals of cholesterol
in one of the depressions of a test-tablet and treat with a drop of concen-
trated 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 Liehermann-Burchard Test. — Dissolve a few crystals of
cholesterol in 2 c.c. of chloroform in a dry test-tube. Now add 10
drops of acetic anhydride and 1-3 drops of concentrated sulphuric acid.
The solution becomes red, then blue, and finally bluish-green in color.
{d) Salkowski's Test. — Dissolve a few crystals of cholesterol in a
little chloroform and add an equal volume of concentrated 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 cholesterol in an evaporating
dish add a few drops of Schiff's reagent.^ 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 directions given
on page 271. 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 filtrate 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 microscope.
{h) 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 directions on
page 271. How does the result compare with that on lecithin?
{d) Place a little cerebrin on platinum foil and warm. Note the
odor.
' Schifif's reagent consists of a mixture of three volumes of concentrated sulphuric acid and
one volume of 10 per cent ferric chloride.
NERVOUS TISSUE. 273
(e) Hydrolysis of Cerehrin. — ^Place the remaining cerebrin in a small
evaporating dish, add equal volumes of water and dilute hydrochloric
acid, and boil for one hour. Cool, neutralize with solid potassium
hydroxide, filter, and test with Fehling's solution. Is there any reduction,
and if so how do you explain it ?
4. Tests for Choline, {a) Rosenheim's Periodide r«5/.— Prepare
an alcoholic extract of the iluid under examination, and after evaporation,
apply Rosenheim's iodo-potassium iodide solution^ to a little of the
residue. In a short time dark brown plates and prisms of choline periodide
begin to form and may be detected by means of the microscope. Oc-
casionally they are large enough to be visible to the naked eye. They
somewhat resemble crystals of haemin (see p. 211). If the slide be
permitted to stand, thus allowing the fluid to evaporate, the crystals
will disappear and leave brown oily drops. They will reappear, how-
ever, upon the addition of fresh iodine solution, v. Stanek claims
that this choline compound has the formula CjIIj^NOLIg.
(b) Rosenheim's Bismuth Test. — Extract the fluid under examination
with absolute alcohol, evaporate, and re-extract the residue. Repeat
the extraction several times. Dissolve the final residue in 2-3 c.c. of
water and add a drop of Kraut's reagent.^ Choline is indicated by the
appearance of a bright brick-red precipitate.
' Prepared by dissolving 2 grams of iodine and 6 grams of potassium iodide in 100 c.c.
water.
^Dissolve 272 grams of potassium iodide in water and add 80 grams of bismuth sub-
nitrate dissolved in 200 grams of nitric acid (sp. gr. 1.18). Permit the potassium nitrate to
crystallize out, then filter it off and make the filtrate up to i liter with water.
18
CHAPTER XVII.
URINE : GENERAL CHARACTERISTICS OF NORMAL AND
PATHOLOGICAL URINE.
Volume. — The volume of urine excreted by normal individuals
during any definite period fluctuates within very wide limits. The
average output for twenty-four hours is placed by German writers
between 1500 and 2000 c.c. This value is not strictly applicable to con-
ditions in America, however, since it has been found that the average
normal excretion of the adult male American falls within the lower
values of 1000-1200 c.c. The volume-excretion is influenced greatly
by the diet, particularly by the ingestion of fluids.
Certain pathological conditions cause the output of urine for any
definite period to depart very decidedly from the normal output. Among
the pathological conditions in which the volume of urine is increased
above normal are the following: Diabetes mellitus, diabetes insipidus,
certain diseases of the nervous system, contracted kidney, amyloid
degeneration of the kidney, and in convalescence from acute diseases in
general. Many drugs such as calomel, digitalis, acetates, and salicylates
also increase the volume of the urine excreted. A decrease from the
normal is observed in the following pathological conditions: Acute
nephritis, diseases of the heart and lungs, fevers, diarrhoea, and vomiting.
Color. ^Normal urine ordinarily possesses a yellow tint, the depth
of the color being dependent in part upon the density of the fluid. The
color of normal urine is due principally to a pigment called urochrome:
traces of hcemato porphyrin, urobilin, and uroeryihrin have also been
detected. Under pathological conditions the urine is subject to pro-
nounced variations in color and may contain many varieties of pigments.
Under such circumstances the urine may vary in color from an ex-
tremely light yellow to a very dark brown or black. Vogel has constructed
a color chart which is of some value for purposes of comparison. The
nature and origin of the chief variations in the urinary color are set
forth in tabular form by Halliburton as follows:
274
URINE.
275
Color.
Nearly colorless
Cause of Coloration.
Pathological Condition.
Dilution, or diminution of
normal pigments.
Nervous conditions: hy-
druria, diabetes insipidus,
granular kidney.
Dark vellow to l)ro\vn-red..
Increase of normal, or oc-
currence of pathological, pig-
ments.
.\cute febrile diseases.
Milkv
Fat globules Chyluria.
Pus corpuscles.
Purulent diseases of the
urinar)- tract.
Orange .
Excreted drugs Santonin, chr}sophanic acid.
Red or reddish.
Hematopx)rphyrin
Unchanged hjemoglobin.
Haemorrhages, or ha?moglo-
binuria.
Pigments in food (logwood,
madder, bilberries, fuchsin).
Brown to brown-black.
Ha;matin
Small haemorrhages.
Methsemoglobin
Methaemoglobinuria.
Melanin
Melanotic sarcoma.
Hydrochinon and catechol Carbolic-acid poisoning.
Greenish-yellow, greenish- Bile-pigments,
brown, approaching black.
Jaundice.
Dirty green' or blue A dark-blue scum on surface,
i with a blue deposit, due to an
I excess of indigo-forming sub-
I stances.
Cholera, tA'phus; seen espe-
cially when the urine is
putrefying.
Brown-yellow to red-brown, I Substances contained in senna,
becoming blood-red upon 1 rhubarb and chelidonium
adding alkalis. I which are introduced into the
I system.
Transparency. — Normal urine is ordinarily perfectly clear and
transparent when voided. On standing for a variable time, however, a
cloud (nubecula) consisting principally of nucleoprotein or mucoid (see
p. 308) and epithelial cells forms. A turbidity due to the precipitation of
phosphates is normally noted in urine passed after a hearty meal.
The urine obtained 2-3 hours after a meal or later is ordinarily free from
turbidity. Permanently turbid urines ordinarily arise from pathological
conditions.
Odor. — The odor of normal urine is of a faint, aromatic type. The
bodies to which this odor is due are not well known, but it is claimed by some
investigators to be due, at least in part, to the presence of minute amounts
of certain volatile orsranic acids. When the urine undergoes decom-
' This dirty green or blue color also occurs after the use of methylene blue in the organism.
276 PHYSIOLOGICAL CHEMISTRY,
position, e. g., in alkaline fermentation, a very unpleasant ammoniacal
odor is evolved. All urines are subject to such decomposition if allowed
to stand for a sufficiently long time. Under normal conditions the urine
very often possesses a peculiar odor due to the ingestion of some certain
drug or vegetable. For instance, cubebs, copaiba, myrtol, saffron, tolu,
and turpentine each imparts a somewhat specific odor to the urine. After
the ingestion of asparagus, the urine also possesses a typical odor.
Frequency of Urination. — The frequency of urination varies
greatly in different individuals but in general is dependent upon the
amount of fluid in the bladder. In pathological conditions an inflam-
matory affection of the urinary tract or any disturbance of the innerva-
tion of the bladder will influence the frequency. Affections of the spinal
cord which lead to an increased irritability of the bladder or a weakening
of the sphincter will result in increasing the frequency of urination.
Reaction. — The mixed twenty-four hour urinary excretion of a
normal individual ordinarily possesses an acid reaction to litmus. This
acidity is now believed to be due to the presence of various acidic radicals
and not to the presence of sodium di-hydrogen phosphate as was formerly
held (see Phosphates, p. 317). This conclusion is reinforced by the
observation that urine may be divided into two portions, one part con-
sisting almost entirely of inorganic matter, including practically all of
the phosphates and having an alkaline reaction, the other containing
practically all of the organic substances and no phosphates and having
an acid reaction. The acidity imparted to the urine by any particular
acid depends entirely upon the extent to which the acid is dissociable,
since it is the hydrogen ion which is responsible for the acid reaction.
The composition of the food is perhaps the most important factor
in determining the reaction of the urine. The reaction ordinarily varies
considerably according to the time of day the urine is passed. For
instance, for a variable length of time after a meal the urine may be
neutral or even alkaline in reaction to litmus, owing to the claim of the
gastric juice upon the acidic radicals to further the formation of hydro-
chloric acid for use in carrying out the digestive secretory function. This
change in reaction is known as the alkaline tide and is common to per-
fectly healthy individuals. The urine may also become temporarily
alkaline in reaction to litmus, as the result of ingesting alkaline car-
bonates or certain salts of tartaric and citric acids which may be trans-
formed into carbonates within the organism. Normal urine upon stand-
ing for some time becomes alkaline in reaction to litmus, owing to the
inception of alkaline or ammoniacal fermentation through the agency of
micro-organisms. This fermentation has no especial diagnostic value
except in cases where the urine has undergone this change within the
URINE.
277
organism and is voided in the decomposed slate. Ammoniacal fermenta-
tion is ordinarily due to cystitis or occurs as the result of infection in the
process of catheterization. A microscopical examination of such urine
(Fig. 86, below) shows the presence of ammonium magnesium phosphate
crystals, amorphous phosphates, and not infrequently ammonium urate.
\Vr •• v,^»^^ \ i •'. ,J V'"'^, ■■■>-« "''. '-T' "J' ' ?. I
Fig. 86. — Deposit in Ammonla.cal Fermentation.
a, Acid ammonium urate; b, ammonium magnesium phosphate; c, bacteria.
Occasionally a urine which possesses a normal acidity when voided,
upon standing instead of undergoing ammoniacal fermentation as above
described will become still more strongly acid in reaction. Such a
phenomenon is termed acid jermentation. Accompanying this increased
—\Si
Fig. 87. — Deposit in Acid Fermentation.
a. Fungus; b, amorphous sodium urate; c, uric acid; d, calcium oxalate.
acidity there is ordinarily a deepening of the tint of the urinary color.
Such urines may contain acid urates, uric acid, jungi, and calcium oxalate
(Fig, 87, above). On standing for a sufficiently long time any urine
which exhibits acid fermentation wall ultimately change in reaction,
2/8
PHYSIOLOGICAL CHEMISTRY.
due to the inception of alkaline fermentation, and will show the microscop-
ical deposits characteristic of such a urine.
Specific Gravity. — The specific gravity of the urine of normal
individuals varies ordinarily between 1.015 and 1.025. This value is
subject to wide fluctuations under various conditions. For instance,
following copious water- or beer-drinking the specific gravity may fall
to 1.003 or lower, whereas in cases of excessive perspiration it may rise
as high as 1.040 or even higher. Where a very accurate determination
of the speciiic gravity is desired use is commonly made
of the pvknometer or of the Westphal hydrostatic balance.
These instruments, however, are not suited for clinical
use. The clinical method of determining the specific
gravity is by means of a urinometer (Fig. 88). This
affords a very rapid method and at the same time is
sufficiently accurate for clinical purposes. The urino-
meter is always calibrated for use at a specific tem-
perature and the observations made at any other tem-
perature must be subjected to a certain correction to
obtain the true specific gravity. In making this cor-
rection one unit oj the last order is added to the ob-
served specific gravity for every three degrees above the
normal temperature and subtracted for every three de-
grees below the normal temperature. For instance, if
in using a urinometer calibrated for 15° C. the specific
gravity of a urine having a temperature of 21° C. is
determined as 1.018 it is necessary to add to the observed
specific gravity two units of the third order to obtain the
real specific gravity of the urine. Therefore the true
specific gravity, at 15° C, of a urine having a specific
gravity of 1.018 at 21° C. is 1.018 -1-0.002 = 1.020.
Fig. 88.— Urin- Pathologically, the specific gravity may be subjected
oMETER AND Cyl- to Very wide variations. This is especially true in dis-
eases of the kidneys. In acute nephritis ordinarily the
urine is concentrated and of a high specific gravity, where.as in chronic
nephritis the reverse conditions are more apt to prevail. In fact, under
most conditions, whether physiological or pathological, the specific
gravity of the urine is inversely proportional to the volume excreted.
This is not true of diabetes mellitus, however, where the volume of
urine is large and the specific gravity is also high, owing to the sugar
contained in the urine.
The amount of solids eliminated in the excretion for twenty-four
hours may be roughly calculated by means oi Long's coefficient, i. e., 2.6.
,1
URINE. 279
The solid content of 1000 c.c. of urintis obtained by mulliplyin«^ the
last two figures of the specific gravity observed at 25° C. by 2.6. To
determine the amount of solids excreted in twenty-four hours if the
volume was 11 20 c.c. and the specific gravity was 1.018 the calculation
would be as follows:
(a) 18X2.6 = 46.8 grams of solid matter in 1000 c.c. of urine.
46.8X1120
(0) -* -= !52.4 grams of solid matter in 11 20 c.c. of urine.
The coefficient of Haser (2.33) which has been in use for years
probably gives values that are inaccurate for conditions existing in
America. This coefficient was calculated on the basis of the specific
gravity determined at a temperature of 15° C.
Freezing-point (Cryoscopy).— The freezing-point of a solution
depends upon the total number of molecules of solid matter dissolved
in it. The determination of the osmotic pressure by this method has
recently come to be of some clinical importance, particularly as an aid
in the diagnosis of kidney disorders. In this connection it is best to
collect the urine from each kidney separately and determine the freezing-
point in the individual samples so collected. By this means considerable
aid in the diagnosis of renal diseases may be secured. The fluids most
frequently examined cryoscopically are the blood (see p. 194) and the
urine. The freezing-point is denoted by A. The value of A for
normal urine varies ordinarily between —1.3° and —2.3° C, the freezing-
point of pure water being taken as 0°. A is subject to very wide fluctua-
tions under unusual conditions. For instance, following copious water-
or beer-drinking A may have as high a value as —0.2° C, whereas on a
diet containing much salt and deficient in fluids the value of A may be
lowered to —3° C. or even lower. The freezing-point of normal blood
is generally about —0.56° C. and is not subject to the wide variations
noted in the urine, because of the tendency of the organism to maintain
the normal osmotic pressure of the blood under all conditions. Variations
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.
Freezing-point determinations may be made by means of the Beck-
mann-Heidenhain apparatus (Fig. 89) or the Zikel pektoscope. The
Beckmann-Heidenhain apparatus consists of the following parts: A
strong battery 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 examination is lowered. A large glass tube (B)
designed as an air-jacket, and formed after the manner of a test-tube is
28o
PHYSIOLOGICAL CHEMISTRY.
introduced through the central aperture in the metal cover and into this
air-jacket is lowered a smaller tube (A) containing the fluid to be tested.
A very delicate thermometer (D), graduated in hundredths of a degree
is introduced 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
surface. 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 pro-
portion 1 : 3 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 equalization of temperature
and observe the course of the mercury column
of the thermometer very carefully. The mer-
cury will gradually fall and this gradual lower-
ing of the temperature will be followed by a
sudden rise. The point at which the mercury
He^dexi^i'nFre^ezing-point rests after this sudden rise is the freezing-point.
Apparatus. {Long.) -phis rise is due to the fact that previous to
Z), a delicate thermometer; ^ ., . ,
C, the containing jar; B, the freezing, a fluid IS always more or less over-
.rwtrwhXt'Su^e ^ofe'i and the thermometer temporarily regis-
to be observed is placed, ters a temperature somewhat below the freezing-
Two stirrers are shown, one . a i n • i r i i
for the cooling mixture in the pomt. As the fluid freezes, howcver, there IS a
jar arid one for the expen- ^ sudden change in the temperature of the
mental mixture. j o i.
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 "inocula-
URINE. 281
tion" causes the fluid to freeze instantaneously. (For details of the
method of determining the freezing-point consult standard works on
physical or organic chemistry.)
Electrical Conductivity. — The electrical conductivity of the urine
is dependent upon the number of inorganic molecules or ions present,
and in this diff'ers 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 rather recently, but from the data secured it seems that
the value generally falls below k=o.ot,. 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, particularly if it is taken in connection with the deter-
mination 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 con-
ductivity, the method by wh'ch it is determined, and the principles
involved consult standard works on physical or electro-chemistry.
Collection of the Urine Sample. — If any dependable data are
desired regarding the quantitative composition of the urine the examina-
tion 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 discarded 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,
\/OH
CHg — CH — CHj,
is a very satisfactory preservative since the excess may be removed 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 diabetic 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 order to further insure the preser-
282 PHYSIOLOGICAL CHEMISTRY.
vation of the urine the cleaned and dried urine receptacle may be rinsed
with an alcoholic solution of thymol and subsequently thoroughly dried
before introducing the urine.
Toluol is also used for the preservation of urine.
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 as well as upon a night sample as above described.
CHAPTER XVTII.
URINE: PHYSIOLOGICAL CONSTITUENTS/
I. Organic Physiological Constituents.
Urea.
Uric acid.
Creatinine.
Creatine. ■
Ethereal sulphuric acids
Hippuric acid.
Oxalic acid.
Indoxyl-sulphuric acid.
Phenol- and />-cresol-sulphuric acids.
Pyrocatechin-sulphuric acid.
Skatoxyl-sulphuric acid.
Xcutral sulphur compounds.
Allantoin.
Aromatic oxvacids .
Cystine.
Chondroitin-sulphuric acid.
Thiocyanates.
Taurine derivatives.
Oxyproteic acid.
Alloxyproteic acid.
Uroferric acid.
Paraoxyphenyl-acetic acid.
Paraoxy phenyl-propionic acid.
Homogentisic acid.
Uroleucic acid.
Oxymandelic acid.
Kynurenic acid.
Amino acids.
Benzoic acid.
Neucleoprotein.
Oxaluric acid.
' 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 physiolog-
ically 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 pres-
ent whether it may be correctly termed a physiological or a pathological constituent.
* Normal constituent of urine of infants and children (see p. 258).
283
284
PHYSIOLOGICAL CHEMISTRY.
f Pepsin.
Enzymes \ Gastric rennin.
[ Amylase.
[ Acetic acid.
Volatile fatty acids ] Butyric acid.
[ Formic acid.
Paralactic acid.
Phenaceturic acid.
^, , . , , f Glycerophosphoric acid.
Pnospnorized compounds < t,, , . . ,
^ ^ [ Pnospnocarmc acid.
[ Urochrome.
Pigments I Uroblin.
[ Uroerythrin.
Ptomaines and leucomaines.
Adenine.
Guanine.
Xanthine.
Epiguanine.
Episarkine.
Hypoxanthine.
Paraxanthine.
Heteroxanthine.
i-Methylxanthine.
2. Inorganic Physiological Constituents.
Ammonia.
Sulphates.
Chlorides.
Phosphates.
Sodium and potassium.
Calcium and magnesium.
Carbonates.
Iron.
Fluorides.
Nitrates.
Silicates.
Hydrogen peroxide.
Purine Bases,
NH.,
UREA, C-O.
NH,
URINE,
28:
Urea is the principal end-product of the metabolism of protein
substances. 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 decrease 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 person as to cause the excretion of total nitrogen to be reduced
to 3-4 grams in 24 hours, only about 60 per cent oj this nitrogen appeared
Fig. 90. — Urea.
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 of decreasing the amount of protein
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 carnivora
and in somewhat smaller amount in the urine of herbivora; 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
286 PHYSIOLOGICAL CHEMISTRY.
in amount after a diet of meat, and least in amount after a diet con-
sisting 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
proteins 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 increased after copious water- or beer-drinking. The
increase is probably due primarily to the washing out of the tissues of
the urea previously formed, but which had not been removed in the
normal processes, and secondarily to a stimulation of protein catabolism.
Urea may be formed in the organism from amino acids such as leucine,
glycocoll, and aspartic acid: it may also be formed from ammonium
carbonate (NHJjCOg or ammonium carbamate, H^N.O.CO.NHj.
There are differences of opinion regarding the transformation 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 may be formed in
other organs or tissues as well.
Urea crystallizes in long, colorless, four- or six-sided, anhydrous, rhom-
bic prisms (Fig. 90, p. 285), which melt at 132° C. and are soluble in
water or alcohol and insoluble in ether or chloroform. If a crystal
of urea is heated in a test-tube, it melts and decomposes with the libera-
tion of ammonia. The residue contains cyanuric acid,
COH
N N
II I
HO.C COH
\^
N
and biuret,
NH3
c=o
\
NH
y
c-o
NH3
The biuret may be dissolved in water and a reddish-violet color obtained
by treating the aqueous solution with copper sulphate and potassium
hydroxide (see Biuret Test, p. 98). Certain hypochlorites or hypo-
bromites in alkaline solution have the power of decomposing urea into
URINE.
2S:
nitrogen, carbon dioxide, and water. Sodium hypobromite brings about
this decomposition, as follows:
CO(NH,), + 3NaOBr-*3NaBr + N2 + CO, + 2H,0.
This property forms the basis for a clinical quantitative determination
of urea (sec page 392).
Urea has the power of forming crystalline compounds with certain
acids; urea nitrate and urea oxalate are the most important of these
compounds. Urea nitrate, C0(NH,),.HN03, crystallizes in colorless,
rhombic or six-sided tiles (Fig. 91, below), which are easily soluble in
water. Urea oxalate, 2.CO(NH2),.H2C204, crystallizes in the form
of rhombic or six-sided prisms or plates (Fig. 93, p. 289): the oxalate
dififers from the nitrate in being somewhat less soluble in water.
Fig. 91. — Urea Xitrate.
A decrease in the excretion of urea is observed in many diseases in
which the diet is much reduced and in some disorders as a result of
alterations in metabolism, e. g., myxoedema, and in others as a result
of changes in excretion, as in severe and advanced kidney disease. A
pathological increase is found in a large proportion of diseases which
are associated with a toxic state.
Experiments ox Urea.
I. Isolation from the Urine. ^ — Place 800 c.c. of urine in a pre-
cipitating jar, add 250 c.c. of baryta mixture,- and stir thoroughly.
' The method based upon the precipitation by nitric acid is also satisfactor)' (see Hoppe-
Seyler's Handbuch der Physiol, und Pathol. Chem. Anal., Eighth edition, 1909, p. 145.)
' Ban-ta mixture consists of a mixture of one volume of a saturated solution of Ba(N03)2
and two volumes of a saturated solution of Ba(OH)2.
PHYSIOLOGICAL CHEMISTRY.
Filter off the precipitate of phosphates, sulphates, urates, and hip-
purates and evaporate the filtrate on a water-bath to a thick syrup. This
syrup contains chlorides, creatinine, 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
filtrate away in a cold place for crystallization. Examine the crystals
under the microscope and compare them with those shown in Fig. 90,
page 285.
2. Solubility. — Test the solubility of urea, prepared by yourself or
furnished by the instructor, in the ordinary solvents (see p. 27) and in
alcohol and ether.
3. Melting-point. — Determine the melting-
point of some pure 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 thermo-
meter as shown in Fig. 92, and suspend the bulb
and its attached tube in a small beaker contain-
ing sulphuric acid. Gently raise the temperature
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 mi-
croscope, and compare them with those reproduced
in Fig. 90, p. 285. Recrystallize 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 care-
fully 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, dissolve the residue
in dilute potassium hydroxide solution, and add very dilute copper sul-
phate solution (see p. 98). The purplish-violet color is due to the pre-
sence of biuret which has been formed from the urea through the appli-
cation of heat as indicated. This is the reaction:
Fig. 92. — Melting-
point Tubes Fastened
TO Bulb of Thermo-
meter.
NH,
2 C=0
URINE.
NH,
I
c=o
289
Urea.
NH + NH,
c=o
NH3
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 of concentrated
nitric acid, and examine under the microscope. Compare the crystals
with those reproduced in Fig. 91, p. 287.
7. Urea Oxalate.— To a drop of a concentrated solution of urea,
prepared as described in the last experiment (6), add a drop of a satu-
FiG. 93. — Urea Ox.-u-ate.
rated solution of oxalic acid. Examine under the microscope and
compare the crystals with those shown in Fig. 93, above.
8. Decomposition by Sodium Hypobromite. — Into a mixture
of 3 c.c. of concentrated sodium hydroxide solution and 2 c.c. of bro-
mine water in a test-tube introduce a crystal of urea or a small amount of
concentrated solution of urea. Through the influence of the sodium hypo-
bromite, NaOBr, 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 effer-
vescence observed. This property forms the basis for one of the methods
in common use for the quantitative determination of urea. Write the
19
290 PHYSIOLOGICAL CHEMISTRY.
equation showing the decomposition of urea by sodium hypobromite.
g. Furfurol Test. — To a few crystals of urea in a small porcelain
dish add 1-2 drops of a concentrated aqueous solution of furfurol and
1-2 drops of concentrated hydrochloric acid. Note the appearance
of a yellow color which gradually changes into a purple. Aliantoin
also responds to this test (see page 305).
HN-C=0
I 1
URIC ACID, O C C - NH
^CO.
HN-C-HN
Uric acid is one of the most important of the constituents of the
urine. It is generally stated that normally about 0.7 gram is excreted
in 24 hours but that this amount is subject to wide variations, particu-
larly under certain dietary and pathological conditions. Yery recently
it has been shown that the average daily excretion of uric acid for ten
men ranging in age from 19 to 29 years and fed a normal mixed diet
w^as 0.597 g^2,m, a value somewhat lower than the generally accepted
average of 0.7 gram for such a period. Uric acid is a diureide and
consequently upon oxidation pelds 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 ammonium urate is difl&cultly soluble. The
acid-alkali urates are more insoluble and form the major portion of the
sediment which separates upon cooling the 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 of one of the older methods for the quantitative deter-
mination of uric acid (Heintz Method, p. 390).
Uric acid is very closely related to the purine bases as may be seen
from a comparison of its structural formula with those of the purine
bases given on page 261. According to the purine nomenclature it is
designated 2-6-8-trioxypurine. Uric acid forms the principal end-
product of the nitrogenous metabolism of birds and scaly amphibians;
in the human organism it occupies the fourth position inasmuch as here
urea, ammonia, and creatinine are the chief end-products of nitrogenous
metabolism. It is generally said that the relarion 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 variations under
pathological conditions; and I'urther th:.t because of the high content of
PT.ATE V.
Uric Acid Crystals. Normal Color. (From Purdy, after Peyer.)
URINE. 291
uric acid in the urine of new-born infants the ratio may be reduced to
1 : 10 or even lower. We now know that this ratio of uric acid to urea
is of little significance under any conditions.
In man, uric acid probably results principally from the destruction
of nuclein material. It may arise from nuclein or other purine material
ingested as food or from the disintegrating cellular matter of the organ-
ism. The uric acid resulting from the first process is said to be of ex-
ogenous origin, whereas the product of the second form of activity is
said to be of endogenous origin. As a result of experimentation, Siv6n,
and Burian and Schur, and Rockwood claim that the amount of endoge-
nous 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 FoHn has taken
exception to the statements of these investigators and claims that, fol-
lowing a pronounced decrease in the amount of protein metabolized,
the absolute quantity of uric acid is decreased but that this decrease is
relatively smaller than the decrease in the total nitrogen excretion and
that the per cent of the uric acid nitrogen, in terms of the total nitrogen,
is therefore decidedly increased.
In birds and scaly amphibians the formation of uric acid is analo-
gous to the formation of urea in man. In these organisms it is derived
principally from the protein 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 excre-
tion 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,
crystalHne, rhombic plates. Uric acid as it separates from the urine
is invariably pigmented, and crystallizes in a large variety of character-
istic forms, e. g.. dumb-bells, wedges, rhombic prisms, irregular rect-
angular or hexagonal plates, whetstones, prismatic rosettes, etc. Uric
acid is insoluble in alcohol and ether, soluble with diflficulty in boiling
water (1:1800) and practically insoluble in cold water (1:39,480, at
18° C). It is soluble in alkalis, alkali carbonates, boiling glycerol,
concentrated sulphuric acid, and in certain organic bases such as ethyl-
amine and piperidine. It is claimed that the uric acid is held in solu-
tion in the urine by the urea and disodium 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
292 PHYSIOLOGICAL CHEMISTRY.
characteristic red or brownish-red precipitate of cuprous oxide is ob-
tained. 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 Nylander's tests.
In addition to being an important urinary constituent uric acid is
normally present in the brain, heart, liver, lungs, pancreas, and spleen;
it also occurs in the blood of birds and has been detected in traces in
human blood under normal conditions.
Pathologically, the excretion of uric acid is subject to wide variations,
but the experimental findings are rather contradictory. 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 i : 50 or higher.
In the study of the influence of X-ray on metabolism Edsall 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 concentrated hydro-
chloric acid, stir thoroughly, and stand the vessel in a cold place for 24
hours. Examine the pigmented crystals of uric acid under the micro-
scope and compare them with those shown in Fig. 106, p. 365 and PI.
v., opposite p. 291.
2. Solubility. — Try the solubility of pure uric acid, furnished by
the instructor, in the ordinary solvents (see p. 27) and in alcohol, ether,
concentrated sulphuric acid and in boiling glycerol.
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 hydro-
chloric 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. 94, p. 293.
4. Murexide Test. — To a small amount of pure uric acid in. a small
evaporating dish add 2-3 drops of concentrated nitric acid. Evapo-
rate to dryness carefully on a water-bath or over a very low flame. A
red or yellow residue remains which turns purplish-red after cooling the
URINE.
293
dish and adding a drop of very dilute ammonium hydroxide. The color
is due to the formation of murexide. If potassium hydroxide is used
instead of ammonium hydroxide a purplish-violet color due to the pro-
duction of the potassium salt is obtained. The color disappears upon
warming; with certain related bodies (purine bases) the color persists
under these conditions,
5. Moreigne's Reaction. — To equal volumes of Moreigne's reagent^
and the solution to be tested add a few drops of concentrated potas-
sium hydroxide. A blue color indicates the presence of uric acid.
6. Schiff's Reaction. — Dissolve a small amount of pure uric acid
in sodium carbonate solution and transfer a drop of the resulting mix-
Fro. 94. — Pt.-RE Uric .\cid.
ture to a strip of filter paper saturated with silver nitrate solution.
A yellowish-brown or black coloration due to the formation of reduced
silver is produced.
7. Ganassini's Test.- — Dissolve a small amount of uric acid in
sodium carbonate. Precipitate the dissolved uric acid by means of
zinc chloride, filter oflf the precipitate, and permit it to stand in contact
with the air. A sky-blue color will develop, a color change which may
be hastened by sunlight. A similar reaction may be obtained by treat-
ing the original precipitate with K2S2O8.
8. Influence upon Fehling's Solution. — Dilute i c.c. of Fehling's
solution \\ith 4 c.c. of water and heat to boiling. Now add slowly, a
few drops at a time, 1-2 c.c. of a concentrated solution of uric acid in
* Moreigne's reagent is made by combining 20 grams of sodium tungstate, 10 grams
of phosphoric acid (sp. gr. 1.13) and 100 c.c. of water. Boil this mixture for twenty minutes,
add water to make the volume of the solution equivalent to the original volume, and acidify
with hydrochloric acid.
* Ganassini: Boll, soc, 1908, No. i.
294 PHYSIOLOGICAL CHEMISTRY.
potassium hydroxide, heating after each addition. From this experiment
what do you conclude regarding the possibiHty of arriving at an erroneous
decision when testing for sugar in the urine by means of Fehling's test ?
9. Reduction of Nylander's Reagent. — To 5 c.c. of a solution 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
I
CREATININE, C = NH
N.(CH3).CH,
Creatinine is the anhydride of creatine and is a constituent of normal
human urine. The theory that creatinine is derived from the creatine
of ingested muscular tissue as well as from the creatine of the muscular
tissue of the organism has recently been proven to be incorrect by Folin,
Klercker, and Wolf and Shaffer. Shaffer believes that creatinine is
the result of some special process of normal metabolism which takes
place to a large extent, if not entirely, in the muscles and further that
the amount of such creatinine elimination, expressed in milligrams per
kilogram body weight, is an index of this special process.^ He further
states that the muscular efficiency of the individual depends upon the
intensity of this process. Under normal conditions about i gram of
creatinine 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 excretion of creatinine under pathological conditions. The creatinine
content of the urine is said to be increased in typhoid fever, typhus,
tetanus, and pneumonia, and to be decreased in anaemia, chlorosis,
paralysis, muscular atrophy, advanced degeneration of the kidneys, and
in leukaemia (myelogenous, lymphatic and pseudo). An increase of
creatinine was also noted in diabetes, an increase probably due to the
creatinine content of the meat eaten. The greater part of the data,
however, relating to the variation of the creatinine excretion under
pathological conditions are not of much value since in nearly every
instance the diet was not sufficiently controlled to permit the collection
of reliable data. And further, until the advent of the Folin method
(see p. 415), there was no accurate method for the quantitative determina-
' He propsoes to designate as the "creatinine coefficient" the excretion of creatinine-nitro-
gen (mgs.) per kilogram of body weight.
^ According to Shaffer the amount excreted by strictly normal individuals is between 7
and II milligrams of crealinine-nitrogen per kilogram of body weight.
URINK.
295
tion of creatinine. ShalTer has very recently called attention to the fact
that a low excretion of creatinine is found in the urine of a remarkably
large number of pathological subjects, representing a variety of conditions,
and that it is therefore evident that the excretion of an abnormally small,
amount of this substance is by no means peculiar to any one disease.
Creatinine crystallizes in colorless, glistening monoclinic prisms
(Fig. 95, belo\v) which are soluble in about 12 parts of cold water; they are
more solube in warm water and in warm alcohol. It forms salts only
with strong mineral acids. One of the most important and interesting
of the compounds of creatinine is creatinine-zinc chloride, (C^H7N30)2-
ZnCU. which is formed from an alcoholic solution of creatinine upon
Fig. 95. — Creatinine.
treatment with zinc chloride in acid solution. Creatinine has the power
of reducing cupric hydroxide in alkaline solution and in this way may
interfere with the determination of sugar in the urine. In the reduction
by creatinine 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. Creatinine
does not reduce alkaline bismuth solutions and therefore does not interfere
with Nylander's and Boettger's tests.
It has recently been shown by Folin that the absolute quantity of
creatinine eliminated in the urine on a meat-free diet is a contsant quantity
different for different individuals, but wholly independent of quantita-
tive changes in the total amount of nitrogen eliminated. Shaffer has
very recently confirmed these findings and has shown that the output of
creatinine under these conditions is constant from hour to hour as well
as from dav to dav.
296
PHYSIOLOGICAL CHEMISTRY.
According to Pekelharing^ muscular tonus increases the creatinine
excretion of the individual whereas muscular exertion does not.
Experiments on Creatinine.
I. 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 CaClj solution
until the phosphates are completely precipitated. Filter off the precipi-
tate, 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
Fig.
Creatinine-zinc Chloride. (Salkowski.)
and allowed to stand in a cold place for 48-72 hours. Creatinine-zinc
chloride (Fig. 96, above) will crystalhze out under these conditions.
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. (Creatine remains undissolved under
these conditions.) The alcoholic extract of creatinine is now evapo-
rated to incipient crystallization and left in a cool place until crystal-
lization is complete. If desired the crystals may be purified by re-
crystallization from water.
2. Weyl's Test. — Take 5 c.c. of urine in a test-tube, add a few
drops of sodium nitro-prusside and render the solution alkaline with
* Pekelharing: Onderzoekingengedaan in het Physiol, Lab. te Utrecht, Vol. 5, No. 12, 1911.
URINE. 297
potassium hydroxide solution. A ruby-red color results which soon
turns yellow. See Legal's test for acetone, page 349.
3. Salkowski's Test. — To the yellow solution obtained in Weyl'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. Dextrose gives a similar red color
but only upon the application of heat. This color reaction observed
when creatinine in alkaline solution is treated with picric acid is the
basic principle of FoHn's colorimetric method for the quantitative
determination of creatinine (see page 415.)
ETHEREAL SULPHURIC ACIDS.
The most important of the ethereal sulphuric acids found in the
urine are phenol-sulphuric acid, p-cre sol-sulphuric acid, indoxyl-sulphuric
acid, and skatoxyl-sidphuric acid. Pyrocatechin-sulphuric acid also
occurs in traces in human urine. The total output of ethereal sul-
phuric acid varies from 0.09 to 0.62 gram for 24 hours. In health the
ratio of ethereal sulphuric acid to inorganic sulphuric acid is about
1:10. These ethereal sulphuric acids originate in part from the phenol,
cresol, indole and skatole formed in the putrefaction of protein material
in the intestine. The phenol passes to the liver where it is conjugated
to form phenol potassium sulphate and appears in this form in the urine
whereas the indole and skatole undergo a preHminary 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 protein material in
the intestine and that therefore a determination of the total ethereal
sulphuric acid content of the urine was an index of the extent to which
these putrefactive processes were proceeding within the organism.
Recently, however, 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 putrefaction, since these
bodies arise only in part from putrefactive processes. He claims that the
ethereal sulphuric acid excretion represents a form of sulphur metabohsm
which is more in evidence upon a diet containing a very small amount of
protein or upon a diet containing absolutely no protein. The ethereal
sulphuric acid content of the urine diminishes as the total sulphur content
diminishes but the percentage decrease is much less. Therefore when
298 PHYSIOLOGICAL CHEMISTRY.
considered from the standpoint 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 experiments also
seem to show that the indoxyl sulphuric acid (indoxyl potassium sulphate
or indican) content of the urine does not originate to any degree from the
metabolism of protein material but that it arises in great part from
intestinal putrefaction and that the excretion of indoxyl sulphuric acid
may alone be taken as a rough index of the extent of putrefactive proc-
esses within the intestine, Indoxyl sulphuric acid,
CH
- /\
HC C-C(0.S03H),
HC C CH
CH NH
therefore, which occurs in the urine as indoxyl potassium sulphate or
indican,
CH
HC C - C(0.S03K),
I II !!
HC C CH
CH NH
is clinically the most important of the ethereal sulphuric acids.
Tests for Indican.^
I. Jaffe's Test. — Nearly fill a test-tube with a mixture composed
of equal volumes of concentrated HCl and the urine under exami-
nation. 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 the tube and contents thoroughly. The chloroform 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.
Repeat this test on some of this same urine to which formaldehyde has
been added. Is there any variation in the reaction from what you
previously obtained ?
' Tlie urine should always be examined /re^/f if this is possible. In any event formaldehyde
should never be used as a preservative for such urines as are to be examined for indican by
means of any test involving hypochlorite or potassium permanganate. The formaldehyde
through its redu( ing power lowers the oxidizing efficiency of the mixture The formation of
formic acid from the aldehyde may also interfere.
URINK. 299
This is the reaction (see also page 1O9):
CI I
HC C - con
2 I II II +2O-
HC C CH
CH NH
Indoxyl, C»H7NO.
CH CH
HC C - CO O.C-C CH
I II I I II I +2H,0
HC C C==C C CH
CH NH NH CH
Indigo-blur, Ci;-,HioNuOj.
2. Obermayer's Test. — Nearly fill a test-tube with a mixture com-
posed of equal volumes of Obermayer's reagent^ and the urine under
examination. Add 2-3 c.c. of chloroform, place the thumb over the end
of the test-tube and shake thoroughly. How does this compare with
Jaffa's test ?
3. Giirber's Reaction. — To dne volume of the urine under ex-
amination and two volumes of concentrated hydrochloric acid in a
test-tube add 2-3 drops of a i per cent solution of osmic acid and 2-3
c.c. of chloroform and shake the tube and contents thoroughly. Com-
pare the color with those obtained in Jaffe's and Obermayer's tests.
An excess of osmic acid does not affect the reaction. Occasionally
better results are obtained if the solution of osmic acid is added directly
to the urine before the addition of the hydrochloric acid. If the urine
under examination be strongly colored or of high specific gravity it
should first be treated with basic lead acetate (one-eighth volume).
The precipitate is then removed by filtration and the resulting filtrate
used in making the test for indican.
4. Rossi's Reaction. — To equal volumes of concentrated hydro-
chloric acid and the urine under examination, in a test-tube, add i
drop of a 10 per cent solution of ammonium persulphate and 2-3 c.c.
of chloroform. Agitate the mixture vigorously and note the color of
the chloroform. Compare this result with those obtained in the other
indican tests.
5. Lavelle's Reaction. — To 10 c.c. of urine in a test-tube add
2-3 c.c. of Obermayer's reagent^ and a similar volume of concentrated
* Obermayer's reagent is prepared by adding 2-4 grams of ferric chloride to a liter of con-
centrated HCl (sp. gr. 1. 19).
300
PHYSIOLOGICAL CHEMISTRY.
sulphuric acid. (During the addition of the acid the tube should be
held under running water in order that the temperature of the mixture
may not rise too high.) Add 2-3 c.c. of chloroform, shake the tube
^dgorously, and observe the depth of color assumed by the chloroform.
The sponsor for this reaction claims it to be the most satisfactory
of the indican tests.
6. Barberio's Reaction/ — To 5 c.c. of the urine in a test-tube
add 2-3 drops of a sodium nitrite solution (i : 2000) and mix well by shak-
ing. Now add 5 c.c. of concentrated hydrochloric acid and 2-3 c.c.
of chloroform and again shake. Note the color of the chloroform.
Compare this test with tests i and 2 on the same urine.
CO.NH.CH^.COOH.
flIPPURIC ACID,
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
Fig. 97. — HipPURic Acid.
by a synthesis of benzoic acid and glycocoU 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. 97, above) the particular form
depending upon the rapidity of crystallization. Pure hippuric acid
melts at 187° C. The most satisfactory method for the isolation of
hippuric acid from the urine in crystalline form is that proposed by
Roaf (see below j. It is easily soluble in alcohol or hot water, and
' Barberio: Policlinico, No. 17, 191 1.
URINE. 301
only slightly soluble in ether. The output of hippuric acid is increased
in diabetes owing probably to the ingestion of much protein and fruit.
It is decreased in fevers and in certain kidney disorders where the synthetic
activity of the renal cells is diminished. Hippuric acid may be deter-
mined quantitatively by means of Dakin's methods (see p. 406).
Experiments on Hippuric Acid.
I. Separation from the Urine, (a) First Method. — Render 500-
1000 c.c. of urine of the horse or cow^ alkaline with milk of lime, boil
for 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 per-
colate the hot solution through thoroughly washed animal charcoal,
being careful to wash out the last portion of the hippuric acid solution
with hot water. Filter, concentrate the filtrate to a small volume and
stand it aside for crystallization. Examine the crystals under the micro-
scope and compare them with those in Fig. 97, page 300. This method
is not as satisfactory as Roaf's method (see below).
(b) Roafs Method. — Place 500 c.c. of urine of the horse or cow^
in a casserole or precipitating jar and add an equal volume of a satu-
rated solution of ammonium sulphate^ and 7.5 c.c. of concentrated
sulphuric acid. Permit the mixture to stand for twenty-four hours and
remove the crystals of hippuric acid by filtration. Purify the crystals
by recrystallization according to the directions given above under First
Method. Examine the crystals under the microscope and compare
them with those given in Fig. 97, p. 300.
If sufficient urine is not available to peimit the use of 500 c.c. a
smaller volume may be used inasmuch as it is possible, by the above
technic, to isolate hippuric acid in crystalline form from as small a
volume as 25-50 c.c. of herbivorous urine. The greater the amount of
ammonium sulphate added the more rapid the crystallization until
at the saturation point the crystals of hippuric acid sometimes form in
about ten minutes.
* 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 con-
veniently accomplished by ingesting 2 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. In case ammonium benzoate is not available
sodium benzoate may be substituted.
* 125 grams of solid ammonium sulphate may be substituted.
302 PHYSIOLOGICAL CHEMISTRY.
2. Melting-point. — Determine the melting-point of the hippuric
acid prepared in the above experiment (see p. 146).
3. Solubility. — Test the solubility of hippuric acid in the ordinary
solvents (page 27) and in alcohol, and ether.
4. Dehn's Reaction. — Introduce about 5 c.c. of the urine or the
solution under examination into a test-tube and add sufficient hypo-
bromite solution^ to impart to the mixture a permanent yellow color.
In the case of urine enough hypobromite should be added to decom-
pose the urea. Heat the mixture to boiling and note the formation
of an orange or brown-red precipitate if hippuric acid is present. If
the solution under examination contains only a trace of hippuric acid
the solution will appear smoky and faintly red in color, whereas if a
larger amount of the acid be present the solution will become opaque
and of an orange or brown-red color. In either case after standing
for some time the solution should clear up and a light, finely divided
precipitate should be deposited. This precipitate consists of earthy
phosphates mixed with an amorphous orange or brown-red substance
of unknown composition. (For some unknown reason this reaction
does not always yield satisfactory results even on pure hippuric acid
solution.)
5. Formation of Nitro-Benzene. — To a little hippuric acid in a
small porcelain dish add 1-2 c.c. of concentrated HNO3 and evapo-
rate 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).
6. 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 sub-
limate of benzoic acid and the odor of hydrocyanic acid.
7. Formation of Ferric Salt. — Render a small amount of a solu-
tion 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.
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 under-
' See note on p. 392.
URINE. 303
Stood. It is eliminated, at least in part, unchanged when ingested, there-
fore 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 thq ingested food supplies a portion of the oxalic
acid found in the urine. There is also experimental evidence that part
of the oxalic acid of the urine is formed within the organism in the
course of protein 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 accom-
panied by a derangement of the oxidation mechanism. An abnormal
increase of oxalic acid is termed oxaluria. 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 oclahedra (Fig. 104, page 363).
Experiments.
Preparation of Calcium Oxalate. First Method. — Place 200-250
c.c. of urine in a beaker, add 5 c.c. of a saturated solution of calcium
chloride, make the urine slightly acid with acetic acid, and stand the
beaker aside in a cool place for 24 hours. Examine the sediment under
the microscope and compare the crystalline forms with those shown in
Fig. 104, p. 363.
Second Method. — ^Proceed as above, replacing the acetic acid by an
excess of ammonium hydroxide and filtering off the precipitate of phos-
phates.
NEUTRAL SULPHUR COMPOUNDS.
Under this head may be classed such bodies as cystine (see p. 80),
chondroitin-sulphuric acid, oxyproteic acid, alloxyproteic acid, uroferric
acid, thiocyanates and taurine derivatives. The sulphur content of the
bodies just enumerated is generally termed loosely combined or neutral
sulphur in order that it may not be confused with the acid sulphur which
occurs in the inorganic 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.
NH.CH.NH
I I .
ALLANTOIN, OC CO.
! I
NH.CO NH
304
PHYSIOLOGICAL CHEMISTRY.
Allantoin has been found in the urine of suckHng calves as well
as in that of the dog and cat, rabbit, monkey, horse and nian.^ 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 mgre abundant in the urine
of women during pregnancy. Underbill also reports the presence of
allantoin in the urine of fasting dogs, an observation which makes it
probable that allantoin is a constant constituent of the urine of such
animals. Allantoin is formed by the oxidation of uric acid and the output
is increased by the feeding of thymus or pancreas to lower animals.
In fact allantoin is considered to be the principal end-product of purine
Fig. 98. — Allantoin, from Cat's Urine.
a and b, Forms in which it crystallized from the urine; c, recrystallized allantoin. (Drawn
from micro-photographs furnished by Prof. Lafayette B. Mendel of Yale University.)
metabolism in such animals. Nothwithstanding certain evidence^ favor-
ing this view it is not generally believed that allantoin is an important
end-product of purine metabolism in man. When pure it crystaUizes
in prisms (Fig. 98, above) and when impure in granules and knobs.
Pathologically, it has been found increased in diabetes insipidus and in
hysteria with convulsions (Pouchet). Mendel and Dakin* have recently
shown that allantoin is optically inactive notwithstanding the fact that it
contains an asymmetric carbon atom. This phenomenon they believe to
be due to tautomeric change. Wiechowski has suggested an excellent
method for the quantitative determination of allantoin.*
* Wiechowski: Biochemische Zeitschtift, 19, 368, 1909.
^Ascher: Biochemische Zeitschrift, 26, 370, 1910; Fairhall and Hawk: Jour. Am. Chem,
Soc, 34, 546, 1912.
^Mendel and Dakin: Jour. Biol. Chem., 7, 153, 1910.
* Wiechowski: Biochemische Zeitschrift, 19, 368, 1909.
URINE. 305
Experiments.
1. Separation from the Urine/ Meissner's Method. — Precipi-
tate the urine with Ijaryta water. Neutralize the filtrate carefully
with dilute sulphuric acid, filter immediately, and evaporate the fil-
trate to incipient crystallization. Completely 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; allantoin
remains undissolved. Bring the allantoin into solution in hot water and
re crystallize.
Allantoin may be determined quantitively by the Paduschka-
Underhill-Kleiner method (see p. 432) or by Loewi's method.^
2. Preparation from Uric Acid.— Dissolve 4 grams of uric acid
in 100 c.c. of water rendered alkaline with potassium hydroxide. Cool
and carefully add 3 grams of potassium permanganate. Filter, immedi-
ately acidulate the filtrate with acetic acid and allow it to stand in a cool
place over night. Filter oiT 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 crystalHzation. Now combine 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. 98,
page 304.
4. Solubility. — Test the solubility of allantoin in the ordinary
solvents (page 27.)
5. Reaction. — Dissolve a crystal in water and test the reaction to
litmus.
6. Furfurol Test. — Place a few crystals of allantoin on a test-tablet
or in a porcelain dish and add 1-2 drops of a concentrated aqueous
solution of furfurol and 1-2 drops of concentrated hydrochloric acid.
Observe the formation of a yellow color which turns to a light purple if
allowed to stand. This test is given by urea but not by uric acid.
7. Murexide Test. — Try this test according to the directions given
on page 292. Note that allantoin fails to respond.
8. Reduction of Fehling's Solution. — Make this test in the us al
way (see p. 32) except that the boiling must be prolonged and excessive.
Ultimately the allantoin will reduce the solution. Compare with the
result on uric acid, page 293.
' The urine of the dog after thymus, pancreas, or uric acid feeding may be employed.
* Archiv fur Experimentelle Pathologie und Pharmakologie, 44, 20, 1900.
306 PHYSIOLOGICAL CHEMISTRY.
AROMATIC OXYACIDS.
Two of the most important of the oxyacids are paraoxyphenyl-acetic
acid.
CH,.COOH,
OH
and paraoxyphenyl-propionic acid,
CH,.CH2.C00H.
OH
They are products of the putrefaction of protein material and tyrosine
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 content may be increased in the same manner
as the phenol content, in particular by acute phosphorous poisoning. A
fraction of the total aromatic oxyacid content of the urine is in combi-
nation 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
CH^.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 Pennsylvania; subsequently Baumann
isolated it and determined its chemical constitution. It occurs in cases
of alcaptonuria. 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 reduces alkaline copper solu-
tions but not alkaline bismuth solutions. Uroleucic acid is similar in
its reactions to homogentisic acid.
Oxymandelic Acid or paraoxyphcnyl-glycolic acid,
URINE. 307
OH
CH(OH).COOH,
has been detected in t|je urine in cases of yellow atrophy of the liver.
Kynurenic Acid or ^-oxy-;9-quinoline carbonic acid,
CH COH
HC C C.COOH,
I II I
HC C CH
\/\/
CH N
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 propor-
tion 1:25. From this acid fluid both the uric acid and the kynurenic
acid separate in the course of 24-48 hours. Filter oflf 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 Capaldi's
method.^
COOH.
/
BENZOIC ACID,
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 compare them
with those reproduced in Fig. 99, p. 308.
3. Sublimation. — Place a little benzoic acid in a test-tube and heat
' Zeitschri/t fiir physiologische Chemie, 23, 92, 1897.
?o8
PHYSIOLOGICAL CHEMISTRY.
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 solution
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 hydroxide to some of the precipitate.
It dissolves and ferric hydroxide is formed. Add a little hydrochloric
Fig. 99. — Benzoic Acid.
acid to another portion of the original precipitate and stand the vessel
away over night. What do you observe ?
NUCLEOPROTEIN.
The nubecula of normal urine has been shown by one investigator
to consist of a mucoid containing 12.7 per cent of nitrogen and 2.3
per cent of sulphur. This body evidently originates in the urinary
passages. It is probably slightly soluble in the urine. Some investigators
believe that the body forming the nubecula of normal urine is nucleo-
protein and not a mucin or mucoid as stated above. A discussion of
nucleoprotein and related bodies occurring in the urine under pathological
conditions will be found on page 339.
NH-CO
OXALURIC ACID, CO I
NH, CO OH.
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. 309
ENZYMES.
Various types of enzymes produced within the organism are excreted
in both the feces and the urine. In this connection it is interesting to
note that pepsin, gastric rennin, and an amylase have been positively
identified in the urine. The occurrence of trypsin in the urine, at least
under normal conditions, 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 principally from the fermentation
of carbohydrates and the putrefaction of proteins. 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 increased 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,
I
PARALACTIC ACID, CH(OH)
COOH.
Paralactic acid is supposed to pass into the urine when the supply
of oxgyen in the organism is diminished through any cause, e. g., after
acute yellow atrophy of the liver, acute phosphorus poisoning, or epi-
leptic attacks. This acid has also been found in the urine of healthy
persons following the physical exercise incident to prolonged marching.
Paralactic acid has been detected in the urine of birds after the removal
of the liver. Underbill reports the occurrence of this acid in the urine
of a case of pernicious vomiting of pregnancy.
CH2.CO.NH.CH2.COOH.
PHENACETURIC ACID,
Phenaceturic acid occurs principally in the urine of herbivorous
animals but has frequently been detected in human urine. It is pro-
[lO PHYSIOLOGICAL CHEMISTRY.
duced in the organism through the synthesis of glycocoll and phenyl-
acetic 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 urochrome, urobilin, and uroeryihrin.
A. UROCHROME.
This is the principal pigment of normal urine and imparts the char-
acteristic yellow color to that fluid. It is apparently closely related
to its associated pigment urobilin since the latter may be readily con-
verted 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.
B. UROBILIN.
UrobiUn, which was at one time considered to be the principal pig-
ment of urine, in reality contributes little toward the pigmentation
of this fluid. It is claimed that no urobilin is present in freshly voided
normal urine but that its precursor, a chromogen called urobilinogen,
is present and gives rise to urobilin upon decomposition through the
influence of light. It is claimed by some investigators that there are
various forms of urobihn, e. g., normal, febrile, physiological, and patho-
logical. Urobilin is said to be very similar to, if not absolutely identical
with, hydrobilirubin (see page 179).
Urobilin may be obtained as an amorphous powder which varies
in color from brown to reddish-brown, red and reddish-yellow, depend-
URINE.
3"
ing upon the way in which it is prepared. It is easily soluble in ethyl
alcohol, amy! alcohol, and chloroform, and slightly soluble in ether,
acetic ether, and in water. Its solutions show characteristic absorption-
bands (see Absorption Spectra, Plate II). Under normal conditions
urobilin is derived from the bile pigments in the intestine.
Urv)bilin is increased in most acute infectious diseases such as ery-
sipelas, malaria, pneumonia, and scarlet fever. It is also increased in
appendicitis, carcifioma of the liver, catarrhal icterus, pernicions ancemia,
and in cases of poisoning by antifebrin, antipyrin, pyridin, and potas-
sium chlorate. In general it is usually increased when blood destruction
is excessive and in many disturbances of the liver. It is markedly
decreased in phosphorus poisoning.
Experiments.
1. Spectroscopic Examination.— Acidify the urine with hydro-
chloric acid 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 spectro-
scope. If urobiHn is present in the fluid the characteristic absorption-
band lying between b and F will be observed (see Absorption Spectra,
Plate II). It 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 lo c.c. of urine with hydrochloric acid 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 precipitate of phosphates and add
a few drops of zinc chloride solution to the filtrate. Observe the pro-
duction 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 chloro-
form and shake well. Separate the chloroform extract and add to
it a few drops of iodine solution (I in KI). Render the mixture alka-
line with dilute solution of potassium hydroxide and note the produc-
tion of a yellow or yellowish-brown color. The solution ordinarily
exhibits a greenish fluorescence.
4. Wirsing's Test. — To 20 c.c. of urine add 3-5 c.c. of chloro-
312 PHYSIOLOGICAL CHEMISTRY.
form and shake gently. Separate the chloroform ^ extract and add
to it a drop of an alcoholic solution of zinc chloride. Note the rose-
red color and the greenish fluorescence. 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 absorp-
tion-band (see Absorption Spectra, Plate II).
6. Ring Test. — Acidify 25 c.c. of urine with 2-3 drops of concen-
trated hydrochloric acid, add 5 c.c. of chloroform and shake the mix-
ture. Separate the chloroform, place it in a test-tube, and add care-
fully 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 be observed.
C. UROERYTHRIN.
This pigment is frequently present in small amount in normal urine.
The red color of urinary sediments is due in great part to the presence
of uroerythrin. It is easily soluble in amyl alcohol, slightly soluble in
acetic ether, absolute alcohol, or chloroform, and nearly insoluble in
water. Dilute solutions of uroerythrin are pink in color while concen-
trated solutions are orange-red or bright red : none of its solutions fluor-
esce. Uroerythrin is increased in amount after strenuous physical exer-
cise, 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. Very little is known, definitely, however, about them.
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 spe-
cific and definite symptom when injected intravenously into a rabbit.
The resulting symptoms are narcosis, sahvation, 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.
PURINE BASES.
The purine bases found in human urine are adenine, carnine, epi-
guanine, episarkine, guanine, xanthine, heteroxanthine, hypoxanthine,
paraxanthine, and i-methylxanthine. The main bulk of the purine
URINE. 313
base content of the urine is made up of paraxanthine, heteroxanthine
and i-methylxanthine which are derived for the most part from the caf-
feine, theobromine, and theophylline of the food. The total purine base
content is made up of the products of two distinct forms of metabolism,
f. e., metabolism of ingested nucleins and purines and metabolism of
tissue nuclein material. Purine bases resulting from the first form of
metabolism are said to be of exogenous origin whereas those resulting from
the second form of metabolism are said to be of endogenous origin. The
daily output of purine bases by the urine is extremely small and varies
greatly with the individual (16-60 milligrams). The output is increased
after the ingestion of nuclein material as well as after the increased destruc-
tion of leucocytes. A well marked increase accompanies leukaemia.
Edsall has shown that the output of purine bases by the urine is in-
creased as a result of X-ray treatment.
Experiment.
I. Formation of the Silver Salts. — Add an excess of magnesia
mixture^ to 25 c.c. of urine. Filter off the precipitate and add am-
moniacal silver solution" to the filtrate. A precipitate composed of
the silver salts of the various purine bases is produced. The purine
bases may be determined quantitatively by means of Kriiger and Schmidt's
method (see p. 429), or Welker's method (see p. 328).
2. Inorganic Physiological Constituents.
Ammonia.
Next to urea, ammonia is the most important of the nitrogenous
end-products of protein metabolism. Ordinarily about 2.5-4.5 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 sidphate. This is due to the fact that combinations of this
sort are not oxidized in the organism to form urea, but are excreted as
such. This explains the increase in the output 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 ultimately appears in the urine as urea.
* Magnesia mixture may be prepared as follows: Dissolve 175 grams of MgS04 and
350 grams of NH^Cl in 1400 c.c. of distilled water. Add 700 grams of concentrated NH^OH,
mix very thoroughly and preserve the mixture in a glass-stoppered bottle.
' Ammoniacal silver solution may be prepared according to directions given on page 430.
314 PHYSIOLOGICAL CHEMISTRY.
Recent experiments^ indicate that the nitrogen in food protein may in
part be replaced by ammonium salts.
Copious water drinking increases the ammonia output. This fact has
been interpreted as indicating a stimulation of the gastric secretion.^
The acids formed during the process of protein destruction within
the body have an influence upon the excretion of ammonia similar to
that exerted by acids which have been administered. Therefore a
pathological increase in the output of ammonia is observed in such
diseases as are accompanied by an increased and imperfect protein me-
tabolism, and especially in diabetes, in which disease diacetic acid and
/3-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 protein metabolism, as measured by the total
nitrogen in the urine, is frequently accompanied by a decreased elimi-
nation of ammonia. The ammonia elimination is therefore probably
determined by other factors than the total protein catabolism as such.
Furthermore, 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 am-
moniacal fermentation (see page 276).
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 cystine, cysteine, taurine, hydrogen
sulphide, ethyl sulphide, thiocyanates, sulphonic acids, oxyproteic acid,
alloxyproteic acid, and uroferric acid. The amount of loosely com-
bined sulphur eliminated is in great measure independent of the extent
of protein decomposition or of the total sulphur excretion. In this
characteristic it is somewhat similar to the excretion of creatinine. The
oxidized sulphur is eliminated in the form of sulphuric acid, principally
as salts of sodium, potassium, calcium, and magnesium; a relatively
small amount occurs in the form of ethereal sulphuric acid. i. e., sulphuric
acid in combination with such aromatic bodies as phenol, indole, skatole,
cresol, pyrocatechin, and hydroquinone. Sulphuric acid in combination
' Grafe and Schlapfer: Zeil. physiol. chem., 77, i, 1912, experiments by Abderhalden in
same journal.
'Wills and Hawk: Jaur. Biol. Chem., 9, xxx, 1911 (Proceedings).
URINE. 315
with Na, K, Ca or Mg is sometimes termqfl inorganic or preformed
sulphuric acid, whereas the ethereal sulphuric acid is sometimes called
cofijiigale sulphuric acid. The greater part of the sul[)hur is eliminated
in the oxidized form, but the absolute percentage of sulphur excreted
as the preformed, ethereal or loosely combined type depends ujjon 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 BaClj, 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 in the urine arises principally from
the oxidation of protein material within the body; a relatively small
amount is due to ingested sulphates. Under normal conditions about
2.5 grams of sulphuric acid is eliminated daily. Since the sulphuric
acid content of the urine has, for the most part, a protein origin and
since one of the most important constituents of the protein 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 proteins 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 as end-products of protein metabolism, is
practically impossible. It has been suggested that the ratio 5 : i expresses
this relation in a general way.
Pathologically, the excretion of sulphuric acid by the urine is in-
creased 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 accompanied by a loss of appetite and a dimin-
ished 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 i c.c. of hydro-
chloric acid and a little barium chloride solution to the filtrate and heat
the mixture to boiling for 1-2 minutes. Note the appearance of a tur-
bidity due to the presence of sulphuric acid which has been separated
from the ethereal sulphates and has combined with the barium of the
BaClj to form BaSO^.
;i6
EEYSIOLOGICAL CHEMISTRY.
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 lead
acetate solution. In a short time the portion of the paper in contact
with the vapors within the test-tube be-
comes blackened due to the formation
of lead sulphide. The nascent hydro-
gen has reacted with the loosely com-
bined or neutral sulphur to form hydro-
gen sulphide and this gas coming in
contact with the lead acetate paper
has caused the production of the black
lead sulphide. Sulphur 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 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. 100,
above.
Fig.
100. — Calcium Sulphate.
{Hensel and Weil.)
Chlorides.
Next to urea, the chlorides constitute the chief solid constituent
of the urine. The principal chlorides found in the urine are those of
sodium, potassium, ammonium, and magnesium, with sodium chloride
predominating. The excretion of chloride 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 chloride. Copious water-drinking in-
creases 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 follows 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 examination 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
URINE. 317
body to maintain this equilibrium, however, and if the fasting organism
be subjected to the influence of diuretics for a time, a point is reached
where the composition of the blood can no longer be maintained and a
gradual decrease in its chloride content occurs which finally results in
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 replace the sodium chloride
of the blood when the latter is decreased in the manner above stated.
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, diarrhoea, certain stomach
disorders, and in acute articular rheumatism.
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 silver nitrate. A white precipitate, due to the formation
of silver chloride, is produced. This precipitate is soluble in am-
monium 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 combination with the alkaline
earths, calcium and magnesium. Phosphates 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, MgPO^, ^
mano-hydrogen, M.HPO^, and di-hydrogen, MH,PO^. 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. Recently, however, it
has been quite clearly shown that the normal 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
* M may be occupied by any of the alkali metals or alkaline earths.
3l8 PHYSIOLOGICAL CHEMISTRY.
combined acidity of all the phosphates present; the excess in the acidity
above that due to phosphates be beleives to be due to free organic acids.
Henderson' maintains that "determinations of hydrogen ionization in
urine and its behavior toward indicators both support the view that in
urine there exists a mixture of m&no- and di-hydrogen phosphates of
sodium, ammonium and other bases." The observation has recently
been made that urine may be separated into two portions, one part con-
sisting almost entirely of inorganic matter including practically all of
the phosphates and having an alkaline reaction, the other containing
practically all of the organic substances and no phosphates and having an
acid reactian.
In bones the phosphates occur principally in the form of the normal
salts of calcium and magnesium. The mono-hydrogen salts as a class
are alkaline in reaction to litmus, and it is to the presence of di-sodium
hydrogen phosphate, Na2HP04, 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 P2O5. Ordinarily the total output is distributed between alkaline
phosphates and earthy phosphates 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-proteins, nucleo-
proteins and lecithins; the phosphorus-containing tissues of the body
also contribute to the total output of this element. Alkaline phosphates
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 intestine
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 ordinary conditions, there can be no absolute relation-
ship between the urinary output of nitrogen and phosphorus. If the
diet is constant, however, from day to day, thus allowing of the prepara-
tion of both a nitrogen and a phosphorus balance,^ a definite ratio may
be established. In experiments upon dogs, which were fed an exclusive
meat diet, the ratio of nitrogen to phosphorus, in the urine and feces,
was found to be 8.1 : i.
' Henderson: Am. Jour. Physiol., 15, 257, 1906.
2 In metabolism experiments, a statement showing the relation existing between the nitro-
gen 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."
URINE.
319
It has been demonstrated by recent investigation that the ingestion of
inorganic phosphorus compounds may give rise to organic phosphorus
compounds such as lecithin, phosphatides, nucleoproteins and phospho-
proteins. This is an instance of an organic substance synthesized from
an inorganic substance. The experiments have been made principally
on ducks* and hens.'
Pathologically the excretion of phosphoric acid is increased in such
diseases of the bones as difluse periostosis, osteomalacia, and rickets;
according to some investigators, in the early stages of pulmonary tuber-
culosis, in acute yellow atrophy of the liver, in diseases which are accom-
panied 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 aflfections, such as the acute infec-
tious diseases; in pregnancy, in the period during which the foetal bones
are forming, and in diseases of the kidneys, because of non-elimination.
Experiments.
I. Formation of "Triple Phosphate." — Place some urine in a
beaker, render it alkaline with ammoniu mhydroxide, add a small
^
\
Fig. ioi. — "Triple Phosphate." (Ogden.)
amount of magnesium sulphate solution and allow the beaker to stand
in a cool place over night. Q-ystals 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. loi, above.
2. "Triple Phosphate" Crystals in Ammoniacal Fermentation.
— Stand some urine aside in a beaker for several days. Ammoniacal
* Fingerling: Biochem. Zeit., 38, 448, IQ12.
^McCollum and Halpin: Jour. Biol. Client., 11, 47 (Proceedings), 1912.
320 PHYSIOLOGICAL CHEMISTRY.
fermentation will develop and "triple phosphate" crystals will form.
Examine the sediment under the microscope and compare the crystals
with those shown in Fig. loi, below.
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 313) 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 was 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 dihydrogen phosphate, NaHgPO^,
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?
Sodium and Potassium.
The elements sodium and potassium are always present in the urine.
Usually they are combined with such acidic radicals as CI, CO3, SO^
and. PO4. The amount of potassium, expressed as KgO, excreted in
24 hours by an adult, subsisting upon a mixed diet, is on the average
2-3 grams, whereas the amount of sodium, expressed as NajO, under
the same conditions, is ordinarily 4-6 grams. The ratio of K to Na is
generally 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 tissues, 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
URINE. 321
principally upon the nature of the diet, aj^^regates on the averaj^e about
I gram and is made up of the phosphates of calcium and magnesium
in the proportion of 1:2. The percentage of calcium salts present in
the urine at any one time forms no dependable index as to the absorp-
tion of this class of salts, since they are again excreted into the intestine
after absorption. It is therefore impossible to draw any satisfactory
conclusions regarding the excretion of the alkaline earths unless we
obtain accurate analytical data from both the feces and the urine.
V^ry 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.
Carbonates.
Carbonates generally occur in small amount in the urine of man
and carnivora under normal conditions, whereas much larger quanti-
ties 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 carbonates. In general a urine containing carbonates
in appreciable amount is turbid when passed or becomes so shortly
after. These bodies ordinarily occur as alkali or alkaline earth com-
pounds and the turbid character of urine containing them is usually
due principally to the latter class of substances. The carbonates of
the alkaline earths are often found in amorphous urinary sediments.
Iron.
Iron is present in small amount in normal urine. It probably occurs
partly in inorganic and partly in organic combination. The iron con-
tained in urinary pigments or chromogens is in organic combination.
According to different investigators the iron content of normal urine will
probably not average more than 0.00 1 gram per day.
Experiment.
Detection of Iron in Urine. — Evaporate a convenient volume
(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 solu-
tion 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 ammo-
nium thiocyanate; a red color indicates the presence of iron, {b) To
the second part of the solution add a little potassium ferrocyanide solu-
tion; aprecipitate of Prussian blue forms upon standing.
322 PHYSIOLOGICAL CHEMISTRY,
Fluorides, Nitrates, Silicates and Hydrogen Peroxide.
These substances are all found in traces in human urine under
normal conditions. Nitrates are undoubtedly introduced into the
organism in the water and ingested food. The average excretion of
nitrates is about 0.5 gram per day, the output being the largest upon a
vegetable diet and smallest upon a meat diet. Nitrites are found only
in urine which is undergoing decomposition and are formed from
nitrates in the course of ammoniacal fermentation. Hydrogen peroxide
has been detected in the urine, but its presence is believed to possess_^no
pathological importance.
CHAPTER XIX.
URINE: PATHOLOGICAL CONSTITUENTS.^
Dextrese.
Proteins
Serum albumin.
Serum globulin.
f Dcutero-proteose.
Proteoses | Hetero-proteose.
"Bence- Jones' protein."
Peptone.
Nucleoprotein.
Fibrin.
Oxyhaemoglobin.
^, , [ Form elements.
Blood <^ ^.
[ Pigment.
Bile f ^'S™'"'^-
1 Acids.
Creatine.^
Acetone.
Diacetic acid.
,5-Oxybutyric acid.
Conjugate glycuronates.
Pentoses.
Fat.
Haema toporphy rin .
Lactose.
Galactose.
Laevulose.
Inosite.
Laiose.
Melanin.
L'rorosein.
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 qualitative
* See note at the bottom of page 283.
* Normal constituent of urine of infants and children.
324 PHYSIOLOGICAL CHEMISTRY.
tests. There are two distinct types of pathological glycosuria, i. e.,
transitory glycosuria and persistent glycosuria. 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 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 Jiigh percentage of sugar possesses a faint yellow color, a high specific
gravity, and a volume which is above normal.
Experiments.
I. Phenylhydrazine Reaction. — Test the urine according to one
of the- following methods: {a) To a small amount of phenylhydrazine
mixture, furnished by the instructor,^ 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 micro-
scopically (Plate III, opposite page 28). 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 osazones are formed from certain
sugars under these conditions, in general each individual sugar giving
rise to an osazone of a definite crystalline form which is typical for that
sugar. It is important to remember in this connection that, of the
simple sugars of interest in physiological chemistry, dextrose and laevu-
lose yield the same osazone, with phenylhydrazine. Each osazone has
a definite melting-point, and as a further and more accurate means of
identification it may be recrystallized and identified by the determina-
tion of its melting-point and nitrogen content. The reaction taking
place in the formation of phenyldextrosazone is as follows :
C„H,,0„ + 2(H3N.NH.C«HJ-^CeH,,0,(N.NH.CeH3)3+2H30-fH,.
Dextrose. Phenylhydrazine. Phenyldextrosazone.
(b) Place 5 c.c. of the urine in a test-tube, add i c.c. of phenylhy-
drazine-acetate solution furnished by the instructor,^ 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. 28).
'The assimilation limit for dextrose has been shown to be between loo and 150 grams
(Brasch: Zeit.filr Biol., 50, 113, 1907.
■■' This mixture is prepared by combining one part of phenylhydrazine-hydrochloride and
two parts of sodium acetate, by weight. These are thoroughly mixed in a mortar.
' 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 phenylhydrazine (the base).
URINE. 325
The phenyl hydrazine 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
phenylhydrazine (the base) and 1/2 c.c. of glacial acetic acid in
a test-tube. Heat the mixture for about one minute over a low flame,
shaking the tube continually to prevent 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 mix-
ture again for a moment and then cool the contents of the tube. Ordi-
narily the crystals form at once, especially if the urine 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 micrscope and compare them with
those shown in Plate III, opposite page 28.
3. Riegler's Reaction.^ — Introduce o.i gram of phenylhydrazine-
hydrochloride and 0.25 gram of sodium acetate into a test-tube, add
20 drops of the urine under examination, and heat the mixture to boiling.
Now introduce 10 c.c. of a 3 per cent solution of potassium hydroxide
and gently shake the tube and contents. If the urine under examination
contains dextrose the liquid in the tube will assume a red color. One
per cent dextrose yields an immediate color whereas 0.05 per cent yields
the color only after the lapse of a period of one-half hour from the time
the alkali is added. If the color appears after the 30-minute interval
the color change is without significance inasmuch as sugar-free urines
will respond thus. The reaction is given by all aldehydes and therefore
the test cannot be safely employed in testing urines preserved by formalde-
hyde. Albumin does not interfere with the test.
4. Bottu's Test.- — To 8 c.c. of Bottu's reagent^ in a test-tube add
I c.c. of the urine under examination and mix the liquids by gentle
shaking. Now heat the upper portion of the mixture to boiling, add
an additional i c.c. of urine and heat the mixture again immediately.
The appearance of a blue color accompanied by the precipitation of
small particles of indigo blue indicates the presence of dextrose in the
urine under examination. The test will serve to detect the presence
of 0.1 per cent of dextrose and is uninfluenced by creatinine or by
ammonium salts.
5. Reduction Tests. — To their aldehyde or ketone structure many
sugars owe the property of readily reducing the alkaline solutions of the
' Riegler: Compt. rend. soc. biol., 66, p. 795.
- Bottu: Cumpt. rend. soc. biol., 66, p. 972.
^ This reagent contains 3.5 grams of o-nitrophenylpropiolic acid and 5 c.c. of a freshly pre-
pared ID per cent solution of sodium hydro.xide per liter.
326 PHYSIOLOGICAL CHEMISTRY.
oxides of metals like copper, bismuth, and mercury; they also possess
the property of reducing ammoniacal silver solutions with the separa-
tion of metallic silver. Upon this property 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 — Cu^O + H^O.
\ Cupric oxide
(black).
OH
Cupric hydroxide
(whitish-blue) .
I
OH
/
Cu
\
OH
-^ 2Cu-0H+H,0-^0.
OTT Cuprous hydroxide
^"- (yellow).
Cu
\
\
OH
Cu
Cu— OH \
I -^ O+H^O.
Cu— OH /
Cu
Cuprous hydroxide Cuprous oxide
(yellow). (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
copper sulphate. Continue the addition until there is a slight perma-
nent 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
URINE. 327
of copper sulphate used is too strong, a small brownish-red precipitate
produced in the presence of a low percentage of dextrose may be entirely
masked. On the other hand, if too little copper sulphate is used a light-
colored precipitate formed by uric acid and purine 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 insufl5cient 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.
Salkowski^ has very recently proposed a modification of the Trom-
mer procedure which he claims is a very accurate sugar test.
(b) Fehling's Test. — To about i c.c. of Fchling's solution" 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 Feh-
ling'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 precipi-
tate 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 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 c&njugate glycuronaies, uric acid, nucleoprotein, and honio-
gentisic acid, when present in sufficient amount, 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 appear-
ance may be mistaken for the cuprous hydroxide. Cupric hydroxide
may also be reduced to cuprous oxide and this in turn be dissolved by
creatinine, 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. According to Laird^
even small amounts of creatinine will retard the reaction velocity of re-
ducing sugars with Fehling's solution.
' Salkowski: Zeit. physiol. Chem., 79, 164, 1912.
^ Fehling's solution is composed of two definite solutions — a copper sulphate solution and
an alkaline tartrate solution, which may be prepared as follows:
Copper sulphate solution = 24.6^ grams of copper sulphate dissolved in water and made
up to 500 c.c.
Alkaline tartrate solution — 12$ 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 bottles and mixed
in equal volumes when needed for use. This is done to prevent deterioration.
'Laird: Journ. Path, and Bad., 16, 398, 1912.
328 PHYSIOLOGICAL CHEMISTRY.
(c) Benedict's Modifications of Fehling's Test. First Modification. —
To 2 c.c. of Benedict's solution^ in a test-tube add 6 c.c. of distilled
water and 7-9 drops (not more) of the urine under examination. Boil
the mixture vigorously for about 15-30 seconds and permit it to cool
to room temperature spontaneously. (If desired this process may be
repeated, although it is ordinarily unnecessary.) If sugar is present
in the solution a precipitate will form which is often bluish-green or
green at first, especially if the percentage of sugar is low, and which
usually becomes yellowish upon standing. If the sugar present exceeds
0.06 per cent this precipitate generally forms at or below the boiling-
point, whereas if less than 0.06 per cent of sugar is present the precipi-
tate forms more slowly and generally only after the solution has cooled.
The greenish precipitate obtained with urines containing small amounts
of sugar may be a compound of copper with the sugar or a compound
of some constituent of the urine with reduced copper oxide instead of
being a precipitate of cuprous hydroxide or oxide as is the case when
the original Fehling solution is reduced.
Benedict claims that, whereas the original Fehling's test will not
serve to detect sugar when present in a concentration of less than o. i per
cent, that the above modification will serve to detect sugar when pre-
sent in as small quantity as 0.015-0.02 per cent. This claim has been
corroborated recently by Harrison.^ The modified solution used in the
above test differs from the original in that 100 grams of sodium car-
bonate is substituted for the 125 grams of potassium hydroxide ordi-
narily used, thus forming a Fehling solution which is considerably less
alkaline than the original. This alteration in the composition of the
Fehling solution is of advantage in the detection of sugar in the urine
inasmuch as the strong alkalinity of the ordinary Fehling solution has a
tendency, when the reagent is boiled with a urine containing a small
amount of dextrose, to decompose sufficient of the sugar to render the de-
tection of the remaining portion exceedingly difficult by the usual technic.
Benedict claims that for this reason the use of his modified solution per-
mits the detection of smaller amounts of sugar than does the use of the
ordinary Fehling solution. Benedict has further modified his solution
for use in the quantitative determination of sugar (see page 385).
' Benedict's modified Fehling solution consists of two definite solutions — a copper sulphate
solution and an alkaline tartrate solution, which may be prepared as follows:
Copper sulphate solution =$4.6$ grams of copper suljjhate dissolved in water anrl made up
to 500 c.c.
Alkaline tartrate solution = ioo grams of anhydrous sodium carbonate and 173 grams of
Rochelle salt dissolved in water anrl made uj) to 500 c.c.
These sfjlutions shoukl be preserved separately in rubber-stoppered bottles and mixed
in equal volumes when needed for use. This is done to prevent deterioration.
^ Harrison: Pharm. Jour., 87, 746, ic;ii.
URINE. 329
Second Modification^ — \'cry recently Benedict has further modi-
fied his solution and has succeeded in obtaining one which does not
deteriorate upon long standing." The following is the procedure for
the detection of dextrose in the urine: To 5 c.c. of the reagent in a
test-tube add eight (not more) drops of the urine to be examined. The
fluid is then boiled vigorously for from one to two minutes and then
allowed to cool sponlaneously. In the presence of dextrose the entire
body of the solution will be filled with a precipitate, which may be red,
yellow, or green in color, depending upon the amount of sugar present.
If no dextrose is present, the solution will either remain perfectly clear,
or will show a very faint turbidity, due to precipitated urates. Even
very small quantities of dextrose in urine (o.i per cent) yield precipi-
tates of surprising bulk with this reagent, and the positive reaction for
dextrose is the filling of the entire body of the solution with a precipitate,
so that the solution becomes opaque. Since amount rather than color
of the precipitate is made the basis of this test, it may be applied, even
for the detection of small quantities of dextrose, as readily in artificial
light as in daylight.
{d) Aliens Modification of Fehling's Test. — The following 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 precipi-
tate of albumin which may be produced, 5 c.c. of the solution of cop-
per sulphate used for preparing Fehling's solution is added. This pro-
duces a precipitate containing uric acid, xanthine, hypoxanthine, phos-
phates, etc. To render the precipitation complete, however, it is desir-
able to add to the liquid, when partially cooled, from i to 2 c.c. of a
saturated solution of sodium acetate having a feebly acid reaction to
litmus.' 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 prepar-
ing Fehling's solution is added, and the liquid boiled for 15-20 seconds.
• Benedict: Jour. .Am. Med. .Iss'n., 57, 11Q3, 191 1.
- Benedict's new solution has the following composition:
Copper sulphate i7-3 S^-
Sodium citrate 173° o^-
Sodium carbonate (anhydrous) 100. o gm.
Distilled water to ." looo.o c.c.
With the aid of heat dissolve the sodium citrate and carbonate in about 600 c.c. of water.
Pour (through a folded filter if necessary) into a glass graduate and make up to 850 c.c. Dis-
sohe the copper sulphate in about 100 c.c of water and make up to 150 c.c. Pour the carbonate-
citrate solution into a large beaker or casserole and add the copper sulphate solution slowly,
with constant stirring. The mixed solution is ready for use, and does not deteriorate upon
long standing.
' 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 mi.xing equal measures of
copper sulphate solution, alkaline tartrate solution and water, adding a little sodium acetate
solution, andhealing the mixture to boiling.
330 PHYSIOLOGICAL CHEMISTRY.
In the presence of more than 0.25 per cent of sugar, separation of cup-
rous oxide occurs before the boiling-point is reached; but with smaller
quantities precipitation takes place during the cooling of the solution,
which becomes greenish, opaque, and suddenly deposits cuprous oxide
as a fine brownish-red precipitate."
(e) Boettger^s Test. — To 5 c.c. of urine in a test-tube add i 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 bismuth. If the test is made with 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 (bis-
muth sulphide).
(/) 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^ and heat for five
minutes in a boiling water-bath.^ The mixture 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, since with albumin a
similar change of color is produced. Dextrose when present to the
extent of 0.08 per cent may be detected by this reaction. 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. Other observers^ have failed to verify the inhibitory action
of the mercuric chloride and have shown that the inhibitory influence of
chloroform may be overcome by raising the temperature of the urine to
the boiling-point for a period of five minutes previous to making the test.
Urines rich in indican, uroerythrin, urochrome or hcsmato porphyrin,
as well as urines excreted after the ingestion of large amounts of certain
medicinal substances, may give a darkening of Nylander's reagent similar
to that of a true sugar reaction. It is a disputed point whether the urine
after the administration of urotropin will reduce Nylander's reagent.'*
Strausz ^ has recently shown that the urine of diabetics to whom
"lothion" (diiodohydroxypropane) has been administered will give a
negative Nylander's reaction and respond positively to the Fehling and
' Nylander's reagent is prepared by digesting 2 grams of bismuth subnitrate 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.
^ Hammarsten suggests that the solution be boiled for 2-5 minutes (according to the sugar
content) over a free flame and the tube then permitted to stand five minutes before drawing
conclusions.
^ Rehfuss and Hawk: Jour. Biol. Chem., 7, 267, 1910; also Zsidlitz: Upsala Lakdre-
foren Fork., N. F., 11, 1906.
* Abt: Archives of Pediatrics, 24, 275, 1907; also Weitbrecht: Schweiz. Woch., 47, 577, 1909.
'Strausz: Miinch med. Woch., 59, 85, 1912.
URINE. 331
polarization tests. "lothion" also interferes with the Xylander test in
vitro whereas KI and I do not.
According to Rustin and Otto the addition of PtCl^ increases the
delicacy of Xylander's reaction. They claim that this procedure causes
the sugar to be converted quantitatively. Xo quantitative method has
yet been devised, however, based upon this principle.
A positive Xylander or Boettger test is probably due to the following
reactions:
{a) Bi(OH)3(XO)3+KOH— Bi(OH)3+KX03.
[h) 2Bi(OH)3-30— Bi, + 3H20.
Bohmansson,^ before testing the urine under examination treats it
(10 c.c.) with 1/5 volume of 25 per cent hydrochloric acid and 1/2
volume of bone black. This mixture is shaken one minute, then filtered,
and the neutralized filtrate tested by X'ylander's reaction. Bohmansson
claims that this procedure removes certain interfering substances, notably
urochrome.
0. Fermentation Test. — Rub up in a mortar about 15 c.c. of the
urine with a small piece of compressed yeast. Transfer the mixture to a
saccharometer (Fig. 3. p. 36) 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 completion of fermentation introduce, by means of a bent pipette, a
little KOH solution into the graduated portion, place the thumb tightly
over the opening in the apparatus and invert the saccharometer. Explain
the result.
The important findings of X^euberg and associates^ recently reported
indicate very clearly that the liberation of carbon dioxide by yeast is not
necessarily a criterion of the presence of sugar. The presence of a new
enzyme called carboxylase has been demonstrated in yeast which has the
power of splitting off CO^ from the carboxyl graup of amino and other
aliphatic acids.
7. Barfoed's Test. — Place about 5 c.c. of Barfoed's solution^ in
a test-tube and heat to boiling. Add the urine under examination slowly,
a lew drops at a time, heating after each addition. Reduction is indi-
cated by the production of a red precipitate. If the precipitate does not
form upon continued boiling allow the tube to stand a few minutes and
examine. XaCl interferes with this test (Welker).
Barfoed's test is not a specific test for dextrose as is frequently stated,
' Bohmansson: Biochem. Zeil., 19, p. 281.
- Neuberg and Associates: Biochem. Zeitsch., 31, 170: 32, 323; 36, (60, 68, 76), 1911.
' Barfoed's solution is prepared as follows: Dissolve 4.5 grams of neutral, cr}-stallized
copper acetate in 100 c.c. of water and add 1.2 c.c. of 50 per cent acetic acid.
332 PHYSIOLOGICAL CHEMISTRY.
but simply serves to detect monosaccharides. Disaccharides will also
respond to the test, according to Hinkel and Sherman/ if the solutionis
boiled sufficiently long in contact with the reagent to hydrolyze the disac-
charide through the action of the acetic acid present in the Barfoed's
solution.
Mathews and McGuigan ^ have also shown that disaccharides will
respond to this test under proper conditions of acidity.
8. Polariscopic Examination. — For directions as to the use of the
polariscope see page 36.
PROTEINS.
Normal urine contains a trace of protein 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 protein urinary constituents. The
following are the more important forms of protein material which have
been detected in the urine under pathological conditions:
(i) Serum albumin.
(2) Serum globulin.
r Deutero-proteose.
(3) Proteoses | Hetero-proteose.
I "Bence- Jones' protein."
(4) Peptone.
(5) Nucleoprotein.
(6) Fibrin.
(7) Oxyhaemoglobin.
ALBUMIN.
Albuminuria is a condition in which serum albumin or serum globulin
appears in the urine. There are two distinct forms of albuminuria, i. e.,
renal albuminuria and accidental albuminuria. Sometimes the terms
"true" albuminuria and "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 pressure in the kidneys, altered
kidney structure, or changes in the composition of the blood entering
the kidneys, thus allowing the albumin to diffuse more readily. In the
accidental form of albuminuria the albumin is not excreted by the kidneys
as is the case in the renal form of the disorder, but arises from the blood,
' Hinkle & Sherman: Journ. Am. Chem. Soc, 29, 1744, 1907.
'^ Mathews and McGuigan: A mer. Journ. Physiol., 19, 175, 1907.
URINE, ^;^s
lymph, or some al])umin-containmg exudate coming into contact with the
urine at some point below the kidneys.
Experiments.
Heller's Ring Test. — Place 5 c.c. of concentrated HNO3 ^^ ^ test-
tube, incline the tube, and, by means of a pipette allow the urine to flow
slowly down the side. The liquids should stratify with the formation of
a while zone of precipitated 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 possible 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 exhibit the formation, at
the point of contact, of a crystalline 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 difficulty.
Various colored zones, due cither to the presence of indican, bile pigments,
or to the oxidation of other organic urinary constituents, may form in
this test under certain conditions. These colored rings should never
be confounded with the white ring which alone denotes the presence of
albumin.
After the administration of certain drugs a white precipitate of resin
acids may form at the point of contact of the two fluids and may cause
the observer to draw wrong conclusions. This ring, if composed of
resin acids, will dissolve in alcohol, whereas the albumin ring will not
dissolve.
Weinberger has recently shown that a ring closely resembling the
albumin ring is often obtained in urines preserved by thymol when sub-
jected to Heller's test. The ring is due to the formation of nitrosothymol
and possibly nitrothymol. If the thymol is removed from the urine by
extraction with petroleum ether^ previous to adding nitric acid, the ring
does not form.
* Accomplished readily by gently agitating equal volumes of petroleum ether and the
urine under examination for two minutes in a test-tube before applying the test.
334 PHYSIOLOGICAL CHEMISTRY.
An instrument called the alhumoscope Qiorismascope) has been devised
for use in this test and has met with considerable favor. The method of
using the albumoscope is described below.
Use oj the Alhumoscope. — This instrument is intended to facilitate
the making of "ring" tests such as Heller's and Roberts'. In making
a test about 5 c.c. of the solution under examination is first introduced
into the apparatus through the larger arm and the reagent used in the
particular test is then introduced through the capillary arm and allowed
to flow down underneath the solution under examination. If a reason-
able amount of care is taken there is no possibility of mixing the two
solutions and a definitely defined white "ring" is easily obtained at the
zone of contact.
2. Roberts' Ring Test. — ^Place 5 c.c. of Roberts' reagent^ in a test-
tube, incline the tube, and, by means of a pipette allow the urine to flow
slowly down the side. The liquids should stratify with the formation of a
while 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. The alhumoscope (see above) may also be used in making
this test.
3. Spiegler's Ring Test. — Place 5 c.c. of Spiegler's reagent^ 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 delicate 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.
Some investigators claim that the delicacy of this test depends 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 i c.c. of 30 per cent
acetic acid and 4 c.c. of Jolles' reagent^ in a test-tube. A white precipitate
indicates the presence of albumin.
• Roberts' reagent is composed of i volume of concentrated HNO3 and 5 volumes of a
saturated solution of MgSO^.
^ Spiegler's reagent has the following composition:
Tartaric acid 20 grams.
Mercuric chloride 40 grams.
Glycerol 100 grams.
Distilled water 1000 grams.
* Jolles' reagent has the following composition:
Succinic acid 4° grams.
Mercuric chloride 20 grams.
Sodium chloride 20 grams.
Distilled water 1000 grams.
URINE. 335
Care should be taken to use the correct amount of acetic acid, since the
use of too small an amount may result in the formation of mercury com-
binations which may cause confusion. In the presence of iodine, mer-
curic iodide will form but may readily be diflerentiated 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 precipitate is due to phosphates it will dis-
appear under these conditions, whereas if it is due to albumin it will
not only fail to disappear but will become more fiocculent 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 pre-
cipitate by reason of its solubility in alcohol.
{h) 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 indicates albumin or phosphates. Acidify
the urine slightly by the addition of 3-5 drops of dilute acetic acid, when
the turbidity, if due to phosphates, will disappear.
Nitric acid is often used in place of acetic acid in these tests. In
case nitric acid 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 5-10 drops of acetic acid. Mix well and
add potassium ferrocyanide drop by drop, until a preciptate forms.
This is a very delicate test. Schmiedl claims that a precipitate of
Fe(Cn)gK2Zn or Fe(Cn)8Zn2 is formed when urines containing zinc
are subjected to this test and that this precipitate resembles the pre-
cipitate secured with protein solutions. In the case of human urine a
reaction was obtained when 0.000022 gram of zinc per cubic centimeter
was present. Schmiedl further found that the urine collected from
rabbits housed in zinc-lined cages possessed a zinc content which was
sufficient to yield a ready response to the test. Zinc is the only interfering
substance so far reported.
7. Tanret's Test. — To 5 c.c. of urine in a test-tube add Tarnet's
reagent^ drop by drop until a turbidity or precipitate forms. This
* 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 soludon up to 60 c.c. with water and add 20 c.c. of glacial acetic
acid to the mixture.
336 PHYSIOLOGICAL CHEMISTRY.
is an exceedingly delicate test. Sometimes the urine is stratified upon
the reagent as in Heller's or Roberts' ring test. According to Repiton,
urates interfere with the delicacy of this test. Tanret, however, claims
that urates do not interfere inasmuch as any precipitate due to urates
may be brought into solution by heat whereas an albumin precipitate
under the same conditions will persist. Tanret further states that
mucin interferes with the delicacy of the test and that it should therefore
be removed from the urine under examination by acidification with
acetic acid and filtration before testing for albumin.
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 pro-
duction 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 (page 335), but they may be easily differentiated
from albumin by means of their solubility in alcohol.
9. Potassium Iodide Test/ — Dilute 5 c.c. of the urine under
examination with 10 c.c. of water and stratify this mixture upon a
potassium iodide solution made slightly acid with acetic acid. In
the presence of 0.01-0.02 per cent of albumin a white ring forms imme-
diately. If the test be allowed to stand two minutes after the stratifi-
cation it will serve to detect 0.005 per cent of albumin.
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 unaccompanied by albumin.
Experiments.
Globulin will respond to all the tests just outlined under Albumin.
If it is desirable to differentiate between albumin 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 magnesium sulphate in
substance to the point of saturation. If the protein 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
' Pharm. Ztg., 54, p. 612.
URINE. 337
if present in the same urine. To do this filter off the globulin after it
has been precipitated by the magnesium sulphate, 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 satu-
rated solution ^of ammonium sulphate. Globulin, if present, will be
precipitated. If no precipitate forms add ammonium sulphate m sub-
stance to the point of saturation. If albumin is present it will be pre-
cipitated 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 pre-
cipitate of ammonium urate may occur when the ammonium 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 pathological con-
ditions such as diphtheria, pneumonia, intestinal ulcer, carcinoma,
dermatitis, osteomalacia, atrophy of the kidneys, and in sarcomata
of the bones of the trunk. "Bence-Jones' protein," a proteose-like
substance, is of interest in this connection and its appearance in the
urine is believed to be of great diagnostic importance in cases of multi-
ple myeloma or myelogenic osteosarcoma. By some investigators this
protein is held to be a variety of hetero-proteose whereas others claim
that it possesses albumin characteristics.
Peptone certainly occurs much less frequently as a constituent of
the urine than does proteose, in fact most investigators seriously ques-
tion 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 protein present was really
proteose.
Experiments.
I. Boiling Test. — Make the ordinary coagulation test according
to the directions given under Albumin, page 335. If no coagulable
338 PHYSIOLOGICAL CHEMISTRY.
protein 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 nucleoprotein which may form.
Now test a few cubic centimeters of the urine for coagulable protein, by
tests 2 and 5 under Albumin, pp. 334-5. If coagulable protein, is present
remove it by coagulation and filtration before proceeding. Introduce
25 c.c. of the urine, freed from coagulable protein, 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. Now
filter this solution, and after testing again for nucleoprotein with very
dilute acetic acid, try the biuret test. If this test is positive the presence
of proteose is indicated.^
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. Alder's Method. — Acidify 10 c.c. of urine with hydrochloric
acid, add phosphotungstic acid until no more precipitate forms and
centrifugate^ the solution. Decant the supernatant 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 con-
tinued 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 positive biuret test indicates the presence of
proteoses.
4. Detection of '' Bence-Jones' Protein." — Heat the suspected
urine very gently, carefully noting the temperature. At as low a tem-
perature as 40° C. a turbidity may be observed, and as the temperature is
raised to about 60° 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 100° 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 dis-
solving at a higher temperature is typical of "Bence- Jones' protein" and
may be used to differentiate it from all other forms of protein material
occurring in the urine.
^ If it is considered desirable to test for peptone the proteose may be removed by satu-
ration with (NHJ2SO4 according to the directions given on page 120 and the filtrate tested
for peptone by the biuret test.
^ It not convenient to use a centrifuge the precipitate may be filtered off and washed on
the fiUer paper with alcohol.
URINE. 339
NUCLEOPROTEIN.
There has been considerable controversy as to the proper classification
for the protein body which forms the "nubecula" of normal urine. By
different investigators it has been called mucin, mucoid, phospho protein,
nuclcoaUnimin, and nuclcoprotein. Of course, according to the modern
acceptation of the meanings of these terms they cannot be synonymous.
Mucin and mucoid arc glycoproteins and hence contain no phosphorus
(see p. 1 12), whereas phosphoproteins and nucleoproteins are phos-
phorized bodies. It may possibly be that both these forms of protein,
i. e., the glycoprotein and the phosphorized type, ocCur in the urine under
certain conditions (see page 308). In this connection we will use the
term nuclcoprotein. The pathological conditions under which the content
of nuclcoprotein is increased includes all affections of the urinary passages
and in particular pyelitis, nephritis, and inflammation of the bladder.
Experiments.
1. Detection of Nucleoprotein. — 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 indication that nucleoprotein 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 nucleoprotein.
2. Ott's Precipitation Test. — Mix 25 c.c. of the urine with an equal
volume of a saturated solution of sodium chloride and slowly add Almen's
reagent.^ In the presence of nucleoprotein a voluminous precipitate
forms.
BLOOD.
The pathological conditions in which blood occurs in the urine m^y be
classified under the two divisions hcpmaturia and hcemoglohinuria. In
haematuria we are able to detect not only the haemoglobin but the unrup-
tured corpuscles as well, whereas in haemoglobinuria the pigment alone is
present. Haematuria is brought about through blood passing into the
urine because of some lesion of the kidney or of the urinary tract below
the kidney. Haemoglobinuria is brought about through haemolysis, i. e.,
the rupturing of the stroma of the erythrocyte and the liberation of the
haemoglobin. This may occur in scur\y, typhus, pyemia, purpura, and
in other diseases. It may also occur as the result of a burn covering a
' 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.
340 PHYSIOLOGICAL CHEMISTRY,
considerable area of the body, or may be brought about through the
action of certain poisons or by the injections of various substances having
the power of dissolving the erythrocytes. Transfusion of blood may also
cause hasmoglobinuria.
Experiments.
1. Heller's Test. — Render lo 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 ordinarily well to make a "control"
experiment using normal urine, before coming to a final decision.
Certain substances, such as cascara sagrada, rhubarb, santonin, 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 pigment 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 sodium chloride 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 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. 59 and
60, page 211. (See Atkinson and Kendall's modification, p. 210.)
3. Heller-Teichmann Reaction.^ — Produce the pigmented pre-
cipitate according to directions given in Heller's test above. If 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,
above.
4. V. Zeynek and Nencki's Haemin Test. — To 10 c.c. of the urine
under examination add acetone until no more 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, immediately place a cover glass in position, and examine
under the microscope. Compare the form of the crystals with those
shown in Figs. 59 and 60, page 211. Haemin crystals produced 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
URINE. 341
a small beaker and heat to 80° C. Add 5 c.c. of the urine under examina-
tion, raise the temperature to 80° C, and stand the mixture aside to cool.
Examine the crystals under the microscope and compare them with those
shown in Figs. 59 and 60, page 211.
6. Guaiac Test. — Place 5 c.c. of urine in a test-tube and by means
of a pipette introduce a freshly prepared alcoholic solution of guaiac
(strength about 1:60) 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. Buck-
master has recently suggested the use of guaiaconic acid instead of the
solution of guaiac. See discussion on page 204 and test on page 209.
7. Schumm's Modification of the Guaiac Test. — To about 5 c.c.
of urine' in a test-tube add about 10 drops of a freshly prepared alcoholic
solution of guaiac. Agitate the tube gently, add about 20 drops of old
turpentine, subject the tube to a thorough shaking, and permit it to stand
for about 2-3 minutes. A blue color indicates the presence of blood in
the solution under examination. In case there is not sufficient blood to
yield a blue color under these conditions, a few c.c. of alcohol should be
added and the tube gently shaken, whereupon a blue coloration will
appear in the upper alcohol-turpentine layer.
A control test should always be made using water in place of urine.
In the detection of very minute traces of blood only 3-5 drops of the
guaiac solution should be employed.
8. Adler's Benzidine Reaction. — This is one of the most delicate of
the reactions for the detection of blood. Different benzidine preparations
vary greatly in their sensitiveness, however. Inasmuch as benzidine
solutions change readily upon contact with light, it is essential that they
be kept in a dark place. The test is performed as follows : To a saturated
solution of benzidine in alcohol or glacial acetic acid add an equal volume
of 3 per cent hydrogen peroxide and i c.c. of the urine under examination.
If the mixture is not already acid, render it so with acetic acid, and note
the appearance of a green or blue color. A control test should be made
substituting water for the urine.
Often when urines containing a small amount of blood are tested by
this reaction, the mixture is rendered so turbid as to make it diflScult to
decide as to the presence of a faint green color. Such urines should be
extracted with an ether-acetic acid solution and the resulting extract
washed with water before the test is applied to it. The sensitiveness of
the benzidine reaction is greater when applied to aqueous solutions than
when applied to the urine.
' Alkaline urine should be made slighdy acid with acetic acid as the blue end-reaction
is very sensitive to alkali.
342 PHYSIOLOGICAL CHEMISTRY.
9. Spectroscopic Examination. — Submit the urine to a spectro-
scopic examination according to the directions given on page 215, looking
especially for the absorption-bands of oxyhemoglobin andmethsemoglobin
(see Absorption Spectra, Plate I.).
BILE.
Both the pigments and the acids of the bile may be detected in the
urine under certain pathological conditions. Of the pigments, bilirubin
is the only one which has been positively identified 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
cause 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.
1. 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, blue,
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. Nakayama's Reaction. — To 5 c.c. of urine in a test-tube add an
equal volume of a 10 per cent solution of barium chloride. Centrifugate
the mixture, pour off the supernatant fluid, and heat the precipitate with
2 c.c. of Nakayama's reagent.^ In the presence of bile pigments the
solution assumes a blue or green color.
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 precipitate, wash it with water, and
transfer to a small beaker. Add alcohol acidified slightly with hydro-
chloric acid and warm upon a water-bath until the solution becomes
colored an emerald green.
According to Steensma, this procedure may give negative results
' Prepared by combining 99 c.c. of alcohol and i c.c. of fuming hydrochloric acid con-
taining 4 grams of ferric chloride per liter.
URINE. 343
even in the presence of the pigments, owing to the fact that the acid-
alcohol is not a sufficiently strong oxidizing agent. He therefore suggests
the addition of a drop of a 0.5 per cent solution of sodium nitrite to the
acid-alcohol mixture before warming on the water-bath. Try this
modification also.
4. 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 hydrochloric acid and, if necessary, shake the tube to bring the
precipitate 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 than Gmelin's test.
Steensma's suggestions mentioned under Huppert's Reaction, above,
apply in connection with this test also.
5. Hammarsten's Reaction. — To about 5 c.c. of Hammarsten's
reagent^ in a small evaporating dish add a few drops 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 obtained.
b. 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 (i : 10) so that the fluids do not
mix. A green ring is observed at the point of contact.
7. Salkowski-Schippers Reaction. — Neutralize the acidity of 10
c.c. of the urine under examination with a few drops of a dilute solution
of sodium carbonate, and add 5 drops of a 20 per cent solution of sodium
carbonate and 10 drops of a 20 per cent solution of calcium chloride.
Filter off the resultant precipitate upon a hardened filter paper and wash
it with water. Remove the precipitate to a small porcelain dish, add
3 c.c. of an acid-alcohol mixture' and a few drops of a dilute solution
of sodium nitrite and heat. The production of a green color indicates
the presence of bile pigments.
8. Bonanno's Reaction.^ — Place 5-10 c.c. of the urine under
examination in a small porcelain evaporating dish and add a few drops
of Bonanno's reagent.* If bile is present an emerald-green color will
' Hammarsten's reagent is made by nuxing i volume of 25 per cent nitric acid and 19
volumes of 25 per cent hydrochloric acid and then adding i volume of this acid mixture
to__4 volumes of 95 per cent alcohol.
- Made by adding 5 c.c. of concentrated hydrochloric acid to 95 c.c. of 96 per cent alcohol.
^ II Tommasi, 2, Xo. 21.
* This reagent may be prepared by dissolving 2 grams of sodium nitrite in 100 c.c. of
concentrated hvdrochloric acid.
344 PHYSIOLOGICAL CHEMISTRY.
develop. Bonanno says the reaction is not interfered with by any known
normal or pathological urinary constituent.
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 sucrose. Now incline 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.
2. Mylius's Modification of Pettenkofer's Test. — To approxi-
mately 5 c.c. of urine in a test-tube add 3 drops of a very dilute (i : 1000)
aqueous solution of furfurol,
HC CH
HC C.CHO.
O
Now incline the tube, run about 2-3 c.c. of concentrated sulphuric 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 sucrose
solution and one or more drops of dilute sulphuric 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.
4. V. Udransky's Test. — To 5 c.c. of urine in a test-tube add 3-4
drops of a very dilute (i : 1000) 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. Hay's Test. — This test is based upon the principle that bile acids
have the property of reducing the surface tension of fluids in which
they are contained. The test is performed as follows: Cool about 10 c.c.
of urine in a test-tube to 17° C. or lower, and sprinkle a little finely
pulverized 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 amount of
bile acids present in the urine. The test is said to react with bile acids
when the latter are present in the proportion i : 120,000.
URINE. 345
Some investigators claim that it is impossible to differentiate between
bile acids and bile pigments by this test.
CH3
ACETONE, C = 0.
I
CH,
It was formerly very generally believed that acetone appeared in
the urine under pathological conditions because of increased protein
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 elimi-
nated has been shown to increase when the subject is fed an abundance
of fat-containing food as well as during fasting, whereas a replace-
ment 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, /9-oxybutyric acid and dia-
cetic 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 ;9-oxybutyric acid
is never found except in conjunction with one or the other of these bodies.
Acetone and diacetic acid arise chiefly from the oxidation of ,5-oxybutyric
acid. The relation existing between these three bodies is shown in the
following reactions:
(a) CH3.CH(OH).CH.,.COOH + 0--CH3CO.CH,.COOH-HH,0.
^-oxybutyric acid. Diacetic acid
ih) CH3CO.CH,.COOH->(CH3)3CO + CO,.
Diacetic acid. Acetone.
Acetone, chemically considered, is a ketone, di-methyl ketone. When
pure it is a liquid which possesses a characteristic aromatic 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 o.or-0.03 gram.
Pathologically, the elimination of acetone is often greatly increased
and at such times a condition of acetonuria is said to exist. This patho-
logical acetonuria may accompany diabetes mellitus, scarlet fever, typhoid
346 PHYSIOLOGICAL CHEMISTRY.
fever, pneumonia, nephritis, phosphorus poisoning, grave anaemias,
fasting, and a deranged digestive function; it also frequently accom-
panies auto-intoxication and chloroform and ether ansesthesia. 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 detection
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 orig-
inal 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 lodofomi Test. — To about 5 c.c. of the urine or
distillate in a test-tube add a few drops of Lugol's solution^ or ordi-
nary iodine solution (I in KI) and enough NH^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 examina-
tion) and note the formation of a yellowish sediment consisting of iodo-
form. Examine the sediment under the microscope and compare the
form of the crystals with those shown in Fig. 7, p. 47. 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 phos-
phate (Fig. 81, p. 242) which mayb e formed in 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 according 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 suggested
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 necessary 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 i : 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 freshly prepared aqueous solution
' Lugol's solution may be prepared by dissolving 4 grams of iodine and 6 grams of potas-
sium iodide in 100 c.c. of distilled water.
URINE. 347
of sodium nitroprussidc and render the mixture alkaline with potassium
hydroxide. A ruby red color, due to creatinine, a normal urinary
constituent, is produced (see Wcyl's test, p. 296). Add an excess of
acetic acid and if acetone is present the red color will be intensified,
whereas in the absence 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 paracrcsol 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. Identify the iodoform by
means of its characteristic odor and its typical crystalline form (see
Fig. 7, p. 47). 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.^
5. Reynolds-Gunning Test.— This test depends upon the solu-
bility of mercuric oxide in acetone and is performed as follows: 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 am-
monium sulphide, (NHJ2S, upon this acid solution. At the zone of
contact a blackish-gray ring of precipitated mercuric 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.
6. Taylor's Test.— To 10 c.c. of the urine or distillate in a test-tube
add a few drops of a freshly prepared aqueous solution of sodium nitro-
prussidc and stratify concentrated ammonium hydroxide upon the mixture.
The production of a magenta color at the point of contact indicates the
presence of acetone in the urine or distillate under examination. Normal
urine yields an orange-red color when subjected to this technic.
Rothera's Reaction.^ — To 5-10 c.c. of urine or distillate in a test-
tube add a little solid ammonium sulphate, 2-3 drops of a freshly pre-
' W'elker reports the production of a pink or red color during the application of this test
to the distillates from pathological urines which had been preserved with powdered thymol.
He found the color to be due to an iodothymol compound which had been previously prepared
synthetically by Messinger and Vortmann.
- Rothera: Jour. Physiol., 37, 491, 1908.
348 PHYSIOLOGICAL CHEMISTRY,
pared 5 per cent solution of sodium nitroprusside and 1-2 c.c. of con-
centrated ammonium hydroxide. The development of a permanganate
color indicates the presence of acetone.
CH3
I
DIACETIC ACID, C = O
CH2.COOH.
Diacetic or acetoacetic acid occurs in the urine only under path-
ological 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 ace-
tonuria, i. e., in fevers, diabetes, etc. (see p. 345). If very little diacetic
acid is formed it may 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 disorders; it is perhaps less fre-
quently observed in adults, but when present, particularly in fevers and
diabetes, it is frequently 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.
Experiments.
I. Gerhardt's Test.- — To 5 c.c. of urine in a test-tube add ferric
chloride solution, drop by drop, until no more precipitate 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 indicates the
possible presence of diacetic acid. Before making a final decision re-
garding the presence of this body make the two following control experi-
ments:
(a) Place 5 c.c. 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.
URINE. 349
(b) Place 5 c.c. of urine in a test-tube, acidify with H2SO^, to free
diacetic acid from its salts, and carefully extract the mixture with ether
by shaking. If diacetic acid is prcsnte it will be extracted by the ether.
Now remove the ethereal solution, evaporate it to dryness, dissolve the
residue in 1-2 c.c. of water and add 3-5 drops of 3 per cent ferric chloride.
Diacetic acid is indicated by the production of the characteristic Bordeaux-
red color. This^color disappears spontaneously in 24-48 hours. Such
substances as antipyrin, kairin, phenacetin, salicylic acid, salicylates,
sodium acetate, thiocyanates, 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.
2. Arnold-Lipliawsky Reaction.^ — This reaction is somewhat more
delicate than Gerhardt's test (i) and serves to detect diacetic acid when
present in the proportion of 1:25,000. It is also negative toward acetone,
^^-oxybutyric acid and the interfering drugs mentioned as causing errone-
ous 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^ in a test-tube, add a few drops of con-
centrated ammonia and shake the tube vigorously. 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. i.ig), 3 c.c. of chloroform, and 2-4
drops of ferric chloride solution and carefully mix the fluids. Diacetic
acid is indicated by the chloroform assuming a violet or blue color; if
diacetic acid is absent the color may be yellow or light red.
H OHH
I ! I
^-OXYBUTYRIC ACID, H-C-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
' This reagent consists of two definite solutions which are ordinarily preserved separately
and mixed just before using. The two solutions are prepared as follows:
(o) One per cent aqueous solution of potassium nitrite.
(b) (Jne gram of />-amino-acetophenon dissolved in loo 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.
350 PHYSIOLOGICAL CHEMISTRY.
either acetone or diacetic acid. Either of these bodies may be formed
from /3-oxybutyric acid under proper conditions. It is present in espe-
cially large 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, /?-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 /?-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 /9-oxybutyric acid is an odorless, transparent syrup, which
is laevorotatory and easily soluble in water, alcohol, and ether; it may be
obtained in crystalline form.
EXPEEIMENTS.
I. Black's Reaction.^ — -Inasmuch as the urinary pigments as well as
any contained sugar or diacetic acid will interfere with the delicacy of this
test when applied to the urine directly the following preliminary procedure
is necessary: Concentrate lo c.c. of the urine under examination to one-
third or one-fourth of its original volume in an evaporating dish at a
gentle heat. Acidify the residue with a few drops of concentrated hydro-
chloric acid, add sufficient plaster of Paris to make a thick paste and allow
the mixture to stand until it begins to "set." It should now be stirred
and broken up in the dish by means of a stirring rod with a blunt end.
Extract the porous meal thus produced twice with ether by stirring and
decantation. Any ^S-oxybutyric acid present will be extracted by the
ether. Evaporate the ether extract spontaneously or on a water-bath,
dissolve the residue in water, and neutralize it with barium carbonate.
To 5 to lo c.c. of this neutral fluid in a test-tube add two to three drops of
ordinary commercial acid hydrogen peroxide. Mix by shaking and add a
few drops of Black's reagent.^ Permit the tube to stand and note the
gradual development of a rose color which increases to its maximum
intensity and then gradually fades. ^
In carrying out the test care should be taken to see that the solution is
cold and approximately neutral and that a large excess of hydrogen peroxide
and Black's reagent are not added. In case but little /?-oxybutyric acid is
present the color will fail to appear or will be but transitory if the oxidizing
' Made by dissolving 5 grams of ferric chloride and 0.4 gram of ferrous chloride in 100
c.c. of water.
^ This disappearance of color is due to the further oxirlation of the diacetic acid.
URINE. 351
agents are added in too great excess. It is preferable to add a few drops of
the reagent and at intervals of a few minutes repeat the process until the
color undergoes no further increase in intensity. One part of ^9-oxybutyric
acid in 10,000 parts of the solution may be detected by this test.
2, Polariscopic Examination. — Subject some of the urine (free
from protein) to the ordinary fermentation test (see page 331). This will
remove dextrose and laevulose, which would interfere with the polariscopic
test. Now examine the fermented fluid in the polariscope and if it is
laevorotatory the presence of /9-oxybutyric acid is indicated. This test is
not absolutely reliable, however, since conjugate glycuronates are also
lasvorotatory after fermentation.
3. Kulz'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 con-
ditions 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 7 2° C. In case very slight traces
of /3-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
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 P^^ cent under normal
conditions. The output may be very greatly increased as the result of the
administration of antipyrin, borneol, camphor, chloral, menthol, morphine,
naphthol, turpentine, etc. The glycuronates as a group are laevorotatory
whereas glycuronic acid is dextro-rotatory. Most of the glycuronates,
reduce alkaline metallic oxides and so introduce an error in the examina-
tion 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.
I. Fermentation-Reduction Test. — Test the urine by Fehling's
test. If there is reduction try Barfoed's test. If negative this indicates
352 PHYSIOLOGICAL CHEMISTRY.
the absence of monosaccharides. A negative fermentation test would
now indicate the presence of conjugate 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 examination, 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 laevorotation in-
dicative of conjugate glycuronates.
2. Tollens' Reaction. — Make this test according to directions given
under Pentoses, p. 353.
PENTOSES.
We have two distinct types of pentosuria, i. e., 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 chronic form of pentosuria, in which the output of pentoses bears
no relation whatever to the quantity and nature of the pentose content of
the food eaten. In occurring in these two forms, pentosuria resembles
glycosuria (see page 324), but it is definitely known that pentosuria bears
no relation to diabetes mellitus and there is no generally accepted theory
to account for the occurrence of the chronic form of pentosuria. The
pentose detected most frequently in the urine is arabinose, the inactive
form generally occurring in chronic pentosuria and the laevorotatory
variety occurring in the alimentary type of the disorder.
#
Experiments.
I. Bial's Reaction.- — To 5 c.c. of Bial's reagent^ in a test-tube add
2-3 c.c. of urine and heat the mixture gently until the first bubbles rise to
the surface.* Immediately or upon cooling the solution becomes green
and a flocculent precipitate of the same color may form.
This test is believed to be more accurate than the orcinol test. It is
claimed that urines containing mew//io^, kreosotal, etc., respond to the orcinol
reaction, but not to Bial's.
' If necessary to differentiate Vjetween lactose and glycuronates apply the mucic acid
test (see p. 354) or the phenylhydrazine reaction (see p. 28).
^ Bial: Deut. med. Woch., 28, 252, i(;o2.
' Orcinol 1.5 gram.
r'uming HCI 500 grams.
Ferric chloride do per cent) 20-30 drops.
* The test may also be performed by adding the urine to the hot reagent. No further
heating should be necessary if pentose is present.
URINE. 353
2. Tollens' Reaction. — To equal volumes of urine and hydro-
chloric acid (sp. gr. 1.09) add a little phloroglucinol and heat the mix-
ture on a boiling water-bath. Pentose, galactose, 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
differentiate between the two latter bodies by the melting-points of their
osazones.
3. Orcinol Test. — Place equal volumes of urine and hydrochloric
acid (sp. gr. 1.09) in a test-tube, add a small amount of orcinol, 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 alco-
holic extract examined spectroscopically. An absorption band between
C and D will be observed.
FAT.
When fat finds its way into the urine through a lesion which brings
some portion of the urinary passages into communication with the lym-
phatic system a condition known as chyluria 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 espe-
cially sulphonal. Such urines ordinarily possess a reddish tint, the depth
of color varying greatly under different conditions.
Experiments.
I. Spectroscopic Examination. — To 100 c.c. of urine add about
20 c.c. of a 10 per cent solution of potassium hydroxide orammonium
hydroxide. 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
23
354 PHYSIOLOGICAL CHEMISTRY.
alcohol acidified with hydrochloric acid. By this process the haematopor-
phyrin is dissolved and on filtering will be found in the filtrate and may be
identified by means of the spectroscope (see page 219, 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.
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 to show the presence of lactose in the urine in a satis-
factory manner, since the formation of the characteristic lactosazone is
not attended with any great measure of success under these conditions.
It is, however, comparatively easy to show that it is not dextrose, for,
while it responds to reduction tests, it does not ferment with pure yeast
and does not give a dextrosazone. An absolutely conclusive test, of
course, is the isolation of the lactose in crystalline form (Fig. 80, p. 238)
from the urine.
On oxidation with nitric acid lactose and galactose yield mucic acid.
This test is frequently used in urine examination to differentiate lactose
and galactose from other reducing sugars.
Experiments.
1. Mucic Acid Test. — Treat 100 c.c. of the urine under examination
with 20 c.c.^ of concentrated nitric acid and evaporate the mixture in a
broad, shallow glass vessel, upon a boiling water-bath until the volume
of the solution is only about 20 c.c. At this point the fluid should be
clear and a fine white precipitate of mucic acid should separate. If the
percentage of lactose in the urine is low it may be necessary to cool the
solution and permit it to stand for some time before the precipitate will
form. It is impossible to differentiate between galactose and lactose by
means of this test, but the reaction does serve to differentiate these two
sugars from all other reducing sugars. A satisfactory differentiation
between lactose and galactose may be made by means of Barfoed's
test, p. 33 T.
2. Rubner's Test. — To 10 c.c. of urine in a small beaker add some
lead acetate, in substance, heat to boiling, and add NH^OH until_^no
* If the specific gravity of the urine is 1020 or over it is necessary to use 25-35 c.c. of nitric
acid. Under these conditions the mi.vture should be evaporated until the remaining volume
is approximately equivalent to that of the nitric acid a !dcd.
URINE. 355
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 laevulose no color at all under the same
conditions.
3. Compound Test. — Try the phenylhydrazine test, the fermentation
test, and Barfoed's test according to directions given under Dextrose,
pages 324, and 331. If these are negative, try Nylander's test, page
330. If this last test is positive, the presence of lactose is indicated.
GALACTOSE.
Galactose has occasionally been detected in the urine, and in particular
in that of nursing infants afflicted with a deranged digestive function.
Lactose and galactose may be differentiated from other reducing sugars
which may be present in the urine by means of the mucic acid test. This
test simply consists in the production of mucic acid through oxidation of
the sugar with nitric acid.
Experiments.
1. Mucic Acid Test. — Treat too c.c. of the urine under examination
with 20 c.c* of concentrated nitric acid and evaporate the mixture in a
broad, shallow glass vessel, upon a boiling water-bath, until the volume of
the solution is only about 20 c.c. At this point the fluid should be clear
and a fine, white precipitate of mucic acid should separate. If the per-
centage of galactose present in the urine is low it may be necessary to cool
the solution and permit it to stand for some time before the precipitate
will form. It is impossible to differentiate between galactose and lactose
by means of this test, but the reaction does serve to differentiate these two
sugars from all other reducing sugars. A satisfactory differentiation
between galactose and lactose may be made by Barfoed's test, p. 331.
2. Tollens' Reaction. — To equal volumes of the urine and hydro-
chloric acid (sp. gr. 1.09) add a little phloroglucinol and heat the mixture
on a boiling water-bath. Galactose, pentose, and glycuronic acid will be
indicated by the appearance of a red color. Galactose may be differen-
tiated from the two latter substances in that its solutions exhibit no absorp-
tion bands upon spectroscopical examination.
LiEVULOSE.
Diabetic urine frequently possesses the power of rotating the plane of
polarized light to the left, thus indicating the presence of a laevorotatory
' If the specific gravity of the urine is 1020 or over it is necessary to use 25-35 ^•^- oi
nitric acid. Under these conditions the mixture should be evaporated until the remainin«^
volume is approximately equivalent to that of the nitric acid added.
356 PHYSIOLOGICAL CHEMISTRY,
substance. The Isevorotation is sometimes due to the presence of laevu-
lose, although not necessarily confined to this carbohydrate, since conju-
gate glycuronates and /3-oxybutyric acid, two other laevorotatory bodies,
are frequently found in the urine of diabetics. Laevulose is invariably
accompanied by dextrose in diabetic urine, but lavulosuria 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. Borchardt's Reaction.— To about 5 c.c. of urine in a test-tube
add an equal volume of 25 per cent hydrochloric acid and a few crystals
of resorcinol. Heat to boiling and after the production of a red color, cool
the tube under running water and transfer to an evaporating dish or
beaker. Make the mixture slightly alkaline with solid potassium hy-
droxide, return it to a test-tube, add 2-3 c.c. of acetic ether, and shake the
tube vigorously. In the presence of laevulose the acetic ether is colored
yellow.
The only urinary constituents which interfere with the test are nitrites
and indican and these interfere only when they are simultaneously present.
Under these conditions, the urine should be acidified with acetic acid and
heated to boiling for one minute to remove the nitrites. In case the
indican content is very large, it will impart a blue color to the acetic ether,
thus masking the yellow color due to laevulose. When such urines are to
be examined, the indican should first be removed by Obermayer's test
(seep. 299). The chloroform should then be discarded, the acid-urine
mixture diluted with one-third its volume of water, and the test applied
as described above. The urine of patients who have ingested santonin
or rhubarb respond to the test. The test will serve to detect laevulose when
present in a dilution of i :2ooo, i. e., 0.05 per cent.
2. Seliwanoff's Reaction. — To 5 c.c. of Seliwanoff's reagent^ in a
test-tube add a few drops of the urine under examination and heat the
mixture to boiling. The presence of laevulose is indicated by the produc-
tion of a red color and the separation of a red precipitate. The latter may
be dissolved in alcohol to which it will impart a striking red color.
If the boiling be prolonged a similar reaction may be obtained with
urines containing dextrose. This has been explained^ in the case of
dextrose as due to the transformation of the dextrose into laevulose by the
catalytic action of the hydrochloric acid. The precautions necessary for
' Seliwanofl's reagent may be prepared by dissolving 0.05 gram of resorcinol in 100 c.c.
of^dilute (i : 2) hydrochloric acid.
* ^ Koenigsfeld: Bioch. Zeit., 38, 311, 1912.
URINE. 357
a positive test for laevulose are as follows: The concentration of the
hydrochloric acid must not be more than 12 per cent. The reaction (red
color) and the precipitate must be observed after not more than 20-30
seconds of boiling. Dextrose must not be present in amounts exceeding
2 per cent. The precipitate must be soluble in alcohol with a bright red
color.
3. Phenylhydrazine Test. — Make the test according to directions
under Dextrose, 3, page 324.
4. Polariscopic Examination. — A simple polariscopic examination,
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 laevorotatory substances, such as conjugate gly-
curonates and ,5-oxybutyric acid.
CHOH
HOHC CHOH
INOSITE, I I
HOHC CHOH
\/
CHOH
Inosite occasionally occurs in the urine in albuminuria, diabetes
mellitus, and diabetes insipidus. It is claimed also that copious water-
drinking causes this substance to appear in the urine. Inosite was at one
time considered to be a sugar but is now known to be hexahydroxybenzene,
as the above formula indicates. It is an example of a non-carbohydrate
in whose molecule the H and O are present in the proportion to form
water. In other words it has the formula of the hexoses, i. e., CgHi^Og.
Inosite occurs widely distributed in the vegetable kingdom, and because
of this fact the theory has been voiced that it represents one of the first
stages in the conversion of a carbohydrate into the benzene ring. It is
found in the liver, spleen, lungs, brain, kidneys, suprarenal capsules,
muscles, leucocytes, testes, and urine under normal conditions.
Experiment.
I. Detection of Inosite. — Acidify the urine with concentrated nitric
acid and evaporate nearly to dryness. Add a few drops of ammonium
hydroxide and a little calcium chloride solution to the moist residue and
evaporate the mixture to dryness. In the presence of inosite (0.00 1 gram)
a bright red color is obtained.
For a more satisfactory test, which is also more time-consuming, see
Salkowski's^ modification of Scherer's test.
' Salkowski: Zeit. physiol. client., 69, 478, 1910.
358 PHYSIOLOGICAL CHEMISTRY.
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 laevorotatory, 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. Ordinarily the freshly passed urine is
clear, but upon exposure to the air the color deepens and may at 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 transformation 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 mistaken for indigo and melanogen for
indican. It is comparatively 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.
2. von Jaksch-PoUak Reaction. — Add a few drops of ferric
chloride solution to to 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 precipi-
tate forms, consisting of phosphates 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 pulmonary tuber-
URINE. 359
culosis, typhoid fever, nephritis, and stomach disorders. 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.
1. Robin's Reaction. — -Acidify lo 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 ID 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^ in a test-tube, mix thor-
oughly by shaking, and quickly add ammonium hydroxide in excess.
The test is positive if both the fluid and the foam assume a red color.
If the tube is allowed to stand a precipitate forms, the upper portion of
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 pres-
ence in the urine this reaction depends is not well understood. Some
investigators claim that a positive reaction indicates an abnormal de-
composition of protein 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 dis-
orders, which is of value diagnostically otily 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, tubercu-
losis, and measles. The reaction has also been obtained in the urine
in various other disorders such as carcinoma, chronic rheumatism,
' Two separate solutions should be prepared and mixed in definite proportions when
needed for use.
(o) Five grams of sodium nitrite dissolved in i liter of distilled water.
(b) Five grams of sulphanilic acid and 50 c.c. of hydrochloric acid in i liter of distilled
water.
Solutions a and b should be preserved in well-stoppered vessels and mixed in the pro-
portion I : 50 when required. Green asserts that greater delicacy is secured by mixing the
solutions in the proportion i : 100. The sodium nitrite deteriorates upon standing and be-
comes unfit for use in the course of a few weeks.
360 PHYSIOLOGICAL CHEMISTRY.
diphtheria, erysipelas, pleurisy, pneumonia, scarlet fever, syphilis,
typhus, etc. The administration of alcohol, chrysarobin, 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) NaNO^ + HCl— HNO^ + NaCl.
NH, N
(b) CeH, +HNO,— CgH, N + 2H2O.
\ \ /
HSO3 SO3
Sulphanilic acid. Diazo-benzenesulphonic acid.
CHAPTER XX.
URINE : ORGANIZED AND UNORGANIZED
SEDIMENTS.
The data obtained from carefully conducted microscopical exami-
nations of the sediment of certain pathological urines are of very great
importance, diagnostically. Too little emphasis is sometimes placed
upon the value of such findings.
Fig. I02. — The Purdv Electric Centrifuge.
Fig. 103. — 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 centrifuge (Fig. 102, above). An older
method, and one sill in 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 sediment 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 perfectly, whereas when the other method is
361
362 PHYSIOLOGICAL CHEMISTRY.
used it is frequently necessary to allow the urine to remain in the con-
ical 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.
Cystine.
Cholesterol.
Hippuric acid.
Leucine ■( ?) and tyrosine.
Haematoidin and biUrubin.
Magnesium phosphate.
Indigo.
Xanthine.
Melanin.
Ammonium Magnesium Phosphate ("Triple Phosphate"). —
Crystals of "triple phosphate" are a characteristic constituent of the
sediment when alkaline fermentation of the urine has taken place either
hejore 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 iil the sediment in two
forms, i. e., prisms and the feathery type. The prismatic form of crystals
(Fig. loi, p. 319) is the one most commonly observed in the sediment; the
feathery form (Fig. loi. p. 319) predominates when the urine is made
ammoniacal with ammonia.
The sediment of the urine in such disorders as are accompanied by a
retention of urine in the lower urinary tract contains "triple phosphate"
crystals as a characteristic constituent. 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. 104, p. 363). 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 phosphate" crystals. They
URINE.
;63
may be differentiated from the phosphate 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,
»
^
• s
^
%
<^
9
## ♦
Fig. 104. — Calcium Oxal.\te. (Ogdeti.)
in organic diseases of the liver, and in various other conditions which are
accompanied by a derangement of digestion or of the oxidation mechan-
ism, 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. Thev occur less fre-
FiG. 105. — Calcium Carbox.a.te.
quently 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 generally crystallizes in the form of granules,
spherules, or dumb-bells (Fig. 105, above). 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.
364 PHYSIOLOGICAL CHEMISTRY.
Calcium Phosphate (Stellar Phosphate). — Calcium phosphate may
occur in the urine in three forms, i. e., amorphous, granular, or crystalline.
The crystals of calcium phosphate are ordinarily pointed, wedge-shaped
formations which may occur as individual crystals, or grouped together
in more or less regularly formed rosettes (Fig. 81, p. 242). Acid sodium
urate crystals (Fig. 107, p. 366) are often mistaken for crystals of calcium
phosphate. We may differentiate between these 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 re-
crystallized 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" (see page 319).
Calciuni Sulphate. — Crystals of calcium sulphate are of quite rare
occurrence in the sediment of urine. Their presence 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. 100,
page 316) 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 oc currence as a constituent of urinary sediment
is of very little clinical significance.
Uric Acid. — Uric acid forms a very common constituent of the sedi-
ment of urines which are acid in reaction. It occurs in more varied forms
than any of the other crystalline sediments (Plate V, opposite page 291,
and Fig, 106, page 365), some of the more common varieties of crystals
being rhombic prisms, wedges, dumb-bells, whetstones, prismatic rosettes,
irregular or hexagonal plates, etc. Crystals of pure uric acid are always
colorless (Fig. 94, page 293), 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
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 sediments from the fact that it is soluble in alkalis,
alkali carbonates, boiling glycerol, concentrated sulphuric acid, and in
certain organic bases such as ethylamine and piperidin. It also responds
PLATE \-I.
Ammontum Urates, showing Spherules and Thorn-apple-shaped Crystals.
(From Os^deii. after Peyer.)
URINE.
365
to the murcxide test (see page 292), SchifT's reaction (see page 293) and
to Moreigne's reaction (see p. 293).
Urates. — The urate sediment may consist of a mixture of the urates of
ammonium, calcium, magnesium, potassium, and sodium. The ammo-
nium 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. There
are two sodium urates, the mono and the di, which may be expressed thus:
Na+\^„ ,, ^ , Na+^
j^ + ^^tl2^4<-'3 and ^^+
^C.H^N.O,.
Both salts dissociate with
the production of an alkaline reaction, the alkalinity being stronger in the
Fig. 106. — Various Forms of Uric Acid.
I, Rhombic plates; 2, whetstone forms; 3, 3, quadrate forms; 4, 5, prolonged into points;
6, 8, rosettes; 7, pointed bundles; 9, barrel forms precipitated by adding hydrochloric acid
to urine.
case of the di-sodium urate. The so-called quadriurate or hemiurate have
no existence as chemical units. ^ The urates of calcium, magnesium, and
potassium are amorphous in character, whereas the urate of ammonium
is crystalline. Sodium urate may be either amorphous or crystalline.
When crystalline it forms groups of fan-shaped clusters or colorless,
prismatic needles (Fig. 107, p. 366). 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). The urates are all soluble in hydrochloric
acid or acetic acid and their acid solutions yield crystals of uric acid upon
standing. They also respond to the murexide test. The clinical signifi-
Taylor: Jour. Biol. Chem., i, 177, 1905.
;66
PHYSIOLOGICAL CHEMISTRY.
cance of urate sediments is very similar to that of uric acid. A considerable
sediment of amorphous urates does not necessarily indicate a high uric
acid content, but ordinarily signifies a concentrated urine having a very
strong acidity.
Fig. 107. — Acid Sodium Urate.
Cystine. — Cystine is one of the rarer of the crystalline urinary sedi-
ments. It has been claimed that it occurs more often in the urine of men
than of women. Cystine crystallizes in the form of thin, colorless, hexa-
gonal plates (Fig. 25, p. 81, and Fig. 108, below) which are insoluble in
water, alcohol, and acetic acid, and soluble in minerals acids, alkalis,
and especially in ammonia. Cystine may be identified by burning it upon
/
Fig. 108. — Cystine. (Ogden.)
platinum foil, under which condition it does not melt but yields a bluish-
green flame.
Cholesterol. — Cholesterol 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 cholesterol
have been found in the sediment in cystitis, pyelitis, chyluria, and nephritis.
URINE. 367
Ordinarily it crystallizes in large regular and irregular colorless, transpar-
ent plates, some of which possess notched corners (Fig. 43, page 166).
Frecjuently, 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, w^hich are colorless needles or prisms
(Fig. 97, page 300) 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 differentiated from uric acid from the
fact that it does not respond to the murexide test and is much more soluble
in water and in ether. The detection of crystals of hippuric acid in
the 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 300).
Leucine and Tyrosine. — Leucine and tyrosine have frequently
been detected in the urine, either in solution or as a sediment. Neither
of them occurs in the urine ordinarily ex-
cept in association wuth the other, /. e.,
whenever leucine is detected it is more
than probable that tyrosine accompanies
it. They have been found pathologi-
cally in the urine in acute yellow atro-
phy of the liver, in acute phosphorus
poisoning, in cirrhosis of the liver, in
severe cases of typhoid fever and small- ^
pox, and in leukaemia. In urinary sedi- Fig. ioq.— Crystals of Impure
, . J- -1 4. u- • Leucine. (Ogden.)
ments leucme ordmaniy crystalhzes in
characteristic spherical masses which show both radial and concentric
striations and are highly refractive (Fig. 109, above). Some investi-
gators claim that these crystals which are ordinarily called leucine are,
in reality, generally urates. This view point has become more general in
recent years. For the crystalline form of pure leucine obtained as a
decomposition product of protein see Fig. 27. p. 85. T}Tosine crystallizes
in urinary sediments in the well-known sheaf or tuft formation (Fig.
24, p. 81). For other tests on leucine and tyrosine see pages ^90
and 91.
Haematoidin and Bilirubin. — There are divergent opinions regard-
ing the occurrence of these bodies in urinary sediment. Each of them
368 PHYSIOLOGICAL CHEMISTRY.
crystallizes in the form of tufts of small needles or in the form of small
plates which are ordinarily yellowish-red in color (Fig. 42, p. 161). Be-
cause 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 crystalline form is the same in each
case, there are certain chemical differences which may be brought out
very strikingly by properly testing. For instance, it has been claimed
that haematoidin may be differentiated from bilirubin through the fact
that it gives a momentary color reaction (blue) 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
haematoidin or bilirubin have been found in the urinary sediment
in jaundice, 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 elon-
gated, 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 stellate needles or occurs as amorphous particles or broken
fragments. 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.
Xanthine. — ^Xanthine 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 xanthine generally
occurs in the form of whetstone-shaped crystals somewhat similar in form
to the whetstone variety of uric acid crystal. They may be differentiated
from uric acid by the great ease with which they may be brought into solu-
tion in dilute ammonia and on applying heat. Xanthine may also form
urinary calculi. The clinical significance of xanthine in urinary sediment
is not well understood.
Melanin. — Melanin is an extremely rare constituent of urinary
sediments. Ordinarily in melanuria the melanin remains in solu-
tion; if it separates it is generally held in suspension as fine amorphous
granules.
URINE. 369
(b) Organized Sediments.
Epithelial cells.
Pus cells.
Hyaline.
Granular.
Epithelial.
Casts. I Blood.
Fatty.
Waxy.
I Pus.
Cylindroids.
Erythrocytes.
Spermatozoa.
Urethral filaments.
Tissue debris.
Animal parasites.
Micro-organisms.
Fibrin.
Foreign substances due to contamination.
Epithelial Cells. — The detection of a certain number of these cells in
urinary sediment is not, of itself, a pathological 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 necessary,
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 particular layer which is being desquamated.
It is frequently a most difficult undertaking, however, to make a clear
differentiation between the various forms of epithelial cells present in the
sediment. If skilfully done, such a miscropical differentiation may prove
to be of very great diagnostic aid.
The principal forms of epithelial cells met with in urinary sediments
are shown in Fig. no, p. 370.
Pus Cells. — Pus corpuscles or leucocytes are present in extremely
small numbers in normal urine. Any considerable increase in the number,
however, ordinarily denotes a pathological condition, generallv an acute
24
o/'
PHYSIOLOGICAL CHEMISTRY.
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 accompany pus corpuscles
in urinary sediment and a careful examination of these associated elements
is necessary in order to form a correct diagnosis as to the origin of the pus.
Protein is always present in urine which contains pus.
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
Fig. no. — 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, squamous epithelium from the
bladder; h, h, neck-of-bladder cells; i, epithelium from prostatic urethra; k, urethral cells;
/, /, scaly epithelium; m, m', cells from seminal passages; n, compound granule cells; o, fatty
renal cell. {Ogden.)
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 corpuscles 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 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 are similar in form.
URINE.
371
Such confusion may be avoided by the addition of iodine solution (I in
KI), a reagent which stains the pus corpuscles a deep mahogany -brown
and transmits to the epithelial cells a light yellow tint. The test proposed
by Vitali often gives very satisfactory results. This simply consists
in acidifying the urine (if alkaline) with acetic acid, then filtering, and
treating th^ sediment on the filter paper with freshly prepared tincture of
guaiac. The presence of i)us in the sediment is indicated if a blue color
is observed. Large numbers of pus corpuscles are present in the urinary
sediment in gonorrhoea, leucorrhoea, chronic pyelitis, and in abscess of
Fig. m. — Pus Corpuscles. (After Ultzmann.)
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 kidney. In addition to the usual constituents found in leucocytes
Mandel and Levene^ claim that pus cells contain glucothionic acid.
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 impor-
tant because of the fact that they generally indicate some kidney disorder;
if albumin accompanies the casts the indication is much accentuated.
Casts have been classified according to their microscopical characteristics
as follows: (a) Hyaline, {b) granular, (c) epithelial, {d) blood, (e) fatty,
ij) waxy, {g) pus.
(a) Hyaline Casts. — These are composed of a basic material which
is transparent, homogeneous, and very light in color (Fig. 112, p. 372).
In fact, chiefly because of these physical properties, they are the most
* Mandel and Levene: Biochemische Zeilschri/t, 4, 78, 1907.
372
PHYSIOLOGICAL CHEMISTRY,
difficult form of renal casts to detect under the microscope. Frequently
such casts are impregnated with deposits of various forms, such as erythro-
cytes, epithelial cells, fat globules, etc., thus rendering the form of the cast
more plainly visible. Staining is often resorted to in order to render the
shape and character of the cast more easily determined. Ordinary iodine
solution (I in KI) may be used in this connection; many of the aniline
dyes are also in common use for this purpose, e. g., gentian-violet, Bis-
marck-brown, methylene-blue, fuchsin, and eosin. Generally, but not
always, albumin is present in urine containing hyaline casts. Hyaline
Fig. 112. — Hyaline Casts.
One cast is impregnated with four renal cells.
casts are common to all kidney disorders, but occur particularly in the
earliest and recovering stages of parenchymatous nephritis and interstitial
nephritis.
{b) Granular Casts. — The common hyaline material is ordinarily the
basic substance of this form of cast. The granular material generally
consists of albumin, epithelial cells, fat, or disintegrated erythrocytes or
leucocytes, the character of the cast varying according to the nature and
size of the granules (Fig. 113, p. 373, and Fig. 114, page 374). Thus
we have casts of this general type classified a.s 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
URINE.
373
probably especially characteristic of the sediment in inflammatory
disorders.
(c) Epithelial Casts. — These are casts bearing upon their surface epi-
• thelial cells from the lining of the uriniferous tubules (Fig. 115, p. 374).
The basic material of this form of cast may be hyaline or granular in
nature. Epithelial casts are particularly abundant in the urinary sedi-
ment in acute nephritis.
(b) Blood Casts. — Casts of this type may consist of erythrocytes
borne upon a hyaline or a fibrinous basis (Fig. 116, p. 374). The occur-
rence of such casts in the urinary sediment denotes renal hemorrhage and
they arc considered to be especially characteristic of acute diflfuse nephritis
and acute congestion of the kidney.
Fig. 113. — Granular Casts. {Mter Peyer.)
((?) Fatty Casts. — Fatty casts may be formed by the deposition of fat
globules or crystals of fatty acid upon the surface of a hyaline or granular
cast (Fig. 117, p. 375). In order to 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
subacute and chronic inflammation of the kidney.
(/) 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 colorless, refractive bodies, but
differ from this form of cast in being, in general, of greater length and
374
PHYSIOLOGICAL CHEMISTRY.
diameter and possessing sharper outlines and a light yellow color (Fig
ii8, p. 375). Such casts occur in several forms of nephritis, but do
not appear to characterize any particular type of the disorder except
amyloid disease, in which they are rather common.
Fig. 114. — ^Granular Casts.
a, Finely granular; b, coarsely granular.
Fig. 115. — Epithelial Casts.
Fig. 116. — Blood, Pus, Hyaline and Epithelial Casts.
a, Blood casts; b, pus cast; c, hyaline cast impregnated with renal cells; d, epithelial casts.
(g) Pus Casts. — Casts whose surface is covered with pus cells or leuco-
cytes are termed pus casts (Fig. 116, above). They are frequently
mistaken for epithelial casts. The differentiation between these two
types is made very simple, however, by treating the cast with acetic acid
URINE.
375
Fig. 117. — Fatty Casts. (After Pcyer.
Fig iiS. — Fatty and Waxy Casts.
<j. Fatty casts; b, waxy casts.
376 PHYSIOLOGICAL CHEMISTRY.
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 hyaline type, but they are ordinarily
fiat in structure with a rather smaller diameter than casts, may possess
forked or branching ends, and are not composed of homogenous material
as are the hyaline casts. Such "false casts" may become coated with
urates, in which event they appear granular in structure. The basic
Fig. 119. — Cylindroids. {Aiier: Peyer.)
substance of cylindroids is often the nucleoprotein of the urine (see Fig.
119, above).
Erythrocytes. — These form elements are present in the urinary
sediment in various diseases. They appear as the normal biconcave,
yellow erythrocyte (Plate IV, opposite page 196) or may exhibit certain
modifications in form, such as the crenated type (Fig. 120, p. 377) which
is often seen in concentrated 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 original 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 sedi-
URINE.
377
ment in diseases of the genital organs, as well as after coitus, nocturnal
emissions, epileptic, and other convulsive attacks, and sometimes in severe
febrile disorders, especially in typhoid fever. In form they consist of an
Fig. I20. — Crexated Erythrocytes.
oval body, to which is attached a long, delicate tail (Fig. 121, below).
Upon examination they may show motility or may be motionless.
Urethral Filaments. — These are peculiar thread-like bodies which
Fig. 121. — Human Spermatozoa.
are sometimes found in urinar}* sediment. They may occasionalh^ be
detected in normal urine and pathologically are found in the sediment in
acute and chronic gonorrhoea and in urethrorrhoea. The ground-sub-
stance of these urethral filaments is, in part at least, similar to that of the
378 PHYSIOLOGICAL CHEMISTRY,
cylindroids (see page 376). The urine first voided in the morning is best
adapted for the examination for filaments. These filaments may ordi-
narily be removed by a pipette since they are generally macroscopic.
Tissue Debris. — Masses of cells or fragments of tissue are frequently
found in the 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 to a final decision as to their origin.
Animal Parasites. — The cysts, booklets, and membrane shreds
of echinococci are sometimes found in the urinary sediments. Other
animal organisms which are more rarely met with in the urine are em-
bryos of the Filaria sanguinis and eggs of the Distoma hamatohium and
Ascarides. Animal parasites in general occur most frequently in the
urine in tropical countries.
Micro-organisms. — Bacteria as well as yeasts and moulds are
frequently detected in the urine. Both the pathogenic and non-patho-
genic forms of bacteria may occur. The non-pathogenic forms most
frequently observed are micrococcus urece, bacillus urea, and staphylococcus
urecB liquefaciens . Of the pathogenic forms many have been observed,
e. g., Bacterium Coli, typhoid bacillus, tubercle bacillus, gonococcus, bacillus
pyocyaneus, and proteus vulgaris. Yeast and moulds are most frequently
met in diabetic urine.
Fibrin. — Following haematuria, fibrin clots are occasionally ob-
served in the urinary sediment. They are generally of a semi-gelatin-
ous consistency and of a very light color, and when examined under
the microscope they are seen to be composed of bundles of highly re-
fractive fibers which run parallel.
Foreign Substances Due to Contamination. — Such foreign sub-
stances as fibers of silk, linen, or wool; starch granules, hair, fat, and
sputum, as well as muscle fibers, vegetable cells, and food particles are
often found in the urine. Care should be taken that these foreign
substances are not mistaken for any of the true sedimentary constituents
already mentioned.
CHAPTER XXI.
URINE : CALCULI.
Urinan' calculi, also called concrelians, or concrements are solid
masses of urinary sediment formed in some part of the urinary tract.
They vary in shape and size according to their location, the smaller
calculi, termed sand or gravel, in general arising from the kidney or the
pelvic portion of the kidney, whereas the large calculi are ordinarily
formed in the bladder. There are two general classes of calculi as
regards composition, i. e., simple and compound. The simple form is
made up of but a single constituent, whereas the compound type con-
tains two or more individual constituents. The structural plan of
most calculi consists of an arrangement of concentric rings about a
central nucleus, the number of rings frequently being dependent upon
the number of individual constituents which enter into the structure
of the calculus. In case two or more calculi unite to form a single calculus
the resultant body will obviously contain as many nuclei as there were
individual calculi concerned in its construction. Under certain condi-
tions the growth of a calculus will be principally in only one direction,
thus preventing the nucleus from maintaining a central location. The
qualitative composition of urinary calculi is dependent, in great part,
upon the reaction of the urine, e. g., if the reaction of the urine is acid the
calculi present will be composed, in great part at least, of substances that
are capable of depositing in acid urine.
According to Ultzmann, out of 545 cases of urinary calculus, uric
acid and urates formed the nucleus in about 81 per cent of the cases;
earthy phosphates in about 9 per cent; calcium oxalate in about 6 per
cent; cystine in something over i per cent, while in about 3 per cent
of the cases some foreign body comprised the nucleus.
In the chemical examination of urinary calculi the most valuable
data are obtained by subjecting each of the concentric layers of the
calculus to a separate analysis. Material for examination may be
conveniently obtained by sawing the calculus carefully through the
nucleus, then separating the various layers or by scraping off from
each layer (without separating the layers) enough powder to conduct
the examination as outlined in the scheme (see page 381).
379
380 PHYSIOLOGICAL CHEMISTRY.
Varieties of Calculus.
Uric Acid and Urate Calculi. — Uric acid and urates constitute the
nuclei of a large proportion (81 per cent) of urinary concretions. Such
stones are always colored, the tint varying from a pale yellow to a
brownish-red. The surface of such calculi is generally smooth but it
may be rough and uneven.
Phosphatic Calculi. — Ordinarily these concretions consist prin-
cipally of "triple phosphate" and other phosphates of the alkaline
earths, with very frequent admixtures of urates and oxalates. The
surface of such calculi is generally rough but may occasionally be rather
smooth. The calculi are somewhat variable in color, exhibiting gray,
white, or yellow tints under different conditions. When composed of
earthy phosphates the calculi are characterized by their friability.
Calcium Oxalate Calculi.^ — This is the hardest form of calculus
to deal with, and is rather difficult to crush. They ordinarily occur
in two general forms, i. e., the small, smooth concretion which is charac-
terized as the hemp-seed calculus and the medium-sized or large stone
possessing an extremely uneven surface which is generally classed as
a mulberry calculus. This roughened surface of the latter form of calcu-
lus is due, in many instances, to protruding calcium oxalate crystals of
the octahedral type.
Calcium Carbonate Calculi.— Calcium carbonate concretions are
quite common in herbivorous animals, but of exceedingly rare occurrence
in man. They are generally small, white, or grayish calculi, spherical
in form and possess a hard, smooth surface.
Cystine Calculi. — The cystine calculus is a rare variety of calculus.
Ordinarily they occur as small, smooth, oval, or cylindrical concretions
which are white or yellow in color and of a rather soft consistency.
Xanthine Calculi. — This form of calculus is somewhat more rare
than the cystine type. The color may vary from white to brownish-
yellow. Very often uric acid and urates are associated with xanthine
in this type of calculus. Upon rubbing a xanthine calculus it has the
property of assuming a wax-like appearance.
Urostealith Calculi. — This form of calculus is extremely rare.
Such concretions are composed principally of fat and fatty acid. When
moist they are soft and elastic, but when dried they become brittle.
Urostealiths are generally light, in color.
Fibrin Calculi. — Fibrin calculi are produced in the process of
blood coagulation within the urinary tract. They frequently occur as
nuclei of other forms of calculus. They are rarely found.
URINE.
381
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382 PHYSIOLOGICAL CHEMISTRY.
Cholesterol Calculi. — An extremely rare form of calculus some-
what resembling the cystine type.
Indigo Calculi. — Indigo calculi are extremely rare, only two cases
having been reported. One of these indigo calculi is on exhibition in
the museum of Jefferson Medical College of Philadelphia.
The scheme, proposed by Heller and given on page 381, will be found
of much assistance in the chemical examination of urinary calculi.
CHAPTER XXII.
URINE : QUANTITATIVE ANALYSIS.
I. Protein.
1. Scherer's Coagulation Method. — The content of coagulablc
protein 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 protein 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 maintained there for a few
minutes in order to insure the complete coagulation of the protein present.
Now filter the urine ^ through a previously washed, dried, and weighed
filter paper, wash the precipitated protein, 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 110° C. Subtract the weight of the
filter paper from the combined weight of the paper and precipitate and
calculate the percentage of protein in the urine specimen.
Calculation. — To determine the percentage of protein present 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 protein by Esbach's reagent" and the apparatus used in the estimation is
Esbach's albuminometer (Fig. 122, p. 384). In making a determin-
ation fill the albuminometer to the point U with urine, then intro-
duce the reagent until the point R is reached. Now stopper the
tube, invert it slowly several times in order to insure the thorough mixing
of the fluids, and stand the tube aside for 24 hours. Creatinine, resin
acids, etc., are precipitated in this method, and for this and other reasons
it is not as accurate as the coagulation method. It is, however, extensively
used clinically. According to Sahli^ the method is "accurate approxi-
' If it is desired the precipitate may be filtered off on an imweighed paper, and its nitrogen
content determined by the Kjeldahl method (see p. 401). In order to arrive at correct figures
for the protein content it is then simply necessary to multiply the total nitrogen content by
6.25 (see p. 438). Correction should be made for the nitrogen content of the filter paper
used unless this factor is negligible.
^ Esbach's reagent is prepared by dissolving 10 grams of picric acid and 20 grams of
citric acid in i liter of water.
^Sahli: Lehrbuch d. klin. Untersuchungs-Methoden, 5th Aufl., 1909.
383
384
PHYSIOLOGICAL CHEMISTRY.
lili
mately to one part per 1000," whereas Pfeiffer^ claims it is not accurate
for less than one-half or for more than five parts per 1000.
Calculatimi. — The graduations on the albuminometer indicate grams
of protein per liter of urine. Thus, if the protein precipitate is level with
the figure 3 of the graduated scale, this denotes that the urine examined
contains 3 grams of protein to the liter. To express the
amount of protein in per cent simply move the decimal
point one place to the left. In the case under con-
sideration the urine contains 0.3 per cent protein.
3. Kwilecki's Modification of Esbach's Method.^
— Add 10 drops of a 10 per cent solution of FeClg to the
acid urine before introducing the Esbach's reagent.
Warm the tube and contents in a water-bath at 72° C.
for 5-6 minutes and make the reading.
II. Dextrose.
I. 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.
Now place 10 c.c. of Fehling's solution-'' in a small
beaker, dilute it with approximately 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 tur-
bidity is produced the Fehling's solution is unfit for use.
Clamp the burette containing the dilute urine immedi-
ately over the beaker and carefully allow from 0.5 to i
c.c. of the diluted urine to flow into the boiling Fehl-
ing's solution. Bring the solution to the boiling-point
after each addition of urine and continue running the urine from the
burette, 0.5-1 c.c. at a time, as indicated, until the Fehling's solution is
completely reduced, i. e., 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
'Pfeiffer: Berl. klin. Woch., 49, 114, 1912.
' Kwilecki: Miittch. Med. Woch., 56, p. 1330.
' Directions for the i)reparalion of Fehling's solution are given in a note at the bottom
of page 32.
Fig. 122. — Es-
bach's Albumin-
ometer. (Ogden.)
urine: quantitative analysis. 385
note the number of cubic centimeters of diluted urine used in the pro-
cess and calculate the percentage of dextrose present, in the sample of
urine analyzed, according to the method given below.
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 co\or finally disappears
is noted. Several means of accurately fixing this point have been sug-
gested, but they are practically all open to objection. As good a "check"
as any, perhaps is to filter a few drops of the solution, through a double
paper, after the blue color has apparently disappeared, acidify the filtrate
with acetic acid and add potassium ferrocyanide. If the copper of the
Fehling's solution has been completely reduced, there will be no color
reaction, whereas the production of a brown color indicates the presence
of unreduced copper. Harrison has recently suggested the following pro-
cedure to determine the e.xact end-point: To about i c.c. of a starch
iodide solution^ 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.
Unreduced copper will be indicated by the production of a purplish-red or
blue color due to the liberation of iodine.
It is ordinarily customary to make at least three determinations by
Fehling's method before 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. ^ If y represents the number 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:
y : 0.05 :: 100 : x (percentage of dextrose).
2. Benedict's Method No. i. — To 30 c.c. of Benedict's solution* in
a small beaker add from 2.5 grams to 5 grams of anhydrous sodium car-
* The starch-iodide solution may be prepared as follows: Mix o.i 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.
- The values for certain other sugars are as follows:
Lactose 0.0676 gram.
Maltose o . 074 gram.
Invert sugar 0.0475 gram.
' Benedict's solution used in the quantitative determination of sugar consists of three
separate solutions. The copper sulphate solution and the alkaline tartrate solution are the same
as those already described in connection with Benedict's qualitative test, see p. ^^. The
third solution is made up as follows:
Potassium ferro-thiocyanate soluticm = i$ grams of potassium ferrocyanide, 62.5 grams
of potassium thiocyanate and 50 grams of anhydrous sodium carbonate dissolved in water
and made up to 500 c.c.
These three solutions should be preserved separately in rubber-stoppered bottles and
mixed in equal volumes when needed for use. This is done to prevent deterioration.
25
386 PHYSIOLOGICAL CHEMISTRY.
bonate^ and heat the mixture to boihng over a wire gauze until the car-
bonate has been brought into solution.
Place the urine under examination in a burette and run it into the hot
Benedict solution rather rapidly^ until the formation of a heavy chalk-
white precipitate is noted and the blue color of the solution lessens per-
ceptibly in its intensity. From this point in the determination from 2 to
10 drops^ of the urine should be run into the boiling Benedict solution at
one time, boiling the solution vigorously for about 15 seconds after each
addition. Complete reduction of the copper is indicated here as in
Fehling's original method, by the complete disappearance of all blue color.
The end-point here, however, is very sharply defined, contrary to the
conditions in the older method.
To prevent the annoying bumping which often interferes with the
titration, a medium-sized piece of washed absorbent cotton* may be
introduced into the solution. This cotton may be stirred about through
the solution as the titration proceeds and the bumping thus eliminated.
Calculation. — Thirty cubic centimeters of Benedict's solution is
completely reduced by 0.073 gram of dextrose. If y represents the num-
ber of cubic centimeters of urine necessary to reduce the 30 c.c. of the
solution we have the following proportion:
y : 0.073 •• i°° • ^ (percentage of dextrose).
Benedict's Method No. 2.^ — "The urine, 10 c.c. of which should be
diluted with water to 100 c.c. (unless the sugar content is believed to be
low), is poured into a 50 c.c. burette up to the zero mark. Twenty-five
cubic centimeters of the reagent" are measured with a pipette into a por-
celain evaporation dish (25-30 cm. in diameter), 10 to 20 grams of
crystallized sodium carbonate (or one-half the weight of the anhydrous
' The amount added depends upon the dilution to which the solution is to be subjected
in titration. For this reason the maximum amount of sodium carbonate should be added
when titrating urines containing a ver}' low percentage of sugar.
^ Not rapidly enough, however, to interfere in any marked degree with the continuous
vigorous })oiling of the solution.
^ The e.xact amount to run in depends upon the intensity of the remaining blue color,
as well as upon the sugar content of the urine. The lo drops should be added at one time
only when urines containing a very low percentage of sugar are under examination.
* Glass wool may be substituted if desired.
* Benedict: Jour. Am. Med. Ass'n., 57, 1193, 191 1.
" Copper sulphate (crystallized) 18 .0 grams.
Sodium carbonate (cn>-stallixed, one-half the weight of the
anhydrous salt may be used) 200 .0 grams.
Sodium or potassium citrate 200.0 grams.
Potassium thiocyanate 125 .0 grams.
Potassium ferrocyanide (5 per cent solution) 5.0 c.c.
Distilled water to make a total volume of 1000. o c.c.
With the aid of heat dissolve the carbonate, citrate and thiocyanate in enough water to
make aViOut 800 c.c. of the mixture and filter if necessary. Dissolve the copper sulphate
separately in about 100 c.c. of water antl pour the solution slowly into the other licjuid, with
constant stirring. Add the ferrocyanide solution, cool and dilute to exactly i liter. Of the
various constituents, the copper salt only need be weighed with exactness. Twenty-five
cubic centimeters of the reagent are reduced by 50 mg. of glucose.
urine: quantitative analysis. 387
salt) arc added, together with a small (juantity of powdered pumice stone
or talcum, and the mixture heated to boiling over a free flame until the
carbonate has entirely dissolved. The diluted urine is now run in from
the burette, rather rapidly until a chalk-white precipitate forms, and the
blue color of the mixture begins to lessen perceptibly, after which the
solution from the burette must be run in a few drops at a time, until the
disappearance of the last trace of blue color, which marks the end-point.
The solution must be kept vigorously boiling throughout the entire
titration. If the mixture becomes too concentrated during the process,
water may be added from time to time to replace the volume lost by
evaporation. The calculation of the percentage of sugar in the original
sample of urine is very simple. The 25 c.c. of copper solution are
reduced by exactly 50 mg. of glucose. Therefore the volume run out of
the burette to effect the reduction contained 50 mg. of the sugar. When
the urine is diluted 1:10, as in the usual titration of diabetic urines, the
formula for calculating the per cent of the sugar is the following:
o.oso . . . , , , . -^ . , ,
^ Xiooo=per cent m origmal sample, wherem X is the number
of cubic centimeters of the diluted urine required to reduce 25 c.c.
of the copper solution."
"In the use of this method chloroform must not be present during the
titration. If used as a preservative in the urine it may be removed by
boiling a sample for a few minutes, and then diluting to its original
volume."
"Like the reagent for qualitative employment, the one for quantitative
work will keep indefinitely after its preparation. As regards the accuracy
of the method, it may be stated that repeated determinations, and com-
parisons with results by the polariscope and by Allihn's gravimetric
process have shown the method to be probably more exact than any other
titration method available for sugar work."
3. Purdy's Method. — Purdy's solution^ 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:
' Purdy's solution has the following composition:
Copper sulphate 4-752 grams.
Potassium hydroxide 23 .5 grams.
Ammonia (U. S. P., sp. gr. 0.9) 35° o c.c.
Glycerol 38.0 c.c.
Distilled water, to make total volume i liter.
In preparing the solution bring the copper sulphate and potassium hydroxide into solution
in separate vessels, mix the two solutions, cool the mixture, and add the ammonia and glycerol.
After this has been done the total volume should be made up to i liter with distilled water.
Thirty-five cubic centimeters of Purdy's solution is exactly reduced by 0.02 gram of
dextrose.
388 PHYSIOLOGICAL CHEMISTRY.
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
introduce 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 oper-
ator as completely as possible.^ Now bring the solution to the boiling-
point and add the urine, drop by drop, untfl the intensity of the blue color
begins to diminish. When this point is reached add the urine somewhat
more slowly 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 to precipitate.
Some investigators consider it to be advisable to dilute the urine
before applying the above manipulation, but ordinarily this is not neces-
sary 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 correction made in the calculation.
Calculation. — -Thirty-five c.c. of Purdy's solution is completely 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).
4. Fermentation Method. — This method consists in the measure-
ment of the volume of carbon dioxide evolved when the dextrose 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 Einhorn's saccharometer (Fig. 3, page 36) is the
apparatus employed is perhaps as satisfactory as any for clinical pur-
poses. The procedure is as follows: Place about 15 c.c. of urine in a
mortar, add about i gram of yeast (1/16 of the ordinary cake of com-
pressed yeast) and carefully crush the latter by means of a pestle. Trans-
fer the mixture to the saccharometer, being careful to note that the
graduated tube is completely filled and that no air bubbles gather at the
* This side tube may also be equipped with a simple air-valve, thus insuring the exclusion
of aiir and thereby contributing to the accuracy of the determination, inasmuch as the cuprous
salts would be reoxidized upon coming in contact with the air. If one is careful to maintain
iJie 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. 389
top. Allow the apparatus to stand in a warm place (30° C.) for 12 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 carbon dioxide are indicated by the graduations
on the side of the saccharometer tube.
The availability of the fermentation procedure as a quantitative aid
has been appreciably lowered through the important findings of Neuberg
and Associates^ recently reported. They show that yeast has the prop-
erty of splitting off carbon dioxide from the carboxyl group of amino and
other aliphatic acids. The active agent in this "sugar-free fermentation"
is an enzyme called carboxylase. Inasmuch as amino acids are always
present in the urine, the error from this source is apparent.
5. 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 lead acetate. The
urine should be well stirred and then filtered through a filter paper which
has not been previously 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 accompanied by other
optically active substances, such as proteins, laevulose, /9-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 36.
III. Uric Acid.
I. Folin-Shaffer Method. — Introduce 100 ex.- of urine into an
Erlenmeyer flask, add 25 c.c. of the Folin-Shaffer reagent* and after
shaking the flask to thoroughly mix the fluids allow the mixture to
stand,' with or without further stirring, until the precipitate has settled
(5-10 minutes). Filter, transfer 100 c.c. of the filtrate to a 200 c.c.
Erlenmeyer flask, add 5 c.c. of concentrated ammonium hydroxide
and allow the mixture to stand for 24 hours. Transfer the precipitated
ammonium urate quantitatively to a filter paper," using 10 per cent
* Neuberg and Associates: Biochemische Zeitschrift, 31, 170; 36 (60, 68, and 76), 191 1.
^ It is preferable to use more than 100 c.c. of urine if the fluid has a specific gravity less
than 1.020.
^ The Folin-Shaffer 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.
* The mi.\ture should not be allowed to stand for too long a time at this point, since uric
acid may be lost through precipitation.
* The Schleicher and Schiill hardened papers or the Baker and Adamson washed, ashless
variety are ver}' satisfactory' for this purpose.
390 PHYSIOLOGICAL CHEMISTRY.
ammonium sulphate to remove the final traces of the urate from the
flask. Wash the precipitate approximately free from chlorides by
means of lo per cent ammonium sulphate solution/ remove the paper
from the funnel, open it, and by means of hot water rinse the precipitate
back through the funnel into the flask in which the urate was originally
precipitated. The volume of fluid at this point should be about loo c.c.
Cool the solution to room temperature, add 15 c.c. of concentrated
sulphuric acid and titrate at once with N/20 potassium permanganate,
K^Mn^O^, solution. The first tinge of pink color which extends through-
out 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 N/20 potassium permanga-
nate 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 permanganate
solution required, we may make the following calculation:
y X 0.00375 = weight of uric acid in 100 c.c. of urine.
Because of the solubility of the ammonium urate a correction of 3
milligrams should be added to the final result.
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 determination of uric acid.
It is believed to be somewhat less accurate than the method just described.
The procedure is as follows: Place 100 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 24 hours. Filter off the uric acid
crystals upon a washed, dried and weighed filter paper and wash them
with cold distilled water, a few cubic centimeters at a time until the
chlorides are removed. Now wash, in turn, with alcohol and with
ether and finally dry the paper and crystals to constant weight at 110° C.
In the process of washing the uric acid free from chlorides an error is
introduced, since every cubic centimeter of water so used dissolves
0.00004 gram of uric acid.^ For this reason a correction is necessary.
' This washing may be conveniently done l^y decantalion if desired, thus retaining the
major portion of the precipitate in the flask.
^ His and Paul: Zeit. physiol. Chem., 31,1, 1900.
URINF. : QUANTITATIVE ANALYSIS. 39I
It has been suggested that the pigment of the crystals is equivalent in
weight to the amount of uric acid dissolved by the first 30 c.c. of water,
and this factor should be taken into account in the computation of the
percentage of uric acid.
Calculation. — Since 100 c.c. of urine was used the corrected weight
of the uric acid crystals, in grams, will express the percentage of uric acid
present.
3. Kriiger and Schmidt's Method. — This method serves for the
detection of both uric acid and the purine bases. The principle in-
volved is the precipitation of both the uric acid and the purine bases
in combination with copper oxide and the subsequent decomposition
of this precipitate by means of sodium sulphide. The uric acid is then
precipitated by means of hydrochloric acid and the purine bases are
separated from the filtrate in the form of their copper or silver com-
pounds. The nitrogen content of the precipitates of uric acid and pur-
ine bases is then determined by means of the Kjeldahl method (see p.
401) and the corresponding values for uric acid and purine bases calcu-
lated. The method is as follows: To 400 c.c. of albumin-free urine ^
in a liter flask," add 24 grams of sodium acetate, 40 c.c. of a solution
of sodium bisulphite' and heat the mixture to boiling. Add 40-80
c.c* of a ID per cent solution of copper sulphate and maintain the tem-
perature of the mixture at the boiling-point for at least three minutes.
Filter off the flocculent precipitate, wash it with hot water until the
wash water is colorless, and return the washed precipitate to the flask
by puncturing the tip of the filter paper and washing the precipitate
through by means of hot water. Add water until the volume in the
flask is appro.ximately 200 c.c, heat the mixture to boiling, and decom-
pose the precipitate of copper oxide by the addition of 30 c.c. of sodium
sulphide solution.^ After decomposition is complete, the mixture should
be acidified with acetic acid and heated to boiling until the separating
sulphur collects in a mass. Filter the hot fluid by means of a filter pump,
wash with hot water, add 10 c.c. of 10 per cent hydrochloric acid and
evaporate the filtrate in a porcelain dish until the total volume has been
reduced to about ten cubic centimeters. Permit this residue to stand
' If albumin is present, the urine should be heated to boiling, acidified with acetic acid
and filtered.
- The total volume of urine for the twenty-four hours should be suflSciently diluted with
water to make the total volume of the solution 1600-2000 c.c.
' .\ solution containing 50 grams of Kahlbaum's commercial sodium bisulphite in 100
A. of water.
* The e.xact amount depending upon the content of the purine bases.
•* This is made by saturating a i per cent solution of soduim hydro.xide with hydrogen
sulphide gas and adding an equal volume of i per cent sodium hydroxide.
Ordinarily the addition of 30 c.c. of this solution is sufficient, but the presence of an excess
of sulphide should be prtrcen by adding a drop of lead acetate to a drop of the solution. Under
these conditions a dark brown color will show the presence of an excess of sodium sulphide.
592
PHYSIOLOGICAL CHEMISTRY.
about two hours to allow for the separation of the uric acid, leaving the
purine bases in solution. Filter off the precipitate of uric acid, using a
small filter paper, and wash the uric acid, with water made acid with
sulphuric acid, until the total volume of the original filtrate and the wash
water aggregates 75 c.c. Determine the nitrogen content of the precipi-
tate by means of the Kjeldahl method (see p. 401) and calculate the uric
acid equivalent.
Calculation. — In calculating the uric acid value from the total nitrogen
simply multiply the latter by three and add 0.0035 to the product as a
correction for the uric acid remaining in solution in the 75 c.c.
IV. Urea.
I. Knop-Hiifner Hypobromite Method (using Marshall's Urea
Apparatus). — Place the thumb over the side opening of the bulbed-
tube of the apparatus (Fig. 123) and care-
fully fill the tube with sodium hypobromite
solution. ' Close the opening in the end of
the tube with a rubber stopper, incline the
tube to allow air-bubbles to escape, and
finally invert the tube and fix the stoppered
end in the saucer-shaped vessel. By means
of the graduated pipette rapidly introduce
I c.c. of urine^ into the hypobromite solu-
tion through the side opening of the bulbed-
tube. Withdraw the pipette immediately
after the urine has been introduced. When
the decomposition of the urea is completed
(10-20 minutes) gently tap the bulbed-tube
with the finger in order to dislodge any gas
bubbles which may have collected on the
inner surface of the glass. The atmospheric
Fig. 123.— Marshall's Urea pressure should now be equalized by attach-
Apparatus. (Tyson.) . i n i -l i
a, Bulbed measuring tube; b, mg the funnel-tube to the bulbcd-tubc at the
^peTitt'funnd-tube? '"'"''' ^^de opening and introducing hypobromite
solution into it until the columns of liquid in
' The ingredients of tlie sodium hypobromite solution should be prepared in the form
of two separate solutions. When needed for use mix one volume of solution a, one volume
of solution b, and 3 volumes of water.
(a) Dissolve 125 grams of sodium bromide in water, add 125 grams of bromine and
make the total volume of the solution i liter.
(b) A solution of sodium hydroxide having a specific gravity of 1.250. This is approxi-
mately a 22.5 per cent solution.
Preserve both solutions in rubber-stoppered bottles.
^ Ordinarily i c.c. of urine is sufficient; more may be used, however, if its content of
urea is very low.
'hiimmiiiiiiiniiM'i
urine: quantitative analysis. 393
the two tubes are uniform in hcij^ht. The graduated scale of the bulbed-
tube should now be read in order to determine the number of cubic
centimeters of nitrogen gas evolved. By means of the appended formula
the weight of the urea present in the urine under examination may be
computed.
Calculatiou.^ — By properly substituting in the following formula
the weight of urea, in grams, contained in the volume of urine decom-
posed (i c.c. or more) may readily be determined:
w
vip-T)
354.5 X 760(1 + 0.003665/)
w; = weight of urea, in grams.
1* = observed volume of nitrogen expressed in cubic centimeters.
/> = barometric pressure expressed in mm. of mercury.
T = tension of aqueous vapor^ for temperature /.
/ = temperature (centigrade).
If we wish to calculate the percentage of urea we may do so by means of
the following proportion in which y represents the volume of urine used
and w denotes the weight of the urea contained in the volume y:
y :w ::x: (percentage of urea) .
Sodium hypobromite solution may also be employed for the deter-
mination of urea in the apparatus devised by Hiifner which is pictured in
Fig. 124, page 394.
2. Knop-Hiifner Hypobromite Method (Using the Doremus-
Hinds Ureometer). — In common wdth the method already described,
this method depends upon the measurement of the volume of nitrogen
gas liberated when the urea of the urine is decomposed by means of sodium
hypobromite solution. The Doremus-Hinds ureometer (Fig. 125, p. 395),
is one of the simplest and cheapest forms of apparatus in general use for
the determination of urea by the hypobromite process. In using this
apparatus proceed as follows: Fill the side tube B and the lumen of the
stopcock C with the urine under examination. Carefully wash out tube
A with water and introduce into it sodium hypobromite solution,^ being
* 0.003665= coefficient of expansion of gases for 1° C. 354.5 = number of c.c. of nitrogen
gas evolved from i gram of urea.
* The values of T for the temperatures ordinarily met with are given in the following
table:
Temp. Tension in mm. Temp. Tension in mm.
15° C 12.677 21° C 18.505
16° C 13
17° C 14
18° C 15
19° C 16
20° C. 17
519 22° C 19675
009 23° C 20.909
351 24° C 22.211
345 25° C 23.582
396
'For directions as to the preparation of this solution see page 392.
394
PHYSIOLOGICAL CHEMISTRY.
careful to fill the bulb sufficiently full to prevent the entrance of air into
the graduated portion. Now allow i c.c. of urine ^ to flow from tube B into
tube A and after the evolution of gas bubbles has ceased (10-20 minutes)
take the reading of the graduated scale on tube A.
In common with all other methods which are based upon the decom-
position of urea by means of hypobro-
mite solution, this method is not abso-
lutely correct. It is, however, suffi-
ciently accurate for ordinary clinical
purposes.
Calculation. — Observe the reading
on the graduated scale of tube A. This
tube is so graduated as to represent the
weight of urea, in grams, per cubic
centimeter of urine. If we wish to com-
pute the percentage of urea present this
may be done very readily by simply
moving the decimal point two places to
the right; e. g., if the reading is 0.02
gram the urine contains 2 per cent of
urea.
3. Folin's Method. — This is one of
the most accurate methods yet devised
for the determination of urea in the
urine. It has, however, been replaced
to a great extent by the very recent
modification of Folin and Pettibone (see
p. 397). The procedure is as follows:
Place 5 c.c. of urine in a 200 c.c. Erlen-
meyer flask and add to it 5 c.c. of con-
centrated hydrochloric acid, 20 grams of
crystallized magnesium chloride, a piece
of paraffin the size of a hazel nut, and
2-3 drops of a I per cent aqueous solution of "alizarin red." Insert a
Folin safety tube (Fig. 126, p. 396) into the neck of the flask and boil the
mixture until each drop of reflow from the safety tube produces a very
perceptible bump; the heat is then reduced somewhat and continued one
and one-half hours. The contents of the flask must not remain alkaline,
and to obviate this, at the first appearance of a reddish tinge in the con-
tents of the flask a few drops of the acid distillate are shaken back into
' If the content of urea in the urine under examination is large, the urine may be diluted
with water before determining the urea. If this is done it must of course be taken into con-
sideration in ( ompuling the content of urea.
Fig. 124. — Hufner's Urea Apparatus.
urine: quantitative analysis.
395
r^
the flask. At the end of i 1/2 hours the contents of the vessel are
transferred to a i-liter flask with about 700 c.c. of distilled water,
about 20 c.c. of 10 per cent potassium hydroxide or sodium hydroxide
solution is added and the mixture distilled into a known volume of N/io
sulphuric acid until the contents of the flask are nearly dry or until the
distillate fails to give an alkaline reaction to litmus, showing the absence
of ammonia. The time devoted to this
])rocess is ordinarily about an hour. Boil
the distillate a few moments to free it from
CO,, then cool and titrate the mixture with
X/io sodium hydroxide, using "alizarin
red" as indicator.
A "check" experiment should always
be made to determine the original am-
monia content of the urine and of the
magnesium chloride, if it is not absolutely
pure, which of course should be subtracted
from the total amount of ammonia as deter-
mined by the above process.
The Folin method is extremely accu-
rate under all conditions except when the
urine contains sugar. When this is the
case the carbohydrate and the urea unite,
upon being heated, and form a very stable
combination. For this reason the Folin
method is not suitable for use in the exam-
ination of such urines. Under such condi-
tions the combination Morner-Sjoqvist-
Folin method which is given below or the
ipethod of Folin and Denis (p. 398) may
be used.
4. Morner-Sjoqvist-Folin Method. — As has already been stated in
the last experiment, this method excels the Folin method in accuracy only
in the determination of urea in the presence of carbohydrate bodies.
Briefly, the procedure is as follows:^ Bring the major portion of 1.5 gram
of powdered barium hydroxide into solution in 5 c.c. of urine in a small
flask, and treat the mixture with 100 c.c. of an alcohol-ether solution,
consisting of two volumes of 97 per cent alcohol and one volume of ether.
Stopper the flask and allow it to stand 12-24 hours. Filter off the pre-
cipitate, wash it with the alcohol-ether mixture and remove the alcohol
' The original description of the method may be found in an article by Morner: Skatj-
dinavisches Archiv Jilr Physiologic, 14, 21)7, 1903.
Fig. 125. — DoREMus-IIixDS
Ureometer.
396
PHYSIOLOGICAL CHEMISTRY.
Q^=£J!fe
and ether from the filtrate by distillation, being careful to keep the tempera-
ture of the mixture below 50° C. ^ Treat the remaining fluid (about 25 c.c.)
with 2 c.c. of hydrochloric acid (sp. gr. 1.124), transfer it carefully to a
200 c.c. flask, and evaporate the mixture to dryness on a water-bath.
Now add 20 grams of crystallized magnesium chloride and 2 c.c. of con-
centrated hydrochloric acid to the residue, and after fitting the flask with
a return cooler boil the mixture on a wire
gauze over a small flame for two hours.
Cool the solution, dilute to 750 c.c. or 1000
c.c. with water, render the mixture alkaline
with potassium hydroxide or sodium hy-
droxide, distil off the ammonia and collect
it in an acid solution of known strength.
Boil the distillate to remove carbon dioxide,
cool and titrate with an alkali of known
strength. In this method, as well as in
Folin's method (see p. 394), correction
must be made for the ammonia originally
present in the urine and in the magnesium
chloride.
5. Benedict's Method.^ — Five cubic
centimeters of urine are introduced into a
rather wide Jena glass test-tube, about 3
grams of potassium bisulphate and 1-2
grams of zinc sulphate^ added, a small
quantity of powdered pumice and a bit of
paraffin are introduced and the mixture
boiled almost to dryness either over a free
flame or by immersion in a sulphuric acid
bath at about 130°. The tubes are then
weighted (a screw clamp is convenient) and
immersed for three-fourths of their length in a bath of sulphuric acid
at a temperature of 162-165° i^^t lower) for one hour.
The contents of the tube are then washed into an 800 c.c. Kjeldahl
distillation flask, diluted to about 400 c.c. with water, made alkaline
by the addition of 15-20 c.c. of 10 per cent KOH (or 25 c.c. 15 per cent
Na2C03) and distilled as usual in the Kjeldahl method (page 401). The
value obtained must be corrected for ammonia.
' There is some decomposition of urea at 60° C.
^ Benedict: Jour. Biol. Chem., 8, 405, igii.
^ An excess of zinc salt is to be avoided as too large quantity tends to cause slight frothing
during the final distillation.
Fig.. 126. — Folin's Urea
Apparatus.
urine: quantitative analysis. 397
Wclker* has suggested an electrical bath for use in the first part of this
method.
6. Method of Folin and Pettibone, No. i.^ — By means of an Ostwald
pipette (see page 403) introduce i c.c. of urine into a Jena test-tube
(20-25 mm. by 200 mm.). Add three good-sized drops of pure phos-
phoric acid, one drop of indicator (alizarin) and a few grains of talcum
powder and concentrate the mixture to one-half its volume by boiling
over a free flamC for 2-3 minutes. At the end of this time heat the test-
tube in a bath of sulphuric acid, oil, or parafhn, for fifteen minutes at
a temperature of 175-180° C.^ By this means the urea is decomposed
with the formation of ammonium phosphate. Dissolve the contents of
the tube in water (1-2 c.c.) with the aid of heat, make alkahne with
potassium hydroxide* (0.5-1 c.c. of a 50 per cent solution) and remove
the liberated ammonia by means of a strong air current (see page 404).
This process requires approximately ten minutes. The ammonia may
be collected in 25 c.c. of Ar/50 hydrochloric acid and the excess acid
titrated with N/ioo sodium hydroxide using alizarin as indicator.
In calculating the urea value a correction must be made for the
ammonia content of the urine.
With the bath previously heated to the proper temperature the above
method may be completed in about one-half hour.
7. Method of Folin and Pettibone, No. 2.^— Dilute the urine so
that I c.c. contains 0.75-1.5 mg. of urea nitrogen. Generally dilutions
of 1:20 or 1:10, depending on the concentration, are satisfactory. By
means of an Ostwald pipette (see page 403) introduce i c.c. of the
diluted urine into a large dry Jena test-tube (20-25 "^"i. by 200 mm.)
which already contains 7 grams of dry ammania-free potassium acetate'
{free from lumps), i c.c. of 50 per cent acetic acid, a small sand pebble
or a little powdered zinc (not zinc dust) to prevent bumping during boiling,
and a temperature indicator.'^
' Wclker: Biochemical Bulletin, i, 439. 1912.
^ Folin and Pettibone: Jour. Biol. Chem., 11, 512, 1912.
^ The bath should be at this temperature when the tubes are introduced. Welker's elec-
tric bath may be used in this connection. (See Biochemical Bulletin, i, 439, 1912).
* Potassium hydroxide is preferred to sodium hydroxide because of the greater solubility
of potassium phosphate.
* Folin and Pettibone: Jour. Biol. Chem., 11, 513, 1912.
* A satisfactory preparation containing less than i per cent of moisture and free from
ammonia may be obtained from J. T. Baker Chemical Co., Phillipsburg, N. J.
^ " This temperature indicator consists of powdered chloride-iodide of mercury (HglCl)
inclosed in a sealed glass bulb not over i mm. in diameter. This salt is bright red at ordinary
temperatures. At 118° C. it turns yellow and melts to a clear dark red liquid at 155° C.
It solidifies again at about 148° C and resumes its red color gradually only in the course of
about twenty-four hours. The melting-point temperature, 153° C, is fortunately a tempera-
ture very readily obtained and maintained by means of potassium acetate and as the acetate
begins to cake and solidify at 160-161° C, there is no danger in this combination of having
either too high or too low a temperature without its being unmistakably apparent.
The HglCl may be prepared by heating, in a drj- state, intimately mixed mercuric chloride
398 PHYSIOLOGICAL CHEMISTRY.
Close the test-tube by means of a rubber stopper carrying an empty
narrow "calcium chloride tube "(i-5 cm. by 25 cm., without bulb) as a
condenser. Suspend the test-tube and condenser above a micro-burner
(see page 403) by means of a burette clamp or some similar device in such
a way that they may be easily raised or lowered. Heat gently, using a
bottomless beaker or some similar device as a wind shield if needed. The
acetate will soon dissolve (two minutes) and the mixture begin to boil.
At this point the indicator begins to melt showing that the desired tem-
perature (153-160° C.) has been reached. Continue the boiling in a
gentle, even manner for ten minutes at the end of which time the decom-
position of the urea is complete. Remove the apparatus from the flame
and dilute the contents with 5 c.c. of water. ^ Add an excess of alkali
(2 c.c. of a saturated solution of sodium hydroxide or potassium carbonate)
and remove the liberated ammonia by means of a strong air current (see
page 404). The ammonia may be caught in a 100 c.c. volumetric flask
which contains about 35 c.c. of ammonia-free water and 2 c.c. of N/10
acid. With a strong air current this process requires only about ten
minutes. Determine the ammonia colorimetrically against i mg. of
nitrogen in the form of ammonium sulphate. For the colorimetric
procedure see the total nitrogen determination, page 402.
8. Method of Folin and Denis. ^ — Sugar interferes with the decompo-
sition of urea. This was formerly believed to be due to the formation of
nitrogenous "melanins,"^ but is more probably due to the formation of
definite, stable ureids."* This difficulty may be overcome by proper dilu-
tion of the urine thus preventing the formation of the ureids. Because
of this great dilution the usual titration procedures are inappHcable, and
the following colorimetric procedure is suggested:
Dilute I c.c. of the urine with 20 to 100 volumes of ammonia-free
water and decompose i c.c. of this dilute urine with potassium acetate
and acetic acid as described under the method of Folin and Pettibone,
No. 2, on page 397.
By means of an air current remove the ammonia to a second test-tube
which contains about 2 c.c. of water and 0.5 c.c. of N/io hydrochloric
acid. Add to the contents of this tube about 2 c.c. of water and 3 c.c. of
the diluted (i : 5) Nessler- Winkler solution (page 404). Wash this colored
and mercuric iodide in molecular proportions at 150-160° C. for 6-8 hours. At the end of the
heating the product should be powdered and used as it is for it cannot be puriiied by the use
of solvents. It should be kept dry until sealed up as indicated." These temperature indi-
cators may be obtained ready prepared in tubes from Eimer & yVmend, New York.
* This water should be added by means of a [ji[)ette through the calcium chloride tube so as
to rinse the sides of the tube and the bottom of the rubber stof)per from any possible traces of
ammonium acetate. Not more than 5 c.c. of water should be used for this purpose.
^ Folin and Denis; Jour. Biol. Chem., 11, 520, igi2.
' Momer: Skand. Arch. Physiol., 14, 319.
* Folin: Am. Jour. Physiol., 13, 46, 1905.
urine: quantitative analysis. 399
solution into a lo c.c. volumetric flask and dilute it to the mark with
ammonia-free water. Transfer the entire volume to a dry cylinder of a
Duboscq colorimeter and determine the dej)th of color against a standard
containing i mg. of nitrogen per loo c.c. of solution. For the detailed
colorimetric procedure see the method for total nitrogen, page 402.
V. Ammonia.
I. Folin's Method. — Place 25 c.c. of urine in an aerometer cylinder,
30-40 cm. in height (Fig. 127, below), add about i 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
Fig. 127. — FoLixs .-ViiMoxiA Apparatus.
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 flask which is intended to absorb the ammonia and for this pur-
pose should contain 20 c.c. of N 10 sulphuric acid, 200 c.c. of ammonia-
free distilled water and a few drops of an indicator (alizarin red or congo
red). To insure the complete absorption of the ammonia the absorption
flask is provided with a Folin improved absorption tube (Fig. 128, p. 400)
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 simi-
lar absorption apparatus to the one just described is attached to the other
side of the aerometer cylinder, thus insuring the passage of ammonia-free
400
PHYSIOLOGICAL CHEMISTRY.
Fig. 1 2 8. — Folin
Improved Absorp-
tion Tube.
air into the cylinder. With an ordinary filter pump and good water pres-
sure the last trace of ammonia should be removed from the cylinder in
about one and one-half hours. ^ The number of cubic
centimeters of the N/io sulphuric acid neutralized by
the ammonia of the urine may be determined by di-
rect titration with N/io sodium hydroxide.
This is one of the most satisfactory methods yet
devised for the determination of ammonia. Steele^
has recently suggested a modification for use on
urines containing triple phosphate sediments. In this
modification .05-1.0 of NaOH and about 15 grams
of NaCl are substituted for the NagCOg of the Folin
method.
Calculation. — Subtract the number of cubic centi-
meters of N/io sodium hydroxide used in the titra-
tion from the number of cubic centimeters of N/io
sulphuric 9,cid taken. The remainder is the number
of cubic centimeters of N/io sulphuric acid neutralized
by the NH^ of the urine, i c.c. of N/io sulphuric
acid is equivalent to 0.0017 ^^«^^^ of NH^. Therefore
if y represents the volume of urine used in the deter-
mination and y' the number of cubic centimeters of N/io sulphuric acid
neutralized by the NH^ of the urine, we have the following proportion :
y : 100 : :y' X0.0017 : x (percentage of NH3 in the urine examined).
Calculate the quantity of NHg in the twenty-four-hour urine specimen.
2. Method of Folin and Macallum.'' — By means of Ostwald pipettes
(page 403) introduce 1-5 c.c. of urine"* into a Jena test-tube (20-25 mm,
by 200 mm.) and add to the urine a few drops of a solution containing
10 per cent of potassium carbonate and 15 per cent of potassium oxalate.
To prevent foaming add a few drops of kerosene or heavy, crude machine
oil. Pass a strong air current (see page 404) through the mixture until
the ammonia has been entirely removed.^ Collect the ammonia in a 100
c.c. volumetric flask containing about 20 c.c. of ammonia-free water and
2 c.c. of N/10 acid.
' With any given filter pump a "check" test should be made with urine or better with a
solution of an ammonium salt of known strength to determine how long the air current must
be maintained to remove all the ammonia from 25 c.c. of the solution.
2 Steele: Jour. Biol. Chem., 8, 365, 1910.
* Folin and Macallum: Jour. Biol. Chem. 11, 523, 1912,
* The volume of urine taken should contain 0.75-1.5 mg. of ammonia nitrogen. With
normal urines 2 c.c. will generally yield the desired amount. With very dilute urines 5 c.c.
may be required, while with diabetic urines rich in ammonium salts i c.c. may be excessive,
thus requiring dilution.
* Ordinarily a period of ten minutes is sufficiently long.
urine: quantitative analysis. 401
Nesslerize as described in the method for total nitrogen, page 402, and
compare with i mg. of nitrogen obtained from a standard ammonium
sulphate solution and similarly Nesslerized.
It has been noted that a trace of something capable of giving a color
with the Nessler- Winkler solution continues to come long after all the
ammonia has been removed from the urine. The nature of this substance
has not yet bopn determined. In actual determinations, by this method,
the influence of this unknown substance, because of the small volume
of urine used, is entirely negligible.
VI. Total Nitrogen.
I. Kjeldahl Method.^— 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 decomposition
of the ammonium sulphate by means of a fixed alkali (NaOH) and the col-
lection 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
computed.
The procedure is as follows: Place 5 c.c. of urine in a 500 c.c. long-
necked Jena glass Kjeldahl flask, add 20 c.c. of concentrated sulphuric
acid and about 0.2 gram of copper sulphate and boil the mixture for some
time after it is colorless (about one hour). Allow the flask to cool and
dilute the contents with about 200 c.c. of ammonia-free water. Add a
little more of a concentrated solution of NaOH than is necessary to neutral-
ize the sulphuric acid" 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 delivery-tube
passes into a vessel containing a known volume (the volume used depend-
ing upon the nitrogen content of the urine) of N/io sulphuric acid, using
care that the end of the delivery-tube reaches beneath the surface of the
fluid.* 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 N/ 10 sulphuric acid solution by
' 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.
- This concentrated sodium hydroxide solution should be prepared in quantity and "check"
tests made to determine the voluine of the solution necessary to neutralize the volume (20 c.c.)
of concentrated sulphuric acid used.
' Powdered zinc may be substituted.
* This deliver}-- tube should be of large caliber in order to avoid the "sucking back"
of the lluid.
26
402 PHYSIOLOGICAL CHEMISTRY.
means of N/io 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 N/io
sodium hydroxide used in the titration from the number of cubic centi-
meters of N/io sulphuric acid taken. The remainder is equivalent
to the number of cubic centimeters of N/io sulphuric acid, neutralized
by the ammonia of the urine. One c.c. of N/io sulphuric acid is equivalent
to 0.0014 gram of nitrogen. Therefore, if }' represents the volume of
urine used in the determination, and v' the number of cubic centimeters
of N/io sulphuric acid neutralized by the ammonia of the urine, we have
the following proportion :
Y : 100 ::y'Xo. 0014 : jc; (percentage of nitrogen in the urine examined).
Calculate the quantity of nitrogen in the twenty-four-hour urine
specimen.
Calculation of Percentage Nitrogen Distribution. — In modern metabol-
ism studies where the various forms of nitrogen are determined, in
addition to the total nitrogen as yielded by the Kjeldahl method, it is
customary to indicate what portion of the total nitrogen was present in
the form of each of the individual nitrogenous constituents. These
percentage values are secured by dividing the weight (grams) of nitrogen
excreted for the day in the form of each individual nitrogenous constituent
by the weight of the total nitrogen output for the same period. For
example, if the total nitrogen excretion is 9.814 grams and the excretion
of urea-nitrogen is 8.520 grams and the excretions of nitrogen in the forms
of ammonia and creatinine are 0.271 gram and 0.639 gram respectively,
the percentage distribution for these forms of nitrogen would be calculated
as follows:
8 . 520 grams urea-nitrogen -^ 9.814 grams total nitrogen = 84.3 percent
0.271 gram ammonia-nitrogen h- g. 8 14 grams total nitrogen = 2.7 percent
0.639 gram creatinine-nitrogen ^ 9,814 grams total nitrogen = 6.5 percent
2. Kjeldahl -Folin-Farmer Colorimetric method.^ — This method
may be considered as a microchemical method based on the Kjeldahl-
Gunning process for decomposing nitrogenous materials and on the
methods of Nessler and of Folin for the determination of ammonia
(see page 399). In the regular Kjeldahl procedure 30-100 mg. of
nitrogen is manipulated whereas in this modification only about i mg. is
utilized. The method is as follows:
Introduce 5 c.c. of urine into a 50 c.c. volumetric flask if the specific
gravity of the urine is over 1018, or into a 25 c.c. flask if the specific
gravity is less than 1018.' Fill the flask to the mark with distilled water
' Folin and P'armer: Jour. Biol. Chem., ir, 493, 1912.
^ The purpose is to so dilute the urine that i c.c. of the diluted lluid shall contain 0.75-1.5
mg. of nitrogen.
urink: quantitative analysis.
403
and invert il several times in order lo guarantee thorough mixing.
Transfer one cubic centimeter* of the diluted urine to a large (20-25
mm. X 200 mm.) Jena-glass test-tube. Add to this i c.c. of cencentrated
sulphuric acid, i gram of potassium sulphate, i drop of 5 per cent copper
sulphate solution and a small, clean, c|uartz pebble or glass bead. (The
pebble or bead is added to prevent bumping.) Hoil the mixture over a
micro-burner.' for about six minutes, i. e., about two minutes after the
M_y
UJ
U
Fig. 129. Fig. ijo.
Figs. 129 and 130. — Forms of Apparatus used in Methods of Folin and Associates for
Determinationof Total Nitrogen, Urea and Ammonia. {From Jour. Bio!. Client. ,\o\. ii, iqi2.)
mixture has become colorless. Allow to cool until the digestion mixture
begins to become viscous. This ordinarily takes about three minutes.
but in any event the mixture must not be permitted to solidify. Add about
6 c.c. of water (a few drops at a time, at first, then more rapidly) to pre-
vent solidification. To this acid solution add an excess of sodium
hydroxide (3 c.c. of a saturated solution is sufficient) and aspirate the
liberated ammonia by means of a rapid air current^ into a volumetric
' This measurement should be made by means of a modified Ostwald pipette (see Ostwald-
Luther: Physiko-Lliemische Messuiigen, 2d. ed., p. 135). Such pipettes may be obtained from
Eimer and Amend, New York.
- A type of burner which has proven satisfactory is Eimer and Amend's No. 2587.
^ Either a vacuum pump or compressed air or a force pump may be used. The com-
pressed air method is rather the more convenient inasmuch as the ammonia may be collected
directly in a volumetric tiask. Inasmuch as the necks of such tlasks (100 c.c.) are not large
enough to permit of the use of a two-hole rubber stopper when suction is used, the ammonia
should be collected in one of the Jena test-tubes previously described which contains 2 c.c. of
N/io hydrochloric acid and about 5 c.c. of ammonia-free water. The ammonium salt is then
transferred to the volumetric flask with 40-50 c.c. of water and Nesslerized as described.
404 PHYSIOLOGICAL CHEMISTRY,
flask (100 c.c.) containing about 20 c.c. of ammonia-free water and 2 c.c.
of N/io hydrochloric acid. (See Figs. 129 and 130, p. 403.) The air
current should be only moderately rapid for the first two minutes but
at the end of this two-minute period the current should be run at its
maximum speed for an interval of eight minutes.
Disconnect the flask, dilute the contents to about 60 c.c. with am-
monia-free water and dilute similarly i mg. of nitrogen in the form of
ammonium sulphate^ in a second volumetric flask. Nesslerize both
solutions as nearly as possible at the same time with 5 c.c. of Nessler-
Winkler solution^ diluted, immediately before using, with about 25 c.c.
of ammonia-free water to avoid turbidity. Immediately fill the two
flasks to the mark with ammonia-free water, mix well and determine the
relative intensity of the two colors by means of a Duboscq colorimeter.^
The color of the unknown should be adjusted to that of the standard
both from above and below the level of the latter. The matching of the
colors is ordinarily very easy. It is desirable to make the readings by
diffused daylight if possible. If electric light must be used, a sheet of
smooth white paper should be interposed between the colorimeter and the
source of light.
Calculation. — The reading of the standard divided by the reading of
the unknown gives the nitrogen in milligrams in the volume oj the urine
taken. Calculate the total nitrogen output for the twenty-four-period.
VII. Amino Nitrogen.
Van Slyke's Method.^ — The method is based on the fact that nitrous
acid in solution spontaneously decomposes with formation of nitric oxide :
2HNO,<riHN03 + NO.
' Care should be taken to secure the pure salt. All ammonium salts contain pyridine
bases which titrate like ammonia but do not react with Nessler's reagent. Pure ammonium
sulphate may be prepared by decomposing a high-grade ammonium salt with sodium hydroxide
and passing the liberated ammonia into pure sulphuric acid. The salt is then pret ipitated by
means of alcohol, then brought into solution in water and re-precipitated by alcohol. The
final product should be dried in a desi?cator over sulphuric acid. Dr. H. L. Emerson of Boston
prepares a salt which is ver)' satisfactory for use in this method.
^Chem. Zeit., 1899, p. 541. The Nessler- Winkler solution has the following formula:
Mercuric iodide 10 grams.
Potassium iodide 5 grams.
Sodium hydro.xide 20 grams.
Water 100 c.c.
The mercuric iodide is rubbed up in a small porcelain mortar with water, then washed into
a flask and the potassium iodide added. The sodium hydroxide is dissolved in the remaining
water and the cooled solution added to the above mixture. The solution cleared by standing
is preserved in a dark bottle.
The 25 c.c. portion of the diluted reagent should be added about one-third at a time to the
contents of the flask. It is very essential that the dilution of the reagent takes place immediately
preceding its use, inasmuch as the diluted reagent deteriorates in a few minutes as is indicated
by the formation of a brick-red precipitate. Fortunately the reagent does not decompose in
this manner in the presence of the ammonium salt.
' The standard may be set at any desired depth but a very satisfactory depth is 20 mm.
The depth should be uniform throughout any series of comparative tests.
* Van Slyke: Jour. Biol. Chem., 9, 185, 191 1.
urine: qu.\>s'titative analysis.
405
This reaction is utilized in displacing all the air of the apparatus with
nitric oxide. The amino solution is then introduced, evolution of nitrogen
mixed with nitric oxide resulting. The oxide is absorbed by alkaline
permanganate solution, and the pure nitrogen measured in a special gas
burette shown in the figure.
The determination of amino nitrogen is carried out as follows:
50 c.c. of the total day's urine are measured into a flask and 2 c.c. of concen-
trated acid added. The acid urine is then placed in an autoclave and
Fig. 131. — Van Slyke's .\iiiNO Nitrogen Appar.\tus.
heated to 175° C. under pressure for an hour and a half. After hydrol-
ysis is complete, a few drops of sodium alizarin sulphonate are added as
indicator and potassium hydroxide added in such quantity as to leave
an excess of the reagent. The solution is then boiled 20-30 minutes in
order to get rid of all the ammonia. After boiling, the solution is approxi-
mately neutralized and the volume made up exactly to 50 c.c. Ten cubic
centimeters of this is then used for each amino acid determination.
The 10 c.c. of urine treated as described are contained in the burette
(B) of the van Slyke apparatus (see Fig. 131). The detailed manipulation
of the urine in order to determine the amino acid content is very simple to
follow. Into the reaction chamber (D) one pours 28 c.c. of sodium ni-
40(l PHYSIOLOGICAL CHEMISTRY.
trite solution (30 grams to 100 c.c. of water) and 7 c.c. of glacial acetic
acid. The stopper is then inserted in (D), and the three-way stop-cock (c)
opened so as to allow the gases to escape into the air. 5 c.c. of water are
now placed in vessel (A) and allowed to run into (D) so as to expell the air
remaining in the apparatus. The cock (c) is closed, (a) left open and the
solution from (D) allowed to back up into (A) until about 5 c.c. are
accumulated there, (D) being shaken so as to get rid of any air dissolved
in the interacting solutions. The gases are again expelled as described
bv opening (c). This process is repeated thus washing the last traces of
air from the apparatus, (a) is now opened, (c) closed and about 25 c.c.
of the solution forced into (A) by the pressure of the nitric oxide formed in
(D). The cock (c) is then opened so that the gas passes into the burette
(F), (a) closed, and the 10 c.c. of urine run into (D). After the reaction
has run for five minutes, (D) is shaken and the remainder of the gas
forced from (D) into the burette by allowing the liquid in (A) to run into
(D). The gases are then run from the burette into the pipette (H),
the latter is thoroughly shaken, till no more gas is absorbed by the alkaline
permanganate solution.^ The pure nitrogen gas is run back into the
burette and measured. The temperature of the gas and barometric
pressure are recorded. Blanks should be run and the slight error due to
the formation of nitrogen gas and oxygen from the interaction of sodium
nitrite and glacial acetic acid accounted for."
Calculation. — As the reaction doubles the amount of nitrogen present
as amino nitrogen, the volume of nitrogen found must be divided by 2.
The results are expressed in milligrams of nitrogen.
VIII. Hippuric Acid.
Dakin's Methods."^ Preliminary Procedure. — ^Place 150 c.c. (or
more; of the urine under examination in a porcelain evaporating dish
and evaporate almost to dryness upon a water-bath. Add about i gram
of sodium dihydrogen phosphate, about 25 grams of gypsum (CaSO^,
2H,Oj and rub up with a pestle and stir with a spatula until a uniform
mixture results. Dry the powder thus produced in a water-oven for
about two hours, at the end of which period it should be rubbed up a
second time, to remove lumps, and transferred to a Schleicher and Schiill
"extraction shell" and extracted in a Soxhlet apparatus in the usual way
' The alkaline permanKanale solution contains 50 grams of potassium permanganate and
25 grams of jxjtassium hyflroxide per liter.
^According to Robinson (Mich. Ag. Exp. Station Tech. Bull., 7, p. 11, 1911), analysis
of the gases formed by the decomposition of sodium nitrite with glacial acetic acid indicates
the presence of small amounts of free oxygen and nitrogen.
'Private communifation to the author from Dr. H. D. Dakin.
urine: quantitative analysis. 407
(see p. 437). The extraction medium is ethyl acetate and the flask con-
taining the acetate should be strongly heated over a sand-bath^ for about
two hours. The ethyl acetate extract is now transferred to a separatory
funnel, and the original flask rinsed with sufficient fresh ethyl acetate
to make the total volume in the separatory funnel'-' about 100 c.c. Wash
the ethyl acetate solution //ir times with a saturated solution of sodium
chloride, usijig 8 c.c. of the sodium chloride solution at each extraction,
shaking vigorously and removing the sodium chloride extract in each case
before adding fresh sodium chloride solution. The sodium chloride
removes the urea completely and the hippuric acid is then determined in the
urea-free solution by the following volumetric or gravimetric procedure:
1. Volumetric Determination. — Transfer the urea-free ethyl acetate
solution, prepared as described above, to a Kjeldahl flask, add about
25 c.c. of water, a small piece of pumice stone to prevent bumping,
attach a condenser and distil off the acetate'' over a free flame. After
practically all of the ethyl acetate has been distilled off, the nitrogen in the
remaining solution should be determined by means of the Kjeldahl
method (see p. 402).
The main source of error in this method is the fact that any nitrogen
present in the form of phenaceturic acid or indole acetic acid is determined
as hippuric acid nitrogen. The error from this source is, however, usually
trifling.
Calculatim. — Calculate as usual for nitrogen determinations, re-
membering that I c.c. oj N/io sulphuric acid is equivalent to 0.0 17Q gram
hippuric acid.
2. Gravimetric Determination. — The urea-free ethyl acetate solu-
tion, contained in the separatory funnel, after washing with sodium
chloride solution, as described under Preliminary Procedure, p. 406,
is washed with 5 c.c. of distilled water to remove the major portion of
the sodium chloride. Transfer the solution from the separatory funnel
to a round-bottomed flask and subject it to a steam distillation in the
usual way. A slow current of steam should be used while the ethyl
acetate is being distilled off and later a more rapid current may be em-
ployed. The distillation should be continued for twenty minutes. Now
add about o.i gram of charcoal to the aqueous solution which is heated
to boiling and filtered hot. Evaporate the solution in a weighed Jena
glass dish on a water-bath until the volume of the solution is reduced to
about 3 c.c. Stand the dish in a warm place until evaporation is complete
' A water-bath cannot be substituted inasmuch as the resuUant extraction would be too
slow.
- This ethyl acetate solution contains hippuric acid, urea, and other substances.
^ The ethyl acetate after separation from the watef}' layer of the distillate may be dried
over calcium chloride and used agian.
4o8 PHYSIOLOGICAL CHEMISTRY.
and a crystalline residue remains. Wash the residue, in turn, with 2
c.c. of dry ether, and i c.c. of water, dry it in an air-bath at 100° C. and
weigh. If it is so desired the residue may be recrystallized from a little
hot water and the melting-point determined. Pure hippuric acid melts at
187° C. Contamination with phenaceturic acid may be detected both by
the melting-point and the microscopical characteristics.
IX. Sulphur.
I. Total Sulphates. Faun's Method. — ^Place 25 c.c. of urine in a
200-250 c.c. Erlenmeyer flask, add 20 c.c. of dilute hydrochloric acid^
(i volume of concentrated HCl to 4 volumes of water) and gently boil the
mixture for 20-30 minutes. To niinimize 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 flask for 2-3 minutes in running water,
and dilute the contents to about 150 c.c. by means of cold water. Add
10 c.c. of a 5 per cent solution of bariuni chloride slowly, drop by drop, to
the cold solution.^ The contents of the flask should not he 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.^
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,* cool and weigh.
Calculation. — Subtract the weight of the Gooch crucible from the
weight of the crucible and the BaSO^ precipitate to obtain the weight of
the precipitate. The weight of Sog^ in the volume of urine taken may
be determined by means of the following proportion.
Mol. wt. Wt. of Mol. wt.
BaSO^ : BaSO^ : : SO3 -.x (wt. of SO3 in grams).
* 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 ordinar)- calcium chloride tube and so
constructed as to deliver 10 c.c. in 2-3 minutes is recommended for use in adding the barium
chloride.
^ If a Gooch crucible is not available, the precipitate of BaS04 may be filtered off upon
a washed filter paper (Schleicher & Schiill's, No. 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 (lame. The ignition may then be carried out in the usual way in the
ordinar}' 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,
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 im-
provised bottom. Ignition should be complete in 10 minutes if no organic matter is present.
* It is considered preferable by many investigators to express all sulphur values in terms
of S rather than SO,.
urine: quantitative analysis. 409
Representing the weight of the BaSO^ precipitate by y and substituting
the proper molecular weights, we have the following proportion :
231.7:^: :79.5 r.Y (wt. of SO3 in grams in the quantity of urine used).
Calculate the quantity of SO3 in the twenty-four-hour specimen of
urine.
To express the result in percentage of SO3 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 hydrochloric acid (i volume of con-
centrated HCl 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 pro-
portionately. Add 10 c.c. of 5 per cent barium chloride slowly, drop by
drop, to the cold solution and from this point proceed as indicated in the
method for the determination of Total Sulphates, page 408.
Calculate the quantity of inorganic sulphates, expressed as SO3, in
the twenty-four-hour urine specimen.
Calculation. — Calculate according to the directions given under Total
Sulphates, above.
3. Ethereal Sulphates. Folin's Method. — Place 125 c.c. of urine in
an Erlenmeyer flask of suitable size, dilute it with 75 c.c. of water and
acidify the mixture with 30 c.c. of dilute hydrochloric acid (i volume of
concentrated HCl to 4 volumes of water). To the coW solution add 20 c.c.
of a 5 per cent solution of barium chloride, drop by drop.^ Allow the
mixture to stand about one hour, then filter it through a dry filter paper.^
Collect 125 c.c. of the filtrate and boil it gently for at least one-half hour.
Cool the solution, filter off the precipitate of BaSO^, wash, dry and ignite
it according to the directions given on page 408.
Calcidation. — The weight of the BaSO^ precipitate should be multi-
plied by 2 since only one-half (125 c.c.) of the total volume (250 c.c.) of
fluid was precipitated by the barium chloride. The remaining calculation
should be made according to directions given under Total Sulphates, page
408.
Calculate the quantity of ethereal sulphates, expressed as SO 3, in the
twenty-four-hour urine specimen.
4. Total Sulphur. Benedict's Method.^— Ten cubic centimeters of
urine are measured into a small (7-8 cm.) porcelain evaporating dish and
' See note (2) at the bottom of page 408.
* This precipitate consists of the inorganic sulphates. If it is desired, this BaSO^ pre-
cipitate may be collected in a Gooch crucible or on an ordinary quantitative filter paper and a
determination of inorganic sulphates made, using the same technic as that suggested
above. In this way we are enabled to determine the inorganic and ethereal sulphates
in the same sample of urine.
' Benedict: Journal of Biological Chemistry, 6, 363, 1909.
4IO PHYSIOLOGICAL CHEMISTRY.
5 c.c.^ of Benedict's sulphur reagent" added. The contents of the dish
are evaporated over a free flame which is regulated to keep the solution
just below the boiling-point, so that there can be no loss through spattering.
When dryness is reached the flame is raised slightly until the entire
residue has blackened. The flame is then turned up in two stages to the
full heat of the bunsen burner and the contents of the dish thus heated to
redness for ten minutes after the black residue {which first fuses) has- become
dry. This heating is to decompose the last traces of nitrate (and chlorate) .
The flame is then removed and the dish allowed to cool more or less
completely. Ten to twenty cubic centimeters of dilute (1:4) hydrochloric
acid is then added to the residue in the dish, which is then warmed gently
until the contents have completely dissolved and a perfectly clear, spark-
ling solution is obtained. This dissolving of the residue requires scarcely
two minutes. With the aid of a stirring rod the solution is washed into^
a small Erlenmeyer flask, diluted with cold, distilled water to 100-150 c.c,
10 c.c. of 10 per cent barium chloride solution added drop by drop, and
the solution allowed to stand for about an hour. It is then shaken up
and filtered as usual through a weighed Gooch crucible.
Calculation. — Make the calculation according to directions given under
Total Sulphates, p. 408. Calculate the quantity of sulphur, expressed
as SO3 or S, present in the twenty-four-hour urine specimen.
5. Total Sulphur. Osborne-Folin Method. — ^Place 25 c.c. of urine*
in a 200-250 c.c. nickel crucible and add about 3 grams of sodium perox-
ide. Evaporate the mixture to a syrup upon a steam water-bath and
heat it carefully over an alcohol flame untfl it solidifies (15 minutes).
Now remove the crucible from the flame and allow it to cool. Moisten
the residue with 1-2 c.c. of water,^ sprinkle about 7-8 grams of sodium
peroxide over the contents of the crucible and fuse the mass over an
alcohol flame for about 10 minutes. Allow the crucible to cool for a few
minutes, add about 100 c.c. of water to the contents and heat at least
one-half hour over an alcohol flame to dissolve the alkali and decompose
the sodium peroxide. Next rinse the mixture into a 400-450 c.c. Erlen-
meyer flask, by means of hot water, and dilute it to about 250 c.c. Heat
the solution nearly to the boiling-point and add concentrated hydrochloric
acid slowly until the nickelic oxide, derived from the crucible, is just
' If llif urine is concentratefi the ([uanlity shouM be slightly increased.
- Crystallizetl copper nitrate, sulphur-free or of known sul])hur content... . 200 grams.
Soflium or potassium chlorate 50 grams.
Distilled water to 1000 C.C.
■'* .Sometimes the ]>orcelain glaze cracks during heating, in which case the solution should
be filtered into the flask.
■* If the urine is very dilute 50 c.c. may be used.
^ This moistening of the residue with a small amount of water is very essential and should
not be neglected.
URINK: (PUANTITATIVE ANALYSIS.
411
brcnight into solution.' A few minutes boiling should now yield a clear
solution. In case too little peroxide or too much water was added for the
final fusion a clear solution will not be obtained. In this event cool the
solution and remove the insoluble matter bv filtration.
Fig. 132. — Bertheldt-Atwatek Ishmb c aiurimeter. K r<)ss-secti(in ok Appar-atcs as
Ready for Use.)
.\, Steel cup or bomb proper; C, collar of steel; G, opening through which o.wgen is forcefi
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.
To the clear solution add 5 c.c. of very dilute alcohol (about 18-20
per cent) and continue the boiling for a few minutes. The alcohol is
added to remove the chlorine which was formed when the solution was
• .\bout 18 c.c. of acid is rec|uired for 8 grams of sodium {)eroxide.
412 PHYSIOLOGICAL CHEMISTRY.
acidified. Add lo c.c. of a lo per cent solution of barium chloride, slowly,
drop by drop,^ to the liquid. Allow the precipitated solution to stand in
the cold two days and then filter and continue the manipulation according
to the directions given under Total Sulphates, page 408.
Calculation. — Make the calculation according to directions given
under Total Sulphates, p. 408. Calculate the quantity of sulphur,
expressed as SO3 or S, present in the twenty-four-hour urine specimen.
6. Total Sulphur. — Sodium Hydroxide and Potassium Nitrate Fusion
Method. — ^Place 25 c.c. of urine in a silver crucible and evaporate to a
thick syrup on a water-bath. Add 10 grams of sodium hydroxide and 2
grams of potassium nitrate to the residue and fuse the mass, over an
alcohol flame, 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 slightly with hydrochloric acid and evaporate it to dry-
ness on a water-bath. Moisten the residue with a few drops of dilute
hydrochloric acid and bring it into solution with hot water. Filter, heat
the filtrate to boiling, and immediately precipitate it by the addition of 10
c.c. of a 10 per cent solution of barium chloride, adding the solution
slowly, drop by drop. Allow the precipitated solution to stand 2 hours
and filter while cold. Ignite, weigh, and calculate according to directions
given under Total Sulphates, p. 408.
Compute the quantity of sulphur, expressed as SO3 or S, present in
the twenty-four-hour urine specimen.
7. Total Sulphur. Sherman's Compressed Oxygen Method."^ —
Evaporate as much urine on an absorbent filter block^ at 55° C. as the
block will conveniently absorb and burn the block so prepared in a bomb-
calorimeter^ using 25-30 atmospheres of oxygen. Connect the bomb
with a wash-bottle containing water, and allow the gas to bubble through
the liquid until the high pressure within the apparatus has been reduced
to atmospheric pressure. Now open the bomb and thoroughly rinse the
interior, using water from the wash-bottle for the first rinsing. Dissolve
any ash found in the combustion capsule in hydrochloric acid and add
this solution to the main solution. Evaporate to 150 c.c, filter, and cool
the filtrate. Add 10 c.c. of a 5 per cent solution of barium chloride to the
cold filtrate, slowly, drop by drop.^ 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
' See note (2) at the bottom of page 408.
^ See Sherman's Organic Analysis, First edition, p. 19.
^ Only a small amount of urine should be added at one time, it being necessary to make
several evaporations before the block contains sufficient urinary residue to proceed with the
combustion.
* The Berthelot-Atwater apparatus (Fig. 132. page 411) is well adapted to this purpose.
* See note (2) at the bottom of page 408.
urine: quantitative analysis. 413
filter il throuj^h a weighed Gooch crucible. Manipulate the precipitate
of BaSO^ according to directions given under Total Sulphates, page 408.
Calculate the quantity of sulphur, expressed as SO3 or S, present in the
twenty-four-hour urine specimen.
X. Phosphorus.
1. 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' 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" until a precipitate ceases to form and a drop of the mix-
ture when removed by means of a glass rod and brought in contact with a
drop of a solution of potassium ferrocyanide on a porcelain test-tablet pro-
duces instantaneously a brownish-red coloration.^ Take the burette
reading and calculate the PjOg content of the urine under examination.
Cakulation. — Multiply the number of cubic centimeters of uranium
acetate solution used by 0.005 to determine the number of grams of PjOg
in the 50 c.c. of urine used. To express the result in percentage of P2O5
multiply the value just obtained by 2, 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 PjOj, the total phosphate content of the twenty-
four-hour urine specimen.
2. Earthy Phosphates. — To 100 c.c. of urine in a beaker 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 magnesium, is precipitated as phosphates
of these metals. Collect 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
' The sodium acetate solution is prepared bv dissolving loo grams of sodium acetate
in Soo c.c. of distilled water, adding loo c.c. of 30 per cent acetic acid to the solution, and
making the volume of the mixture up to r liter with water.
- This uranium acetate solution may be prepared by dissolving about 34. grams of uranium
acetate in one liter of water. One c.c. of this solution should now be made equivalent to
0.005 gram of PjO,, phosphoric anhydride. It 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 o.i gram of PoO^, 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 i c.c. of the uranium solution should precipitate 0.005 gr^i^"' '^^ P20,-„ exactly
20 c.c. of the uranium solution should be required to precipitate 50 c.c. of the standard phos-
phate solution. If the two solutions do not bear this raeltion to each other they may be
brought into pro[)cr relation by diluting the uranium solution with distilled water or by in-
creasing its strength.
' .\ lo per cent solution of potassium ferrocyanide is satisfactory.
414 PHYSIOLOGICAL CHEMISTRY.
solution, and determine the P20g 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 number of grams of P2O5
in the 100 c.c. of urine used. Since 100 c.c. of urine was taken this value
also expresses the percentage of P2O5 present.
Calculate the quantity of earthy phosphates, in terms of P2O5, 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.
Total Phosphorus. — Sodium Hydroxide and Potassium Nitrate
Fusion Method.— Vlsice 25 c.c. of urine in a large silver crucible and evapo-
rate to a syrup on a water-bath. Add 10 grams of NaOH and 2 grams of
KNO3 to the residue and fuse the mass until all organic matter has dis-
appeared 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, dissolve it in hot water, and
transfer to a beaker. Now add an equal volume of molybdic solution^ and
keep the mixture at 40° C. for twenty-four hours. Filter off the precipi-
tate, wash it with dilute molybdic solution, and dissolve it in dilute
ammonia. Add dilute hydrochloric acid to the solution, being careful to
leave the solution distinctly ammoniacal. Magnesia mixture^ (lo-i 5 c.c.)
should now be added and after stirring thoroughly and making strongly
ammoniacal with concentrated ammonia the solution should be 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, inciner-
ate, and weigh, as magnesium pyrophosphate, Mg2P207, in the usual
manner.
In this method the phosphoric acid of the urine is precipitated as
ammonium magnesium phosphate and in the process of incineration this
body is transformed into magnesium pyrophosphate.
Calculation. — The quantity of phosphorus, expressed in terms of
PjOj, in the volume of urine taken may be determined by means of the
following proportion :
.Mol. wt. Wt. of Mol. wt.
Mg,P,0,:Mg,P,0,::PA:^ (wt. of PA in grams).
ppt.
' Directions for the preparation of tlie solution are given on p. 64.
^ Directions for the preparation of magnesia mixture may be found on p. 313.
urine: quantitative analysis. 415
If V represents the weight of the Mg^PjO^ precipitate and we make
the proper substitution we have the following proportion :
221.1 : V : :i40.9 :.v (wt. of P^Oj. in grams, in the ciuantity of urine used.)
To express the result in percentage of FjOj simply divide the value of
-v. as just determined, by the quantity of urine used.
XI. Creatinine.
Folin's Colorimetric Method.— This method is based upon the
characteristic property possessed alone by creatinine, of yielding a
certain definite color-reaction in the presence of picric acid in alkaline
solution. The procedure is as follows: 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 hydro.xidc. shake thoroughly
and allow the mixture to stand for 5 minutes. During this interval
pour a little N/2 potassium bichromate solution^ 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 accuracy 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 reading, which is apt to be less accurate than the succeeding
readings. In time as one becomes proficient in the technic it is
perfectly safe to take the average of \.\v€: first two readings.
At the end of the 5-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, and replaced
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 creatinine is above 15 mg. or below 5 mg. the determi-
nation should be repeated with a volume of urine selected according to
the content or creatinine. This vaiiation in the volume of urine according
to the content of creatinine is quite essential, since the method loses in
accuracy when more than 15 mg. or less than 5 mg. of creatinine is
present in the solution of unknown strength.
Calculation. — By experiment it has been determined that 10 mg.
' This solution contains 24.55 grams of potassium bichromate to the liter.
4l6 PHYSIOLOGICAL CHEMISTRY.
of pure creatinine, when brought into solution and diluted to 500 c.c.
as explained in the above method, yields a mixture 8.1 mm, of which
possesses the same colorimetric value as 8 mm. of a N/2 solution of
potassium bichromate. Bearing this in mind the computation is readily
made by means of the following proportion in which y represents the
number of millimeters of the solution of unknown strength equivalent to
the 8 mm. of the potassium bichromate solution:
}' : 8.1 : : 10 : x (mgs. of creatinine 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 determination. The
10 represents 10 mg. of creatinine which gives a color equal to 8.1 mm.,
whether dissolved in 5, 10, or 15 c.c. of fluid.
Calculate the quantity of creatinine in the twenty-four-hour urine
specimen.
XII. Creatine.
Folin-Benedict and Myers Method.^ — To 20 c.c. of urine in a
50 c.c. volumetric flask, add 20 c.c. of normal hydrochloric acid and
place the flask in an autoclave at a temperature of 117-120° C. for
one-half hour. Add distilled water until the volume of the acid-urine
mixture is exacdy 50 c.c, close the flask by means of a stopper, and
shake it thoroughly. Approximately neutralize 25 c.c. of this mixture,
introduce it into a 500 c.c. volumetric flask and determine its creatinine
content according to Folin's Method (see p. 415).
Calculation. — Calculate as explained on p. 415, and from this value
subtract the value for the original content of creatinine before hydrolysis.
The difference between these two values will be the creatine content of the
original urine in terms of creatinine.
' . XIII. Indican.
Ellinger's Method. — This method for the quantitative determin-
ation of indican is based upon the principle underlying Jaffe's test for
the detection of indican (see p. 298). The method is as follows: To
50 c.c. of urine ^ in a small beaker or casserole add 5 c.c. of basic lead
acetate solution,^ mix thoroughly, and filter. Transfer 40 c.c. of the
filtrate to a separatory funnel, add an equal volume of Obermayer's
reagent (see p. 299) and 20 c.c. of chloroform, and extract in the usual
* Benedict and Myers: Am. J . Phys., 18, 397, 1907.
* If the urine under examination is neutral or alkaline in reaction it should be made faintly
acid with acetic acid before adding the basic lead acetate.
^ For preparation of basic lead acetate solution see Appendix.
urine: quantitative analysis. 417
manner. This cxtnulion with chloroform shcnild he repeated until
the chloroform solution remains colorless. Shake up the combined
chloroform extracts 2-3 times with distilled water in a separating funnel
and complete the purification by extracting with very dilute sodium
hydroxide (1:1000). Remove all traces of alkali by washing with water.
Now filter the combined chloroform extracts through a dry filter pajjcr
into a drv Erlenmever flask. Distil off the chloroform, heat the residue
on a boiling water-bath for 5 minutes in the open flask, and wash the dried
residue with hot water\ Add 10 c.c. of concentrated sulphuric acid to
the washed residue, heat on the water-bath for 5-10 minutes, dilute with
TOO c.c. of water, and titrate the blue solution with a very dilute solution of
potassium permanganate. ■ The end-point is indicated by the dissipation
of all the blue color from the solution and the formation of a pale yellow
color.
Beautiful plates of indigo blue sometimes appear in the chloroform
extract of urines containing abundant indican. In urines preserved
by thymol the determination of indican is interfered with unless great
care is taken in washing the chloroform extract with dilute alkali. Care
should be taken, therefore, to make the indican determination upon
fresh urine, before the addition of the preservative.
Plasencia* has recently suggested a method which is shorter than
EUinger's and according to its sponsor, just as accurate.
Calculation. — Ellinger claims that one-sixth of the amount deter-
mined must be added to the value obtained by titration in order to secure
accurate data. This correction should always be made.
XIV. Chlorides.
Dehn-Clark Method.^— In this method the organic compounds,
that hold the chlorine too firmly for its quantitative precipitation with
silver nitrate, are destroyed by oxidation with sodium peroxide. Sodium
peroxide in the presence of water gives off nascent oxygen according to the
following equation: *
Na^O. + H,0— 2NaOH -^ O.
The oxygen then attacks the organic matter and the chlorine is left
as sodium chloride. The procedure is as follows: To 10 c.c. of urine
' The washing should be continued until the wash water is no longer colored. Ordi-
narily two or three washings are sufficient. If a separation of indigo particles takes place
during the washing process, the wash water should be filtered, the indigo extracted with chloro-
form, and the usual method applied from this point.
-A-" stock solution" of potassium permanganate containing 3 grams per liter should be
prepared, and when needed for titration purposes a suitable volume of this solution should
be diluted with 40 volumes of water. The potassium permanganate solution should be
standardized with pure indigo.
' Plasencia: Revista de Medicina y Cirugia., 17, i, 191 2.
'Private communication to the author from Mr. S. C. Clark.
27
4l8 PHYSIOLOGICAL CHEMISTRY.
in a 75-100 c.c. casserole, add i. 0-1.2 gram of sodium peroxide and
evaporate the mixture to dryness on a boiling water-bath. In case the
residue is not pure white, thus indicating that insufficient sodium peroxide
has been added, the residue should be moistened with distilled water,
additional sodium peroxide added, and the mixture again evaporated to
dryness. When the oxidation is complete, treat the mass with 10-20 c.c.
of distilled water and stir until it has practically all been brought into
solution. Then introduce a bit of litmus paper and add dilute nitric acid
(1:1) until the litmus paper turns red and all effervescence ceases. Now
place the casserole on a hot plate or on a gauze and heat the contents
almost to the boiling-point.^ To the hot solution add a standard solution
of silver nitrate (see page 419) in slight excess.^ Filter off the silver
chloride while the solution is still hot and wash the precipitate thoroughly
with distilled water. To the filtrate, add i c.c. of a saturated solution of
ferric ammonium sulphate and then titrate with a standard solution of
ammonium thiocyanate (see page 420) until the clear, slightly yellow
fluid (or the opalescent, milky fluid, in case there is much excess of silver
nitrate) changes to a slight reddish-brown color. The color of the end-
point varies with the individual. The exact end-point reached is not so
important as is the securing of the same end-point in a series of deter-
minations as that obtained in the standardization of the standard solutions
used.
Calculation.- — The standard solution of silver nitrate should be
made up so that i c.c. equals o.oio gram of sodium chloride and i c.c.
of the ammonium thiocyanate should be equivalent to i c.c. of the silver
nitrate solution (see p. 419). Then, if the number of cubic centimeters
of ammonium thiocyanate used be subtracted from the number of cubic
centimeters of silver nitrate, the difference is the number of cubic centi-
meters of silver nitrate actually used in the precipitation of chlorine as
silver chloride. This number, multiplied by 0.0 10, gives the weight in
grams of the sodium chloride in the 10 c.c. of urine used. If it is desired to
express the result in percentage of sodium chloride, move the decimal
point one place to the right.
In a similar manner the weight or percentage of chlorine may be com-
puted, using the factor 0.006 as explained in Mohr's method, below.
Calculate the quantity of sodium chloride and of chlorine in the twenty-
four-hour urine specimen.
2. Mohr's Method. — To 10 c.c. of urine in a small platinum or porce-
' If there is a slight precipitate, due to silicic acid from the casserole, this is filtered off
and the filtrate collected in a 200 c.c. beaker.
^ This point is most easily recognized by keeping the solution hot and in constant agitation
while adding the silver nitrate so that the silver chlori le formed coagulates and sinks, leaving
a clear, supernatant fluid.
urine: quantitative analysis. 419
lain crucible or dish add about 2 grams of chlorine-free potassium nitrate
and evaporate to dryness at 100° C. (The evaporation may be con-
ducted over a low tlame provided care is taken to prevent loss by sj)urting.)
By means of crucible tongs hold the crucible or dish over a free flame until
all carbonaceous 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 acidified 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, add 2-5 drops of neutral potassium chromate
solution to the mixture, and titrate with a standard silver nitrate solution.^
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 chlor-
ine and sodium chloride in the urine examined.
Calculation. — Since i c.c. of the standard silver nitrate solution is
equivalent to 0.0 10 gram of sodium chloride, to obtain iheweight, 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.
3. 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. i . 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 a known volume of the
standard silver nitrate' solution (20 c.c. is ordinarily used) in order to
precipitate the chlorine and insure the presence of a.n excess oi silver nitrate.
The mixture should be continually shaken during the addition of the
' The cessation of eflervescence and the presence of some undecomposed calcium car-
bonate at the bottom of the vessel are the indications of neutralization.
' Standard silver nitrate solution may be prepared by dissolving 29.042 grams of silver
nitrate in i liter of distilled water. Each cubic centimeter of this solution is equivalent to
O.OIO gram of sodium chloride or to 0.006 gram of chlorine.
420 PHYSIOLOGICAL CHEMISTRY.
standard solution. Allow the flask to stand lo minutes, then fill it to the
loo ex. graduation with distilled water and tlioroughly mix the contents.
Now filter the mixture through a dry filter paper, collect 50 c.c. of the
filtrate and titrate it with standardized ammonium thiocyanate solution.^
The first permanent tinge of red-brown indicates the end-point. Take
the burette reading and compute the weight of sodium chloride in the
10 c.c. of urine used.
Calculation. — The number of cubic centimeters of ammonium thio-
cyanate solution used indicates the excess of standard silver nitrate
solution in the 50 c.c. of filtrate titrated. Multiply this reading by 2, in-
asmuch as only one-half of the filtrate was employed, and subtract this
product from the number of cubic centimeters of silver nitrate (20 c.c.)
originally used, in order to obtain the actual number of cubic centimeters
of silver nitrate utilized in the precipitation 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
silver nitrate solution, actually utilized in the precipitation, 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.
In a similar manner the weight, or percentage of chlorine may be com-
puted using the factor 0.006 as explained in Mohr's method, page 418.
Calculate the quantity of sodium chloride and. chlorine in the twenty-
four-hour urine specimen.
4, Volhard-Harvey Method.- — Introduce 5 c.c. of urine into a
small porcelain evaporating dish or casserole and dilute with about 20 c.c.
of distilled water. Precipitate the chlorides by the addition of 10 c.c. of
standard silver nitrate solution^ and add 2 c.c. of acidified indicator.*
Now run in a standard ammonium thiocyanate solution^ from a burette
* This solution is made of such a strength that i c.c. of it is equal to i c.c. of the standard
silver nitrate solution used. To prepare the solution dissolve 13 grams of ammonium
thiocyanate, NH^SCN, in a little less than a liter of water. In a small flask place 20 c.c.
of the standard silver 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 ammonium thiocyanate solution from a burette until a per-
manent red-brown tinge is produced. This is the end-reaction and indicates that the last
trace of silver nitrate has been precipitated. Take the burette reading and calculate the
amount of water necessary to use in diluting the ammonium thiocyanate in order that 10 c.c.
of this solution may be exactly equal to 10 c.c. of the silver nitrate solution. Make this dilution
and titrate again to be certain that the solution is of the proper strength.
^ Harvey: Archives of Internal Medicine, 6, 12, 1910,
^ See p. 419.
* This is prepared as follows: To 30 c.c. of distilled water add 70 c.c. of 33 per cent
nitric acid (sp. gr. 1.2) and dissolve 100 grams of crystalline ferric ammonium sulphate in this
dilute acid solution. Filter and use the filtrate which is a saturated solution of the iron salt.
This single reagent takes the place of the nitric acid and ferric alum as used in Volhard-
Arnold method, and insures the use of the proper quantity of acid.
* This is a solution of ammonium thiocyanate of such a strength that 2 c.c. is equivalent
to I c.c. of the silver nitrate solution. First make a concentrated solution by dissolving 13
urine: quantitative analysis. 421
until a faint red-brown tint is visible throughout the mixture. This
point may be determined readily by permitting the precipitate to settle
somewhat. Calculate the sodium chloride value so indicated below.
(If a red tint is produced when the first drop of thiocyanate is added
an additional to c.c. of the standard silver nitrate solution must be in-
troduced. The titration should then proceed as above described and
proper allo^'ance made in the calculation for the extra volume of silver
nitrate employed.)
Calculation. — Since 2 c.c. of the ammonium thiocyanate solution is
equivalent to i c.c. of the silver nitrate solution, divide the burette reading
by 2 and subtract the quotient from 10 c.c, the quantity of silver nitrate
solution taken. This value is the number of cubic centimeters of silver
nitrate solution actually used in the precipitation of the chlorides. As
1 c.c. of the silver nitrate solution is equivalent to o.oi gram of sodium
chloride, the number of cubic centimeters of silver nitrate solution used
multiplied by o.oi gram will give the weight of sodium chloride in the 5
c.c. portion of urine used. The weight of chlorine may be computed by
using the factor 0.006 as explained under Mohr's method, page 418.
Calculate the weight of sodium chloride and chlorine in the twenty-
four-hour urine specimen.
A "short cut" method of calculating the twenty-four-hour output of
sodium chloride consists in subtracting the burette reading from 20 c.c,
multiplying this value by the total urine volume and pointing off three
places.
XV. Acetone and Diacetic Acid.
I. Folin-Hart Method. ^This method serves the same purpose as
the Messinger-Huppert Method, i. e., the determination of both acetone
and diacetic acid in terms of acetone. It is, however, much simpler and
less time-consuming. The method includes the transformation of the
diacetic acid into acetone and carbon dioxide by means of heat and the
subsequent removal of the acetone thus formed, as well as the preformed
acetone, by means of an air current as first suggested by Folin (see p.
399). The procedure is as follows: Introduce into a wide-mouthed bottle
grams in one liter of water. To determine the requisite dilution to make such a solution that
2 c.c. shall be equivalent to i c.c. of the silver nitrate solution proceed as follows: Introduce
10 c.c. of the silver nitrate solution into a small porcelain evaporating .dish or casserole, add
30-50 c.c. of distilled water, 2 c.c. of the acid indicator and titrate as described above with
the ammonium thiocyanate solution. The total volume of the concentrated thiocyanate
solution including that used in this titration is divided by ten, and the result multiplied by the
difference between this burette reading and 20 c.c. This will give the volume of distilled water
which must be added to the concentrated thiocyanate solution to render 2 c.c. equivalent to
I c.c. of the silver nitrate solution.
422
PHYSIOLOGICAL CHEMISTRY.
2CO c.c. of water, an accurately measured excess of N/io iodine solution^
and an excess of 40 per cent potassium hydroxide. Prepare an aerometer
cylinder containing alkaline hypoiodite solution to absorb any acetone
which may be present in the air of the laboratory, and between the cylinder
and bottle suspend a test-tube about two inches in diameter. This
large test-tube should contain 20 c.c. of the urine under examination, 10
drops of a 10 per cent solution of phosphoric acid, 10 grams of sodium
chloride, and a little petroleum, and should be raised sufficiently high to
facilitate the easy application of heat to its bottom portion. The con-
nections on the side of the tube should be provided with bulb-tubes
containing cotton. When the apparatus is arranged as described, it
should be connected with a Chapman pump and an air current passed
through for twenty-five minutes. During this period the contents of
the test-tube are heated just to the boiling-point and after an interval of
five minutes again heated in the same manner. By this means the
diacetic acid is converted into acetone and at the end of the twenty-five-
minute period this acetone, as well as the preformed acetone, will have
been removed from the urine to the absorption bottle and there retained
as iodoform.
The contents of the absorption bottle should now be acidified with
concentrated hydrochloric acid,^ and titrated with N/io sodium thio-
sulphate and starch as in the Messinger-Huppert method (see below).
2. Messinger-Huppert Method.^ — ^Place 100 c.c. of urine in a dis-
tillation flask and add 2 c.c. of 50 per cent acetic acid. Connect the
flask with a condenser, properly arrange a receiver, 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 resid-
' Proceed as follows in order to obtain a rough idea regarding the amount of N/io iodine
solution to be used: Introduce into a test-tube lo c.c. of the urine under examination and
I c.c. of a solution of ferric chloride made by dissolving loo grams of ferric chloride in loo c.c.
of distilled water. After permitting the mixture to stand for two minutes, compare the
color with that of an equal volume of the ferric chloride solution in a test-tube of similar
diameter. If the two solutions be of approximately the same color intensity, 20 c.c. of the
urine under examination will yield sufficient acetone to require nearly 10 c.c. of N/io iodine
solution. In case the mixture is darker in color than is the ferric chloride solution, the former
should be diluted with distilled water until it it of approximately the same intensity as the
ferric chloride solution. From this data the amount of N/io iodine solution required may be
roughly estimated by means of the following table:
Urine c.c.
Ferric chloride.
10
1
ID
I
10
I
10
I
Water.
N/io Iodine required c.c.
10
10
20
20
35
30
50
* An excess of iodine is indicated by the development of a brown color
^ This method serves to determine bolk acetone and diacetic acid in terms of acetone.
urine: quantitativk analysis. 423
ual portion of the mixture to a second distillation. Test this fluid for
acetone and if the presence of acetone is indicated add about roo c.c. of
water to the residue and again distil. Treat the united acetone distillates
with I c.c. of dilute (12 per cent) sulphuric acid and redistil, collecting
this second distillate in a glass-stoppered flask. During distillation, how-
ever, 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 attached 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 N/io 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 N/io iodine
solution until an excess is obtained. Retitrate this excess of iodine with
N/io 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 ^'isible. This is the end-
reaction.
Calculatim. — Subtract the number of cubic centimeters of N/io
thiosulphate solution used from the volume of N/io iodine solution
employed. Since i c.c. of the iodine solution is equivalent to 0.967
milligram of acetone, and since i c.c. of the thiosulphate solution is
equivalent to i c.c. of the iodine solution, if we multiply the remainder
from the above subtraction 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.
XVI. Acetone.
I. Folin's Method. — The same type of apparatus is used in this
method as that described in Folin's method for the determination of
ammonia (see p. 399). The procedure is as follows: Introduce 20-25
c.c. of the urine under examination into the aerometer cylinder and
add 10 drops of 10 per cent phosphoric acid,^ 8-10 grams of sodium
chloride,^ and a little petroleum. Introduce into an absorption flask, ^
' Oxalic acid (0.2 — 0.3 gram) may be substituted if desired.
- Acetone is insoluble in a saturated solution of sodium chloride.
^Folin's improved absorption tube (see Fig. 128, p. 400) should be used in this connec-
tion inasmuch as the original type embracing the use of a rubber stopper is unsatisfactory be-
cause of the solvent action of alkaline hypoiodite on rubber.
424 PHYSIOLOGICAL CHEMISTRY.
such as is used in the ammonia determination (see p. 399), 150 c.c. of
water, 10 c.c. of a 40 per cent solution of potassium hydroxide, and an
excess of a N/io iodine solution. Connect the flask with the aerometer
cylinder, attach a Chapman pump, and permit an air current, slightly
less rapid than that used for the determination of ammonia, to be drawn
through the solution for 20-25 minutes. All of the acetone will, at this
point, have been converted into iodoform in the absorption flask. Add
10 c.c. of concentrated hydrochloric acid (a volume equivalent to that of
the strong alkali originally added), to the contents of the latter and
titrate the excess of iodine by means of N/io sodium thiosulphate solution
and starch, as in the Messinger-Huppert method (see p. 422),
Folin has further made suggestions regarding the simultaneous deter-
mination of acetone and ammonia by the use of the same air current.^
This is an important consideration for the clinician inasmuch as urines
which contain acetone and diacetic acid are generally those from which
the ammonia data are also desired. The procedure for the combination
method is as follows: Arrange the ammonia apparatus as usual (see p.
399), and to the aerometer of the ammonia apparatus attach the acetone
apparatus set up as described above. Regulate the air current with
special reference to the determination of acetone and at the end of 20-25
minutes disconnect the acetone apparatus and complete the determination
of the acetone as just described. The air current is not interrupted, and
after having run one and one-half hours the ammonia apparatus is de-
tached and the ammonia determination completed as described on page
399-
If data regarding diacetic acid are desired, the result obtained by
Folin's method may be subtracted from the result obtained by the Mes-
singer-Huppert method (see p. 422), inasmuch as the latter method
determines both acetone and diacetic acid. Under all conditions the
determination of acetone should be as expeditious as possible. This
is essential, not only because of the fact that any diacetic acid present
in the urine will become transformed into acetone, but also because of the
rapid spontaneous decomposition of the alkaline hypoiodite solution used
in the determination of the acetone. It has been claimed that alkaline
hypoiodite solutions are almost completely converted into iodate solutions
in one-halj hour. Folin states, however, that the transformation is not
so rapid as this, but he nevertheless emphasizes the necessity of rapidity
of manipulation. At the same time it should be remembered that the
air current must not be as rapid as for ammonia, inasmuch as the alkaline
hypoiodite solution will not absorb all the acetone under those conditions.
' These determinations may even be made on the same sample of urine if the sample is
too small for the double determination. -
urine: quantitative analysis. 425
XVII. Diacetic Acid.
1. Folin-Hart Method. — Arrange the ap])aratus as described under
the Folin-Hart method for the determination of acetone and diacetic acid
(see p. 421). Start the air current in the usual way and permit it to run
25 minutes •without the application of heat to the urine under examination.
Under these conditions the preformed acetone present in the solution is
all removed (^ee p. 423). Immediately attach a freshly prepared absorp-
tion bottle or introduce fresh alkaline hypoiodite solution into the original
bottle. Apply heat to the large test-tube as already described (see p. 422),
in order to convert the diacetic acid into acetone, permit the air current to
continue for the usual 25-minute period, and determine the diacetic acid
value in terms of acetone by the usual titration procedure (see p. 422).
2. Folin-Messinger-Huppert Method. — Determine the combined
acetone and diacetic acid, in terms of acetone, by the Messinger-Huppert
method (see p. 422), and subsequently determine the acetone by Folin's
method (see p. 423) , Subtract the value determined by the second method
from that obtained in the first method to secure data regarding the diacetic
acid content of the urine, in terms of acetone.
XVIII. ^5-Oxybutyric Acid.
I. Shaffer's Method.— Introduce 25-250 c.c. of urine ^ into a 500
c.c. volumetric flask and add an excess of basic lead acetate and 10 c.c.
of concentrated ammonium hydroxide. Dilute the mixture to the 500
c.c. mark, shake the flask thoroughly and filter. Transfer 200 c.c. of
the filtrate to an 800 c.c. Kjeldahl distilling flask, add 300-400 c.c. of
water, 15 c.c. of concentrated sulphuric acid and a little talcum and
distil the mixture until 200 to 250 c.c. of distillate has been collected (A).^
To this distillate (A), which contains acetone (both preformed and that
produced from diacetic acid), 3^x16. volatile fatty acids is added 5 c.c. of 10
percent potassium hydroxide and the distillate redistilled in order to
remove the volatile fatty acids. ^ This second distillate (A,) is then
titrated with standard iodine and thiosulphate (see p. 423). The urine-
sulphuric acid residue from which distillate A was obtained is again
' The amount used depends upon the expected yield of ^-o.xj-butyric acid. In the case
of urines which give a strong ferric chloride reaction for diacetic acid, or when 5-10 grams
or more of /J-oxybutyric acid is expected, it is unnecessary- to use more than 25-50 c.c. of
urine. However, in case only a trace of /^-o-xybutyric acid is expected, the volume should be
much larger as indicated. Under all conditions, the amount specified is sufficient for duplicate
determinations. It is desirable to use such a volume of urine as contains the proper amount of
/9-oxybutyric acid to yield 25-50 milligrams of acetone.
* This distilling flask should be provided with a dropping tube, by means of which water
may be introduced in order to prevent the contents of the flask from becoming less than
400 c.c. in volume. Care should be taken to use a good condenser in the distillation, but it
is not necessary to cool the distillate with ice.
^ Formic acid is one of the most troublesome.
426 PHYSIOLOGICAL CHEMISTRY.
distilled, 400-600 c.c. of a 0.1-0.5 P*^^ cent potassium bichromate solu-
tion, being added, by means of the dropping tube, during the process of
distillation.^ In adding the bichromate, care should be taken not to
add it faster than the distillate collects except in cases where the boiling
fluid assumes a pure green color, thus indicating that the bichromate is
being used up more rapidly. After about 500 c.c. of distillate (B) has
collected, 20 c.c. of a 3 per cent solution of hydrogen peroxide and a
few cubic centimeters of potassium hydroxide solution are added and
the mixture (B) subjected to redistillation. Distil off about 300 c.c. and
titrate this distillate (B2) as usual with iodine and thiosulphate (see p. 423.)
Calculation. — The author advises the use of solutions of thiosulphate
and iodine, which are a trifle stronger than N/io; i. e., 103. 4 N/io.
Each cubic centimeter of an iodine solution of this strength is equivalent
to one milligram of acetone or to 1.794 milligrams of /?-oxybutyric acid.
The thiosulphate solution is accepted as the standard and should be
restandardized, from time to time, by a N/io solution of potassium
bi-iodate.
2. Black's Method. — Render 50 c.c. of the urine under examination,
faintly alkaline with sodium carbonate and evaporate to one-third the
original volume. Concentrate to about 10 c.c. on a water-bath, cool the
residue, acidify it with a few drops of concentrated hydrochloric acid^
and add plaster of Paris to form a thick paste. Permit the mixture to
stand until it begins to "set," then break it up with a stout glass rod
having a blunt end and reduce the material to the consistency of a fairly
dry coarse meal.^ Transfer the meal to a Soxhlet apparatus and extract
with ether for two hours. At the end of this period evaporate the ether-
extract either spontaneously or in an air current. Dissolve the residue
in water, add a little bone-black, if necessary, filter until a clear solution
is obtained and make up the filtrate to a known volume (25 c.c. or less)
with water. The /?-oxybutyric acid should then be determined by means
of the polariscope.
3. Darmstadter's Method. — This method is based on the fact that
crotonic acid is formed from /5-oxybutyric acid under the influence of
concentrated mineral acids. The method is as follows: Render 100 c.c.
of urine slightly alkaline with sodium carbonate and evaporate nearly to
dryness on a water-bath. Dissolve the -esidue in 150-200 c.c. of 50-55
per cent sulphuric acid, transfer the acid solution to a i-liter distillation
flask and connect it with a condenser. Through the cork of the flask
' Generally the addition of 0.5 gram of potassium bichromate is sufficient. In case
the urine contains a high concentration of sugar or when a large volume of urine is used,
it may be necessary to use 2-3 grams of the bichromate.
^ The residue should give a distinct red color with litmus paper.
* Before this is accomplished it may, in some cases, be necessary to add a little more
plaster of Paris.
urine: quantitative analysis. 427
introduce the stem of a dropping 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 litjuid in the distillation
flask constant by the addition of water from the dropping funnel as the
distillate collects. Ordinarily it will take about 2-2 1/2 hours to collect
this amount of distillate. Extract the distillate three times ^ with ether
in a separatopy 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 N/io sodium hydroxide solution, using phenolphthalcin as
indicator.
Calciilatimi. — One c.c. of N/io sodium hydroxide solution equals
0.0086 gram of crotonic acid, i part of crotonic acid equals 1.2 1 part of
/?-oxybutyric acid, and i c.c. of N/io sodium hydroxide solution equals
0.0 104 1 gram of ;9-oxybutyric acid. To compute the quantity of fi-
oxybutyric acid, in grams, multiply the number of cubic centimeters of
N/io sodium hydroxide solution used by 0.01041.
4. Bergell's Method. — Render 100-300 c.c. of sugar-free' 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 phos-
phoric acid (being careful to keep the mixture cool), 20-30 grams of
finely pulverized, anhydrous copper sulphate, and 20-25 grams of flne
sand. Mix the mass thoroughly, place it in a paper extraction thimble^
and extract the dry mixture with ether in a Soxhlet apparatus (Fig. 136,
page 437). Evaporate the ether, dissolve the residue in about 25 c.c. of
water, decolorize the fluid with animal charcoal, if necessary, and deter-
mine the content of .-^-oxybutyric acid by a polarization test.
5. 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 water. 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 laevo-rotatory substances except /3-oxybutyric acid will
have been removed and the ItEvo-rotation now exhibited by the urine will
be due entirely to that acid.
XIX. Acidity.
Folin's Method. — The total acidity of urine may be determined as
follows: Place 25 c.c. of urine in a 200 c.c. Erlenmcyer flask and add
' Shaffer has recently called attention to the fact that it is extremely difficult to extract
all of the crotonii acid if but three extractions are made
- If sugar is present it must he removed by fermentation.
' The Schleicher and Schiill fat-free extraction thimble is very satisfactory.
428 PHYSIOLOGICAL CHEMISTRY.
15-20 grams of finely pulverized potassium oxalate and 1-2 drops of a i
per cent phenolphthalein solution to the fluid. Shake the mixture
vigorously for 1-2 minutes and titrate it immediately with N/io sodium
hydroxide until a faint but unmistakable pink remains permanent on
further shaking. Take the burette reading and calculate the acidity of
the urine under examination.
Calculation. — If y represents the number of cubic centimeters of
N/io 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 calculated by means of the following proportion:
2~,:y::y' -.x (acidity of 24-hour urine expressed in cubic centimeters of
N/io sodium hydroxide).
Each cubic centimeter of N/io sodium hydroxide contains 0.004
gram of sodium hydroxide, and this is equivalent to 0.0063 gram of
oxalic acid. Therefore, 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 i^ i^ i^ desired to express the total acidity in grams
of oxalic acid.
XX. Purine Bases.
I. Welker's Modification of the Methods of Arnstein and of
Salkowski.' — -Four hundred cubic centimeters of urine, free from
protein, are treated with 100 c.c. of magnesia mixture and 600 c.c. of
water. This is then filtered and of the clear filtrate a measured quantity
(600-800 c.c.) is treated with an excess (10 c.c.) of a 3 per cent silver
nitrate solution. Concentrated ammonium hydroxide is added in
small quantities, with stirring, until all the chlorides have dissolved.
Allow the flocculent precipitate of the silver purine compounds to settle
to the bottom, then pass the supernatant liquid through the filter before
disturbing the precipitate. Finally transfer the precipitate quantita-
tively to the paper which must be of known nitrogen content. The
precipitate is washed with dilute (r per cent) ammonium hydroxide.
The paper with the precipitate is then transferred to a Kjeldahl flask
and about 100 c.c. of water and a small quantity (about o.i gram) of
magnesium oxide are added. The water is then boiled until all the
ammonia has been driven off. Test the steam with litmus paper.
The material in the flask is then digested by means of the usual
Kjeldahl method (see p. 401). The digestion must be watched care-
' Private communication from Dr. W. H. Welker.
urine: quantitative analysis. 429
fully at the time the sulphuric acid reaches sufficient concentration to
affect the filter paper, inasmuch as the SOj produced causes consider-
able frothing. The total nitrogen (purine base, uric acid and filter-
paper nitrogen) is now determined in the usual way (see Kjeldahl Method,
p. 401). This result minus the uric acid and filter-paper nitrogen will
give the figure for the purine-base nitrogen.
2. Kriiger. and Schmidt's Method. — This method serves for the
determination of both uric acid and the purine bases. The principle
involved is the precipitation of both the uric acid and the purine bases
in combination with copper oxide and the subsequent decomposition
of this precipitate by means of sodium sulphide. The uric acid is then
precipitated by means of hydrochloric acid and the purine bases are
separated from the filtrate in the form of their copper or silver com-
pounds. The nitrogen content of the precipitates of uric acid and
purine bases is then determined by means of the Kjeldahl method (see
p. 401) and the corresponding values for uric acid and purine bases
calculated. The method is as follows: To 400 c.c. of albumin-free
urine ^ in a liter flask,' add 24 grams of sodium acetate, 40 c.c. of a solu-
tion of sodium bisulphite' and heat the mixture to boiling. Add 40-
80 c.c.^ of a 10 per cent solution of copper sulphate and maintain the
temperature of the mixture at the boiling-point for at least three minutes.
Filter off the flocculent precipitate, wash it with hot w^ater until the
wash water is colorless, and return the washed precipitate to the flask
by puncturing the tip of the filter paper and washing the precipitate
through by means of hot water. Add water until the volume in the
flask is approximately 200 c.c, heat the mixture to boiling and decom-
pose the precipitate of copper oxide by the addition of 30 c.c. of sodium
sulphide solution.^ After decomposition is complete, the mixture
should be acidified with acetic acid and heated to boiling until the sepa-
rating sulphur collects in a mass. Filter the hot fluid by means of a filter-
pump, wash with hot water, add 10 c.c. of 10 per cent hydrochloric acid
and evaporate the filtrate in a porcelain dish until the total volume has
been reduced to about 10 c.c. Permit this residue to stand about two
hours to allow' for the separation of the uric acid, leaving the purine
' If albumin is present, the urine should be heated to boiling, acidified with acetic acid,
and filtered.
- The total volume of urine for the twenty-four hours should be sufficiently diluted with
water to make the total volume of the solution 1600-2000 c.c.
' A solution containing 50 grams of Kahlbaum's commercial sodium bisulphite in 100 c.c.
of water.
* The exact amount depending upon the content of the purine bases.
* This is made by saturating a i per cent solution of sodium hydroxide with hydrogen
sulphide gas and adding an equal volume of i per cent sodium hydroxide.
Ordinarily the addition of 30 c.c. of this solution is sufficient, but the presence of an excess
of sulphide should be proven by adding a drop of lead acetate to a drop of the solution. Under
these conditions a dark brown color will show the presence of an excess of sodium sulphide.
43© PHYSIOLOGICAL CHEMISTRY.
bases in solution. Filter off the precipitate of uric acid, using a small
filter paper, and wash the uric acid, with water made acid with sulphuric
acid, until the total volume of the original filtrate and the wash water
aggregates 75 c.c. Determine the nitrogen content of the precipitate by
means of the Kjeldahl method (see p. 401), and calculate the uric acid
equivalent. ^
Render the filtrate from the uric acid crystals alkaline with sodium
hydroxide, add acetic acid until faintly acid and heat to 70° C. Now
add I c.c. of a 10 per cent solution of acetic acid and 10 c.c. of a sus-
pension of manganese dioxide^ to oxidize the traces of uric acid which
remain in the solution. Agitate the mixture for one minute, add 10 c.c.
of the sodium bisulphite solution^ and 5 c.c. of a 10 per cent solution of
copper sulphate and heat the mixture to boiling for three minutes. Filter
off the precipitate, wash it with hot water, and determine its nitrogen
content by means of the Kjeldahl method (see p. 401). Inasmuch as the
composition and proportion of the purine bases present in urine is variable,
no factor can be applied. The result as regards these bases must there-
fore be expressed in terms of nitrogen.
Benedict and Saiki* report cases in which the total purine nitrogen
by this method was less than the uric-acid nitrogen as determined by
the Folin-Shaffer method. The inaccuracy was found to lie in the
Kriiger and Schmidt method. To obviate this they advise the addition
of 20 c.c. of glacial acetic acid for each 300 c.c. of urine employed, the
acid being added before the first precipitation.
3. Salkowski's Method. — ^Place 400-600 c.c. of protein-free urine
in a beaker. Introduce into another beaker 30-50 c.c. of an ammoni-
acal silver solution^ with 30-50 c.c. of magnesia mixture, ° add some
ammonium hydroxide and if necessary some ammonium chloride to
clear the solution. Now add this solution to the urine, stirring con-
tinually with a glass rod, and allow the mixture to stand for one-half
hour. Collect the precipitate on a filter paper, wash it with dilute
ammonium 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.
' This may be done by multiplying the nitrogen value by three and adding three and
one-half milligrams to the product as a correction for the uric acid remaining in solution
in the 75 c c.
^ Made by heating a 0.5 per cent solution of potassium permanganate with a little alcohol
until it is decolorized.
' To dissolve the excess of manganese dioxide.
* Benedict and Saiki: Jour. Biol. Chetn., 7, 27, 1909.
'•" Prepared by dissolving 26 grams of silver nitrate in about 500 c.c. of water, adding enough
ammonium hydroxide to redissolve the prec ipitate which forms upon the first addition of the
ammonia and making the balance of the mixture up to i liter with water.
• Directions for preparation may be found on page 313.
urine: quantitative analysis.
431
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 ammoni-
acal and precipitate the purine 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 thiocyanate according to the Volhard-
Arnold method (see p. 419). Calculate the content of purine bases in
the urine examined, bearing in mind that in an equal mixture of the
silver salts of the purine bases, such as we have here, one part of silver
corresponds to 0.277 gram of nitrogen or to 0.7381 gram of the bases.
XXI. Purine Nitrogen.
Hall's Purinometer.'^ — By means of the instrument shown in Fig.
133, urine may be examined for total purine nitrogen, i. e., nitrogen in
the form of purine bases, urates and uric acid. The method does
not give an absolutely accurate measure of the purine
values. It is, however, of considerable service clini-
cally. The principle of the method is the preliminary
precipitation of the phosphates present followed by the
precipitation of the purine bodies in the form of their
silver compounds by means of an ammoniacal silver
nitrate solution. The volume of this silver precipitate
is then determined and its nitrogen value interpolated
by means of a table of equivalent values. In using
the purinometer proceed as follows: Collect the
twenty-four-hour urine and mix it thoroughly. Take
TOO c.c. of the urine and if albumin is present make
slightly acid with acetic acid and boil and filter.
Close the stopcock of the instrument and introduce
Qo c.c. of urine and 20 c.c. of a modified magnesia
mixture.* Turn the stopcock and permit the pre-
cipitated phosphates to pass into the lower chamber
of the instrument. After an interval of ten minutes
has elapsed the stopcock should be closed and suffi-
FiG. 133. — Hall's
Purinometer.
' Hall: The Purine Bodies, Philadelphia, 1904.
-This is prepared as follows: Dissolve 10 grams of magnesium chloride in about 75 c.c.
of water and add 10 grams of ammonium chloride. Introduce 100 c.c. of concentrated
ammonium hydroxide into this mixture If a precipitate forms add ammonium hydroxide
until a clear solution is obtained. Make the volume 200 c.c. by means of water and add 10
grams of purified talcum.
432 PHYSIOLOGICAL CHEMISTRY.
cicnt ammoniacal silver nitrate solution^ added to make the total
volume in the upper chamber loo c.c. The precipitate of the silver
compounds of the purine bodies should be pale yellow. Any silver
chloride present may be brought into solution in the strong ammoniacal
solution by the repeated inversion of the purinometer. In case the chloride
does not dissolve it should be brought into solution by the addition of
further ammonium hydroxide. Place the purinometer in a dark room
for twenty-four hours and at the end of this time read the volume of the
purine precipitate. Interpolate the value in terms of purine nitrogen by
means of the following table:
^ . . Purine nitrogen
Precipitate p^^. ^^^^
^'''' (grams in loo c.c.)
4 o . 0078
2 o . 0097
6 0.0117
7 0.0136
8 0.0156
9 0.0175
10 0.0185
II 0.0195
12 o . 0205
13-
0.0218
14 0.0225
15 0.0234
16 0.0249
xy 0.0260
18 0.0265
19 0.0270
20 0.0275
21 0.0283
22 o . 0286
23 0.0299
24 0.0312
25 0.0325
Calculation. — Multiply the purine nitrogen percentage by the total
volume of urine and divide by 100 to obtain the total purine nitrogen
value. For example, if the precipitate was found to be 12 c.c. and the
total volume of the twenty-four-hour urine was 1300 c.c. the calculation
would be as follows:
12 c.c. =0.0205 per cent purine nitrogen.
0.0205 X13.0 =0.2665 gram purine nitrogen.
XXII. Allantoin.^'
Paduschka-Underhill-Kleiner Method. — To 50-100 c.c. of urine
m a beaker add basic lead acetate until no more precipitate forms.
' This solution has the following formula:
Silver nitrate i gram
Ammonium hydroxide (sp. gr. 0.90) 100 c.c.
Talcum 5 grams
Distilled water 100 c.c.
' A much more accurate method has been devised by Wiechowski {Biochemische Zeitschrift,
19, 368, 1909.)
urine: quantitative analysis. 433
Filter and pass hydrogen sulphide gas through an ahquot portion of the
filtrate to remove the excess of lead. ^ Filter again, drive off the hydrogen
sulphide by heat and treat an aliquot portion of the filtrate with a lo per
cent solution of silver nitrate until precipitation is complete." Filter oflf
this precipitate, wash it with water and determine its nitrogen content by
means of the Kjeldahl method (see p. 401). This is the "purine nitro-
gen." Rendjer an aliquot portion of the filtrate faintly alkaline,^ with
a I per cent solution of ammonium hydroxide and add 50-100 c.c. of a 10
per cent solution of silver nitrate. If allantoin be present a white,
flocculent precipitate will form and gradually sink to the bottom of the
solution. Filter, wash the precipitate free from ammonium hydroxide
by means of a i per cent solution of sodium sulphate and determine its
nitrogen content by the Kjeldahl method (see p. 401).
XXIII. 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 ammoniacal, 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 Jiot 15 per cent
solution of hydrochloric acid. By means of a separatory funnel extract
the solution with 150 c.c. of ether which contains 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 fil-
trate, 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. Concentrate 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 oxalate
on a washed filter paper,* 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 of urine taken
may be determined by multiplying the weight of CaO by the factor
1.607 1.
' In the original method of Paduschka sodium sulphate is used for this purpose.
- Ordinarily from 20-30 c.c. is required
' Using litmus as the indicator.
* Schleicher and Schiill, No. 589, is satisfactorj-.
28
434 PHYSIOLOGICAL CHEMISTRY.
XXIV. 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 evapo-
ration at an increased temperature, either under atmospheric conditions
or in vacuo, are attended with error.
■ Shackell's method^ which entails the vacuum desiccation of the frozen
sample is extremely satisfactory and should be used in all biological work
where the greatest accuracy is desired.
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 multiplying the second and third
decimal figures of the specific gravity by 2.6. This gives us the number
of grams of solid matter in i 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 calculation would be as
follows:
{a) 16 X2.6 =46.8 grams of solid matter in i liter of urine.
46.8X1120
(b) =^2.4. grams of solid matter in 11 20 c.c. of urine,
icoo ^ ^
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.
' Shackell: American Journal of Physiology, 24, 325, 1909.
CHAPTER XXIII.
QUANTITATIVE ANALYSIS OF MILK, GASTRIC JUICE, AND
BLOOD.
)C.C.
(a) Quantitative Analysis of Milk.
1. Specific Gravity. — This may be determined conveniently by means
of a Soxhlet, \'eith, or Quevenne lactometer. A lactometer reading of
32° denotes a specific gravity of 1.032. The determination should be
made at about 60° F. and the lactometer reading cor-
rected by adding or subtracting 0.1° for every degree F.
above or below that temperature.
Fat. (a) Babcock's Centrifugal Method. — The princi-
ple of this method is the destruction of organic matter
other than fat by sulphuric acid and the centrifugation
of the acid solution in the special tube shown in Fig.
134 and the subsequent reading of the percentage of fat
by means of the tube's graduated neck. The method is
one of the most satisfactory in common use and is
accurate to within 0.5 per cent. Proceed as follows:
By means of a special narrow pipette introduce milk
into the tube up to the 5 c.c. mark. Now add sufficient
sulphuric acid (sp. gr. i. 83-1. 834) to fill the body of the
tube and rotate the tube to secure a homogeneous acid-
milk solution. Fill the neck of the tube with an acid-
alcohol mixture.^ Centrifuge the tube and contents for
one to two minutes and read off the percentage of fat by
means of the graduated neck of the tube. If the top
of the fat column is not at zero it may be brought there
by the addition of water and a moment's recentrifugation.
In case very rich milk (over 5 per cent fat) is under
examination, it may be diluted with an equal volume
of water before examination and the fat percentage
multiplied by 2. In the examination of cream it is
customary to dilute the sample with four volumes of water and multiply
the resultant fat value by 5.
2. Fat. — (6) Quantitative Determination of Fat in Milk by the Meigs^
' This mixture consists of equal volumes of amyl alcohol and concentrated hydrochloric
acid.
* Original paper by Dr. Arthur V. Meigs in Philadelphia Medical Times, July i, 18S2.
435
Fig. 134. — Bab-
cock Tube.
436
PHYSIOLOGICAL CHEMISTRY.
Method with Modification and Improved Apparatus by Croll. ^ — The method
as stated by Dr. Meigs is: Approximately lo c.c. of milk is carefully
weighed and transferred to an ordinary loo c.c. glass-stoppered graduated
cylinder. Twenty c.c. each of distilled water and ether (0.720) are
added, the ground-glass stopper tightly inserted in the bottle, and the
whole shaken vigorously for five minutes.
Then the bottle is carefully unstoppered,
20 c.c. 95 per cent alcohol added, the
stopper reinserted and again shaken for
five minutes. The bottle is now placed
on a table and the contents will separate
into two distinct strata, the upper of
which contains practically all the fat.
This stratum is carefully removed by a
small pipette and transferred to a carefully
weighed glass evaporating dish. The thin
ether layer remaining is washed by the
addition of 5 c.c. of ether. This is re-
moved by pipetting off. This washing is
repeated four times. On each addition
the sides of the bottle should carefully be
washed down by the fresh ether. Finally,
the pipette is rinsed with a little ether.
The evaporating dish with contents is now
placed on a safety water-bath and the ether
evaporated. The drying is continued in a
hot-air oven at a temperature below 100°
C. and finally completed in a desiccator to
constant weight.
Croll's modification consists of subse-
quent repeated extraction of the end-
product of evaporation with absolute ether. The combined extracts
are filtered and the small filter paper is washed repeatedly with absolute
ether. The combined extracts and washings are evaporated and dried
as before and then weighed.
The piece of apparatus shown in Fig. 135, above was also devised
by Croll to do away with the use of the pipette. On closing the top
with a finger and blowing into the mouth-piece, the upper stratum is
forced out into the dish. The bottle is washed by simply pouring the
ether into the tube. This lessens the possibility of accidental loss.
The accuracy of the method compared with that of the Soxhlet method,
* Private Communication.
Fig. 135. — Croll's Fat Apparatus.
QUANTITATIVE ANALYSIS Ok' MILK.
437
using the paper-coil modification and extracting until fresh portions of
absolute ether gave no further trace of extractive material, is shown by
the average difference on twelve samples of human milk being only 0.017
per cent less than by the Soxhiet and on seven samples cow's milk being
onlv 0.0 TQ per cent less. The extreme differences in case of the human
milk were —0.004 per cent and —0.044 per
cent and in case of the cow's milk— 0.006
per cent and— 0.068 per cent.
(c) Adams' Paper-coil Method.- — Intro-
duce about 5 c.c. of milk into a small
beaker, quickly ascertain the weight to
centigrams, stand a fat-free coil' in the
beaker, and incline the vessel and rotate
the coil in order to hasten the absorption
of the milk. Immediately upon the com-
plete absorption of the milk remove the
coil and again quickly ascertain the
weight of the beaker. The difference in
the weights of the beaker at the two
weighings represents the quantity of milk
absorbed by the coil. Dry the coil care-
fully at a temperature below 100° C. and
extract it with ether for 3-5 hours in a
Soxhiet apparatus (Fig. 136, p. 437.)
Using a safety water-bath, heat the flask
containing the fat to constant weight at a
temperature below 100° C.
Calculatio-n. — Divide the weight of fat,
in grams, by the weight of milk, in
grams. The quotient is the percentage
of fat contained in the milk examined.
{d) Approximate Determination by Feser'sLactoscope. — 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. 137) may be used for this purpose.
Proceed as follows: By means of the graduated pipette accompanying
the instrument introduce 4 c.c. of milk into the lactoscope. Add water
gradually, shaking after each addition, and note the point at which the
black lines upon the inner white glass cylinder are distinctly visible.
Observe the point on the graduated scale of the lactoscope which is level
with the surface of the diluted milk. This reading represents the per-
* Verj- satisfactorj' coils are manufactured by Schleicher and Schull.
SoxHLET Apparatus.
43S
PHYSIOLOGICAL CHEMISTRY.
-I
centage of fat present in the undiluted milk. Pure milk should contain at
least 3 per cent of fat.
3. Total Solids/ — Introduce 2-5 grams of milk into a weighed fiat-
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°-ioo° C.
until the weight is constant. (If platinum dishes are employed this residue
may be used in the determination of ash according to the method described
below.)
Calculation} — Divide the weight of the residue, in grams, by the
weight of milk used, in grams. The quotient is the
percentage 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* until a white or light gray ash is ob-
tained. Cool the dish in a desiccator and weigh. (This
ash may be used in testing for preservatives according to
directions on page 244.)
5. Proteins. — Introduce a known weight of milk
(5-10 grams) into a 500 c.c. Kjeldahl digestion flask
and add 20 c.c. of concentrated sulphuric acid and
about 0.2 gram of copper 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 determination according to the directions given
under Kjeldahl Method, page 401.
Calculation. — Multiply the total nitrogen content by the factor 6.37^
to obtain the protein content of the milk examined.
6. Caseinogen. — 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 caseinogen
' Shackell's method for the vacuum desiccation of frozen preparations may be used where
great accuracy is desired (see American Journal of Physiology, 24, 325, 1909).
2 Lead foil dishes, costing only about one dollar per gross, make a very satisfactory substi-
tute for the platinum dishes.
^ 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:
5=0.25 L + 1.2 F4-O.I4
S = total solids.
L = lacometer reading.
F = fat content.
* Great care should be used in this ignition, the dish at no time being heated above a faint
redness, as chlorides may volatilize.
■'' The usual factor employed for the calculation of protein from the nitrogen content is
6.25 and is based on the assumption that proteins contain on the average 16 per cent of nitrogen.
This special factor of 6.37 is used here to calculate the protein content from the total nitrogen,
since the principal protein constituents of milk, i. e., caseinogen and laclalbumin , contain 15.7
per cent, of nitrogen.
J
Fig. 137. — Feser's
Lactoscope.
QUANTITATIVK ANALYSIS OF MILK. 439
admixed with a little fat and lacto-globulin. Filter off the precipitate,
wash it thoroughly with a saturated solution of magnesium sulphate/
transfer the filter paper and precipitate to a Kjeldahl digestion flask, and
determine the nitrogen content according to the directions given in the
previous experiment.
Calculation. — Multiply the total nitrogen by the factor 6.37 to obtain
the casein content.
7. Hart's Caseinogen Method.- — Introduce 10.5 c.c. of milk into
a 200 c.c. Erlcnmcyer llask and add 75 c.c. of distilled water and 1-1.5
c.c. of 10 per cent acetic acid.^ Mix the contents by giving the flask a
vigorous rotary motion. The precipitated caseinogen is now filtered off
upon a 9-1 1 cm. filter paper.^ Wash out the adsorbed and loosely
combined acetic acid by means of cold water. Continue the washing
of both the caseinogen on the filter and that adhering to the flask, until
the wash water has reached a volume of at least 250 c.c.
Now return the precipitate and paper to the original Erlenmeyer flask,
add 75-80 c.c. of neutral (carbon dioxide-free) water, 10 c.c. of N/io
potassium hydroxide and a few drops of phenolphthalein. Stopper the
flask and shake it vigorously, by hand or machine, until the caseinogen
has been brought into solution.* Rinse the stopper with neutral (carbon
dio.xide-free) water and titrate the alkaline caseinogen solution at once
with N/io hydrochloric acid until there is a disappearance of all red
color. °
Calculation. — Subtract the corrected^ acid reading from the 10 c.c.
of alkali used. The difference is the percentage of caseinogen in the
milk. For example, if it takes 6.7 c.c. of N/io hydrochloric acid to
titrate the alkaline solution to the end point and the check test was
equivalent to 0.2 c.c. N/ 10 acid the caseinogen value would be obtained
as follows:
10 —(6.7 + 0.2) =3.1 per cent caseinogen.
8. Lactalbumin. — To the filtrate and washings from the determi-
' Preserve the filtrate and washings for the determination of lactalbumin (Expt. 8).
- Hart: Jour. Biol. Client., 6, 445, 1909.
' In general 1.5 c.c. of acetic acid gives a clear solution which filters nicely but occasionally,
when the milk has a low caseinogen value it is advisable to use less acetic acid.
* The process of filtration may be retarded through the packing of the caseinogen mass upon
the filter paper. In this case conduct a fine stream of cold water against the upper point of
contact of filter paper and caseinogen. By this means the caseinogen precipitate is loosened
and gathers in the apex of the filter. This procedure is very essential. It is not necessary to
remove the caseinogen which adheres to the interior of the flask.
^ Solution is indicated by the disappearance of the white caseinogen particles which would
otherwise settle to the bottom of the flask.
' .\ check test should be run parallel with the entire determination. Even with special
precautions as to neutrality, it is generally found that an acid check of 0.2-0.3 c.c. will be
obtained. This check titration should be added to the volume of acid used in titration.
440 PHYSIOLOGICAL CHEMISTRY.
nation of caseinogen, in Experiment 6, add Almen's^ reagent until no
more precipitate forms. Filter off the precipitate and determine the
nitrogen content according to the directions given under Proteins, above.
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 proteins 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 384. I' 'I
Calculation. — Make the calculation according to directions given under
Fehling's Method, p. 384, bearing in mind that 10 c.c. of Fehling's solution
is completely reduced by 0.0676 grams 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 volu-
metric determination of (i) 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 dishes.^ Label the
vessels A, B and C, respectively, and proceed with the analysis according
to the directions given below.
I. Total Acidity.^ — Add 3 drops of a i per cent alcoholic solution
of phenolphthalein^ to the contents of vessel A and titrate with N/io
sodium hydroxide solution until a faint pink color is produced which
cannot be deepened by further addition of a drop of N/io sodium
hydroxide. Take the burette reading and calculate the total acidity.
Calculation. — The total acidity may be expressed in the following ways :
* Alm6n'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.
^ For a discussion of combined acid see chapter on Gastric Digestion.
^ If suflScient gastric juice is not available it may be diluted with water or a smaller amount,
e. g., 5 c.c. taken for each determination.
* This includes free and combined acid and acid salts.
' One gram of phenolphthalein dissolved in 100 c.c. of 95 per cent alcohol.
QUANTITATIVE ANALYSIS OF MILK. 44 1
1. The number of cubic centimeters of N/io sodium hydroxide
solution necessary to neutralize 100 c.c. of gastric juice.
2. The weight (in grams) of sodium hydroxide necessary to neutralize
TOO 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, /. e., percentage of hydrochloric acid.
The forms of expression most frequently employed are i and 3,
preference bemg given to the former.
In making the calculation note the number of cubic centimeters of
N/io sodium hydroxide 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 hydrochloric acid, by
weight, multiply the value just obtained by 0.00365.^
2. Combined Acidity." — Add 3 drops of sodium alizarin sulphonate
solution^ to the contents of vessel 5 and titrate with N/io sodium hydroxide
solution until a violet color is produced. In this titration the red color,
which appears after the tinge of yellow due to the addition of the indicator
has disappeared, must 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 N/io 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 calculated
according to the directions given under Total Acidity, page 440.
3. Free Acidity/ — Add 4 drops of di-methyl-amino-azobenzene
(Topfer's reagent) solution^ to the contents of the vessel C and titrate
with N/io sodium hydroxide solution until the initial red color is replaced
by lemon yellow.^ Take the burette reading and calculate the free
acidity.
Calculatimi. — The indicator used reacts only to free acid, hence
the number of cubic centimeters of N/io sodium hydroxide used in-
dicates 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 440.
' One c.c. of N/io hydrochloric acid contains 0.00365 gram of hydrochloric acid.
- Hydrochloric acid combined with protein material.
' One gram of sodium alizarin sulphonate dissolved in 100 c.c. of water.
* Hydrochloric acid not combined with protein material.
^ One-half gram dissolved in 100 c.c. of 95 per cent alcohol.
• If the lemon yellow color appears as soon as the indicator is added it denotes the absence
of free acid.
442 PHYSIOLOGICAL CHEMISTRY.
4. Acidity Due to Organic Acids and Acid Salts. — This value
may be conveniently calculated by subtracting the number of cubic
centimeters of N/io sodium hydroxide used in neutralizing the contents
of vessel C from the number of cubic centimeters of N/io sodium hydrox-
ide solution used in neutralizing the contents of vessel B. The remainder
indicates the number of cubic centimeters of N/io 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 100 c.c. of
gastric juice may be calculated according to directions given under
Total Acidity, page 440.
(c) Quantitative Analysis of Blood.
For the methods involved in the quantitative examination of blood
see Chapter XII.
APPENDIX.
Almen's Reagent/ — 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.
Ammoniacal Silver Solution.' — Dissolve 26 grams of silver nitrate
in about 500 c.c. of water, add enough ammonium hydroxide to redis-
solve the precipitate which forms upon the first addition of the ammonium
hydroxide and make the volume of the mixture up to i liter with water.
Arnold-Lipliawsky Reagent.^ — 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.
Barfoed's Solution. '—Dissolve 4.5 grams of neutral, crystallized
copper acetate in 100 c.c. of water and add 1.2 c.c. of 50 per cent 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.
Basic Lead Acetate Solution." — This solution possesses the following
formula:
Lead acetate 180 grams.
Lead oxide (Litharge) no grams.
Distilled water to make 1000 grams.
Dissolve the lead acetate in about 700 c.c. of distilled water, with boiling.
Add this hot solution to the finely powdered lead oxide and boil for one-
half hour with occasional stirring. Cool, filter and add sufl&cient dis-
tilled water to the filtrate to make the weight one kilogram.
Benedict's Solutions.^ — First Modification. — Benedict's modified
' Ott's precipitation test, p. 339. Determination of lactalbumin, p. 439.
* Salkowski's method, page 430.
' Arnold-Lipliawsky reaction, page 349.
* Barfoed's test, pages 36 and 331.
' Isolation of urea from urine, page 287.
* Indican determination, page 416.
' Benedict's modifications of Fehling's test, pages 328 and 329, and Benedict's Method No.
I, page 385.
443
444 PHYSIOLOGICAL CHEMISTRY.
Fehling solution consists of two definite solutions — a copper sulphate
solution and an alkaline tartrate solution, which may be prepared as
follows:
Copper sulphate solution =34.65 grams of copper sulphate dissolved
in water and made up to 500 c.c.
Alkaline tartrate solution =100 grams of anhydrous sodium carbonate
and 173 grams of Rochelle salt dissolved in water and made up to 100
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.
Second Modification.- — \'ery recently Benedict has further modified
his solution and has succeeded in obtaining one which does not deteriorate
upon long standing. It has the following composition:
Copper sulphate 17.3 grams.
Sodium citrate 173° grams.
Sodium carbonate loo .o grams.
Distilled water to make i liter.
With the aid of heat dissolve the sodium citrate and carbonate in about
600 c.c. of water. Pour (through a folded filter paper if necessary) into
a glass graduate and make up to 850 c.c. Dissolve the copper sulphate
in about 100 c.c. of water and make up to 150 c.c. Pour the corbonate-
citrate solution into a large beaker or casserole and add the copper
sulphate solution slowly, with constant stirring. The mixed solution is
ready for use and does not deteriorate upon long standing.
Benedict's solution as used in the quantitative determination of sugar
(Method No. i) consists of three separate solutions, the two mentioned
under First Modification and in addition a potassium ferro-thiocyanate
solution. This third solution contains 15 grams of potassium ferrocyanide,
62.5 grams of potassium thiocyanate and 50 grams of anhydrous sodium
carbonate dissolved in water and made up to 500 c.c. In preparing the
Benedict's solution for quantitative work the three solutions mentioned are
combined in equal parts.
Benedict's Sugar Reagent (Method No. 2).^
Copper sulphate (crj'stallized) 18.0 grams.
Sodium carbonate (crystallized, one-half the weight of the
anhydrous salt may be used) 200 .0 grams.
Sodium or potassium citrate 200.0 grams.
Potassium thiocyanate 125 .0 grams.
Potassium ferrocyanide (5 per cent solution) 5.0 c.c.
Distilled water to make a total volume of 1000. o c.c.
With the aid of heat dissolve the carbonate, citrate and thiocyanate
in enough water to make about 800 c.c. of the mixture and filter if necessary.
* Quantitative determination of sugar, page 385.
APPENDIX, 445
Dissolve the copper sulphate separately in about loo c.c. of water
and pour the solution slowly into the other liquid, with constant stirring.
Add the ferrocyanide solution, cool and dilute to exactly i liter. Of the
various constituents, the copper salt only need be weighed with exactness.
Twenty-live cubic centimeters of the reagent are reduced by 50 mg. of
glucose.
Bial's Reagent.' •
On inol 1.5 grams.
Fuming HCl 500 . 00 grams.
Ferric chloride (10 per cent) 20-30 drops.
Benedict's Sulphur Reagent.
Crystallized copper nitrate, sulphur-free or of known sulphur
content 200 grams.
Sodium or potassium chlorate 50 grams.
Distilled water to 1000 c.c.
Black's Reagent.- — Made by dissolving 5 grams of ferric chloride
and 0.4 gram of ferrous chloride in 100 c.c. of water.
Blood Serum. — This may easily be obtained in quantity by the
procedure described under Hemagglutination in the chapter on Blood.
Boas' Reagent.^ — Dissolve 5 grams of rcsorcinol and 3 grams of
sucrose in 100 c.c. of 50 per cent alcohol.
Bonnano's Reagent. — Dissolve 2 grams of sodium nitrite in 100
c.c. of concentrated hydrochloric acid.
Bottu's Reagent. — To 3.5 grams of (7-nitrophenylpropiolic acid
add 5 c.c. of a freshly prepared 10 per cent solution of sodium hydroxide
and make the volume of the solution one liter with distilled water.
Combined Hydrochloric Acid (Protein Salt). — To prepare so-
called combined hydrochloric acid simply add a soluble protein such as
Witte's peptone to free hydrochloric acid of the desired strength until
it no longer responds to free acid tests (see chapter on Gastric Digestion).
For example, if 0.2 per cent combined acid is required the protein would
be added to 0.2 per cent free hydrochloric acid.
Strictly speaking there is no such thing as "combined" acid in this
sense. When the protein is added a protein salt of the acid is formed which
ionizes differently from the free acid.
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.
Cross and Bevan's Reagent. — Combine tivo parts of concentrated
hydrochloric acid and one part of zinc chloride by weight.
' Test for pentose, page ^^2.
• Black"* reaction, page 350.
' Test for free acid, page 130.
* Test for free acid, page 130.
446 PHYSIOLOGICAL CHEMISTRY.
Ehrlich's Diazo Reagent.^— Two separate solutions should be
prepared and mixed in definite proportions when needed for use.
{a) Five grams of sodium nitrite dissolved in i liter of distilled water.
{b) Five grams of sulphanilic acid and 50 c.c. of hydrochloric acid in
I liter of distilled water.
Solutions a and b should be preserved in well-stoppered vessels and
mixed in the proportion i : 50 when required. Green asserts that greater
delicacy is secured by mixing the solutions in the proportion i : 100.
The sodium nitrite deteriorates upon standing and becomes unfit for
use in the course of a few weeks.
Esbach's Reagent.^ — Dissolve 10 grams of picric acid and 20 grams
of citric acid in i liter of water.
Fehling's Solution.^ — ^Fehling's solution is composed of two definite
solutions — a copper sulphate solution and an alkaline tartrate solution,
which may be prepared as follows :
Copper sulphate solution = 2)A-^S grams of copper sulphate dissolved
in water and made up to 500 c.c.
Alkaline tartrate solution =12^ 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
bottles and mixed in equal volumes when needed for use. This is done
to prevent deterioration.
Ferric Alum Solution/ — A cold saturated solution.
Folin-Shaffer Reagent/ — 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 i c.c. of furfurol to 1000 c.c. of distilled
water.
Gallic Acid Solution/ — A saturated alcoholic solution.
Gies' Biuret Reagent. — This reagent consists of 10 per cent KOH
solution to which enough 3 per cent CuSO^ solution has been added
to impart a slight though distinct blue color to the clear liquid. The
CuSO^ should be added drop by drop with thorough shaking after
each addition.
Guaiac Solution/ — Dissolve 0.5 gram of guaiac resin in 30 c.c.
of 95 per cent alcohol.
' Ehrlich's diazo reaction, page 359.
^ Esbach's method, page 383.
' Fehling's method, page 384. Fehling's test, pages 32 and 327.
* Volhard-Arnold method, page 419.
' Folin-Shaffer method, page 389.
" Mylius's modification of Pettenkofer's test, pages 164 and 344. v. Udransky's test, pages
164 and 344.
' Gallic acid test, page 243.
* Guaiac test, pages 186, 209 and 240.
APPENDIX. 447
Giinzberg's Reagent/ — Dissolve 2 grams of phloroglucinol and i
gram of vanillin in 100 c.c. of 95 per cent alcohol.
Hammarsten's Reagent." — Mix i volume of 25 per cent nitric
acid and 19 volumes of 25 per cent hydrochloric acid and add i volume
of this acid mixture to 4 volumes of 95 per cent alcohol. It is perfer-
able that the acid mixture be prepared in advance and allowed to stand
until yellow in.color before adding it to the alcohol.
Hayem's Solution. — This solution has the following formula:
Mcrcuri ■ chlori le o 25 grams.
Sodium chl )ri le 0.5 grams.
Sodium suli)liate 2.5 grams.
DiililL'd water loo.o grams.
Hopkins-Cole Reagent.^ — To one liter of a saturated solution of
oxalic acid add 60 grams of sodium amalgam and allow the mixture
to stand until the evolution of gas ceases. Filter the dilute with 2-3
volumes of water.
Hopkins-Cole Reagent (Benedict's Modification). — Ten grams
of powdered magnesium are placed in a large Erlenmcyer flask and
shaken up with enough distilled water to liberally cover the magnesium.
Two hundred and fifty cubic centimeters of a cold, saturated solution
of oxalic acid is now added slowly. The reaction proceeds very rapidly
and with the liberation of much heat, so that the flask should be cooled
under running water during the addition of the acid. The contents
of the flask arc shaken after the addition of the last portion of the acid
and then poured upon a filter, to remove the insoluble magnesium oxalate.
A little wash water is poured through the filter, the filtrate acidified with
acetic acid to prevent the partial precipitation of the magnesium on long
standing, and made up to a liter with distilled water. This solution
contains only the magnesium salt of glyoxylic acid.
Hypobromite Solution.^ — The ingredients of this solution should
be prepared in the form of two separate solutions which may be united
as needed.
(a) Dissolve 125 grams of sodium bromide in water, add 125 grams
of bromine and make the total volume of the solution i liter.
{b) A solution of sodium hydroxide having a specific gravity of
1.25. This is approximately a 22.5 per cent solution.
Preserve both solutions in rubber-stoppered bottles and when needed
for use mix one volume of solution a, one volume of solution b, and 3
volumes of water.
' Test for free acid, page 130.
^ Hammarsten's reaction, pages 163 and 343.
' Hopkins-Cole reaction, page q8.
* Methods for determination of urea, page 392.
448 PHYSIOLOGICAL CHEMISTRY,
Iodine Solution.^ — Prepare a 2 per cent solution of potassium
iodide and add sufficient iodine to color it a deep yellow.
Iodine-Zinc Chloride Reagent.- — Dissolve 20 grams of zinc chloride
in 8.5 c.c. of water. Cool, and introduce iodine solution (3 grams KI +
1.5 gram I in 60 c.c. of water) drop by drop until iodine begins to
precipitate.
JoUes' Reagent.^ — This reagent has the following composition:
Succinic acid 40 grams.
Mercuric chloride 20 grams.
Sodium chloride 20 grams.
Distilled water 1000 grams.
Kantor and Gies' Biuret Paper.* — Immerse filter paper in Gies'
Biuret Reagent (p. 99) then dry and cut into strips.
Kraut's Reagent.^ — Dissolve 272 grams of potassium iodide in
water and add 80 grams of bismuth subnitrate dissolved in 200 grams
of nitric acid (sp. gr. 1.18). Permit the potassium nitrate to crystallize
out, then filter it off and make the filtrate up to i liter with water.
Lugol's Solution.*' — Dissolve 4 grams of iodine and 6 grams of
potassium iodide in 100 c.c. of distilled water.
Magnesia Mixture.' — Dissolve 175 grams of magnesium sulphate
and 350 grams of ammonium chloride in 1400 c.c. of distilled water.
Add 700 grams of concentrated ammonium hydroxide, mix thoroughly,
and preserve the mixture in a glass-stoppered bottle.
Millon's Reagent.^ — Digest i part (by weight) of mercury with
2 parts (by weight) of nitric acid (sp. gr. 1.42) and dilute the resulting
solution with 2 volumes of water.
Molisch's Reagent.''' — ^A 15 per cent alcoholic solution of a-naphthol.
Molybdic Solution.^" — Molybdic solution is prepared as follows,
the parts being by weight
Molybdic add i part.
Ammonium hydroxide (sp. gr. o . 96) 4 parts.
Nitric acid (sp. gr. 1.2) 15 parts.
Moreigne's Reagent.^* — Combine 20 grams of sodium tungstate,
' Iodine test, page 50.
^ .A.myloid formation, p. 54.
' Jolles' reaction, pages 105 and 334.
* Protein tests, p. 332.
* Rosenheim's bismuth test for choline, page 273.
" Gunning's iodoform test, page 346, and Bardach's reaction, page loi.
' Sodium hydroxide and potassium nitrate fusion method for determination of total phos-
phorus, page 414.
" Millon's reaction, page 97.
■ Molisch's reaction, page 27.
'" Sodium hydroxide and potassium nitrate fusion method for determination of total phos-
phorus, page 414.
' ' Moreigne's reaction, page 293.
■APPENDIX. 449
lo grams of phosphoric acid (sp. gr. 1.13) and 100 c.c. of water. Boil
the mixture for twenty minutes, add water to make the volume of the
solution equivalent to the original volume, and acidify with hydrochloric
acid.
Morner's Reagent.'— Thoroughly mix i volume of formalin, 45
volumes of distilled water, and 55 volumes of concentrated sulphuric
acid.
Nakayama's Reagent.-- — Prepared by combining 99 c.c. of alcohol
and I c.c. of fuming hydrochloric acid containing 4 grams of ferric
chloride per liter.
Nessler-Winkler Solution.
Mercuric iodide .' lo grams.
Potassium iodide 3 grams.
Sodium hydroxide 20 grams.
Water 100 c.c.
The mercuric iodide is rubbed up in a small porcelain mortar with
water, then washed into a tlask and the potassium iodide added. The
sodium hydroxide is dissolved in the remaining water and the cooled solu-
tion added to the above mixture. The solution cleared by standing is
preserved in a dark bottle.
Neutral Olive Oil.^ — Shake ordinary olive oil w^ith a lo 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.'* — Digest 2 grams of bismuth subnitrate
and 4 grams of Rochelle salt in 100 c.c. of a 10 per cent solution of potas-
sium hydroxide. The reagent should then be cooled and filtered.
Obermayer's Reagent.^ — Add 2-4 grams of ferric chloride to a
liter of hydrochloric acid (sp. gr. 1.19).
Oxalated Plasma.^ — Allow arterial blood to run into an equal
volume of 0.2 per cent ammonium oxalate solution.
Para-dimethylaminobenzaldehyde Solution.'' — This solution is
made by dissolving 5 grams of para-dimethylaminobenzaldehyde in
100 c.c. of 10 per cent sulphuric acid.
Para-phenylenediamine Hydrochloride Solution.^ — Two grams
dissolved in too c.c. of water.
Phenolphthalein.® — Dissolve i gram of phenolphthalein in 100
c.c. of 95 per cent alcohol.
' Morner's test, page 91.
'Nakayama's reaction, pages 162 and 342.
* Emulsification of fats, page 143.
* Nylander's test, pages 34 and 330.
* Obermayer's test, page 299.
* Experiments on blood plasma, page 214.
^ Herter's para-dimethylaminobenzaldehyde reaction, page 176.
* Detection of hydrogen peroxide, page 244.
' Topfer's method, page 440.
29
450 PHYSIOLOGICAL CHEMISTRY.
Phenylhydrazine Mixture/ — This mixture is prepared by com-
bining I part of phenylhydrazine-hydrochloride and 2 parts of sodium
acetate by weight. These are thoroughly mixed in a mortar.
Phenylhydrazine-acetate Solution.- — This solution is prepared
by mixing i volume of glacial acetic acid, i volume of water, and 2
volumes of phenylhydrazine (the base).
Purdy's Solution.^ — Purdy's solution has the following composition:
Copper sulphate 4-752 grams.
Potassium hydroxide 23.5 grams.
Ammonia (U. S. P., sp. gr. o. 9) 35° • o c.c.
Glycerol 38 . o c.c.
Distilled water, to make total volume i liter.
Roberts' Reagent/ — Mix i volume of concentrated nitric acid
and 5 volumes of a saturated solution of magnesium sulphate.
Rosenheim's lodo-Potassium Iodide Solution/ — Dissolve 2 grams
of iodine and 6 grams of potassium iodide in 100 c.c. of water.
Salted Plasma/' — Allow arterial blood to run into an equal vol-
ume of a saturated solution of sodium sulphate or a 10 per cent solu-
tion of sodium chloride. Keep the mixture in the cold room for about
24 hours.
Schiff's Reagent/ — This reagent consists of a mixture of three
volumes of concentrated sulphuric acid and one volume of 10 per cent
ferric chloride.
Schweitzer's Reagent/ — Add potassium hydroxide to a solution
of copper sulphate which contains some ammonium chloride. Filter
off the precipitate of cupric hydroxide, wash it, and bring 3 grams of
the moist cupric hydroxide into solution in a liter of 20 per cent ammo-
nium hydroxide.
Seliwanoff's Reagent/ — Dissolve 0.05 gram of resorcinol in 100
c.c. of dilute (1:2) hydrochloric acid.
Sherrington's Solution/*^ — This solution possesses the following
formula:
Methylene-blue o . i gram.
Sodium chlorirle 12 gram.
Neutral potassium o.xalate 1.2 gram.
Distilled water 300 .0 grams.
Sodium Acetate Solution/^ — Dissolve loo grams of sodium acetate
' Phenylhydrazine reaction, pages 28 and 324.
- Phenylhydrazine reactiDn, pa^es 28 and 324.
^ Purdy's method, page 387.
■* Roberts' ring test, pages 104 and 334.
^ Rosenheim's periorlide test, page 273.
"Experiments on blood plasma, page 214.
^ Schiff's reaction, pages i66 and 272.
* Schweitzer's solubility test, page 54.
* Seliwanoff's reaction, pages 40 and 356.
'""Blood counting," page 224.
" Uranium acetate method, page 413.
APPENDDC. 451
in 800 c.c. of distilled water, add too c.c. of 30 per cent acetic acid to the
solution, and make the volume of the mixture up to i liter with distilled
water.
Sodium Alizarin Sulphonate.' — Dissolve i gram of sodium aliz-
arin sulphonate in 100 c.c. of water.
Sodium Sulphide Solution.- — Saturate a i per cent solution of
sodium hydcoxide with hydrogen sulphide gas and add an equal volume
of I per cent sodium hydroxide.
Solera's Test Paper. ^ — Saturate a good cjuality of filter paper
with 0.5 per cent starch paste to which has been added sufficient iodic
acid to make a i per cent solution of 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.'' — This reagent has the following composition:
Tartaric acid 20 grams.
Mercuric chloride 40 grams.
Glycerol 100 grams.
Distilled water 1000 grams.
Standard Ammonium Thiocyanate Solution.^ — This solution is
made of such a strength that i c.c. of it is equal to i c.c. of the standard
silver nitrate solution mentioned below. To prepare the solution dissolve
12.9 grams of ammonium thiocyanate, NH^SCN, in a little less than a
liter of water. In a small flask place 20 c.c. of the standard silver nitrate
solution, 5 c.c. of a cold saturated solution of ferric alum 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 ammonium
thiocyanate solution from a burette until a permanent red-brown tinge is
produced. This is the end-reaction and indicates that the last trace
of silver nitrate has been precipitated. Take the burette reading and
calculate the amount of water necessary to use in diluting the ammonium
thiocyanate in order that 10 c.c. of this solution may be exactly equal
to 10 c.c. of the silver nitrate solution. Make the dilution and titrate
again to be certain that the solution is of the proper strength.
Standard Silver Nitrate Solution." — Dissolve 29.042 grams of
silver nitrate in i liter of distilled water. Each cubic centimeter of this
solution is equivalent to 0.0 1 gram of sodium chloride or to 0.006 gram
of chlorine.
Standard Uranium Acetate Solution.^ — Dissolve about 34 grams of
' Topfer's method, page 440.
^ Kniger and Schmidt's method, pages 391 and 429.
' Solera's reaction, page 64.
* Spiegler's ring test, pages 104 and 334.
* Volhard-.Arnold method, page 419, and Dehn-Clark method, page 417.
* Volhard- Arnold method, page 419, Mohr's method, page 418, and Dehn-Clark method,
page 417.
' Uranium acetate method, page 413.
452 PHYSIOLOGICAL CHEMISTRY.
uranium acetate in i liter of water. One c.c. of such a solution should now
be made equivalent to 0.005 gram of PzOg, phosphoric anhydride. It
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 o.i gram of PgOg, add 5 c.c. of the sodium acetate solution
mentioned on p. 450 and titrate with the uranium solution to the correct
end-reaction as indicated in the method proper on p. 413. Inasmuch as
I c.c. of the uranium solution should precipitate 0.005 gram of PjOj,
exactly 20 c.c. of the uranium solution should be required to precipitate
the 50 c.c. of the standard phosphate solution. If the two solutions do
not bear this relation to each other they must be brought into the proper
relation by diluting the uranium solution with distilled water or by in-
creasing its strength.
Starch Iodide Solution/ — 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 continuously. Cool the starch paste, add
20-25 grams of potassium iodide and dilute the mixture to 50 c.c. This
solution deteriorates upon standing, and therefore must be freshly pre-
pared as needed.
Starch Paste. ^ — 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 approxi-
mate I per cent starch paste which is a very satisfactory strength for
general use.
Stokes' Reagent.^ — ^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.
Suspension of Manganese Dioxide.^ — Made by heating a 0.5 per
cent solution of potassium permanganate with a little alcohol until it is
decolorized.
Tanret's Reagent.^ — 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.
' Fehling's method, page 384.
- Fehling's method, page 384.
' HLaemoglobin, page 216. Haemochromogen, page 219.
* Kriiger and Schmidt's method, pages 391 and 429.
* Tanret's test, pages 104 and 334.
APPENDIX. 453
Tincture of Iodine.* — Dissolve 70 grams of iodine and 50 grams of
potassium iodide in i liter of 95 per cent alcohol.
Toison's Solution.^ — This solution has the following formula:
Methyl violet o .025 gram.
Sodium chloride : i .0 gram.
Sodium sulphate 8.0 grams.
Glycerol 30 .0 grams.
Distilled water 160.0 grams.
Topfer's Reagent.^ — Dissolve 0.5 gram of di-methylaminoazobenzene
in 100 c.c. of 95 per cent alcohol.
Tropaeolin 00.^ — Dissolve 0.05 gram of tropasolin 00 in 100 c.c.
of 50 per cent alcohol.
Ufifelmann's Reagent.^ — Add a 5 per cent solution of ferric chloride
to a I per cent solution of carbolic acid until an amethyst-blue color is
obtained.
' Smith's test, pages 163 and 343.
^ " Blood counting," page 224.
' Topfer's method, page 440.
* Test for free acid, page 130.
* Uffelmann's reaction, page 136.
NDEX.
Acacia solution, fwrmation of emulsion by, 141
Acetone, 323, 345
formula for, ,14s
Gunning's iodoform test for, 346
Legal's iodoform test for, 346
Lieben's test for, 347
quantitative determination of, 423
Reynolds-Gunning test for, 347
Rothera's reaction for, 347
Taylor's test for, 347
Acholic stool, 179
Achroo-dextrins, 48, 61, 65
a-achroo-dextrin, 61
^-achroo-dextrin, 61
r-achroo-dextrin, 61
Acid, acetic, 284, 309
alloxyproteic, 283, 303, 359
amino-acetic, 58, 74, 77
amino-butyric, 171
amino-ethyl-sulphonic, 159, 260
a-amino-;9-hydroxy-propionic, 74. 78
a-amino-/3-imidazol-propionic, 73, 82
o-amino-iso-butyl-acetic, 74, 84
a-amino-;S-methyl ,3-ethyl-propionic, 73, 85
a-amino-normal glutaric, 74, 87
o-amino-propionic, 73, 77
amino-succinic, 74, 86
amino- valerianic, 171
a-amino-iso-valerianic (see Valine), 74, 83,
a-diamino-^-dithiolactyl 73, 80
aspartic, 74, 86
benzoic, 168, 283, 307
butyric, 8, 238, 243, 284, 309
caproic, 235, 238
carbamic, 196
cholic, 159
chondroitin-sulphuric. 250, 283, 303
citric, 23s
combined hydrochloric (protein salt), 61, 126,
441
cyanuric, 286
a-e-di-amino-caproic, 73, 85
diaminotrihydroxydodecanoic, 73, 89
diazo-benzene-sulphonic, 360
ethereal sulphuric, 169, 283, 297
fatty, 139, 140, 14s, 284, 383
formic, 284, 309
free hydrochloric, 61, 66, 130, 441
glucothionic, 371
glutamic, 74, 87
glycocholic, 159
glycosuric, 306
glycuronic, 42, 353
glycerophosphoric, 268, 269, 284. 3 to
glyoxylic, 97, 98
guanidine-a-amino- valerianic, 73, 83
hippuric, 168, 283, 300, 406
Acid, homogentisic, 32, 283, 306, 327
iniinazalpropionic, 171
indolacetic, 171,407
indole- rt-amino-propionic, 73, 82
indoxyl-sulphuric, 169, 297
inosinic, 25s, 260
kynurenic, 283, 307
lactic, 45, 126, 136, 138, 235, 256
lauric, 235
mucic, 41, 45, 354, .355
myristic, 235
nucleic, 94, 112
osmic, 271, 299
oxalic, 283, 302
oxaluric, 283, 308
oxy-a-pyrrolidine-carboxylic, 73, 89
oxymandelic, 283, 306
oxyproteic, 283, 303, 359
palmitic, 140, 145, 151.
para-cresol-sulphuric, 283, 297
para-oxyphenyl-aceti(i 169, 171, 17s. 283,
303
para-oxy-;3-phenyl-a-amino-propionic, 74, 79
para-oxyphenyl-propionic, 169, 171, I7 5i
283, 303
paralactic, 23s, 256, 284, 309
phenaceturic, 284, 309. 4°?
phenol-sulphuric, 283, 297
phenylacetic, 171
phenyl- a-amino propionic, 74, 78
phenylpropionic, 171
phosphocamic, 25s, 260, 284, 310
phosphoric, 3 17
pyrocatechin-sulphuric, 283, 297
a-pyrrolidine-carboxylic, 73, 88
sarcolactic, 256
skatole acetic, 82
skatole-carbonic, 174, 175
skatoxyl-sulphuric, 283, 297
stearic, 269
succinic, i 7 i
sulphanilic, 360
tannic, 50, 53
taurocholic, 159
uric, 32. 255, 277, 283, 290, 362, 368
uroferric, 283, 303. 359
uroleucic, 283
volatile fatty, 169, 172, 284, 309
Acid albuminate. See Acid metaprotein.
Acid infraprotein. See Acid metaprotein.
Acid metaprotein, 1 16
coagulation of, 1 1 6
experiments on, 116
precipitation of, 116
preparation of, 116
solubility of, 116
sulphur content of, 116
455
456
INDEX.
Acidity of gastric juice, quantitative determina-
tion of, 440
urine, cause of, 276, 317
quantitative determination of, 427
Acidosis, cause of, 350
Acid-haematin, 219
Acree-Rosenheim formaldehyde reaction, 100
Acrolein, formation of, from olive oil, 143
from glycerol, 146
Activation, 6, 150
Activation by calcium salts, 150
^dam's paper coil method for determination of
fat in milk, 43 7
Adamkiewicz reaction, 97
Adaptation, 62
Adenase, 4
Adenine, 4, 261, 284, 312
Adipocere, 142
Adler's benzidine reaction for blood, i8s, 204,
209, 341
Agar-agar, 26, 55, 56, 180
Agglutination, 197
Alanine, 73, 77, 171
Albumin, egg, 107
powdered, preparation of, 107
tests on, 107
serum, 93, 95, 194, 323, 332
Albumin in urine, 323, 332
acetic acid and potassium ferrocyanide
test for, 33 5
coagulation or boiling test for, 33s
Heller's ring test for, 333
JoUes' reaction for, 334
Roberts' ring test for, 334
sodium chloride and acetic acid test for, 336
Spiegler's ring test for, 334
Tanret's test for, 335
tests for, 333
Albumins, 93, 95, 96
Albuminates. See Metaproteins.
Albuminates, formation of, by metallic salts,
102, 103
Albuminoids, 93, 112
Albumoscope, 104, 334
Albumoses (see Proteoses, p. 119)
Alcohol-soluble proteins. See Prolamins.
Aldehyde, 25, 30
Aldehyde group, 44
Aldehyde test for alcohol, 47
v. Aldor's method of detecting proteose in
urine, 338
Aldose, 25
Aliphatic nucleus, 73, 74
Alkali albuminate. See Alkali metaprotein.
Alkali-hffimatin, 212, 219
Alkali metaprotein, 94, 116, 117
experiments on, 117
precipitation of, 117
preparation of, 117
sulphur content of, 117
Alkaline tide, 276
AUantoin, 283, 303
crystalline form of, 304
experiments on, 305
formula for, 303 '
preparation of, from uric acid, 305
quantitative determination of, 432
separation of, from urine, 305
Allen's modification of Fehling's test, 329
Almen's reagent, preparation of, 339
Alloxyproteic acid, 283, 303, 359
Aloin -turpentine test for "occult blood," 181,
i8s
Amandin, 93
Amide nitrogen, 69
Amidulin. See Soluble starch, 18, 48, 6i
Amino acids, 69, 93, 149, 159, 171, 283
group, 95, 98
a-amino-^-hydroxy-propionic acid, 74, 78
a-amino-/3-imidazol-propionic acid, 73, 82
a-amino-iso-butyl-acetic acid, 74, 84
a-amino-normal-glutaric acid, 74, 87
404
Amino-butyric acid, 171
Amino-nitrogen, quantitative determination of,
Amino-succinic acid, 74, 86
Amino-valerianic acid, 171
a-amino-iso-valerianic acid, 74, 83
Ammonia, 69, 76, 108
Ammonia in urine, 284, 313
quantitative determination of, 399
Ammoniacal silver solution, preparation of, 430
Ammoniacal-zinc chloride test for urobilin, 311
Ammonium magnesium phosphate (" Triple
phosphate"), 277, 319
in urinary sediments, 362
Ammonium urate, 290, 36s, 389
crystalline form of, Plate VI, opposite
P-, 36s
Amphopeptone, 95, 120
Amylase, pancreatic, 4, 10, 150
digestion of dry starch by, 151, 156
inulin by, 157
experiments on, 10, 155
influence of bile upon action of, 156
metallic salts, upon action of, 156
most favorable temperature for action of,
iS6
salivary, 4, 10, 60, 126
activity of, in stomach, 61, 126
experiments on, 10, 65
inhibition of activity of, 61, 66
nature of action of, 61
products of action of, 61
vegetable, 4, 10
Amylases, 3, 4, 10, 60, 150
experiments on, 10, 6s, iSS
Amyloid, 54, 113
Amylolytic enzymes. See Amylases.
quantitative determination of activity
of, 18
Animal parasites in feces, 181, 183, 184
in urinary sediments, 369, 378
Antialbumid, 130
Antienzymes, 9
experiments on, 17
Antimony pentachloride as cellulose solvent,
SS
Antimony trichloride as cellulose solvent, ss
Antipepsin, 9, 17
Antipeptone, 120
Antirennin, 9
Antithrombin, 204
antitrypsin, 9, 18
Aporrhegmas, 72, 170
Appendix, 443
INDEX.
457
Arabinose, 25, 42, 352
Bial's reaction for, 42, 352
orcinol test on, 43, 353
phenylhydrazine test on, 43
ToUens' reaction on, 42, 353
Arginase, 4
Arginine, 4, 72, 73. 83, i49
Amold-Lipliawsky reaction for diacetic acid, 349
reagent, preparation of, 349
Aromatic oxyacids, 283, 306
Ascaris, 17,18
Asparagine, 86
Aspartic acid, 69, 72. 74. 86
crystalline form of, 86
formula for, 86
Ash of milk, quantitative determination of, 438
Assimilation limit, 27, 41
Assimilation limit of dextrose, 27, 324
galactose, 41
Atkinson and Kendall's haemin test, 210
Autol>-tic enzymes, 3
Babcock fat method, 435
tube, 435
Bacteria in feces, 181
quantitative determination of, 191
Barberio's reaction for indican, 300
Bardach's reaction, loi
Barfoed's reagent, preparation of, 14, 36, 331
Barfoed's test for monosaccharides, 36, 331
Baryta mixture, preparation of, 287
Basic lead acetate solution, 416, 443
Bayberry tallow, saponification of, 144
source of, 144
Bayberry wax. See Bayberry tallow, 144
Bead test (Einhom), 189
Beckmann-Heidenhain apparatus, 280
" Bence Jones' protein," detection of, 338
Benedict's methods for quantitative deter-
mination of sugar, 38s
Benedict's method for quantitative deter-
mination of sulphur, 409
Benedict's method for quantitative deter-
mination of urea, 396
Benedict's modifications of Fehling's test, 33, 328
solutions, preparation of, 33, 328
solution, for use in quantitative deter-
mination of sugar, preparation of, 385
sulphur reagent, preparation of, 409
Benzidine reaction, Adler's, for blood, 185, 204,
209, 341
Benzoic acid, 168, 283, 307
crystalline form of, 308
experiments upon, 307
formula for, 307
solubility of, 307
sublimation of, 307
Berthelot-Atwater bomb calorimeter, 411
Bergell's method for determination of ,3-oxy-
butyric acid, 427
Bial's reaction for pentoses, 42, 352
Bial's reagent, preparation of, 42, 352
Bile, 158, 323, 342
constituents of, 159
daily secretion of, 158
freezing-point of, 159
influence on digestion, gastric, 136
pancreatic, 151, 155, 156
Bile, inorganic constituents of, 159, 162
nucleoprotein of, 162
reaction of, 158, 162
secretion of, 158
specific gravity of, 159
Bile acids, 159
Guerin's reaction for, 164
Hay's test for, 164
Mylius's test for, 164
Neukomm's test for, 164
Pettenkofer's test for, 163
tests for, 163
V. Udransky's test for, 164
Bile acids in feces, detection of, 187
Bile acids in urine, 323, ^44
Hay's test for, 344
Mylius's test for, 344
Neukomm's test for, 344
Pettenkofer's test for, 344
tests for, 344
V. Udransky's test for, 344
Bile pigments, 160
Gmelin's test for, 162
Hammarsten's reaction for, 163
Huppert's reaction for, 162
Rosenbach's test for, 162
Smith's test for, 163
tests for, 162
Bile pigments in urine, 323, 342
Gmelin's test for, 342
Hammarsten's reaction for, 343
Huppert's reaction for, 342
Nakayama's reaction for, 342
Rosenbach's test for, 342
Salkowski's test for, 343
Salkowski-Schipper's reaction for,
343
Smith's test for, 343
tests for, 342
Bile salts, 7, 159
crystallization of, 159, 164
Biliary calculi, 161
analysis of, 165
Bilicyanin, 160
Bilifuscin, 160
Bilihumin, 160
Biliprasin, 160
Bilirubin, 160
crystalline form of, 161
in urinary sediments, 362, 367
Biliverdin, 160, i6i
" Biological" blood test, 205
Bismuth test for choline, 273
Biuret, 99, 286
formation of, from urea, 99, 286
Biuret paper of Kantor and Gies, 99
Biuret potassium cupric hydroxide. See Cupri-
potassium biuret, 99
test, 98
Posner's modification of, 100
Biuret reagent (Gies), preparation of, 99
Black's method for determination of fi-O'x.y-
butyric acid, 426
reaction for 5-oxybutyric acid, 350
reagent, preparation of, 350
Blood, 194. 323, 339
agglutination of, 197
Bordet test for, 205
458
INDEX.
Blood, clinical examination of, 220
coagulation of, 195, 203
Howell's theory of, 203
constituents of, 194, 196
defibrinated, 206
detection of, 204, 209, 215
erythrocytes of, 194, 197, 212
experiments on, 206
form elements of, 194
guaiac test for, 186, 204, 209
haemin test for, 204, 210
"occult," in feces, 181, 185
oxyhemoglobin of, 197, 216
in urine, 283, 339
leucocytes of, 202
medico-legal tests for, 204
microscopical examination of, 196, 204, 206,
nucleoprotein of, 194, 195
pigment of, 197
plaques, 203
plasma, 194, 214
plates, 203
platelets, 203
preparation of haematin from, 212
preparation of "laky," 207
quantitative analysis of, 220
reaction of, 194, 206
serum, 195, 213
specific gravity of, 194, 206
spectroscopic examination of, 215
test for iron in, 207
total amount of, 194
V. Zeynek and Nencki's hsemin test for, 210
Blood casts in urine, 369, 373
Blood corpuscles, 194, 196, 202
"counting," 224, 228
Blood dust, 194, 203
Blood in urine, 323, 339
Adler's benzidine reaction for, 341
guaiac test for, 341
Teichmann's hsemin test for, 340
Heller's test for, 340
Heller-Teichmann reaction for, 340
Schalfijew's hsemin test for, 340
Schumm's modification of guaiac test
for, 342
spectroscopic examination of, 342
tests for, 340
V. Zeynek and Nencki's haemin test for,
340
Blood plasma, 194, 214
constituents of, 194
crystallization of oxyhsemoglobin of,
201, 214
effect of calcium on oxalated, 214
experiments on, 214
preparation of fibrinogen from, 214
oxalated, 214
salted, 214
Blood serum, 195, 213
coagulation temperature of, 213
constituents of, 195
experiments on, 213
precipitation of proteins of, 213
separation of albumin and globulin of,
2'.?
sodium chloride in, 213
Blood serum, sugar in, 213
Blood stains, examination of, 215
Boas' reagent, as indicator, 132
preparation of, 132
Boekelman and Bouma's method for deter-
mination of /?-oxybutyric acid, 427
Boettger's test for sugar, 34, 330
Bomb calorimeter, Berthelot-Atwater, 411
Bonanno's reaction, 163, 343
Bonanno's reagent, preparation of, 163, 343
Bone, constituents of, 251
ossein of, preparation of, 251
quantitative composition of, 252
Bone ash, scheme for analysis of, 253
Borchardt's reaction for laevulose, 40, 356
Bordet test, detection of human blood by, 205
Boric acid and borates in milk, detection of, 244
Bottu's reagent, preparation of, 29, 325
Bottu's test, 29, 325
Bromelin, 4
Buccal glands, 59
Buffy coat, formation of, 196
Bunge's mass action theory, 1 26
Biirker's hsemocytometer, 228
Butter, composition of, 141, 238
Butyric acid, 8, 238, 243, 284, 309
Butyrin, 141, 238
Bynin, 93, 112
Cadaverin, 8 s
Calcium and magnesium in urine, 284, 320
carbonate in urinary sediments, 362, 363
casein, 128, 236
oxalate, 362
in urinary sediments, 362
phosphate in urinary sediments, 362, 364
in milk, 235, 242
sulphate in urinary sediments, 362, 364
Calculi, biliary, 161, 165
urinary, 379
calcium carbonate in, 380
oxalate in, 380
cholesterol in, 382
cystine in, 360
fibrin in, 380
indigo in, 382
phosphates in, 380
uric acid and urates in, 380
urostealiths in, 380
xanthine in, 3-80
Calliphora, larvae of, formation of fat from
protein by, 143
Cane sugar (see Sucrose, p. 46)
Canton silk, 64, 74
Caproic acid, 23s, 238
Carbamic acid, 196
Carbocyclic nucleus, 73, 74
Carbohydrates, 25
classification of, 25
composition of, 25
review of, 57
scheme for detection of, 58
variation in solubility of, 26
Carbonates in urine, 284, 321
Carbon moiety of protein molecule, 142
Carbon monoxide, haumoglobin, 216
tannin test for, 217
Carboxyl group, 4, 75, 95
INDEX.
459
Carboxylase, 4, 9, 36, .5,?i, 389
Carnine, 255
Carnitine, 255
formula for, 260
Camomuscarine, 255
Camosine, 255, 260
Cartilage, 250
constituents of, 250
experiments on, 250
Hopkins-Cole reaction on, 251
loosely combined sulphur in, 251
Millon's reactiorPon, 251
preparation of gelatin from, 251
solubility of, 250
xanthoproteic test on, 251
Casein, 72, 128, 236
calcium, i 28. 236
decomposition of, 72
quantitative determination of, 438, 439
soluble, 128, 236
Caseinogen, 94, 95, 128, 236, 438
action of rennin upon, 128, 236
biuret test on, 241
Millon's test on, 241
precipitation of, 241
preparation of, 241
quantitative determination of, Hart's method
for, 439
solubility of, 241
test for loosely combined sulphur in, 241
test for phosphorus in, 242
Casts, 369. 371
blood, 369, 3 73
epithelial, 369, 373
fatty, 369, 3 73
granular, 369, 372
hyaline, 369, 371
pus, 369, 3 74
waxy. 369, 373
Casts in urinary sediments, 369, 371
Cat gut, 13s
Catalase, 4, 17, 23
experiments on, 17, 23
quantitative determination of, 23
Catalysis, 2
Cellulose, 26, S3
action of Schweitzer's reagent on, 54
hydrolysis of, 54
iodine test on, 54
solubility of, 54
solvents, 55
utilization by animals, 53
Cellulose group, 26
Cerebrin (cerebroside), 268, 272
experiments on, 272
hydrolysis of, 272
microscopical examination of, 273
preparation of, 272
solubility of, 272
Cerebro-spinal fluid, choline in, 269
Cerebrosides, 268
Charcot-Leyden crystals, 18 1
form of, 181
Chlorides in urine, 284, 316
detection of, 317
quantitative determination of, 417
Cholecyanin, 16 1
Choleprasin, 160
Cholera-red reaction for indole, 176
Cholesterol, 162, 165, 268. 271, 362, 366
crystalline form of, 1 66
formula for, 270
iodine-sulphuric acid test for, 165, 272
isolation of, from biliary calculi, 165
Liebermann-Burchard test for, 165, 272
occurrence of, in urin^y sediments, 362, 366
origin of, 271
preparation of, from nervous tissue, 271
Salkowski's test for, 166, 272
SchifT's reaction for, 166, 272
tests for, i6s, 272
Choletelin, 160
Choline, 269, 273
formula for, 269
tests for, 273
Chondrigen, 1 13
Chondroalbumoid, 250
Chondromucoid, 113, 250
Chondroitin, 250
Chondroitin-sulphuric acid, 250, 283, 303
Chondrosin, 250
Chromoproteins, see Hemoglobins, 94, 95
Chyle, 206
CipoUina's test, 29, 325
Clark's modification of Dehn's method for
determination of chlorides, 417
Cleavage products of protein (see Decompo-
sition products), 69, 72
Clupeine, 72, 93, 95
Coagulated proteins, 94, 117
biuret test on, 1 19
digestion of, 20
formation of, 1 1 7
Hopkins-Cole reaction on, 1 19
Millon's reaction on, 119
solubility of, 119
xanthoproteic reaction on , 119
Coagulation of blood, 203
Howell's theory of, 203
Coagulation of proteins, 106, 117
changes in composition during, 117
fractional, 106, 117
Coagulation temperature of proteins, 106, 117
apparatus used in determining, 106
method employed in determining, 106
Co-enzyme, 7,12
Collagen, 93, 112, 247
experiments on, 247
percentage of, in ligament, 249
in tendon, 246
production of gelatin from, 248
solubility of. 248
transformation of, 247
Collodion dialyzer, 30
Colloidal solution, 23s
Colloids, 235, 2S5
tissue, 268
Colostrum, 236, 238
microscopical appearance of, 239
Combined hydrochloric acid (protein salt), 61, 126
130, I3S, 445
preparation of, 445
tests for, 130
Compound test for lactose in urine. 355
Congealing-point of fat, 147
Congo red, as indicator, 131, 132
460
INDEX.
Congo red, preparation of, 132
Conjugated proteins, 94, 95, 112
classes of, 94, 95, 112
nomenclature of, 94, 112
occurrence of, 112
Conjugate glycuronates, 32, 323, 327, 351
fermentation-reduction test for, 351
Tollens' reaction on, 352
Connective tissue, 245
Constipation, aid in, 56, 180
Cowie's guaiac test, 186
Creatine, 196, 255, 257, 283, 323, 416
crystalline form of, 254
formula for, 260
quantitative determination of, 416
separation of, from meat extract, 264
Creatinine, 32, 255, 283, 294, 415
coefficient, definition of, 294
crystalline form of, 295
daily excretion of, 294
as influenced by muscular tonus,
296
experiments on, 296
formula for, 260, 294
Jaffe's reaction for, 297
quantitative determination of, 415
Salkowski's test for, 297
separation of, from urine, 296
Weyl's test for, 296
Creatinine-zinc chloride, formation of, 295, 296
Cresol, para, 169
tests for, 177
Cross and Sevan's reagent, 54
preparation of, 54
solubility test, S4
Cryoscopy, 279
Cul-de-sac, 125
Cupri-potassium biuret, formation of, 99
formula for, 99
Cyanuric acid, 286
formula for, 286
Cylindroids in urinary sediments, 369, 376
a-Cyprinine, 73
Cystine, 72, 74, 80, 362, 366
crystalline form of, 81
detection of, 366
formula for, 80
in urinary sediments, 363, 366
Cytoglobulin, 94, 95
Cytosine, 113
Wheeler- Johnson reaction for, 113
Dakin's methods for quantitative determination
of hippuric acid, 406
Dare's haemoglobinometer, 222
description of, 222
determination of haemoglobin by, 222
Darmstadter's method for determination of
^-oxybutyric acid, 426
Deamidizing enzyme, 3, 4
Decomposition products of proteins, 68, 69, 72, 76
crystalline forms of, 77-89
experiments on, 89
isolation of, 89
Degradation products of protein (see Decompo-
sition products, 68)
Dehn-Clark method for chlorides, 417
Dehn's reaction for hippuric acid, 300
Delusive feeding experiments, 125
Derived proteins, 94, 114
Detection of preservatives in milk, 243
boric acid and borates, 244
formaldehyde, 243
hydrogen peroxide, 244
salicylic acid and salicylates, 243
Deuteroproteose, 94, 95, 121
Dextrin, 26, 48, 52
achroo-, 48, 61
a-achroo-, 61
/3-achroo-, 61
/•-achroo- 61
erythro-, 48, 61
action of tannic acid on, 53
diffusibility of, 53
Fehling's test on, 53
hydrolysis of, 53
iodine test on, 52
solubility of, 52
Dextrosazone, crystalline form of, Plate III,
opposite p. 28
Dextrose, 25, 27, 323
Allen's modification of Fehling's test for,
329
Barfoed's test on, 36, 331
Boettger's test on, 34, 330
Bottu's test on, 29, 325
CipoUina's test on, 29, 325
Benedict's modification of Fehling's test,
33, 328
diffusibility of, 30
experiments on, 27, 324
Fehling's test on, 32, 327
fermentation of, 35, 331
formula for, 27
iodine test on, 29
Molisch's reaction on, 27
Moore's test on, 30
Nylander's test on, 34, 330
phenylhydrazine test on, 28, 324
quantitative determination of, 384
reduction tests on, 30, 325
Riegler's reaction, 29, 325
solubility of, 27
Trommer's test on, 31, 326
Dextrosazone, crystalline form of, Plate III,
opposite p. 28
Diacetic acid, 323, 348
Amold-Lipliawsky test for, 349
formula for, 348
Gerhardt's test for, 348
quantitative determination of, 425
Diamino acid nitrogen, 69
Diaminotrihydroxydodecanoic acid, 72, 73, 89
a-£-di-amino-caproic acid, 73, 8s
Dialysis, 30
Dialyzers, preparation of, 30
Diastase (see Vegetable amylase, 3, 4, 10)
Diazo-benzene-sulphonic acid, 359
reagent, preparation of, 359
Diazo reaction (Ehrlich's), 359
Differentiation between pepsin and pepsinogen,
i27> 134
Digestion, gastric, 124
pancreatic, 148
salivary, 59
Di-iodo-hydroxypropane (lothion), 35, 330
INDEX.
461
Di-methyl-amino-azobenzene (see Topfer's re-
agent), 13 a
Dipeptides, 62, 71, 75, 95
Disaccharides, 25, 43
classification of, 25
Dissociation products of protein (see Decom-
position products, 68)
Doremus-Hinds ureometer, 395
Drying method for determination of total solids
in urine, 434
Duodenum, epithelial cells of, 148
Earthy phosphates in urine, 284, 317
quantitative determination of, 413
Edestan, 20, 94, 115
experiments on, 115
Edestin, 72, 93, 109
coagulation of, in
crystalline forms of, no
decomposition of, 72
microscopical examination of, in
Millon's test on, in
preparation of, no
solubility of, in
tests on crystallized, in
filtrate of, in
Ehrlich's diazo-benzene-sulphonic acid reagent,
preparation of, 359
Ehrlich's diazo reaction, 359
Ehrlich's mechanical eye-piece, use of, 228
Einhom's bead test, 189
Einhom's saccharometer, 36
Elastin, 93, 112, 249
adsorption of pepsin, by, 249
experiments on, 249
preparation of, 249
solubility of, 249
Electrical conductivity of urine, 281
Electrolj-tes, influence on enzyme activity, 7, 151
Embryos, glycogen in, 256
Emulsin, 4
Enterokinase, 4, 150
Enzymes, i
activation of, 6
adsorption of, 6
classification of, 4
definition of, 2
experiments on, 10
influence of electrolj-tes, 7, 151
list of, 4
preparation of, 5
properties of, 5
reference books, 9
Epiguanine, 284, 312
Episarkine, 284, 312
Epithelial cells in urinary sediments, 369
casts in urinary sediments, 369, 370
Epithelial tissue, 24s
experiments on, 245
Erepsin, 4, 15, 152
experiments on, 15
Erythrocytes, 194, 196, 197, 224
counting the, 224, 228
diameter of, 196
form of, 196
influence of osmotic pressure on, 208
in urinary sediments, 369, 376
number of, per cubic mm., 197
Erythrocytes of diflerent species, 196
stroma of, 201
variation in number of, 197
Erythro-dextrin, 48, 6 r
Esbach's albuminometer, 384
method for determination of albumin, 384
reagent, preparation of, 384
Ester, definition of, 139
hydrochloric acid, of ha;matin, 212
sulphuric acid, of haematin, 212
Ethereal sulphates, 283, 297
quantitative determination of, 409
Ethereal sulphuric acid, 169, 283, 297
Ethyl butyrate test for pancreatic lipase, 157
Euglobulin, 194
Excelsin, no
crystalline form of, no
Extractives of muscular tissue, 255
nitrogenous, 255
non-nitrogenous, 255
Fatigue substances of muscle, 260
Fats, 139
absorption of, 141
apparatus for determination of melting-
point of, 146
chemical composition of, 139, 140
congealing-point of, 146
crystallization of, 141, 144
digestion of, 141
emulsification of, 141, 143
experiments on, 143
formation of from protein, 142
formation of acrolein from, 143
hydrolysis of, 140
in milk, 23s, 238, 242
in urine, 323, 353, 369, 373
melting-point of, 146
nomenclature of, 140
occurrence of, 139
permanent emulsions of, 141, 143
quantitative determination of, in milk, 435
rancid, 141
reaction of, 141
saponification of, 140, 144
solubiUty of, 141, 143
transitory emulsions of, 141, 143
Fat-splitting enzymes (see Lipases, 3, 4, 12)
Fatty acid, 139, 140, 145
Fatty casts in urinary sediments, 369, 373
Fatty degeneration, 142
Fecal amylase, quantitative determination of, 189
Fecal bacteria, 181, 191
Fecal bacteria, quantitative determination of, 191
Feces, 178
agar-agar, influence of, 180
bacteria in, 181, 191
blood in, 181, 185
daily excretion of, 178
detection of albumin and globulin in, 188
bile acids in, 187
bilirubin in, 187
caseinogen in, 187
cholesterol in, 185
hydrobilirubin in, 186
inorganic constituents of, 188
nucleoprotein in, 187
proteose and peptone in, 188
462
INDEX.
Feres, enzymes of, 182
experiments on, 184
"fasting," 183
form and consistency of, 180
hydrogen ion concentration of, 180
macroscopic constituents of, 181
microscopic constituents of, 181
nitrogen of, 182
odor of, 179
parasites and ova in, 183
pigment of, 178
reaction of, 1 80
Scybala form of, 180
separation of, importance of, 180, 189
separation of, experiment on, 189
Fehling's method for determination of dextrose,
384
Benedict's modifications of,
38s
solution, preparation of, 32, 327
test, 32, 327
Allen's modification of, 329
Benedict's modifications of, 3 28
Ferments, classification of, 4
Fermentation, " sugar-free," 9, 36
Fermentation • method for determination of
dextrose, 388
Fermentation-reduction test for conjugate
glycuronates, 351
Fermentation test, 36, 331
Ferric chloride test for thiocyanate in saliva, 64
Fibrin, 19s, 203, 214, 369, 378
in urinary sediments, 369, 378
separation of, from blood, 195, 203
solubility of, 214
Fibrin ferment, 19s, 203
Fibrin-heteroproteose, 73
Fibrinogen, 19s, 203
Fibroin, Tussah silk, 73
Fischer apparatus, 80
photograph of, 80
Fleischl's hsemometer, 220
description of, 220
determination of ha;moglobin by, 220
Pleischl-Miescher hsemometer, 221
Fluorides in urine, 284, 322
Fly-maggots, experiments on, 142
Folin-Hart method for determination of com-
bined acetone and diacetic acid, 421
for determination of diacetic acid, 425
Folin-Messinger-Huppert method for deter-
mination of diacetic acid, 425
Folin's method for determination of acetone, 423
acidity of urine, 427
ammonia, 399
creatinine, 415
ethereal sulphates, 409
inorganic sulphates, 409
total sulphates, 408
urea, 394
Folin and Denis' method for urea, 398
Folin and Macallum's method for ammonia, 400
Folin and Pettibone's method for urea (No. i),
397
Folin and Pettibone's method for urea (No. 2),
397
Folin, Benedict and Myers' method for deter-
mination of creatine, 416
Folin-Shaffer method for determination of uric
acid, 389
Foreign substances in urinary sediment, 369, 3 78
Formation of methylphenylkevulosazone, 40
Form elements of blood, 194
Formic acid, 284, 309
Fractional coagulation of proteins, 117
Free hydrochloric acid, 126
tests for, 130
Freezing-point of bile, 159
blood, 194
milk, 23 s
pancreatic juice, 149
urine, 279
Fuchsin-frog experiment, 262
Fuld and Levison's method for peptic activity,
20
Fundus glands, 125
Furfurol solution, preparation of, 164
Fusion mixture, preparation of, 271
Galactans, 26, 55
Galactase, 239
Galactose, 25, 41, 323, 355
experiments on, 41
Gallic acid test for formaldehyde, 243
Ganassini's test, 293
Gastric digestion, 124
conditions essential for, 134
general experiments on, 134
influence of bile on, 136
influence of water on, 124
influence of different temperatures on,
134
most favorable acidity for, 134
power of different acids in, 13s
products of, 127, 130
Gastric fistula, 125
Gastric juice, 125
acidity of, 126
influence of water on, 124
artificial, preparation of, 129
composition of, 125
enzymes of, 125
lactic acid in, test for, 136
origin of hydrochloric acid of, 126
quantitative analysis of, 440
quantity of, 125
reaction of, 126
secretion of, 124
influence of water on, 124
specific gravity of, 125
Gastric lipase, 129
Gastric protease, 1 1
Gastric rennin, 125, 128, 136
action of, upon caseinogen, 128, 136, 236,
241
experiments on, 136, 241
influence of, upon milk, 136, 241
in gastric juice, absence of, 129
nature of action of, 128, 236
occurrence of, 128, 236
Gelatin, 72, 74, 247, 248
coagulation of, 248
decomposition of, 72
experiments on, 248
formation of, 247
Hopkins-Cole reaction on, 248
INDKX.
463
Gelatin, Millon's reaction on, J48
precipitation of, by alcohol, 249
alkaloidal reagents, 248
metallic salts, 248
precipitation of, by mineral acids, 248
preparation of, from cartilage, 251
from collagen, 248
salting-out of, 248
solubility of, 248
Gerhardt's test for diacetic acid, 348
Gerhardt's test for urobilin, 311
Gies' biuret reagent, preparation of, 99
Gliadin, 93, 112
decomposition of, 72
Globin, 72, 93, 197
decomposition of, 72
Globulins, 93, 95, 109
experiments on, 109
preparation of, 109
serum, 93. 194. 3^i. 332
in urine, 323, 332
tests for, 332
vegetable, 109
Glucoproteins (see Glycoproteins, p. 94, 112)
Glucose (see Dextrose, p. 25, 27, 323)
r.lucothionic acid, 371
Glutamic acid, 74, 87, 149
formula for, 87
Glutelins, 93, 95, 1 1 1
Glutenin, 93, 95, m
Glycerol, 139, 146
borax fusion test on, 146
experiments on, 146
formula for, 143
Glycerol extract of pig's stomach, preparation
of, 130
(jlycerophosphoric acid, 268, 269, 284, 310
Glycocholic acid, 159
Glycocholic acid group, 159
Glycocoll, 74, 77, 159, 168
crystalline form of, 167
formula for, 77, 159, 168
preparation of, 167
Glycocoll ester hydrochloride, crystalline form
of, 7 7
Glycogen, 26, 52, 255, 256
experiments on, 263
hydrolysis of, 264
in embryos. 256
influence of saliva on, 264
iodine test on, 263
preparation of, 263
Glycogenase, 4
Glycol>'tic enzymes, 4
Glycoproteins, 94, 112, 247
experiments on, 247
hydrolysis of, 247
Glycosuria, alimentary, 27
Glycosuric acid, 306
(jlycuronates, conjugate, 32, 323, 327, 351
Glycuronic acid, 42, 353
Glycyl-glycine, formation of, 75
Glycyl-tryptophane test, 15, 154
Glyoxylic acid, 97, 98
formula for, 97, 98
Gmelin's test for bile pigments, 162, 342
Rosenbach's modification of, 162, 342
Granular casts in urinary sediments, 369, 372
Granulose, 48
Green stools, cause of, 179
Gross' method for quantitative determination of
tryptic activity, 22
Guaiac solution, preparation of, 446
Guaiac test on blood, 204, 209
on feces, 186
milk, 240
pus, 371
in urine, 341
Guaiac test, Schumm's modification of, 209
Guanase, 4
Guanidine-a-amino- valerianic acid, 73, 83
Guanidine-residue, 69
Guanine, 4, 255, 261
Gum arabic, 26, 55
Gums and vegetable mucilage group of carbo-
hydrates, 26
Gunning's iodoform test for acetone, 346
Giinzberg's reagent, as indicator, 131
preparation of, 131
Gurber's reaction for indican, 299
Haemagglutination, 197, 208
Haemagglutinin, 197, 208
Haematin, 114, 197, 212
^cid-, 219
alkali-, 218
preparation of, 212
reduced alkali-, 218
Haematoidin, 160, 161, 179
crystalline form of, 161, 179
in urinary sediments, 362, 367
Hsematuria, 339
Haematoporphyrin, 202, 219, 323, 353
in urine, 274, 323, 353
Hsemin crystals, form of, 204
test, 210
Haemochromogen, 114, 197, 213
Haemocyanin, 94, 95, 114
Hjemocytometer, Biirker's, 228
Thoma-Zeiss, 224
Hasmoconein (see Blood dust, 194, 203)
Haemoglobin, 94, 95, 112, 114
carbon monoxide, 202, 216
decomposition of, 197
diffusion of, 209
met, 202, 2t8
oxy, 197, 201, 216
quantitative determination of, 220, 222
reduced. 197, 216
Haemoglobins, 94, 112
Haemoglobinuria, 316
Haemolysis, 194, 207
Hffiser's coefficient, 279, 434
Hair, human, 74, 245
Hammerschlag's method for determination of
specific gravity of blood, 206
Hammarsten's reaction, 163, 343
reagent, preparation of, 163, 343
Hart's caseinogen method, 439
Hayem's solution, 229
Hay's test for bile acids, 164, 344
Heintz method for determination of uric acid,
290, 390 1^
Helicoprotein, 94
Heller's test for blood in urine, 340
Heller-Teichmann reaction for blood in urine, 340
464
INDEX.
Heller's ring test for protein, 104, 333
Hemicellulose, 26, ss
experiments on, 56
utilization of, by animals, 55
Hemiurate, 365
Herter's naphthaquinone reaction for indole, 175
Herter's para-dimethylaminobenzaldehyde reac-
tion, 176
Heterocyclic nucleus, 73, 74
Heteroproteose, 95
Heteroxanthine, 284, 313
Hexone bases, 8s
Hexosans, 26, 55
Hexoses, 25, 26
Hippuric acid, 168, 283, 300, 406
crystalline form of, 300
Dakin's method for quantitative determi-
nation of, 406
Dehn's reaction for, 302
experiments on, 168, 277, 301
formula for, 168, 300
in urinary sediments, 367
melting-point of, 302
Roaf's method for crystallization of, 301
separation of, from urine, 301
solubility of, 302
sublimation of, 302
synthesis of, 168
Histidine, 73, 82, 149
hydrochloride, crystalline form of, 83
Knoop's color reaction for, 82
Histones, 93, 95
Hoffmann's reaction for tyrosine, 91
Homogentisic acid, 32, 283, 306, 327
formula for, 306
Hopkins-Cole reaction, 98
on solutions, 98
on solids, 108
Hopkins-Cole reagent, preparation of, 98
Hopkins-Cole reagent (Benedict modification),
preparation of, 98
Hordein, 73, 93, 112
Horismascope (see Albumoscope, 104)
Hormones, definition and discussion of, 148, 237
Hopkins' thiophene reaction for lactic acid, 137
Hiifner's urea apparatus, 394
Human fat, composition of, 140
hair, composition of, 245
Huppert's reaction for bile pigments, 162, 342
Hurthle's experiment, 267
Hyaline casts in urinary sediments, 369, 371
Hydrobilirubin, detection of, in feces, 179
extraction of, 186
Hydrochloric acid of the gastric juice, 126
origin of, theories as to, 1 26
seat of formation of, 125
Hydrochloric acid test for formaldehyde (Leach) ,
243
Hydrogen peroxide in urine, 284, 322
detection of, in milk, 244
Hydrolysis of cellulose, 54
cerebrin, 273
dextrin, 53
glycogen, 264
inulin, 51
proteins, 69
starch, 50
sucrose, 47
Hyperacidity, 126
Hypoacidity, 126
Hypobromite solution, preparation of, 392
Hypoxanthine, 255, 261, 265, 284, 312
formula for, 261
Hypoxanthine silver nitrate, crystalline form of,
26s
Ichthulin, 94
Ignotine, 255
formula for, 260
Imide bonds, 75
Iminazolethylamine, 171
Iminazolpropionic acid, 171
Indican, 169, 283, 297, 416
Barberio's reaction, 300
formula for, 169, 298
Gurber's reaction for, 299
Jaffe's test for, 298
Lavelle's reaction for, 299
Obermayer's test for, 299
origin of, 169, 297 •
Rossi's reaction for, 299
Indicators, theory of, 130
table of, 131
Indigo-blue, 170, 299
formula for, 170, 299
Indigo in urinary sediments, 362, 368
Indolacetic acid, 171, 407
Indole, 169, 17s, 179
formula for, 169
origin of, 169, 179
test for, 17s
Indole- a-amino-propionic acid, 73, 82
Indolpropionic acid, 171
Indoxyl, 169, 299
formula for, 169, 299
origin of, 169, 298
potassium sulphate (see Indican, pp. 169,
283, 297, 416
Indoxyl-sulphuric acid, 169, 298
formula for, 169, 298
Infraproteins (see Metaproteins, 94, 95, ns)
Inorganic physiological constituents of urine, 284,
313
Inosinic acid, 255, 260
formula for, 260
Inosite, 25, 323, 357
formula for, 357
in urine, 323, 357
Intestinal juice, 150
enzymes of, 152
preparation of, 13
Inulase, 51
Inulin, 26, so
action of amylolytic enzymes on. Si. 65
Fehling's test on, 51
hydrolysis of, si
iodine test on, 51
reducing power of, 51
solubility of, 51
sources of, 50
Inversion, 46, 48
Invertase (see Sucrase, s. 46)
Invertases, experiments on, 13
Invertin (see Sucrase, 4, 5, 46)
Inverting enzymes, 3
Invert sugar, 46
INDKX.
405
Iodide of dextrin, s»
of starch, 50
Iodine test, 29, 50, S«i S3. 54. 56
Iodine-sulphuric acid test for cholesterol, 165, 272
lodine-zinc-chloride reaction, 54
Iodoform test for alcohol, 47
lodothymol compound, 347
"lothion," 35, 330
Iron in blood, 207
detection of, 207
in bone ash, 253
detection of, 253 "*
in protein, 68
in urine, 284, 321
detection of, 321
Isoleucine, 73, 85
Isomaltose, 25, 44, 61
Iso valerianic acid, 171
Jacoby-Solms method, 21
Jaffe's reaction for creatinine, 297
Jaflfe's test for indican, 298
V. Jaksch-Pollak reaction for melanin, 358
Jejunum, epithelial cells of, 148
Jolles' reaction for protein, 105, 334
reagent, preparation of, 105, 334
Juice, gastric, 124, 125, 127
pancreatic, 148, 150
intestinal, 150
Kantor and Gies's biuret paper, 99
Kastle's peroxidase reaction, 240
Kephalin, 268, 270
Kephyr, 45
Keratin, 93, 112, 245, 246
experiments on, 246
solubility of, 246
sources of, 245
sulphur content of, 245, 246
Ketone, 25, 30
Ketose, 25
Kjeldahl method for determination of nitrogen,
401
Kjeldahl-Folin-Farmer nitrogen method, 402
Knoop's color reaction for histidine, 82
Knop-Htifner hypobromite method for determi-
nation of urea, 392, 393
Konto's reaction for indole, 176, i88
Koppe's electrolytic dissociation theory. 1 26
Koumyss, 45
Kraut's reagent, preparation of, 273
Kreosotal, 352
Kruger and Schmidt's method for the quantita-
tive determination of
purine bases, 429
of uric acid,.39i
Kiilz's test for )9-oxybutyric acid, 351
Kwilecki's modification of Esbach's method. 384
Kynurenic acid, 283, 307
formula for, 307
isolation of, from urine. 307
quantitative determination of, 307
Laccase, 4
Lactalbumin, 93, 235, 238
quantitative determination of, 439
Lactase, 4, 14, 152
experiments on, 14
30
Lactic acid, 45, 136, 256
ferric chloride test for, 137
Hopkins' thiophene reaction for, 137
in muscular tissue, 256
in stomach contents, 136, 137
tests for, 136
Uffelmann's test for, 136
Lacto-globulin, 235, 238
Lactometer, determination of specific gravity of
milk by, 435
Lactosazone, crystalline form of, Plate III, oppo-
site p. 28
Lactoscope, Feser's, 438
Lactose, 25, 45, 23s. 237
experiments on, 45
fermentation of, 45
in urine, 323, 354
quantitative determination of, 440
Lactosin in milk, 238
Laevo- a-proline, 88
Laevulosazone, crystalline form of, Plate III, op-
posite p. 28
Ljevulose, 25, 39
Borchardt's reaction for, 40
in urine, 323, 355
methyl-phenylhydrazine test for, 40
Seliwanoflf's reaction for, 40
Laiose in urine, 323, 358
"Laked" blood, 194, ao?
Laky blood, 207
Laurie acid, 235
Laurin, 139
Lavelle's reaction for indican, 299
Leach's hydrochloric acid test for formaldehyde,
243
Lecithans, 94
Lecithin, 94, 196, 268, 269
acrolein test on, 271
decomposition of, 269
experiments on, 271
formula for, 269
microscopical examination of, 271
osmic acid test on, 271
preparation of, 271
test for phosphorus in, 271
Lecithoproteins, 94, 112
Legal's reaction for indole, 176
test for acetone, 346
Leucine, 72, 74, 84, 91, 127, 149. 362, 367
crystalline form of impure, 367
pure, 8s
experiments on, 91
formula for, 84
in urinary sediments, 362, 367
microscopical examination of, 91
separation of, from tyrosine, 90
solubility of, 91
sublimation of, 91
Leucocytes, 194, 202
counting the, 227
number of, per cubic mm., 202
size of, 302
variation in number of, 202
Leucocytosis, 202
Leucosin, 103
Leucyl-alanyl-glycine, formation of, 75
Leucyl-glycyl-alanine, 62
Leucyl-leucine, formation of, 75
466
INDEX.
Lichenin, 26, 52
Lieben's test for acetone, 347
Lieberkuhn's jelly (see Alkali metaprotein, p.
117)
Liebermann-Burchard test for cholesterol, 165,
272
Liebermann's reaction, 100
Lipase, gastric, 125
Lipase, pancreatic, 4, 12, 149, 151
action of, in dilution, 151
experiments on, 12, 157
ethyl-butyrate test for, 157
litmus-milk test for, 157
Lipases, 4,12
experiments on, 12
Lipeses, 3
Lipins, 268
Lipoids of nervous tissue, 268, 271
Lipolytic enzymes (see Lipases, p. 4, 12).
"Litmus-milk" test for pancreatic lipase, 157
Long's coefficient, 278, 434
Lugol's solution, preparation of, 10 1
Lymph, 194, 205
Lysine, 72, 73, 85, 127, 149
Lysine picrate, crystalline form of, 86
Magnesia mixture, preparation of, 313, 414, 448
Magnesium in urine, 284, 320
phosphate in urinary sediments, 362, 368
Maltase, 4, 15, 44, 62, 152
experiments on, 15
Maltosazone, crystalline form of, Plate III, op-
posite p. 28
Maltose, 25, 44
experiments on, 44
structure of, 44
Marshall's urea apparatus, 392
Melanin in urine, 323, 358
urinary sediments. 362, 368
Melting-point apparatus, 146
of fats, determination of, 146
Messinger-Huppert method for determination of
combined acetone and diacetic acid, 422
Metaproteins, 94, 115
acid, 1 16
alkali, 1 17
experiments on, 116
precipitation of, 116
sulphur content of, 116
Methaemoglobin, 202, 218
Methylene blue, 135
Methyl-mercaptan, 169
Methyl-pentose (see Rhamnose, p. 25)
Methylphenylhydrazine. 40
Methylphenyllaivulosazone, formation of, 40
i-methylxanthin, 284, 312
Mett's method for determination of peptic
activity, 19
Mett's tubes, preparation of, 20
Micro-organisms in urinary sediments, 369, 378
Milk, 23s
citric acid in, 235
detection of calcium phosphate in, 242
lactose in, 242
preservatives in, 243
difiference between human and cow's, 237
experiments on, 239
formation of film on, 236, 239
Milk, freezing-point of, 235
guaiac test on, 240
influence of rennin on, 241
isolation of fat from, 242
Kastle's peroxidase reaction on, 240
Lactose in, 235, 237, 242
crystalline form of, 238
microscopical appearance of, 236, 239
preparation of caseinogen from, 241
properties of caseinogen of, 241
quantitative analysis of, 43 s
reaction of, 23s, 239
separation of coagulable proteins of, 242
specific gravity of, 235, 239
Millon's reaction, 97
reagent, preparation of, 97
Mohr's method for determination of chlorides,
418
Molisch's reaction, 27
Molybdic solution, preparation of, 64
Monamino acid nitrogen, 69
Monosaccharides, 25, 26
Barfoed's test for, 36, 331
classification of, 25
Moreigne's reaction for uric acid, 293
reagent, preparation of, 293
Morner-Sjoqvist-Folin method for determination
of urea, 395
Morner's reagent, preparation of, 91
test for tyrosine, 91
Motor and functional activities of the stomach,
136
Mucic acid, 41, 45, 354, 355
test, 41, 45, 354, 355
Mucin, 60, 63, 94, 112
biuret test on, 63
hydrolysis of, 64
isolation of, from saliva, 63
Millon's reaction on, 63
Mucins, 94, 112
Mucoid, 94, 112, 246, 247, 249
experiments on, 247
hydrolysis of, 247
in urine, 308, 323, 319
preparation of, from tendon, 247
Mucoids, 94, 1 12
Murexide test, 292
Muscle plasma, 254, 262
formation of myosin clot in, 254, 262
fractional coagulation of, 254, 261
preparation of, 262
reaction of, 254, 261
Muscular tissue, 254
ash of, smooth and striated, 259
commercial extracts of, 260
experiments on "dead," 263
"living," 261
extractives of, 255, 264
fatigue substances of, 260
formulas of nitrogenous extractives of,
260
glycogen in, 256, 263
involuntary, 254
lactic acid in, 255, 256
nonstriated, 254
pigment of, 260
preparation of glycogen from, 263
muscle plasma from, 261, 262
INDEX.
467
Muscular tissue, proteins of, 254
reaction of living, 257
rigor mortis of, 254
separation of extractives from, 264
striated, 254
voluntary, 254
Myohaematin, 260
Myosan, 94
formation of, 26,?
Myosin, 254
biuret test gn, 26,?
coagulation of, 26,5
preparation of, 26.5
solubility of, 26.?
Myosinogen, 254
Myristic acid, 235
Myristin, 139
Myrtle wax (see Bayberry tallow. 144)
Nakayama's reaction for bile pigments, 162. 342
reagent, preparation of, 162, 342
Nencki and Sieber's reaction for urorosein, 359
Neosine, 255
formula for, 260
Nervous tissue. 268
constituents of, 268
experiments on lipoids of, 271
lipoids of, 268, 27 I
percentage of water in, 268
phosphorized fats of, 268
proteins of, 268
Nessler-Winkler solution, 404
Neurokeratin, 268
Neutral fats, 139, 141, 143
Neutral olive oil, preparation of, 143
Neutral sulphur compounds, 283, 303
Nitrates in urine, 284, 322
Nitrilase,3
Nitrilese, 3
Nitrites in saliva, test for, 64
Nitrogen, 68
forms of in protein molecule, 69
importance of, in sustaining life, 69
in urine, quantitative determination of, 401
Nitrogen distribution, calculation of, 402
Nitrogen iodide, formation of, 346
Nitrogenous extractives of muscular tissue, 255,
264
formulas for, 260
Nitroso-indole nitrate test, i 76
Nitrosothymol, formation of in Heller's test, ^i^:^
Non-nitrogenous extractives of muscular tissue,
355
Normal urine, 274
characteristics of, 274
constituents of, 283
experiments on, 287
Novaine. 255
formula for, 260
Nubecula, 308, 339
Nuclease, 4
Nucleic acid, 94, 113
Nucleins, 113, 130
Nucleohistone, 93, 95
Nucleoproteins. 94, 95, 112, 162, 283, 308. 323,
339
in bile, 162
in feces, 187
Nucleoproteins in nervous tissue, 268
in urine, 283, 308, 323, 339
lest for, 339
occurrence of, i 1 3
Ott's precipitation test for, 339
Xylander's reagent, preparation of, 34, 330
test, 34, 330
Obermayer's test for indican, 299
reagent, preparation of, 299
Oblitine, 255
"Occult" blood in feces, 181, 185
tests for, 185
Olein, 140, 235
Olive oil, 143
emulsification of. 143
neutral, preparation of, 143
Opalisjn in milk, 238
Optical methods, 39, 194
Orcinol test. 43, 353
Organic physiological constituents of urine, 283
Organized ferments, i
Organized urinary sediments, 361, 369
Ornithine, 70, 171
Osborne-Folin method for determination of total
sulphur in urine, 410
Ossein, 25 i
preparation of, 251
Osseoalbumoid, 251
Osseomucoid, 94, 113, 251
chemical composition of, 113
Osseous tissue, 251
experiment on, 252
Ott's precipitation test for detection of nucleo-
protein in urine, 339
Ovalbumin, 93
Ovoglobulin, 93
Oxalated plasma, preparation of, 214
Oxalic acid. 283, 302. 433
formula for, 302
in urine, 283, 302
quantitative determination of, 433
Oxaluria, 303
Oxaluric acid, 283, 308
Oxamide, 98
Oxidases, 4, 239
Oxyacids, 169, 177, 283, 306
tests for, 177
;J-oxybutyric acid, 323, 349, 42s
Black's method for determination of,
426
Black's reaction for, 350
formula for, 349
Kiilz's test for, 351
origin of, 350
polariscopic examination for. 351
quantitative determination of. 425
Shaflfer's method for determination of,
425
Oxyha;moglobin, 69
Reichert's method for crystallization of, 214
crystalline forms of, 198-201
Oxymandelic acid, 283, 306
Oxyproline, 72, 73, 88
Oxyproteic acid, 283, 303, 359
Paduschka-Underhill-Kleiner method for quanti-
tative determination of allantoin. 432
468
INDEX.
Palmitic acid, 140, 145
crystalline form of, 145
experiments on, 14s
formula for, 140, 151
preparation of, 145
Palmitin, 140, 23s
Pancreatic amylase, 4, 150, 155
digestion of dry starch by, 151, is6
inulin by, 157
experiments on, 155
influence of bile upon action of, 136
metallic salts upon action of, 156
most favorable temperature for action
of, iSS
Pancreatic digestion, 148
general experiments on, 154
products of, 149, 153
Pancreatic insufficiency, Schmidt's nuclei test for,
188
Pancreatic juice, 140, 148, 149, 150
artilcial, preparation of, iS3
daily excretion of, 149
enzymes of, 149
freezing-point of, 149
mechanism of, secretion of, 148
reaction of, 148
solid content of, 149
specific gravity of, 149
Pancreatic lipase, 4, 140, 150, 157
experiments on, 12, 157
ethyl-butyrate test for, i s 7
litmus-milk test for, i s 7
Pancreatic protease (see Trypsin, p. 11).
Pancreatic rennin, s, 149, 152
experiments on, 157
Papain, s, 11
Paracasein, 237
Para-cresol-sulphuric acid, 283, 297
Paradimethylamino benzaldehyde solution, prep-
aration of, 176
Paralactic acid, 236, 284, 309
Paramyosinogen, 2S4
Paranucleoprotagon, 268, 271
Paraoxyphenylacetic acid, 169, 171, 175, 283, 303
Paraoxy- ^-phenyl- o-amino-propionic acid, 74, 79
Paraoxyphenylpropionic acid, 169, 171, i7S. 283,
303
Paraphenylenediamine hydrochloride, 244
Parasites, 181, 183, 369, 378
Paraxanthine, 284, 312
Parietal cells, 125
Parotid glands, characteristics of saliva secreted
by, 59
Pathological constituents of urine, 323
Pathological urine, 274, 323
constituents of, 323
experiments on, 324
Pektoscope, 379
Pentamethylenediamine, 1 7 1
Pentapeptides, 71, 9S
Pentos3S, 25, 41
experiments on, 42
in urine, 323, 352
tests for, 352
Pentosans, 26, 41, 55
Pepsin (see Gastric Protease), i, s. n, 12S. 127
action of, influence of bile upon, 127, 136
influence of different acids upon, 135
Pepsin, action of, influence of metallic salts upon,
135
temperature upon, 134
conditions essential for action of, 134
differentiation of, from pepsinogen, 127, 134
digestive properties of, 127
formation of, 127
most favorable acidity for action of, 134
presence of, in intestine, 128
proteolytic action of, 127
Pepsin-hydrochloric acid, 134
Pepsin -rennin controversy, 129
Pepsinogen, 6, 127, 130
differentiation of, from pepsin, 127, 134
formation of, 127
extract of, preparation of, 130
Peptic activity, Fuld and Levison's method for
determination of, 20
Mett's method for the determination of,
19
Rose's method for determination of, 21
Peptic proteolysis, 127
products of, 127
relation of, to tryptic proteolysis, 128
Peptides, 69, 71, 95, 121
Peptone, 69, 71, 9s, 119
ampho, 9S, 120
anti, 120
differentiation of, from proteoses, 120
experiments on, 120, 121
in urine, 323, 337
tests for, 337
separation of, from proteoses, 120
Periodide test for choline, 273
Peroxidases, 5, 239
Pettenkofer's test for bile acids, 163, 344
Mylius's modification of, 164,
344
Neukomm's modification of,
164, 344
Phenaceturic acid, 284, 309
Phenol, 169
tests for, 177
Phenolphthalein as indicator, 131, 133
preparation of, 133
test for blood in feces, 185
Phenol-sulphuric acid, 283, 297
Phenylacetic acid, 171
Phenyl- a-amino propionic acid, 74, 78
Phenylalanine, 69, 72, 74, 78
Phenyldextrosazone, 28
crystalline form of, Plate III, opposite p. 28
Phenylethylamine, 171
Phenylhydrazine, 28, 29
acetate solution, preparation of, 28
mixture, preparation of, 28
reaction, 28, 324
Cipollina's modification of, 29, 325
Phenyllactosazone, crystalline form of, Platd 111,
opposite p. 28
Phenylmaltosazone, crystalline form of Plate III,
opposite p. 28
Phenylpotassium sulphate, 297
Phenylpropionic acid, 171
Phosphates in urine, 276, 284, 317
detection of, 320
experiments on, 319
quantitative determination of, 413
INDEX.
469
Phosphatase, 3
Phosphatese, 3
Phosphatides. 94, 114. iS9. 27°
Phosphocamic acid, ass, 260, 284, 310
Phosphoproteins, 94, 9s. m. "4
Phosphorized compounds in urine, 284, 310
Physiological constituents of urine, 283
Phj-tasc, s
Pigments of urine, 274, 284, 310
Pine wood test for indole, 176
Piria's test for tyrosine, 91
Polariscope, use of, 36
in detection of conjugate glycuronates, 352
in determination of dextrose, 36, 332
^-oxbutyric acid, 3Si
Polypeptides, 69, 71, 95
Polysaccharides, 2s, 47
classification of, 25
properties of, 47
Posner's modification of biuret test, 100
Potassium in urine, 284, 320
Potassium indoxyl-sulphate (see .Indican, pp.
169, 283, 297, 416.)
formula for, 169, 298
origin of, 168, 297
tests for, 298
Potassium iodide test for albumin, 105, 336
Primary protein derivatives, 94, 114
Primary proteoses, 121
Products of protein hydrolysis, 69, 72, 73, 74
Prolamins. 93, iii
classification of, 93
Proline. 69, 72, 73. 88. iii, 127, 149
crystalline form of laevo-o-, 88
crystalline form of copper salt of, 89
Prosecretin, 148
Protagon, 268, 269
preparation of, 271
structure of, 270
Protamines, classification of, 93, 95
Proteans, 94, i is
Protease, gastric, 11
experiments on, 11
pancreatic, ii
experiments on. 11
vegetable, 11
Proteases, 1 1
experiments on. 1 1
Proteins, 68, 92, 323, 332
acetic acid and potassium ferro-cyanide
test for, 105
Acree-Rosenheim test on, 100
action of alkaloidal reagents on, 104
action of metallic salts on, 103
mineral acids, alkalies and organic acids
on, 103
Adamkiewicz reaction on, 97
Bardach's reaction on, 10 1
biuret test on, 98
chart for use in review of, 1 33
chemical composition of, 68
classification of, 93, 9s
coagulation, influence of salts upon, 117
coagulation or boiling test for, io6
color reactions of, 97
conjugated, 94, 95, 112
decomposition of, 68, 72, 73, 74
by hydrolysis, 69
Proteins, decomposition of, by oxidation, 69
products of, 69, 72, 73, 74, 76
experiments on, 89
separation of, 89
study of, 69, 72, 89
derived, 94, 114
formation of fat from, 142
formulas of, 69
Heller's ring test on, 104
importance of, to life, 68
Hopkins-Cole reaction on. 98
in urine, 323, 332
test for, 333
Liebermann's reaction on, 100
Millon's reaction on, 97
molecular weights of, 69
Posner's reaction on, 100
precipitation of, by alcohol, 107
alkaloidal reagents, 104
metallic salts, 103
mineral acids, 103
precipitation reactions of, T02
quantitative determination of, in milk, 438,
439
review of, 122
Robert's ring test on, 104
salts of, 102
salting-out experiments on, 106
scheme for separation of, 123
simple, 93, 95
synthesis of, 71, 75
xanthoproteic reaction on, 97
coagulated, 94, 117
biuret test on, 119
formation of, 117
Hopkins-Cole reaction on, 119
Millon's reaction on, 119
solubility of, 119
xanthoproteic reaction on, 119
Protein-coagulated enzymes, 3, 128, 152, is6
Proteins, conjugated, 94, 112
classes of, 94, 112
experiments on, 63, 113, 209, 241, 247
nomenclature of, 94, 112
occurrence of, 112
Protein-cystine, 8i
Protein derivatives, primary, 70, 94, 114
secondary, 70, 94, 119
Proteins of milk, 235, 236, 238
quantitative determination of, 438, 439
Proteolytic enzymes (see Proteases, p. ix)
Proteolysis, peptic, 127
tryptic, 128, 149
Proteose, 69, 94, 9S. i'9
v. Aldor's method for detection of, 338
biuret test on, 120
coagulation test on, 120
deutero, 94, 95, 120
differentiation of, from peptone, 1 20
experiments on, 120, 121
hetero, 95, 120
in urine, 3231, 33 7
test for, 337
potassium ferrocyanide and acetic test on,
121
powder, preparation of, 121
precipitation of, by nitric acid, lai
by picric acid, 121
470
IXDEX.
Proteose, precipitation of, by potassio-mercuric
iodide, 121
by trichloracetic acid, 1 2 1
primary, 121
proto, 94, 95, 121
Schulte's method for detection of, 338
secondary, 121
separation of, from peptones, 120
Protoproteose, 94, 95, 121
Proteoses and peptones, 94, 95, 119, 120
separation of, 1 20
tests on, 121
Proteose-peptone, 120
Proteose-peptone, coagulation test on, 120
experiments on, 120
Millon's reaction on, 120
precipitation of, by nitric acid, 120
by picric acid, 120
Prothrombin, 203, 204
Pseudo-globulin, 194, 195
Ptomaines and leucomaines in urine, 284, 312
Ptyalin (see Salivary amylase, 4, 60)
Purdy's method for determination of dextrose, 387
solution, preparation of, 387
Purine bases, 113, 284, 312, 428
in urine, quantitative determination of, 428
Pus casts in urinary sediments, 369, 374
Pus cells in urinary sediments, 369
Putrefaction, indican as an index of, 169, 297
Putrefaction mixture, preparation of a, 171
Putrefaction products, 169
experiments on, 171
most important, 169
tests for, 1 75
Pyloric glands, 124
Pyrocatechin-sulphuric acid, 283, 297
a-pyrrolidine-carboxylic acid (see Proline, pp. 69,
Quadriurate, 365
Qualitative analysis of the products of salivary
digestion, 67
stomach contents, 137
Quantitative analysis of blood, 442
of gastric juice, 440
of milk, 435
of urine, 383
Quantitative determination of ammonia in urine,
.599
amylolytic activity, 18
acetone in urine, 423
acetone and diacetic in urine, 421
acidity of urine, 427
allanto'in in urine, 432
amino nitrogen, 404
ash of milk, 438
caseinogen of milk, 438, 439
catalase, 23
chlorides in urine, 417
creatine in urine, 416
creatinine, 415
dextrose in urine, 384
diacetic acid in urine, 425
fat in milk, 435
fecal amylase, 189
fecal bacteria, 191
hippuric acid in urine, 406
indican in urine, 416
Quantitative determination of lactalbumin in
milk, 439
lactose in milk, 440
nitrogen in urine, 401
oxalic acid in urine, 433
^-oxybutyric acid in urine, 425
peptic activity, 19
phosphorus in urine, 413
protein in milk, 438
protein in urine, 383
purine bases in urine, 428
purine nitrogen, 431
sulphur in urine, 408
total solids in milk, 438
total solids in urine, 434
tryptic activity, 22
urea in urine, 392
uric acid in urine, 389
Quevenne lactometer, determination of specific
gravity of milk by, 438
Raflfinose, 25, 47
Rancid fat, 141
Raw and heated milk tests, 240
Reaction of the urine, 276, 317
Reduced alkali-hcematin, 219
Reduced haemoglobin, 216
Reductases, 239
Reichert's method for crystallization of oxy-
haemoglobin, 214
Remont's method for detection of salicylic acid
and salicylates, 243
Rennin, gastric, 125, 128
action of, upon caseinogen, 128, 236
experiments on, 136, 241
influence of, upon milk, 128, 236
in gastric juice, absence of, 129
nature of action of, 128, 236
occurrence of, 128
Rennin, pancreatic, 149, 152
experiments on, 157
Rennin-pepsin controversy, 129
Reticulin, 112
Reversibility of enzyme action, 8, 62
Reynolds-Gunning test for acetone, 347
Rhamnase, s
Rhamnose, 25, 43
Ricin, 12, 208
Riegler's reaction, 29, 325
Rigor mortis, 254
Ring test for urobilin, 312
Roaf's method for crystallizing hippuric acid, 301
Robin's reaction for urorosein, 359
Robert's ring test for protein, 104, 334
reagent, preparation of, 104, 334
Rosenheim's bismuth test for choline, 273
Rosenheim's periodide test for choline, 273
Rose's method for determination of pepsin, 21
Rossi's reaction for indican, 299
Rothera's reaction for acetone, 347
Rubner's test for lactose in urine, 354
Saccharide group, 26
Saccharose (see Sucrose)
Sahli's desmoid reaction, 135
Saliva, 59
alkalinity of, 60
amount of, 60
INDEX.
471
Saliva, bacteria in, 6.{
biuret test on, 6.5
calcium in, 64
chlorides in, 64
constituents of, 60
diKCStion of dry starch by, 65
diKestion of inulin by, 65
digestion of starch paste by, 61, 65
dilution of, influence on digestion, 61
enzymes contained in, 60
excretion of potassium iodide in, 67
inor»;anic matter in, tests for, 64
Millon's reaction on, 63
mucin from, preparation of, 63
nitrites in, test for, 64
phosphates in, test for, 64
potassium thiocyanate in, 60
reaction of, 60, 63
secretion of, 59
specific gravity of, 60, 63
sulphates in, test for, 64
tests for. 63
thiocyanates in. 60, 64
tripeptide-splitting enzymes in, 62
Salivary amylase, i, 4, 10, 60, 126
activity of, in stomach, 61, 126
inhibition of activity of, 61
nature of action of, 60, 61
products of action of, 6t
Salivary digestion, 59
influence of acids and alkalis on, 61, 66
dilution on, 61, 65
metallic salts on, 66
temperature on, 65
nature of action of acids and alkalis on,
66
qualitative analysis of products of, 67
Salivary digestion in stomach, 61, 126
Salivary glands. 59
Salivary stimuli, 59
Salkowski-Autenrieth-Barth method for deter-
mination of oxalic acid in urine. 433
Salkowski's method for determination of purine
bases. 430
Salkowski-Schippers reaction for bile pigments,
■ 63, 34,i
Salkowski's test for cholesterol, 166, 272
for creatinine, 297
Salmine, 85, 86, 72, 73, 93, 95
Saponification, 140, 144
of lard, 146
Salted plasma, preparation of, 214
Salting-out experiments on proteins, 103, 106
Sarcolactic acid, 256
Scallops, preparation of glycogen from, 263
Schalfijew's method for preparation of ha;niin,
212
Schema for "blood counting," 232
Scheme for analysis of biliary calculi, 165
bone ash, 253
stomach contents, 138
urinary calculi. 381
separation of carbohydrates, 58
of proteins, 1 23
Scherer's coagulation method for determination
of albumin in urine, 383
Schiffs reaction for cholesterol, 166, 272
for uric acid, 293
Schifl's reagent, preparation of, 166, 272
Schmidt's nuclei test for pancreatic insufficiency,
188
Schmidt's test for hydrobilirubin, 186
Schulte's method for detection of proteose in
urine, 338
Schumm's modification of the guaiac test, 209
Schutz's law, statement of, 8, 20
Schweitzer's reagent, action of, on cellulose, S4
preparation of, 54
Scleroproteins, 95 (see Albuminoids)
Scombrine, 72, 93
Scombrone, 93, 95
Scybala, 56, 180
Secondary protein derivatives, 70, 94, 119
Secondary proteoses, 1 2 1
Secretin, 148
Seliwanoff's reaction, 40, 356
reagent, preparation of, 40. 356
Separation of feces, importance of, in nutrition
and metabolism experiments, 180, 189
Serine, 69, 72, 74, 78
crystalline form of, 78
formula for, 78
Seromucoid, 94, 113, 196
Serum albumin, 93, 95, 194. 3^3, 33^
in urine. 323, 332
test for, 333
Serum globulin. 93, 194, 323, 332
in urine, 323, 336
test for, 336
Shackell's method for vacuum desiccation, 434
Shaffer's method for determination of jJ-oxy-
butyric acid, 425
Sherman's compressed oxygen method for de-
termination of total sulphur in urine, 4 1 2
Sherrington's solution, preparation of, 225
Silicates in urine, 284, 322
Skatole, 169, 171, 176, 179
tests for, I 76
Skatole-carbonic acid, 174
test for. 177
Smith's test for bile pigments. 163, 343
Soap, salting-out of , 145
Sodium and potassium in urine, 284, 320
Sodium alizarin sulphonate as indicator, 131,
133
preparation of. 133
Sodium chloride, crystalline form, 213
Sodium chloride in urine, 284, 316, 417
Sodium hydroxide and potassium nitrate fusion
method for determination of total sulphur and
phosphorus in urine. 412, 414
Sodium hypobromite solution, preparation of, 392
Sodium sulphide solution, preparation of, 429
Solera's reaction for detection of thiocyanate in
saliva, 64
test paper, preparation of, 64
Soluble starch, 10, 48, 61
Soxhlet apparatus for extraction of fat, 437
Soxhlet lactometer, determination of specific
gravity of milk by, 435
Specificity of enzyme action, 7
Spectroscope, use of in detection of blood. 215
Spermatozoa in urinary sediments. 369. 376
microscopical appearance of human. 376
Spiegler's ring test for protein. 104, 334
reagent, preparation of, 104, 334
472
INDEX.
Spongin, 74
Sprigg's method for determination of peptic
activity, 19
Standard ammonium thiocyanate solution, prepa-
ration of, 420
silver nitrate solution, preparation of, 419
uranium acetate solution, preparation of, 413
Starch, 26, 48
action of alcohol on iodide of, 50
action of alkali on iodide of, 50
heat on iodide of, 50
dry, digestion of, by pancreatic amylase,
isi, 156
dry, digestion of, by salivary amylase, 65
experiments on, 48
iodine test for, $0
microscopical characteristics of, 48
microscopical examination of, 48
potato, preparation of, 48
soluble, 10, 48, 6i
solubility of, 48
various forms of, 49
Starch group, 26
Starch paste, action of tannic acid on, 50
diffusibility of, 50
digestion of, by pancreatic amylase, 150,
iSS
by salivary amylase, 61, 65
Fehling's test on, 50
hydrolysis of, 50
iodic acid paper, 64
preparation of, 50
Steapsin (see Pancreatic lipase, 4, 140, 150, 157)
Stearic acid, 269
Stearin, 140, 23 s
Stellar phosphate, 242, 362, 364
Stercobilin, 179
Stokes' reagent, action of, 216
preparation of, 216
Stomach, motor and functional activities of, 136
Stomach contents, lactic acid in tests for, 136
peptide-splitting enzyme in, 128
qualitative analysis of, 137
Stone-cystine, 81
Sturine, 72, 73, 93
Sublingual glands, characteristics of saliva secreted
by, 59
Submaxillary glands, characteristics of saliva
secreted by, 59
Substrate, 2, 47
Succinic acid, 171
Sucrase, 5, 13, 152
experiments on, 13
vegetable, 13
Sucrose, 25, 46
experiments on, 46
inversion of, 46
production of alcohol from, 47
structure of, 46
Sulphanilic acid, 359
Sulphates in saliva, test for, 64
Sulphates in urine, 284, 315
experiments on, 315
ethereal, 283, 297
quantitative determination of, 409
inorganic, 284, 314
quantitative determination of, 409
total, quantitative determination of, 408
Sulphocyanides (see Thiocyanates, 60, 64, 420)
Sulphur in protein, 68, 108
loosely combined, tests for, 108
in urine, quantitative determination of, 408
acid, 108
lead blackening, 108
mercaptan, 108
neutral, 283, 303
oxidized, 108, 109
unoxidized, 108
Suspension of manganese dioxide, 430
Synaptase (see Emulsin, 4)
Tallow bayberry, saponification of, 144
Tallquist's haemoglobin scale, determination of
haemoglobin by, 224
Tannic acid, influence of, on dextrin, S3
on starch, 50
Tannin test for carbon monoxide hasmoglobin,
217
Tanret's reagent, preparation of, 105, 33s
Tanret's test, 105, 33S
Tartar, formation of, 60
Taurine, 159, 166, 255, 260, 283, 303
derivatives, 283, 303
formula for, 159, 260
preparation of, 166
Taurocholic acid, 159
group, 1 59
Taylor's test for acetone, 347
Teichmann's crystals, form of (see Haemin
crystals, p. 211)
test, 210, 340
Tendomucoid, 94, 112, 113, 247
biuret test on, 247
chemical composition of, 113
hydrolysis of, 247
loosely combined sulphur in, test for, 247
preparation of, 247
solubility of, 247
Tetrapeptides, 71, 95
Tetramethylene-diamine, 171
Thiocyanates in saliva, significance of, 60
ferric chloride test for, 64
Solera's reaction for, 64
Thiocyanates in urine, 280, 303
Thiophene reaction, 137
Thoma-Zeiss haemocytometer, 224
Thrombin, 5, 203
Thromboplastine, 204
Thymus histone, 93
Thymol, formula for, 281
interference of, in Lieben's acetone test, 347
interference in Heller's ring test, 333
use of, as preservative, 281
Tincture of iodine, preparation of, 453
Tissue, adipose, experiments on, 128, 253
conne«tive, 245
■white fibrous, 246
composition of, 246
experiments on, 247
yellow elastic, 249
composition of, 249
experiments on, 249
epithelial, 245
experiments on, 245
muscular, 254
experiments on, 261
INDEX.
473
Tissue, nervous, 268
experiments on, 271
osseous, 2 5 I
experiments on, 252
Tissue debris in urinary sediments, 369, 378
Titanium tetrachloride as cellulose solvent, 53
Toison's solution, preparation of, 225
ToUen's reaction on conjugate glycuronates, 352
arabinose, 4 a
galactose, 41
pentoses in uripe, 353
Topfer's method for quantitative analysis of
gastric juice, 440
Topfer's reagent, as indicator, 131, 132
preparation of, 132
Total solids, of milk, quantitative determination
of, 438
of urine, quantitative determination
of, 434
Total sulphur of urine, quantitative determination
of, 409-413
phosphorus of urine, quantitative deter-
mination of, 414
Trehalase, s
Trichloracetic acid, precipitation of protein by,
104
Trimethyl-oxyethyl-ammonium hydroxide (see
Choline, 269)
Trioses, 25
Tripeptides, 71, 95
Triple phosphate, 277, 319, 362, 380
crystalline form of, 319 ;
formation of, 319
Trisaccharides, 2s, 47
Trommer's test, 31, 326
Tropaeolin 00, as indicator, 131, 132
preparation of, 132
Trj-psin (see also Pancreatic protease, 5, 11, 22,
128, 149, 154
action of, upon proteins, 69, 128, 149, 154
experiments on, is4
influence of alkalis and mineral acids upon,
149
nature of, 149
pure, preparation of, 149
Trypsinogen, 4, 6, 149
activation of, 6, 149
Tryptic digestion, 128, 149
influence of bile on, 155
metallic salts on, 154
most favorable reaction for, 154
temperature for, 154
products of, 149, 153
Tryptic proteolysis, 128, 149
Tryptophane, is, 69, 72, 73, 82, 149, 154
bromine water test for, is4
formula for, 82
group in the protein molecule, 97, 98
Hopkins-Cole reaction for, 98
occurrence of, as a decomposition product of
protein, 69, 72, 73, 82,
occurrence of, as an end-product of pan-
creatic digestion, 149, 154
Tussah silk fibroin, 73
"Twinning" of oxyhemoglobin crystals, 202
Tyrosinase, 5
Tyrosine, s. 69i 72, 74i 79, 90i 97. I27> 149, 362, 367
crystalline form of, 8 r
Tyrosine, experiments on, 90
formula for, 79
Hoffmann's reaction for, 91
in urinary sediments, 362, 367
microscopical examination of, 90
Morncr's test for, 91
occurrence of, 69, 72, 74, 79, 149
Piria's test for, 91
salts of, 79
separation of, from leucine, 90
solubility of, 91
sublimation of, 91
Tyrosine-sulphuric acid, 91
V. Udrdnsky's test for bile acids, 164, 344
Uflelmann's reagent, preparation of, 137
reaction for lactic acid, 136
Unknown substances in urine, 323, 359
Unorganized ferments, i
sediments in urine, 361, 362
Uranium acetate method for determination of
total phosphates in urine, 413
Uracil, 113
Wheeler- Johnson reaction for, 113
Urate, ammonium, crystalline form of, Plate VI,
opposite p. 365
sodium, crystalline form of, 366
Urates in urinary sediments, 362, 365
Urea, 196, 255, 283, 284
crystalline form of, 284
decomposition of, by sodium hypobromite,
286, 289
excretion of, 28s, 287
experiments on, 287
formation of, 286
formula for, 284
furfurol test for, 290
isolation of, from the urine, 287
melting-point of, 288
quantitative determination of, 392-399
Urea nitrate, 287, 289
crystalline form of, 287
formula for, 287
oxalate, 287, 289
crystalline form of, 289
formula for, 287
Urease, s
Urethral filaments in urinary sediments, 369, 377
Uric acid, 32, 196, 255, 277, 283, 290, 362, 364,
389
crystalline form of, pure, 293
endogenous, 291
exogenous, 291
experiments on, 292
formula for, 290
Oanassini'e test, 293
in leukaemia, 293
in urinary sediments, 362, 364
crystalline form of Plate V, oppo-
site p. 291, 36s
isolation of, from the urine, 292
Moreigne's reaction for, 293
murexide test for, 292
origin of, 291
quantitative determination of, 389
Folin-Schaffer method for, 389
Heintz method for, 390
474
INDEX.
Uric acid, quantitative determination of, Kriiger
and Schmidt's method for, 391
reducing power of, 32, 293, 327
Schiff's reaction for, 293
Uricase, 5, 16
Uricolytic enzymes, 3, 5, 16
experiments on, 16
Urinary calculi, 379
calcium carbonate in, 380
oxalate in, 380
cholesterol in, 382
compound, 379
cystine in, 380
fibrin in, 380
indigo in, 382
phosphates in, 380
scheme for chemical analysis of, 381
simple, 379
uric acid and urates in, 380
urostealiths in, 380
xanthine in, 380
Urinary concrements (see Urinary calculi, p. 379)
Urinary concretions (See Urinary calculi, p. 379)
Urinary sediments, 361
ammonium magnesium phosphate in,
362
animal parasites in, 369, 378
calcium carbonate in, 362, 363
oxalate in, 362
calcium phosphate in, 362,364
sulphate in, 362, 364
casts in, 369, 371
cholesterol in, 362, 366
collection of, 361
cylindroids in, 369, 376
cystine in, 362, 366
epithelial cells in, 369
erythrocytes in, 369, 376
fibrin in, 369, 378
foreign substances in, 369, 378
haematoidin and bilirubin in, 362, 367
hippuric acid in, 362, 367
indigo in, 362, 368
leucine and tyrosine in, 362, 367
magnesium phosphate in, 362, 368
melanin in, 362, 368
micro-organisms in, 369, 378
organized, 361, 369
pus cells in, 369
spermatozoa in, 369, 376
tissue ddVjris in, 369, 378
unorganized, 361, 362
urates in, 362, 36.";
urethral filaments in, 369, 377
uric acid in, 362, 364
xanthine in, 362, 368
Urination, frequency of, 276
Urine, 274-434
acetone in, 323, 34s
acidity of, 276, 31 7
acid fermentation of, 277
albumin in, 323, 332
alkaline fermentation of, 276
alantoin in, 283, 303
amino acids, 283
ammonia in, 276, 284, 313, 399
aromatic oxyacids in, 283, 306
benzoic acid in, 283, 307
Urine, bile in, 323, 342
blood in, 323, 339
calciuna in, 284, 320
carbonates in, 284, 321
chlorides in, 284, 316
collection of, 281
conjugate glycuronates in, 323, 351
color of, 274
creatine, 258, 283, 323
creatinine in, 283, 294
dextrose in, 323
diacetic acid in, 323, 348
electrical conductivity of, 281
enzymes in, 284, 309
ethereal sulphuric acid in, 283, 297
fat in, 323, 353
fluorides in, 284, 322
freezing-point of, 279
galactose in, 323, 355
general characteristics of, 274
globulin in, 323, 336
Haser's coefficient for solids in, 279, 434
hasmatoporphyrin in, 323, 353
hippuric acid in, 283, 300, 367
hydrogen peroxide in, 284, 322
inorganic physiological constituents of, 28
313
inosite in, 323, 357
iron in, 284, 321
lactose in, 323, 354
laevulose in, 323, 355
laiose in, 323, 358
leucomaines in, 284, 312
Long's coefficient for solids in, 27S, 434
magnesium, in, 284, 320
melanin in, 323, 358
neutral sulphur compounds in, 283, 303
nitrates in, 284, 322
nucleoprotein in, 283, 308, 323, 339
odor of, 27s
organic physiological constituents of, 283
oxalic acid in, 283, 302
oxaluric acid in, 283, 308
/3-oxybutyric acid in, 323, 349, 425
pathological constituents of, 323
paralactic acid in, 256, 284, 309
pentoses in, 323, 352
peptone in, 323, 337
phenaceturic acid in, 284, 309
phosphates in, 284, 317
phosphorized compounds in, 284, 310
physiological constituents of, 283
Ijigments of, 274, 284, 310
potassium in, 284, 320
proteins in, 323, 352
proteoses in, 323, 332, 337
ptomaines in, 284, 312
purine bases in, 284, 312, 428
quantitative analysis of, 383-434
reaction of, 276, 317
silicates in, 284, 322
sodium, in, 284, 320
solids of, 278, 434
specific gravity of, 278
sulphates in, 284, 314, 408
transparency of, 275
unknown substances in, 323, 359
urea in, 283, 284
INDEX.
475
Urine, uric acid in, 2.S.}, jgo
urorosein in, ^j.i, 3sH
volatile fatty acids in, 284. ,509
volume of, 274
Urobilin, 274, 284, .?'o
tests for, i 1 1
Urochrome, 274, 284, 310, 330
Uroerythrin, 274, 284, 3>o. 330
I'roferric acid, 283, 303, 3S9
I'roleucic acid, 283, 306
Urorosein, 323, 358^
tests for, 3 59
Valine, 69, 72, 74, 83
Van Slyke's method for determination of amino
nitrogen, 408
Vegetable amylase, 4,10
lipase, 4,12
protease, 1 1
sucrase, 13
Vegetable globulins, 93, 95, 109
Vegetable gums, 26
With lactometer, determination of specific
gravity of milk by, 43 5
Viscosity test, 64
Vitellin, 94, 95
Volatile fatty acids, 169, 172, 284. 309
Volhard-Amold method for determination of
chlorides, 4 1 9
Volhard-Harvey method for determination of
chlorides, 420
Volume of the urine, 274
Water at meals, influence of, 61, 62, 124. 151, 180,
182, 183, 314
softened, 62
Wax myrtle, 144
Waxy casts in urinary sediments, 369, 373
Weber's guaiac test for blood in feces, 186
Weinland, formation of fat from protein, T43
Welker's electrical bath, 397
modified method for purine bases, 428
Weyls test for creatinine, 296
Wheeler-Johnson reaction for uracil and cytosine,
113
White fibrous connective tissue, 246
experiments on, 247
Wiechowski's method for determination of
allantoin, 304, 432
Wilkinson and Peters' test, 240
Wirsing's test for urobilin, 31 1
Witche's milk, 237
Wohlgemuths' method for quantitative deter-
mination of amylolytic activity, 18
Author's modification of, 189
Xanthine, 255, 258, 261, 284, 312
crystalline form of, 258
formula for, 261
in urinary sediments, 362, 368
isolation of, from meat extract, 266
Weidel's reaction for, 266
bases (see Purine bases, pp. 113, 284, 312,
428)
Xanthine silver nitrate, 265, 266
crystalline form of, 266
test, 266
Xanthoproteic reaction, 97
Xantho-oxidase, 5
Xylose, 25, 43
orcinol reaction on, 43
phenylhydrazine reaction on. 43
Tollens' reaction on, 43
Yellow elastic connective tissue, 249
composition of, 249
experiments on, 249
Zappert slide, 227
Zein, 72, 74, 93, 95, 112
decomposition of, 72, 74
Zeller's test for melanin, 358
v. Zeynek and Nencki's haemin test, 21 2, 340
Zikel pektoscope, 279
Zymase, classification of, 5
preparation of, 2
Zymo-exciter, 6
Zymogen, 6, 127,
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