THE LIBRARY

OF

THE UNIVERSITY
OF CALIFORNIA

LOS ANGELES

DIRECTIONS

FOR

LABORATORY WORK

IN

PHYSIOLOGICAL CHEMISTRY.

BT

HOLMES C. JACKSON, PH.D.,

Professor of Physiology, University and Bellevue Hospital Medical College,
New York City.

SECOND EDITION, REWRITTEN AND ENLARGED.
SECOND THOUSAND.

NEW YORK:

JOHN WILEY & SONS.

LONDON: CHAPMAN & HALL, LIMITED.

1911

Copyright, 1902, 1908,

BT

HOLMES C. JACKSON.

•3

PREFACE.

THE second edition of this Manual appears as the result
of a thorough revision of the original edition, which was issued
last year ; numerous additions to the subject-matter have also
been inserted.

In its preparation the author has had iiv mind to provide
a guide for systematic work in a course in Physiological
Chemistry such as is given in the majority of the American.
Medical Schools. An attempt has been made to present, in
experimental form, all of the essentials of the subject. At
the same time, the experiments are simple and of a form
which would not overstep the possibilities of equipment and
time available in the average laboratory in physiological chem-
istry. Those facts which do not allow of simple experimen-
tal study have been ignored. The text preceding the experi-
ments has been limited to an explanation merely sufficient for
a correct understanding and performance of the work indi-
cated, with the expectation that the laboratory course will be
supplemented by lectures showing the important relationship
of the pure chemistry of the subject to physiological and
pathological processes.

The description of the experiments has been set down with
considerable detail and, it is hoped, clearness, since the suc-

iii

578896

iv PREFACE.

cessful termination of tests and experiments depends to such
a large extent upon the exact manner in which they are con-
ducted.

Direct explanation of the results of the experiments is pur-
posely omitted, with the end in view that the student might
be led to reason these out for himself.

Further, it has been presumed that the student taking this
work does it in preparation for the practice of medicine. Par-
ticular emphasis has therefore been laid on that point, espe-
cially in the part devoted to urine analysis, where those meth-
ods have been presented which adapt themselves particularly
to clinical work.

In conclusion the author claims little originality in the
experiments, and acknowledges the use of all text-books and
manuals which were available.

H. C. J.

TABLE OF CONTENTS.

PAGE

CARBOHYDRATES 1

Pentoses 2

Monosaccharides 3

Disaccharides 5

Polysaccharides 7

FATS 9

PROTEINS 15

Simple proteins 20

Conj ugate proteins , 25

Derived proteins 27

MUSCULAR TISSUE 29

Proteins 29

Nitrogenous extractives 30

Non-nitrogenous extractives 34

BONE 36

NERVOUS TISSUE 38

Lipoids 38

Cerebrosides 40

Cholesterols 41

SALIVARY DIGESTION 43

GASTRIC DIGESTION 46

Peptic proteolysis 51

PANCREATIC DIGESTION 56

Tryptic proteolysis 57

Amylolysis 60

Lipolysis 60

Pancreatic rennin 61

INTESTINAL PUTREFACTION 62

BILE 67

Conjugate acids 68

Pigments 69

V

vi TABLE OF CONTENTS.

PAOE

BLOOD 72

Blood serum 73

Blood plasma 74

Form elements 75

Spectroscopic examination 77

MILK 80

Quantitative separation 81

URINE. . 85

Normal constituents 88

Quantitative ueierininations 103

Pathological constituents 121

SEDIMENTS 134

APPENDIX 137

INDEX 145

LABORATORY WORK

IN

PHYSIOLOGICAL CHEMISTRY.

THE CARBOHYDRATES.

The term carbohydrate is usually considered as embrac-
ing those compounds which contain the elements C, H, and 0,
the H and 0 being present in the same ratio as they exist in
water. This definition, strictly followed, would include in the
group substances such as lactic acid, acetic acid, and inosit,
which are obviously not carbohydrates. The carbohydrates
may be conveniently divided into three groups; namely,
Monosaccharides or Glucoses, Disaccharides or Saccharoses,
and Polysaccharides or Amyloses; but a more strictly chem-
ical classification would designate them according to the
number of carbon atoms present in the molecule, the prefix
aldo- or keto- denoting to what general class of compounds
(aldehydes or ketones) they belong: thus trioses, tetroses,
pentoses, hexoses, heptoses, etc., and aldohexose, ketopen-
tose, etc. Of the greatest physiological importance are the
pentoses, hexoses, hexobioses (Disaccharides), and polyoses
(Amyloses).

Note the general character and appearance of the van-

2 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY

OILS specimens of carbohydrates which are presented for
study.

Test for the constituent elements as follows:

(a) Carbon. — 1. Heat cautiously some of the substance
on a platinum foil. The piece will char, owing to the sepa-
ration of the carbon in the substance. Further heating ren-
ders the carbon capable of combining with the oxygen of the
air, with the result that the former passes off as C02, and
in the case of carbohydrates, where combustion is com-
plete, no residue is obtained. A substance containing oxy-
gen in sufficient quantities to form C02 with all the carbon
present will not carbonize.

2. Mix thoroughly some of the dried substance with
powdered CuO and place the mixture in the bottom of a dry
test-tube. Upon warming, the carbon of the substance is
oxidized by the oxygen of the CuO and escapes as C02.
This C02 may be detected by holding a glass rod moistened
with lead acetate at the mouth of the test-tube.

(6) Hydrogen. — 1. In the latter experiment, moisture will
have collected on the cold part of the test-tube. The hydro-
gen of the substance, in the presence of heat, has combined
with the oxygen supplied by the CuO, forming H20.

PENTOSES, C5H1005.
ARABINOSE. XYLOSE.

The pentoses do not occur free in nature, but exist in the
form of pentosanes — bodies which are found in the fruits and
polysaccharide gums (e.g., gum arabic, cherry gum) and from
which the pentoses may be obtained by hydrolysis with
weak acids. The ingestion of pentosanes also causes pento-
ses to appear in the urine. Chemically they are aldehydes
and as such reduce Fehling's solution. With regard to their

THE CARBOHYDRATES. 3

action on polarized light, they exist in three modifications,
but those derived from the pentosanes are dextrogyrate.
Pentose solutions when heated with phloroglucinol or orcinol
and HC1 (sp. gr. 1.09) acquire respectively a cherry- red or
green color and posses characteristic absorption spectra.
The pentoses are non-fermentable and yield with phenyl-
hydrazin osazones having characteristic properties. Boil
some cherry gum for some hours with 1 per cent H2S04.
Test the solution for pentose as follows:

(a) Saturates c.c. of HC1 (sp. gr. 1.09) with phloroglucinol
by warming on the boiling-water bath. A slight excess of
the phloroglucinol is advantageous. Add to this a few c.c.
of the pentose solution and continue the heating. Grad-
ually a cherry-red color develops in the solution and the
latter shows characteristic absorption bands between D
and E.

(6) Try the same test using orcinol instead of phloro-
glucinol. The solution turns reddish, then reddish blue, and
finally green, with a precipitate of that color settling out of
the solution. This precipitate is soluble in amyl alcohol, in
which solvent it also has characteristic absorption bands
between C and D.

(c) Prepare pentosazones after the method employed
on p. 5.

HEXOSES OR MONOSACCHARIDES, C6H1206.
DEXTROSE. LEVULOSE. GALACTOSE.

The monosaccharides, in general, are soluble, crystalline,
and optically active bodies which yield, upon boiling with
alkalies, oxidation products and caramel; they reduce metal-
lic oxides in alkaline solution; ferment with yeast and with
bacterium lactis ; and give, with phenylhydrazin, osazones

4 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

with characteristic crystalline forms, solubilities, and melt-
ing-points.

GLUCOSE = DEXTROSE = GRAPE-SUGAR.

Of the monosaccharides, dextrose holds first place in
importance in the animal economy. Examine and taste the
dry substance. Test its solubility in water and in hot and
cold alcohol. In the following tests make use of a 1 per cent
solution:

(a) Moore's Test. — To 5 c.c. of the dextrose solution add
an equal volume of NaOH, and heat. The mixture becomes
yellow and finally brown, due to the formation of caramel.
This test lacks delicacy and reliability in examining urine.

(6) Trommer's Test. — To 5 c.c. of the dextrose solution
add an equal volume of NaOH. Then add, drop by drop, a
dilute solution of CuS04 (so dilute that the green color is just
visible) until a trace of permanent precipitate remains. The
solution should be deep blue in color. Warm the upper part
of the solution and note result. Write all the equations for
the reactions taking place in this experiment.

(c) Fehling's Test. — Heat 5 c.c. of Fehling's solution just
to boiling and add a few drops of the dextrose solution. Con-
tinue the boiling until the solution commences to respond as
with Trommer's test. Compare the reactions of this test
with those of the previous ones.

(d) Barfoed's Test. — To 5 c.c. of Barfoed's reagent add a
few drops of the dextrose solution and boil. Note the result
and write equations.

(e) Nylander's Test. — To 5 c.c. of the dextrose solution add
10 drops of Nylander's reagent and boil. The solution grad-
ually turns yellow and finally black, bismuth being precipi-
tated.

THE CARBOHYDRATES. 5

(/) Silver Nitrate. — To 5 c.c. of AgN03 solution -in a clean
test-tube add dilute NH4OH, drop by drop, until the precipi-
tate, which is at first formed, dissolves. Then add a few
drops of the dextrose solution and warm on the water-bath.
Note the formation of a metallic mirror on the side of the
test-tube. Explain the chemical changes.

(g) Phenylhydrazin. — In a test-tube prepare a mixture
of 5 drops of phenylhydrazin, 10 drops of glacial acetic acid,
and 1 c.c. of a saturated solution of NaCl; then add 5 c.c. of
the dextrose solution and boil for a few minutes. Yellow
phenylglucosazone crystals will appear on cooling.

Under the microscope these appear as fine yellow needles,
usually arranged in sheath-shaped bundles. If determina-
tions are made of the composition and melting-point of these
crystals, this test furnishes most characteristic and conclusive
evidence for dextrose. Write the equations for the forma-
tion of the osazones.

(h) Fermentation. — In a test-tube shake 20 c.c. of the
dextrose solution with a small piece of compressed yeast.
Place the mixture in a saccharometer and allow it to stand
in a thermostat at 40° C. A gas collects at the top of the
tube and its volume is in a direct ratio to the amount of dex-
trose in the solution. What is the character of the chemical
changes which have taken place?

(i) Polarization. — See demonstration and make a read-
ing on the instrument of the degree of right-handed rotation.

HEXABIOSES OR DISACCHARIDES, C12H22On.
SACCHAROSE. LACTOSE. MALTOSE.

The disaccharides possess, with the exception of saccha-
rose, the same general properties as the simple sugars. As
their name implies, upon hydrolysis they take up a molecule

6 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

of water and break down into two molecules of a monosac-
charide.

This decomposition in the case of saccharose has assumed
the name of inversion from the fact that when a dextro-
rotatory solution of cane-sugar suffers hydrolysis, the result-
ant mixture is Isevogyrate. This is caused by the strong
laevo-rotation of the Isevulose more than overcoming the
dextrogyrate polarization of the dextrose.

Saccharose = dextrose +lsevulose.
SACCHAROSE = CANE-SUGAR = SUCROSE.

Sa3charose differs from the other members of this group
in not reducing metallic oxides in alkaline solution and in
not forming osazones with phenylhyclrazin. It is not
directly fermentable with yeast, but only after previous
inversion by the ferment invertin, secreted by the yeast-cell.
A slight reduction which is sometimes obtainable with
Fehling's test may be explained by the inverting action of
the strong alkali.

Use a 1 per cent solution of cane-sugar.

Tests: (a) Fehling's; Moore's; Nylander's; Barfoed's;
using in each case 5 c.c. of the saccharose solution. Com-
pare these results with those obtained for dextrose.

(6) To 10 c.c. of the saccharose solution add 1 c.c. concen-
trated HC1 and boil several minutes. Allow this to cool
and then neutralize with NaOH. Use this solution in making
Fehling's, Nylander's, and Barfoed's tests. Write the equa-
tions for the chemical changes which have taken place and
determine the character of the carbohydrate formed.

Inversion and Fermentation.

1. Examine the saccharose solution in the polariscope
and determine the degree of rotation.

THE CARBOHYDRATES. 7

2. Place 10 c.c. of the saccharose solution in a test-tube
with 5 c.c. of 1 per cent HC1. Allow the mixture to remain
two hours at 37° C. Finally examine this in the polariscope
and test its reducing power. To what condition in the body
is this comparable?

3. To 10 c.c. of the saccharose solution add some invertin
and allow the test-tube to remain at 37° C. Test for invert
action.

4. See demonstration of the formation of alcohol and
C02 from a fermentation of cane-sugar by yeast. The C02
as it forms is caught in passing out of the flask by a valve of
Ba(OH)2. The alcohol is distilled off from the mixture in
the flask.

MALTOSE will be studied under Salivary Digestion and
LACTOSE under Milk.

POLYOSES OR POLYSACCHARIDES, (C6H1005)«-

STARCHES. DEXTRINS. GLYCOGEN. CELLULOSES.
VEGETABLE GUMS.

The polysaccharides, as a class, are amorphous, more or
less insoluble substances which do not diffuse through animal
membranes. They are optically active, and with the excep-
tion of the dextrins do not reduce Fehling's solution. By
hydration with weak acids or with enzymes the polysac-
charides are converted into monosaccharides ; dextrins and
disaccharides being the intermediate products. The polysac-
charides are incapable of undergoing fermentation unless
previously inverted.

STARCHES.

The starches are insoluble bodies which, with hot water,
form opalescent colloidal solutions or pastes. These, if suffi-

8 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

ciently concentrated, gelatinize upon cooling. The most
characteristic reaction of the starches is their behavior toward
iodine, with which they form blue compounds whose color
disappears upon heating, but returns as the solution cools.

(a) Examine under the microscope and sketch the follow-
ing starch granules: potato, corn, wheat, rice, and arrowroot.

(6) Place some starch in a test-tube half full of water and
shake thoroughly until tho starch is finely divided. Heat
water to boiling in another test-tube and to this add enough
of the cold starch mixture to make a translucent solution
(about 2 per cent). What takes place upon pouring the
suspended starch into the hot water? The solution of starch
thus obtained is called a paste, and in making experiments
with this substance such a paste must always be prepared.

(c) To 5 c.c. of starch paste add a drop of iodine solution.
Warm gradually and then allow to cool. Note changes.

(d) Boil 10 c.c. of starch paste with 1 c.c. of concentrated
HC1 for 5 minutes. Observe the change which the solution
undergoes. Neutralize part of this cold solution with NaOH,
and apply Fehling's and Barfoed's tests. (If no reduction
appears, continue the boiling of the original acid solution for
some minutes longer and repeat the neutralization and tests.)
Determine the character of the sugar causing this reduction.

For the DEXTRINS see Salivary Digestion; for GLYCOGEN
see Muscular Tissue.

THE FATS.

The neutral fats are glycerol esters of fatty acids, in that
three hydrogens of the carboxyl groups of three fatty acids
are replaced by the glycerol radical. A general formula for
a neutral fat is represented thus:

CH2-0-OC-R
CH-0-OC-R'

CH2-0-OC-R"

in which R, R', and R" stand for three of the same or
different hydrocarbon radicals. In the molecule of the
ordinary animal fats R, R', and R" may be represented
by three hydrocarbon residues of palmitic, of stearic, of
oleic, or of butyric acid, the compounds being designated
respectively as tripalmitin, tristearin, etc.

The specific character of a fat is dependent upon the nature
of the fatty acid in the molecule; and since, in general, the
melting-points of the saturated fatty acids increase with
their carbon content, the lower compounds remain liquid at
ordinary temperature and the character of tributyrin (R,
R', and R" =C3H7) is that of a soft fat, while tristearin (R, R',
and R" = CiyH35) possesses considerable firmness.

Observe the olive oil, butter, mutton and beef fats pre-
sented for study. Pure neutral fats are devoid of color,
odor, and taste, and their low specific gravity and insolubility

10 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

allow them to float on the surface of water. They are solu-
ble in ether, chloroform, and benzol; hot alcohol also dis-
solves them, but upon cooling they separate out usually in
crystalline form. Under certain conditions fats undergo a
peculiar physical change called emulsification, which is beau-
tifully illustrated in the natural state of the fat in milk.
Under the influence of superheated steam, acids, or ferments,
fats are broken down into their component parts — fatty
acids and glycerol. When an alkali or sodium alcoholate is
employed, soaps and glycerol are formed and the process
is called saponification. Another form of decomposition is
effected when fats remain for any length of time in contact
with the oxygen of the air. They then become rancid by
the liberation and subsequent oxidation of the fatty acid
from the molecule and the formation of lower volatile acids
which cause unpleasant odors and tastes.

Try the following tests, making use of olive oil :
(a) Test its solubility in water; ether; chloroform; alco-
hol.

(6) Test its reaction. What is the normal reaction of a
fat? Express by equations what occurs when butter becomes
rancid.

(c) In a test-tube warm a few drops with potassium bisul-
phate. Notice odor. What is the reaction taking place?

(d) Let a drop of an ether solution of a fat fall upon paper.

(e) Dissolve a little lard in 10 c.c. of a mixture of equal
parts of alcohol and ether. Allow this to remain uncovered
until crystals begin to form.

(/) Saponification (Bayberry Wax — Tripalmitin).

Place a piece of the wax, half the size of a walnut, in an
evaporating-dish which is half full of water. Then add about

THE FATS. 11

10 c.c. NaOH and boil until the substance is dissolved, adding
water from time to time as the solution evaporates, so that
the original volume is constantly maintained. Write the
reaction which is taking place. After complete solution, add
carefully dilute H,S04 until the reaction just becomes acid and
then cool. A greenish crust of free fatty acid will form on
the surface of the liquid, so that the solution may be poured
off. (Keep this solution.) Break the fatty acid crust into
small pieces and wash with tap water; finally dry between
filter-paper. Dissolve part of the fatty acid in 50 c.c. of hot
95 per cent alcohol, filter hot through a dry funnel and allow
the filtrate to cool slowly.

Palmitic acid separates out in snowy-white crystalline
form. Examine some under the microscope and sketch the
crystals.

(g) Saponification (Lard),

Place about 100 c.c. of alcoholic potash in a flask contain-
ing 25 grams of lard. Warm the mixture upor. the water-
bath until a drop let fall into water is perfectly soluble.
Then pour the solution into an evaporating-dish containing
100 c.c. water and evaporate until the alcohol has been driven
off. While still hot acidify with dilute H2S04 and then
cool. Write the chemical equations for the various steps in
the procedure. The fatty acid rises to the surface and may
be easily removed from the liquid beneath. Unite this solu-
tion with the similar one in Experiment 1 and, after neutrali-
zation with Na2C03, evaporate it down to about 5 c.c. What
substances are present in this solution? Retain this.

12 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

FATTY ACIDS AND SOAPS.

Since the character of the fatty acid determines the
character of the fat, the properties and solubilities of the
fatty acids and fats must be similar, as the tests will
prove.

The soaps are salts of the fatty acids; the sodium salts
being solid and hard, while those of potassium are soft.
Both are soluble in water. The remaining metallic salts
are insoluble in water, but generally dissolve in alcohol.
The lead combination with oleic acid (lead plaster) differs
from the corresponding one with palmitic and stearic acids
in being soluble in ether.

Perform the following reactions with the fatty (oleic) acid:

(a) Test its solubility in ether; chloroform; water;
alcohol.

(6) Warm some in a dry test-tube with potassium bisul-
phate.

(c) Shake up a few pieces of the fatty acid with 25 c.c.
water and add NaOH, drop by drop, until the acid is all dis-
solved. Write the reaction. What is formed?

Use this soap solution for the following reactions:

(a) Add 5 drops to a saturated solution of NaCl.

(6) To 5 c.c. of lead acetate add a few c.c. of the soap
solution. A sticky precipitate results (lead oleate), which is
soluble in ether. Write the reaction. What is the impor-
tance of this compound?

(c) To 50 c.c. of tap water in a flask add the soap solu-
tion, drop by drop, alternately shaking until a permanent
lather is obtained. The amount of soap solution required
to obtain the lather is in direct proportion to the hardness of
the water. Explain the chemistry of this experiment.

THE FATS. 13

Emulsification of Fats.

Certain poorly understood factors enter into the forma-
tion and preservation of an emulsion. Apparently the
viscosity of the menstruum and the condition of the
surface tension existing between that menstruum and the
globules form the fundamental physical requisites for the
obtaining of a permanent emulsion. Solutions of gums,
proteins, and soaps emulsify fats with varying degrees of
permanency. The mere presence of a soap in the solution does
not seem to render the fat particularly prone to emulsify;
the necessary condition is apparently the intermolecular forma-
tion of a soap in the solution as exemplified upon the addi-
tion of an alkali to fat containing some free fatty acid. An
emulsion formed in this manner is the most permanent of all
those induced by artificial means; a similar procedure takes
place in the emulsification of fat in the intestine.

Make up the following mixtures in test-tubes:

(a) 2 drops of neutral olive oil + 10 c.c. water.

(6) 2 drops of neutral olive oil + 10 c.c. water + a few
drops of Na2C03.

(c) 2 drops of neutral olive oil + 10 c.c. of the soap solu-
tion.

(d) 2 drops of neutral olive oil + 10 c.c. of an egg-albumin
solution.

(e} 2 drops of rancid olive oil + 10 c.c. of water + a
few drops of Na-jCOg.

Shake each tube approximately the same length of time
and compare the relative permanence of the various emul-
sions. Which conditions are the most favorable, and
why?

14 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

CH2OH

GLYCEROL, CHOH
CH2OH.

Make use of the solution obtained from the two saponifica-
tions for the following tests :

(a) Note the taste and try its solubility in alcohol; ether;
water.

(6) Add some dry potassium bisulphate and warm.
Compare this with the same reaction under Fats. Write the
equation.

(c) Test with Fehling's solution.

(d) To some dilute CuS04 add the remainder of the glycerol
solution; then drop by drop add dilute NaOH. What causes
the blue color of the solution?

THE PROTEINS.

The proteins are complex compounds of C, H, 0, N, and S
(in some cases P and Fe) occurring widely distributed in the
plant and animal kingdoms. The members of this class of
bodies, although differing greatly in chemical and physical
characteristics, possess in common certain definite properties
and chemical reactions which allow of classification and sub-
division. The proteins form the chief type of the food-
stuffs, since the nitrogen present in them is absolutely essen-
tial in order to sustain life.

They may be divided as follows:

The simple proteins, those giving upon decomposition
indole, skatole, tyrosine, cystine, amino acids, pyrrol deri-
vatives, etc.

The conjugate proteins, composed of a simple protein and
a complex non-protein component.

The derived proteins, derivatives of the proteins and
formed through hydrolytic changes.

Reactions General to all Proteins.

For the following tests make use of dried egg-white or
casein :

Test the substance for carbon and hydrogen.

Nitrogen. — (a) Make an intimate mixture of the sub-
stance with soda-lime and place it in a dry test-tube. Warm
gently. Hold a piece of moistened red litmus paper or a

15

16 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

glass rod moistened with HC1 at the mouth of the test-tube.
What is the reaction which takes place?

(6) Warm together carefully in a dry test-tube a few
particles of the dry substance and a 3-4 mm. cube of metallic
sodium. (Caution: Do not place the tube in the flame.
Gases are evolved which are explosive unless the temperature
is kept moderate.) When the fusion' is complete and the
tube has cooled somewhat plunge the end into a small amount
of water placed in a suitable vessel, preferably a conical glass.
The glass of the test-tube will probably break, and unless the
sodium has been completely fused a slight explosion will
result. When the water has thoroughly impregnated the
fused mass, filter and to the filtrate add a few drops of ferric
chloride and of ferrous sulphate solution. Upon acidifying
with HC1 a blue precipitate of Prussian blue is obtained.
Write all the reactions which take place in this manipulation.

Sulphur. — The sulphur in the protein molecule exists in
part in such a form that upon boiling with caustic soda it is
easily split off as sodium sulphide. This in the presence of
a lead salt forms black lead sulphide. On this account such
sulphur has assumed the name of loosely combined or lead-
blackening sulphur. If the quantity of sulphur obtained in
the above manner be multiplied by |, the product is the
amount of sulphur considered to be present in the cystine
nuclei of the given protein. In some proteins — keratin,
serum albumin, serum globulin — the total sulphur and cystine
sulphur content are the same and we are justified in assum-
ing that all the sulphur in the molecule exists in the form of
cystine. In other proteins only £— | of the total sulphur is to
be accounted for as cystine S; hence it must be concluded
that sulphur exists in some atomic complex which yields, upon
the decomposition of the molecule, substances like a-thio-
lactic acid, mercaptans, ethyl sulphide, etc. Sulphur of this
type may be styled, in contradistinction, -firmly combined
sulphur. The sum of the loosely and firmly combined sul-
phur equals the total sulphur of the substance.

WHE PROTEINS. 17

Test for lead-blackening sulphur as follows:

Place about 5 c.c. of dilute NaOH in a test-tube with a
small quantity of the substance, and add two drops of lead
acetate. Boil the mixture for a few minutes and note the
changes. The depth of color may be said to correspond
roughly to the amount of neutral sulphur present in the sub-
stance.

Test for total sulphur as follows :

Mix some of the substance with double its quantity of the
fusion mixture (Na2CO3 + KN03). Place the whole in a
small porcelain crucible and warm cautiously until the mix-
ture becomes colorless. (If the fusion begins to sputter,
remove the flame.) The residue (fused mass) should be
nearly white. It is then dissolved in a small quantity of
water, filtered, and the filtrate, after the addition of a few
drops of HC1, is brought to boiling and treated with BaCl3
solution. What is this white precipitate? Write the equa-
tion.

Phosphorus. — Heat some casein with the fusion mixture
as in the previous experiment. Dissolve the fused mass in
10 c.c. of water acidified with HN03. Filter and add 5 c.c.
of ammonium molybdate solution. Warm some minutes at
about 80° C. What is the yellow precipitate?

Iron. — Incinerate a small quantity of haemoglobin in a
porcelain crucible. The ash should be red. Dissolve out
the ash with 10 c.c. of dilute HC1. Filter. Test this solu-
tion for Fe with potassium sulphocyanide or potassium
ferrocyanide.

Color Reactions.

The following tests are based upon reactions taking place
between the reagent employed and certain more or less well-
defined groups or atomic complexes of the protein molecule.
These groups are present in varying proportions in the dif-
ferent proteins and in some cases even certain complexes

18 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

may be lacking altogether, facts which explain the individual
differences in the strengths of the reactions obtained.

For the following tests make use of the egg-white solution
as an example of a typical protein:

(a) Xanthoproteic Test. — To 5 c.c. of the protein solution (or
dry substance) add 5 c.c. of HNO3. Note the white precipi-
tate. Boil until the precipitate is dissolved and the solution
becomes light yellow. Cool, and add an excess of NH4OH.
The color changes to orange. Other substances may give a
yellow solution with HN03, but their solutions do not respond
orange upon neutralization with NH4OH. What are the
chemical changes of this reaction and the atomic complexes
involved?

(6) Millon's Test. — Add a few c.c. of Millon's reagent to
5 c.c. of the protein solution. The precipitate which forms
turns red slowly upon heating. This reaction is suitable for
solids, or liquids when the quantity of salts in the solution is
not excessive. What atomic complex in the protein molecule
causes this reaction? What simple substance also responds
to this test?

(c) Biuret Test. — Suited for testing solutions only. — Place
5 c.c. of the protein solution in a test-tube and add an equal
volume of NaOH. Then add, drop by drop, a CuS04 solu-
tion so dilute that the color is hardly visible. The color
obtained will vary from blue violet to reddish violet, accord-
ing to the nature of the protein in solution. If too much
CuS04 is added the solution becomes green. A specific
atomic complex in the protein molecule is also accountable
for this reaction. What simple substance reacts positively
to this test?

(d) Adamkiewicz Test. — To 3 c.c. of a glyoxylic acid
solution (Hopkins-Cole reagent) add 1-2 c.c. of the protein
solution. Stratify this mixture upon 5 c.c. of cone. H2S04.

THE PROTEINS. 19

A red to reddish- violet color develops at the junction of the
two liquids. Of what important complex does this reaction
show the presence?

Precipitation Reactions.

The precipitation reactions may be divided into three
general classes according as the protein acts as a base, as an
acid, or as a neutral body. To the first class belong those
reactions in which the weak organic acids combine with the
protein as a base and an insoluble salt results. The second
class of reactions embrace those between neutral salts and
the protein in which an albuminate of the base (metal) is
formed. In the third class the protein does not take part
in the reaction. Precipitation occurs when the reagent
which is added (in this case a salt such as MgS04, Na^SO^,
(NH4)2S04) has appropriated to itself sufficient of the solvent
to throw the protein out of its solution (salted out).

(a) Heller's Test. — If 5 c.c. of HN03 is placed in a test-
tube and a few c.c. of the albumin solution allowed to flow
gently down the side of the test-tube and stratify itself on
top of the HN03, a white ring of precipitated albumin will
form where the two liquids meet. HC1 and H2S04 will also
give the same reaction, but the precipitates are soluble in an
excess of the reagents.

(6) Acetic acid and potassium ferrocyanide. — Make 5 c.c.
of the protein solution acid with acetic acid and add, drop
by drop, a dilute solution of potassium ferrocyanide. Note
result.

(c) Make 25 c.c. of the protein solution slightly acid,
using dilute acetic acid.

1. To 5 c.c. of this solution add 2 drops of tannic acid
solution.

20 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

2. To 5 c.c. of this solution add 2 drops of picric acid
solution.

3. To 5 c.c. of this solution add 3 volumes of 95 per cent
alcohol.

4. To 5 c.c. of this solution add MgS04 (in substance)
to saturation.

5. To 5 c.c. of this solution add (NH4)2S04 (in substance)
to saturation.

Note results and see if the precipitation is complete in each
case.

(d) Acidify 10 c.c. of the protein solution with dilute HC1.

1. To 5 c.c. of this solution add 2 drops of phosphotungstic
acid.

2. To 5 c.c. of this solution add 2 drops of potassio-
mercuric-iodide.

(e) To successive portions of 5 c.c. of the protein solution
add a few drops of CuS04; neutral and basic lead acetate;
HgCl2; trichloracetic acid (2-5 per cent solution); Fe2Cl6.

Make careful notes of the results of the above reactions
and where possible write equations. Decide as to which
class of reaction each belongs.

The use of metallic salts as antidotes in cases of poisoning
is based upon these precipitation reactions.

SIMPLE PROTEINS.

The group of simple proteins allows of a further subdi-
vision into the simple native proteins and the albuminoids.

The native proteins, as far as it can be ascertained, exist
in the fluids and tissues of the body in the same or at least a
similar form in which they appear in the laboratory. The
albuminoids are chemically closely related but are charac-
terized by great insolubility in all neutral solvents. To

THE PROTEINS. 21

-the former sub-group belong the albumins, the globulins,
histones, and protamines; to the latter the keratins,
elastins, collagen, recticulin, and the skeletins.

ALBUMINS.

The albumins present the most characteristic type of the
proteins since they give a positive response to all the typical
protein reactions.

Remember that an albumin (egg-white) solution was
employed for all the protein tests.

(a) To 10 c.c. of the albumin solution add (NH4)2SO4 to
saturation. Note result and compare with experiment (c) 5,
p. 20. Filter off the precipitate and test the filtrate with the
biuret test.

(6) To 10 c.c. of the albumin solution add MgS04 to sat-
uration. Compare this result with that of experiment (c) 4,
p. 20. Now add 2 drops of acetic acid. What is the precipi-
tate?

(c) Test the albumin solution for lead-blackening sul-
phur.

Coagulation.

Certain of the proteins in the presence of water and heat
undergo a change which is confined to an intramolecular
rearrangement of the atoms in the molecule. This trans-
formation, which is termed coagulation, brings about physical
differences in the behavior of the proteins. They become
insoluble in all of the ordinary protein solvents. Under simi-
lar conditions the degree of temperature at which a definite
protein will coagulate is fairly constant, a fact which is
employed to ascertain not only the character but also the
purity of an unknown protein.

22 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

(d) Heat 5 c.c. of the albumin solution to boiling and
then add one or two drops of very dilute acetic acid. The
albumin separates out in an insoluble, flocky (coagulated)
form. Why is the addition of the acetic acid necessary and
why is it added after boiling? Try to dissolve the coagulum
in some of the ordinary protein solvents. Make 5 c.c. of the
albumin solution faintly alkaline, and heat. Note differ-
ences.

(e) The temperature of coagulation is determined as
follows:'

Fill a test-tube (one-third of its capacity) with the clear,
very slightly acidulated solution of the protein. By means
of a bored cork fasten into the test-tube a thermometer in
such a manner that its bulb is entirely immersed in the solu-
tion. Suspend the test-tube in a large beaker of water which
rests upon a wire gauze. By cautious heating, the tempera-
ture of the water in the beaker may be raised slowly and the
point on the thermometer noted at which the first flocks of
coagulated protein appear in the solution. This point is
considered as the coagulation temperature of the substance
under these conditions, but it varies with the reaction of
the solution and the nature and quantity of the salts
present.

Another form of coagulation is induced by the action of
certain ferments, such as the fibrin ferment of the blood, by
the action of which the soluble fibrinogen is transformed into
insoluble fibrin.

(/) Heat some dried albumin in a dry test-tube to about
100° C. After cooling the tube try to dissolve the substance
in water. Why does not the albumin coagulate by the
heat?

THE PROTEINS. 23

GLOBULINS.

As a class the globulins are characterized by their insolu-
bility in water and solubility in weak salt solutions (5-10 per
cent). A suitable solution of a typical globulin (edestin) is
easily prepared by extracting finely ground hemp-seed for
an hour with a 10 per cent solution of NaCl, and finally
filtering the mixture through paper. The edestin may be
obtained in crystalline form if the extraction of the seed is
made with a 5 per cent solution of NaCl at 55° C. and the
extract filtered through a hot-water funnel. Upon cooling
the protein separates out of the solution in well-formed and
characteristic crystals, hexagonal in shape.

(a) Try two protein color reactions and three precipita-
tion reactions. Test the coagulability of the solution.

(6) Pour some of the solution, drop by drop, into a beaker
of water. A precipitation of the globulin occurs owing to
the decrease in the percentage of salt in the solution by dilu-
tion. A similar precipitation may also be accomplished by
removing the salts by dialysis.

(c) Start dialysis experiment. A very simple form of
dialyzer can be arranged as follows:

Select a beaker and a funnel whose stem has been removed
at the neck and whose diameter is somewhat greater than
that of the beaker. Allow the funnel to hang in the beaker
resting upon its rim. Cut and fold some well-moistened
parchment paper in the manner of a filter and place it in the
funnel. Fill the beaker nearly to the top with water and
pour into the parchment filter enough of the solution to be
dialyzed so that it about half fills it. The levels of the inner
and outer fluids should correspond.

In 24 hours note carefully any changes which may have
occurred. What has caused them?

(d) Saturate 10 c.c. of the globulin solution with MgS04.

24 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Filter off the precipitate and test the filtrate for protein.
Compare this reaction with the similar one for albumin.

(e) Cool 10 c.c. of the globulin solution to about 0° C.
and let C02 gas bubble through it. Note result.

(/) Take 10 c.c. of blood-serum and saturate it with
MgS04. What is this precipitate? Filter, and to the fil-
trate add two drops of acetic acid. What is this second
precipitate? Test both precipitates for protein.

ALBUMINOIDS.

The albuminoids form a class of substances of hetero-
geneous nature closely allied to the proteins, but exhibiting
also marked differences. They form the chemical basis of
the skeletal and epidermal structures and the variations in
the properties, and characteristics which are observed in
them are probably dependent upon morphological changes.
In general they occur in an insoluble form and are charac-
terized by their resistance to reagents which tend to dissolve

them.

COLLAGEN.

Collagen occurs as the chief constituent of connective
tissue (e.g., cartilage) and also as the organic matrix of bone
(ossein). It is easily hydrated by boiling with water or
weak acids and is then converted into gelatin, which on
this account may be considered as the hydrate of collagen.

GELATIN.

Gelatin possesses the characteristic of forming, with hot
water, solutions which set in a jelly upon cooling, if the con-
centration is greater than 1 per cent.

Finely cut tendons or bones from which the salts have

THE PROTEINS. 25

been previously removed by allowing them to remain in weak
HC1 for several days, serve as excellent material for the
preparation of gelatin.

Place the substance in an evaporating-dish half full of
slightly acidulated water and continue to boil until the
material is dissolved. Do not allow the solution to become
concentrated. Prolonged boiling will also cause the gelatin
to be converted into gelatoses which do not possess the
power of gelatinization.

Make the following tests, dissolving the jelly in hot water
as it is needed:

(a) Biuret; (6) Millon's; (c) Acetic acid and potassium
ferrocyanide; (d) Tannic acid; (e) HC1 or H2S04; (/) Satu-
ration with (NH4)2S04; (g) Lead-blackening sulphur; (h)
Adamkiewicz. Note and compare the results carefully with
those obtained with proteins.

KERATINS.

These form the chief constituent of hair, nails, hoofs, horns,
feathers, etc. Their main characteristic is insolubility, and the
relatively large percentage of sulphur which they contain.
This is probably present in a cystine nucleus in the molecule,
and the largest part is given off in the lead-blackening form.
Use horn shavings for the following reactions:
(a) Lead-blackening sulphur; (6) Millon's; (c) Xantho-
proteic; (d) Adamkiewicz; (e) Try its solubility in water,
dilute acids, and alkalies.

26 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

CONJUGATE PROTEINS.

These substances are much more complex in their chem-
ical constitution than the simple proteins. They may be
subdivided into —

1. The Glycoproteins — compounds of a protein component
and a carbohydrate complex.

2. The Nucleoproteins — compounds of a protein compo-
nent and nucleins or nucleic acids.

3. The Hcemoglobins and related bodies — compounds of
a protein component and a pigment which is chiefly respira-
tory in character.

4. The Lecithoproteins — compounds of a protein com-
ponent with lecithins.

5. The Phosphoproteins — compounds of a protein com-
ponent and a phosphorus containing substance other than
nucleic acid or lecithins.

GLYCOPROTEINS.

In a certain sense some of the simple proteins may be
considered as glycoproteins, in that they yield, upon decom-
position, reducing substances of undoubted carbohydrate
nature; in the case of the true glycoproteins, however, the
carbohydrate nucleus is obtained without interference with
the integrity of the component protein molecule. The
mucins, mucoids, and chondroproteins form the chief classes
of the glycoproteins.

Of the mucins, that obtained from the saliva is selected
as a typical example for study. They do not contain phos-
phorus, are acid in nature, and are mainly characterized by
forming viscous colloidal solutions. The mucins dissolve in
weak alkali solutions and may be reprecipitated on the addi-
tion of a weak acid. Upon boiling with a dilute acid they split
off a carbohydrate nucleus which reduces Fehling's solution.

THE PROTEINS. 27

Add 100 c.c. of saliva to 200 c.c. of 95 per cent alcohol.

Filter off the precipitated mucin and make the following
tests :

(a) Try some color-reactions for proteins.

(6) Dissolve some of the precipitate in weak NaOH and
then add dilute acetic acid, drop by drop. Note results.

(c) Boil the remaining precipitate for 10 minutes in a
small flask with 50 c.c. of 5 per cent HC1, cool, neutralize
with NaOH, and test for a reducing body with Fehling's
solution.

THE NUCLEOPROTEINS AND NUCLEINS.

As the name indicates, the nucleoproteins originate in
and are derived from the so-called mitoplasm or chromatin
of the cell nucleus and consequently exist in greater quanti-
ties in the glandular organs than elsewhere in the body.
Their individual nomenclature is founded upon their place
of origin; thus the pancreas, spleen, and yeast nucleopro-
tein. By boiling with a weak acid they are decomposed
into a protein component and the nucleins which, in turn,
upon further boiling, split up into another protein part and
the nucleic acids. The nucleoproteins and nucleins only
differ, therefore, in the quantity of the protein component
which they contain, and since the nucleic acid radical is the
carrier of all the phosphorus which is present in the com-
pound, it follows that the nucleins are chemically distin-
guishable from the nucleoproteins by their larger phosphorus
content. The two terms are sometimes used interchangea-
bly. The nucleins are acidic in character, soluble in alkalies,
but are precipitated by acids and alcohol. Upon treatment
with caustic alkalies, nucleic acids result, — substances which
contain phosphorus in the form of phosphoric acid. The

28 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

nucleic acids vary according to their content of carbohydrate
nucleus, purine and pyrimidine bases.

Impure nucleins may be prepared for study by thorough
digestion of a nucleoprotein-containing tissue with pepsin-
hydrochloric acid and extraction of the residue with NH4OH.
The nuclein is then precipitated from the solution with
dilute HC1.

(a) Try the solubility of the substance in water, dilute
alkali and alcohol.

(6) Try three color protein tests.

(c) Boil some with 10 per cent H2S04 for 5 minutes,
cool, add an excess of NH4OH, and finally a few drops of
AgN03 solution. Of what is the precipitate indicative?

(d) Treat a little of the substance in a crucible with some
fusion mixture. Test the fused mass for phosphorus.

DERIVED PROTEINS.

These substances may be subdivided into primary and
secondary derivatives according as the hydrolytic change is
slight or more deep seated.

1. PRIMARY PROTEIN DERIVATIVES.
(A) Metaproteins. — Products of the action of acids and
alkalies whereby the protein molecule is so far altered as to
form products soluble in very weak acids and alkalies but
insoluble in neutral fluids.

ACID AND ALKALI ALBUMINATES.

The albuminates are modified proteins derived from
either albumins or globulins by the action of acids and alkalies
with the aid of heat. They are non-coagulable by heat
except when held in suspension in the solutions from which
they have been precipitated by neutralization respectively

THE PROTEINS. 29

with acids or alkalies. In the formation of alkali albuminate
neutral sulphur is split off from the protein molecule.

To 25 c.c. of the albumin solution add 5 drops of very
dilute HC1 and warm for 15 minutes at 40° C. Cool.

(a) Exactly neutralize one-half of this solution with very
dilute NaOH. What is this precipitate? Shake up the pre-
cipitate and divide the solution into two parts. To the first
add a drop of NaOH and heat. Heat the other part and
then add a drop of NaOH. Explain the differences in results.

(6) Try three characteristic protein tests on the remain-
ing half of the solution.

To 25 c.c. of the albumin solution add 10 drops of NaOH
and warm for 15 minutes at 40° C. Cool.

(c) Try the effect of heating some of this solution.

(d) Neutralize the solution and repeat the procedure
employed under (a), using HC1 instead of NaOH.

(e) Try three characteristic protein tests.
Explain the results.

(B) Coagulated Proteins. — Insoluble products which result
from (1) action of heat on their solutions, (2) action of alco-
hols on the protein. Heat coagulation has been considered
on p. 21.

2. SECONDARY PROTEIN DERIVATIVES.
Products of a more pronounced hydrolytic cleavage of
the protein molecule.

(A) Proteases, see p. 52.

(B) Peptones, see p. 53.

(C) Peptides.— These are definitely characterized com-
binations of two or more amino-acids, the carboxyl group of
one being united with the arnino group of the other with the
elimination of a molecule of water. Some of the more com-
plex polypeptides give the biuret reaction.

MUSCULAR TISSUE.

The chief chemical constituents of muscular tissue may
be divided as follows:

The proteins of which the two most important are myogen,
an albumin (not typical;, and myosin, a globulin.

The nitrogenous extractives, comprising creatine, purine
bases, uric acid, carnine, and urea.

The non-nitrogenous extractives, made up of glycogen,
dextrose, lactic acid, inosit, fats, and inorganic salts.

Reaction. — Test the reaction of living and dead muscle to
litmus and Congo-red paper. Explain the differences in the
results obtained.

PROTEINS.

MYOGEN.

Place 25 grms. of hashed fresh muscle in a beaker with 75
c.c. of water and allow it to stand, with frequent stirring, for
one hour. Strain off the muscle through some cheese-cloth
(keep the residue), and filter the solution.

Use this solution for the following tests:

(a) What is the reaction of the solution?

(6) Test the coagulability of the protein in the solution.

(c) Perform three color protein tests.

(d) Is the protein precipitated by MgSO4 or (NH4)2S04?
Why is myogen not a typical albumin or globulin?

30

MUSCULAR TISSUE. 31

MYOSIN.

Digest the above meat residue with 100 c.c. of 15 per cent
NH4C1 solution for 24 hours in a covered beaker. Then filter
and use the filtrate for the following reactions:

(a) Pour a little of the solution, drop by drop, into a
beaker filled with water. Compare this reaction with a simi-
lar one under Globulins.

(6) Heat a few c.c. of the filtrate. Filter and test the fil-
trate for Ca. Is myosin a coagulable protein ?

(c) Saturate 10 c.c. of the filtrate with MgS04. Filter and
test the filtrate for protein.

(d) Try Adamkiewicz, Biuret, and Xanthoproteic tests.

NITROGENOUS EXTRACTIVES.

These substances, together with the non-nitrogenous
extractives, form the chief constituents of a hot-water extract
of muscular tissue, such as is known commercially as Liebig's
Extract. The most important of the nitrogenous extract-
ives are creatine, xanthlne, hypoxanthine, guanine, uric
acid, and urea. The latter two are only present in traces.
The purine bases represent in all probability an intermediate
stage in the nuclear metabolism of the muscle-cell between
the nucleins on the one hand and uric acid or urea on the
other.

/NH2

CREATINE, HN = C< /CH3

N\ //°

XCH2-C<

XOH

Extract 500 grms. chopped beef with 500 c.c. water for
half an hour over the water-bath at 50° C. Strain as dry as
possible through muslin and make a second extraction in the

32 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

same way, with an equal amount of water (save residue).
Unite the two extracts, and after concentration to about 200
c.c. acidify the solution with 2 or 3 drops of acetic acid.
Remove the coagulated proteids by filtration, and to the fil-
trate * add basic lead acetate, carefully avoiding any excess;
the precipitate consists of phosphates, chlorides, sulphates, etc.
Allow this to settle and then filter. Warm the filtrate and
pass H2S through it to remove the excess of lead. Filter hot.
The filtrate, which should be water-clear, is then concen-
trated on a water-bath to a thin syrup. Upon standing sev-
eral days in a cool place crystals of creatine will deposit. Filter
off the crystals, and wash them with 88 per cent alcohol.
(Keep the filtrate for the separation of the purine bases.)

Place some of the crystals in a small flask with 10 c.c.
dilute H2SO4, and heat for half an hour on the water-bath,
keeping the volume constant. While still warm add BaC03,
in substance, to neutralization. Filter and evaporate the
filtrate to 10 c.c. The creatine has been changed to creatinine.
Write the equation. Perform the following tests with the
solution:

(a) Place 2 drops of the solution upon a watch-glass and
add to it a few drops of an alcoholic solution of ZnCl2; allow
it to stand for several days and then examine the crystals
under the microscope.

(6) Weyl's Reaction. — To 2 c.c. of the creatinine solution
add three drops of a freshly prepared dilute solution of
sodium nitroprusside. Then add, drop by drop, dilute
NaOH. A ruby-red color is produced which quickly changes
to yellow. If the solution is now acidified with acetic acid

* This filtrate corresponds to Liebig's extract, and if the latter is used
for study instead of chopped beef, the procedure of separation is taken
up at this point.

MUSCULAR TISSUE. 33

and heated, a green color is obtained, and upon continued
boiling a precipitate of Prussian blue settles out.

(c) Jaffe's Reaction. — Treat some of the solution with a
dilute solution of picric acid, and make it faintly alkaline
with NaOH. The solution immediately becomes a deep red
Acetone responds to this test, but gives merely a yellowish-
red color.

PURINE BASES.

(6)

(1)

(2) HC (5)C— NI

I I (7)

C—

H (8)

(3) N - C -

(4) (9)

This represents structurally the compound, purine, the
nucleus from which all the purine bases may be derived. The
structure of the bases is easily obtained by substitution in
the nucleus at the position indicated by number.
Hypoxanthine = 6-Oxypurine.
Guanine = 2-Amino-6-oxypurine.
Xanthine = 2,6-Dioxypurine.
Adenine = 6-Aminopurine.

The purine bases in greater part exist as constituent groups
in the more complex nucleic acid molecule. In some tissues,
however, they have been found in the free state. They form
crystalline salts with the mineral acids and are all precipitated
from acid solutions by means of phosphotungstic acid.
Upon the addition of ammoniacal silver nitrate they sepa-
rate from their solutions as silver combinations. Copper
acetate combines with them to form insoluble compounds.

The separation of the bases from the creatine filtrate (see
p. 31) takes place as follows:

34 LABORATORY WORK IX PHYSIOLOGICAL CHEMISTRY.

After removal of the alcohol, the filtrate is made ammo-
niacal with NH4OH and ammoniacal AgN03 solution added
until no further precipitation occurs. This precipitate is
composed of the double silver salts of all the purine bases.
These silver combinations, after their removal from the
solution by filtration, are dissolved in boiling-hot HX03
(sp. gr. 1.1) and the mixture filtered hot.

In this reaction the silver nitrate compounds of the bases
are formed, all of which are soluble in the hot nitric acid solu-
tion. Upon cooling, the guanine, adenine, and hypoxan-
thine combinations crystallize out, but the xanthine remains
in the solution, from which, after filtration and treatment
with NH4OH in excess, it is thrown down as a reddish
precipitate (xanthine silver oxide). This precipitate, sus-
pended in its solution, is next treated with hydrogen sulphide
and the mixture warmed and filtered. The xanthine will
crystallize out of the filtrate upon concentration.

The precipitate of the three other bases is suspended in
water and treated with hydrogen sulphide, warmed and
filtered hot in the same manner as the xanthine. After con-
centration the solution is saturated with NB^OH and digested
on the water-bath. The guanine remains insoluble, while
the other two bases pass into solution. Filter the mixture
hot. From this filtrate when freed from the ammonia and
allowed to cool, the adenine separates, leaving the hypoxan-
thine in the solution.

(a) Try some of the crystals with murexide test.

(6) Examine the various precipitates for crystals. Make
sketches of them.

(c) Warm some of the xanthine crystals with bromine-
water and evaporate the solution to dryness on the water-
bath. Allow ammonia vapors to come in contact with the
residue; this becomes red.

MUSCULAR TISSUE. 35

NON-NITROGENOUS EXTRACTIVES.
GLYCOGEN (CftH1005)n.

This polysaccharide, sometimes called animal starch on
account of its similarity in function to plant starch, exists
wherever living protoplasm is present and is therefore
classed as one of the primary constituents of the cell. It is
especially abundant in the liver, where it apparently repre-
sents the carbohydrate surplus of the organism and in the
muscle, where it serves as a ready source of energy. Glyco-
gen forms a tasteless white powder, insoluble in alcohol and
ether, but forming with water an opalescent solution which
is dextrorotatory. It does not reduce metallic oxides in
alkaline solution. By the action of hydrolytic agents or
enzymes it is converted into maltose and dextrose similarly
to starch.

The common scallop serves as the most convenient and
prolific source of glycogen from muscular tissue. By the
simple extraction of the tissues with boiling water, slightly
acidulated, a solution is obtained for the following reactions:

(a) Notice the color of the solution. Add 2 drops of the
iodine solution to 5 c.c. of the glycogen solution. Warm
gently and allow to cool. Note changes.

(6) Test 5 c.c. of the solution with Fehling's solution.

(c) To 5 c.c. of the solution add three volumes of 95 per
cent alcohol.

(d) To 5 c.c. of the solution add 5 drops of concentrated
HC1 and boil for some minutes. Neutralize cold with NaOH
and test with Fehling's solution. What is this reducing
substance? How could you prove it?

(e) To 5 c.c. of the solution add a few drops of filtered
saliva, and warm at 40° C. for a few minutes. Notice changes
in the solution. Determine the character of the final reducing
body formed.

BONE.

Bone consists of an organic matrix, ossein, which is identi-
cal with collagen obtained from the white fibrous connective
tissue. This matrix is impregnated with insoluble inorganic
salts, which serve to give strength and stability to the tissue.
Ossein when hydrated with weak acids is converted into
gelatin.

MINERAL CONSTITUENTS.

Incinerate 10 grms. of bone under the hood. Extract the
grayish residue with 25 c.c. of hot dilute HN03, filter, and
use the filtrate for the following tests:

(a) Phosphoric Acid. — To 10 c.c. of the solution add 10 c.c.
of the molybdic solution and warm in the water-bath at 75°
C. until a canary-colored precipitate settles out. What is
this? Filter it off on a small filter, wash once with very
dilute HN03, and then dissolve the precipitate upon the
filter by adding dilute NH4OH,' drop by drop. To the fil-
trate add a few c.c. of magnesium mixture. What is this
precipitate? Write the equations for all the reactions in
this procedure.

(6) Chlorides. — To a few c.c. of the solution add a few
drops of AgN03. What results?

(c) Calcium. — Make 10 c.c. of the solution alkaline with
NH4OH. What is the precipitate? Filter, and to the filtrate
add ammonium oxalate. The white precipitate of calcium

36

BONE. 37

oxalate is proof of the presence of calcium combined other-
wise than with phosphoric acid.

(d) Magnesium. — To 15 c.c. of the solution add a slight
excess of NH4OH. Then make the mixture acid with acetic
acid; this should dissolve the greater part of the precipitate.
The slight insoluble substance is to be filtered off, dissolved
in HC1 and tested for iron by means of potassium ferro-
cyanide. The acetic acid filtrate is treated with a few c.c.
of ammonium oxalate and the resultant precipitate filtered
off. This new filtrate is made alkaline and a few c.c. of a
disodium phosphate solution added. Why does a precipitate
at this point prove the presence of magnesium? Write the
equations.

(e) Carbonates. — Did you notice an effervescence when
you added HN03 after incineration? What was the cause?

NERVOUS TISSUES.

Nervous tissue presents for chemical study the following
classes of substances:

1. Proteins — two ordinary cell globulins coagulating at
45° C. and 75° C., and a nucleoprotein.

2. Lipoids — bodies of a fatty nature, including the leci-
thins, cerebrosides, and protagon. It is somewhat question-
able whether the latter substance is a unit or a mixture of
lecithins, cer^brin, and proteins.

3. Cholesttrols and fatty acids.

4. Neurokeratin — a substance apparently related to ker-
atin.

5. Extractives — the same as those of muscular tissue.

LIPOIDS.

The lipoids, together with the cholesterols, are usually
classed as the myelin bodies from the fact that they exist as
a mixture in the so-called myelin substance of the medulla.
They contain fatty acid radicals in their molecule, and on
this account are closely related to the fats and sometimes
are classified under them.- For the sake of convenience in
isolation for study they will be considered here, although it
must not be forgotten that the lecithins are widely distributed
in all forms of living tissues and belong to the primary con-

38

NERVOUS TISSUES. 39

stituents of cells in general. The cerebrosides are found in
plant as well as animal tissues. So little that is positive
is known about ' ' protagon " that no notice need be taken of it.

LECITHINS.

CH-O-R'

CH2-0-

PO— 0-C2H4\

(CH3)=N
OH OH/

R and R'=the same or different fatty acid radicals.

The structure of the lecithin molecule is proved by the
•decomposition products which are obtained from it, viz.,
chohne, glycerophosphoric acid, and fatty acids. From the
fact that various fatty acid radicals enter into the composi-
tion of the molecule and that the nitrogenous complex may
also differ, it follows that the existence of many lecithins is
possible. As a class they are soluble in ether and alcohol
and from such solutions may be precipitated by cooling to
0° C. or by the addition of acetone. The lecithins combine
with acids and bases as well as with proteins and other bodies
to form more or less loose combinations, such as the lecitho-
proteins. With water they swell up, giving off long fila-
ments (myelin forms), and finally pass into an emulsion.

Various lecithins can be separated from the yolk of the
egg, from yeast, or from brain-tissue. The procedure for the
isolation of the brain-lecithins is as follows: The finely
divided brain-tissue, which may or may not have been dehy-
drated by boiling with acetone for some hours previously, is

40 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

allowed to stand for three days in cold ether. The solution
is then filtered, and to the filtrate, which contains the lecithins
and cholesterol, acetone is added. The precipitated leci-
thins may be removed by filtration and the ether-acetone
filtrate evaporated to dryness. (Keep this cholesterol.)
Try the following reactions with the lecithin:
(a) Place a particle of the substance on a glass slide and
add a drop or two of water. Examine this under the micro-
scope.

(6) Shake some of the substance in water and examine
microscopically.

(c) To some of the substance add a drop of osmic acid.
What is the effect?

(d) Test the substance for phosphorus by fusion.

(e) Test for the glycerol radical.

CEREBROSIDES.

This class of non-phosphorized nitrogenous substances,
whose exact composition is not known, is characterized by a
glucoside constitution yielding upon decomposition a sugar
which is identical with galactose. Fatty acids in relatively
large quantities can also be isolated from the cleavage prod-
ucts.

CEREBRIN.

This is the most important substance in this group.
Whether it exists uncombined in the tissue is still an un-
decided question. It is insoluble in water, dilute alkalies,
or baryta-water. It dissolves in boiling alcohol, from which
solution it separates as a flaky precipitate upon cooling.
This precipitate is made up of microscopic globules.

NERVOUS TISSUES. 41

Cerebrin is usually prepared according to the following
procedure: A small amount of the finely divided brain-tissue
is boiled for one-half hour in a casserole with twice its weight
of baryta-water. The solution is filtered off and the residue
extracted with boiling 95 per cent alcohol. From this
extract, if filtered while still hot, the cerebrin will separate
in small crystals. The cholesterol should stay in the alco-
holic solution, but if any becomes insoluble with the cere-
brin it is easily removed by washing the precipitate with
ether in the filter.

Test the substance as follows:

(a) Try to detect the presence of nitrogen and phos-
phorus.

(6) Boil some of the substance for one hour with 5 per
cent H2S04. After cooling the mixture, neutralize it and
test for a reducing action on Fehling's solution.

(c) Warm some of the substance on a platinum foil.
Notice the odor. To what is it similar?

THE CHOLESTEROLS.

These bodies belong to the class of primary cell con-
stituents. They are studied here on account of the existence
of suitable material at hand. Cholesterol usually exists
free, as in the case of the brain-tissue and gall-stones, but it
is -also found in combination with fatty acids, as esters in
the blood-plasma. The best known cholesterol is a mon-
atomic alcohol with C27H45OH as the empirical formula. At
one time it was termed a " non-saponifiable " fat. It has
the general solubilities of the fats and crystallizes readily
from an alcohol or ether solution in the form of superimposed

42 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

rhombic plates. The bile-acids also have a solvent action
on cholesterol.

Make use of the cholesterol prepared in the course of the
separation of lecithin (p. 39).

(a) Try the solubility of the substance in water.

(6) Salkowski's Test. — Dissolve a small quantity of the
cholesterol in a few c.c. of chloroform and add an equal
volume of concentrated H2S04. The acid solution takes on
a greenish fluorescence and the chloroform becomes red.

(c) Liebermann's Test. — Dissolve a crystal of cholesterol
in about 2 c.c. of chloroform and add first 10 drops of acetic
anhydride and then concentrated H2S04, drop by drop. The
mixture will become red, then blue, and finally green.

SALIVARY DIGESTION.

As usually obtained for study, the saliva forms a mixture
of the secretion of the submaxillary, sublingual, parotid,
and the mucous glands of the mouth. It presents a viscid,
slightly opalescent appearance, possessing either alkaline
or acid reaction according to the indicator employed, and
containing about 0.5-1 per cent solids. Besides the proteins
and the potassium sulphocyanide, its chief and essential
organic constituent is the enzyme, ptyalin, which gives to
the secretion its importance in the digestion of carbohydrates.
A physical function of the saliva consists in rendering the
food moist, which action, in conjunction with the presence of
the mucin, allows the food-bolus to be more readily swal-
lowed.

Chemical Characteristics.

Collect about 50 c.c. of filtered saliva.

(a) Test its reaction with litmus paper and phenol-
phthalein. What causes this apparent discrepancy?

(6) To a few c.c. of saliva add acetic acid, drop by drop,
until a precipitate forms. (See under Mucin.) Filter off
the mucin and test the filtrate for protein. What is the
result?

(c) Allow a drop of saliva to fall in the centre of a piece
of filter-paper. Then add a drop of ferric chloride to the
paper where it is moist. Note color. Now add a drop of

43

44 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

mercuric chloride. What is the result? For what substance
are these tests? >

PTYALIN.

This substance belongs to the class of amylolytic enzymes
in that its function consists in the conversion of starch and
glycogen into maltose. Its action is maximum at 46° C.
and is destroyed at 65-70° C. Alkalies and free acids tend
to retard its activity. Apparently it acts best in a neutral
solution and in the presence of proteins with which the
acids or alkalies may combine; in this way a greater degree
of acidity or of alkalinity may be borne without injury
than in a pure solvent. This is important to -remember
when considering the possibility of the continuance of saliva
digestion in the stomach.

AMYLOLYSIS.

The amylolytic action of ptyalin on starch is a hydrolytic
change in which successive products are evolved until the
final stage .of maltose or isomaltose is reached. The end
product of a similar cleavage by means of weak acids is
dextrose. Thus from starch is formed successively soluble
starch or amygdulin, then erythrodextrin, the achroodextrins,
maltodextrin and maltose, and, in case of acids, dextrose.
There are indications that at each step of the cleavage small
quantities of maltose are produced. These facts can be
shown by the following experiment:

(a) Prepare some starch paste and allow it to cool to
40° C. Dilute the saliva five times and regulate the water-
bath for 40° C. Take 15 c.c of the paste; add to it 1 c.c. of
the diluted saliva and place the test-tube in the water-bath.
Watch the mixture until the turbidity disappears and the
solution becomes transparent. Then pour out into a clean

SALIVARY DIGESTION. 45

test-tube a few c.c. of the solution and add a drop of iodine
solution. What color is obtained? Replace the mixture
in the bath and as the amylolysis proceeds remove successive
portions (2 c.c.) and test with the iodine solution. What
successive variations in color do you obtain? Finally the
iodine fails to give a color to the solution. This is called the
achromic point; why? Test the remainder of the solution
with Fehling's solution. What is the character of the sugar
present? What is the cause of these changes indicated by
the iodine reaction? Make a scheme of the successive
hydrolytic products formed in the digestion.

Influence of Conditions upon the Activity of Ptyalin.

(6) Make up the following mixtures in separate test-tubes
and keep them in the water-bath at 40° C., using in each case
5 c.c. of the diluted saliva and 10 c.c. of the starch paste.

(1) Starch paste + saliva.

(2) Starch paste + saliva first boiled and then cooled.

(3) Starch paste + saliva exactly neutralized with 0.2%

HC1.

(4) Starch paste + saliva made acid with 0.2% HC1.

(5) Starch paste + saliva made alkaline with 0.5%

Na,C03.

In all these test-tubes note carefully the changes which
are taking place from time to time, testing the rapidity of
digestion by means of the iodine reaction. Determine the
varying lengths of time necessary for the achromic point to
be reached.

After some time it will be noticed that in experiments (4)
and (5) the starch paste has not changed; change the reac-'
tion in each to that corresponding to the normal saliva and
again allow them to digest. What result?

Make deductions from these test-tube experiments.

GASTRIC DIGESTION.

Pure mixed gastric juice such as is obtained from a gastric
fistula forms a clear, colorless, and odorless solution with
an acid reaction and low specific gravity (1.004). The
total solids amount to only 2 per cent, of which the following
are the chief.

Inorganic: hydrochloric acid, the chlorides of sodium,
potassium, and calcium, with traces of the phosphates of
magnesium and iron.

Organic: pepsin, rennin or chymosin, mucin, traces of
other proteins, and sometimes lactic acid.

HYDROCHLORIC ACID.

In all probability the most important function of the
hydrochloric acid is that of a germicide preventing putrefac-
tion and the formation of obnoxious gases. Secondarily, it
gives to the gastric contents a more favorable condition for
the action of the pepsin, although the establishment of an
acid reaction is not absolutely necessary for peptic digestion.
The hydrochloric acid also seems to possess some power of
inverting cane-sugar.

The following experiments are intended to simulate possi-
ble gastric mixtures which may be encountered in testing
stomach contents for HC1. The acid may appear alone or
together with lactic acid and digestive products. The relia-
bility and sensitiveness of the various indicators employed
under the different conditions will also be shown.

46

GASTRIC DIGESTION. 47

Test each of the following solutions with all the indicators
mentioned below, and tabulate the results whether positive or
negative.

(a) 0.3 per cent HC1; (6) 0.05 per cent HC1; (c) 0.8 per
cent lactic acid; (d) a mixture containing equal volumes of
"a" and "c"; (e) a mixture containing equal volumes of "b"
and a 2 per cent albumose solution. Before using, warm the
last mixture for a few minutes at 40° C.

Indicators.

An indicator is a substance which possesses a different
color when dissolved in an alkaline solution from what it
does in an acid one. They are slightly dissociable acids or
bases, and the change in color is due to the appearance or
disappearance of colored ions.

1. Dimethylaminoazobenzene, N(CH3)2-C6H4-N=N-C6H5.
Add one or two drops directly to the solution to be tested.
Free mineral acid is indicated by a carmine-red color.

2. TropceolinOO, NH(C6H5) - C6H4 - N = N - C6H4 -
S03Na. Add one or two drops directly to the solution to
be tested. Free acid is indicated by a red or reddish- violet
color. The reaction becomes more delicate when performed
in a similar manner to that suggested for Giinzburg's reagent.

3. Congo-red. — Use Congo-red paper, prepared by dipping
filter-paper into the alkaline indicator solution and drying.
Free acid is indicated by the blue color.

4. Gunzburg's Reagent. — Evaporate 2 drops of the solution
to be tested with 1 drop of the indicator, in a porcelain dish,
carefully, over a water-bath. Upon dryness the presence of
free HC1 is indicated by the development of a rose-red color.

5. Boas' Reagent. — Mix 3 drops of the solution to be tested
with the same amount of indicator and evaporate cautiously

48 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

in a porcelain dish. Free acid is shown by a rose or vermil-
ion color.

/XX /(OH),

6. Alizarin, C6H4< >C6H<

XXX \S03Na

Add 2 or 3 drops of the indicator directly to the solution.

The color will be yellow in acid and acid salt solutions, but
red to violet in alkaline. This indicator is especially suited
for the determination of the acidity of the urine by titration.

/C6H4OH

7. Phenolphthak'in, C6H

Add 4 drops of the indicator directly to the solution.

The indicator is colorless in neutral and acid reactions, but
becomes red in the presence of alkalies. It shows the pres-
ence of free, combined, mineral, and organic acids, and acid
salts of all kinds. It therefore gives the total acidity. It
may indicate an acid reaction when dimethylaminoazoben-
zene shows alkaline. Explain this.

The Inverting Action of HCL f

To 5 c.c. of a 5 per cent cane-sugar solution add 5 c.c. of
1 per cent HC1 and place the mixture in the water-bath for
an hour at 40° C. At the end of this time cool, neutralize,
and test the reducing power of the mixture. Explain the
result.

LACTIC ACID, CH3.CH(OH)-C^OH-

Although this substance is commonly considered as an
abnormal constituent of the stomach contents it may exist

GASTRIC DIGESTION. 49

as a normal product formed during the digestion of a meal
rich in carbohydrates.

The following experiments are designed to show the ordi-
nary tests for lactic acid and the effect upon the reliability
of these tests, of the simultaneous presence of bodies likely to
be found in a stomach contents.

(a) To successive portions of 5 c.c. of Uffelmaris reagent
add a few drops of solutions a, c, and d, under Hydrochloric
Acid. Note carefully color changes and make deductions.

Make a very dilute solution of Fe2Cl6 in which the yellow
color is hardly visible. Such a reagent is much more sensi-
tive than Uffelman's. Use 5 c.c. of the dilute ferric chloride
solution in testing each of the following:

(6) Solutions a, c, and d (under HC1).

(c) A solution of H2NaP04.

(d) Alcohol, 5 per cent.

(e) A 1 per cent solution of saccharose; glucose.

Make deductions as to the value of the test applied
directly to gastric contents.

In order that all chances for error may be avoided, lactic
acid can be easily separated from disturbing conditions by
shaking the stomach contents or gastric juice with ether in
which the acid is soluble. Such an ether extract is evapo-
rated carefully on the water-bath, the residue taken up with
water and tested with the dilute Fe2Cl6 solution.

As this ether extract cannot contain any of the above
substances, the presence of which in the stomach contents
might interfere with a correct diagnosis, a positive test for
lactic acid in this case is decisive evidence of its presence.

PEPSIN AND PEPSINOGEN.

Pepsin is apparently not secreted as such, but appears
first in the gastric mucosa in an antecedent form, or zymogen,

50 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

pepsinogen. This latter substance shows considerable resist-
ance to reagents (dilute alkalies) which destroy pepsin.
Pepsinogen is itself inactive proteolytically, but seems to be
converted into active pepsin by the action of the HC1 of the
gastric juice. Pepsin belongs to the proteolytic enzymes,
converting proteins into proteoses, peptones, amino acids,
and diamines. The temperature of its optimum activity is
from 35-45° C. It is destroyed at 65° C.

Pepsin is only active in acid solutions, and the amount of
acidity required for its maximum activity varies according to
the character of the acid employed and the form of protein
to be digested. Pepsin is easily destroyed by weak alkalies
(0.01 per cent), and less readily by strong acids (5-10 per
cent).

The following set of experiments should prove these
facts:

1. A glycerol extract of a pig's gastric mucosa contains
pepsinogen.

2. A 0.2% HC1 extract of a pig's gastric mucosa contains
pepsin HCL

3. A watery extract of a pig's gastric mucosa contains
pepsin.

Make use of the above extracts numbered 1, 2, and 3
respectively, and in each test-tube add a piece of fibrin,
keeping all at 40° C. in the water-bath.

(a) Fibrin + 5 c.c. of 0.2% HC1.

(b) " +5 c.c. of solution 3.

(c) " -f one drop of solution 1 + 5 c.c. of water.

(d) " + 5 c.c. of solution 2.

(e) " + one drop of solution 1 + 5 c.c. of 0.2% HC1.
(/) " + 5 c.c. of solution 2 (the latter having pre-
viously been heated to boiling and again cooled).

(#) Fibrin + one drop of solution 1 (the latter having

GASTRIC DIGESTION. 51

previously been heated to boiling with 5 c.c. water and again
cooled) + 5 c.c. of 0.2% HC1.

(h) Fibrin + 5 c.c. of solution 3 + 5 c.c. of 0.8% lactic
acid.

(&) Fibrin + 5 c.c. of solution 3 + 5 c.c. of 1% oxalic acid.

(ro) " +5 " " " 3 + 5 " "5%HC1.

(n) " +5" " " 3 + 5" "0.5%Na,CO,.

(p) " +5 " " " 3 + 3 " "bile.

Note carefully, in each case, the relative rapidity with
which the flock of fibrin is disintegrated.

PEPTIC PROTEOLYSIS.

The previous experiments have indicated that the action
of pepsin is directed toward the transformation of the proteins
by a process of cleavage into soluble and diffusible products.
The question concerning the characterization, separation,
and identification of these soluble digestive products appears
at present to rest in a state of uncertainty, but in a general
way they are conventionally divided into the proteoses, pep-
tones, and a mixture of nitrogenous substances ("amino"
bodies) only characterized by not giving the biuret reaction.
The current method for the separation of these bodies is
based, first, upon the precipitation of the proteoses by com-
plete saturation of their solution with (NH4)2S04, and, second,
upon the precipitation of the peptones in the filtrate (from
the proteoses) by means of iodo-potassium iodide,, The
remaining substances are removed from their solution
by the addition of phosphotungstic acid. The proteoses
allow of a further separation by means of fractional precipi-
tation with (NHJ2S04, and the peptones are divided into
two fractions by 95 per cent alcohol. This method isolates

52 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

three fractions of proteoses and two of peptones, the pro-
cedure for which is the following:

For the purposes of study, a pepsin-hydrochloric acid
digestion of meat or fibrin should be prepared, using 0.3%
HC1 and 0.5 grm. pepsin per liter (Grubler's purissimum or
Parke-Davis' scale pepsin). Allow the digestion to proceed
at 40° C. for three days. After filtration of the digestion
mixture and exact neutralization with dilute NaOH the
solution is ready for use. Heat a little of it to boiling to
show that no coagulable proteins exist in the mixture.

To a given quantity of the digestion mixture add an
equal volume of a saturated solution of (NH4)2S04. Stir
the solutions together and allow the mixture to stand until
the resulting precipitate has settled sufficiently to allow
of considerable decantation. Filter the remainder. This
precipitate obtained by half saturation of the mixture repre-
sents

Fraction I. — It may be further separated into two parts:
one soluble in 95 per cent alcohol — protoproteose,
and the other remaining insoluble — heteroproteose.

In the constitution of protoproteose the aromatic
groups predominate, while in heteroproteose the
fatty acid radicals are chiefly found.

To the filtrate from Fraction I add one-half its volume of
a saturated solution of (NH4)2S04. Allow this to stand
and proceed as before. This second precipitate obtained
by two-thirds saturation corresponds to

Fraction II. — It consists of Deuteroproteose. A and contains
the greater part of the lead-blackening sulphur
which was present in the original proteid molecule.
On this account it may be termed Thioproteose.

Completely saturate the filtrate from Fraction II with

GASTRIC DIGESTION. 53

(NH4)2S04 (in substance), allow the mixture to stand, and
treat as previously.

Fraction III. — This precipitate is termed Deuteroproteose B or
Synproteose. It contains the greater part of the car-
bohydrate nucleus of the original protein molecule
and is, therefore, sometimes called Glycoproteose.

To the filtrate from Fraction III add TV its volume of a

N

— H2S04 solution, which is saturated with (NH4)2S04. Pro-
ceed with the precipitation and filtration as before.

Fraction IV. — This fraction, which in the case of some
proteins is absent, is designated as Deuteroproteose C.

The acid-saturaled filtrate from Fraction IV is treated
with a solution of iodo-potassium iodide (saturated with
(NH4)2SO4) until no further precipitate results. This is
allowed to settle and is then filtered off. This precipitate
upon treatment with 95 per cent alcohol subdivides into a
soluble and insoluble fraction. If the insoluble residue
is filtered off it forms

Fraction V, denoted as Peptone A; and

Fraction VI, which consists of the alcoholic filtrate from
Fraction V, is called Peptone B.

Very little is known regarding the character of
these two peptone fractions. Undoubtedly they
contain peptides of varying complexity mixed with
amino acids. Fraction V gives the biuret reaction
while Fraction VI does not; hence the former frac-
tion must possess the most complex polypeptides.

The "amino" bodies are precipitable with phospho-
tungstic acid and remain in greater part in the filtrate sub-
sequent to the treatment with iodo-potassium iodide.

54 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

More or less of acid albuminate may be formed during
the initial stage of the digestion (first half -hour), but this
quickly disappears and apparently does not participate
in the future formation of the proteoses, peptones, and
"amino" bodies.

Perform the following tests with the different fractions:

(a) Try the biuret reaction on each fraction.

(6) Test Fractions I and // for lead-blackening sulphur.

(c) Boil Fraction III with 10 per cent H2S04 for an hour.
Cool, neutralize, and test the mixture with Fehling's solu-
tion.

(d) Show, first, that the solutions of Fractions I, II, IIIt
IV are precipitated by nitric, picric, and trichloracetic acids,
and that the precipitates so produced disappear when heat
is applied and reappear upon cooling; and, second, that
they are non-coagulable by heat and respond to the acetic
acid and potassium ferrocyanide reaction.

(e) Prove that the peptones (Fractions V and VI) are
not precipitated by nitric or trichloracetic acids; that Millon's
and xanthoproteic reactions give indecisive results; that
lead-blackening sulphur is absent; and that both tannic and
picric acids combine to form insoluble bodies.

It must be finally emphasized that these various fractions
are not to be considered as unit substances. The method of
separation just outlined simply attempts to isolate certain
mixtures ("fractions") each of which contains in greater
part some characteristic component group or complex
broken off during the peptic cleavage of the original protein
molecule. Thus in Deuteroproteose A the lead-blackening
or cystine nucleus predominates, while Deuteroproteose B
is characterized by containing the larger part of the carbohy-
drate complex.

GASTRIC DIGESTION. 55

RENNIN OR CHYMOSIN.

This body is classed as a coagulating enzyme acting
upon caseinogen with the formation of an albumose-like
body and soluble casein. The latter in the presence of cal-
cium salts is precipitated in the form of a coagulum or curd
which is termed (insoluble) casein.

Rennin is affected by varying conditions (temperature,
reaction, etc.) in a way similar to pepsin.

Make use of the rennin solution given and prepare five
test-tube mixtures as follows, using in each case about 5 c.c.
of milk and holding them at 40° C. for fifteen minutes;
in each experiment add the rennin solution last.

(a) Milk + 15 drops of 0.3% HC1.

(6) " +1 c.c. of rennin solution.

(c) " +1 " " " " +2 cc. ammonium
oxalate.

(d) Milk + 2 c.c. of rennin solution + 5 drops 0.3%
HC1.

(e) Milk + 2 c.c. of rennin solution + 10 drops 0.5%
Na2C03.

Note the results carefully. If (c) has not clotted, heat
it to boiling to kill the enzyme. Cool and add a few drops
of calcium chloride solution. What is this precipitate
which settles out? What are the effects of acids and alka-
lies on the milk alone? Why did not the milk clot in experi-
ment c?

PANCREATIC DIGESTION.

The composition and character of pancreatic juice varies
greatly under different conditions. Thus far it has been
practically impossible to collect the juice in a manner which
will permit of the assumption that it is comparable to that
secreted into the intestine.

The juice as obtained from a temporary fistula is clear,
viscous, and alkaline in reaction, the alkalinity corresponding
to about a 0.5 per cent Na2C03 solution. It differs from
the gastric juice hi being rich in proteins, having a sp. gr.
of about 1.030. The character and quantity of the enzymes
which may be present hi the fluid varies considerably accord -
ing to the nature of the diet.

The following have been shown to exist in different
extracts of the gland and in the secretion itself at certain
times.

1. Trypsin — a proteolytic enzyme appearing first in the
gland as a zymogen, trypsinogen, entirely comparable to
pepsinogen.

2..Amylopsin or pancreatic diastase — an amylolytic en-
zyme almost identical with ptyalin.

3. Steapsin or lipase — a lipolytic enzyme probably not
characteristic of the pancreatic juice.

4. Pancreatic rennin — a coagulating enzyme acting on
the caseinogen of milk in a way somewhat similar to the
gastric rennin. Various extracts of the pancreatic gland

56

PANCREATIC DIGESTION. 57

have been prepared each with a view to obtaining predominat-
ing reactions for the specific enzyme. (See Appendix.)

TRYPSIN AND TRYPSINOGEN.

Trypsin is active in an alkaline, faintly acid or neutral
solution, but its maximum reaction occurs in a 1 per cent
solution of Na2C03. It is killed at a temperature about
55° C.

Use the proteolytically active pancreatic extract and
prepare test-tube digestions in the water-bath at 40° C., as
under gastric digestion.

(a) Fibrin + 5 c.c. of the pancreatic extract.

(6) " +5" " " " " + 2 c.c. 0.5%

Na2C03.

Compare the manner of action of the trypsin upon the
fibrin to that of the pepsin.

(c) Fibrin + 5 c.c. of 0.5% Na2CO3.

(d) " + 5 " " " " li +5 c.c. pancreatic ex-
tract which has been previously boiled and cooled.

TRYPTIC PROTEOLYSIS.

As regards its proteolytic activity, the enzyme trypsin
is more energetic and far-reaching than pepsin. Conse-
quently in pancreatic digestion there takes place a more
rapid and deep-seated cleavage of the protein molecule
than is possible in the stomach. Proteoses and peptones
are formed in the middle stages of the digestion, but these
are rapidly carried over into substances with a relatively
small molecular weight which do not give the biuret reaction
and consequently cannot be termed peptones. It is now
considered that the end products of a long-continued tryptic
digestion of the ordinary proteins comprise the amino-acids,

58 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

viz., leucine, tyrosine, aspartic and glutamic acids; the hexone
bases, viz., arginine, lysine, and histidine; the so-called trypto-
phane or proteinochromogen, which is an indole derivative ; and
probably other bodies whose nature is not well understood.

Prepare a pancreatic digestion as follows:

Place in a large flask (5-10 liters) 500-1000 grms. of
fibrin, a liter of an infusion of Kuhne's dried pancreas, and
1000-2000 c.c. of 0.25 per cent Na2C03; add plenty of chloro-
form or powdered thymol, and allow the mixture to digest at
40° C. for at least a week.

At the end of this time the solution is exactly neutralized
with dilute H2S04 and concentrated to about one-half its
volume; it is then filtered. From the filtrate the proteoses
are removed by saturation with (NH4)2S04 in a nearly
boiling solution. These are filtered off and tho filtrate still
further concentrated. During this procedure relatively
large quantities of leucine and tyrosine should crystallize out
of the mixture along with the (NH4)2S04. These must be
filtered off and the (NH4)2S04 removed from the filtrate by
means of Ba(OH)2 and BaC03. The BaS04 is again re-
moved by filtration and to the new filtrate dilute H2S04
added to just precipitate the excess of Ba in the solution.
After filtration the solution will contain the hexone bases.

Compare the quantities of proteoses and peptones ob-
tained in this digestion with those from the peptic proteolysis.

TYROSINE, CH2-CH-COOH.

Use the crystals obtained in the pancreatic digestion.

(a) Examine them under the microscope.

(6) Try to dissolve some in cold, then warm water. Then

PANCREATIC DIGESTION. 59

add about 5 c.c. of Millon's reagent to the test-tube and
heat. Why is this result positive?

(c) Piria's Test. — Place a few of the tyrosine crystals upon
a small watch-glass with two drops of concentrated H2S04 and
warm for half an hour on the water-bath. Then transfer it
with about 15 c.c. water to a test-tube and neutralize with
BaC03 in substance. Filter and add a few drops of weak
ferric chloride solution (neutral). A positive test is evidenced
by the formation of a violet .color.

(d) Morner's Test. — To a little of the powder placed in a
test-tube add a few c.c. of Morner's reagent, and heat care-
fully to boiling. A green color develops which is quite
lasting.

CHJX /NH*

LEUCINE, >CH-CH,-CH „

PTT / \ /}J

X)H

Make use of the substance obtained from the pancreatic
digestion.

(a) Examine some crystals under the microscope.

(6) Place a little in a clean dry test-tube and warm care-
fully. The leucine sublimes, without melting, on the cold
parts of the tube. If the powder is heated too high or
suddenly, the substance is decomposed and the odor of
amylamine is given off. Write the equation for the formation
of this substance.

(c) Scherer's Test. — Heat a little leucine with two drops of
concentrated HN03 upon a platinum foil over a free flame
until a colorless residue is obtained. Then add two drops
of NaOH and evaporate carefully in the flame. The mass
becomes dark red in color and rolls around on the foil like
an oil-drop.

60 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

AMYLOLYSIS.

Amylopsin is stronger in its action than ptyalin; other-
wise the conditions which influence its activity are practically
the same as the salivary diastase.

Make use of the glycerol or alcoholic extract of the pan-
creas.

To 10 c.c. of starch paste add 5 c.c. of pancreatic extract
and place in the water-bath at 40° C. Test from time to
time with the iodine solution as you did under Salivary
Digestion. Do you get an achromic point? Finally test for
a reducing sugar. What is the character of this?

LIPOLYSIS.

Steapsin is the least stable of all the pancreatic enzymes,
and is particularly sensitive to the action of acids, being
destroyed easily by all except the higher fatty acids.

Its action on neutral fats tends toward a breaking down
of the fat molecule into fatty acids and glycerol.

For the following experiments use the alkaline glycerol
extract of the gland.

(a) To 10 c.c. of "litmus milk" add 5 c.c. of the pan-
creatic extract. Place the test-tube in the water-bath at
40° C. What is the cause of the change which takes place?

(6) Repeat experiment (a) with the exception that
boiled pancreatic extract is used. Note any change?

(c) Instead of "litmus milk," try the experiment with
neutral ethyl butyrate. What is the equation for the reac-
tion which takes place?

PANCREATIC DIGESTION. 61

PANCREATIC RENNIN.

The ability of pancreatic juice to cause the clotting of
milk has long been known; but only of late has the action
been ascribed to a definite enzyme, which, on account of the
similarity in its function to the milk-clotting enzyme of the
stomach, has been called pancreatic rennin. This sub-
stance also possesses the power of bringing about a peculiar
changed condition of the caseinogen of the milk by which
the protein is rendered liable to coagulation. The coagu-
lable protein is termed metacasein, and its appearance is
denoted as the metacasein reaction. Metacasein is appar-
ently an intermediate stage in the action of the pancreatic
rennin upon caseinogen, in which pancreatic casein is the
end product. Pancreatic casein differs in some slight degree
from true casein.

(a) To 5 c.c. of milk add 1 c.c. of the pancreatic extract
and place the mixture at 40° C. Note the formation of the
clot.

(6) To 10 c.c. of the milk add 1 c.c. of the pancreatic ex-
tract diluted with 2 volumes of water. Place the test-tube
at 40° C. and from time to time remove portions (1 c.c.) of
the mixture and heat each to boiling. In one of them
coagulation in fine flakes will occur. Note the time from the
beginning of the experiment.

(c) Make an exact duplicate of the preceding experi-
ment, and at the time at which the appearance of the meta-
casein reaction would be expected, judging from the last
experiment, take out the tube from the bath. Examine
a drop under the microscope. Stand the tube in a cool
place and note the formation of a clot. This will dissolve
when replaced in the water-bath.

INTESTINAL PUTREFACTION.

The chief interest in the study of the effects produced
by bacteria upon protein matter lies in the direction of the
additional insight which can be obtained into the consti-
tution of the protein molecule. This becomes possible
.since the microorganisms effect a cleavage of the mole-
cule which, while similar in character to that exerted by
the digestive enzyme, is more vigorous and deep-seated
than even trypsin itself; and the products obtained are so
simple in composition and structure that they have yielded
easily to investigation.

The most important substances which result from bac-
terial decomposition of proteins may be divided as follows :

1. Tyrosine and its derivatives, the phenols, aromatic
acids, and oxy-acids (e.g., oxyphenylpropionic and oxy-
phenylacetic acids).

2. Indole and its derivatives, skatole, skatole-carbonic
acid, and tryptophane.

3. Fatty acids and derivatives, leucine, etc.

4. Sulphur derivatives and gases, hydrogen sulphide,
methyl mercaptan, CEU, CC>2, and NHs.

5. Proteoses and " peptones " are present in small
amounts as intermediary products.

The following method is employed for the preparation
and separation of the various products. It lends itself also
for demonstration purposes, and the different distillates
can be studied individually.

62

INTESTINAL PUTREFACTION. 63

Prepare a mixture of lean chopped beef and coagulated
egg-white in equal amounts, and add thereto about 1 liter
of water to each pound of material employed.

If cultures of colon bacillus are available, sterilize the
flask and its contents and add some of the culture. Other-
wise make the mixture alkaline (add to each liter 100 c.c.
of 10% Na2C03), and inoculate with some meat which has
been allowed to putrefy. After shaking thoroughly stop the
flask with a bored cork which is provided with a glass tube
to which is attached a valve (wash-bottle) of mercuric
cyanide (3 per cent). This serves to collect the methyl
mercaptan and to render the flask less odorous. Allow the
flask to remain at 40° C. for two to three weeks. The
separation of the products takes place as follows:

Distil the putrefactive mixture with steam until the
distillate equals one-half the volume of the original mixture.
There is thus provided, 1, a distillate (A) ; and, 2, a distil-
lation residue (A), which will be treated separately.

Distillate A.

Acidify with weak HC1 and redistil (usually one-half volume).

Distill

llate B. Distillation-residue B.

Make alkaline with (NH4C1.)

KOH and again distil.

Distillate C. Distillation-residue C.

Keep the first few c.c. Saturate with CO, gas

of this distillation sep- and redistil,

arate from the rest
(Indole and Skatole).

ite D. Distillation-residue D.

(Phenols.) (Fatty adds.)

64 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.
Distillation-residue B.

Upon evaporation this can be shown to contain NH4C1
formed from the NH3 of the putrefaction product and the
HC1 added in the manipulation.

Distillate C.

Contains indole and skatoie (see Conjugate Sulphates).

The first distillation carries over skatoie; this may be
tested as follows:

(a) To 2 c.c. add a drop of HNO3 and a drop or two of
KN02 solution (2 per cent). Notice the turbidity.

(6) Add some concentrated HC1 to some of the distillate.

Try the succeeding tests with the main distillate (indole}.

(a) Moisten a piece of pine wood with concentrated HC1
and dip it into the distillate. A cherry-red color appears
on the wood.

(6) Legal's reaction. — To a few c.c. of the distillate add
5 drops of a freshly prepared solution of sodium nitroprusside ;
then make it alkaline with 2 drops of NaOH. A violet color
results, which resolves itself into deep blue upon acidifica-
tion with glacial acetic acid.

(c) Acidify the distillate with HN03 and add 2 drops
of KNO2 solution (see Skatoie). A red precipitate of nitroso-
indole nitrate results.

Distillate D.

Contains phenol and p.cresol. (Make very faintly alkaline
with KOH and concentrate to a small volume.)

For the reactions of phenol see p. 96.

p.cresol gives with ferric chloride solution a dirty-green
coloration.

INTESTINAL PUTREFACTION. 65

Distillation-residue D.

Acidify this with concentrated HC1 and shake it out with
a small amount of ether. Upon evaporation of the ether
extract, the volatile fatty acids remain.

Distillation-residue A.
Concentrate and filter; then extract with ether.

r~ ""i

Ether extract. Residual solution.

Remove the ether by evaporation Concentrate until crys-

and extract the residue with tallization begins; filter,
warm water; filter.

Crystalline precipitate.
(Leucine and tyrosine.)

Filtrate B.
/Proteoses, peptonesA
1 tryptophane and I
\aromatic acids. /

1 1

rate A, Residue.

(Oxy-acids and skatole-
carbonic acid.)

(non- volatile fatty acids).

Filtrate A.

This contains p-oxyphenylacetic acid, p-oxyphenylpro-
pionic acid, and skatole-carbonic acid.

(a) Try the ferric chloride reaction (see Phenol) .

(6) Try Millon's reaction. Which of the three sub-
stances are these tests for?

(c) Perform test (c) under Indole and compare the result-
ing colors and precipitates with those for indole and skatole.

*

Residue.
See Fatty Acids for tests.

66 LABORATORY WORK L\ PHYSIOLOGICAL CHEMISTRY.

Crystalline Precipitate.
See Leucine and Tyrosine.

Filtrate B.

See Proteoses and "Peptones."

Tryptophane, indoleaminopropionic acid (proteinochro-
mogen). This substance is formed as one of the products in
the pronounced cleavage of proteins as in tryptic digestion
and putrefaction. Its presence in the protein molecule is of
considerable importance to the animal economy. It is sup-
posed to stand as the mother substance of the various pig-
ments of the body. Add a few c.c. of chlorine or bromine
water to the solution; a reddish-violet precipitate results
(proteinochrome).

Finally examine the mercuric cyanide valve which was
attached to the flask containing the putrefactive mixture.

Hydrogen sulphide has produced a black precipitate of
HgS.

Methyl mercaptan is indicated by the presence of a gray-
ish-green precipitate.

BILE.

The function of the bile is mainly excretory in character,
consisting in the removal by way of the intestine of various
relatively insoluble substances, such as cholesterol and leci-
thin. Secondarily, it exerts an emulsifying power upon
the fats, and fat absorption is markedly decreased in its
absence. In this sense it may be classed under the digestive
juices.

Bile as it is secreted by the liver forms a clear, limpid
solution of a color either yellowish-red, brown, or green ac-
cording to the species of animal from which it is obtained.
It reacts alkaline to litmus, but acid to phenolphthalein and
possesses a decidedly bitter taste and an odor of musk. By
its stay in the gall-bladder the character of the fluid is
changed considerably. Absorption of water and admix-
ture of mucus-like substances derived from the walls of the
bladder increase the specific gravity from 1.010 to 1.035.
When taken from the gall-bladder, bile, therefore, presents
a ropy, viscous appearance and contains about 10 per cent
of solids. Bile holds the following substances in solution:
the salts of the bile-acids, the bile-pigments, mucin or phospho-
protein, cholesterol, lecithin, inorganic salts, and traces of
fat, soaps, and urea.

Note the color, consistency, and reaction of the samples
presented.

Notice the differences in color between the ox and dog

bile.

67

68 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY-

Use the diluted bile for the following reactions:

(a) Add acetic acid drop by drop to 5 c.c. of the bile.

Is this precipitate mucin? How could you prove it? Filter

and test the filtrate for protein. What is the most suitable

test?

(6) To 1 c.c. of olive-oil add 10 c.c. of water and shake

thoroughly. Allow it to stand in the rack.

(c) To 1 c.c. of olive-oil add 10 c.c. of diluted bile. Allow
this to stand. Compare the permanency of the emulsion.

(d) To 10 c.c. of a gastric digestion mixture add diluted
bile, drop by drop. What is this precipitate and its signifi-
cance?

CONJUGATE BILE ACIDS.

These are present as the sodium salts of taurocholic and
glycocholic acids, and are formed by the combination of
cholalic acid with taurin and glycocoll respectively. Tauro-
cholic acid, by virtue of the taurin, contains sulphur and
has a solvent action on the more insoluble glycocholic acid.
It is more abundant in the bile of carnivora, while glyco-
cholic acid predominates in that of man and herbivora.
Together they tend toward rendering the cholesterol and
lecithin more soluble and exert a distinctly inhibitory action
on the heart, slowing its rhythm.

Pettenkofer's Test. — Place 5 c.c. of concentrated H2S04 in
a clean dry test-tube. In another test-tube place 5 c.c. of
diluted bile to which has been added a few drops of a 2 per
cent cane-sugar solution or a solution of furfurol 1 : 1000. Pour
the diluted bile carefully down the sides of the tube con-
taining the H2S04 so that the two fluids do not mix. (Method
of stratification.) Notice the coloration at the line of con-
tact of the two solutions. Shake the tube slightly, allowing
a little more of the bile to come in contact with the H2S04.

BILE. 69

The temperature must never rise above 70° C; to avoid
this, cool the tube under the tap. Upon careful mixing
and cooling as described above, the whole solution finally
becomes cherry-red or reddish purple. Such a solution
shows a definite and characteristic spectrum which distin-
guishes it from other substances, giving the same reaction,
such as phenol, petroleum, fusel-oils, pyrocatechinol, choles-
terol, and proteins.

Crystallization of the Bile Salts (Phttner's}.

Mix 20 c.c. of the bile with sufficient animal charcoal
to form a thick paste and allow the mass to evaporate on
the water-bath to dryness. Grind up the dry residue and
extract it in a flask with 25 c.c. of absolute alcohol on the
water-bath for 15-20 minutes. Filter and to the filtrate add
ether until a slight precipitate is visible. Cover the vessel and
set it away for a few days. Examine the crystals. Dissolve
some in alcohol and try Pettenkofer's Test.

BILE PIGMENTS.

The differences in color which were noticed in the various
samples of bile are dependent upon the presence in pre-
dominating amounts of certain pigments of which the two
most important are bilirubin and biliverdin. The former
is chiefly present in the bile of carnivora, while that of the
herbivora contains biliverdin in greater quantities. The
bile pigments are closely connected generically and probably
chemically with the blood and urinary pigments.

Bilirubin is insoluble in water, somewhat soluble in alcohol
and ether, and dissolves readily in chloroform, benzene, and
acids and alkalies. It oxidizes in the air to biliverdin.

Biliverdin is soluble in alcohol, partly soluble in ether,
and insoluble in chloroform and water.

70 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

The pigments possess the chemical characteristics of
weak acids and are present in biliary calculi as calcium salts.

Use diluted bile for the following tests:

Gmelin's Test. — Stratify 5 c.c. of yellow HN03 and 5 c.c.
of diluted bile as explained in the previous experiment.
Notice the play of colors at the junction of the two liquids
— green, then blue, violet, red, and yellow. To what are
these colors due, and what specific substances do they indi-
cate?

Smith's Test. — Stratify 5 c.c. of diluted bile and 3 c.c. of
tincture of iodine. Notice the bright green ring.

Huppert's Test.— To 10 c.c. of bile add an equal volume
of milk of lime and shake thoroughly. Filter off and after
washing once with water remove the precipitate to a small
beaker with 25 c.c. of alcohol acidulated with a few drops of
HC1. Warm the beaker in a water-bath until the alcohol
begins to assume an emerald-green color. What is the chem-
istry of this reaction?

Hammarsten's Test. — To 5 c.c. of Hammarsten's reagent
add five drops of diluted bile. A green color immediately
develops. Upon the further addition of the reagent in small
quantities to the mixture, the same play of colors may be
obtained as in Gmelin's test.

Analysis of a Biliary Calculus.

Place some of the pulverized calculus in a clean dry test-
tube with 15 c.c. of ether. Shake thoroughly. Filter off the
ether through a dry filter and funnel into a porcelain dish
and allow it to evaporate in the air. What is the residue ?
Test it.

If any of the calculus still remains in the test tube wash
it upon the filter paper with some ether; wash the residue of
the calculus now on the filter paper with a 10 per cent solu-

BILE. 71

tion of HC1; then wash twice with water and dry the paper
in the funnel.

When dry pass through the filter again and again about
10 c.c. of chloroform until this assumes a yellowish color,
caused by its solvent action on the bilirubin. Finally allow
a few c.c. of fresh chloroform to flow through the funnel, and
uniting this with the previous extract, let it evaporate in the
air.

Treat the residue of the calculus still on the filter-paper
with about 10 c.c. of alcohol. What does this extract?
Evaporate off the alcohol on the water-bath. Notice the
color of the residue and compare it with that of bilirubin.

What was the necessity for the use of the hydrochloric
acid in the analysis?

Perform the following tests with the residue of bilirubin:

(a) Gmelin's test.

(6) Dissolve some of the substance in a few c.c. of chloro-
form and shake it with a dilute solution of Na2C03. Notice
the changes which occur in the aqueous solution.

BLOOD.

Blood may be considered as composed of a fluid plasma
in which are suspended the form elements, i.e., the red
corpuscles, the leucocytes, and the platelets. When the
plasma coagulates, there separates from it the insoluble
fibrin or the clot, and the fluid which remains or is pressed
out by the contraction of the clot, is designated as the serum.
The reaction of the blood is alkaline to litmus, the alkalinity
being equivalent to about 0.25 per cent Na2C03. As was the
case with the other body fluids, it is also acid to phenol-
phthalein, since it contains sodium bicarbonate and dihy-
drogen phosphate.

The specific gravity of the blood varies within rather
small limits, 1.055-1.066, and the molecular concentration,
as indicated by the depression of the freezing point, is under
normal conditions almost constant. Approximately 60 per
cent by weight of the blood is composed of corpuscles and
the remaining 40 per cent of plasma.

General Reactions.

Test the reaction with litmus paper previously moistened
with a concentrated solution of NaCl. To what is this reac-
tion due? Specific gravity — see Hammarsten, p. 223, Ham-
merschlag's method.

(a) Examine a drop under the microscope.

(6) To 5 c.c. of blood add 10 c.c. of water. Notice
changes in the solution and examine a drop under the micro-
scope. What is laky blood?

(c) To 5 c.c. of blood add 10 c.c. of an 0.8 per cent NaCl
solution. Examine a drop of this also. What is meant by

a solution isotonic with blood?

73

BLOOD. 73

(d) Add a few drops of bile, chloroform, and ether to
successive portions of 5 c.c. of blood.

(e) To 5 c.c. of blood add 1 c.c. of hydrogen peroxide.
To what is the frothing due?

(/) To about 10 c.c. of water add a few drops of blood
and enough freshly prepared tincture of guaiacum to cause
a slight turbidity. Then add to the mixture a few c.c. of
hydrogen peroxide. Explain the effects produced.

(g ) Add 10-12 drops of a saturated glacial acetic acid solu-
tion of benzidine to 2-3 c.c. of 3% hydrogen peroxide. Mix and
to this add 2 or 3 drops of very dilute blood. Note change
in color. Try the same reaction using dilute blood which
has previously been boiled and then cooled.

BLOOD SERUM.

Blood serum presents a clear yellow liquid with a specific
gravity of 1.027-1.032. It consists of a watery solution of
serum albumins, globulins, enzymes, dextrose, fats, fibrin
ferment, inorganic salts, and a yellow coloring matter belong-
ing to the class of luteins or lipochromes. In addition it
holds in solution traces of nearly every soluble substance
which has been found in the body tissues. Of the 8-9 per
cent of total solids in the serum, 7 per cent is made up of the
proteins. NaCl is present to the extent of about 65 per
cent of the inorganic salts.

PROTEINS.

(a) Heat about 25-30 c.c. of serum to boiling with the
addition of a drop or two of acetic acid. Filter and test
filtrate and precipitate for protein (Millon's and biuret
reactions). Retain the filtrate.

(6) Saturate 15 c.c. of the serum with MgS04. Remove
the precipitate by filtration, and to the filtrate add two
drops of acetic acid. The filtrate from this second pre-

74 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

cipitate should be protein-free. Test it. What is the dif-
ference between the two precipitates? What is another
method for the separation of albumins and globulins?

(c) To 5 c.c. of the serum add 10 c.c. of 95 per cent alcohol.

(d) " 5 " " " " at 30° C add anhydrous Na2S04
to saturation.

(e) To 5 c.c. of the serum add a solution of uranium
acetate until no further precipitate is obtained.

After the removal of the precipitates in experiments
(c), (d), and (e), test the nitrates for protein.

SUGAR AND SODIUM CHLORIDE.

Use the protein-free filtrate from experiment a (under
Proteins).

(a) Test a few c.c. of the filtrate with Fehling's solution.

(6) Allow a few c.c. to evaporate on a watch-glass over
the water-bath. Examine the glass under the microscope.

(c) Test for chlorides.

BLOOD PLASMA.

(a) To a few c.c. of oxalated plasma add a few drops of
a 2 per cent CaCl2 solution. What is the result? Why?

(6) Dilute 2 c.c. of salted plasma with 10 volumes of
water. What happens?

(c) Do the same to some serum. What is the difference,
and why?.

(d) To 5 c.c. of the salt-plasma add an equal volume of
a saturated NaCl solution. What is the precipitate?

FIBRIN.

This substance is formed by the action of the fibrin fer-
ment upon the globulin, fibrinogen, which is present in all
the coagulable fluids of the body.

BLOOD. 75

Note the character of the substance presented. It has
been obtained by whipping the blood before it has had time
to set in a solid mass.

Apply three color protein tests.

Recall the action of 0.2 per cent HC1, pepsin, and trypsin
upon fibrin.

FORM ELEMENTS.

These consist of the red blood cells, the leucocytes, and
the platelets. Chemically the solid matter of the red corpus-
cles consists of 90 per cent hemoglobin, 8 per cent proteins
and nucleins, and the remainder cholesterol, lecithin, and
inorganic salts. The predominating base of these salts is
potassium.

The leucocytes, being typical animal cells, contain those
substances which are intimately connected with the term
protoplasm. Such are proteins (especially nucleoproteins),
lecithins, cholesterols, fats, carbohydrates, inorganic salts,
and water. Little is known concerning the composition of
the blood-platelets. They probably contain a protein of the
globulin type and a nucleoprotein.

OXYHJEMOGLOBIN.

Oxyhaemoglobin or haemoglobin belongs to the class of
conjugate proteins often spoken of as the respiratory pig-
ments, and forms the most important constituent of the
form elements. When its watery solution is heated to 70° C
it decomposes with the formation of an iron-containing pig-
ment, hcematin, and an albuminous body, globin.

The function' of the oxyhremoglobin is that of an oxygen
carrier. The compound holds the oxygen in a rather loose
combination, and in all probability the iron of the hsematin
molecule is directly concerned in the process. This instability

76 LABORATORY WORK IX PHYSIOLOGICAL CHEMISTRY.

of the oxygen combination renders the compound particularly
liable to the action of reducing agents. A reduction of this
kind takes place in the capillaries of the tissues, hemoglobin
or reduced hemoglobin being formed, the presence of which
in predominating quantities gives to the venous blood its
characteristic dark red color.

Hemoglobin is very prone to combine to form derivatives
with certain compounds such as carbon monoxide, nitric
oxide, and hydrogen sulphide. These simple substances,
having a greater affinity for the hemoglobin molecule than
the oxygen possesses, replace the latter readily and form
stable and more or less toxic compounds which cause death
by oxygen starvation. Again, partial decomposition prod-
ucts of hemoglobin showing characteristic spectra are easily
formed under certain conditions; such are methemoglobin,
hemochromogen, hematoporphyrin, etc. Some slight phys-
ical and chemical differences seem to exist between the oxy-
hemoglobins obtained from the blood of various animals, a
fact which explains why the coloring matter from the blood
of different animals does not crystallize in the same form or
with the same facility.

Place on a glass slide one drop of defibrinated dog's blood.
To this add one drop of water and mix with a platinum wire.
Allow the mixture to evaporate at room temperature until the
edges of the drop have begun to dry. Then place a cover-
glass on the slide and examine under the microscope. Sketch
the crystals of oxyhemoglobin.

The proof for the presence of oxyhemoglobin is usually
adduced by showing under the microscope crystals of hemin.

Hcemin — Teichmann's Crystals.
Hemin is the HC1 ester of the anhydride of hematin.
Place one drop of NaCl solution upon a microscopic slide

BLOOD. 77

and allow it to evaporate to dryness. Then add a very small
drop of blood and two drops of glacial acetic acid and cover
with a glass. Warm cautiously until bubbles of gas begin to
form in the mixture under the cover-glass. Examine and
sketch under the microscope. The hsemin crystals are rhom-
bic plates, brown in color by transmitted light. In large
masses they have a metallic lustre and appear steel-blue by
reflected light.

Spectroscopic Examination.

(a) Oxyhcemoglobin. — Dilute 1 c.c. of blood with 200 c.c.
of water. Examine spectroscopically. At this dilution one
broad absorption-band is seen extending from the D line
(588) to b (518). The violet end of the spectrum is also
absorbed as far as the F line (486). Upon again diluting
this solution with an equal volume of water it is noticed that
the broad band has resolved itself into two, the one next to D
being narrower and more intense than the broader one to the
right. Between the two bands is a green interspace. Less
of the violet end is now absorbed. Upon still further dilu-
tion, the bands become narrower and finally disappear simul-
taneously.

(6) Hcemoglobin (Reduced Hcemoglobiri). — Prepare some
Stokes' reagent as follows: Dissolve 3 grams of ferrous sul-
phate in a small quantity of water and add to it in watery
solution 2 grams of tartaric acid. Make up the mixture to
100 c.c. and just before using add NH4OH until the precipi-
tate which at first forms is dissolved. This solution of
ammonium ferrotartrate is a reducing agent, removing the
oxygen which is in weak combination with the oxyhaBmoglo-
bin, and thus forming hemoglobin. To the blood, 200 times
diluted, add a few drops of Stokes' reagent. Notice the
change in color. Examine in the spectroscope. A broad,

78 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

less sharply defined absorption-band is seen occupying as
much space on the spectrum as the two bands of oxyhsemo-
globin, but the haemoglobin band is moved further to the left.
If this solution is shaken in the air the color returns to that
of oxyhsemoglobin and the latter's characteristic spectrum
also reappears.

(c) Methcemoglobin. — Add to blood (diluted 1:15) two
drops of a freshly prepared solution (10%) of potassium
ferricyanide. The color of the blood becomes brown. The
spectrum, in addition to two bands corresponding nearly to
those of oxy haemoglobin, which, however, are only faintly
seen, shows a band in the red near C. If to such a solution,
while still in position before the spectroscope, a drop or two
of Stokes' reagent is added, the characteristic absorption-
bands of oxyhasmoglobin appear for a second and are then
quickly replaced by those of haemoglobin. Shaking in the
air causes the latter to be reoxidized to oxyhsemoglobin with
the consequent spectral change.

(d) Hcemochromogen (Reduced Hcematiti). — To blood
(diluted 1 : 15) add two or three drops of strong NaOH and
warm gently until the color changes to a brownish green.
Then cool and add two drops of Stokes' reagent. Such a solu-
tion shows in the spectrum two very dark bands coinciding
apparently with those of oxyhaemoglobin ; upon careful
examination, however, it will be seen that green light appears
on the left of the left band and consequently both bands must
be moved further to the right than the oxyhsemoglobin ones.

(e) Hcematoporphyrin (Iron-free Hcematiri). — Place a few
c.c. of concentrated H2S04 in a test-tube and add a drop of
blood, mixing well. The color changes to wine-red, and spec-
troscopically the solution shows two bands on the opposite
side of the D line. The one to the left is narrower and
weaker, while that to the right is much more intense,

BLOOD. 79

broader, and more sharply defined. This spectrum is very
characteristic.

(/) Carbon Monoxide Hcemoglobin. — Carbon monoxide
haemoglobin may be easily prepared by passing ordinary coal-
gas through defibrinated blood until the latter assumes a
carmine or cherry-red color characteristic of the combination
of CO with haemoglobin. Examined spectroscopically such a
solution shows two bands similar to oxyhaemoglobin except
that the bands of the C0-ha3moglobin spectrum are nearer to
the violet end. Add Stokes' reagent to the solution; the
spectrum remains unchanged.

Make the following tests, first with diluted blood (oxy-
haemoglobin) and then with diluted CO-hsemoglobin; note the
differences carefully:

(a) Add to 5 c.c. of the blood 3 c.c. (NH4)2S.

(6) « «5« « „ « 10 c.c. of Stokes' reagent.

(c) Shake with air.

What is characteristic and important in the combination of
CO with haemoglobin as shown by the above experiments?
Make drawings of all the spectra seen and compare them with.
those in the text-books.

MILK.

Milk physiologically considered stands as a secretion
in the strictest sense of the word, since its organic constitu-
ents are specific products of the activity of the cells of the
mammary gland. Chemically it presents a perfect emul-
sion of fat whose menstruum holds in solution three proteins
(lactalbumin, lactglobulin, and caseinogen), lactose, inor-
ganic salts (chiefly Ca), gases, and traces of creatinine, leci-
thin, cholesterol, urea, and citric acid. In the comparison
of the composition of human and cow's milk, the chief point
to be noted is the relatively low percentage of total solids,
protein and fat, and high percentage of lactose in human
as against cow's milk. Fresh milk possesses either an ampho-
teric or alkaline reaction, but with phenolphthalein it also
has a certain acidity. Upon standing, it develops a definite
acid reaction by the change of the lactose into lactic acid.
This throws the caseinogen out of solution and the milk is
said to be sour. Fresh milk does not coagulate by heat, but
forms a scum supposed to consist of some form of caseinogen
and calcium salts.

The specific gravity of human and cow's milk is approxi-
mately the same (1.028-1.034). It is increased by the re-
moval of the fat (cream), which has a specific gravity lower
than that of water.

General Reactions.

(a) Examine under the microscope fresh milk, skimmed
milk, and colostrum. What are the differences?

80

MILK. 81

(6) Take the specific gravities and explain the variations
in the results.

(c) Test the reaction with litmus paper.

(d) Saturate 10 c.c. of milk with MgSO4. What is the
precipitate?

(e) Shake 5 c.c. of milk with ether. What takes place?
Now add a few drops of NaOH. Explain this change.

(/) Heat some milk in an evaporating-dish. Notice the
scum.

(</) Heat 5 c.c. of milk and 5 c.c. of NaOH to boiling.
The yellowish-brown coloration is caused by the action of
the alkali on the lactose.

(/i) Try the guaiac reaction described under blood, using
milk instead. Explain the result.

QUALITATIVE SEPARATION OF THE CONSTITUENTS
OF MILK.

Dilute 200 c.c. of milk with 800 c.c. of water and add weak
acetic acid, drop by drop, until a white flocky precipitate
separates out, leaving a fairly clear solution. Allow the pre-
cipitate to settle; then filter (Filtrate A) it off and let it
drain as dry as possible. Remove the white precipitated
caseinogen from the paper and place it in a flask with 200 c.c.
of a mixture one-half ether and one-half alcohol. Allow the
precipitate to stand in contact with the ether for an hour,
shaking from time to time. Then filter off the ether mix-
ture and let it evaporate spontaneously. The residue con-
sists of most of the fat of the milk, mechanically precipitated
adherent to the caseinogen. Keep both the caseinogen and
the fatty residue.

Place the filtrate A in an evaporating-dish and heat over
a free flame ; the solution being acid, any coagulable protein

82 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

not precipitated with the acetic acid will be rendered insolu-
ble. When the solution has concentrated to about one-
half its original volume, this may be filtered off and the clear
filtrate again set evaporating until crystals begin to appear,
separating out of the solution (now about 20 c.c.). Keep
the coagulable protein precipitate.

After allowing the solution to cool, filter off the crystals of
calcium phosphate and again place the filtrate over the water-
bath and allow it to evaporate to a thick syrup. Of what is
this composed?

From the milk have now been isolated—

1. Caseinogen. 2. Fats. 3. Coagulable protein. 4. Cal-
cium phosphate. 5. Lactose.

These require separate study.

CASEINOGEN.

This substance is a non-coagulable protein of the class of
phosphoproteins. It contains about 0.8% phosphorus and
0.7-1.2% sulphur. Caseinogen deports itself chemically
as a weak acid combining with bases to form soluble salts
or caseinogenates. The acid itself is insoluble. It exists
in the milk in the form of Ca-caseinogenate (apparently not
in a true solution, however), and when stronger acids are
added caseinogen is formed and falls out of solution.

(a) Try three color-protein tests.

(6) Test for phosphorus and sulphur by fusion.

(c) Dissolve some in very dilute NH4OH and reprecipitate
with acetic acid.

(d) Test for lead-blackening sulphur.

(e) Grind up a little of the substance in a mortar with
CaC03 and water, then filter and in the filtrate test for case-
inogen. What chemical reaction has taken place here?

MILK. 83

FATS.

The fats of milk are in general those which occur in
adipose tissue. There are present also in small quantities
fats formed from the volatile fatty acids, particularly butyric
and caproic acids. These give to milk-fat (butter) its charac-
teristic odor when from any cause the fatty acids are liberated
(rancidity). The proportion of the various fats is roughly
as follows: triolein, -|; tripalmitin, ^ ; tristearin, £; tributyrin
and tricaproin, ^.

Use the fatty residue in the evaporating-dish (see method
of separation). Dissolve the fat in 10 c.c. of ether and add
to it 3-5 c.c. of sodium alcoholate. Notice what takes place
and compare this saponification with those performed under
fats. Evaporate off the ether and replace it with water.
The precipitate should dissolve. Now add dilute H2S04 to
a distinct acid reaction.

Note the odor. To what is it due? Write the equations
for the reactions.

COAGULABLE PROTEIN.

Lactalbumin and lactglobulin together occur to the
extent of 1 per cent in cow's milk. They are closely related
to, though not identical with, serum albumin and serum
globulin.

Try three color-protein tests.

CALCIUM PHOSPHATE.

In the inorganic salts, phosphoric forms the chief acid
and calcium the base, except in human milk, where potas-
sium seems to predominate. Strangely enough, iron is only
present in traces.

Dissolve some in dilute HN03.

84 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY

(a) Add an equal volume of ammonium molybdate and
warm to 70° C. What is the precipitate? Filter it off, dis-
solve in NH4OH and reprecipitate as ammonio-magnesium
phosphate.

(6) Make a portion of the HN03 solution alkaline with
NH4OH and reacidify with acetic acid. Add a few c.c. of
ammonium oxalate. Examine the precipitate microscopic-
ally.

LACTOSE.

Lactose occurs only in milk, if the small amounts which
are occasionally present in the blood and urine of pregnant
women are disregarded. It is a disaccharide and breaks
down into a molecule of dextrose and one of galactose. It
undergoes yeast fermentation very slowly, but is easily
attacked by bacterium lactis, in which process lactic acid is
formed. With phenylhydrazin, lactose yields osazones, which
crystallize in needles and melt at 200° C. Lactose is soluble
in water, but does not dissolve in either alcohol or ether.
Its "solutions are dextrogyrate.

Make use of the syrup derived from the last evaporation
in the milk-separation.

(a) Try Fehling's, Moore's, and Ny lander's tests.

(6) Apply the fermentation test.

(c) Try the phenylhydrazin test (see p. 5).

(d) To 5 c.c. of the lactose solution add sufficient freshly
prepared indigo-carmine solution to render the mixture
decidedly blue; then make it alkaline with dilute Na2C03
solution. Upon warming, the mixture becomes red, yellow,
and finally colorless. What do these changes indicate?

URINE.

Considered from the physiological point of view, the
urine forms the chief exit for those soluble substances which,
either on account of their toxicity under certain conditions
or from their lack of available potential energy, are unsuited
for utilization and further retention in the organism. Chem-
ically, the urine presents a watery solution of organic and
inorganic bases and acids, whose degree and manner of com-
bination is dependent upon the conditions (affinity, mass-
action, etc.) existent in the solution. The compounds
present in the urine represent, on the one hand, the end
products of combustion and cellular metabolism, and, on
the other, bodies excreted in the same or similar form in
which they were ingested. It is this double origin of the
constituents of the urine which necessitates a comprehensive
knowledge of the daily ingesta, if rational or accurate de-
ductions are to be made from the composition of the urine.
Again, as the character and composition of the urine ex-
creted at different periods of the day varies within rather
wide limits, it is absolutely essential in every examination
of urine that the sample presented for analysis should repre-
sent a definite part, if not the whole, of a well-mixed 24-hours
excretion. This in part affords an explanation of the now
current method of considering the constituents of the urine
from the standpoint of weight per diem (grammes) rather than
concentration (per cent). Percentage data neglect one

85

86 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

important factor (volume), the consideration of which is
indispensable, especially when extreme values are met with.
It is unnecessary to add that the urine should be ex-
amined as fresh as possible, or, in the event of this not being
feasible, the fluid must be well supplied with antiseptic agents
such as powdered thymol, chloroform, NaF, etc.

Physical Properties.

The following comprise the important physical properties
of urine to be specially noted in an examination:

Color. — Aromatic alcohols, salol and allied compounds
cause the urine to become brown upon standing. Rhubarb
and senna impart a reddish color, which, if the urine is
alkaline, may be confounded with the appearance of blood.
The ingestion of quinine in large quantities gives to the
urine a greenish opalescence and methylene blue is followed
by the appearance of urine of a decided bluish-green tinge.

It is usual to refer to Vogel's color chart, but unless the
color is other than that of some shade of yellow, little can be
deduced from the determination of this property.

Odor. — Normal (urinous), ammoniacal, or that peculiar to
some substance not normally present, such as acetone,
methyl mercaptan, etc.

Transparency. — Clear or cloudy. Usually upon standing,
a cloud of mucous-like substance separates, suspending itself
in some part 'of the urine.

Reaction. — Acid, neutral, or alkaline.

As is true of any acid solution, the acidity of the urine
is due to the presence of dissociated hydrogen atoms (ions).
It is usually assumed that these acid ions are derived from
the acid salts, such as H2NaP04 or HNa-jPC^ (mineral acidity),
but in all probability the H ions of the organic acids in the
urine also add a not inconsiderable part to the. total acidity.

URINE. 87

Free mineral acids are never present. By a proper relationship
between the dihydrogen and monohydrogen phosphates, a
neutral or amphoteric reaction may prevail (litmus). Under
normal conditions the 24-hour urine is never alkaline; but
portions of urine drawn a few hours after digestion may
react alkaline, due to the withdrawal of acidic radicals
(acid ions) from the blood in the formation of the acid gastric
juice. Alkaline urine may be caused by free or fixed alkalies
and when this is the case the urine is always cloudy or turbid.
Care should be taken that the observed alkalinity is not a
result of bacterial contamination. Volatile alkalies such
as ammonia react blue to litmus, but upon warming the
paper the red color returns ; this will not occur if the reaction
is caused by a fixed alkali. Organic acids are burnt in
the body to carbonates and the latter decrease the acidity,
sometimes, in fact, causing an alkaline reaction to appear.

Volume and Specific Gravity. — Under normal conditions
these two factors vary inversely with each other; a large
volume of urine is usually accompanied by a low specific
gravity and vice versa. The volume depends upon the
amount of water ingested and that excreted by the bowels,
skin, and lungs; it varies between 800 and 1500 c.c. (aver-
age 1250 c.c.). The specific gravity may fall between 1.010
and 1.030 (average 1.017-1.020).

The volume should be measured in a graduated cylinder
capable of holding two liters. Should it be required to
retain the urine for some time, thymol must be added and
the fluid placed in a stoppered vessel.

Determination of the specific gravity is made by means
of the urmometer (hydrometer). If there is a sediment the
urine must be warmed, filtered, and allowed to cool.

Place the cylinder upon the table and fill it to within an
inch of the top with urine; then immerse the urinometer.

88 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Read off on the graduation of the spindle the mark where
the meniscus of the fluid cuts it.

Sediments. — Notice should be taken as to their color,
amount, character, etc. The method for the determination
of the identity of such will be taken up later.

Total Solids. — These may be calculated approximately by
multiplying the second and third decimals of the specific
gravity by Haser's coefficient = 2. 33. This gives the number
of grammes in 1000 c.c. of the urine, from which must be
calculated the total amount in the twenty-four hours.

THE NORMAL CONSTITUENTS OF THE URINE.
INORGANIC COMPOUNDS.

Concerning the exact nature of the combinations formed
in the urine by the various bases and acids (ions) but little
is known at present. The compounds are present in rather
dilute solution and as such are amenable to all the chemical
and physical laws of salts in like solutions, complicated,
however, by the fact that the condition is not one of a single
salt in a simple solution but of an exceedingly complex mix-
ture of salts both inorganic and organic. The dissociation
of one salt is influenced by the presence of others, also disso-
ciated, and the particular combinations of the various (ions)
bases or acids depend upon their relative masses and avidities.
Again, as regards two compounds with like ions each de-
creases the solubility of the other, while the solubility of
unlike ions is increased by their mutual presence.

These factors, dissociation, mass action, and affinity deter-
mine the character of the ionic equilibrium which is present
in the urine, but as to the exact nature of such equilibrium,
our present methods tell next to nothing. The later work

URINE. 8&

along physical lines has been in this direction, in the hope
that the results will be of value for diagnostic purposes.

At present it is customary to determine the quantity
of a given base or acid radical and then to express it
empirically in terms of a compound of this radical, the
quantity of which is considered to predominate in the mix-
ture. Thus chlorine is expressed as NaCl, potassium as K20,
etc. The ordinary inorganic acidic radicals (anions) of the
urine are Cl', SO/', PO/", and CO/' and the basic radicals;
(kations), NH/, 1C, Na', Ca", Mg", with traces of Fe".

ACIDIC RADICALS.

(a) Chlorides. — These are present combined principally
with Na and K. Acidify some of the urine with HN03 and
add a drop of AgN03. Prove that this precipitate is AgCl.

(&) Sulphates. — These occur in two forms, viz., preformed
or sulphate-sulphuric acid and ethereal or combined sulphuric
acid. The former precipitates directly from the urine by the
addition of BaCl2 in an acid reaction, the latter only after the
previous boiling of the urine with a mineral acid. Acidify
some urine with acetic acid and add an excess of BaCl2.
Prove that this precipitate is BaS04. Filter (filtrate must
be clear), and to this add a few c.c. of concentrated HC1 and
boil a few minutes. A turbidity is indicative of the presence
of ethereal sulphates. Explain this.

Sulphur in the organic or lead-blackening form may also
be present in the urine as a constituent of taurine, cystine,
KSCN, etc. Place some urine in a flask with a few pieces of
pure zinc and add enough HC1 to cause a gentle evolution of
gas. Partially close the mouth with a piece of filter-paper
moistened with lead acetate. In the presence of organic sul-
phur the paper is blackened. Explain this.

90 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

(c) Phosphates. — These are present combined with NH4,
Na, K, Ca, and Mg as primary, secondary, or tertiary com-
pounds.

1. Make some urine alkaline with NH4OH. What is the
precipitate? Filter and to the clear filtrate add two drops
of BaCl2. .What is this new precipitate? Filter this off
through the same filter as the first and dissolve the whole
precipitate in dilute HN03. Test for phosphates.

2. Make the urine acid with acetic acid and add a few
drops of uranium nitrate solution. Of what is the precipitate
composed?

3. Acidify the urine with a few c.c. of HN03 and add
some molybdic solution. Warm at 80° C. What is this
precipitate?

4. Boil about 10 c.c. of the urine and add one-quarter its
volume of Fehling's solution. Notice the color of the pre-
cipitate. To what is it due? Then boil 10 c.c. of Fehling's
solution and add one-quarter its volume of urine. What
occurs? Contrast these two experiments.

Write the equations for the reactions in the above experi-
ments.

BASIC RADICALS.

With the exception of NH4 these have little or no chemi-
cal significance. The proof for the presence of ammoniacal
compounds will be taken up under the quantitative deter-
minations.

URINE. 91

ORGANIC COMPOUNDS.
NH2

UREA, OC<

XNH2

Urea or carbamide is present in the urine in about a
2 per cent solution or 30 grms. for 24 hours. It is very
soluble in water and alcohol and crystallizes in long rhombic
prisms. It possesses weakly basic properties and forms com-
binations with acids analogous to double salts. When
heated gently, urea yields biuret. The Micrococcus urece
splits it into NH3 and C02.

Prepare some urea from the urine as follows-:

Evaporate one-half a liter of urine to syrupy consistency
and exhaust the residue with hot alcohol. Filter off the
latter and evaporate the nitrate to dryness. This residue
is extracted on the water-bath with successive portions of
pure acetone, which must be filtered hot. The mixed acetone
filtrates are then evaporated slowly to a small volume and
allowed to cool. The crystals which separate out may be
filtered off and washed with cold acetone.

Make use of these urea crystals for the following reac-
tions :

(a) Dissolve some urea in a few c.c. 01 water. Place in
each of two watch-glasses one drop of the solution. To one
add one drop of dilute HNO3 and to the other a drop of dilute
oxalic acid solution. Allow them to stand and then examine
the crystals under the microscope. What are these com-
pounds?

(6) Warm carefully a few crystals of urea in a dry test-
tube. The substance melts, giving off NH3. Continue heat-

92 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

ing until the fused mass solidifies. Cool the test-tube and
add a few c.c. of water and NaOH drop by drop until
the residue is all in solution. Add now a drop or two of
very dilute CuS04 solution. To what reaction does this
correspond? What substance is formed from the urea?
Write the equation.

(c) To 5 c.c. of NaOH add some bromine water and
drop in a crystal of urea. What is the reaction which takes
place?

HN— CO

I I H
URIC ACID, CO C— N\

>CO

HN— C— X

II

Uric acid is a diureide and structurally is composed of
two urea groups attached to a 3-carbon chain. It is closely
related to the purine bases being built up from the "purine"
type.

Uric acid acts as a weak dibasic acid and forms three
types of salts — neutral, biurate, and quadriurate.

The neutral salts which are formed by the replacement
of two hydrogen atoms of the acid by two atoms of the base
are very unstable.

The biurates have only one of the hydrogens replaced
and form very stable compounds. They present the chief
type in which uric acid exists in the urine.

The quadriurates stand between the other urates as re-
gards stability. They are formed by a weak combination
of uric acid and biurate molecule for molecule. They
therefore contain in the molecule one-fourth the quantity
of base that exists in the neutral urate. When uric acid
separates out spontaneously from a urine, it is caused by an

URINE. 93

inter-reaction between the acid phosphates and the biurates
in which quadriurates are formed; the latter immediately
break up into uric acid and biurate, and the former is
thrown out of solution.

Pure uric acid forms a white powder. As it separates
out from urine in the presence of impurities, it assumes a great
diversity of crystalline forms, all of which are characteristic,
however; the crystals are always tinged with pigment de-
rived from the urine.

Uric acid is practically insoluble in hot and cold water,
alcohol or ether. The sodium, potassium, and lithium salts
are soluble, especially the latter. The ammonium, calcium,
and magnesium salts are insoluble. The quantity excreted
in 24 hours varies according to the individual and the diet,
but usually amounts to 0.5 — 1.0 grms. (average 0.7 grm.).
If urine is made sufficiently acid with HC1 to react strongly
to litmus (about 30 c.c. cone. HC1 to the liter) and allowed
to stand, uric acid will separate out in crystals of a dark
red color.

For the following tests make use of the uric acid prepared
in this way:

(a) Examine the crystals under the microscope. Sketch
as large a variety as can be found.

(6) Test the solubility in water, NaOH and NH4OH.

(c) Dissolve some of the crystals in a few c.c. of dilute
NaOH and then add NH4C1 to saturation. What is the pre-
cipitate?

(d) Heat a crystal on platinum-foil.

(e) Dissolve some uric acid in a small quantity of dilute
NaOH. Add concentrated H2S04 carefully, drop by drop,
until the solution is too warm to touch; then add a few c.c.
of potassium permanganate solution. What is the reaction
which takes place?

94 LABORATORY WORK IX PHYSIOLOGICAL CHEMISTRY.

(/) Make a concentrated solution of uric acid and pour it
into 10 c.c. of Fehling's solution which has previously been
brought to boiling. Observe and note result.

(g) Dissolve some uric acid in dilute Na2C03 and place
a, couple of drops on some filter-paper previously moistened
with AgN03. To what is the blackening due?

(h) Murexide Test. — Place a few crystals in a clean and
dry evaporating-dish and pour upon them two drops of
concentrated HN03. Evaporate to dryness very carefully
over a free flame. A yellowish residue results which upon
cooling and the addition of a drop of NH4OH becomes purple-
red. If NaOH instead be used, the color will be purple-
violet. What is the chemistry of the reaction?

PURINE BASES.

These substances as they appear in the urine have a
double origin. They may be the result of ingested nu-
cleins (exogenic origin) and also the end products of the
nuclear metabolism of the tissues (endogenic origin). Al-
though ten different bases have been isolated, it is question-
able if many of them are not laboratory products formed
during the complicated processes of their isolation. Xan-
thine, hypoxanthine, guanine, adenine, epiguanine, para-
xanthine, and carnine are said to be present in the urine.
Taken together the quantity daily excreted is only about
20-100 mg. or about 10 per cent of the uric acid. They are
capable of forming insoluble compounds with Ag, Cu, phos-
photungstic acid, etc.

To 20 c.c. of urine add an excess of the magnesium mix-
ture. Filter, and to the filtrate add ammoniacal silver
nitrate solution. The precipitate is composed of the Ag
compounds of all the bases. This is filtered off, suspended
in water, decomposed with H2S, and the Ag2S removed by
filtration. The clear filtrate is evaporated to dryness. This

URINE 95

residue is treated with 3% H2S04 which dissolves the purine
bases and leaves the uric acid undissolved. The dissolved
purine bases may be reprecipitated with silver nitrate solution.

XNH CO

CREATININE, HN=C\

XN-CH3-CH2

Creatinine appears in the urine as the result of the inges-
tion of meat which contains creatine. The amount is also
somewhat dependent upon the nitrogenous metabolism,
being decreased in starvation. For the relationship be-
tween creatine and creatinine and the reactions and tests,
see under Muscle. On a mixed diet about 0.1 grm. is ex-
creted in 24 hours.

Creatinine allows of a separation from the urine as
follows :

To 50 c.c. of urine add 3 c.c. of a saturated solution of
sodium acetate and then 10 c.c. of a saturated solution of
HgCl2. Filter off the precipitated urates, sulphates, and
phosphates, and set the nitrate aside for 24 hours. The
mercury compound of creatinine separates out in the form
of spherical globules. Examine some under the microscope.
This compound of creatinine is readily decomposed by acids
or by H2S. Try Weyl's or Jaffe's test directly on the urine
and then on the creatinine isolated by the above method.
Test its reducing power.

CONJUGATE SULPHATES.

Of the toxic substances which are formed as the result of
intestinal putrefaction of the proteins, phenol, p-cresol,
indole, and skatole appear in the urine in the form of alkali
salts of non-toxic ethereal combinations with sulphuric
acid; pyrocatechinol and hydrochinol are also excreted in
the same way. Taken together these compounds are denoted

96 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

as the ethereal or conjugated sulphates. The strength of
combination and manner of detection of the sulphate radical
has been studied under the inorganic salts. The amount
of the conjugate sulphates varies from 0.09 to 0.6 grm.,
being dependent, as it would be expected from their origin,
upon the extent of putrefaction in the intestine.

The Phenol and p-Cresol sulphates exist in normal
urine in relatively small amounts, about 30 mg. for twenty-
four hours. They may be isolated and detected as follows:

Treat the urine with | its volume of 25 per cent H2S04
and distil off TV of the volume of the solution. Use this
distillate for the succeeding reactions:

(a) Add some Millon's reagent to a little of the solution.
Warm. Compare this with the test under protein and
tyrosine.

(6) To a few c.c. of the solution add a trace of neutral
ferric chloride solution. When did you use this reaction
before? Add a drop of HC1.

(c) To 10 c.c. of the distillate add some bromine-water.
Note the whitish crystalline precipitate of tribromphenol.

The combinations with Pyrocatechinol and hyurochinol
originate after the ingestion of these bodies or of phenol.
The urine containing them becomes brown on standing ("car-
bolic urines ").

Indole and Skatole (/3-methyl indole) do not unite with
the sulphuric acid directly as such, but first suffer an oxida-
tion by which indoxyl and skatoxyl are formed; they then
combine and appear in the urine as potassium indoxyl or
skatoxyl sulphate. The former is called animal indican
and is not to be confounded with the indican of plants. The
skatoxyl compound is only present in minimal quantities
and not infrequently is absent entirely.

URINE. 07

INDICAN= POTASSIUM INDOXYL SULPHATE,
C.H/NH^H

The following tests are based upon the oxidation of the
indican to indigo, and the solvent action of chloroform upon
the latter.

(a) Jaffe's Test. — To about 10 c.c. of urine add 2 or 3 c.c.
of chloroform and mix well with an equal volume, 10 c.c., of
concentrated HC1. Then add drop by drop, shaking well
between each drop, a concentrated solution of chloride of
lime. The indigo is dissolved by the chloroform. Note the
color changes.

(6) Obermayer's Test. — Perform the test in the same man-
ner except instead of concentrated HC1 and lime add an equal
volume of Obermayer's reagent.

(c) Hammarsteris Test. — The same test, using Hammar-
sten's reagent. Compare results.

c

OXALIC ACID,

\OH

Oxalic acid is present in the normal urine in the form of
calcium oxalate, which is held in solution by the presence of
the acid phosphates. Frequently the salt separates out in
crystalline form (see sediments) and then lays the foundation
for renal and vesical calculi. The quantity averages about
30 mg. for 24 hours. In certain conditions which are not
well understood the amount is largely increased (oxaluria).
The oxalic acid originates in part from the oxalates ingested

98 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

in vegetable food, but as it does not disappear in starvation,
some must arise from a particular phase of protein metabolism,
possibly the nucleins.

The following is the procedure employed for its isolation:
The urine is acidified (20 c.c. of HC1 sp. gr. 1.12 for every
1000 c.c.) and extracted with a 10 per cent solution of alcohol
in ether. Three extractions are united, evaporated to 10-20
c.c., filtered and the filtrate made slightly alkaline with
NH4OH; to this is added a few c.c. of 1 per cent CaCl2 solu-
tion and the mixture is rendered faintly acid with acetic
acid. Collect the precipitate on a small filter and examine
it under the microscope. Heat some on a platinum foil.

HIPPURIC ACID, C6H6-CO-NH-CH2-C/QH

This substance is formed in the renal cells by a syn-
thesis of benzoic and aminoaceticacid or glycocoll. Normally
it is present as a hippurate to the amount of 0 • 7 grm. in
24 hours. The quantity is markedly augmented when ben-
zene derivatives such as exist in plants and fruits are inges! ed.
An increase in amount may also result from excessive putre-
faction of vegetable material in the intestine. Thus it is that
larger quantities of hippuric acid are always found in the
urine of herbivora than in that of carnivora. It readily crys-
tallizes in long rhombic prisms or needles formed in rosettes,
and is soluble in water and alcohol. If urine containing
hippuric acid is acidified. (20-30 c.c. of cone. HC1 to the liter)
and evaporated on the water-bath to a small volume, crystals
of this substance will form upon cooling. They may be col-
lected on a filter and washed- with a little alcohol saturated
with ether.

(a) Heat a few crystals in a dry test-tube. They will
melt at 187° C., and at still higher temperatures will decom-

URINE. 99

pose with the formation of a red coloration (decomposition
of the glycocoll) and an odor resembling that of the oil of
bitter almonds (benzonitrile).

PIGMENTS.

Variation in the yellow color of the urine noticed under
certain conditions, is attributable to the presence of different
pigments, the most important of which are urochrome,,
urobilin, and uroerythrin.

UROCHROME.

This is the name of the substance which gives to normal
urine its characteristic yellow color. Of its properties and
constitution next to nothing is known. It is isolated from
urine when the latter is saturated with (NH4)2S04 and the
filtrate extracted with alcohol. The pigment is soluble
in the alcohol and the entire color of the urine passes into
the solvent. Urochrome, when acted upon by mild reducing
agents, yields a second pigment which is apparently identical
with urobilin. The name uroerythrin is applied to the pig-
ment which imparts to the brick-dust sediment of urates
(sedimentum lateritium) its pinkish coloration. Solutions
of this pigment are rapidly decolorized by light.

UROBILIN.

Although not ordinarily present to such an extent as
urochrome, urobilin is frequently increased in diseased
conditions, and its importance lies in the knowledge which
has been gained concerning its origin from and relation to
the biliary pigments. The substance is extremely soluble,
dissolving in all ordinary solvents. It exhibits in alcoholic

100 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

solutions a green fluorescence which is greatly augmented
if ammonia and alcoholic ZnCl?, solution are added. Acid
alcoholic solutions of urobilin also show a distinct absorption-
band between 6 and F.

In constitution urobilin possesses the same acidic proper-
ties which characterize all the animal pigments. It forms
insoluble compounds with bases, and is completely precipi-
tated from its solution by saturation with (NH4)2S04 (differ-
ing from urochrome).

(a) To 50 c.c. of urine faintly acidified with H2S04 add
ammonium sulphate (in substance) to saturation. Filter off
the precipitate and dissolve it in alcohol containing a few
drops of concentrated HC1. What sort of a body is urobilin?

(6) To 50 c.c. of urine add an equal mixture of neutral
and basic lead acetate until the precipitation is complete.
Filter and allow the precipitate to drain as dry as possible.
Then place it and the filter-paper in an evaporating-dish
half full of 95 per cent alcohol acidulated with HC1. Warm
over the water-bath until the alcohol is well colored. Filter
and examine the solution in the spectroscope. Now add a
few drops of NH4OH and a few c.c. of an alcoholic zinc
chloride solution. Note the fluorescence. In what way is
the coloring matter of the urine precipitated?

(c) Shake 25 c.c. of urine with 2 to 3 drops of pure HC1
and 5 c.c. of chloroform. Remove the chloroform and
stratify upon it a few c.c. of a solution of zinc acetate in
alcohol. Notice the green ring; by shaking, the solution
becomes fluorescent.

CARBOHYDRATES AND RELATED BODIES.

Under apparently normal conditions, reducing sub-
stances of a carbohydrate nature appear in the urine in small

URINE. 101

and variable quantities. The most important of these are
the pentoses, glycuronates, and possibly dextrose. It is
still debatable whether dextrose is present in the urine under
absolutely normal conditions, or, better, under conditions
which do not permit of the slightest suspicion that the renal
function is impaired. If it does exist in the urine in such
cases, it is present in amounts which escape detection by the
usual methods.

PENTOSES.

As the methods for isolation and identification of the
pentoses improve, the presence of these substances in the
urine is being more frequently noted. Xylose, Arabinose,
and Rhamnose (methyl-pentose) have been described. They
originate in the organism after the ingestion of cherries,
plums, grapes, etc., which contain the mother substances
of the pentoses, the pentosanes. The pentoses are not
readily assimilable, and when ingested appear in part un-
changed in the urine. It must be remembered that the
tests which were given for pentoses (p. 3) are not abso-
lutely characteristic for these substances, since glycuronic
acid and related bodies also react positively with them.
The reactions may be used, nevertheless, as confirmatory tests
if the polarization and fermentative power of the urine are
also determined. The pentoses found in the urine are non-
fermentable and inactive toward polarized light, while the
glycuronates are Isevogyrate.

(a) Test the urine in the polariscope.

(6) Try the orcinol and phloroglucinol tests with 10 c.c.
of urine.

(c) Heat to boiling 4-5 c.c. of Bial's reagent and then
add a few drops of the urine.

102 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

GLYCURONIC ACID.

Glycuronic acid originates as a product of the inter-
mediary metabolism of the carbohydrate nucleus in the
protein molecule. Ordinarily it is present in the urine in
small amounts combined with phenol, indoxyl, and skatoxyl
as conjugate glycuronates; but under certain conditions,
either as the result of an abnormal increase in the quantity
of these bodies just mentioned, or the ingestion of aromatic
substances, such as the camphors, chloral, and naphthol,
etc., the conjugate glycuronates appear in such quantities
as to give to the urine a left-handed polarization and a
decided reducing power on Fehling's solution. (Some glycu-
ronates do not reduce.) Glycuronic acid is dextrogyrate,,
does not ferment, and gives positive results with the orcinol
and phloroglucinol tests (see the pentoses). As glycuronic
acid itself is never present in the urine, and since the gly-
curonates are said to respond negatively to the orcinol test,
this test has been used as a means of differentiating between
the pentoses and the glycuronates. This is only true, how-
ever, of the glycuronates which do not split off the glycuronic
acid component during the performance of the orcinol test
(boiling on the water-bath with HC1). Glycuronic acid
forms with parabromphenylhydrazin an osazone which
melts at from 200-216° C. and which, if dissolved in pyridin
and alcohol, shows a left-handed rotation of 7° 25' or 369°
(a)D.

A Isevogyrate urine which, upon treatment with Fehling's
solution, gives a yellowish precipitate and partial reduction
of the copper may be more than suspected of containing
conjugate glycuronates.

Examine a sample of urine in the same way as under
pentoses.

URINE. 103

QUANTITATIVE DETERMINATIONS.

PHYSICAL PROPERTIES.

Determinations of certain physical properties of the
urine have come into prominence of late as a means for
furnishing additional data concerning the general state
of equilibrium which obtains in the fluid. The urine, being
in fact merely a somewhat dilute solution of organic and
inorganic salts, must possess the same properties and respond
to the same physical laws which govern such solutions. The
most important property whose determination is attempted
is that of the depression of the freezing-point below that of
the solvent, water.

The law states that the presence bf substances dissolved
in a solvent causes the freezing-point of the solution to be
lower than that of the solvent, and that the amount of the
depression is in direct proportion to the number of molecules
•or (when dissociation has taken place) ions in the solution.

The depression of the freezing-point, which is represented
by the Greek letter J, is therefore dependent upon the
molecular concentration.

The only substances which are present in the urine in a
quantity sufficient to exert any influence on J are the urea
and sodium chloride and possibly the phosphates and sul-
phates, although the effect of the latter two, even if it did lie
outside of the limit of experimental error, could not suffice
to cause differences which would influence deductions. The
other factors which affect the J are those which cause varia-
tions in the specific gravity of the urine, and it is stated that
..a relationship exists between the two which only varies
-within very small limits. A priori, this seems impossible

104 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

when it is remembered that solutions of urea and sodium
chloride with the same specific gravity would have widely
differing J, since urea is non-dissociable, while sodium chlo-
ride in the strength in which it exists in the urine disso-
ciates to a considerable degree.

The freezing-point determination is made by the use of
the Beckmann apparatus (see demonstration). The manip-
ulation is simple, rapid, and requires little experience.

Another property of the urine which is dependent upon
the presence of electrolytes in the solution is termed the
electrical condiictivity. Electrolytes are substances which
become capable of conveying electricity in virtue of the fact
that they become dissociated in solution into (electrically)
active, positive or negative parts or ions. Therefore the
electrical conductivity is in direct proportion to the degree
of dissociation or ionization of the urine. This property is
unaffected by variations in the content of urea in the
urine. Such determinations are accomplished by the use
of a rather complicated apparatus based upon the theory
of the Wheatstone bridge. (See demonstration.) The im-
perfect knowledge which exists in regard to the conditions
obtaining in the various fluids of the body does not permit
of exact interpretations of the results acquired by these
methods.

It must suffice, therefore, to merely mention that various
factors are derived from a combination of the estimation of
the J and that of urinary substances, such as NaCl, total
nitrogen, etc., and that such factors are constantly increas-
ing in value as data serving to present a clearer picture of the
conditions under which a given sample of urine may have
been excreted.

URINE. 105

ACIDITY OF THE URINE.

It is quite generally understood that the degree of acidity
of the urine is due to the excess of the dihydrogen (acid)
over the monohydrogen phosphates, the relation to the
total phosphates usually being about 60 per cent of the
former to 40 per cent of the latter. Based upon this view,
the methods for the quantitative determination of the acidity
have consisted in the estimation of the total P205 and the
P205 present in the form of the acid phosphates, and from
which the degree of relative acidity of the urine was derived.
Many difficulties arise in the procedure for the quantitative
separation and determination of the two types of phosphates,
and the present methods have been shown to be full of
errors. Theoretical objections have also been raised, and
attempts made to show that the ' ' organic " acidity, as opposed
to the " mineral" (phosphate) acidity, plays a not incon-
siderable role in determining the total acidity. How un-
important the estimation of the organic acidity really is
can be understood when it is" noted that organic acids appear-
ing in a solution of mixed phosphates are able to remove the
base from the monohydrogen phosphates, and thus pro-
duce an almost equivalent increase in the quantity of the
acid phosphates. The presence of the organic acids, there-
fore, only serves to increase the acid phosphates, and the
latter still remain theoretically the most accurate indica-
tion of the total acidity. The technical difficulties have
led to the reliance upon the method of direct titration of

the urine with r^ alkali. This procedure has the advantage

of rapidity and possesses some value, perhaps, where com-
parisons only are required. Absolute values in the present
state of the question are unattainable.

106 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Quantitative Estimation.

To 25 c.c. of urine in a flask add 15-20 grms. of powdered
potassium oxalate and two drops of phenolphthalein. Allow
the oxalate to partly dissolve by shaking and titrate the

mixture immediately with ^ NaOH until the first faint

pink coloration is discernible. The acidity in this case is

N
expressed in terms of j~ acid for 100 c.c. of urine.

CHLORIDES.

The quantity of chlorine excreted in the urine during the
24 hours is subject to great variations. On a mixed diet it
usually amounts to from 10 to 15 grms., calculated as NaCl.
Under normal conditions the quantity eliminated is depend-
ent upon the quantity of chlorine and water ingested; but
in pathological states, such as the formation and subsequent
absorption of exudates, it is respectively markedly decreased
and then increased. In starvation the amount becomes
minimal.

Quantitative Estimation.

The principle of the method is the following:
To the urine is added an excess of AgN03 over and above
what is necessary to precipitate all the chlorides present.
The excess of Ag is then determined by means of a sulpho-
cyanide solution, using iron alum as an indicator.
Reagents necessary:

1. A AgN03 solution, each c.c. of which precipitates 0.01
grm. NaCl (29.075 grms. AgNO3 in a liter).

2. A saturated solution of iron alum.

3. Chlorine-free HN03 of a specific gravity 1.2.

URINE. 107

4. A potassium sulphocyanide solution of which 2 c.c.
corresponds to 1 c.c. of the known AgN03 solution.

Method: Prepare a clean and dry graduate. By means
of a pipette measure off accurately 5 c.c. of urine and run it
into the graduate. Add 3 c.c. of the HN03 and dilute with
water to about 25 c.c. Then allow exactly 10 c.c. of the
known AgN03 solution to flow in. Add water until the
volume equals 50 c.c. ; then mix thoroughly. Transfer this
now to a clean and dry beaker and clean and dry the gradu-
ate again. Prepare also a clean and dry funnel with paper
and filter the mixture in the beaker into the graduate. When
25 c.c. of a water-clear nitrate has passed through remove the
funnel to a test-tube. Add 10 drops of the iron alum to the
graduate and titrate with the known potassium sulphocya-
nide until the first tinge of pink appears in the solution.

Calculate the amount of chlorine or Nad in the 5 c.c. of
the urine used, and then in the 24-hour sample.

SULPHATES =S03.

A variable fraction (80 to 90 per cent) of the total sulphur
of the urine exists in a completely oxidized form as the salts of
sulphuric acid, usually denoted and calculated as S03. Of the
total S03, about nine-tenths is combined with bases (preformed)
anc? one-tenth with aromatic radicles (conjugate sulphates).
The "organic " sulphur appears as taurine, cystine, KSCN, etc.

Since the food contains merely minimum quantities of
S03, that found in the urine must originate as a product
of protein metabolism, in which the sulphur of the molecule
becomes oxidized. Thus it is that ordinarily the S03 output
mav be considered as indicative of the amount of protein
burned in the body. About 1.5-3.0 grms. (average 2.5
grms.) SOs are excreted during 24 hours.

108 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Quantitative Estimation.

The procedures for the determination of these various
units rest upon the following facts:

Boiling the urine with dilute HC1 liberates all the sul-
phate radicles present in such a form that they may be
precipitated with BaCl2 as BaS04. This gives the amount of
total sulphate. If acetic acid be used instead of HC1, the
resulting precipitate with BaCl2 will be made up of the pre-
formed or sulphate-sulphate. This may be filtered off and
the filtrate treated as for the total sulphates, the result being
the amount of ethereal sulphates present. The difference
between this and the total will be the amount of the pre-
formed sulphate.

TOTAL S03.

In a beaker place 50 c.c. of filtered urine and dilute with
100 c.c. of water, adding 5 c.c. of HC1. Heat to boiling, add
very slowly 20 c.c. of the BaCl2 solution and allow it to cool
and stand covered in a cool place for 24 hours. Filter through
a small ash-free filter; the precipitate must be removed quan-
titatively from the beaker to the paper by means of warm
water. Now wash the white precipitate with water until the
washings give no test for chlorine; then dry at 100° C.
When dry, slip out the paper from the funnel, fold it up so
that the contained sulphate cannot fall out and place it in a
porcelain crucible which has previously been ignited and
weighed. Ignite the paper in 'the crucible carefully and
burn until the residue becomes white. Cool the crucible and
weigh. The difference in the two weights will give that of the
BaS04 from 50 c.c. of urine, from which may be calculated
the S03 in the total 24-hour sample.

URINE. 109

CONJUGATE S03.

To 75 c.c. of urine in a beaker add an equal volume of
water; acidulate with about 10 drops of acetic acid, heat
to boiling and a,dd 10 c.c. of the BaCl2 solution. Place the
covered beaker in a cool place for about 24 hours. The
precipitate is then collected on a filter and in the filtrate
and combined washings the S03 is determined as outlined
under total S03. In this case the result corresponds to
the S03 of the conjugate sulphates.

ORGANIC SULPHUR.

Evaporate 50 c.c. of urine down to .dryness, add the
fusion mixture and fuse with an alcoholic flame until a white
residue remains. Take this up with hot water strongly
acidulated with HC1. Determine the amount of S03 present
in this solution and calculate the amount for the 24 hours,
from which may be subtracted the total S03 which has been
previously estimated in another sample of the same urine.
The difference will give the organic sulphur expressed as
S03 in the 24-hour sample.

PHOSPHATES = P205.

Phosphorus is present in the urine to the greatest extent
as phosphoric acid combined with bases, but a small quantity
(2-2.5 per cent) exists as organic P.; e.g., glycerophosphoric
acid, lecithin, etc. It has been stated that the amount of
this latter form of phosphorus is a true indication of the ni-
trogenous metabolism taking place in the body. The phos-
phoric acid phosphorus is expressed as P205, and during
24 hours about 1.5-3.5 grms. (average 2.5 grms.) P206

110 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

are excreted. This amount is made up of alkaline and
earthy P205 in decidedly varying proportions; of the alkaline
P205 about 60 per cent consists of the dihydrogen (acid)
phosphate and the remaining 40 per cent as the monohydrogen
phosphate.

TOTAL P205.

The procedure is based upon the quantitative precipita-
tion of all the phosphates in the urine with uranium nitrate,
as uranium phosphate, using potassium ferrocyanide as
indicator. The phosphates must be present as acid salts.

Quantitative Estimation.
Reagents necessary:

1. A uranium nitrate solution 1 c.c. of which equals 0.005
grm. P205 (35.461 grms. in a liter). How is this calculated?

2. An accessory solution (100 grms. of sodium acetate and
300 grms. of acetic acid in a liter).

3. A solution of potassium ferrocyanide. What is the
reaction which indicates the absolute precipitation of all
the P205?

Method: Place 25 c.c. of urine in a large evaporating-dish,
add 5 c.c. of the accessory solution (why?), and warm gently
over an asbestos board. Keeping the solution warm, add the
known uranium nitrate solution from a burette, from time to
time removing a drop of the urine on the end of a glass rod
and adding it to a drop of the potassium ferrocyanide which
has been placed upon a white porcelain dish. When all the
P205 has been precipitated by the uranium and the first ex-
cess of the latter appears in the urine solution, the drop of
indicator on the plate when tested as above will take on a
faint reddish-brown color. The test should be repeated after
a minute; if it again causes a color, the titration is complete,

URINE. Ill

otherwise more of the uranium solution must be added and
retested.

From the number of c.c. of the uranium nitrate solution
required calculate the P205 in the 25 c.c. of urine employed
and from that determine the total P205 in the 24-hour
urine.

EARTHY P205.

In the precipitation of the earthy phosphates by means
of NH4OH various errors creep in which render the determina-
tion of doubtful value. Thus, in whatever form the earthy
bases (Ca or Mg) were present in the urine, they would form
insoluble phosphates when the fluid was made alkaline, and
the precipitate would therefore contain P205 not originally
combined with the alkaline earths.

The method is given because it is still employed.

Quantitative Estimation.

Place 50 c.c. of urine in a beaker and make it alkaline with
NH4OH. A precipitation of the earthy phosphates occurs.
Allow this to stand for a couple of hours and then collect the
precipitate upon a small filter. After having washed the
precipitate with very dilute NH4OH transfer it quantitatively
to an evaporating-dish by means of dilute acetic acid, which
dissolves the P205. Dilute the solution to about 25 c.c. and
titrate as outlined under total phosphates. The difference
between this amount and that of the total will be the amount
of alkaline P205.

ORGANIC PHOSPHORUS.

The method employed for the determination of the organic
phosphorus in the urine is based upon the same principle as
that in use for organic sulphur. The total phosphorus is

112 LABORATORY WORK IX PHYSIOLOGICAL CHEMISTRY.

estimated after fusion as P205. From this amount is de-
ducted the total P205 determined on another sample of the
same urine, and the difference corresponds to the organic
phosphorus calculated in terms of P205.

TOTAL NITROGEN (KJELDAHL).

A well-nourished man eliminates under ordinary con-
ditions with a mixed diet 10-16 grms. nitrogen. The amount
is dependent upon the body weight, the diet, and various
factors which may influence the metabolism of protein in the
organism. Muscular work has no effect upon the nitrogenous
excretion. In round numbers 85 per cent of the nitrogen is
in the form of urea; 4 to 5 per cent, NH3; 1 to 2 per cent,
uric acid; and the remaining extractives, etc., 8 to 10 per
cent.

Quantitative Estimation.

The Kjeldahl method for the determination of the total
nitrogen of the urine has come into almost universal use.

The principle consists in the decomposition of all the or-
ganic matter by heating with sulphuric acid, whereby all of
the carbon and hydrogen become oxidized to C02 and H20,
and the nitrogen of such compounds which contain it in
combination with hydrogen (such as =NH, NH2, NH3),
but not with oxygen, appears as ammonia. This is lib-
erated from the acid solution by saturation with NaOH;
the gas is then distilled over into a known quantity of acid,
the amount of which thus neutralized being determined by
the titration of the acid still remaining.

Reagents necessary (all nitrogen-free):

1. Concentrated H2S04 (2 parts fuming: 3 parts pure
concentrated).

2. Potassium sulphate (powdered).

URINE. 113

3. Solution of sodium hydrate, sp. gr. 1.23 (two-thirds
saturated).

4. Solution of ^ NaOH.

N

5. Solution of ^ H2S04.

Detailed Method. — 5 or 10 c.c. of urine (according to con-
centration) are measured out by a pipette and placed in a
long-neck digestion (Kjeldahl) flask. To this is added 10 c.c.
of the cone. H2S04 and one-half of a teaspoonful of potassium
sulphate. The mixture is allowed to boil over a sand-bath or
wire gauze until the solution becomes water-clear (3 to 6 hrs.).
After the flask has cooled, the contents are removed quan-
titatively to an Erlenmayer flask (content, 1 liter), using
about 400 c.c. of water, and to this solution is added without
mixing 40-50 c.c. of the strong NaOH solution. The flask
is now quickly connected by means of a rubber cork with a
condenser-tube, the other end of which is immersed in a known

N
quantity of ^ H2S04 (50-200 c.c. according to the estimated

amount of nitrogen in the urine employed). The vessel

N
used to hold the ^ H2S04 is usually a small Erlenmayer

flask. The contents of the large flask are well mixed and
a flame placed beneath a wire gauze upon which the flask
must rest. After the beginning of ebullition, the boiling
should be continued for 45 minutes. This must be regulated
so that the NH3 comes over gradually. At the end of the
time the small flask is removed so that the end of the con-
denser still remains in the flask but is not in contact with
the fluid. It must be held in this position for some minutes
to allow the condenser-tube to be washed inside with the
water still distilling over, and outside with a stream from

114 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

the wash-bottle. Then turn oft the flame. The amount of

N N

^-r acid still unneutralized is titrated with ^ NaOH. The

difference between this and the amount originally placed

N

in the flask will represent the amount of the ^ acid neu-
tralized by the NH3 which was distilled over.

N

1 c.c. -JQ H2S04= 0.0014 grm. N., from which may be

calculated the quantity of N. in the 10 c.c. used and then
in the 24-hour sample.

UREA (HUFFNER'S METHOD).

For the determination of urea, the following method,
whose use has become almost universal, seems sufficiently
accurate to warrant its continuance in favor. The rapidity
with which it can be performed offsets the disadvantage
of the mere approximation of results and renders it pre-
eminently suited for clinical purposes.

Only about 92 per cent of the nitrogen of the urea actually
present in the urine is obtained as a gas, although the theoret-
ical reaction is quantitative,

CO/

NH2

+ 3NaOBr= N2+C02+2H20+3NaBr
NH2

(see experiment (e) under Urea), but the deficit is partially
diminished by the fact that other substances (e.g., uric acid)
also yield up some of their nitrogen as a gas by the decompo-
sition with hypobromite.

_ The more accurate methods (Morner-Sjoqvist or Folin's)
will be demonstrated.

URINE. 115

Quantitative Estimation.

Reagents necessary:

Hypobromite solution (see Appendix).

In making use of the Doremus or similar ureometers, it is
advisable to so dilute the urine that the content of urea
will not exceed approximately 0.5 per cent.

Rinse the ureometer first with water and then fill it with
the hypobromite solution, so that when the apparatus is per-
pendicular and no air is at the top, the amount of fluid in
the bulb covers the opening from it to the upright tube.
Then draw up into the pipette the exact amount of urine,
and placing it under the surface of the solution with the pipette
tip well into the space below the upright tube, force the urine
out of the pipette slowly, noting that the bubbles generated
all pass upward into the closed tube. Care must be taken
that the last drop of urine is expelled from the pipette with-
out allowing any air to escape from it also. Allow the reac-
tion and collection of gas to go on for half an hour, then make
a reading on the scale on the areometer of the apparatus.
The readings become more accurate when the whole appara-
tus is immersed in a beaker of water in such a manner that
the levels of the fluids in the two vessels correspond. The
figures on the scale of the instrument indicate grammes of
urea in 1 c.c. of the urine.

URIC ACID (HOPKINS-FOLIN METHOD).

The following method seems to combine considerable
accuracy with simplicity and rapidity of performance. It
is based upon the fact of the precipitation of the soluble
urates by saturation with ammonium salts (Hopkins);
and additional accuracy has been obtained by the previous
removal of certain substances, e.g., mucoids, phosphates

116 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

(Folin). The urates are finally titrated with -^ potassium

permanganate .

Quantitative Estimation.
Reagents necessary:

1. 500 grms. (NH4)2S04, 5 grms. uranium acetate, and
60 c.c. of 10 per cent acetic acid dissolved in water and the
mixture made up to a liter.

2. KTT K2Mn208 titrated against a ^ solution of oxalic

acid.

Place 300 c.c. of urine in a beaker, add 75 c.c. of the
ammonium sulphate reagent and mix thoroughly. After
the resulting precipitate has settled sufficiently (5 minutes),
the mixture is filtered through a double-folded filter. When
250 c.c. of the filtrate have passed through, this volume is
divided into two portions of 125 c.c. each, to serve as dupli-
cates. To each portion add 5 c.c. of concentrated NH4OH,
mix thoroughly, and allow them to stand for 24 hours. The
precipitated ammonium urate is then transferred quanti-
tatively to a filter, using a 10 per cent (NH4)2SO4 solution
to remove the last portions of the precipitate from the beaker.
After removing the filter-paper from the funnel and opening
it up, the precipitate is washed with about 100 c.c. of water
into the same beaker in which the ammonium urate was
precipitated; to this 15 c.c. of cone. H2S04 is added and the

N
mixture immediately titrated with the ^: K2Mn208 solution,

which is added from a burette until the first permanent

N
tinge of pink color appears. 1 c.c. of ^ K2Mn208 solution =

3.75 mg. uric acid. Calculated the quantity of uric acid in
the 24-hour sample.

URINE. 117

AMMONIA.

Ammonia in the form of ammonium salts is present in
the urine of carnivora to the extent of 0.3-1.2 (average 0.7)
grms. for 24 hours. Its function in the body consists in
combining with and thus rendering non-toxic the mineral
and organic acids which appear in the organism, either after
their ingestion or their formation in metabolism. The
amount found in the urine is, therefore, markedly increased
in certain pathological states (e.g. diabetes) in which more
or less large quantities of organic acids make their appear-
ance in the blood as a result of disordered metabolism.

Quantitative Estimation.

Prepare two 500-cc. wide-mouthed Erlenmeyer flasks and
one tall cylinder (100 c.c. graduate) ; into the neck of each
insert a two-hole rubber stopper, one hole of which is pro-
vided with an L-shaped glass tube, each arm being about 3
inches long, the other hole provided with an L tube, the long
arm extending to within one-quarter inch of the bottom of
the vessels. A Folin absorption tube may be employed in-
stead of the long arm in the absorption flasks.

Place 25 c.c. of urine in the tall cylinder with about one
gram of dry sodium carbonate; cover the urine with one-half
inch of crude petroleum to prevent foaming.

To each flask add 20 c.c. ^ H2S04, 200 c.c. of distilled

water and a few drops of an indicator (Congo red or alizarin
red). Place the three bottles in series with the cylinder in
the middle, and connected in such a manner that the short
arm of one bottle is attached to the long arm of the succeeding
one. The long arm of the first flask is open to the air; the
short arm of the last bottle must be connected with a suction
pump. The purpose of the first flask is to insure the passage

118 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

of ammonia-free air into the cylinder; the acid in the second
flask absorbs the ammonia liberated by the urine and carried
over by the current of air sucked through the system. Suc-
tion should be continued for about two hours.

N
The collection flask should finally be titrated with ^ NaOH

and the amount necessary for neutralization subtracted from
the number of cubic centimeters of acid originally taken.
The remainder, when multiplied by 0.0017, gives the amount
(grams) of ammonia in 25 c.c. of urine.

PROTEIN.

Quantitative Estimation.

Of the numberless methods which have been suggested
for the quantitative determination of the total protein in
the urine, the following has received perhaps the most marked
approbation from the clinicians. It is based upon the quan-
titative precipitation by picric acid in the presence of an
organic acid, of all forms of proteins which appear in the
urine under abnormal conditions. The results obtained
are sufficiently accurate if the urine does not contain over
0.4 per cent protein. When more than this is present, the
urine must be diluted with water in such amounts as to allow
the percentage to fall below that figure.

The procedure is as follows :

Fill the albuminometer to the mark "u" with urine acidi-
fied with a few drops of dilute acetic acid, and add Esbach's
reagent to "R ". Stop the end of the tube with a cork. By
inverting several times the contents of the tube can be thor-
oughly mixed without producing any froth on the top of the
mixture. Allow the corked tube to stand upright for 24
hours and then read off the height of the precipitate on the
scale, which indicates directly the number of grammes of dry
protein contained in a liter of the urine.

URINE. U9

DEXTROSE.

All of the methods for the quantitative estimation of
dextrose in the urine are founded upon the following proper-
ties of this carbohydrate:

1. Reducing action on copper and bismuth.

2. Effect on the plane of polarized light.

3. Fermentation by yeast.

1. The methods of this type assume that the total reducing
power of the urine may be ascribed to dextrose. This is
probably true in most instances, but the fact of the pos-
sible presence in appreciable quantities of other copper-
reducing bodies must not be neglected in critical examina-
tions, especially when the reduction is small and at the same
time atypical.

The Allihn method is by far the most accurate in results
and satisfactory in its performance. Its accuracy, how-
ever, is entirely dependent upon the strict attention with
which all the details of the procedure are followed out. The
cuprous oxide produced by the reducing action of an unknown
amount of dextrose upon a definite volume of Fehling's
solution is filtered off and further reduced to metallic cop-
per by a stream of hydrogen gas. This metallic copper is
then weighed. The quantity of sugar which corresponds
to this weight of copper is found upon consultation of tables.
The details are too numerous to allow of their explanation
in a limited space, but the method is to be recommended
highly.

The most common clinical method depends upon the
complete reduction as indicated by the entire loss of color,
of a given quantity of Fehling's solution, by means of titra-
tion with diluted urine.

120 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Method: 10 c.c. of quantitative Fehling's. solution are
placed in an evaporating-dish and diluted with 40 c.c. of
water. The solution is brought to boiling and kept so while
the urine (usually diluted ten times) is run in slowly from
a burette, until the blue color of the copper solution has.
entirely disappeared. An accurate determination of the end
point of the reaction requires considerable experience.

Since 10 c.c. of Fehling's solution are completely reduced
by 0.05 grm. of dextrose, the quantity of urine required
to produce the end reaction must contain that quantity of
dextrose. From this can be easily calculated the total
amount of dextrose in the 24-hour urine.

2. The possible simultaneous appearance in the urine
of laBvogyrate bodies (e.g. glycuronates, /?-oxybutyric acid)
renders the application of methods of this type of doubtful
value. Even when substances of this kind are apparently
absent, comparative values obtained by polarization and
reduction methods seldom agree within wide limits. When-
ever polarization determinations are made, they must be-
followed by a second estimation after fermentation.

The urine may be decolorized by the addition of a crystal
or two of lead acetate and subsequent stirring and filtration
(see Polarization, p. 5).

3. For rough approximations this method, using Einhorn's
saccharometer, serves the purpose better than any other..
The procedure is described under Monosaccharides, p. 5.
A method of this type has also been suggested, based upon
the difference of the specific gravity of urine observed before
and after fermentation.

URINE. 121

PATHOLOGICAL URINARY CONSTITUENTS.

PROTEINS.

Representatives of nearly every class of proteins have
been detected in urine at various times. Normal urine un-
doubtedly contains traces of these substances; thus, for
example, in the "mucous cloud" which separates out from
many urines upon standing a phosphoprotein can be demon-
strated. Of the proteins which appear under well-defined
morbid conditions, the most important are the albumins,
the globulins, and the proteoses. So-called peptone has
also been noticed. Since, however, the conception of the
properties of the mixture which passes under this name is
so ill-defined and changeable, it is probably better in the
future to dismiss the term peptonuria. Haemoglobin and
related substances may also escape from the blood into the
urine.

ALBUMIN AND GLOBULIN.

Serum albumin and serum globulin usually appear to-
gether in the urine under the name of albuminuria; but
concerning the relative quantities of the two bodies under
the various conditions very little is known. The two
proteins allow of separation in the same manner as was
employed under Blood Proteins, p. 73.

In testing urines suspected of containing protein, the
fluid should always be perfectly clear. This can be accom-
plished either by repeated filtration through paper or asbestos
or by shaking with magnesia.

The following tests are best suited for ordinary conditions:

(a) Heat Test. — Heat 5 c.c. of clear urine to boiling and add

122 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

1 to 3 drops of dilute acetic acid. If the urine contains more
than a small amount of albumin, this will settle out in flocks
after the addition of acid. When mere traces are present the
solution may only become turbid and should then be com-
pared to the urine before heating, but to which the same
amount of acid has been added. This test is only of value as
a positive one. When a negative result is obtained other
tests should be tried with a view to confirmation.

In some cases the addition of acetic acid to boiled urine
may give rise to a faint precipitate or turbidity which disap-
pears upon shaking. This is caused by the formation of acid
albumin, which may be salted out by one-third saturation
with NaCl after the addition of more acetic acid to keep
the phosphates in solution. Faintly alkaline or amphoteric
urine may sometimes give on heating a precipitate due to
phosphates, which is sometimes difficult to distinguish from
a precipitate of albumin. Again, such a urine may remain
perfectly clear and still contain albumin. The addition of a
small amount of acid will, in the first case, dissolve the
phosphates, and, in the second, precipitate any albumin
remaining soluble. Phosphoproteins nor proteoses do not
react similarly, since both bodies are soluble in hot acid
solutions; but a precipitate settling out upon cooling may
point to the presence of such substances. Resins resulting
from the administration of petroleum, turpentine, oil of
sandal-wood, tolu-balsam, etc., may be present in the urine,
and if so, will be precipitated by the acid. Such precipitates
easily dissolve in alcohol.

(6) Heller's Test.— Place 5 c.c. of cone. HN03 in a test-tube
and allow a few c.c. of the urine which is being filtered to flow
from the funnel down the sides of the tube. The urine will
thus stratify itself on top of the acid, and at the surface
of contact of the two liquids the albumin will be precipitated

URINE. 123

and will appear as a white ring. As the HN03 upon standing
diffuses upwards into the urine the ring may become broader.
This test is delicate enough for ordinary clinical purposes,
but will not show the presence of traces. Urines containing
an excessive amount of urea may form a crystalline precip-
itate in this test, but such a precipitate cannot be confused
with albumin. Colored rings may also form, due to the
oxidation of the urinary pigments, and a blue coloration is
produced by indican. Substances mentioned under the heat
test, such as resins, etc., may form a cloud, but simple tests
such as those indicated will exclude them.

(c) Roberts' Modification of Heller's Test— This test is
performed similarly to the last by the stratification of a few
c.c. of the urine upon 5 c.c. of Roberts' reagent. It has
some optical advantages and colored rings never appear in its
use.

(d) Acetic Acid and Potassium Ferrocyanide Test. — Acidify
5 c.c. of urine with two drops of acetic acid and add, drop by
drop, a dilute solution of K4FeCN6. In the presence of
albumin a white precipitate occurs which dissolves in a
large excess of the reagent. Traces of albumin may be de-
tected with this reaction. Should the precipitate dissolve
upon heating, proteoses may be suspected. The presence of a
considerable amount of mucin or phosphoprotein in the urine
may sometimes give rise to a precipitate with acetic acid
alone. This must be removed by filtration before the ferro-
cyanide test can be completed.

(e) Trichlor acetic Acid Test. — Stratify a few c.c. of a con-
centrated aqueous solution of this reagent with 5 c.c. of
urine. A white ring sharply defined indicates the presence
of albumin. The precipitate may also be proteoses, but in
this case the ring dissolves with cautious warming. This test
is more delicate than Heller's, and by its use smaller quanti-

124 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

ties of albumin are demonstrable in urines with which the
more common tests yield negative results.

(/) Spiegler's Test. — Stratify 5 c.c. of urine which has
been slightly acidified with acetic acid, upon a few c.c. of
Spiegler's reagent. In the presence of albumin a white ring
appears at the line of contact of the two liquids. This
test is very sensitive, showing albumin in a dilution 1 : 250,000.
In fact most normal urines indicate protein with the reagent.
This must be borne in mind in making deductions from
its use. Urines containing iodides give a precipitate of HgL.

(g) Tanret's (Bouchardai) Test. — This reagent is added,
drop by drop, to 5-10 c.c. of the urine until a turbidity or
precipitate appears. The reagent precipitates besides al-
bumin, mucin, peptone, and alkaloids. In cases where the
presence of alkaloids in the urine is suspected, the peptone
and alkaloids may be dissolved in potassio-mercuric iodide
and the solution shaken out with ether, whereby the alkaloid
is dissolved.

PROTEOSES.

Non-coagulable bodies which are precipitated by satura-
tion with (NH4)2S04 and which give the biuret reaction,
have been demonstrated and isolated from urines under
different conditions. These substances react positively to
nearly all of the proteose reactions, and it seems definitely
decided that true proteoses actually appear at certain times
in the urine. Of especial interest is the presence of an
albumose-like substance (Bence-Jones' body) associated with
multiple myelomata of the bone.

The identification of these bodies follows from the use of
the same tests employed under Digestion, p. 54.

URINE. 125

DEXTROSE.

Before testing for dextrose in the urine, protein, if present,
must be removed by heat and acetic acid. The following
tests depend upon the power of dextrose to reduce metallic
oxides, as evidenced by the formation of precipitates or color
changes. It must be remembered that the urine may also
contain other bodies such as creatinine, uric acid, allantoin,
hydroquinol, alkaptonic acid, urine and bile pigments, and
conjugate glycuronic acids, which also reduce metallic
oxides to a slighter degree, however. It is, therefore, better
never to base a decision entirely upon reduction tests.

Never allow the urine to boil more than a few seconds in
performing the tests. This will tend to eliminate the possi-
bility of a reduction caused by the above-mentioned sub-
stances.

(a) Trommer's, Fehling's, and Fermentation Tests. — Per-
form these tests as suggested under Monosaccharides (p. 4),
using the urine instead of the dextrose solution.

(6) Benedict's Modification of Fehling's Test. — To 5 c.c. of
Benedict's solution add five to eight drops of urine. Boil
the mixture vigorously for one or two minutes and allow the
test tube and contents to cool on the rack. A precipitate
will form in the solution, the color of which may be red,
yellow, or green, according to the amount of dextrose present.
This test is more sensitive than Fehling's and has the advantage
that the test solution does not deteriorate upon longstanding^

(e) Nylander's Test. — To 10 volumes of urine add 1 volume
of Nylander's reagent and heat. The presence of sugar is
indicated by a dark coloration of the urine followed by a
separation of a black precipitate. By this very sensitive test
the reducing character of some normal urines may be shown.

126 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

Urines containing sulphides cannot be tested by this method.
Why?

(d) Phenylhydrazin Test. — Perform this test as given under
the Monosaccharides, using 15 c.c. of urine. As other sub-
stances in the urine may give a precipitate with the reagent,
a mere separation of an insoluble body is not sufficient
evidence for the presence of a sugar. The precipitate must
be yellow and must be examined carefully and critically
under the microscope. If sufficient quantities are obtainable
for a melting-point determination, this procedure should be
carried out. Phenylglucosazone melts at 204-205° C.

(e) Polarization Test. — As it was stated in the discussion
of the value of this test for quantitative purposes, the results
obtained are apt to be misleading, especially when no polar-
ization of the urine can be detected. When taken as evidence
confirmatory of other tests it may serve a useful purpose. The
dextro-rotation must completely disappear aftei fermentation.

BILE.

The presence of bile imparts to the urine a saffron color,
•which upon standing takes on a greenish tinge. Icteric
urine is usually cloudy or turbid and the sediment, if any, is
rather strongly colored.

In order to insure a positive identification of icteric urine
by means of the bile acids it is necessary to separate them
from the urine by a long and laborious procedure and then to
perform Pettenkofer's test. Though this is the more accurate
method, still it is more usual clinically to perform tests for
the presence of the biliary pigments directly on the urine.

(a) Perform Gmelin's, Smith's, Hammarsten's, and Hup-
pert's tests.

(6) Rosenbach's Modification of Gmelin's Test.— Filter

URINE. 127

some icteric urine and to the moistened paper add one drop of
HN03. Colored rings around the drop correspond in color
and arrangement to those obtained in Gmelin's test. Cau-
tion : Impure filter-paper may give this test, so make a con-
trol test in using strange paper.

(c) Haycraft's Test.— Fill a test-tube half full of fresh
urine and sprinkle on the surface powdered sulphur. If the
sulphur sinks to the bottom the presence of bile salts is indi-
cated. This can only be used as a confirmatory test.

BLOOD AND BLOOD PIGMENTS.

Blood, as such, may be present in the urine (haBmaturia).
In these cases the urine is cloudy, brownish-red in color, and
contains serum albumin and serum globulin. Upon microscop-
ical examination blood corpuscles are found in the sediment.

In ha3moglobinuria the form elements are absent and
the urine holds the oxyhsemoglobin in solution; metha3-
moglobin often accompanies it, and hsematin appears under
some conditions. In a number of diseases, but especially
after the use of sulphonal and similar therapeutic agents,
hsematoporphyrin has been observed in large quantities.
Reduced hemoglobin is never present.

OXYH^EMOGLOBIN.

(a) Notice the color of the urine. If fresh it has a reddish
tinge and is turbid. Try the benzidine reaction, (g) p. 73.

(6) Examine it with the spectroscope. If the urine is not
fresh be on the lookout' for metha3moglobin. Warm a por-
tion of the urine with an excess of NaOH; filter and add a
few drops of (NH4)2S. Oxy haemoglobin is changed to hasmo-
chromogen with its characteristic spectrum.

(c) Heller's Test — Make the urine strongly alkaline with

128 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

NaOH and heat it. The oxyhamoglobin is split into hsematin
and protein, and the earthy phosphates being precipitated,
drag down the hsematin with them. Filter off the precipi-
tate, which should be of a brownish color, and after drying it,
try to obtain Teichmann's crystals (p. 76).

(d) Struve's Test. — Make a portion of the urine alkaline
with NaOH and precipitate with tannic acid. Test this pre-
cipitate for hsemin.

METH^EMOGLOBIN.

Examine the urine with the spectroscope, add a few drops
of NH4OH and filter; a two-banded spectrum appears similar
to that of oxyhsemoglobin. Do not confound the spectra of
methsemoglobin and of hsematin in acid solution.

HvEMATIN.

1. Examine in the spectroscope. If a single band is
present add (NH4)2S to the urine, filter, and again examine it.
The two bands of reduced hsematin should appear.

2. Make tests c and d under Oxy hemoglobin.

ILEMATO PORPHYRIN.

The preparation of this substance from the urine for the
purposes "of identification and spectroscopic examination is
as follows:

Method of Garrod.—To 100 c.c. of urine add 20 c.c. of
10 per cent NH4OH. The phosphates of the earthy metals
are precipitated and with them the haematoporphyrin. This
precipitate is filtered off, washed, and warmed in a flask with
acidulated alcohol. The pigment goes into solution. Use

URINE 129

this for the spectroscope. Upon the addition of a small
amount of water the alcoholic extract will exhibit a red
fluorescence. For tests see under Blood, p. 78.

CH2-S— S-CH2

CYSTINE, CH-NH2 CH-NH,.

This substance has come into prominence of late as a
protein decomposition product containing sulphur in the
neutral or lead-blackening form. In fact, the cystine which
can be obtained in the decomposition of certain proteins
contains an amount of lead-blackening sulphur which corre-
sponds closely to the total sulphur of the original protein
molecule. This would seem to imply that the sulphur of
some proteins was present in the molecule as a cystine
nucleus, and that proteins containing large amounts of
neutral sulphur have present large numbers of cystine groups
in the molecule. Cystine is also closely related to Taurine,

CH2.S03H
NH2

•^A-^^

t.

a substance which contains oxidized sulphur in organic form
and which probably represents an intermediate stage of
oxidation between the neutral sulphur atom of cystine and
the mineral sulphates of the urine. Cystine, upon standing,
separates out of the urine as colorless six-sided plates, insolu-
ble in water, acetic acid, alcohol, and ether. It sometimes is
found in the form of calculi. Cystineuria has repeatedly
been observed as an apparently anomalous metabolism of

130 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

the sulphur, peculiar to certain families, all the members of
which excrete normally relatively large quantities of the sub-
stance (0.5-1.0 grm. for 24 hours).

The appearance of the characteristic crystals and an
abnormal amount of lead-blackening sulphur in the urine
is sufficient proof of the presence of cystine.

FATTY ACID DERIVATIVES.

Associated with certain peculiar metabolic disturbances,
three closely related substances (/9-oxybutyric acid, diacetic
acid, and acetone) may appear in the urine either separately
or together. They have been noticed in severe cases of
diabetes, scarlet fever, cachexia, etc., and are the cause of
that aromatic fruity (apple-like) odor which is so frequently
present in diabetic urines. /3-oxybutyric acid only appears
in conjunction with acetone or diacetic acid, but the latter
two are often found alone in the urine.

^-OXYBUTYRIC ACID, CH3-CH(OH) -C

It is only necessary to test for this substance in urine
which contains diacetic acid. Since this body is Ia3vogyrate
and non-fermentable, the urine after fermentation should
turn the plane of polarized light to the left. This is not
sufficient evidence, however, since the conjugate glycuronates
also are Ia3vogyrate after fermentation. The following test
had better be added as confirmatory of #-oxy butyric acid.

Evaporate some fermented urine to a syrup and after
the addition of an equal volume of concentrated H2S04
distil directly without cooling; a-crotonic acid CH3— CH

is formed and distils over, and the acid

URINE. 131

crystallizes out from the distillate. The crystals are soluble
in ether and melt at 72° C.

CH2-COOH
DIACETIC ACID, |

0=C-CH3

The urine must be tested soon after voiding, as this body
disappears upon standing.

Strongly acidify some urine with H2S04 and shake it out
with ether. Separate the ether from the solution and shake-
out the former with water which is just colored with Fe2Cl6.
The watery solution becomes violet and upon the addition of
more Fe2Cle turns bordeaux-red.

The ferric chloride solution may also be added to the urine
directly. In this case the phosphates must be completely
removed by the Fe2Cl6 and filtration. Then add more Fe2Cl6,
to the filtrate.

Salicylic acid and salicylates give a similar reaction.
When these bodies are, present they may be removed from
the acidified urine by shaking it out with chloroform or ben-
zene in which the diacetic acid is not soluble. The urine is
then treated with ether as above.

CH3
ACETONE, |

0=C- CH3

Distil 100 c.c. of urine to which has been added 2 c.c. of
50 per cent acetic acid. Take the first 50 c.c. of the distillate,,
add 1 c.c. of concentrated H2S04 diluted 8 times and redistil
over 25 c.c. Make the following tests with this solution:

(a) Lieben's Test. — Place some of the solution in a test-
tube and make it alkaline with sodium carbonate. Add

132 LABORATORY WORK IN PHYSIOLOGICAL CdE.tliX

enough iodo-potassium iodide to give the solution a decided
yellow color Warm at 65° C. for 5 minutes and allow to
cool. A yellow precipitate of iodoform settles out, recogniza-
ble by its odor and its hexagonal crystals. This test is also
given by alcohol.

(6) Gunning's Modification. — In this test ammonia is
substituted for the sodium carbonate and a tincture of iodine
in place of the iodopotassium iodide. A black precipitate of
iodide of nitrogen is first formed, but this gradually disappears
on standing, leaving the iodoform visible, if present. Alcohol
or aldehydes do not give this reaction.

(c) LegaVs Test. — To a few c.c. of the distillate add a few
drops of a freshly prepared solution of sodium nitroprusside
and make the solution alkaline with NaOH. A ruby-red
color is produced which quickly disappears. Creatinine also
gives this reaction. If the alkaline acetone solution is treated
with a large excess of acetic acid, the color becomes red,
whereas in the case of creatinine it is changed to green and
then blue.

(d) Lange's Modification of LegaVs Test. — To 15 c.c. of
urine add one c.c. of glacial acetic acid, and two or three drops
of a freshly prepared solution of sodium nitroprusside. Mix
this solution well and carefully stratify upon it concentrated
ammonium hydroxide. At the line of contact a violet ring
will appear. This reaction is not given with creatinine,
alcohols or aldehydes.

EHRLICH'S DIAZO REACTION.

Diazobenzenesulphonic acid comes into prominence as a
reagent in various connections.

It is employed in the detection of sugar, protein, bilirubin,
and an unknown chromogen of the urine which is so com-
monly present associated with certain pathological conditions
(typhoid, pulmonary tuberculosis, etc.). It is especially
as a test for this unknown substance or substances that

URINE. 133

diazobenzenesulphonic acid was used by Ehrlich and has
since been designated by the clinicians as Ehrlich's Diazo-
reac ion. The fact must not be forgotten, however, that, as
a test for bilirubin this reagent has also been employed by
Ehrlich, and by others to determine the presence of sugar
and protein in the urine. The color obtained in the reaction
in the case of these two latter substances does not differ
greatly from that of the so-called Ehrlich's reaction, and on
this account the possibility of error in interpretation must
always be borne in mind.

Ehrlich's diazo-reaction is usually performed as follows:
An equal volume of a freshly prepared solution of diazo-
benzenesulphonic acid is added to the urine and the mixture
is then rendered alkaline with an excess of NH4OH. An
orange color develops in ordinary cases, but in certain
urines there results a red color which may vary from a car-
mine to a deep ruby-red; upon shaking the solution the
froth also partakes of the color. Sometimes a green or
violet precipitate will settle out upon standing.

The formation of the fresh diazobenzenesulphonic acid is
effected by the previous preparation of the two following
solutions :

1. 1 grm. sulphanilic acid and 50 c.c. of cone. HC1 dis-
solved in a liter of water.

2. 5.0 grm. sodium nitrite dissolved in a liter of water.
Just previous > to use these two solutions are mixed in

the proportion 40 : 1 and the mixture used as above.

The free nitrous acid which is liberated by the action of
the HC1 upon the NaN02, reacts with the sulphanilic acid
with the formation of diazobenzenesulphonic acid, according
to the equation

/NH2 /N^

C8H/ +HN02=C6H/
XHS03 XSO

SEDIMENTS.

UNORGANIZED.

Separating from a urine which is acid in reaction, the fol-
lowing sediments may be present :

1. CRYSTALLINE TYPE.*

(a) Uric Acid, — Color, golden brown. To what is this
due? Does not dissolve upon warming. Soluble in NaOH
and re precipitated by HC1. Responds to the murexide
test. Very characteristic crystalline form under the micro-
scope.

(6) Calcium Oxalate. — Usually present mixed with uric
acid. Colorless. Dissolves easily in HC1, but is insoluble in
acetic acid. (See Triple Phosphate, with which there is the
possibility of confusion.) Under the microscope the crystals
are transparent, refractive, octahedral (envelope shape).

(c) Bilirubin and Hcematoidin. — The former crystallizes in
golden or brown rhombic plates or needles. Dissolves easily
in alkalies and chloroform and gives Gmelin's reaction. The
latter is similar in crystalline form, but does not dissolve
in alkali and gives a blue coloration with HN03.

(d) Cystine. — Under the microscope it appears as super-
imposed six-sided plates, which are insoluble in acetic acid,
but soluble in NEUOH (differing from uric acid).

(e) Tyrosine, Leucine, and Xanthine. — Very rare. For
teste see under these substances.

134

SEDIMENTS. 135

(/) Phosphates. — 1. Magnesium Phosphate. Rhombic
plates, soluble in acetic acid, slightly attacked by ammonium
.carbonate. 2. Calcium Phosphate. Soluble in acetic acid.
Crystals wedge-shaped, seldom found. 3. Ammonio-magne-
sium Phosphate (Triple Phosphate). These separate only
when the reaction is weakly acid or amphoteric.

(g) Potassium Sulphate. — Long colorless needles, insoluble
in NH4OH or acids; seldom found.

2. AMORPHOUS TYPE.

(a) Uric Acid Salts (Acid Urates). — Brick-red or brown-
ish-red in color. Dissolves upon warming and gives the
murexide test. Upon the addition of a mineral acid, free
uric acid separates out in small crystalline form.

(6) Calcium Oxalate. — Dumb-bell shape. See above for
detection.

(c) Calcium Sulphate. — Dumb-bell shape, insoluble in HC1.

(d) Fat. — Strongly refracting round drops, soluble in ether.

Sediments separating from an alkaline reacting urine:

1. CRYSTALLINE TYPE.

(a) Triple Phosphate. — Dissolves easily in acetic acid;
unchanged by ammonium carbonate (see Magnesium Phos-
phate) ; appears under the microscope as large colorless prisms
(coffin-cover shape). Upon warming gives off NH3.

(6) Ammonium Urate. — Dissolves in HC1 or acetic acid,
followed by the separation of free uric acid crystals (rhom-
bic form). Forms dark balls with needles radiating from the
circumference (chestnut-burs). Gives off NH3 upon heating
on a platinum foil.

(c) Magnesium Phosphate. — See under Acid Urine.

136 LABORATORY WORK IN PHYSIOLOGICAL CHEMISTRY.

2. AMORPHOUS TYPE.

(a) Earthy Phosphate. — Dissolves in acetic acid without
the development of gas.

(6) Earthy Carbonate. — Dissolves in acetic acid with effer-
vescence.

(c) Calcium Carbonate. — Dumb-bell shape. Soluble in
acetic acid, with an escape of gas. (Compare Calcium
Oxalate.)

SCHEME FOR IDENTIFYING SEDIMENTS.

On heating the sediment on a platinum foil it

Does not char.

Does char.

Fresh sediment treated
with HC1

Fresh sediment gives the
murexide test.

Does not effervesce.

Effervesce, ^^

Sediment treated with
NaOH gives

Fresh sediment gently
heated and then treated
with HC1

Ammonia.

|I

No ammonia.

Srf

_; J2.

q o

fl

Does not effervesce.

Effervesce, Calcium

Fresh sediment moist-
ened with NaOH

NH3

la
P

NoNH3

1*

is

fj?

APPENDIX.

The methods for the preparation of the solutions, reagents,
and material of which use is made in the laboratory work
outlined in the preceding pages, are as follows:

SOLUTIONS.

Hydrochloric acid, 0.2 per cent; contains 6 c.c. of cone.
HC1 to the liter of water.

Acetic acid, 20 per cent in water.

Silver nitrate, 2 per cent in distilled water.

Barium chloride, 10 per cent in distilled water.

Picric acid, a cold saturated solution.

Tannic acid, 5 per cent in water.

Phosphotungstic acid, 10 per cent in water.

Zinc acetate, 1 per cent in 95 per cent alcohol.

Zinc chloride, saturated water solution, diluted with
95 per cent alcohol to a sp. gr. 1.20; filtered.

Ammonium oxalate, a cold saturated solution.

Alcoholic potash, 10 per cent KOH in 95 per cent alcohol.

Iodine solution, 0.5 per cent solution of potassium iodide
saturated with iodine.

Sodium alcoholate, sodium dissolved in absolute alcohol.

Potassium permanganate -TTJ, 3.16 grms. in a liter.

137

138 APPENDIX.

Potassium mercuric iodide, same as Tanret's reagent
without the addition of acetic acid.

REAGENTS.

Ammonium Molybdate.

50 grms. molybdic acid dissolved in 200 grms. 10 per cent
NH4OH; pour this solution slowly into 750 grms. HNO3
(sp. gr. 1.2); allow the mixture to stand for several days;
then filter.

Ammoniacal Silver Nitrate.

2.5 per cent in water; to this is added NH4OH until the
precipitate is completely dissolved.

Barfoed's Reagent.

4 per cent copper acetate in water; the solution made
.faintly acid with acetic acid and filtered.

Benedict's Solution.

Dissolve with heat 173 grams of sodium citrate and 100
grams of anhydrous sodium carbonate in about 600 c.c. of
water. Dissolve 17.3 grams of cupric sulphate in about 150
c.c. of water. Mix the two solutions by slowly pouring the
cupric sulphate into the carbonate-citrate solution. Make
the volume up to 1000 c.c.

Bial's Reagent.

500 c.c. of 30 per cent HC1 in which are dissolved 1 grm.
of orcinol and 25 drops of a 63 per cent solution of crystalline
ferric chloride.

Esbactis Reagent.

10 grms. picric acid and 20 grms. citric acid dissolved
in a liter of water.

APPENDIX. 139

Fehling's Solution.

A mixture in equal volumes of the following solutions:

1. Copper sulphate solution, 69.28 grms. crystalline
CuS04 dissolved in water and made up to a liter.

2. Alkaline tartrate solution, 346 grms. crystalline Ro-
chelle salts dissolved in 350 c.c. of water and 250 grms.
NaOH dissolved in 300 c.c. of water; these two mixed and
the volume made up to a liter.

Hammarsten's Reagent.

I volume HN03 and 19 volumes HC1 (both acids about
25 per cent). This acid mixture should be kept at least a
year. Then mix with it four times its volume of 95 per
cent alcohol.

Hopkins-Cole Reagent.

60 grms. sodium amalgam added to 1 liter of a saturated
solution of oxalic acid. After the development of gas has
ended, filter and dilute with 2 or 3 volumes of water.

Hypobromite Solution.

Mix 100 c.c. of each of the following solutions and dilute
with 300 c.c. of water.

1. 125 grms. bromine and 125 grms. sodium bromide
dissolved in water and the volume made up to 1000 c.c.
Keep in stoppered bottle.

2. A solution of sodium hydrate with a sp. gr. 1.250.

Magnesium Mixture.

100 grms. MgS04 and 200 grms. NH4C1 dissolved in 800 c.c.
of water; to this add 400 grms. concentrated NH4OH.
Mix thoroughly and keep in glass-stoppered bottle.

140 APPENDIX.

Milloris Reagent.

To a given amount of metallic mercury add twice its
weight of HN03 (sp. gr. 1.42); after the evolution of gas has
ceased, warm slightly and then dilute the mixture with 2 vol-
umes of water. Allow this to stand at least 24 hours; then
filter.

Morner's Reagent.

1 volume formalin, 45 volumes distilled water and 55
volumes concentrated H2S04; thoroughly mixed.

Nylander's Reagent.

2 grms. bismuth subnitrate and 4 grms. Rochelle salts
digested on the water in 100 c.c. of 10 per cent NaOH; cool
and filter.

Obermayer's Reagent.

1 liter of fuming concentrated HC1 to which has been
added 2-4 grms. ferric chloride.

Roberts' Reagent.

1 volume concentrated HN03 mixed with 5 volumes of a
saturated solution of MgS04.

Spiegler's Reagent.

8 grms. mercuric chloride, 4 grms. tartaric acid, and
20 grins, glycerol dissolved in 200 c.c. of water.

Tanret's Reagent (Potassium-Mercuric-Iodide).
3.32 grms. potassium iodide dissolved in 20 c.c. of water;
to this add 1.35 grms. mercuric chloride also dissolved in
20 c.c. of water; dilute the mixture to 60 c.c. and mix with
it 20 c.c. glacial acetic acid.

APPENDIX. 141

Uffelmann's Reagent.

1 per cent solution of carbolic acid in water colored
faintly amethyst with a solution of ferric chloride.

INDICATORS.

Alizarin.
1 grm. dissolved in 100 c.c. of water and filtered.

Boas' Reagent.

5 grms. resorcinol and 3 grms. saccharose dissolved in
100 c.c. 95 per cent alcohol.

Congo Red.

1 grm. dissolved in 90 c.c. of water to which is added
10 c.c. of 95 per cent alcohol.

Dimethylaminoazobenzene.
0.05 grm. dissolved in 100 c.c. 95 per cent alcohol.

Gunzburg's Reagent.

2 grms. phloroglucinol and 1 grm. vanillin dissolved in
100 c.c. 95 per cent alcohol. (Does not keep well.)

Phenolphthalein.
1 grm. dissolved in 100 c.c. 95 per cent alcohol.

Tropceolin 00.
0.05 grm. dissolved in 100 c.c. 50 per cent alcohol.

142 APPENDIX.

MATERIAL.

Fusion Mixture.

5 parts Na^COa in substance and 1 part KN03 in sub-
stance, intimately mixed.

Litmus Milk.

Add a strong filtered solution of litmus to fresh milk
until the latter is decided bluish in color.

Neutral Olive-oil.

Ordinary olive-oil thoroughly shaken with a 10 per cent
solution of Na^COg; then extract the mixture with ether.
The residue after the evaporation of the ether is neutral fat.

Oxalated Blood Plasma.

As the blood flows from an artery, allow it to mix thor-
oughly in a beaker with an equal volume of 0.2 per cent
ammonium oxalate solution.

Salted Blood Plasma.

Allow the blood flowing from an artery to mix thoroughly
with an equal volume of a saturated solution of Na2S04 or 10
per cent NaCl. Place the mixture away in a cold place and
let it remain for 24 hours.

Pancreatic Extracts.

Proteolytically active: finely divided gland digested for
three days with water containing 5-10 c.c. of chloroform
to the liter, or with a saturated solution of NaCl.

Amylolytically active: water or glycerol extract of the
gland previously allowed to stand exposed to the air for

APPENDIX. 143

24 hours; or finely divided gland digested with saturated
solution of NaCl; or digestion of the finely comminuted
gland at 40° C. in 0.7 per cent NaCl.

Lipolytically active: slightly alkaline watery infusion of
the gland; or an alkaline glycerol extract of the fresh gland
(9 parts glycerol and 1 part 1 per cent Na^COg solution).

Rennetically active: finely minced fresh gland digested
with 4 times its weight of 25 per cent alcohol for 4 to 5 days.
Then filter after the addition of a trace of acetic acid.

INDEX.

Acetone 131

Acetic acid and potassium

ferrocyanide test 19, 123

Achroodextrin 44

Acid albuminate 54

Acidic radicals in urine 89

Acidity of urine, quantitative

determination of 105

Adamkiewicz' test 18

Albumin 21

, coagulation of 21

in urine 121-124

Albuminates, acid and alkali. 24

Albuminoids 27, 28

Alcohol as protein precipitant 20

Alizarin as indicator 48

, preparation of 141

Amino bodies 54

Ammonia in urine 117

, quantitative deter-
mination of .... 117
Ammoniacal silver nitrate,

preparation of 138

Ammonium molybdate, prep-
aration of .... 138
sulphate as pro-
tein precipitant 20

urate 135

Amygdalin 44

Amylolysis 44, 60

Amylopsin 56, 60

Amyloses 1, 7, 8

Arabinose 2

Appendix 137

Bacterium Micrococcus urece . 91

Barfoed's test 4

reagent, prepara-
tion of 138

PAOB

Basic radicals in urine 90

Baybenywax,saponification 10

Benedict's reagent 138

test for dextrose. 125

Benzidine reaction 73, 127

Bial's reagent 101, 138

Bile _ 67

acids, conjugate 68

pigments, tests for 69, 70

salts, crystallized (Platt-

ner's) 69

Biliary calculus, analysis of . . 70

Bilirubin 69

in sediments 134

Biliverdin 69

Biurates 92

Biuret test 18

Blood, general 72

pigments 75

in urine 127

, spectroscopic
examination

of 77

plasma 74

, oxalated 74

, prepara-
tion of 142

, salted 74

, prepara-
tion of 142

platelets 75

Blood proteins 73

in urine 127

Boas' reagent, test for HC1 . . 47
, preparation of. 141

Boettger's test 125

Bone... 36

, mineral constituents . . 36

of 36

145

146

INDEX.

Calcium carbonate in sedi-
ment 136

oxalate in sediment. 134
phosphate in bone

ash . . . . 36

phosphate in milk . . 83

in sediment 135

sulphate in sediment 135

Cane-sugar 6

Carbohydrates 1

in urine 100

Carbon, test for 2

Carbonates in bone ash 37

, earthy, in sediment 136

Casern 55

, pancreatic 61

Caseinogen, test for 82

Celluloses 7

Cerebrin 40

Cerebrosides 38, 40

Chlorides in bone ash 36

in urine 89

, quantitative de-
termination of . 106

Cholesterol in bile 70

in biliary calculus 70
in nervous tissue. 41
Chymosin (see Rennin).
Coagulation of proteins .... 21
, method of de-
termination 22

CO-haemoglobin 79

Collagen 27

Color reactions for proteins . 17

of urine 86

Conductivity, electrical, of

urine 104

Congo-red as indicator 47

, preparation of ... 141
Conjugate sulphates .... 95, 109
proteins. ... 15, 25-27
Copper sulphate as protein

precipitant 20

Creatine in muscle 31

, transformation into

creatinine 31

Creatinine, preparation from

creatine 31

in urine 95

Cresol, p- 64, 96

Cystine in urine 129

in sediment 134

PA8B-

Depression of freezing-point

of urine 103

Deuteroproteoses 52

Dextrin 7

Dextrose, tests for 4

in blood 74

in urine 125

Diacetic acid in urine 131

Dialysis experiment 23

Digestion, gastric 46

, peptic 51

, pancreatic 56

, tryptic 57

Dimethylaminoazobenzene as

indicator 47

, preparation of 141

Disaccharides 1, 5, 7"

Edestin 23

Ehrlich's diazo reaction . 132, 134
Electrical conductivity of

urine 104

Emulsification of fats 13

Enzymes, preparation of. ... 142

Erythrodextrin 44

Esbach's reagent, preparation

of 138

Fat 9

, emulsification of - 13

in milk 83

in sediment 135

, saponification of . ... 10, 11
Fatty acids, derivatives in

urine 130

in nervous tissue 38

, perparation of. 11

, reactions for . . 12'

Fehling's test 4

solution, prepara-
tion of 139

Fermentation of dextrose ... 5

of saccharose. . 7

Fibrin 74

Form elements in blood 75

Freezing-point, depression of,

in urine 103:

Fusion mixture,preparation of 142

Galactose $

Garrod's method for haemato-

porphyrin 128

INDEX.

147

Gastric digestion 46

juice 46

Gelatin, tests for 28

Globulin in urine 121

, tests for 23

Glucose (see Dextrose).

Glycerol 14

Glycocholic acid 68

Glycogen in muscle 34

, tests frr: , „ 35

Gl y coprotein 25

Glycoproteose 53

Glycuronic acid 102

Gmelin's test for bile pigments 70
Guaiac reaction for blood ... 73

for milk 81

Gum arabic 2

cherry 2

Gunnings' modification of

Legal's test for acetone. . . 132
Giinzburg's reagent, test 'for

HC1 . . 47
, prepara-
tion of 141

Hsematin 75

in urine 125

, reduced 78

Haematoidin in sediment 134

Haematoporphyrin 78

in urine . . 1 28

Hsematuria 128

Ha?min crystals, test for blood 76

Haemochromogen 78

Haemoglobin 75, 77

carbon monoxide
(see CO-hemoglobin).

Haemoglobinuria 127

reagent, prep-
aration of. 139
Hammarsten's test for bile

pigments 70
for indican 97

Haycraft's test 127

Heller's test for protein 19

for oxyhaemoglo-

bin 127

Heteroproteose 52

Hexoses 1

Hippuric acid 98

Hopkins-Cole reagent, prepa-
ration of . . 139

Hopkin's-Fplin method for

uric acid 115

Hilfner's method for urea ... 114

Huppert's test for bile pig-
ments 70

Hydrochinol, conjugate in

urine 96

Hydrochloric acid in gastric

juice 46

Hydrogen, test for 2

Hypobromite solution, prepa- .

ration of 139

Hypoxanthine 32

Indican 97

Indicators 47

Indigo-carmin test for lactose 84
Indole in intestinal putrefac-
tion 62-64

conjugate in urine ... 96

Inorganic compounds in urine 88

Intestinal putrefaction 62

Inversion 6

Invertin 6

Iron in bone ash 37

in protein 17

, qualitative test for .... 17

Jaffe's test for creatine 37

for indican 97

Keratin, test for 28

Lactic acid, test for, in stom-
ach contents 47, 48

Lactose 5

in milk 84

Lard, saponification of 11

Lead oleate 12

Lecithins 39

Lecithoproteins 15

Legal's reaction for acetone . 132

for indole ... 64

Leucine, tests for 59

in sediment 134

Levulose 3

Lieben's test for acetone .... 131
Liebermann's test for choles-
terol 42

Lipoids of nervous tissue .... 38

Lipolysis 60

Litmus milk 60

, preparation of . 142

148

IXDEX.

9A.GE

Magnesium in bone ash 37

mixture, prepara-
tion of 139

Magnesium sulphate as pro-
tein precipitant 20

Maltodextrin 44

Maltose 5, 44

Metacasein reaction 61

Methsemoglobin 78

Milk, general 80

, qualitative separation

of the constituents of. 81
Millon's reagent, preparation

of 140

test for proteins .... 18

Mineral constituents of bone . 36

Monosaccharides 1, 3, 5

Moore's test for hexoses .... 4
Morner's reagent, preparation

of 140

test 59

Mucins in bile 67

in saliva 43

, tests for 25

Murexide test 94

Muscular tissue 29

, nitrogenous

extractives of 30
, non-nitroge-
n o u s e x -

tractives of 34

, proteins of . . 29

Myogen 29

Myosin 30

Nervous tissue 38

, cerebrin of . . 40
, cholesterol of. 38, 41
, fatty acids of. 38
, lecithins of . . 39
, lipoidsof. . . . 38
, neurokeratin

of 38

, proteins of . . 38

Neurokeratin 38

Neutral fats (see Fats).

Neutral olive-oil 13

, preparation of 142
Nitrogen, qualitative tests for 15
, total, quantitative
determination of
(Kjeldahl).. 112-114

Nitrogenous extractives

of muscular tissue 30

of nervous tissue 38

Nucleins , 26

, tests for 27

Nucleoproteins 26

Nylander's reagent, prepara-
tion of 140

test 4, 125

Obermeyer's reagent, prepar-

tion of 140

test for indican . 97

Odor of the urine 86

Oleate, lead 12

Oleicacid 12

Olive-oil, neutral 13

, preparation of .... 141

Orcinol as test for pentoses. . . 3

Ossein of bone 27, 36

Oxalic acid 97

preparation from urine 98

/J-oxybutyric acid in urine ... 130

OxyhaBmoglobin 75, 127

Palmitic acid 11

Pancreatic digestion 56

extracts, prepara-
tion of 138

rennin 56, 61

Pentosanes 2

Pentoses, tests for 2

in urine 101

Pepsin 49-51

Pepsinogen 50

Peptic proteolysis 51

Peptones 51, 53, 54

in intestinal putre-
faction 62

Pettenkofer's test for bile

acids 68

Phenol, conjugated in urine . 96
Phenolphthalein as indicator. 48
, preparation

of 140

Phenylhydrazin reaction. . 5, 126
Phloroglucinol, as test for pen-

tose 3

Phosphates, earthy, in urine .111

in sediment 135

in urine . 109

INDEX.

149

PAGE

Phosphates, quantitative de-
termination of 109, 110

Phosphoric acid in bone ash . . 36

Phosphorus in protein 17

, organic, in urine. 110
, qualitative test

for, in urine ... Ill

Phosphoprotein 26, 122

Phosphotungstic acid, as pro-
tein precipitant 20

Picric acid as protein precipi-
tant 20

Pigments, biliary 69

, blood, in urine ... 127

, urinary 99

Piria's test for tyrosine 59

Plattner's crystallized bile . . 69

Polarization 5

of urine 126

Polysaccharides 1, 7, 8

Potassium mercuric iodide . . 20
sulphate in sedi-
ment 135

sulphocyanide in

saliva 43

Precipitation reactions 19

Protagon 38

Proteins 15

, coagulation of. . . 21, 22
, compound . . 15, 25, 27

in blood serum 73

in milk 83

in nervous tissue ... 29

in urine 121

, quantitative deter-
mination of 118

Proteoses 51, 124

in intestinal putre-
faction 62

in urine 124

Protoproteose 52

Ptyalin 44

Purine bases in muscle 32

in urine 94

, method of sep-
aration 33

Putrefaction experiment and

separation 63

Pyrocatechinol, conjugated in

Quadiurates 92

PAGE

Reaction of the urine 86

Reagents, preparation of ... 138

Reduced hsematin 78

hemoglobin 77

Rennin 55

Rhamnose 101

Robert's modification of Hel-
ler's test 123

reagent, preparation

of 140

Rosenbach's modification of
Gmelin's test 126

Saccharose 1, 5-7

Salivary digestion 43

Salkowski's test for choleste-
rol 42

Saponification of bayberry

wax 10

of lard 11

Scherer's test for leucine .... 59
Schlosing's method for ammo-
nia 117

Sediments in acid reaction . . 134
in alkaline reaction 135
, scheme for identi-
fying 136

unorganized 134

Serum albumin 73

globulin 73

Skatole-carbonic acid .... 62, 65
conjugated in urine . . 96
in intestinal putre-
faction 62-64

Soaps 12

Sodium alcoholate 83

chloride in blood-

cerum 74

Solutions, preparation of .... 137
Smith's test for bile pigments 70
Specific gravity of the urine . . 87
Spectroscopic examination of

the blood 77

Spiegler's reagent, prepara-
tion of 140

test 124

Starches 7

Starch paste 8

Steapsin 56, 60

Stokes' reagent 77

Struve's test for oxyhaemoglo-
bin.. . 128

150

INDEX.

Sugar in blood 74

Sulphates, conjugate, in urine 109
, quantitative deter-
mination of .... 107

, total 108

Sulphur in urine neutral .... 89

, lead-blackening .... 15

, oxidized 16

, qualitative test for . 15

Synproteose 53

Tannic acid, as protein pre-
cipitant i 19

Tanret's reagent, preparation

of 140

test 124

Taurocholic acid 68

Teichmann's crystals, test for

blood 75

Tetroses 1

Thioproteose 53

Total solids in urine 88

Trichloracetic acid, test for

proteins 20, 123

Trioses 1

Trommer's test for hexoses . . 4

Tropseolin OO as indicator. . . 47

, preparation of 141

Trypsin 56, 57

Trypsinogen 57

Tryptic proteolysis 57

Tryptophane 62, 66

Tyrosine, test for 58

in sediment 134

Uffelman's reagent,test for

lactic acid 49
, preparation

of 141

Urates 92

, ammonium, in sedi-
ments 135

PASS

Urea 91

, preparation of 91

, quantitative determi-
nation of 114

Uric acid 92

, preparation of 93

, quantitative deter-
mination 115

Uric acid in sediment 134

, tests for 93

Urine 85

, acidity of 105

, quanitative
determina-
tion of . 86

, color of 86

, odor of 86

, physical properties of . 86
, quantitative deter-
mination of. 103, 104

, reaction of 86

, sediments in 88, 134

, specific gravity of .... 87
, tests for normal con-
stituents of 88

, total solids of 88

, transparency of 86

, volume of 87

Urinary pigments 99

Urobilin 99

, test for 100

Urochrome 99

Volume of the urine 87

Water, test for hardness .... 12
Weyl's test for creatinine ... 32

Xanthine 32

in sediments 134

Xanthoproteic reaction 12

Xylose 8

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