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By the same Author.

A TEXTBOOK OF CHEMICAL PHYSIOLOGY
AND PATHOLOGY.

With 104 Illustrations. Svo. 28s.

LONGMANS, GREEN, & CO., 89 Paternoster Row
London, New York, and Bombay.

‘ Jo

CHEMICAL PHYSIOLOGY,

FOR THE USE OF STUDENTS

W. D. HALLIBURTON, M.D., F.RS.

FELLOW OF THE ROYAL COLLEGE OF PHYSICIANS
PROFESSOR OF PHYSIOLOGY IN KING’S COLLEGE, LONDON
AUTHOR OF ‘TEXT-BOOK OF CHEMICAL PHYSIOLOGY AND PATHOLOGY’

FOURTH EDITION

LONGMANS, GREEN, AND CO.
39 PATERNOSTER ROW, LONDON
NEW YORK AND BOMBAY

1901

PREFACE

THE FOURTH EDITION

THE general arrangement and plan of this book remain the same
as in previous editions. The subject matter has been brought up
to date.

I have endeavoured to keep the work within a moderate

compass, but, with the advance of the science, even its essentials
expand.

The principal additions now introduced are new sections
relating to the chemistry of nervous tissues and of the purine
compounds and to the important subject of osmosis. The
description of the phenomena of polarised light has also been
considerably expanded.

I have to thank Dr. H. Wittoveusy Lyte, who has again
assisted me in reading proof-sheets and preparing the Index.

W. D. HALLIBURTON.
Kixe’s Contece: May 1901.

CONTENTS

I it Soh ne SS

ELEMENTARY COURSE

Tue CaRBOHYDRATES AND Fats
Tue Prorems .

Tue Prorems (continued)
Foops :
Saniva . “ ‘
Peptic DicEstTion
Pancreatic DIGESTION .
Bre

Tse Broop

UrnnxE. : ;
Uninz (continued) .

Hew RAdaeaenel

ParnHotocicaL URINE 3 3 2
Scueme For Detecrinc PuysioLocican ProxmmatTe PRINcIPLEs

ADVANCED COURSE
- Exrnopvortox ‘:

CARBOHYDRATES

XIU.

XIV. Acrion or Mar upon Srarcu
XV. CrysTanuisaTion or Eco ALBUMIN

XVI

; CoaeuLation or Minx
q XVII. Tue Axscmoses

PAGE

118

139

140
143
144
145
147

Vill ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON
XVIII. Dicestion

XIX. Hamocuosin AnD rts DERIVATIVES .
XX. Srrvum
XXI. Coacunation or Buoop
XXII. Muscrz anp Nervous Tissvzs
XXIII. Urea anp CHLormes 1n URINE
XXIV. Estimation or PHospHaTE AND SULPHATES IN URINE
XXYV. Uric Actp anp CREATININE .

XXVI. PicMENTS oF THE URINE

APPENDIX

H®MACYTOMETERS |.
H@M0GLOBINOMETERS
PoLaRISATION OF LIGHT .
POLARIMETERS

Tue SPECTRO-POLARIMETER

RELATION BETWEEN CIRCULAR POLARISATION AND CHEMICAL CONSTITUTION

Mercurian Arr Pumps .
ANALYSIS OF GASES
Kseipanw’s Mretuop or Esrimatinc NitrRoGEN

Sonutions, Dirrusion, Dratysis, Osmosis

INDEX .

PAGE

149
151
155
157
159
166
171
174
177

209

REPRE ESESRERKE EER S 6 a pep» eed

LIST OF ILLUSTRATIONS

Dextrose CRYSTALS
IyostTE CrysTALs

Misx-sucar CRYSTALS

Section oF Pea, sHowrnc SrarncH GrRarNs

Far Cents
Snorere Warm Batu
D1aLysER

Dracram oF 4 CELL

Mrx

asain CoRPUSCLES .
Atyveour or SeRovus GLAND

Moucovs CELis
Supmaxmnary Guanp
Yeast Cems .
ScurzoMycetTes
Bacttius ANTHRACIS
Carpiac GLAND
Pynortic Gianp

Carprac GLAND

Atvzeonts or Pancreas .

Leverne OrysTaus .
Tyrostye Orystats .
Hzmatorwm CrysTats
CHoLEsTERIN CrYsTAis

Schafer

- Yeo
Heidenhain

. Langley

. Langley
Heidenhain
Yeo’s Physiology
After Zopf
Koch

Klein

PAGE

49,

56.
57.

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Vintus or Rar Kizrep purine Far Apsorprion . Schéifer
Mucous Mremprane or Froe’s INTESTINE DURING Fat

ABSORPTION . : < F ‘ . Schafer
Frermn Fruaments AND Bioop TAsuets . Schiifer
Action or REAGENTS oN Buoop CoRPUscLES . Schiifer
OxyH@MOGLOBIN CRYSTALS «© «© «© « « Quain’s Anatomy
Hain Crystats . ‘ . ‘ Preyer
Diacram or SPECTROSCOPE . ine ;
Figure or SPECTROSCOPE AND ACCESSORIES . . - McKendrick

ARRANGEMENT oF Prisms IN Direct-viston SprctRoscopE Gscheidlen

Sranp For DrrEct-vision SPECTROSCOPE

Apsorption SPECTRA Rollet
ABSORPTION SPECTRA ‘ ‘ ‘ ‘
Dupre’s Urea APPARATUS Gamgee
URINOMETER . 3 ‘ ‘ ; : : ; . McKendrick
Urea CRYSTALS : : : é Frey
Urea NITRATE AND OXALATE Frey
TripLe PHospHATE CRYSTALS . Frey
Uric Acrp CrysTaLs Frey
Hiepuric Acip CrysTALs . : . . : ‘ Frey
CREATINE CRYSTALS . Frey
CREATININE CRYSTALS Frey
Acip Soprum UrnatE Frey
Acrm Ammontum URATE Frey
ENVELOPE CRYSTALS OF Cancrum OXALATE Frey
Cystmn CRYSTALS Frey
TripLE PHospHATE CRyYsTALs . Bryant’s Surgery
Caucium PHospHATE CRYSTALS Bryant's Surgery
ALBUMINOMETER OF EsBacH z
Two Burerres oN STAND Sutton
Hor-atr Oven with Gas ReeutaTor. ; j ; Gscheidlen

OsazONE CRYSTALS .

Apsorption SpecTRA oF HamoaLosm, &c.

Coloured plate to face

58. Pxorocraraic Spectrum or HmMoGLopin AND OxyHaMOGLOBIN Gamgee

PAGE

78
83
88
89
91
93
93
94
95
95
96
106
108
108
110
117
119
123
124
124
126
126
126
126
127
127
129
130
140
141
152
153

LIST OF ILLUSTRATIONS

ProtocrapHic SPEcTRUM OF OxYHZMOGLOBIN AND METHzMO-

AE ae eee ea aera es ° - Gamgee
CentrirucaL MacHINE ~. " : j : = ° ° -
Assorption Spectra OF MyYOH2MATIN . F “ . ‘ peg
MowmCOATOR, = ww eS Be are : : Gscheidlen
Assorption Spectra or Urtnary PIGMENTS .- : . After Hopkins
Gowers’ H2MACYTOMETER . 5 : -

Onrver’s H2MACYTOMETER . : ; ~ :

Gowers’ H2MOGLOBINOMETER . : S - ‘

Fueiscny’s HeMoMETER. . .

Otrver’s HzmocLosINoMETER.

Mopet to Inuustrate Ponarisep Licut : 3 °

Mover to InnustraTe Powarisep Licut . 3 Z

Move. to Iniustrate Ponarisep Licut ‘ ‘ P ‘ ° ;

Diacram To Expnarm Ponarisation or Licut

Discram To Expram Ponarisation or Licut

Sonem’s SaccHARIMETER é ‘ . :

Dracram oF Optica ARRANGEMENTS IN Sonem’s SaccHARIMETER .
Lavrent’s PoLaRmeter

SPECTRO-POLARIMETER OF VON FLEISCHL.

Dracram or AsyMMetTRic Carson AToMS

DracramM or PriiicEr’s Pump 2 3 - é . : °

L. Hoz’s Am Powe. ‘ ‘ ; : . 2 ‘
Watter’s Apparatus ror Gas ANALYSIS “ : : - Waller
KseLpanu’s MrtHop P ‘ ; 2 Waller, after Argutinsky

Dracram To InnustraTe Osmosis . .

161
179
181
182
183
184
185
187
187
188
189
190
191
191
192
193
195
196
197
198
199
203

ESSENTIALS

CHEMICAL PHYSIOLOGY

INTRODUCTION

; Chemical Physiology or Physiological Chemistry, as it is some-
times termed, deals with the chemical composition of the body and
_ with the chemical changes it undergoes; it deals also with the com-
_ position of the food which enters, and the excretions which leave, the
_ body.
q When a chemist examines living things he is placed at a dis-
‘ advantage when compared with an anatomist; for the latter can
with the microscope examine cells, organisms, and structures in the
living condition. The chemist, on the other hand, cannot at present
state anything positive about the chemical structure of living matter,
because the reagents he uses will destroy the life of the tissue he
is examining. There is, however, no such disadvantage when he
examines non-living matter, like food and urine, and it is therefore
in the analysis of such substances that chemical physiology has made
very important advances, and the knowledge so obtained is of the
_ greatest practical interest to the student and practitioner of medicine.
_ The animal organism is in its earliest embryonic state a single
cell ; as development progresses it becomes an adherent mass of
“simple cells. In the later stages various tissues become differen-
tiated from each other by the cells becoming grouped in different
ways, by alterations in the shape of the cells, by deposition of inter-
llular matter between the cells, and by chemical changes in the
iving matter of the cells themselves. Thus in some situations the
lls are grouped into the various epithelial linings; in others the

B
7 S .

2 ESSENTIALS OF CHEMICAL PHYSIOLOGY

cells become elongated, and form muscular fibres ; in the connective
tissues we have a preponderating amount of intercellular material,
which may become permeated with fibres, or be the seat of the deposi-
tion of calcareous salts, as inbone. Instances of chemical changes in
the cells themselves are seen on the surface of the body, where the
superficial layers of the epidermis become horny (2.e. filled with the
chemical substance called keratin); in the mucous salivary glands,
where the cells become filled with mucin, which they subsequently
extrude; and in adipose tissue, where they become filled with
fat.

In spite of these changes, the variety of which produces the great
complexity of the adult organism, there are many cells which still
retain their primitive structure ; notable among these are the white
corpuscles of the blood.

A cell may be defined as a mass of living material containing in
its interior a more solid structure called the nucleus. The nucleus
exercises a controlling influence over the nutrition and subdivision
of the cell.

The living substance is usually pervaded with granules; one of
these minute particles called the centrosome exercises an attractive
influence on the granules and fibrils of the protoplasm in its neighbour-
hood, and the appearance so produced is called the attraction sphere.
The attraction sphere becomes especially prominent, and divides into
two when the cell is about to divide; this usually precedes the
division of the nucleus.

Living material is called protoplasm, and protoplasm is charac-
terised by (1) its power of movement (seen in amoeboid movement,
ciliary movement, muscular movement) ; (2) its power of assimila- —
tion, that is, it is able to convert into protoplasm the nutrient —
material or food which is ingested; (8) its power of growth—this —
is a natural consequence of its power of assimilation ; (4) its power —
of reproduction—this is a variety of growth; and (5) its power to
excrete, to give out waste materials, the products of its other
activities.

Of all the signs of life, those numbered 2 and 6 in the foregoing
list are the most essential. Living material is in a continual state of
unstable chemical equilibrium, building itself up on the one hand,
breaking down on the other ; the term used for the sum total of these
intra-molecular rearrangements is metabolism. The chemical sub-
stances in the protoplasm which are the most important from this.
point of view are the complex nitrogenous compounds called Proteids.
So far as is at present known, proteid material is never absent from —

re INTRODUCTION 3

living substance, and is never present in anything else than that
_ which is alive or has been formed by the agency of living cells. It
_ may therefore be stated that Proteid Metabolism is the most essential
characteristic of vitality.
The chemical structure of protoplasm can only be investigated
_ after the protoplasm has been killed. The substances it yields are
{1) Water; protoplasm is semi-fluid, and at least three-quarters of
its weight, often more, are due to water. (2) Proteids. These are
the most constant and abundant of the solids. A proteid or albu-
_minous substance consists of carbon, hydrogen, nitrogen, oxygen,
_ with sulphur and phosphorus in small quantities only. In nuclein,
a proteid-like substance obtained from the nuclei of cells, phosphorus
is more abundant. The proteid obtained in greatest abundance from
the cell-protoplasm is nucleo-proteid, that is a compound of proteid
with varying amounts of nuclein. White of egg is a familiar instance
_ of an albuminous substance or proteid, and the fact (which is also
- familiar) that this sets into a solid on boiling will serve as a reminder
that the greater number of the proteids found in nature have a
_ similar tendency to coagulate under the influence of heat and other
agencies. (3) Various other substances occur in smaller proportions,
_ the most constant of -which are lecithin, a phosphorised fat; choles-
_ terin, a monatomic alcohol; and inorganic salts, especially phos-
_ phates and chlorides of calcium, sodium, and potassium.
: It will be seen from this rapid survey of the composition of the
_body how many are the substances which it is necessary we should
‘study; the food from which it is built up is also complex, for animals
_ do not possess to such an extent as plants do the power of building
"up complex from simple materials.
We may now proceed to an enumeration of the chemical con-
stituents of the animal body, and group them in a systematic way.
The substances out of which the body is built consist of
chemical elements and of chemical compounds, or unions of these
_ elements.
The elements found in the body are carbon, hydrogen, njtrogen,
‘oxygen, sulphur, phosphorus, fluorine, chlorine, iodine, silicon,
ium, potassium, calcium, magnesium, lithium, iron, and occa-
sionally manganese, copper, and lead.
_ Of these very few occur in the free state. Oxygen (to a small extent)
and nitrogen are found dissolved in the blood-plasma ; hydrogen is
ormed by putrefaction in the alimentary canal. With some few ex-
ceptions such as these, the elements enumerated above are found
mbined with one another to form compounds.

4 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The compounds, or, as they are generally termed in physiology,
the proximate principles, found in the body are divided into—

(1) Mineral or inorganic compounds.

(2) Organic compounds, or compounds of carbon.

The inorganic compounds present are water, various acids (such
as hydrochloric acid in the gastric juice), ammonia (as in the urine),
and numerous salts, such as calcium phosphate in bone, sodium
chloride in blood and urine, and many others.

The organic compounds are more numerous; they may be sub-
divided into—

1. Various groups of alcohols and organic acids, and their
derivatives, such as the fats and carbohydrates.

2. Various derivatives of ammonia, amides, amines, urea, &c.

3. Aromatic bodies, or derivatives of benzene.

4. Proteids, the most important of all, and substances allied to —

proteids like the albuminoids, pigments, and ferments.
A more convenient practical method of grouping the proximate
principles of the body and of food is the following :-—

Fats—e.g. butter, fats of adipose tissue.

Carbohydrates—e.g. sugar, starch.

Simple organic bodies—e.g. alcohol, choles-
terin, vegetable acids, and salts, lactic acid.

( Water.
Inorganic . Salts—e.g. chlorides and phosphates of sodium
{ and calcium.
acace2i 6 sania sat eee
ape | Albuminoids—e.g. gelatin, keratin. :
Bssropenous | Simpler siteipeni bodies—e.g. lecithin,
Organic 4 creatine, urea.

Non-nitrogenous |

Many of the substances enumerated above only occur in small
quantities. The most important are the inorganic substances, water
and salts; and the organic substances, proteids, carbohydrates, and
fats. It is necessary in our subsequent study of the principles of
chemical physiology that we should always keep in mind this simple
classification ; the subdivision of organic substances into proteids,
fats, and carbohydrates forms the starting point, the A B C, as one
might say, of chemical physiology.

I will conclude this introductory chapter by giving a list of the
apparatus and reagents necessary for a practical study of the subject,
and some tables which it will be often found convenient to refer to.

The following set of reagents conveniently contained in 4 to 6 oz. glass
stoppered bottles should be provided for each two students :—
Sulphuric acid, concentrated.
. » 25 per cent.
0-1

INTRODUCTION

Nitric acid, concentrated.
Fuming nitric acid.
Hydrochloric acid, concentrated.
ee s 0°2 per cent.!
Acetic acid, glacial.
a Pe “i per cent.

Glyoxylic saa.
Caustic potash, 20 per cent.

” ” O1 ”
Ammonia.
Sodium carbonate, 1 per cent.
Ammonium sulphide solution.
Ammonium sulphate, saturated solution.
Silver nitrate, 1 per cent.
Barium chloride, saturated solution.
Ammonium molybdate solution.
Millon’s reagent.*
Solution of ferrocyanide of potassium.

- litmus.

a sodium phosphate.

iodine in potassium iodide.

Methylated spirit.
Ether.
Esbach’s reagent.°
Solution of copper sulphate, 1 per cent.
Fehling’s solution.

The following additional reagents will be required by those taking the
advanced course :—

Solution of mercuric chloride.
- potassium ferricyanide.
Sodium carbonate, saturated solution.
» chloride, saturated solution.
= 10-per-cent. solution.
Magnesium sulphate, saturated solution.
Lime water.
mixture.*
Sodium acetate solution.®
Phosphoric acid, 0°5 per cent.
Absolute alcohol.

In addition to these, there should be kept in stock in the laboratory, to be
_ given out for the lessons in which they are used, the following :—

Solid sodium chloride.
ss Magnesium sulphate.

_! Made by adding 994 ¢.c. of water to 6 c.c. of the concentrated hydrochloric
acid of the British Pharmacopeia.
2 Mercury is dissolved in ‘te own weight of strong nitric acid. The solution so
obtained is diluted with twice its volume of water. The decanted clear liquid is
- Millon’s reagent.
8% Ten grammes of picric acid and 20 grammes of citric acid are dissolved in
800 to 900 c.c. of boiling water, and then sufficient water added to make up a litre.
* Made by mixing 1 volume of barium nitrate solution with 2 of barium hydrate
solution, both saturated in the cold.
| __ * Prepared as follows :—Sodium acetate, 100 grammes; water, 900 c.c.; glacial

acetic acid, 100 c.c.

6 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Solid ammonium sulphate.
», sodio-magnesium sulphate.
Standard solution of uranium acetate or nitrate for estimating

phosphates.’
Standard solution of mercuric nitrate for estimating urea.
” ” wa? ” ” chlorides.
” ” silver ” ” ”
Caustic soda, 40 per cent.
Bromine. :

Solution of potassium bichromate.
Phenyl hydrazine hydrochloride.
Solid sodium acetate.
Phospho-tungstic acid.

Glacial phosphorie acid.

Each student should be provided with—

A Bunsen burner.

1 dozen test-tubes in test-tube stand.

2 or 3 4-oz. flasks.

2 flat porcelain dishes.

2 or 4 4-0z. beakers.

2 small glass funnels and a funnel stand.

A glass stirring rod and a small pipette.

1 burette.

An iron tripod with wire gauze.

Filter papers and litmus papers.

A 100-c.c. cylindrical measuring glass.

A thermometer marked in degrees Centigrade.
A urinometer.

A tin can on a stand to be used as a water-bath.

Apparatus which is not so frequently used, such as that employed in
generating carbonic anhydride, carbonic oxide, or sulphuretted hydrogen,
may be given out as required. The laboratory should also possess a good
balance, with its accessories, water and air baths, kept at various temperatures,
retorts, and analytical apparatus generally. The microscope, polarimeter,
spectroscope, dialyser, are also frequently employed in chemico-physiological
investigations.

WEIGHTS AND MEASURES

The weights and measures usually employed in science are those of the
metric system, but as in this country the practical physician still largely
uses English grains and ounces, we may compare the two systems in the
following way :—

Weights
(English System.)

1 grain = 0°0648 gramme
1 ounce = 487°5 grains = 28°3595 grammes
1 Ib. = 16 oz. = 7,000 grains = 458°5925 9

The scruple = 20 grains = 1:296 gramme, and the drachm = 60 grains = 3°888
grammes, are retained in use, but neither is an aliquot part of the ounce;
though for practical purposes an ounce is considered to consist of 8 drachms. —

' Instructions how to make standard solutions will be given in the lessons
where they are used,

INTRODUCTION 7

(Metric System.)

1 milligramme = 0-001 gramme = 0°015432 grain

1 centigramme = 0-01 oF = 0°154323—,,

1 decigramme = 01 Ee = 1°543235 ,

1 gramme =s 15°43235 grains

1 decagramme = 10 grammes = 1543235 "
1hectogramme= 100 oy = 1543-235 BE ci
1 kilogramme = 1,000 Be = 15432°35 ve

2 Ib. 3 oz. 1198 ,,

Measures of Length
(English System.)
1 inch = 25°4 millimetres
1 foot = 12 inches = 304°8 millimetres
(Metric System.)

The standard of length is the metre ; subdivisions and multiples of which,
with the prefixes milli-, centi-, and deci-, on the one hand, and deca-, hecto-,
and kilo-, on the other, have the same relation to the metre as the subdivi-
_ sions and multiples of the gramme, in the table just given, have to the

_ gramme, thus: .

1 millimetre = 0°001 metre = 0°03937 inch
lcentimetre=0°01 ,, = 03937 ,,

1 decimetre = 0-1 » = 38°93707 inches
1 metre = 39°37079 ,,

Measures of Capacity
(English System.)

1 minim = 0059 cubic centimetre
1 fluid drachm = 60 minims = 8°549 cubic centimetres
1 fluid ounce =8 fluid drachms = 28°398 s

1 pint = 20 fluid ounces = 567°936 a

1 gallon =8 pints = 4°54837 litres

ry (Metric System.)

Tn the metric system the measures of capacity are intimately connected
| with the measures of length; we thus have cubic millimetres, cubic centi-
_ metres, and so forth. The standard of capacity is the litre, which is equal to
_ 1,000 cubic centimetres ; and each cubic centimetre is the volume of 1 gramme
_ of distilled water at 4° C.t

1 cubic centimetre (generally written c.c.)} |§=16-931 minims.
1 litre = 1,000 ¢.c. =1 pint 15 oz. 2 drs. 11 m. = 35°2154 fiuid ounces.
1 cubic inch = 16386 c.c.

sar

THERMOMETRIC SCALES

_ The scale most frequently used in this country is the Fahrenheit scale ; ;
‘in this the freezing-point of water is 32°, and the boiling-point 212°. On the
tinent the Réaumur scale is largely employed, in which the freezing-

__ ' 4° C. is the temperature at which water has the greatest density. For prac-
tical purposes measures are more often constructed so that a cubic centimetre holds
@ gramme of water at 16° C., which is about the average temperature of rooms.
The true cubic centimetre contains only 0-999 gramme at 16° C.

8 ESSENTIALS OF CHEMICAL PHYSIOLOGY

point is 0°, and the boiling-point 80°. In scientific work the Centigrade
scale has almost completely taken the place of these; in this system the
freezing-point is 0°, and the boiling-point 100°.

To convert degrees Fahrenheit into degrees Centigrade, subtract 82 and
multiply by §, or C=(F—82)3. Conversely, degrees Centigrade may be con-
verted into degrees Fahrenheit by the following formula: F = 3C + 82.

TENSION OF AQUEOUS VAPOUR IN MILLIMETRES OF.
MERCURY FROM 10° TO 25° C.

10°. 9126 14°. 11-882 18°. 15°351 22°. 19°675
11°. 9°751 15°. 12°677 19°. 16°345 23°. 20-909
~ 12°. 10°421 16°. 13.519 20°. 17°396 24°, 22°211
13°. 11:130 17°. 14-409 21°. 18°505 25°. 23°582

TABLE OF THE DENSITY OF WATER AT TEMPERATURES
BETWEEN 0° AND 30° C.

0°. 0:99988 8°. 0°99988 16°. 0°99900 24°. 0:99738
1°. 0°99993 9°. 0°99982 17°. 0°99884 25°. 0:99714
2°. 0°99997 10°. 0°99974 18°. 099866 26°. 0°99689
3°. 0°99999 11°. 099965 19°. 0°99847 27°. 0°99662
4°, 1-00000 12°. 099955 20°. 0:99827 28°. 099635
5°. 0°99999 13°. 0°99942 21°. 0:99806 29°. 0°99607
6°. 0°99997 14°. 0°99930 22°. 0°99785 80°. 099579
7°. 0°99994 15°. 099915 23°. 0:99762
SYMBOLS AND ATOMIC WEIGHTS OF THE PRINCIPAL
ELEMENTS
Aluminium Al 27:02 Fluorine F 191 Phosphorus P 30°96
Antimony Sb 120-0 Gold Au 1970 Platinum Pt 1943
Arsenic As 749 Hydrogen H 10 Potassium K 39°04
Barium Ba 1368 Iodine I 126:53 Silver Ag 107°66
Bismuth Bi 2080 Tron Fe 559 Silicon Si 28°3
Boron B 109 Lead Pb 2064 Sodium Na =. 28°05

Bromine Br 79°75 Magnesium Mg 24:0 Strontium Sr 873
Cadmium Cd 112°0 Manganese Mn 55:0 Sulphur 8 31-98
Calcium Ca 39°99 Mercury Hg 199°8 Tj s

Carbon C 120 Nickel Ni 586 1” pie
Chlorine Cl 35:45 Nitrogen N 1401 Tungsten W 1836
Copper Cu 63°2 Oxygen O 160 Zine Zn 1178

ELEMENTARY COURSE

LESSON I
THE CARBOHYDRATES AND FATS

1. Note the general appearance of the specimens of grape sugar or dex-
trose, cane sugar, dextrin, and starch which are given round.

2. Put some of each into cold water. Starch is insoluble; dextrose, cane
sugar, and dextrin dissolve after a time, but more readily in hot water.

3. Trommer’s test for dextrose ——Put a few drops of copper sulphate solu-
tion into a test-tube, then solution of dextrose, and then strong caustic potash.
On adding the caustic potash a precipitate is first formed, which, owing to
the presence of the sugar, rapidly redissolves, forming a blue solution. On
boiling this a yellow or red precipitate (cuprous hydrate or oxide) forms.

Fehling’s test for dextrose—Fehling’s solution is a mixture of copper
sulphate, caustic soda, and Rochelle salt of a certain strength. It is used for
estimating dextrose quantitatively (see Lesson XII.). It may be used as a
qualitative test also. Boil some Fehling’s solution ; if it remains clear itis
in good condition ; add to it an equal volume of solution of dextrose and boil
again. Reduction, resulting in the formation of cuprous hydrate or oxide,
takes place as in Trommer’s test.

4, Cane Sugar.—(a) The solution of cane sugar when mixed with copper
sulphate and caustic potash gives a blue solution. But on boiling no reduc-
tion occurs.

(6) Take some of the cane sugar solution and boil it with a few drops of
25-per-cent. sulphuric acid. This converts it into equal parts of
dextrose and levulose. It then gives Trommer's or Fehling’s test
in the typical way.

(c) Boil some of the cane sugar solution with an equal volume of con-
centrated hydrochloric acid. A deep red solution is formed. Dex-
trose, lactose, and maltose do not give this test.

5. Starch.—(a) Examine microscopically the scrapings from the surface
of a freshly cut potato. Note the appearance of the starch grains with their
_ concentric marki
(6) On boiling starch with water an opalescent solution is formed,

which, if strong, gelatinises on cooling.

(c) Add iodine solution. An intense blue colour is produced, which dis-
appears on heating, and if not heated too long reappears on cooling.
N.B.—Prolonged heating drives off the iodine, and consequently
no blue colour returns after cooling. :

(@) Conversion into dextrin and dextrose. To some starch solution in
flask add a few drops of 25-per-cent. sulphuric acid, and boil for
15 minutes. Take some of the liquid, which is now clear, and show
the presence of dextrin and dextrose.

10 ESSENTIALS OF CHEMICAL PHYSIOLOGY

6. Dextrin.—Add iodine solution to solution of dextrin, and a reddish-
brown solution is produced. The colour disappears on heating and reappears
on cooling.

7. Glycogen.—Solution of glycogen is given round: (a) it is opalescent
like that of starch.

(6) With iodine it gives a brown colour very like that given by dextrin.
The colour disappears on heating and reappears on cooling.
(c) By boiling with 25-per-cent. sulphuric acid for 15 to 20 minutes it is
converted into grape sugar.
8. Lard is given round as an example of a fat. (a) It is insoluble in
water. :
(6) By boiling with potash it yields a solution of soap.
(c) Add to this solution a few drops of 25-per-cent. sulphuric acid. On
heating a layer of fatty acid collects on the surface.
(d) Shake up some lard with ether. It dissolves, leaving little or no
residue.

CARBOHYDRATES

The carbohydrates are found chiefly in vegetable tissues, and
many of them form important foods. Some carbohydrates are,
however, found in or formed by the animal organism. The most
important of these are glycogen, or animal starch; dextrose; and
lactose, or milk sugar. x

They may be for the greater part arranged into three groups,
according to their empirical formula. The names and formulz of
these groups, and the most important members of each, are as
follows :—

The + and — signs in the above list indicate that the substances
to which they are prefixed are dextro- and levo-rotatory respectively
as regards polarised light.'

The carbohydrates may be conveniently defined as compounds of
carbon, hydrogen, and oxygen, the two last-named elements being in
the proportion in which they occur in water. This definition is,
however, only a rough one, and if pushed too far would include

' For a description of polarised light and polarimeters see Appendix. This and
the other matter in the Appendix are placed there for convenience, not because

they are unimportant. Students are therefore urged to refer to and carefully
study these subjects.

_ water and in alcohol. It is crystalline

THE CARBOHYDRATES AND FATS il

several substances like acetic acid, lactic acid, and inosite, which
are not carbohydrates.

The formule given above are merely empirical ; and there is no
doubt that the quantity 7 in the starch group is variable, and often
large; hence the name polysaccharides that is given to the group.
Research has, moreover, shown thai the glucoses are either aldehydes
or ketones of hexatomic alcohols having the general formula
C,H,(OH),. Thus dextrose is the aldehyde of sorbite, levulose the
ketone of mannite, and galactose the aldehyde of dulcite. The
amyloses may be regarded as the anhydrides of the glucoses [nCgH.0,
— nH,O = (C,H,,0;),]. The sucroses are condensed glucoses—z.e.
they are formed by the combination of two molecules of glucose with
the loss of one molecule of water (Cg,H,.O, + CgH,,0, — H,O
=C,.H,.0,,); hence the term disaccharide. The following are the
chief facts in relation to each of the principal carbohydrates :—

MONOSACCHARIDES

Dextrose or Grape Sugar.—This carbohydrate is found in fruits,
honey, and in minute quantities in the blood (0°12 per cent.) and
numerous tissues, organs, and fluids of the

body. It is the form of sugar found in ae
large quantities in the blood and urine %. \
_ in the disease known as diabetes. >
} Dextrose is soluble in hot and cold 0

(see fig. 1), but not so sweet as cane sugar.

When heated with strong potash certain Y

complex acids are formed which have a

yellow or brown colour. This constitutes >

Moore’s test for sugar. In alkaline solu- F1G. 1.—Dextrose crystals.

_ tions dextrose reduces salts of silver,

_ bismuth, mercury, and copper. The reduction of cupric to cuprous

oxide constitutes Trommer’s test, which has been already described

at the head of the lesson. On boiling it with an alkaline solution of

picric acid, a dark red opaque solution due to reduction of the picric

_ to picramic acid is produced. Another important property of grape

sugar is that under the influence of yeast it is converted into alcohol
and carbonic acid (C;H,,0, = 2C,H,O + 2CO,).

q Dextrose may be estimated by the fermentation test, by the

polarimeter, and by the use of Fehling’s solution. The last method

_is the most important; it rests on the same principles as Trommer’s

12 ESSENTIALS OF CHEMICAL PHYSIOLOGY

test, and we shall study it and other methods of estimating sugar
in connection with diabetic urine (see Lesson XII).

Levulose.—When cane sugar is treated with dilute mineral acids
if undergoes a process known as inversion—+.e. it takes up water and
is converted into equal parts of dextrose and levulose. The pre-
viously dextro-rotatory solution of cane sugar then becomes leyo-
rotatory, the levo-rotatory power of the levulose being greater than
the dextro-rotatory power of the dextrose formed. Hence the term
inversion. Similar hydrolytic changes are produced by certain
ferments, such as the invert ferment of the intestinal juice.

Pure levulose can be crystallised, but so great is the difficulty of
obtaining crystals of it that one of its names was unerystallisable
sugar. Small quantities of levulose have been found in blood, urine,
and muscle. It has been recommended as an article of diet in
diabetes in place of ordinary sugar; in this disease it does not
appear to have the harmful effect that other sugars produce.
Levulose gives the same general reactions as dextrose.

Galactose is formed by the action of dilute mineral acids
or inverting ferments on lactose or
milk sugar. It resembles dextrose
in being dextro-rotatory, in reducing
cupric hydrate in Trommer’s test,
and in being directly fermentable
with yeast. When oxidised by means
of nitric acid it, however, yields an
acid called mucic acid (CH, 90x),
which is only sparingly soluble in
water. Dextrose when treated this
way yields an isomeric acid—+z.e. an
acid with the same empirical formula,
called saccharic acid, which is readily
soluble in water.

Fic. 2.—Inosite crystals.

Inosite, or muscle sugar, is found in muscle, kidney, liver, and other parts
of the body in small quantities. It is also largely found in the vegetable
kingdom. It is a crystallisable substance (see fig. 2) and has the same
formula (C,H,,0,) as the glucoses. It is, however, not a sugar. It gives
none of the sugar tests, and careful analysis has shown it has quite a different
chemical constitution from the true sugars. It belongs to the aromatic
series, and is only included here for convenience.

DISACCHARIDES

Cane Sugar.—This sugar is generally distributed throughout the
vegetable kingdom in the juices of plants and fruits, especially the

THE CARBOHYDRATES AND FATS 13

4 sugar cane, beetroot, mallow, and sugar maple. It is a substance of
great importance as a food. After abundant ingestion of cane sugar
traces may appear in the urine, but the greater part undergoes
_ inversion in the alimentary canal.
Pure cane sugar is crystalline and dextro-rotatory. It holds
cupric. hydrate in solution in an alkaline liquid—that is; with
Trommer’s test it gives a blue solution. But no reduction occurs on
boiling. After inversion it is strongly reducing.

Inversion may be brought about readily by boiling with dilute
mineral acids, or by means of an inverting ferment, such as that
occurring in the succus entericus or intestinal juice. It then takes
up water and is split into equal parts of dextrose and levulose (see
p. 12).

C,2H..0;,; + HO = C;H,.0, +C,H)20,
[cane sugar] [dextrose] [levulose]

With yeast, cane sugar is first inverted by means of a special
soluble ferment produced by the yeast cells, and then there is an
_ alcoholic fermentation of the glucoses so formed.

Lactose, or milk sugar, occurs in milk. It has also been described
as occurring in the urine of women in the early days of lactation or
after weaning.

It erystallises in rhombie prisms (see fig. 3). It is much less
_ soluble in water than cane sugar or dextrose,

and has only a slightly sweet taste. It is ER
insoluble in alcohol and ether; aqueous

solutions are dextro-rotatory. Y 2
Solutions of lactose give Trommer’s
_ test, but when the reducing power is tested Ns Ui My
quantitatively by Fehling’s solution it is

found to be a less powerful reducing agent

than dextrose. If it required seven parts pig. 3—witk-sugar crystals.
of a solution of dextrose to reduce a given

quantity of Fehling’s solution, it would require ten parts of a solution
of lactose of the same strength to reduce the same quantity of
_ Fehling’s solution.

Lactose, like cane sugar, can be hydrolysed by the same agencies
as those already enumeraied in connection with cane sugar. The
_ glucoses formed are dextrose and galactose.

C,2H.0,,; + H,O0 = C,H,.0; + C>H,2.0,
[lactose] [dextrose] {galactose]
With yeast it is first inverted, and then alcohol is formed. This,
however, occurs slowly.

14 ESSENTIALS OF CHEMICAL PHYSIOLOGY

With the lactic-acid organisms which bring about the souring of
milk the lactic-acid fermentation is produced. This may also occur
as the result of the action of putrefactive bacteria in the alimentary
canal. The two stages of the lactic-acid fermentation are represented
by the following equations :-—

(1) C),H».0;, + H,O = 4C;H,0;
[lactose] - [lactic acid]
(2) 4C,H,O; == 2C,H,0, + 4CO, + 4H,

[lactic acid] —_ [butyric acid]

Maltose is the chief end product of the action of malt diastase on
starch, and is also formed as an intermediate product in the action
of dilute sulphuric acid on the same substance. It is also the chief
sugar formed from starch by the diastatic ferments contained in the
saliva (ptyalin) and pancreatic juice (amylopsin).’ It can be ob-
tained in the form of acicular crystals ; it is strongly dextro-rotatory.
It gives Trommer’s test; but its reducing power, as measured by
Fehling’s solution, is one-third less than that of dextrose.

By prolonged boiling with water, or, more readily, by boiling with
a dilute mineral acid, or by means of an inverting ferment, such as
occurs in the intestinal juice, it is converted into dextrose.

C\2H2,0,, + H,O = 20,H).0,
{maltose] {dextrose]

It undergoes readily the alcoholic fermentation.

The three important physiological sugars (dextrose, lactose, and
maltose) may be distinguished from one another by their relative
reducing action on Fehling’s solution (1:0 : 0°71 : 0°63), or by the
phenyl-hydrazine test described in Lesson XIII.

POLYSACCHARIDES,

Starch is widely diffused through the vegetable kingdom. It
occurs in nature in the form of microscopic grains, varying in size
and appearance, according to their source. Each consists of a
central spot (/iilwm) round which more or less concentric envelopes
of starch proper or granulose alternate with layers of cellulose.
Cellulose has very little digestive value, but starch is a most
important food.

Starch is insoluble in cold water; it forms an opalescent solution
in boiling water, which if concentrated gelatinises on cooling. Its
most characteristic reaction is the blue colour it gives with iodine.

On heating starch with dilute mineral acids dextrose is formed.

' An isomeric sugar called iso-maltose (see Lesson XIII.) is also formed under
these circumstances.

THE CARBOHYDRATES AND FATS 15

By the action of diastatic ferments, maltose is the chief end product.
In both cases dextrin is an intermediate stage in the process.

Before the formation of dextrin the starch solution loses its opal-
escence, a substance called soluble starch or amidulin being formed.
This, like native starch, gives a blue colour with iodine. Although the
molecular weight of starch is unknown, :
the formula for soluble starch is probably
(CgH1005)200- Equations that represent
_ the formation of sugars and dextrins
_ from this are very complex, and are at
present hypothetical.

Dextrin is the name given to the
intermediate products in the hydration
of starch, and two chief varieties are
distinguished — erythro-dextrin, which
gives a reddish-brown colour with tS
iodine; and achroé-dextrin, which does Fic. 4.—Section of pea showing starch

and aleurone grains embedded in the
not. protoplasm of the cells: a, aleurone

It is readily soluble in water, but pings Bae ior ates)
insoluble in alcohol and ether. It is
_ gummy and amorphous. It does not give Trommer’s test, nor
_ does it ferment with yeast. It is dextro-rotatory. By hydrating

agencies it is converted into glucose.

Glycogen, or animal starch, is found in liver, muscle, and colour-
less blood corpuscles. It is also abundant in all embryonic
tissues.

Glycogen is a white tasteless powder, soluble in water, but it
_ forms, like starch, an opalescent solution. It is insoluble in alcohol
| and ether. It is dextro-rotatory. With Trommer’s test it gives a
_ blue solution, but no reduction occurs on boiling.

With iodine it gives a reddish or port-wine colour, very similar to
that given by erythro-dextrin. Dextrin may be distinguished from
glycogen by (1) the fact that it gives a clear, not an opalescent,
_ solution with water; and (2) it is not precipitated by basic lead
acetate as glycogen is. It is, however, precipitated by basic lead
acetate and ammonia. (3) Glycogen is precipitated by 55 per cent.
_ of alcohol ; the dextrins require 85 per cent. or more.

_ Cellulose.—This is the colourless material of which the cell-walls
-and woody fibres of plants are composed. By treatment with
strong mineral acids it is, like starch, converted into glucose, but
’ with much greater difficulty. The various digestive ferments have
’ little or no action on cellulose; hence the necessity of boiling starch

16 ESSENTIALS OF CHEMICAL PHYSIOLOGY

before it is taken as food. Boiling bursts the cellulose envelopes of
the starch grains, and so allows the digestive juice to get at the
starch proper. Cellulose is found ina few animals, as in the test or
outer investments of the Tunicates.

For further information regarding the carbohydrates see Lesson XIII.

‘THE FATS

Fat is found in small quantities in many animal tissues. It is,
however, found in large quantities in three situations, viz. bone
marrow, adipose tissue, and milk. The consideration of the fat in
milk is postponed to Lesson IV.

Vic. 5.—A few cells from the margin of a fat lobule ; 7. g., fat globule distending fat cell; n, nucleus ;
m, membranous envelope of the cell; c7., bunch of crystals within a fat cell; c, capillary vessel ;
vy, venule ; ¢. ¢., connective tissue cell. The fibres of the connective tissue are not represented.

The contents of the fat cells of adipose tissue are fluid during life,
the normal temperature of the body (37° C., or 99° F.) being con-
siderably above the melting-point (25° C.) of the mixture of the fats
found there. These fats are three in number, and are called palmitin,
stearin, and olein. They differ from one another in chemical com-
position and in certain physical characters, such as melting-point
and solubilities. Olein melts at —5° C., palmitin at 45° C., and stearin
at 53-66° C. Thus, it is olein which holds the other two dissolved at
the body temperature. Fats are all soluble in hot alcohol, ether, and
chloroform, but insoluble in water.

Chemical Constitution of the Fats—The fats are compounds of

THE CARBOHYDRATES AND FATS 17

- fatty acids with glycerin, and may be termed glycerides or glyceric
ethers. The term hydrocarbon, applied to them by some authors, is

_ wholly incorrect.

The fatty acids form a series of acids derived from the monatomic

_ alcohols by oxidation. Thus, to take ordinary ethyl alcohol, C,H;HO,

the first stage in oxidation is the removal of two atoms of hydrogen

_ to form aldehyde, CH;.COH: on further oxidation an atom of oxygen

' is added to form acetic acid, CH;.COOH.

"i A similar acid can be obtained from all the other alcohols

» thus :—

- From methyl alcohol CH;.HO, formic acid H.COOH sis obtained

» ethyl ve C.H,.HO, acetic , CH,.COOH i

» propyl ,, C,;H,.HO, propionic ,, C,H;.COOH a

So Oabyl = |, C,H,.HO, butyric ,, C;H;.COOH we

» amyl ,, ©;H,,-HO, valeric ,, C,H,.COOH a

» hexyl ,, OC H,3-HO, caproic ,, C;H,,.COOH pa
and so on.

Or in general terms :—
q From the alcohol with formula C,H,,,.;.HO the acid with formula
C,_,H,,_;-CO.OH is obtained. The sixteenth term of this series has
the formula C,;H;,.CO.0H, and is called palmitic acid; the
_ eighteenth has the formula C,;H;;.CO.OH, and is called stearic acid.
Bach acid, as will be seen, consists of a radicle, C,_,H.,_,CO, united
to hydroxyl (HO).
. Oleic acid, however, is not a member of the fatty acid series
| proper, but belongs to a somewhat similar series of acids known as
| the acrylic series, of which the general formula is C,_,H»,_;COOH.
_ Itis the eighteenth term of the series, and its formula is C,;,H;3;.CO.OH.
Glycerin or Glycerol is a triatomic alcohol, C;H;(OH),—~.e. three
_ atoms of hydroxy] united to a radicle glyceryl (C;H;). The hydrogen
in the hydroxyl atoms is replaceable by other organic radicles. As
"an example take the radicle of acetic acid called acetyl (CH;.CO).
“The following formule represent the derivatives that can be obtained
by replacing on, two, or all three hydroxyl hydrogen atoms in this
way :—

OH OH (OH (0.CH,.CO
| oH OH oH, | OF C;H,-0.CH;.CO C,H;/0.CH,.cO
j OH 0.CH;.CO (0.CH;.CO (0.CH,.CO

- [glycerin] [monoacetin] [diacetin] [triacetin]

Triacetin is a type of a neutral fat; stearin, palmitin, and olein
ought more properly to be called tristearin, tripalmitin, and triolein
Cc

18 ESSENTIALS OF CHEMICAL PHYSIOLOGY

respectively. Hach consists of glycerin in which the three atoms of
hydrogen in the hydroxyls are replaced by radicles of the fatty acid.
This is represented in the following formule :—

Acid Radicle Fat
Palmitic acid C,,H,,.COOH Palmityl C,,H,,.CO Palmitin C,H,(OC,,H,,.CO),
Stearic acid C,,H,..COOH Stearyl C,,H,;.CO Stearin C,H.(OC,,H,..CO),
Oleic acid C,-H,,.COOH Oleyl C,,H,,.CO Olein ©,H,(OC,,H,,.CO),

Decomposition Products of the Fats—The fats split up into the
substances out of which they are built up.

Under the influence of superheated steam, mineral acids, and in —
the body by means of certain ferments (for instance, the fat-splitting
ferment steapsin of the pancreatic juice), a fat combines with water
and splits into glycerin and the fatty acid. The following equation —
represents what occurs in a fat, taking tripalmitin as an example :—

C3;H;(0.C,;H3;CO);+3H,O=C;H;(OH);+3C,,;H;,;C0.0H
[palmitin—a fat] [glycerin] [palmitic acid—a fatty acid]

In the process of saponification much the same sort of reaction
occurs, the final products being glycerin and a compound of the base
with the fatty acid, which is called a soap. Suppose, for instance,
that potassium hydrate is used; we get— :

C,H;(0.C,;H3,CO);+3KHO=C;H;(OH),;+3C,,;H3,;CO.0K
[palmitin—a fat] [glycerin] [potassium palmitate—a soap]

Emulsification.—Another change that fats undergo in the body is —
very different from saponification. It is a physical rather than a
chemical change ; the fat is broken up into very small globules, such ~
as is seen in the natural emulston—amilk.

Lecithin (C,,H,,NPO,).—This is a very complex fat, which
yields on decomposition not only glycerin and a fatty (stearic)
acid, but phosphoric acid, and an alkaloid [N.(CH3),;C,H,0o]
called choline in addition. Lecithin is found to a great extent in
the nervous system,! and to a small extent in bile. Together with —
cholesterin, a crystallisable, monatomic alcohol (C.,H,;.HO), which
we shall consider more at length in connection with the bile, it is
found in. small quantities in the protoplasm of all cells.

1 See further under Nervous Tissues, Lesson XXII.

LESSON II
THE PROTEIDS

L 1. Tests for Proteids.—The following tests are to be tried with a mixture of
" one part of white of egg to ten of water. (Egg-white contains a mixture of
_ albumin and globulin.)

q (a) Heat Coagulation.—Faintly acidulate with a few drops of 2-per-cent.
_ acetic acid and boil. The proteid is rendered insoluble (coagulated proteid).
(0) Precipitation with Nitric Acid.—The addition of strong nitric acid to
the original solution also produces a white precipitate.

(ec) Xanthoproteic Reaction.—On boiling the white precipitate produced by
nitrie acid it turns yellow; after cooling add ammonia; the yellow becomes

(d) Millon’s Test.—Millon’s reagent (which is a mixture of the nitrates of
| mercury containing excess of nitric acid; see p. 5) gives a white precipitate,
_ which turns brick-red on boiling.
| _— (e) After the addition of a few drops of 20-per-cent. acetic acid, potassium
| ferrocyanide gives a white precipitate.
(f) Piotrowski’s test—Add a drop of a 1-per-cent. solution of cupric
_ sulphate to the original solution and then caustic potash, and a violet solution
is obtained.

Repeat experiment (f) with a solution of commercial peptone, and note
_ that a rose-red solution is obtained. This is called the biwret reaction.
: (g) Adamkiewicz’s Reaction (Hopkins’s modification).—Add the albumin
_ solution to dilute glyoxylic acid, and then strong sulphuric acid; an intense
violet colour is obtained. Most commercial specimens of glacial acetic acid
contain glyoxylic acid and may be used in place of pure glyoxylic acid in this
test

2. Action of Neutral Salts—(a) Saturate the solution of egg-white with
“magnesium sulphate by adding crystals of the salt and grinding it up
thoroughly in a mortar. A white precipitate of egg-globulin is produced.
Filter. The filtrate contains egg-albumin. The precipitate of the globulin
is very small.
q (6) Half saturate the solution of egg-white with ammonium sulphate.
_ This may be done by adding to the solution an equal volume of a saturated
solution of ammonium sulphate. The precipitate produced consists of the
_ globulin ; the albumin remains in solution.
(c) Completely saturate another portion with ammonium sulphate by
adding crystals of the salt and grinding in a mortar—a precipitate is pro-
_ duced both the globulin and albumin. Filter. The filtrate contains no
_ protei
__ (d) Repeat the last experiment (c) with a solution of commercial peptone.
A precipitate is produced of the albumoses or proteoses it contains. Filter.
The filtrate contains the true peptone. This gives the biuret reaction (see
above), but large excess of strong potash must be added on account of the
‘presence of ammonium sulphate. Ammoniwm sulphate precipitates all
proteids except peptone.

20 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON III
THE PROTEIDS (continued)

1. Action of Acids and Alkalis on Albumin.—Take three test-tubes and
label them A, B, and C.

In each place an equal amount of diluted egg-white, similar to that used
in the last lesson.

To A add a few drops of 0°1-per-cent. solution of caustic potash.

To Badd the same amount of 0°1-per-cent. solution of caustic potash.

To C add a rather larger amount of 0-1-per-cent. sulphuric acid.

Put all three into the warm bath’ at about the temperature of the body
(36-40° C.).

After five minutes remove test-tube A, and boil. The proteid is no

FG. 6.— Simple warm bath, as described in footnote.

longer coagulated by heat, having been converted into alkali-albwmin. After
cooling, colour with litmus solution and neutralise with 0-1-per-cent. acid.
At the neutral point a precipitate is formed which is soluble in excess of
either acid or alkali.
Next remove B. This also now contains alkali-albumin. Add to it afew

' A convenient form of warm bath suitable for class purposes may be made by
placing an ordinary tin pot half full of water over a bent piece of iron which
acts as a warm stage as in the figure. The stage is kept warm by a small gas
flame. Such a warm bath may be placed between every two or three students.

THE PROTEIDS 21

drops of sodium phosphate, colour with litmus, and neutralise as before.
Note that the alkali-albumin now requires more acid for its precipitation
than in A, theacid which is first added converting the sodium phosphate into
acid sodium phosphate.

_ Now remove C from the bath. Boil it. Again there is no coagulation,
‘the proteid having been converted into acid-albwmin, or syntonin. After
cooling, colour with litmus and neutralise with 0-1-per-cent. alkali. At the
neutral point a precipitate is formed, soluble in excess of acid or alkali.
(Acid-albumin is formed more slowly than alkali-albumin, so it is best to
_ leave this experiment to the last.)

2. Take some gelatin and dissolve it in hot water. On cooling, the solu-
tion sets into a jelly (gelatinisation).

Take a dilute solution of gelatin, and try all the proteid tests with it
enumerated on p. 19. Carefully note down your results.

3. Add a few drops of acetic acid to some saliva. A stringy precipitate
_ of mucin is formed.

4. A tendon has been soaked for a few days in lime water. The fibres
_ are not dissolved, but they are loosened from one another owing to the solu-
tion of the interstitial or ground substance by the lime water. Take some
of the lime-water extract and add acetic acid. A precipitate of mucin is
obtained. The fibres themselves consist of collagen, which yields gelatin on
boiling. Vitreous humour or the Whartonian jelly of the umbilical cord is
‘much richer in ground substance than tendon, and, if treated in the same
way, a much larger yield of mucin is obtained.

The Proteids are the most important substances that occur in
animal and vegetable organisms; none of the phenomena of life
occur without their presence; and though it is impossible to state
‘positively that they occur as such in living protoplasm, they are
invariably obtained by subjecting living structures to analytical
processes.

Proteids are highly complex compounds of carbon, hydrogen,
oxygen, nitrogen, and sulphur occurring in a solid viscous condition
or in solution in nearly all parts of the body. The different members
of the group present differences in chemical and physical properties.
‘They all possess, however, certain common chemical reactions
and are united by a close genetic relationship. :

| The various proteids differ a good deal in elementary composition.
| Hoppe-Seyler gives the following percentages :—

C H N s O
From 51:5 69 15°2 03 20°9
To 545 73 17:0 2-0 23°5

We are, however, not acquainted with the constitutional formula
of proteid substances. There have been many theories on the
“su bject, but practically all that is known with certainty is that many
different substances may be obtained by the decomposition of pro-
teids. How they are built up into the proteid molecule is unknown

22 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The decompositions that occur in the body are, moreover, different
from those which can be made to occur in the laboratory ; hence the
conclusion that living protoplasm differs somewhat from the non-
living proteid material obtainable from it.

(1) In the body. Carbonic acid, water, and urea' are the chief
final products. Glycocine, leucine, creatine, uric acid, ammonia, &c.,
are probably intermediate products. Carbohydrates (glycogen)
and fats may also originate from proteids.

(2) Outside the body. Various strong reagents break up proteids
into ammonia, carbonic acid, amines, fatty acids, amido-acids like
leucine, arginine, and glycocine, and aromatic compounds like
tyrosine.

TESTS FOR PROTEIDS

Solubilities.—All proteids are insoluble in alcohol and ether.
Some are soluble in water, others insoluble. Many of the latter are —
soluble in weak saline solutions. Some are insoluble, others soluble
in concentrated saline solutions. It is on these varying solubilities
that proteids are classified. ;

All proteids are soluble with the aid of heat in concentrated
mineral acids and alkalis. Such treatment, however, decomposes as
well as dissolves the proteid. Proteids are also soluble in gastric and
pancreatic juices; but here, again, they undergo a change, being
converted into a hydrated variety of proteid of smaller molecular
weight called peptone. The intermediate substances formed in this
process are called proteoses or albumoses. Commercial peptone con-
tains a mixture of proteoses and true peptone.

Heat Coagulation—Many of the proteids which are soluble in
water or saline solutions are rendered insoluble when those solutions
are heated. Thisis true for most of the proteids that occur in nature.
The solidifying of white of egg when heated is a familiar instance of
this. The temperature of heat coagulation differs in different pro-
teids: thus myosinogen and fibrinogen coagulate at about 56° C.;
serum albumin and serum globulin at about 75° C.

The proteids which are coagulated by heating their solutions come
for the most part into two classes—the albumins and the globulins.
The full distinction between these we shall see immediately. We
may, however, state here that the albumins are soluble in distilled
water; the globulins are not, but require salts to hold them in
solution.

' Recent research has shown that urea can be also obtained from proteids by
analytical methods outside the body (see under Urine).

THE PROTEIDS 23

Indiffusibility The proteids (peptones excepted) belong to the
class of substances called colloids by Thomas Graham ; that is, they
pass with difficulty, or not at all, through animal membranes. In
the construction of dialysers, vegetable parchment is very largely
used (see figs. 7 and 8).
) Proteids may thus be separated from diffusible (crystalloid) sub-
stances like salts, but the process is a somewhat tedious one. If
some serum or white of egg is placed in a dialyser, and distilled
water outside, the greater amount of the salts passes into the water
through the membrane; the two proteids, albumin and globulin,

= Fic. 8.—In this form of dialyser
the substance to be dialysed
Fic. 7.—Dialyser. The lower opening of the bell jar is placed within the piece of

in water is tightly covered with parchment tubing suspended in the
paper. The fiuid to be dialysed is placed within this larger vessel of water. The
vessel ; the crystalloids pass out into the distilled water tubing is made of parchment
outside through the parchment paper. paper.

Temain inside. The globulin is, however, precipitated, as the salts
which previously kept it in solution have been removed.

The terms ‘ diffusion’ and‘ dialysis’ should be distinguished from each other.
If water is carefully poured on the surface of a solution of any substance,
this substance gradually spreads through the water, and the composition of
the mixture becomes uniform in time. The time occupied is short for
_ substances like sodium chloride, and long for substances like albumin. The
- phenomenon is called diffusion. If the solutions are separated by a mem-
_ brane the term ‘dialysis’ isemployed. The word osmosis is properly restricted
to the passage of water through membranes, and can be best studied
when semi-permeable membranes are employed. See fully article Osmosis
in Appendix.

Crystallisation.— Hemoglobin, the red pigment of the blood, is a
_ proteid substance, and is crystallisable (for further details, see THE
_ Buoop). Like other proteids it has an enormously large molecule ;

¥
24 ESSENTIALS OF CHEMICAL PHYSIOLOGY

though crystalline, it is not crystalloid in Graham’s sense of
that term. Blood pigment is not the only crystallisable proteid..

Long ago crystals of proteid (globulin or vitellin) were observed in ~

the aleurone grains of many seeds, and in the similar proteid occur-
ring in the egg-yolk of some fishes and amphibians. By appropriate
methods these -have been separated and re-crystallised. Further,
egg-albumin itself has been crystallised. Ifa solution of white of egg
is diluted with half its volume of saturated solution of ammonium
sulphate, the globulin present is precipitated and is removed by
filtration. The filtrate is now allowed to remain some days at the
temperature of the air, and as it becomes more concentrated from
evaporation, minute spheroidal globules and finally minute needles,
either aggregated or separate, make their appearance (Hofmeister).
-Crystallisation is much more rapid and perfect if a little acetic or
sulphuric acid is added (Hopkins). Serum albumin (from horse and
rabbit) has also been similarly crystallised (Giirber).

Action on Polarised Light.—All the proteids are leyo-rotatory,
but the amount of rotation they produce varies with the kind of
proteid. See Appendix.

Colour Reactions.—The principal colour reactions: (1) the
xanthoproteic; (2) Millon’s; (3) the violet colour with copper
sulphate and caustic potash, have been already given in the heading
to this lesson. The first two colour reactions depend on the presence
of an aromatic radicle in the proteid molecule. Peptones behave
differently from the native proteids in this last test. They give a
rose-red colout instead of a violet, if only a trace of copper sulphate
is used. The albumoses act in this respect like the peptones. This.
rose-red colour is also given by the substance called biuret ;! hence
the test is called the biwret reaction.

Precipitants of Proteids——Proteids are precipitated by a large
number of reagents; the peptones and albumoses are exceptions in
many cases, and will be considered separately afterwards (see
Lesson VI.).

Solutions of the proteids are precipitated by—

1. Strong acids, like nitric acid.

2. Pieric acid.

3. Acetic acid and potassium ferrocyanide.

4, Acetic acid and excess of neutral salts like sodium sulphate.

“ ' Biuret is formed by heating solid urea ; ammonia passes off and leaves biuret.
us :—

2CON,H,- NH, = 0,0,N,H,

(urea) {ammonia} {biuret].

THE PROTEIDS 25.

5. Salts of the heavy metals, like copper sulphate, mercuric
chloride, lead acetate, silver nitrate, &e.
+ 6. Tannin.

7. Alcohol.

8. Saturation with certain neutral salts, such as ammonium
sulphate.

It isnecessary that the words coagulation and precipitation should;
in connection with the proteids, be carefully distinguished. The term
coagulation is used when an insoluble proteid (coagulated proteid) is
formed from a solubleone. This may occur—

1. When the proteid is heated—heat coagulation.

; 2. Under the influence of a ferment; for instance, when a curd is
formed in milk by rennet or a clot in shed blood by the fibrin ferment
—ferment coagulation.
3. When an insoluble,precipitate is produced by the addition of
certain reagents (nitric acid, picric acid, tannin, &c.).
There are, however, other precipitants of proteids in which the
precipitate formed is readily soluble in suitable reagents, like saline
_ solution, and the proteid continues to show its typical reactions. This
precipitation is not coagulation. Such a precipitate is produced by
saturation with ammonium sulphate. Certain proteids, called
globulins, are more readily precipitated by such means than others.
Thus, serum globulin is precipitated by half-saturation with
ammonium sulphate. [Full saturation with ammonium sulphate
precipitates all proteids but peptone. The globulins are precipitated
_ by certain salts like sodium chloride and magnesium sulphate, which
_do not precipitate the albumins. The precipitation of proteids by
salts in this way is conveniently termed ‘ salting out.’

The precipitation produced by alcohol is peculiar in that after a
time it becomes a coagulation. Proteid freshly precipitated by
_ alcohol is readily soluble in water or saline media; but after it has
_ been allowed to stand some weeks under alcohol it becomes more
and more insoluble. Albumins and globulins are most readily
rendered insoluble by this method; albumoses and peptones are
apparently never rendered insoluble by the action of alcohol. This
fact is of value in the separation of these proteids from others.

CLASSIFICATION OF PROTEIDS

Proteids may be of animal or vegetable origin, and both animal
and vegetable proteids may be subdivided in the same way. We
Shall, however, be chiefly concerned with the animal proteids.

26 ESSENTIALS OF CHEMICAL PHYSIOLOGY

If we use the term ‘proteid’ in the widest sense, the first main sub-
division of these substances is into

A. The simple proteids.

B. The compound or conjugated proteids.

c. The albuminoids.

p. The protamines. These substances are regarded by Kossel as
the simplest proteids, and though it is doubtful whether this view
will be ultimately accepted, it will be convenient to consider them
here.

We may now take these four classes one by one.

A. The Simple Proteids

Class I. Albumins.—These are soluble in water, in dilute saline
solutions, and in saturated solutions of sodium chloride and mag-
nesium sulphate. They are, however, precipitated by saturating
their solutions with ammonium sulphate. Their solutions are
coagulated by heat, usually at 70-73° C. The following are
instances :—

(a) Serum albumin. (8) Egg albumin. (c¢) Lact-albumin (see
Mik).

Class II. Globulins.—These are insoluble in water, soluble in
dilute saline solutions, and insoluble in concentrated solutions of
neutral salts like sodium chloride, magnesium sulphate, and am-
monium sulphate. A globulin dissolved in a dilute saline solution
may therefore be precipitated—

1. By removing the salt—by dialysis (see p. 23).

2. By increasing the amount of salt. The best salts to employ
are ammonium sulphate (half saturation) or magnesium sulphate
(complete saturation).

The globulins are coagulated by heat; the temperature of heat
coagulation varies considerably. The following are instances :— _

(a) Fibrinogen

(6) Serum globulin (paraglobulin)

(d) Myosinogen in muscle.

(ec) Orystallin in the crystalline lens.

If we compare together these two important classes of proteids,
we find that they all give the same general tests, that all are coagu-
lated by heat, but that they differ in solubilities. This difference in
solubility may be stated in tabular form as follows :—

in blood plasma.

THE PROTEIDS 27

Reagent Albumin Globulin

Water. be alia soluble insoluble |
Dilute saline solution ‘ | soluble soluble
Saturated solution of magnesium sul-

phate or sodium chloride. soluble insoluble |
Half-saturated solution of ammo- -

nium sulphate . soluble insoluble
Saturated solution of ammonium

sulphate... te | insoluble -—iinsoluble_—si

Class III. Proteoses ) These products of digestion will be studied
Class IV. Peptones in Lessons VI. and VII.
Class V. Coagulated Proteids.—There are two principal subdivi-
sions of these :— 4
(a) Proteids in which coagulation has been produced by heat;
they are insoluble in water, saline solutions, weak acids, and weak
alkalis ; soluble after prolonged boiling in concentrated mineral acids ;
dissolved by gastric and pancreatic juices, they give rise to peptones.
(6) Proteids in which Sonerane has been produced by fer-
ments :—
i. Fibrin (see Buoop). ii. Myosin (see Muscue). iii. Casein (see
_ Mire).
q Directions for testing for and separating proteids will be found at
the end of this elementary course.

Albuminates

Albuminates are compounds of proteid (albumin or globulin) with
mineral substances. Thus, if a solution of copper sulphate is added
| toasolution of albumin, a precipitate of copper albuminate is obtained.

_ Similarly, by the addition of other salts of the heavy metals, other
metallic albuminates are obtainable.

The albuminates which are obtained by the action of dilute acids
_and alkalis on either albumins or globulins are of considerable
_ physiological interest because they are formed during digestion, and
it is to these we shall chiefly confine our attention. The general
_ properties of the acid-albumin, or syntonin, and the alkali-albwmin
_which are thereby respectively formed will be gathered from the
_ practical exercise which stands at the head of this lesson. They are
insoluble in pure water, but are soluble in either acid or alkali, and
_ are precipitated by neutralisation unless disturbing influences like the
_ presence of sodium phosphate are present. It may also be added
| that, like globulins, they are precipitated by saturation with such

28 ESSENTIALS OF CHEMICAL PHYSIOLOGY

neutral salts as sodium chloride and magnesium sulphate. If dis-
solved in acid or alkali, they are not coagulated by heat.

A variety of alkali albumin (probably a compound containing a
large quantity of alkali) may be formed by adding strong potash to
undiluted white of egg. The resulting jelly is called Lieberkithn’s
jelly. A similar jelly is formed by adding strong acetic acid to un-
diluted egg-white.

The halogens (chlorine, bromine, and iodine) also form albumi-
nates, and may be used for the precipitation of proteids.

B. The Compound Proteids

The compound proteids are compounds of albuminous substances
with other organic materials, which are as a rule also of complex
nature.

They may be divided into the following groups :—

1. Hemoglobin and its allies. These are compounds of proteid
with an iron-containing pigment, and will be fully considered under
Buoop.

2. Gluco-proteids. These are compounds of proteids with members
of the carbohydrate group. This class includes the mucins, and
substances allied to the mucins, called mucoids.

Dr. Pavy has shown that a small quantity of a similar carbo-
hydrate can be split off from various other proteids, which we have
already classified as simple proteids.

Mucin is a widely distributed substance, occurring in “epithelial
cells, or shed out by them (mucus, mucous glands, goblet cells) ; and
in connective tissue where it forms the chief constituent of the
ground substance or intercellular material.

The mucin obtained from different sources varies in composition
and reactions. There are probably several mucins; they all agree
in the following points :—

(a) Physical character. Viscid and tenacious.

(b) Precipitability from solutions by acetic acid; they all dissolve
in dilute alkalis like lime water.

(c) They are all compounds of a proteid with a carbohydrate
provisionally called animal gum, which by treatment with dilute
mineral acid can be hydrated into a reducing but non-fermentable
sugar, the nature of which is at present uncertain.

The mucoids differ from the mucins either in being non-precipitable
from alkaline solutions by acetic acid, or in being readily soluble in
excess of acetic acid. One of these, called ovo-mucoid, is found in

THE PROTEIDS 29

white of egg, and others called pseudo-mucin and para-mucin are
sometimes found in the fluid of ovarian cysts, and dropsical
effusions.

3. Nucleins and nucleo-proteids.—These are compounds of proteid
with a complex organic acid called nucleic acid, which contains
phosphorus.

Nucleo-proteids—Compounds of proteids with nuclein. They
are found in the nuclei and protoplasm of cells. Caseinogen of milk
and vitellin of egg-yolk are similar substances. In physical charac-
ters they often closely simulate mucin; in fact, the substance called
mucin in the bile is in some animals a nucleo-proteid. They may be
distinguished from mucin by the fact that they yield on gastric
digestion not only peptone but also an insoluble residue of nuclein
which is soluble in alkalis, is precipitable by acetic acid from such a
solution, and contains a high percentage (10-11) of phosphorus.

Some of the nucleo-proteids also contain iron, and it is probable
that the normal supply of iron to the body is contained in the nucleo-
proteids, or hematogens (Bunge), of plant and animal cells.

The relationship of nucleo-proteids to the coagulation of the blood
is described under Bioop.

Nucleo-proteids may be prepared from cellular structures like
testis, thymus, kidney, &c., by two methods :—

1. Wooldridge’s method.—The organ is minced and soaked in
water for twenty-four hours. Acetic acid added to the aqueous
extract precipitates the nucleo-proteid,
or, as Wooldridge called it, tissue fibri-
Dingo,

2. Sodiwm chloride method.— The
minced organ is ground up in a mortar
with solid sodium chloride ; the result-
ing viscous mass is poured into excess
of distilled water, and the nucleo-proteid
rises in strings to the top of the water. _

The solvent usually employed for a *" asm compose! of qpoaricplonn

; ; Sate d byaloplasm
_ nucleo-proteid, whichever method it is Intranets wotaaik aes

prepared by, is a 1-per-cent. solution of Stee
sodium carbonate.

Nuclein is the chief constituent of cell-nuclei. Its physical
_ characters are something like mucin, but it differs chemically in
containing a high percentage of phosphorus. Nuclein is identical
_ with the chromatin of histologists (see fig. 9).
_ Oni decomposition nuclein yields a complex organic acid called

30 ESSENTIALS OF CHEMICAL PHYSIOLOGY

nucleic acid, together with a variable amount of proteid. Nucleic
acid on decomposition yields phosphoric acid and various bases of
the xanthine group. Some forms of nuclein, called pseudo-nuclein,
such as are obtained from casein and vitellin, differ from the true
nucleins in not yielding these xanthine or, as they are sometimes
termed, alloxuric or purine bases.

Further particulars concerning the alloxuric bases will be given
under Uric Actin, to which they are closely related.

The following diagrammatic way of representing the decomposi-
tion of nucleo-proteid will assist the student in remembering the
relationships of the substances we have just been considering :—

Nucte0-PRoTEIp

subjected to gastric digestion yields
|

Proteid Uneer into Nuclein, which remains as an insoluble
peptone, which goes residue. If this is dissolved in alkali
into solution. and then hydrochloric acid added it

yields ‘
Cones ty) |

Proteid—converted into A precipitate consisting of nucleic acid.

acid albumin in solu- If this is heated in a sealed tube with

tion. hydrochloric acid, it yields a number
of imperfectly known substances like
thymic acid and in some cases a re-
ducing sugar. But the best known .
and constant products of its decom-
position are

Phosphoric acid. Purine bases, viz :
Adenine
Hypoxanthine
Guanine
Xanthine

The nuclein obtained from the nuclei or heads of the spermatozoa
consists of nucleic acid without any proteid admixture. In fishes’
spermatozoa, however, the nucleic acid is united to protamine, the
chemical properties of which we shall be considering immediately.

THE PROTEIDS 31

C. The Albuminoids

The albuminoids form a heterogeneous group of substances which,
though similar to the proteids in many particulars, differ from them
in certain other points. The principal members of the group are the
following :— ,

1. Collagen, the substance of which the white fibres of connective
tissue are composed. Some observers regard it as the anhydride of
gelatin.

2. Ossein.—This is the same substance derived from bone:!

3. Gelatin. —This substance is produced by boiling collagen with
water. It possesses the peculiar property of setting into a jelly when
a solution made with hot water cools. It gives most of the proteid
colour tests. Many observers state, however, that it contains no
sulphur. On digestion it is like proteid converted into peptone-like
substances, and is readily absorbed. Though it will replace in diet
a certain quantity of proteid, acting as what is called a ‘ proteid-
sparing’ food, it cannot altogether take the place of proteid as a food.
’ Animals fed on gelatin instead of proteid waste rapidly. Chondrin,
the very similar substance obtained from hyaline cartilage, appears
to be a mixture of gelatin with mucinoid materials.

4, Elastin —This is the substance of which the yellow or elastic
fibres of connective tissue are composed. It is a very insoluble
material. The sarcolemma of muscular fibres and certain basement
membranes are very similar.

5. Keratin, or horny material, is the substance found in the
surface layers of the epidermis, in hairs, nails, hoofs, and horns. It
is very insoluble, and chiefly differs from proteids in its high
percentage of sulphur. A similar substance, called newrokeratin, is
found in neuroglia and nerve fibres. In this connection it is inter-
esting to note that the epidermis and the nervous system are both
formed from the same layer of the embryo—the epiblast.

6. Chitin and similar substances found in the exo-skeleton of
many invertebrates.

1 In round numbers the solid matter in bone contains two-thirds inorganic or
earthy matter, and one-third organic or animal matter. The inorganic constituents
are calcium phosphate (84 per cent. of the ash), calcium carbonate (13 per cent.),
and smaller quantities of calcium chloride, calcium fluoride, and magnesium
phosphate. The organic constituents are ossein (this is the most abundant),
elastin from the membranes lining the Haversian canals, lacune, and canaliculi,
and proteids and nuclein from the bone corpuscles. There is also asmall quantity
_ of fat even after removal of all the marrow. Dentine is like bone chemically, but
the proportion of earthy matter is rather greater. Hmamiel is the hardest tissue in
_ the body ; the mineral matter is like that found in bone and dentine; but the
organic matter is so small in quantity as to be practically non-existent. (Tomes.)
Enamel is epiblastic, not mesoblastic like bone and dentine.

82 _ ESSENTIALS OF CHEMICAL PHYSIOLOGY

D. The Protamiues

These are basic substances which are combined with nuclein in
the heads of the spermatozoa of certain fishes. They resemble pro-
teids in many of their characters—e.g. they give Piotrowski’s reaction
and some of the other tests for proteids. They are regarded by
Kossel as the simplest proteids. On hydrolytic decomposition, they
first yield substances analogous to the peptones, which are called
protones, and then they split up into simpler materials which are
mainly bases containing six atoms of carbon, and called in con-
sequence the hexone bases. These bases have the following names

and formule :—

Histidine C,H,N;0,
Arginine C,H 1 4N,0,
Lysine C,H,4N.O0.

Protamines differ in their composition according to their source,
and yield these products in different proportions. Thus salmine (the
protamine from the salmon) has the empirical formula C3)H;,N,;0,,
and stwrine (from the sturgeon) C3, HN Oz.

A good deal of work still remains to be done at these substances,
but the five which have hitherto been most fully worked at are
salmine, sturine, scombrine (from the mackerel), cyclopterine (from
‘Cyclopterus lumpus), and clupeine (from the herring).

Of these salmine and clupeine appear to be identical; the
principal products of the decomposition of salmine, clupeine, and
‘scombrine are arginine, amido-valerianic acid, and a small quantity of
an unknown residue.

Sturine yields these same products with lysine, and histidine in
addition.

Cyclopterine yields the same products as sturine, with the addition
of an aromatic substance.

The more complex proteids and albuminoids yield hexone bases,
and so Kossel considers that all these substances contain a prota-
mine nucleus. The more complex proteids, however, yield many

other products of decomposition in addition to these bases, of which —

leucine (CgH,;NO.) and tyrosine (CyH,,NO3), an aromatic substance,
may be specially mentioned. Cyclopterine, which also yields an
aromatic substance, is thus an important chemical link between the

other protamines and the proteids proper. It is interesting to note —
how many of these decomposition products contain six carbon atoms, —

and it reminds one that in the sugars obtained from starch there are
also six atoms of carbon.

683

LESSON IV _
FOODS

A. Milk. 1. Examine a drop of milk with the microscope.

2. Note the specific gravity of fresh milk with the lactometer; compare

_ this with the specific gravity of milk from which the cream has been removed

(skimmed milk). The specific gravity of skimmed milk is higher owing to
the removal of the lightest constituent—the cream.

8. The reaction of fresh milk is neutral or slightly alkaline.

4. Warm some milk in a test-tube to the temperature of the body, and

add afew drops of rennet. After standing, a curd is formed from the con-
version of caseinogen, the chief proteid in milk, into casein. The casein
entangles the fat globules. The liquid residue is termed whey. No curdling
' is produced if the rennet solution is previously boiled, because heat kills
ferments. ;
5. Take some milk to which 0°2 per cent. of potassium oxalate has been
added ; warm to 40° C. and add rennet. No curdling takes place because
_ the oxalate has precipitated the calcium salts which are necessary in the
coagulation process.

Take a second specimen of oxalated milk and add a few drops of 2-per-
cent. solution of calcium chloride, and then rennet; curdling or coagulation

takes place if the mixture is kept warm in the usual way.
. 6. To another portion of warm milk diluted with water add a few drops
| of 20-per-cent. acetic acid. A lumpy precipitate of caseinogen entangling
the fat is formed.
4. Filter off this precipitate, and in the filtrate test for lactose or milk
sugar by Trommer’s test (see Lesson I.) ; for lact-albumin by boiling, or by
Millon’s reagent (see Lesson II.); and for earthy (that is, calcium and
-magnesium) phosphates by ammonia, which precipitates these phosphates.
Phosphates may also be detected by adding nitric acid and ammonium
molybdate and boiling; a yellow crystalline precipitate is formed.
8. Fat (butter) may be extracted from the precipitate by shaking it with
ether; on evaporation of the ethereal extract the fat is left behind, forming
a greasy stain on paper. The presence of fat may also be demonstrated by
the black colour produced by the addition of osmic acid to the milk.
9. Caseinogen, like globulins, is precipitated by saturating milk with
sodium chloride or magnesium sulphate, and by half saturation with
ammonium sulphate, but differs from the globulins in not being coagulated
by heat. The precipitate produced by saturation with salt floats to the
surface with the entangled fat, and the clegr salted whey is seen below after
an hour or two.
B. Flour.—Mix some wheat flour with a little water into a stiff dough.
Wrap this up in a piece of muslin and knead it under a tap or in a capsule of
water. The starch grains come through the holes in the muslin (identify by
iodine test), and an elastic sticky mass remains behind. This is a proteid
called gluten. Suspend a fragment of gluten in water; add nitric acid and
boil ; it turns yellow ; cool and add ammonia; it turns orange (xanthoproteic
reaction). Boil another fragment with Millon’s reagent ; it turns a brick-red

34 ESSENTIALS OF CHEMICAL PHYSIOLOGY

C. Bread contains the same constituents as flour, except that some of the
starch has been converted into dextrin and dextrose during baking (most
flours, however, contain a small quantity of sugar). Extract breadcrust.
with cold water, and test the extract for dextrin (iodine test) and for dextrose
(Trommer’s test). If hot water is used, starch also passes into solution.

D. Meat.—This is our main source of proteid food. Cut up some lean
meat into fine shreds and grind these up with salt solution. Filter and test
for proteids.

THE PRINCIPAL FOOD-STUFFS
We can now proceed to apply the knowledge we have obtained of

the proteids, carbohydrates, and fats to the investigation of some im-
portant foods. We do not actually use as food the various organic

proximate principles in the pure condition ; it is necessary that in a

suitable diet these should be mixed in certain proportions, and in
nature we find them already mixed for us. The chief proximate
principles in food are :—
1. Proteids }
2. Carbohydrates ‘organic.

3. Fats |
4, Water F
5. Salts j Inorganic.

In milk and in eggs, which form the exclusive foods of young
animals, all varieties of these proximate principles are present in suit-
able proportions. Hence they are spoken of as perfect foods. Eggs,
though a perfect food for the developing bird, contain too little carbo-

hydrate fora mammal. In most vegetable foods carbohydrates are —
in excess, while in animal food, like meat, the proteids are predomi- ©

nant. Ina suitable diet these should be mixed in proper proportions,
which must vary for herbivorous and carnivorous animals. We
must, however, limit ourselves to the omnivorous animal, man.
A healthy and suitable diet must possess the following characters :—
1. It must contain the proper amount and proportion of the
various proximate principles.

2. It must be adapted to the climate, to the age of the individual, |

and to the amount of work done by him.

3. The food must contain not only the necessary amount of proxi-
mate principles, but these must be present in a digestible form. As
an instance of this many vegetables (peas, beans, lentils) contain even
more proteid than beef and mutton, but are not so nutritious, as they
are less digestible, much passing off in the feces unused.

The nutritive value of a diet depends chiefly on the amount of
carbon and nitrogen it contains in a readily digestible form. A man
doing a moderate amount of work will eliminate, chiefly from the

FOODS 35

_ lungs in the form of carbonic acid, from 250 to 280 grammes of
carbon per diem. During the same time he will eliminate, chiefly
in the form of urea in the urine, about 15 to 18 grammes of nitrogen.
These substances are derived from the metabolism of the tissues,
and various forms of energy, work and heat being the chief, are
simultaneously liberated. During muscular exercise the output of
carbon greatly increases ; the increased excretion of nitrogen is not
nearly so marked. Taking, then, the state of moderate exercise, it is
necessary that the waste of the tissues should be replaced by fresh
material in the form of food; and the proportion of carbon to
nitrogen should be the same as in the excretions: 250 to 15, or 16°6
tol. The proportion of carbon to nitrogen in proteid is, however,
_ 53 to 15, or 35 to 1. The extra supply of carbon must come from
- non-nitrogenous foods—viz. fat and carbohydrate.
Moleschott gives the following daily diet :—

Proteid 120 grammes.
Fat 90 ‘“
Carbohydrate 333 -
Ranke’s diet closely resembles Moleschott’s ; it is—
' Proteid 100 grammes.
Fat 100 ;.

Carbohydrate 250 sa

' In preparing diet tables, such adequate diets as those just given

‘should be borne in mind. The following dietary (from G. N.
Stewart) will be seen to be rather more liberal, but may be taken as
fairly typical of what is usually consumed by an adult man in the
| twenty-four hours, doing an ordinary amount of work.

Grammes of
Food-stuff Quantity a
. Ni Car-| Pro-
prsons bon | teids | Fats hydrates Salts
Metric English
system weights
| Leanmeat | 250 grammes 9oz. | 8 33, 55 | 85) OO. 4
Bread .| 500 ,, is, |6 |112| 40] 75] 245 | 65
| Milk 608) 2 5, 3 pint | 3 35; 20/20 | 25 | 35
Butter 30 * 1 oz 0 20 0 | 27 0 | 05
| Fat with
meat . 30 pe i ie 0 22 0 |30 0 10
Potatoes .| 450 ,, 16), | 15 47; 10/0 | 9 | 45
Oatmeal .| 75 ,, » | 27 | 80] 10/4] 48 |2
20-2 |299| 135 |97 | 413 21

36 ESSENTIALS OF CHEMICAL PHYSIOLOGY

MILK

Milk is often spoken of as a ‘ perfect food,’ and it is so for infants.
For those who are older it is so voluminous that unpleasantly large
quantities of it would have to be taken in the course of the day to
insure the proper supply of nitrogen and carbon. Moreover for adults
it is relatively too rich in proteid and fat. It also contains too little
iron (Bunge); hence children weaned late become anzemice.

The microscope reveals that it consists of two parts: a clear fluid
and a number of minute particles that float in it. These consist of
minute oil globules, varying in diameter from 0°0015 to 0:005 milli-
metre.

The milk secreted during the first few days of lactation is called
colostrum. It contains very little caseinogen, but large quantities of
globulin instead. Microscopically, cells from the acini of the

oo?

2,
co]

2
goes

Fic. 11.—a, 6, colostrum
corpuscles with fine
and coarse fat globules
respectively ; ¢, a, ¢,
pale cells devoid of
fat. (Heidenhain.)

Fic. 10.—Microscopie appearance of milk in the
early stage of lactation, showing colostrum
corpuscles in addition to fat globules. ( Yeo.)
mammary gland are seen, which contain fat globules in their interior ;
they are called colostrum corpuscles,

Reaction and Specific Gravity.—The reaction of fresh cow’s milk
and of human milk is generally neutral or slightly alkaline. In
carnivora the milk is acid. All milk readily turns acid or sour as the
result of fermentative change, part of its lactose being transformed
into lactic acid (see p. 37). The specific gravity of milk is usually

ascertained with the hydrometer. That of normal cow’s milk varies

from 1028 to 1034. When the milk is skimmed the specifie gravity

rises, owing to the removal of the light constituent, the fat, to 1033 to”
'1037. In all cases the specific gravity of water with which other
substances are compared is taken as 1000.

FOODS 37

_ Composition.—Frankland gives the following table, contrasting the
milk of woman, ass, and cow :—

—* Woman ' Ass Cow
; Per cent. | Percent. | Per cent.
| Proteids (chiefly caseinogen) . 27 17 42
| Butter (fat) - z . 4 35 | 13 38 |
CS eM ST 50 || 45 38
ee oe 05 0-7

Hence, in feeding infants on cow’s milk, it will be necessary to
dilute it, and add sugar to make it approximately equal to natural
human milk.

The Proteids of Milk.—The principal proteid in milk is ; called
caseimogen ; this is the one which is coagulated by rennet to form
casein. Cheese consists of casein with the entangled fat. The other
) proteid in milk is an albumin. Itis present in small quantities only ;
| it differs in some of its properties (specific rotation, coagulation

temperature, and solubilities) from serum-albumin ; it is called ies:
albumin.
The Coagulation of Milk.—Rennet is the agent usually aaibed
for this purpose: it is a ferment secreted by the stomach, especially
y sucking animals, and is generally obiained from the cali.
The curd consists of the casein and entangled fat: the liquid
Tesidue called whey contains the sugar, salts, and albumin of the
milk. There is also a small quantity of a new proteid called whey-
proteid, which differs from caseinogen by not being convertible into
casein. It is produced by the decomposition of the caseinogen
molecule during the process of curdling.
The curd formed in human milk is more finely divided than that
in cow's milk ; hence it is more digestible. In feeding children and
invalids on cow’s milk, the lumpy condition of the curd may be
Riisvintoa by the addition of lime water or barley water to the milk.
Considerable discussion has taken place as to whether the
caseinogen of human milk may not be a different proteid from that
Mf cow's milk, especially in relation to the amount and manner of
combination of its phosphorus. The differences, however, appear to
3 explicable on the hypothesis that they are due to variations in
= amounts of calcium salts and of citric acid which are present.
_ Caseinogen itself may be precipitated by acids such as acetic-acid

or by saturation with neutral salts like sodium chloride. This, how-
er, is not coagulation, but precipitation. The precipitate may be
ed and dissolved in lime water; the addition of rennet then

38 ESSENTIALS OF CHEMICAL PHYSIOLOGY

produces coagulation in this solution, provided that a sufficient
amount of calcium salts is present.

The addition of rennet produces coagulation in milk, provided that
a sufficient amount of calcium salts is present. If the calcium salts
are precipitated by the addition of potassium oxalate, rennet causes
no formation of casein. The process of curdling in milk is a double
one ; the first action due to rennet is to produce a change in caseino-
gen; the second action is that of the calcium salt, which precipitates —
the altered caseinogen as casein. In blood also, calcium salts are
necessary for coagulation, but there they act in a different way,
- namely, in the production of fibrin-ferment (see CoAGULATION OF
Buoop).

Caseinogen is often compared to alkali-albumin. The atter,
however, does not clot with rennet, and is, unlike caseinogen, readily
soluble in excess of acids. Caseinogen is not a globulin, though it
is, like globulins, precipitated by neutral salts. It differs from a
globulin in not being coagulated by heat. It is a nucleo-proteid :
that is, a compound of a proteid with the proteid-like but phosphorus-
rich material called nuclein (see p. 29).

The Fats of Milk——The chemical composition of the fat of milk
(butter) is very like that of adipose tissue. It consists chiefly of
palmitin, stearin, and olein. There are, however, smaller quantities
of fats derived from fatty acids lower in the series, especially butyrin
and caproin. The relation between these varies somewhat, but the
proportion is roughly as follows :—Olein, #; palmitin, 3; stearin, ¢;
butyrin, caproin, and caprylin, 7. The old statement that each fat
globule is surrounded by a membrane of caseinogen has been shown
to be incorrect. Milk also contains small quantities of lecithin, a —
phosphorised fat; of cholesterin, an alcohol which resembles fat in —
its solubilities (see Brine), and a yellow fatty pigment or lipochrome.

Milk Sugar or Lactose—This is a saccharose (C,;2H..0),). Its ©
properties have already been described in Lesson I. p. 13.

Souring of Milk.—When milk is allowed to stand, the chief change
which it is apt to undergo is a conversion of a part of its lactose into
lactic acid. This is due to the action of micro-organisms, and would
not occur if the milk were contained in closed sterilised vessels.
Equations showing the change produced are given on p. 14. When
souring occurs, the acid which is formed precipitates a portion of the
caseinogen. This must not be confounded with the formation of
casein from caseinogen which is produced by rennet. There are,
however, some bacterial growths which produce true coagulation like ©
rennet.

: oo rye aor

FOODS 39

_ Aleoholic Fermentation in Milk.—When yeast is added to milk,
‘the sugar does not readily undergo the alcoholic fermentation.
Other somewhat similar fungoid growths are, however, able to
produce the change, as in the preparation of koumiss; the milk
sugar is first inverted, that is dextrose and galactose are formed from
it (see p. 13), and it is from these sugars that alcohol and carbonic
acid originate.

_ _ The Salts of Milk.—The chief salt present is calcium phosphate ;
-a small quantity of magnesium phosphate is also present. The
other salts are chiefly chlorides of sodium and potassium.

zt EGGS

. Am this country the eggs of hens and ducks are those particularly
_ selected as foods. The shell is made of calcareous matter, especially
_ealcium carbonate. The white is composed of a richly albuminous
- fluid enclosed in a network of firmer and more fibrous material. The
amount of solids is 13°3 per cent.; of this 12-2 is proteid in nature.
The proteids are albumin, with smaller quantities of egg-globulin
(see p. 26) and ovo-mucoid (p. 28). The remainder is made up of
_ sugar (0°5 per cent.), traces of fats, lecithin and cholesterin, and 0°6
per cent. of inorganic salts. The yolk is rich in food materials for
_ the development of the future embryo. In it there are two varieties
of yolk-spherules, one kind yellow and opaque (due to admixture
' with fat and a yellow lipochrome), and the other smaller, transparent
and almost colourless: these are proteid in nature, consisting of the
nucleo-proteid called vitellin {pp. 29, 30). Small quantities of sugar,
lecithin, cholesterin, and inorganic salts are also present.

The nutritive value of eggs is high, as they are so readily diges-
tible ; but the more an egg is cooked the more insoluble do its proteid
constituents become.

MEAT

This is composed of the muscular and connective (including
adipose) tissues of certain animals. The flesh of some animals is
“not eaten ; in some eases this is a matter of fashion ; some flesh, like
that of the carnivora, is stated to have an unpleasant taste; and in
other cases (e.g. the horse) it is more lucrative to use the animal as a
beast of burden. 7

_ Meat is the most concentrated and most easily assimilable of
nitrogenous foods. It is our chief source of nitrogen. Its chief solid
constituent is proteid, and the principle proteid is myosin. In addi-
tion to the extractives and salts contained in muscle, there is always

40 ESSENTIALS OF CHEMICAL PHYSIOLOGY

a certain percentage of fat, even though all visible adipose tissue is
dissected off. The fat-cells are placed between the muscular fibres,
and the amount of fat so situated varies in different animals. It is
particularly abundant in pork; hence the indigestibility of this form
of flesh: the fat prevents the gastric juice from obtaining ready
access to the muscular fibres.

The following table gives the chief substances in some of the.
principal meats used as food.

Constituents Ox Calf Pig | Horse | Fowl Pike
Water : oo) MOG 75°6 72°6 74:3 708 79°3
Solids : ; 23°3 24°4 27°4 25°7 29°2 20°7
Proteids and

gelatin... 20°0 1G*4 19°9 21°6 22°7 18°3
Fat . A ; 15 2°9 6:2 2°5 4°1 0°7
Carbohydrate . 0-6 0-8 66 0°6 1:3 09
Salts . r ; 12 1:3 i i 1:0 11 0-8

The large percentage of water in meat should be particularly
noted ; if a man wished to take his daily minimum of 100 grammes.
of proteid entirely in the form of meat, it would be necessary for him
to consume about 500 grammes (i.e. a little more than 1 lb.) of meat
per diem.

FLOUR

The best wheat flour is made from the interior of wheat grains,
and contains the greater proportion of the starch of the grain and
most of the proteid. Whole flour is made from the whole grain
mimus the husk, and thus contains not only the white interior but —
also the harder and browner outer portion of the grain. This outer —
region contains a somewhat larger proportion of the proteids of the —
grain. Whole flour contains 1 to 2 per cent. more proteid than the
best white flour, but it has the disadvantage of being less readily }
digested. Brown flour contains a certain amount of bran in
addition ; it is still less digestible, but is useful as a mild laxative, —
the insoluble cellulose mechanically irritating the intestinal canal as —
it passes along.

The best flour contains very little sugar. The presence of sugar
indicates that germination has commenced in the grains. In the
manufacture of malt from barley this is purposely allowed to go on.

When mixed with water, wheat flour forms a sticky adhesive
mass called dough. This is due to the formation of gluten, and the
forms of grain poor in gluten cannot be made into dough (oats, rice,

FOODS 41

&e.). Gluten does not exist in the flour as such, but is formed on
the addition of water from the pre-existing soluble proteids (e.g.
_ globulins) in the flour.

. The following table contrasts the composition of some of the more
important vegetable foods :—

Constituents | Wheat | Barley | Oats | Rice | Lentils | Peas | Potatoes |
! |
Water . .- ./| 186 | 188 | 124 | 131 | 125 | 148 | 760
Proeid . . - 124 | 111 | 104 | 79 | 248 Es |- 20
Wat. Cw tC 4 |) 2] | OD | 19) 16 | 02
Starch . . . 679 | 649 578 765 | 548 (493 206
Cellulose. . . 25 | 53/112 | 06 | 36 | 75 | OF
Mineral salts - .| 18 | 27) 80 | 10 {| 24 | 81 | 10

We see from this table—
1. The great quantity of starch always present.
2. The small quantity of fat; that bread is generally eaten with
_ butter is a popular recognition of this fact.
; 3. Proteid, except in potatoes, is pretty abundant, and especially
so in the pulses (lentils, peas, &c.). The proteid in the pulses is not
gluten, but consists of vitellin and globulin-like substances.
In the mineral matters in vegetables, salts of potassium and
_ magnesium are, as a rule, more abundant than those of sodium and
calcium.

BREAD

Bread is made by cooking the dough of wheat flour mixed with
| yeast, salt, and flavouring materials. A ferment in the flour acts at
_ the commencement of the process when the temperature is kept a
little over that of the body, and forms dextrin and sugar from the
starch, and then the alcoholic fermentation, due to the action of the
yeast, begins. The bubbles of carbonic acid, burrowing passages
_ through the bread, make it light and spongy. This enables the
digestive juices subsequently to soak into it readily and affect all
parts of it. During baking the gas and alcohol are expelled from the
bread, the yeast is killed, and a crust forms from the drying of
the outer portions of the dough.

White bread contains, in 100 parts, 7 to 10 of proteid, 55 of carbo-
hydrates, 1 of fat, 2 of salts, and the rest water.

COOKING OF FOOD

_ The cooking of foods is a development of civilisation, and much
Telating to this subject is a matter of education and taste rather than

42 ESSENTIALS OF CHEMICAL PHYSIOLOGY

of physiological necessity. Cooking, however, serves many useful
ends :—

1. It destroys all parasites and danger of infection. This relates
not only to bacterial growths, but also to larger parasites, such as
tapeworms and trichine.

2. In the case of vegetable foods it breaks up the starch eae
bursting the cellulose and allowing the digestive juices to come into
contact with the granulose.

3. In the case of animal foods it converts the insoluble collagen
of the universally distributed connective tissues into the soluble
gelatin. The loosening of the fibres is assisted by the formation of
steam between them. By thus loosening the binding material, the
more important elements of the food, such as muscular fibres, are
rendered accessible to the gastric and other juices. Meat before it is
cooked is generally kept a certain length of time to allow rigor mortis
‘to pass off.

Of the two chief methods of cooking, roasting and boiling, the
former is the more economical, as by its means the meat is first
surrounded with a coat of coagulated proteid on its exterior, which
keeps in the juices to a great extent, letting little else escape than the
dripping (fat). Whereas in boiling, unless both bouillon and bouilli
are used, there is considerable waste. Cooking, especially boiling,
renders the proteids more insoluble than they are in the raw state, but
this is counterbalanced by the other advantages that cooking possesses.

Beef Tea.—In making beef tea and similar extracts of meat it is
necessary that the meat should be placed in cold water, and this is
gradually and carefully warmed. In cooking a joint itis usual to put
the meat into boiling water at once, so that the outer part is coagulated,
and the loss of material minimised.

An extremely important point in this connection is that beef tea
and similar meat extracts should not be regarded as foods. They
are valuable as pleasant stimulating drinks for invalids, but they
contain very little of the nutritive material of the meat, their chief
constituents, next to water, being the salts and extractives (creatine,
creatinine, lactic acid, &c.) of flesh.

Many invalids restricted to a liquid diet get tired of milk, and
imagine that they get sufficient nutriment by taking beef tea instead.
It is very important that this erroneous idea should be corrected.
One of the greatest difficulties that a physician has to deal with in
these cases is the distaste which many adults evince for milk. It is
essential that this should be obviated as far as possible by preparing
the milk in different ways to avoid monotony. Some can take
koumiss ; but a less expensive variation may be introduced in the

FOODS 43

shape of junkets, which, although well known in the West of
England, are comparatively unknown in other parts. The prepara-
tion of a junket consists in adding to warm milk in a bowl or dish a
small quantity of essence of rennet (Clark’s essence is very good for
this purpose) and flavouring material according to taste. The mix-
ture is then put aside, and in a short time the milk sets into a- jelly
(coagulation of casein), which may then be served with or without
cream.

Soup contains the extractives of meat, a small proportion of the
proteids, and the principal part of the gelatin. The gelatin is usually
increased by adding bones and fibrous tissue to the stock. It is the
presence of this substance which causes the soup when cold to gela-

ACCESSORIES TO FOOD

Among these must’ be placed alcohol, the value of which within
moderate limits is not as a food but as a stimulant; condiments
(mustard, pepper, ginger, curry powder, &c.), which are stomachic
stimulants the abuse of which is followed by dyspeptic troubles ;
and tea, coffee, cocoa, and similar drinks. These are stimulants
_ chiefly to the nervous system ; tea, coffee, maté (Paraguay), guarana
(Brazil), cola nut (Central Africa), bush tea (South Africa), and a
few other plants used in various countries all owe their chief pro-
perty to an alkaloid called theine or caffeine (CsH,)N,O2); cocoa
to the closely related alkaloid, theobromine (C;H,N,O2); coca to
cocaine. These alkaloids are all poisonous, and used in excess, even
in the form of infusions of tea and coffee, produce over-excitement,
loss of digestive power, and other disorders well known to physicians.
Coffee differs from tea in being rich in aromatic matters; tea contains
a bitter principle, tannin. To avoid the injurious solution of too
much tannin, tea should only be allowed to infuse (draw) for a few
‘minutes. Cocoa is not only a stimulant but a food as well; it con-
tains about 50 per cent. of fat, and 12 per cent. of proteid. But
cocoa as usually taken in the form of an infusion does not contain
‘much of these food substances.

Green vegetables are taken as a palatable adjunct to other foods,
rather than for their nutritive properties. Their potassium salts are,
however, abundant. Cabbage, turnips, and asparagus contain 80 to
92 water, 1 to 2 proteid, 2 to 4 carbohydrates, and 1 to 1°5 cellulose
percent. The small amount of nutriment in most green foods ac-
counts for the large meals made by, and the vast capacity of the
alimentary canal of herbivorous animals.

44 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON V
SALIVA

1. To a little saliva in a test-tube add acetic acid. Mucin is precipitated
in stringy flakes.

2. Filter some fresh saliva to separate cells and mucus, and apply the
xanthoproteic or Millon’s test to the filtrate; the presence of proteid is
shown.

3. Put some 0°5-per-cent. starch solution into two test-tubes. Add some
filtered saliva to one of them, and put both in the water-bath at 40° C.
After five minutes remove them and test both fluids with iodine and
Trommer’s test. The saliva will be found to have converted the starch into
dextrin and sugar (maltose).

4, The presence of potassium sulphocyanide (KCNS) in saliva may be
shown by the red colour given by a drop of ferric chloride. This colour is
discharged by. mercuric chloride.

5. The reaction of saliva is alkaline to litmus paper.

The saliva is the first digestive juice to come in contact with the
food: it is secreted by three pairs of salivary glands, the parotid,
submaxillary, and sublingual. The secretions from these differ
somewhat in composition, but they are mixed in the mouth, the
secretion of the minute mucous glands of the mouth and a certain
number of epithelial cells and débris being added to it. The so-
called ‘ salivary corpuscles’ are derived either from the glands them-
selves or from the tonsils.

The secretion of saliva is a reflex action; the taste or smell of
food excites the nerve endings of the afferent nerves (glossopharyngeal
and olfactory) ; the efferent or secretory nerves are contained in the
chorda tympani (a branch of the seventh cranial nerve) which
supplies the submaxillary and sublingual, and in a branch of the
glossopharyngeal which supplies the parotid. The sympathetic
branches which supply the blood vessels with constrictor nerves
contain in some animals secretory fibres also.

The parotid gland is called a serous or albuminous gland; before
secretion the cells of the acini are swollen out with granules; after
secretion has occurred the cells shrink, owing to the granules haying
been shed out to contribute to the secretion (see fig. 12).

SALIVA 45

The submaxillary and sublingual glands are called mucous
glands: their secretion contains mucin. Mucin is absent from
parotid saliva. The granules in the cells are larger than those of
the parotid gland ; they are composed of mucinogen, the precursor
_ of mucin (see fig. 13).

In a section of a mucous gland prepared in the ordinary way the
_mucinogen granules are swollen out, and give a highly refracting
appearance to the mucous acini (see fig. 14).

COMPOSITION OF SALIVA

On microscopic examination of mixed saliva a few epithelial
scales from the mouth and salivary corpuscles from the salivary
glands are seen. The liquid is transparent, slightly opalescent, of
slimy consistency, and may contain lumps of nearly pure mucin.
On standing it becomes cloudy owing to the precipitation of calcium
carbonate, the carbonic acid which held it in solution as bicarbonate
escaping.
Of the three forms of saliva which contribute to the mixture
found in the mouth, the sublingual is richest in solids (2°75 per cent.).
_ The submaxillary saliva comes next (2:1 to 2°5 per cent.). When
artificially obtained by stimulation of nerves in the dog the saliva
_ obtained by stimulation of the sympathetic is richer in solids than
that obtained by stimulation of the chorda tympani. The parotid
saliva is poorest in total solids (0°3 to 0°5 per cent.), and contains no
mucin. Mixed saliva contains in man an average of about 0-5 per
cent. of solids: it is alkaline in reaction, due to the salts in it; and
has a specific gravity of 1002 to 1006.

The solid constituents dissolved in saliva may be elassified
thus :—-
_ ¢ a. Mucin: this may be precipitated by acetic acid.

b. Ptyalin: an amylolytic ferment.

ec. Proteid: of the nature of a globulin.
_ d. Potassium sulphocyanide.

e. Sodium chloride: the most abundant salt.

f. Other salts: sodium carbonate, calcium phosphate

and carbonate; magnesium phosphate; potassium
chloride.

THE ACTION OF SALIVA
The action of saliva is twofold, physical and chemical.
_ The physical use of saliva consists in moistening the mucous
‘membrane of the mouth, assisting the solution of soluble substances

46 ESSENTIALS OF CHEMICAL PHYSIOLOGY |

4
in the food, and in virtue of its mucin lubricating the bolus of food —
to facilitate swallowing.

Fig. 12,—Alveoli of serous gland: A, loaded before secretion; B, after a short period of active
secretion; O, after a prolonged period. (Langley.)

Fia. 13.—Mucous cells from a fresh submaxillary gland of dog: a, loaded with mucinogen granules
before secretion ; b, after secretion: the granules are fewer, especially at the attached border of
the cell; a’ and 6’ represent cells in a loaded and discharged condition respectively which have
been irrigated with water or dilute acid. The mucous granules are swollen into a transparent

mags of mucin traversed by a network of protoplasmic cell-substance. (Foster, after Langley.)

Fig. 14.—Section of part of the human submaxillary gland. (Heidenhain.) To the right is a group
of mucous alveoli, to the left a group of serous alveoli. ‘

The chemical action of saliva is due to its active ‘principle,
ptyalin. This substance belongs to the class of wnorganised ferments

SALIVA 47
or enzymes, and to that special class of unorganised ferments which
are called amylolytic (starch splitting) or diastatic (resembling
| diastase, the similar ferment in germinating barley and other grains).
__ A general description of ferments will be found at the end of this
lesson. _

The starch is first split into dextrin and maltose; the dextrin is
_ subsequently converted into maltose also: this occurs more quickly
with erythro-dextrin, which gives a red colour with iodine, than with
the other variety of dextrin called achroé-dextrin, which gives no
_ colour with iodine. Brown and Morris give the following equa-
tion :—

10(C,H,,0;), + 4nH,0

{starch] [water]
= 4nC,9H..0;, + (C>H1905), + (CeHi095),
[maltose] [achrow-dextrin] _ [erythro-dextrin]

Ptyalin acts in a similar way, but more slowly on glycogen ; it
has no action on cellulose; hence it is inoperative on uncooked
_ starch grains, for in these the cellulose layers are intact.

Ptyalin acts best at about the temperature of the body (35-40°),
and in a neutral medium; a small amount of alkali makes but
little difference; a very small amount of acid stops its activity.
The conversion of starch into sugar by saliva in the stomach con-
tinues for 15 to 30 minutes; the hydrochloric acid which is poured
out by the gastric glands first neutralises the saliva and combines
_ with the proteids in the food; but immediately free hydrochloric
acid appears the ptyalin is destroyed, so that it does not resume work
_ even when the semi-digested food once more becomes alkaline in the
duodenum.

FERMENTS

The word fermentation was first applied to the change of sugar
into alcohol and carbonic acid by means of yeast. ’
The evolution of carbonic acid causes frothing
and bubbling; hence the term ‘fermentation.’
The agent yeast which produces this is called
the ferment. Microscopic investigation shows
that yeast is composed of minute rapidly growing
- unicellular organisms (torule) belonging to the
: fungus group of plants. Fi. 15.—Cells of the yeast

. ‘ 2 eet process of bud-

The souring of milk, the transformation of g, between which
x : J . pr some bacteria.

- urea into ammonium carbonate in decomposing (Yeo’s ‘ Physiology.’),.
urine, and the formation of vinegar (acetic acid) from alcohol are pro-

duced by the growth of very similar organisms. The complex series

48 ESSENTIALS OF CHEMICAL PHYSIOLOGY

of changes known as putrefaction which are accompanied by the
formation of malodorous gases and which are produced by the growth
of various forms of bacteria, also come into the same category. .
That the change or fermentation is produced by these organisms
is shown by the fact that it occurs only when the organisms are
present, and stops when they are removed or killed by a high tem-

a
e

= |

Fic. 16.—Typical forms of Schizomycetes (after Zopf): a, micrococcus : b, Macrococcus ; ¢, bac-
terium 3 d, bacillus ; e, chlostridium ;/, Monas Okenii; g, leptothrix ; 4, i, vibrio; &, spirillum;
}, spirulina ; m, spiromonas ; 7, spirochaete ; 0, cladothrix,

perature or by certain substances (carbolic acid, mereuric chloride,

&e.) called antiseptics. The organisms probably produce fermenta-

tive effects by shedding out soluble ferments or enzymes.

The ‘ germ theory ’ of disease explains the infectious diseases by
considering that the change in the system is of the nature of fermen-
tation, and, like the others we have mentioned, produced by microbes ;_
the transference of the bacteria or their spores from one person to
another constitutes infection. The poisons produced by the growing
bacteria appear to be either alkaloidal (ptomaines) or proteid in

SALIVA 49

The existence of poisonous proteids is a very remarkable

and those which are not poisonous, but which are useful as foods.
Snake venom is an instance of a very virulent poison of proteid
_ There is another class of chemical transformations which at first
sight differ very considerably from all of these. They, however,
resemble these fermentations in the fact that they occur indepen-
dently of any apparent change in the agents that produce them.
The agents that produce them are not living organisms, but chemical
substances, the result of the activity of living cells. The change of
starch into sugar by the ptyalin of the saliva is an instance.

| Bic. 17.Bacilus anthracis, the agent that produces anthrax or splenic fever (Koch): A, bacilli,

mingled with blood corpuscles from the blood of guinea-pig, some of the bacilli dividing : B, the
same after three hours’ cnlture in a drop of aqueous humour. They grow out into long leptothrix-
like filaments, which subsequently divide up, and spores are developed in the segments.

Ferments may therefore be divided into two classes :—

1. The organised ferments—torule, bacteria, &e.

-_ 2. The unorganised ferments or enzymes—like ptyalin.

Each may be again subdivided according to the. nature of the
chemical change produced.

In digestion, the study of which we are just commencing, it is the
norganised ferments with the action of which we have chiefly to
deal. The unorganised ferments may be classified as follows :—

(a) Amylolytic—those which change amyloses (starch, glycogen)
nto sugars. Examples: ptyalin, diastase, amylopsin.

ete) + 4

50 ESSENTIALS OF CHEMICAL PHYSIOLOGY

(b) Proteolytic—those which change proteids into proteoses and
peptones. Examples: pepsin, trypsin.

(c) Steatolytic or lipolytic—those which split fats into fatty acids
and glycerin. An example, steapsin, is found in pancreatic juice.

(d) Inversive—those which convert saccharoses (cane sugar,
maltose, lactose) into glucose. Examples: invertin of intestinal
juice and of yeast cells,

(e) Coagulative—those which convert soluble into insoluble
proteids. Examples: rennet, fibrin ferment, myosin ferment.

Most ferment actions are hydrolytic—7.e. water is added to the
material acted on, which then splits into new materials. Thisis seen
by the following examples :— |

1. Conversion of cellulose into carbonic acid and marsh gas
(methane) by putrefactive organisms.

(C,H,,0;)n+nH,0=3nCO,+3nCH,

{cellulose} [water] {carbonic [methane]
acid]

2. Inversion of cane sugar by the unorganised ferment invertin :—
C,H 2.0), +H,O=C,H),05+ CH 120, -
{cane sugar] {water] [dextrose] [levulose]

It appears also that some enzymes are oxygen carriers and
produce oxidation. They are termed oxidases. Very little is known
of these at present.

A remarkable fact concerning the ferments is that the substances
they produce in time put a stop to their activity ; thus in the case
of the organised ferments the alcohol produced by yeast, the phenol,
eresol, &c., produced by putrefactive organisms from proteids, first
stop the growth of, and ultimately kill, the organisms which produce
them. In the case of the unorganised ferments, the products of their
activity hinder and finally stop their action, but on the removal of
these products the ferments resume work.

This fact suggested to Croft Hill the question whether ferments
will act in the reverse manner to their usual action; and in the
case of one ferment, at any rate, he found this to be the case.
Inverting ferments, as we have just seen, usually convert a
disaccharide into monosaccharides. One of these inverting
ferments, called maltase, coverts maltose into dextrose. If,
however, the ferment is allowed to act on strong solutions of
dextrose, it converts a small proportion of that sugar back into
maltose again. ‘Reversible action’ has since this been shown to
occur in the case of other enzymes.

Ferments act best at a temperature of about 40° C. Their

SALIVA 51

activity is stopped, but the ferments are not destroyed, by cold; it is
stopped and the ferments killed by too great heat. A certain amount
of moisture and oxygen is also necessary ; there are, however, certain
‘micro-organisms that act without free oxygen; these are called
-anaérobic, in contradistinction to those which require oxygen, and
which are therefore called aérobic.

The chemical nature of the enzymes is very difficult to investigate ;

they are substances that elude the grasp of the chemist to a great
extent. So far, however, research has taught us that they are either
-proteid in nature or are substances closely allied to the proteids.
_ The distinction between organised ferments and enzymes is more
apparent than real; for the micro-organisms exert their action by
enzymes that they secrete. By crushing yeast cells Buchner has
_ succeeded in obtaining from them an enzyme that produces alcoholic
fermentation.

52 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON VI
PEPTIC DIGESTION

1. Half fill four test-tubes—
A with water. B with 0°2-per-cent. hydrochloric acid. C with 0:2-
per-cent. hydrochloric acid. D with solution of white of egg (1 to
10 of water).
2. To A add a few drops of glycerin extract of stomach' (this contains —
pepsin) and a piece of a solid proteid like fibrin.
To B also add pepsin solution and a piece of fibrin.
To C add only a piece of fibrin.
To D add a few drops of pepsin solution and fill up the tube with 0°2-per- —
cent. hydrochloric acid.
3. Put the tubes into the water-bath at 40° C., and observe them care-

In A the fibrin remains unaltered.

In B it becomes swollen, and gradually dissolves.

In C it becomes swollen, but does not dissolve.

4. After half an hour examine the solution in test-tube B.

(a) Colour some of the liquid with litmus and neutralise with dilute
alkali. Acid-albumin, syntonin, or parapeptone is precipitated.

(b) Take another test-tube, and put into it a drop of 1-per-cent. solution of
copper sulphate ; empty it out so that the merest trace of copper sulphate :
remains adherent to the wall of the tube; then add the solution from test-
tube B and a few drops of strong caustic potash. A pink colour (biuret re-
action) is produced. This should be carefully compared with the violet tint
given by unaltered albumin.

(ec) To a third portion of the fluid in test-tube B adda drop of nitric acid ;
albumoses or propeptones are precipitated: This precipitate dissolves on
heating and reappears on cooling.

5. Repeat these three tests with the digested white of egg in test-tube D.

6. Examine an artificial gastric digestion which has been kept a week.
Note the absence of putrefactive odour; in this it contrasts very forcibly
with an artificial pancreatic digestion under the same circumstances.

THE SECRETION OF GASTRIC JUICE

The juice secreted by the glands in the mucous membrane of the
stomach varies in composition in the different regions, but the mixed
gastric juice, as it may be termed, is a solution of a proteolytic fer-
ment called pepsin in a saline solution, which also contains a little
free hydrochloric acid.

1 Benger’s liquor pepticus may be used instead of the glycerin extract 0
stomach.

PEPTIC DIGESTION

HN)
Nitod

Fic. 19.—A p

neck ;

land from a

2’s stomach
n,

f tubule cut

ric g
mouth

ylo
? .
ion o

(Ebstei

section of the do
n)
tr,a deep port

versely.

duct or mouth of the

on the right the base of a tubule is more hi

’

or fundus of one of its tubules
3 Pp, parietal ceil.

¢, central cell

Fic. 18.—A cardiac gland from the dog’s stomach (Klein)

54 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The gastric juice can be obtained during the life of an animal by
means of a gastric fistula. Gastric fistule have also been made in
human beings, either by accidental injury or by surgical operations.
The most celebrated case is that of Alexis St. Martin, a young
Canadian who received a musket wound in the abdomen in 1822.
Observations made on him by Dr. Beaumont formed the starting-
point for our correct knowledge of the physiology of the stomach
and its secretion.

We now make artificial gastric juice by mixing weak hydeocblons
acid (0°2 per cent.) with a glycerin or aque-
ous extract of the stomach of a recently
killedanimal. This artificial juice acts like
the normal juice.

Two kinds of glands are distinguished
in the stomach, which differ from each other
in their position, in the character of their
epithelium, and in their secretion. The
cardiac glands are those situated in the
cardiac half of the stomach: their ducts
are short, their tubules long in proportion.
The latter are filled with polyhedral cells,
only a small lumen being left; they are
more closely granular than the correspond-
ing cells in the pyloric glands. They are
called principal_or central cells. Between
them and the basement membrane of the
tubule are other cells which stain readily
with aniline dyes. They are called parietal
or oxyntic (i.e. acid-forming) cells. . The
pyloric glands, in the pyloric half of the
stomach, have long ducts and short tubules
Fig, 20.—A cardiac gland of simple lined with cubical cells. There are no

form from the bat’s stomach. :
Osmic acid preparation (Lang- parietal cells.
ley): ¢, columnar epithelium

of the surface ; n, neck of the The central cells of the cardiac glands

land, with central and parietal
falls:; baseorfandusoveapied and the cells of the pyloric glands are

iich hibit the granules ae, loaded with granules. During secretion
Cr the alana **s the Tumen they discharge their granules, those that
remain being chiefly situated near the

lumen, leaving in each cell a clear outer zone (see fig. 20). These
are the cells that secrete the pepsin. Like secreting cells generally,
they select certain materials from the lymph that bathes them ;

these materials are worked up by the protoplasmic activity of the

PEPTIC DIGESTION 55

cells into the secretion, which is then discharged into the lumen of
the gland. The most important substance in a digestive secretion is
the ferment. In the case of a gastric juice this is pepsin. We
can trace an intermediate step in this process by the presence of the
granules. The granules are not, however, composed of pepsin, but
of a mother-substance which is readily converted into pepsin. We
shall find a similar ferment precursor in the cells of the pancreas,
and the term zymogen is applied to these ferment percursors. The
zymogen in the gastric cells is called pepsinogen. The rennet-
ferment or rennin that causes the curdling of milk is distinct from
_ pepsin, and is preceded by another zymogen ; it is, however, formed
_ by the same cells.
The parietal cells undergo merely a change of size during secre-
tion ; at first they are somewhat enlarged, and after secretion they
shrink. They are also called oxyntic cells, because it is believed
that they secrete the hydrochloric acid of the juice. Heidenhain
succeeded in making in one dog a cul-de-sac of the fundus, in another
of the pyloric region of the stomach; the former secreted a juice
containing both acid and pepsin; the latter, parietal cells being
absent, secreted a viscid alkaline juice containing pepsin. The for-
mation of a free acid from the alkaline blood and lymph is an
important but puzzling problem. There is no doubt that it is formed
from the chlorides of the blood and lymph, and of the many theories
advanced as to how this is done, Maly’s is, on the whole, the most
‘satisfactory. He considers that the acid originates by the interac-
tion of the calcium chloride with the disodium hydrogen phosphate
_ of the blood, thus :—
2Na,HPO,+3CaCl,=Ca;(PO,),+4NaCl+2HCl

[disodium [calcium [calcium [sodium [bydro-

hydrogen chloride] phosphate] chloride] chloric
phosphate] acid]

or more simply by the interaction of sodium chloride and sodium di-
hydrogen phosphate, as is shown in the following equation :—

NaH,PO,+NaCl=Na.HPO,+HCl

[sodium di- {sodium [disodium [hydro-
hydrogen chloride] hydrogen chloric
phosphate] phosphate] acid]

The sodium dihydrogen phosphate in the above equation is pro-
_bably derived from the interaction of the disodium hydrogen phos-
_ phate and the carbonic acid of the blood, thus :—

) Na,HPO,+CO,+H,O0=NaHCO;+NaH,PO,

_. But, as Professor Gamgee has pointed out, these reactions can

56 ESSENTIALS OF CHEMICAL PHYSIOLOGY

hardly be considered to occur in the blood generally but rather in —
the oxyntic cells, which possess the necessary selective powers in —
reference to the saline constituents of the blood, and the hydro-
chloric acid, as soon as it is formed, passes into the secretion of the: q
gland in consequence of its high power of diffusion. |

COMPOSITION OF GASTRIC JUICE

The following table gives the percentage composition of the —
gastric juice of man and the dog :—

Constituents Human Dog
Water ‘ ; 99°44 97°30
Organic substances (chiefly
pepsin) . : 0°32 1-71

HCl . ‘ : : 4 0-20 | 0°30
CaCl, . : , . xf 0-006 0-06
NaCl . . : : zt 014 0°25
KCl . ‘ ‘ ‘ ; 0°05 0-11
NH,Cl . ‘ ‘ ; — 0°05
Ca,(PO,). ; : a 1 0°17
Mg,(PO,), . : ‘ - | 0-01 0°02
Bae le ies | J 0-008

One sees from this how much richer in all constituents the gastric
juice of the dog is than that of man. Carnivorous animals have
always a more powerful gastric juice than other animals: they have
more work for it to do; but the great contrast seen in the table is,
no doubt, partly due to the fact that the persons from whom it has
been possible to collect gastric juice have been invalids. In the
foregoing table one also sees the great preponderance of chlorides
over other salts: apportioning the total chlorine to the various
metals present, that which remains over must be combined with
hydrogen to form the free hydrochloric acid of the juice.

Pepsin stands apart from nearly all other ferments by requiring
an acid medium in order that it may act. Probably a compound of
the two substances called pepsin-hydrochloric acid is the really
active agent. Other acids may take the place of hydrochloric acid,
but none act so well. Lactic acid is often found in gastric juice:
this appears to be derived by fermentative processes from the
food.

Some recent very valuable work performed by Pawlow on dogs
has enabled him not only to show that the secretory fibres for the
gastric glands are contained in the vagus or pneumogastric nerves,
but also to obtain a gastric juice free from any admixture with saliva

PEPTIC DIGESTION ae)

or food. The main facts in relation to this pure juice are as.
- follows :—It is clear and colourless ; it has a specific gravity of 1003 to.
- 1006. It is feebly dextro-rotatory, gives no biuret reaction, but gives.
the ordinary proteid reactions. It contains from 0-4 to 0°6 per cent.
of hydrochloric acid. It is strongly proteolytic, and inverts cane
sugar. When cooled to 0° C. it deposits a fine precipitate of pepsin ;
_ this settles in layers, and the layers first deposited contain most of
_ the acid, which is loosely combined with and carried down by the
| pepsin. Pepsin is also precipitable by saturation with ammonium
_ sulphate (Kiihne). Elementary analysis gave the following results :—

Pepsin precipitated by cold— Precipitated by Am,So,
Carbon . 3 . 50°73 per cent. | 50°37
Hydrogen ‘ (oe 6°88
Chlorine . | 1-01 to 1-17 * 0°89
Sulphur . ; 0-98 1:34

| Nitrogen. P - not estimated 14°55 to 15-0
_ Oxygen . : . the remainder. the remainder.

More recently Pawlow has by a very ingenious surgical operation
"succeeded in the dog in separating off from the stomach a diverticulum
’ which pours its secretion through an opening in the abdominal wall.
The following are his main results :-—

1. Increase in thé quantity of food given causes an increase in
the amount of gastric juice secreted.

2. The juice is most abundant in the early periods of digestion,
but it continues to be secreted in declining quantity as long as any
_ food remains to be dealt with. When there is no food given there
is no juice. But sham feeding or chewing will cause it to flow.

3. The amount of pepsin secreted is similarly proportional to the
needs of thé animal.

4. The larger the proportion of proteid in the diet, the more
abundant and active is the juice secreted.

THE ACTION OF GASTRIC JUICE

} The principal actions of the gastric juice have been already
_ practically studied : the action of pepsin in converting the proteids.
of the food into the diffusible peptones is its chief action. The
eurdling of milk by rennet will be found described in Lesson IV.
There is a still further action—that is, the gastric juice is anti-
septic ; putrefactive processes do not normally occur in the stomach,
_and the organisms that produce such processes, many of which are
“swallowed with the food, are in great measure destroyed, and thus
the body is protected from them. The acid is the agent in the aes
_ that possesses this power.

58 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The formation of peptones is a process of hydrolysis; peptones
may be formed by other hydrating agencies like superheated steam
and heating with dilute mineral acids. There are certain inter-
mediate steps in this process ; the intermediate substances are called
propeptones or proteoses. The word ‘proteose’ is the best to em-
ploy: it includes the albumoses (from albumin), globuloses (from
globulin), vitelloses (from vitellin), &c. Similar substances are also
formed from gelatin (gelatoses) and elastin (elastoses).

Another intermediate step in gastric digestion is called para-
peptone: this is acid-albumin or syntonin. In classifying the pro-
ducts of digestion it will be convenient to take albumin as our
example, but we must remember that globulin, myosin, and all the
other proteids form corresponding products. The products of
digestion may be classified according to the order in which they are
formed as follows :—

1. Parapeptone or acid-albumin.

( (a) Proto-albumose

The primary albumoses, 2.e.
2. Propeptone ° Hetero-albumose

those which are formed

first.
(c) Deutero-albumose

3. Peptone.

It is very doubtful whether all the albumin passes through the
acid-albumin stage.

Peptones.—These are the final products of the action of gastric ~

juice on native proteids.
They are soluble in water, are not coagulated by heat, and are

not precipitated by nitric acid, copper sulphate, ammonium sulphate, —

and a number of other precipitants of proteids. They are precipi-

tated but not coagulated by alcohol. They are also precipitated by —

tannin, picric acid, potassio-mercuric iodide, phospho-molybdie acid,
and phospho-tungstic acid.

They give the biuret reaction (rose-red, with a trace of copper
sulphate and caustic potash or soda).

Peptone is readily diffusible through animal membranes. The
utility of the formation of diffusible substances during digestion is
obvious.

Proteoses.—These are the intermediate products in the hydrolysis
of native proteids into peptones.

They are not coagulated by heat; they are precipitated but not
coagulated by alcohol; like peptone they give the biuret reaction.
They are precipitated by nitric acid, the precipitate being soluble on

—aS eS aera

PEPTIC DIGESTION 59

heating, and reappearing when the liquid cools. This last is a

distinctive property of proteoses. They are slightly diffusible.
The following table will give us at a glance the chief characters
_ of peptones and proteoses in contrast with those of native proteids
like albumin and gobele: —

Variety of | Actionof | Actionof | Action of Action of | copper | pitrusi-
Pra . ammonium | sulphate | 4...
proteid heat | alcohol | nitric acid sulphate qaubenustic bility
| | potash
} | |
| | PS ae
Albumin | Coagulated | Precipitated, | Precipitated Precipitated | Violet | Wit
hen coagu-| inthe cold ; by complete colour |
i | not readily saturation |
| soluble on i
| | heating | j
Globulin Ditto | Ditto Ditto Precipitated Ditto Ditto
} by half satu- |
} ration; also |
: precipitated |
by satura-
t tion with }
| y
} '
| Proteoses | Not coagu- | Precipitated,| Precipitated Precipitated Rose-red | Slight
(albu- lated but notco-| inthecold;| by saturation) colour
moses) agulated readily so- | | (biuret
luble on reaction)
heating ; |
the precipi- | |
tate reap-| t
on |
cooling *
| Peptones | Not coagu- | Precipitated, a eet Not precipi- Rose-red Great
lated but not co-| ed tated colour
| agulated | | (binret

> reaction) |

The difference between the different wabcnans is § akidy one of
‘solubility. The primary proteoses (proto- and hetero-) are precipitated
by saturation with magnesium sulphate or sodium chloride. Deutero-
‘proteose is not; it is, however, precipitated by saturation with
ammonium sulphate. Proto- and deutero- proteoses are soluble in
water ; hetero-proteose is not; it requires a salt to hold it in
solution.

_ To sum up: the main action of the gastric juice is upon the pro-
teids of the food, converting them into more soluble and diffusible
products. The fats are not chemically altered in the stomach;

their proteid envelopes are, however, dissolved, and the solid fats
are melted. Starch is unaffected ; but cane sugar is inverted. The
inversion of cane sugar is largely due to the hydrochloric acid of the
juice, and is frequently assisted by inverting ferments contained in
the vegetable food swallowed.

_ * In the case of deutero-albumose this reaction only oceurs in the presence of
excess of salt.

60 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON VII
PANCREATIC DIGESTION

1. A 1-per-cent. solution of sodium carbonate, to which a little glycerin
extract of pancreas! has been added, forms a good artificial pancreatic fluid.
2. Half fill three test-tubes with this solution.
A. To this add half its bulk of diluted egg-white (1 in 10).
B. To this add a piece of fibrin.
C. Boil this; cool; then add fibrin.
8. Put all into the water-bath at 40° C. After half an hour, test A and B
for alkali-albumin by neutralisation, for albumoses by nitrie acid, and for
albumoses and peptone by the biuret reaction.

4. Note in B that the fibrin does not swell up and dissolve, as in gastric

digestion, but that it is eaten away from the edges to the interior.

5. In © no digestion occurs, as the ferments have been destroyed by
boiling.

6. Take a solution of starch, equal quantities in three test-tubes.

D. To this add a few drops of glycerin extract of pancreas (without

the sodium carbonate).
E. To this add a few drops of bile.
F. To this add both bile and pancreatic extract.

7. Put these into the water-bath, and test small portions of each every
half-minute by the iodine reaction. It disappears first in F; then in D;

while E undergoes no change. Test D and F for maltose by Fehling’s
solution.

8. Shake up a few drops of olive oil with artificial pancreatic juice
(extract of pancreas and sodium carbonate). A milky fiuid (emulsion) is
formed, from which the oil does not readily separate on standing.

9. The foregoing experiments illustrate the action that pancreatic juice
has on all three classes of organic food.

i. On Proteids.—Fibrin, albumin, &c. are converted into proteoses and
peptone by the ferment trypsim in an alkaline medium.

ii. On Carbohydrates.—Starch is converted into sugar (maltose) by the
ferment amylopsin, especially in presence of bile.

iii. On Fats—These are emulsified. In the body they are also split into
fatty acid and glycerin by the ferment steapsin; but this cannot be shown
with the artificial juice, as steapsin is not soluble in glycerin.

The pancreas is a compound racemose gland, like the parotid.
There are, however, histological points of difference between them.
The secretory nerve fibres are contained in the vagus (Pawlow).
Microscopic examination of the gland cells in different stages of

1 Benger’s liquor pancreaticus diluted with two or three times its volume of
1-per-cent. sodium carbonate may be used instead.

ee ini ee ee ae

PANCREATIC DIGESTION 61

activity reveals changes comparable to those already described in the
_ ease of salivary and gastric cells. Granules indicating the presence of
a zymogen which is called trypsinogen (that is, the precursor of
trypsin, the most important ferment of the pancreatic juice) crowd
the cells before secretion: these are discharged during secretion, so
that in an animal whose pancreas has been powerfully stimulated to
secrete, as by the administration of pilocarpine, the granules are seen
only at the free border of the cells (see fig. 21).

As in the case of gastric juice, experiments on the pancreatic
_ secretion are usually performed with an artificial juice, made by
_ mixing a weak alkaline solution (1-per-cent. sodium carbonate) with
an extract of pancreas. The pancreas should be kept some time

Fie. 21.—Part of an alveolus of the rabbit's pancreas: A, before discharge: B, after.
(From Foster, after Kiihne and Lea.)

before the extract is made, or treated with dilute acid so as to ensure
that the transformation of trypsinogen into trypsin has taken place.

Quantitative analysis of human pancreatic juice givesthe following
_ results :-—

Water ; E ‘ : 97°6 per cent.
Organic solids . : : 1:8 *s
Inorganic salts . : ; 0°6 *

Dog’s pancreatic juice is considerably richer in solids.

The organic substances in pancreatic juice are—

__ (a) Ferments. These are the most important both quantitatively
and functionally. They are four in number :— :
| i. Trypsin, a proteolytic ferment.

ii. Amylopsin or pancreatic diastase, an amylolytic ferment.

iii. Steapsin, a fat-splitting ferment.

iv. A milk-curdling ferment.

(6) A small amount of proteid matter, coagulable by heat.

62 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The inorganic substances in pancreatic juice are—

Sodium chloride, which is the most abundant, and smaller quanti-
ties of potassium chloride, and phosphates of sodium, calcium, and
magnesium. The alkalinity of the juice is due to phosphates and
carbonates, especially of sodium.

ACTION OF PANCREATIC JUICE

The action of pancreatic juice, which is the most powerful and
important of all the digestive juices, may be described under the
headings of its four ferments.

1. Action of Trypsin.—Trypsin acts like pepsin, but with certain
differences, which are as follows :—

(a) It acts in an alkaline, pepsin in an acid medium.

(b) It acts more rapidly than pepsin; deutero-proteoses can be
detected as intermediate products in the formation of peptone.
Primary (i.e. proto- and hetero-) proteoses have not been found ; the
action is apparently too rapid to admit of their detection.

(d) It acts more powerfully on certain albuminoids (such as elastin)
which are difficult of digestion in gastric juice. Collagen, however,
is not digested.

(e) Acting on solid proteids like fibrin, it eats them away from
the surface to the interior; there is no preliminary swelling as in
gastric digestion.

(f) Trypsin acts further than pepsin, on prolonged action partly
decomposing the peptone which has left the stomach into simpler
products, of which the most important are leucine and tyrosine.

Besides leucine and tyrosine, smaller quantities of aspartic acid
[amido-succinic acid, C.H;(NH,)(COOH),], glutamic acid [amido-
pyrotartaric acid, C,;H,(NH,.)(COOH),], lysine, arginine, ammonia,
and a substance of doubtful nature called tryptophan are also formed ;
tryptophan gives a reddish-violet colour with chlorine or bromine
water.

Kiihne considered that the peptones which leave the stomach may
be arranged into two classes: (1) hemipeptone, which is split by the
prolonged action of pancreatic juice into the substances (leucine,
tyrosine, &c.) just enumerated ; and (2) antipeptone, which resists
this further action.

This theory held the field for many years, but recent researches
have shown that it has little or no foundation in fact.

We know that the action of proteolytic enzymes is by the process

Mra s'ah pm

PANCREATIC DIGESTION 63-

of hydrolysis to split the heavy proteid molecule into smaller and
smaller molecules ; first we get proteoses, then peptones, and finally
simple substances like leucine and tyrosine: the formation of
leucine, tyrosine, &c., does not occur to any great extent in normal
_ digestion, for these simple materials are of little or no nutritive value,
_ The substance formerly called antipeptone is really not a peptone at
all. Siegfried showed it is a substance of low molecular weight, and
according to him it is identical with a substance he had previously
separated from muscle, and called carnic acid (C,9H,;N;0s5).
Further work by Kutscher has shown that Siegfried is probably
_ wrong in regarding antipeptone as a single substance, but that it is in
reality a mixture of several substances of which he has separated out
the hexone bases arginine and histidine (see p. 32) and aspartic acid.

Atany rate antipeptone is not on the same level as the true peptones,
_ which are capable of utilisation by the organism, but is in the same
category as leucine and tyrosine and similar materials of low mole-
cular weight.

These facts can all be easily accounted for on the supposition that

_ a variable fraction of the proteid molecule is broken off with com-
parative ease, and appears as leucine, tyrosine, and other amido-acids.
_ This is more easily performed by the more powerful tryptic enzyme
than by the comparatively feeble agent pepsin. Pepsin, however,
is not entirely inactive in this direction, for although leucine and
tyrosine cannot as a rule be found in a gastric digestion, yet there
are analogous substances of low molecular weight (aspartic acid and
the hexone bases) which were incorrectly grouped together by the
_ earlier workers as a peptone (antipeptone).
1 2. Action of Amylopsin.—The conversion of starch into maltose
_ isthe most powerful and rapid of all the actions of the pancreatic
juice. It is much more powerful than saliva, and will act even on
unboiled starch. The absence of this ferment in the pancreatic juice
of infants is an indication that milk, and not starch, is their natural
diet.

3. Action on Fats.—The action of pancreatic juice on fats is a
‘double one: it forms an emulsion, and it decomposes the fats into
fatty acids and glycerin by means of its fat-splitting ferment steapsin.
The fatty acids unite with the alkaline bases to form soaps (saponifica-
tion). The chemistry of this is described on p. 18. The fat-splitting
power of pancreatic juice cannot be studied with a glycerin extract,
as steapsin is not soluble in glycerin: either the fresh juice or a
watery extract of pancreas must be used.

The formation of an emulsion may be studied in this way. Shake

64 ESSENTIALS OF CHEMICAL PHYSIOLOGY

‘up olive oil and water together, and allow the mixture to stand; the
finely divided oil globules soon separate from and float on the
surface of the water; but if a colloid matter like albumin or gum is
first mixed with the water, the oil separates more slowly. A more
permanent emulsion is formed by an alkaline fluid, and especially
when a small amount of free fatty acid is being continually liberated ;
the acid combines with the alkali to form a soap; the soap was
formerly stated to form a thin layer on the outside of each oil globule,
which prevented them running together again. This is not now
regarded as correct; the oil globules are prevented from running
together because of differences in the surface tension between them
and the surrounding fluid. Pancreatic fluid possesses, however, all
the necessary qualifications for forming an emulsion :—i. It is
alkaline; ii. It is viscous from the presence of proteid; iii. It has
the power of liberating free acids.

4. Milk-curdling Ferment.—The addition of pancreatic extracts
to milk causes clotting, which differs in some of its details from the
ecurdling produced by rennin; but this action can hardly ever be |
called into play, as the milk upon which the juice has to act hasbeen —
already curdled by the rennin of the stomach.

INTESTINAL DIGESTION

The pancreatic juice does not act alone on the food in the intes-
tines. There are, in addition, the bile, the succus entericus
(secreted by the crypts of Lieberkiihn), and bacterial action to be
considered.

The bile, as we shall find, has little or no digestive action by
itself, but combined with pancreatic juice it assists the latter in all
its actions. In our practical exercises we have already seen this is
true for the digestion of starch. It is also true for the digestion of
proteid, and very markedly so for the digestion of fat. Occlusion of |
the bile-duct by a gall-stone or by inflammation prevents bile entering —
the duodenum. Under these circumstances the faces contain a —
large amount of undigested fat. In some animals the bile contains
a ferment which is able to convert starch into sugar. The chief —
value of the bile in digestion is to act as a solvent of fats and fatty —
acids. This property it owes to the bile salts.

The suceus entericus has no action on fats or proteids, but it
appears to have to some extent the power of converting starch into
sugar; its most important action, however, is due to a ferment it
. gontains called invertin, which inverts saccharoses—that is, it con-
verts cane sugar and maltose into glucose. The original use of the

PANCREATIC DIGESTION 65

term ‘inversion’ has been explained on p.12. It may be extended
to include the similar hydrolysis of other saccharoses, although there
_ may be no formation of levorotatory substances. There are probably
several inverting ferments in the succus entericus, one of which acts
on cane sugar, one on maltose, and one on milk sugar.

Bacterial Action.—The gastric juice is an antiseptic; the-pan-
_creatic juice is not. A feebly alkaline fluid like pancreatic juice is
_ just the most suitable medium for bacteria to flourish in. Even in
an artificial digestion the fluid is very soon putrid, unless special
precautions to exclude or kill bacteria are taken. It is often difficult
to say where pancreatic action ends and bacterial action begins, as
‘many of the bacteria that grow in the intestinal contents, having
reached that situation in spite of the gastric juice, act in the same
way as the pancreatic juice. Some form sugar from starch, others
peptone, leucine, and tyrosine from proteids, while others, again,
break up fats. There are, however, certain actions that are entirely
due to these putrefactive organisms.

' i. On carbohydrates. The most frequent fermentation they set
| ‘up is the lactic acid fermentation: this may go further and result in
| the formation of carbonic acid, hydrogen, and butyric acid (see p. 14).
» Cellulose is broken up into carbonic acid and methane. This is the
' chief cause of the gases in the intestine, the amount of which is
increased by vegetable food.
ti. On fats. Im addition to acting like steapsin, lower acids
(valeric, butyric, &c.) are produced. The formation of acid products
from fats and carbohydrates gives to the intestinal contents an acid
reaction. Recent researches show that the contents become acid
‘much higher up in the small intestine than was formerly considered
‘to be the case. These organic acids do not hinder- pancreatic
digestion to any appreciable extent.
iii. On proteids. Fatty acids and amido-acids, especially leucine
tyrosine, are produced ; but these putrefactive organisms have a
ial action in addition, producing substances having an evil odour,
like indole, skatole, and phenol. There are also gaseous products in
some cases.
If excessive, putrefactive processes are harmful ; if within normal
hi mits, they are useful, helping the pancreatic juice and, further, pre-
venting the entrance into the body of poisonous products. It is
ssible that, in digestion, poisonous alkaloids are formed. Certainly
his is so in one well-known case. Lecithin, a material contained in
small quantities in many foods, and in large quantities in egg-yolk
‘and brain, is broken up by the pancreatic juice into glycerin, phos-
F

66 ESSENTIALS OF CHEMICAL PHYSIOLOGY

phorie acid, stearic acid, and an alkaloid called choline. We are, |
however, protected from the poisonous action of choline by the
bacteria, which break. it up into carbonic acid, methane, and

ammonia.
LEUCINE AND TYROSINE

These two substances’ have been frequently mentioned in the
preceding pages. As types of the decomposition products of proteids
they are important, though probably only small quantities are
normally formed during digestion.

They belong to the group of amido-acids. On p. 17 we have
given a list of the fatty acids; if we replace one of the hydrogen

On ty,
eat)

Fic. 22.—Leucine crystals. Fic, 23.—Tyrosine erystals.

atoms in a fatty acid by amidogen (NH,), we obtain what is called
an amido-acid. Take acetic acid: its formula is CH;.COOH; replace
one H by NH,, and we get CH,.NH,.COOH, which is amido-acetic
acid or glycocine. If we take caproic acid—a term a little higher
in the series—its formula is C;H,,.COOH; amido-caproic acid is
C;H).NH,.COOH, which is also called lewcine. There are, however,
several isomeric amido-caproic Acids. It was thought until. quite
recently that leucine was the amido-acid of normal caproie acid, but
it has been shown to be a-amido-iso-butylacetic acid. The difference |
in the structure of these two compounds may be represented by the
following graphic formule :

PANCREATIC DIGESTION 67

(OH, (CH, CH,
eg oa Teobatyl 3
raat 7 ‘ so-buty
fe \CH, Bie oft). cH,
r CH.NH, acetic acid CH.NH,
COOH. COOH

Tyrosie is a little more complicated, as it is not only an amido-acid,
but also contains an aromatic radicle. Propionic acid has the
formula C,H;.COOH ; amido-propionie acid is C.H,.NH,.COOH,
and is called alanine. If another H in this is replaced by oxyphenyl
' (C;H,-OH), we get C,H;.NH,.C;,H,OH.COOH, which is oxyphenyl-
amido-propionic acid, or tyrosine. Figs. 22 and 23 represent the
crystalline forms of leucine and tyrosine.

EXTIRPATION OF THE PANCREAS

Complete removal of the pancreas in animals and diseases of the
‘pancreas in man produce a condition of diabetes, in addition to the
) loss of pancreatic action in the intestines. Grafting the pancreas
from another animal into the abdomen of the animal from which the
pancreas has been removed relieves the diabetic condition.
. How the pancreas acts otherwise than in producing the pancreatic
) juice is not known. It must, however, have other functions related
to the general metabolic phenomena of the body, which are disturbed
jy removal or disease of the gland. This is an illustration of a
‘universal truth—viz. that each part of the body does not merely do
its own special work, but is concerned in the great cycle of changes
which is called general metabolism. Interference with any organ
‘upsets not only its specific function, but causes disturbances through
the body generally. The interdependence of the circulatory and
Tespiratory systems is a well-known instance. Removal of the
thyroid gland upsets the whole body, producing widespread changes
Known as myxcedema. Removal of the testis produces not only a
loss of the spermatic secretion, but changes the whole growth and
appearance of the animal. Removal of the greater part of the
Kidneys produces rapid wasting and the breaking down of the tissues
to form an increased quantity of urea. The precise way in which.
these glands are related to the general body processes is, however, a
Subject of which we know as yet very little. The theory at present
most in favour is that certain glands produce an internal secretion,

ister to parts elsewhere. Removal of such glands as the thyroid
: F2

a ESSENTIALS OF CHEMICAL PHYSIOLOGY
0

r suprarenal produces disease or death because this internal secre- —
tion can no longer be formed. In the case of the pancreas,
Professor Schifer has propounded the theory that the external
secretion of the pancreas (that is, pancreatic juice) is formed by
the cells lining the acini, and that the internal secretion, stoppage —
of which in some way leads to diabetes, is produced in the curious.
islets of epithelium-like cells scattered through the organ, and which
the reader will find described in works on histology.

LESSON VIII
BILE

1. Ox bile is given round. Observe its colour, taste, smell, and reaction
to litmus paper.

2. Acidulate a little bile with 20-per-cent. acetic acid. A stringy precipitate
of a mucinoid substance is obtained. Filter this off and boil the filtrate ;
no proteid coagulable by heat is present.

3. Add a few drops of bile to (a) acid-albumin prepared as described in
Lesson III., and (6) solution of proteoses to which half its volume of
_ 0-2-per-cent. hydrochloric acid has been added. A precipitate occurs in each
case. Bile salts precipitate the unpeptonised proteid which leaves the stomach.
4. Pettenkofer’s Test for Bile Salts.—To a thin film of bile in a capsule

add a drop of solution of cane sugar and a drop of concentrated sulphuric
acid. A purple colour is produced. This occurs more quickly on the
application of heat. The test may also be performed as follows :—Shake up
-some bile and cane sugar solution in a test-tube until a froth is formed.
Pour concentrated sulphuric acid gently down the side of the tube ; it produces
a purple colour in the froth.
5. Gmelin’s Test for Bile Pigments.—On to a little fuming nitric acid
_ (i.e. nitric acid containing nitrous acid in solution) in a test-tube pour gently
a little bile. Notice the succession of colours—green, blue, red, and yellow
—at the junction of the two liquids. This test may also be performed in a
Place a drop of fuming nitric acid in the middle of a thin film of
bile ; it becomes surrounded by rings of the above-mentioned colours.
6. Examine crystals of cholesterin microscopically.
7. To a few cholesterin crystals add a drop of iodine solution and a drop
_ of concentrated sulphuric acid. A play of colours (red, green, blue) is
produced. Other tests for cholesterin are described on p. 74.

Bile is the secretion of the liver which is poured into the duo-
denum ; it has been collected in living animals by means of a biliary
fistula ; the same operation has occasionally been performed in human
beings. After death the gall bladder yields a good supply of bile
which is more concentrated than that obtained from a fistula.

Bile is being continuously poured into the intestine, but there is
an increased discharge immediately on the arrival of food in the duo-
denum ; there is a second increase in secretion a few hours later.

_ Though the chief blood supply of the liver is by a vein (the portal
vein), the amount of blood in the liver varies with its needs, being
increased during the periods of digestion. This is due to the fact

70 ESSENTIALS OF CHEMICAL PHYSIOLOGY

that in the area from which the portal vein collects blood—stomach,
intestine, spleen, and pancreas—the arterioles are all dilated, and the
capillaries are thus gorged with blood. Further, the active peristalsis
of the intestine and the pumping action of the spleen are additional
factors in driving more blood onwards to the liver.

The bile is secreted from the portal blood at a much lower
pressure than one finds in glands, such as the salivary glands, the
blood supply of which is arterial. Heidenhain found that the
pressure in the bile duct of a dog averaged 15 mm. of mercury,
which is about double that in the portal vein.

The second increase in the flow of bile—that which occurs some
hours after the arrival of the semi-digested food (chyme) in the intestine
—appears to be due to the effect of the digestive products carried by
the blood to the liver stimulating the hepatic cells to activity; this
is supported by the fact that proteid food increases the quantity of
bile secreted, whereas fatty food which is absorbed, not by the portal
vein, but by the lacteals, has no such effect.

The chemical processes by which the constituents of the bile are

formed are obscure. We, however, know
a ) 7 that the biliary pigment is produced by

. & = 6, the decomposition of hemoglobin. Bili-
a om” zg = 9° rubin is, in fact, identical with the iron-
v ¢ o® “Sp free derivative of hemoglobin called
ties y em Y. hematoidin, which is found in the form
@ AL a of crystals in old blood-clots such as
EB Ye occur in the brain after cerebral heemor-
Cp 8H ee hage (see fig. 24)
B78 BY BD 2 rhage (see fig. 5
& ~

An injection of hemoglobin into the
portal vein, or of substances like water
which liberate hemoglobin from the red blood corpuscles, produces an
increase of bile pigment. If the spleen takes any partin the elabora-
tion of bile pigment, it does not proceed so far as to liberate heemo-
globin from the corpuscles. No free hemoglobin is discoverable in
the blood plasma in the splenic vein.

The amount of bile secreted is differently estimated by different
observers ; the amount secreted daily in man appears to vary from
500 c.c. to 1 litre (1,000 c.c.).

Fic, 24.—Heematoidin crystals.

THE CONSTITUENTS OF BILE

The constituents of the bile are the bile salts proper (taurocholate
and glycocholate of soda), the bile pigments (bilirubin, biliverdin), a
mucinoid substance, small quantities of fats, soaps, cholesterin,

ipa, = 2
ae ee Ja

BILE 71

lecithin, urea, and mineral salts, of which sodium chloride and the
phosphates of iron, calcium, ahd magnesium are the most important.
Bile is a yellowish, reddish-brown, or green fluid, according to
the relative preponderance of its two chief pigments. It has a musk-
like odour, a bitter-sweet taste, and a neutral or faintly alkaline re-
action.
The specific gravity of human bile from the gall bladder is 1026 to
1032; that from a fistula, 1010 to 1011. The greater concentration
_ of gall-bladder bile is partly but not wholly explained by the addition
to it from the walls of that cavity of the mucinoid material.
/ The amount of solids in bladder bile varies from 9 to 14 per cent.,
in fistula bile from 1-5 to 3 per cent. The following table shows that
_ this low percentage of solids is almost entirely due to want of bile salts.
_ This can be accounted for in the way first suggested by Schiff—that
_ there is normally a bile circulation going on in the body; a large
quantity of the bile salts that passes into the intestine is first split
up, then reabsorbed and again secreted. Such a circulation would
_ obviously be impossible in cases where all the bile is discharged to the
exterior.
The following table gives some important analyses of human
bile :—

Fistula bile | Fistula bile (case z :
$ (healthy woman. Normal bile |
Consti f 2 Pr
‘tuents | Copeman a0 d | tt) py oat Sig and (Frerichs) |
|
|
Sodium glycocholate . |} : 0-165 1) 2
Retigivimdrichointe .|f °°? 1 0-055 } wat
“sys , lecithin, fat. | 0-0990 0-038 118
ucinoid material . <8 0-1725 }
Pigment . : ‘ 1 0.0725 | j eat aa
Inorganic salts . ‘ 0°4510 | 0-878 0-78
| Total solids . : 1-4230 1-284 -14:08
Water (by difference) ; 98-5570 | 98-716 85°92
| : }
100-0000 _ 100-000 | 10000 |
|

Bile Mucin.—There has been considerable diversity of opinion as
to whether bile mucin is really mucin. The most recent work in
‘Hammarsten’s laboratory shows that differences occur in different
animals. Thus in the ox there is very little true mucin, but a great
amount of nucleo-proteid ; in human bile, on the other hand, there is
very little if any nucleo-proteid : the mucinoid material present there
is really mucin. (On the general characters of Mucrn and Nucngo-
Proreips see pp. 28 and 29.)

72 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The Bile Salts—The bile contains the sodium salts of complex
amido-acids called the bile acids. The two acids most frequently
found are glycocholic and taurocholic acids. The former is the more
abundant in the bile of man and herbivora; the latter in carnivorous
animals, like the dog. The most important difference between the
two acids is that taurocholie acid contains sulphur, and glycocholic
acid does not.

Glycocholic acid (C.,H,;NO,) is by the action of dilute acids and
alkalis, and also in the intestine, hydrolysed and split into glycocine
or amido-acetic acid and cholalic acid.

C,,H,,NO,+H,O=CH,.NH».COOH+C,H,,0;
[glycocholic acid] {glycocine] [cholalic acid}

The glycocholate of soda has the formula C.,H,,.NaNOg. .

Taurocholic acid (C,,H,,;NO,S) similarly splits into taurine or
amido-ethyl-sulphonic acid and cholalic acid.

C,,H,,NO;S+ H,O=C,H,.NH,.HS80; + Co,H4 0;
(taurocholic acid] {taurine] [cholalie acid]

The taurocholate of soda has the formula C.,H,,NaNO,S.

The colour reaction called Pettenkofer’s reaction, described in the
practical exercises at the head of this lesson, is due to the presence
of cholalic acid. The sulphuric acid acting on sugar forms a small
quantity of a substance called furfuraldehyde, in addition to other
products. The furfuraldehyde gives the purple colour with cholalic acid.

The Bile Pigments.—The two chief bile pigments are bilirubin
and biliverdin. Bile which contains chiefly the former (such as dog’s
bile) is of a golden or orange-yellow colour, while the bile of many
herbivora, which contains chiefly biliverdin, is either green or bluish
green. Human bile is generally described as containing chiefly bili-
rubin, but there have been some cases described in which biliverdin was
inexcess. The bile pigments show no absorption bands with the spec-
troscope ; their origin from the blood pigment has already been stated
(p. 70).

Bilirubin has the formula C,,H,,N,O;: it is thus an iron-free
derivative of hemoglobin. The iron is apparently stored ‘up in the
liver cells, perhaps for future use in the manufacture of new hemo-
globin. The bile contains only a trace of iron.

Biliverdin has the formula C,,H,,N.O, (i.e. one atom of oxygen
more than in bilirubin): it may occur as such in bile; it may be
formed by simply exposing red bile to the oxidising action of the
atmosphere ; or it may be formed as in Gmelin’s test by the more
vigorous oxidation produced by fuming nitric acid.

eS ee ee ee

BILE 73

Gmelin’s test consists of a play of colours—green, blue, red, and
finally yellow, produced by the oxidising action of fuming nitric acid
(that is, nitric acid containing nitrous acid in solution). The end or
yéllow product is called choletelin, C,~H,,N2O¢.

Hydrobilirubin.—If a solution of bilirubin or biliverdin in dilute
alkali is treated with sodium amalgam or allowed to putrefy, a brown-
ish pigment is formed called hydrobilirubin, C3.H,,N,O;. With the
spectroscope it shows a dark absorption band between 0 and F, anda
fainter band in the region of the D line.

Urobilin.— Hydrobilirubin isinteresting because a similar substance
is formed from the bile pigment by reduction processes in the intestine,
and constitutes stercobilin, the pigment of the feces. Some of this
is absorbed and ultimately leaves the body in the urine as one of its
pigments called wrobilin. A small quantity of urobilin is sometimes
found preformed in the bile. The identity of urobilin and stercobilin
has been frequently disputed, but the recent work of Garrod and
Hopkins has confirmed the old statement that they are the same
substance with different names. Urobilin has a well-marked absorption
band in the region of the F line, and when partially precipitated from
an alkaline solution by acidification, it also shows an absorption band
in the region of the E line. Hydrobilirubin differs from urobilin in
containing much more nitrogen in its molecule (9-2 instead of 41 per
cent.), and is probably a product of less complete reduction than
urobilin. (See further Lesson XXVI.)

Cholesterin.—This substance is contained not only in bile, but
very largely in nervous tissues. Like lecithin, it is an abundant
constituent of the white substance of
Schwann. It is found also in blood cor-
puscles. In bile it is normally present in
small quantities only, but it may occur in
excess, and so form the concretions known
as gallstones, which are generally more or
less tinged with bilirubin.

Though its solubilities remind one of a
fat, cholesterin is not a fat. It is, in fact,
chemically speaking, a monatomic alcohol.
lis formula is C,,H,;.HO.

From alcohol or ether containing water
_ it erystallises in the form of rhombic tables, which contain a molecule
__ of water of. crystallisation: these are easily recognised under the
microscope (fig. 25). It gives the following tests :—

Fic. 25.—Cholesterin crystals.

74 ESSENTIALS OF CHEMICAL PHYSIOLOGY

1. With iodine and concentrated sulphuric acid the crystals give
a play of red, green, and blue.

2. Heated with sulphuric acid and water (5 : 1) the ‘iden of the
crystals turn red.

3. A solution of cholesterin in chloroform, gently shaken with an
equal amount of concentrated sulphuric acid, turns red, and ultimately
purple, the subjacent acid acquiring a green fluorescence. (Salkow-
ski’s reaction.)

‘The mode of origin of cholesterin in the body has not been clearly
made out. Whether it is formed in the tissues generally, in the
blood, or in the liver, is not known ; nor has it been determined con-
clusively that it is derived from albuminous or nervous matter. It is
also doubtful if we are to regard it as a waste substance of no use to
the body, as its presence in the blood-corpuscles, in nervous matter,
in the egg, and in vegetable grains, points to a possible function of a
histogenetic or tissue-forming character.’ (McKendrick.)

A substance called iso-cholesterin, isomeric with ordinary chole-
sterin, is found in the fatty secretion of the skin (sebum) ; it is largely
contained in the preparation called lanoline made from sheep’s-wool
fat. It does not give Salkowski’s reaction with chloroform and sul-
phuric acid just described.

THE USES OF BILE

One of the most remarkable facts concerning the bile is its
apparently small use in the digestion of food. It is doubtless, to a
large extent, excretory. Some state that in some animals it has a slight
action on fats and carbohydrates, but it appears to be rathera coadjutor
to the pancreatic juice (especially in the digestion of fat) than to have
any independent digestive activity (see p. 64).

Bile is said to be a natural antiseptic, lessening the putrefactive
processes in the intestine. This is also very doubtful. Though the
bile salts are weak antiseptics, the bile itself is readily putrescible,
and the power it has of diminishing putrescence in the intestine is due
chiefly to the fact that by increasing absorption it lessens the amount
of putrescible matter in the bowel.

When the bile meets the chyme the turbidity of the latter is in-
creased, owing to the precipitation of unpeptonised proteid. This is an
action due to the bile salts, and it has been surmised that this con-
version of the chyme into a more viscid mass is to hinder somewhat
its progress through the intestines; it clings to the intestinal wall,
thus allowing absorption to take place. The neutralisation of the acid
gastric juice by the bile also allows the alkalinity of the pancreatic

BILE 15

q juice to have full play. Bile isa solvent of fatty acids, and assists
_ the absorption of fat (see p. 80).

THE FATE OF THE BILIARY CONSTITUENTS

We have seen that fistula bile is poor in solids as compared with
_ normal bile, and that this is explained on the supposition that the
normal bile circulation is not occurring—the liver cannot excrete
what it does not receive back from the intestine. Schiff was the first
to show that if the bile is led back into the duodenum, or even if the
animal is fed on bile, the percentage of solids in the bile excreted is at
once raised. It is on these experiments that the theory of a bile
circulation is mainly founded. The bile circulation relates, however,
chiefly, if not entirely, to the bile salts: they are found but sparingly
| in the feces; they are only-represented to a slight extent in the urine :
| hence it is calculated that seven-eighths of them are re-absorbed from
| the intestine. Small quantities of cholalic acid, taurine, and glycocine
_ are found in the feces; the greater part of these products of the decom-
_ position of the bile salts is taken by the portal vein to the liver, where
they are once more synthetised intothe bile salts. Some of the taurine is
_ absorbedand excreted as tauro-carbamic acid (C,H,NHCO.NH,HSO;)
_ intheurine. Some of the absorbed glycocine may be excreted as urea
or uric acid. The cholesterin and mucus are found in the feces ; the
_ pigment is changed into stercobilin, a substance like hydrobilirubin.
_ Some of the stercobilin is absorbed, and leaves the body as the urinary
- pigment, urobilin.

THE FECES

The feces are alkaline in reaction, and contain the following sub-
stances :—

1. Water: in health from 68 to 82 per cent.; in diarrhea it is
_ more abundant still.

2. Undigested food ; that is, if food is taken in excess, some escapes
_ the action of the digestive juices. On a moderate diet unaltered
-proteid is never found.

3. Indigestible constituents of the food: cellulose, keratin, mucin,
_ chlorophyll, gums, resins, cholesterin.

4. Constituents digestible with difficulty : uncooked starch, ten-
_ dons, elastin, various phosphates, and other salts of the alkaline earths.
5. Products of decomposition of the food: indole, skatole, phenol,
acids such as fatty acids, lactic acid, &c.; hematin from hemoglobin ;
insoluble soaps like those of calcium and magnesium.

76 ESSENTIALS OF CHEMICAL PHYSIOLOGY

6. Bacteria of all sorts and débris from the intestinal wall; cells,
nuclei, mucus, &c.

7. Bile residues : mucus, cholesterin, traces of bile acids and their
products of decomposition, stercobilin from the bile pigment."

MECONIUM

Meconium is the name given to the greenish-black contents of the
intestine of new-born children. It is chiefly concentrated bile, with
débris from the intestinal wall. The pigmentis a mixture of bilirubin
and biliverdin ; it is not stercobilin.

ABSORPTION ;

Food is digested in order that it may be absorbed. It is absorbed
in order that it may be assimilated—that is, become an integral part
of the living material of the body.

Having now considered the action of digestive juices, we can study
the absorption which follows. In the mouth and cesophagus the
thickness of the epithelium and the quick passage of the food through
these parts reduce absorption to aminimum. Absorption takes place
more rapidly in the stomach: the small intestine with its folds and
villi to increase its surface is, however, the great place for absorption ;
and although the villi are absent from the large intestine, absorption
occurs there also, but to a less extent.

Foods such as water and soluble salts like sodium chloride are
absorbed unchanged. The organic foods, however, are considerably
changed, colloid materials like starch and proteid being converted re-
spectively into the diffusible materials sugar and peptone.

There are two channels of absorption, the blood vessels (portal
capillaries) and the lymphatic vessels or lacteals.

Absorption, however, is no mere physical process of diffusion and
filtration. We must also take into account the fact that the cells
through which the absorbed substances pass are living, and in virtue
of their vital activity not only select materials for absorption, but also
change*those substances while in contact with them. These cells
are of two kinds—(1) the columnar epithelium that covers the
surface ; and (2) the lymph cells in the lymphoid tissue beneath. It
is now generally accepted that of the two the former, the columnar
epithelium, is the more importans. When these cells are removed or
rendered inactive by sodium fluoride, absorption practically ceases,
though the opportunities for simple filtration or diffusion would be by
such means increased.

' Stercobilin may originate also from the hematin of the food. (MacMunn.)

BILE 77

Absorption of Carbohydrates—Though the sugar formed from
starch by ptyalin and amylopsin is maltose, that found in the blood is
glucose. Under normal circumstances little if any is absorbed by the
lacteals. The glucose is formed from the maltose by the succus
entericus, and perhaps also by the vital action of the epithelial cells.
Cane sugar and milk sugar are also converted into glucose before
absorption.

The carbohydrate food which enters the blood as glucose is taken
to the liver, and there stored up in the form of glycogen—a reserve
store of carbohydrate material for the future needs of the body.
Glycogen, however, is found in animals which take no carbohydrate
food. It must, then, be formed by the protoplasmic activity of the
liver cells from their proteid constituents. The carbohydrate store
leaves the liver in the blood of the hepatic vein as glucose (dextrose)
once more.

The above is a brief statement of the glycogenic functions of the
liver as taught by Claude Bernard, and accepted by the majority of
physiologists. It has always been strongly contested by Dr. Pavy,
who has in a recently published book revived the question. Dr.
Pavy’s theory is that the glycogen formed in the liver from the sugar
of the portal blood is never during life reconverted into sugar, but is
used in the formation of other substances like fat and proteid ; in sup-
port of this he has shown that proteids contain a carbohydrate radicle.
He denies that the post-mortem formation of sugar from glycogen that
occurs in an excised liver is a true picture of what occurs during life.

Absorption of Proteids——A certain amount of soluble proteid is
absorbed unchanged. Thus, after taking a large number of eggs,
egg albumin is found in the urine. Patients fed per rectwm derive
nourishment from proteid food, though proteolytic ferments are not
present in this part of the intestine.

Most proteid, however, is normally absorbed as peptone and
proteose (albumose). Peptones and proteoses are absent from the
blood under all circumstances, even from the portal blood during the
most active digestion. Im other words, during absorption the
epithelial cells change the products of proteolysis (peptones and
proteoses) back once more into native proteids (albumin and
globulin).

The greater part of the proteid absorbed passes into the blood; a
little into the lymph vessels also; but this undergoes the same change.

When peptone (using the word to include the proteoses also) is
injected into the blood stream, poisonous effects are produced, the
coagulability of the blood is lessened, the blood pressure falls,

78 ESSENTIALS OF CHEMICAL PHYSIOLOGY

secretion ceases, and in the dog 0°3 gramme of ‘ peptone’ per kilo-
gramme of body weight is sufficient to kill the animal.
The epithelial cells of the alimentary canal thus protect us from

Fic. 26.—Section of the villus of a rat killed during fat-absorption (H. A. Schifer) :
ep, epithelium ; str, striated border ; c, lymph cells; c’, lymph cells in the epithelium ;
1, central lacteal containing disintegrating lymph cells.

those poisonous effects by converting the harmful peptone into the
useful albumin and globulin.

Absorption of Fats.—The fats undergo in the intestine two
changes: one a physical change (emulsification), the other a chemi-
cal change (saponifica-
tion). The lymphatic
vessels are the great
channels for fat absorp-
tion, and their name,
lacteals, is derived from
the milk-like appearance
of their contents (chyle)
during the absorption of
Fic. 27.—Mucous membrane of frog’s intestine during z fat fat.

goaciplnn Ce eehite epettation sy etdnted:: The way ims Meio Se
minute fat globules pass
from the intestine into the lacteals has been the subject of much

BILE 79

controversy. The course they take may be studied by killing animals
at varying periods after a meal of fat, and making osmic acid micro-
scopic preparations of the villi. Figs. 26 and 27 illustrate the
appearances observed by Professor Schiifer.

The columnar epithelium cells become first filled with fatty globules
of varying size, which are generally larger near the free border. The
globules pass down the cells, the larger ones breaking up into
smaller ones during the journey; they are then transferred to the
amoeboid cells of the lymphoid tissue beneath: these ultimately
penetrate into the central lacteal, where they either disintegrate or
discharge their cargo into the lymph stream. The globules are
by this time divided into immeasurably small ones, the molecular
basis of chyle. The chyle enters the blood stream by the thoracic
duct, and after an abundant fatty meal the blood plasma is quite
milky; the fat droplets are so small that they circulate without
hindrance through the capillaries. The fat in the blood after a
meal is eventually stored up in connective tissue cells of adipose
tissue. It must, however, be borne in mind that the fat of the body
is not exclusively derived from the fat of the food, but it may

_ originate also both from proteid and from carbohydrate.

As, however, the fat globules were never seen penetrating the
striated border of the epithelial cells, there was a great difficulty in
understanding how they reached the interior of these cells; the cells
will not take up other particles, and it appears certain that they do
not in the higher animals protrude pseudopodia from their borders
_ (this, however, does occur in the endoderm of some of the lower
_ invertebrates).

) Recent research has in a great measure solved this difficulty. In
the first place particles may be present in the epithelium and lym-
_ phoid cells while no fat is being absorbed. These particles are pro-
toplasmic in nature, as they stain with reagents that stain proto-
| plasmic granules; they however also stain darkly with osmic acid,
and so are apt to be mistaken for fat. There is, however, no doubt
that the particles found during fat absorption are composed of fat.
There is also no doubt that the epithelial cells have the power of
_ again forming fat out of the fatty acids and glycerin into which it has
_ been broken up in the intestine. Munk, who has performed a large
_ number of experiments on the subject, showed that the splitting of
fats into glycerin and fatty acids occurs to a much greater extent
_ than was formerly supposed; these substances being soluble pass

) readily into the epithelium cells; and these cells perform the syn-
) thetic act of building them into fat once more; the fat so formed

80 ESSENTIALS OF CHEMICAL PHYSIOLOGY

appears in the form of small globules, surrounding or becoming
mixed with the protoplasmic granules that are ordinarily present.
Another remarkable fact which he made out is that after feeding an
animal on fatty acids the chyle contains fat. The necessary glycerin
must have been formed by protoplasmic activity during absorption.
The more recent work of Moore and Rockwood has shown that fat is.
absorbed entirely as fatty acid or soap; and that preliminary emulsi-
fication, though advantageous for the formation of these substances,
is not essential.

We thus see how with increase of knowledge, due to improved
methods of research, a complete change has come over the ideas
which physiologists have held regarding this subject. It is not so
many years ago, that the physical change—emulsification—which
fats undergo in the intestine, was considered to be more important than
the chemical changes—fat-splitting and saponification. In fact the
small amount of chemical change which was supposed to occur was
regarded as quite subordinate, and of value merely in assisting the
process of emulsification. We now know that the exact converse is.
the truth; the chemical change is the important process, and emul-
sification the subordinate one.

Bile aids the digestion of fat, in virtue of its being a solvent of fatty
acids, and it probably assists fat absorption by reducing the surface
tension of the intestinal contents; membranes moistened with bile
allow fatty materials to pass through them more readily than would
otherwise be the case. In cases of disease in which bile is absent
from the intestines, a large proportion of the fat in the food passes.
into the feces.

LESSON IX!
THE BLOOD

Blood Plasma

1. The coagulation of the blood has been prevented in specimen A by the
_ addition of neutral salt (an equal volume of saturated sodium-sulphate
solution, or a quarter of its volume of saturated magnesium-sulphate solu-
_ tion). The corpuscles have settled, and the supernatant salted plasma has
_ been siphoned off. >

2. The coagulation of the blood in specimen B has been prevented by the
addition of an equal volume of a 0°4-per-cent. solution of potassium oxalate
- in normal saline solution. :
3. Put a small quantity of A into three test-tubes and dilute each with

about ten times its volume of liquid :

A 1. With distilled water.
A 2. With solution of fibrin ferment containing a little calcium chloride.*
A38. With the same. :
4. Put A 1 and A 2 into the water-bath at 40° C.; leave A 3 at the tem-
_ perature of the air. A 1 coagulates slowly or not at all; A 2 coagulates
_ rapidly; A 3 coagulates less rapidly than A 2.
5. Add to some of B a few drops of dilute (2 per cent.) calcium chloride
_ solution: it coagulates, and more quickly, if the temperature is 40° C.

Blood Serum

Blood serum is the fluid residue of the blood after the separation of the
clot ; it is blood plasma minus fibrin. The general appearance of fibrin
obtained by whipping fresh blood will already be familiar to the student, as
he has used it in experiments on digestion (Lessons VI. and VII.).

Serum has a yellowish tinge due to serum lutein, but as generally obtained
it is often contaminated with a small amount of oxyhemoglobin, and so looks
reddish. It contains proteids (giving the general tests already studied in
Lesson II.), extractives, and salts in solution. The proteids are serum
albumin and serum globulin. The fibrin ferment is also a proteid-like sub-
stance. It is present in only small quantities, and in the following experi-
ments is precipitated with serum globulin.

1 This lesson may conveniently be divided into two, the first dealing with
| plasma and serum, and the second with hemoglobin.
| 7? An easy way of preparing an impure but efficient solution of fibrin ferment
is to take 5 c.c. of blood serum and dilute it with a litre of distilled water. A
ial precipitation of globulin takes place,,and carries down the ferment with it.
After a few hours pour off the supernatant fluid and dissolve the precipitate in
half a litre of tap water to which a few drops of 2 per cent. solution of calcium
oride have been added. This solution can be then given round to the class as
fibrin ferment.

82 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Separation of the serum proteids.—(a) Dilute serum with fifteen times
its volume of water. It becomes cloudy owing to the partial precipitation of
serum globulin. Add a few drops of 2 per cent. acetic acid; the precipitate
becomes more abundant, but dissolves in excess of the acid.

(b) Pass a stream of carbonic acid through serum diluted with twenty
times its bulk of water. A partial precipitation of serum globulin occurs.

the salt and grinding in a mortar. A precipitate of serum globulin is produced.

(d) Half saturate the serum with ammonium sulphate by adding to it an
equal volume of a saturated solution of the salt. Serum globulin is.
precipitated.

(e) Completely saturate the serum with ammonium sulphate by adding
crystals of the salt and grinding in a mortar; a precipitate is produced of
both the globulin and the albumin. Filter through a dry filter paper; the
filtrate contains no proteid.

Hemoglobin

6. Direct the spectroscope to the window and carefully focus Fraun-
hofer’s lines. Note especially D in the yellow, and E, the next well-marked
line, in the green.

7. Direct the spectroscope to a luminous gas flame: these lines are absent..
Place a little sodium chloride in the flame. Notice the bright yellow line in
the position of the D line.

8. Take a series of six test-tubes of about equal size. Fill the first with
diluted defibrinated ox-blood (1 part of blood to 31 of water); then fill the
second tube with the same mixture diluted with an equal bulk of water
(1 in 64) ; half fill the third tube with this and fill up the tube with an equal
bulk of water (1 in 128), and so on. The sixth tube will contain 1 part of
blood to 1,024 of water, and will be nearly colourless.

9. Into another series of six test-tubes put a few drops of ammonium
sulphide ; then pour in some of the contents of each of the first series and
warm very gently.

10. Examine the tubes with the spectroscope and map out on a chart the
typical absorption bands of oxyhzemoglobin in the first series, and of (reduced)
hemoglobin in the second series. Notice that in the more dilute specimens
of hemoglobin the bands are no longer seen, whereas those of oxyhemoglobin
in specimens similarly diluted are still visible.

11. Take a tube which shows the single band of reduced hemoglobin
and shake it with the air; the bright red colour returns to it, and it shows.
spectroscopically the two bands of oxyhemoglobin for a short time. Con-
tinue watching the two bands, and note that they fade and are replaced by a
single band as reduction again occurs.

12. Mix a drop of defibrinated rat’s blood on a slide with a drop of water,
or mount it in a drop of Canada balsam. Examine the crystals of oxyhemo-
globin as they form.

13. Smear a little blood, obtained by pricking the finger, on a slide and
allow it to dry; cover, and run glacial acetic acid under the cover glass, and.
boil. Examine microscopically for the dark brown crystals of hemin.

COAGULATION OF BLOOD

Microscopic investigation of vertebrate blood shows that it consists
of a fluid which holds in suspension large numbers of solid bodies—
the red and the white corpuscles and the blood tablets.

THE BLOOD 83

After blood is shed it rapidly becomes viscous and then sets into
- a solid red jelly. The jelly soon contracts and squeezes out a straw-
coloured fluid called the serum, in which the shrunken clot ultimately
floats.
_ With the microscope, filaments of fibrin are seen forming a net-
| work throughout the plasma, many radiating from small clumps of
_ blood tablets. It is the formation of fibrin which is the essential act
_ of coagulation: this entangles the corpuscles and forms the clot.
Fibrin is formed from the plasma, and may be obtained free from
' corpuscles when blood plasma is allowed to clot, the corpuscles hav-
ing previously been removed. It may be also obtained from blood by
_ whipping it with a bunch of twigs; the fibrin adheres to the twigs
and entangles but few corpuscles. These may be removed by

_ Fic. 28.—Fibrin filaments and blood tablets : A, network of fibrin shown after washing away the
corpuscles from a preparation of blood that has been allowed to clot. Many of the filaments
radiate from small clumps of blood tablets; B (from Osler), blood corpuscles and blood tablets
within a small vein.

. washing with water. Serum is plasma minus fibrin. The relation
of plasma, serum, and clot can be seen at a glance in the following
scheme of the constituents of the blood :—

Plasma { seis
Clot

Corpuscles

Blood

_ It may be roughly stated that in 100 parts by weight of blood 60-65
parts consist of plasma and 35-40 of corpuscles.
_ The buffy coat is seen when blood coagulates slowly, as in horse’s
blood. The red corpuscles sink more rapidly than the white, and
the upper stratum of the clot (buffy eoat) consists mainly of fibrin
and white corpuscles.
Coagulation is hastened by—
1. A temperature a little over that of the body.
2. Contact with foreign matter.
3. Injury to the vessel walls.
4. Agitation.
5. Addition of calcium salts.

84 ESSENTIALS OF CHEMICAL PHYSIOLOGY

6. Injection of nucleo-proteid produces intravascular clotting.
Very minute doses, however, produce the opposite effect—namely
delay of coagulation.

Coagulation is hindered or prevented by—

1. A low temperature. In a vessel cooled by ice, coagulation —
may be prevented for an hour or more.

2. The addition of a large quantity of neutral salts like sodium —
sulphate or magnesium sulphate.

3. Contact with the living vascular walls.

4. Contact with oil.

5. Addition of a soluble oxalate (e.g. potassium oxalate): this
precipitates the calcium necessary for coagulation as insoluble calcium
oxalate.

6. Injection of commercial peptone (which consists chiefly of
proteoses) into the circulation of the living animal.

7. Addition of leech extract. This acts in virtue of a proteose it
contains.

The theory generally received which accounts best for the coagu-
lation of the blood is that of Hammarsten, and it may be briefly
stated as follows :—

When blood is within the vessels one of the constituents of the plasma,
a proteid of the globulin class called fibrinogen, exists in a soluble
form.

When the blood is shed the fibrinogen molecule is split into two
parts : one part is a globulin, which remains in solution, the other is
the insoluble material fibrin.

This change is brought about by the activity of a special un-
organised ferment called the fibrin ferment.

This ferment does not exist in healthy blood contained in healthy
blood vessels, but is one of the products of the disintegration of the
white corpuscles and blood tablets that occwrs when the blood leaves
the vessels or comes into contact with foreign matter.

To this may be added, as the result of recent research, that a
soluble calcium salt is essential for the formation of the ferment; that —
the fibrin ferment belongs to the class of nucleo-proteids; that other
nucleo-proteids (Wooldridge’s tissue-fibrinogens, see p. 29) obtained
from most of the cellular organs of the body produce intravascular
clotting when injected into the circulation of a living animal.

The fibrin ferment may very conveniently be called thrombin.
Like other ferments it is preceded by a mother substance or zymogen ~
which may be called prothrombin ; the action of calcium salts is to ©

THE BLOOD 85

convert prothrombin into thrombin. We may therefore represent
_ the process of clotting in the following tabular way.

In the plasma a proteid substance From the colourless corpuscles a
exists, called nucleo-proteid is shed out, called
FIBRINOGEN. PROTHROMBIN.
By the action of calcium salts
prothrombin is converted into fibrin-

ferment, or
THROMBIN.
i

Thrombin acts on fibrinogen in such a way that two new substances are
formed,

ee my
= Sr

One of these is unimportant, viz. The other is important, viz.
_ @ globulin (fibrino-globulin) which FF rerin, which entangles the cor-

| remains in solution. Its amount is puscles and so forms the Cuor.

| very small.

THE PLASMA AND SERUM

The liquid in which the corpuscles float may be obtained by
employing one or other of the methods already described for pre-
venting the blood from coagulating. The corpuscles, being heavy,
| sink, and the supernatant plasma can then be removed by a pipette
or siphon; the separation can be effected more thoroughly by the
_ use of a centrifugal machine (see fig. 60, Lesson XXI_).
On counteracting the influence which has prevented the blood from
coagulating, the plasma then itself coagulates. Thus plasma obtained
by the use of cold clots on warming gently ; plasma which has been
_ decalcified by the action of a soluble oxalate clots on the addition of
_ acalcium salt; plasma obtained by the use of a strong solution of
salt coagulates when this is diluted by the addition of water, the
addition of fibrin ferment being necessary in most cases; where co-
-agulation occurs without the addition of fibrin ferment, no doubt some
is present from the partial disintegration of the corpuscles which has
| already occurred. Pericardial and hydrocele fiuids resemble pure
plasma very closely in composition. Asa rule, however, they contain
few or no white corpuscles, and do not clot spontaneously, but after
‘the addition of fibrin ferment or liquids like serum that contain
fibrin ferment they always yield fibrin.
_ Pure plasma may be obtained from horse’s veins by what is known
) as the ‘living test-tube’ experiment. If the jugular vein is ligatured
) in two places, so as to include a quantity of blood within it, then

86 ESSENTIALS OF CHEMICAL PHYSIOLOGY

removed from the animal and hung in a cool place, the blood will not
coagulate for many hours. The corpuscles settle, and the supernatant
plasma can be removed with a pipetite.

The plasma is alkaline, yellowish in tint, and its specific gravity
is about 1026 to 1029.

Its chief constituents may be enumerated as follows :—

1,000 parts of plasma contain—

Water ; : : : ; : - 902-90
Solids ; ; : , ; : i DELO
Proteids : 1, yield of fibrin i : ‘ 4:05

2, other proteids ‘ . - 78°84
Extractives (including fat) ; : : 5°66
Inorganic salts . ; : : 8°55

In round numbers plasma contains 10 per cent. of solids, of which
8 per cent. are proteid in nature. The small quantity of fibrinogen
as indicated by the amount of fibrin formed should be carefully noted.

Serum contains the same three classes of constituents—proteids,
extractives, and salts. The extractives and salts are the same in the
two liquids. The proteids differ, as is shown in the following
table :—

Proteids of Plasma Proteids of Serwm

Fibrinogen Serum globulin

Serum globulin Serum albumin

Serum albumin | Fibrin ferment (nucleo-proteid)
| Fibrino-globulin

The gases of the plasma and serum are small quantities of
oxygen, nitrogen, and carbonic acid. The greater part of the oxygen —
of the blood is combined in the red corpuscles with hemoglobin ;
the carbonic acid is chiefly combined as carbonates (see RESPIRATION).

We may now consider one by one the various constituents of the
plasma and serum.

A. Proteids—/ibrinogen.—This is the substance acted on by
fibrin ferment. It yields, under this action, an insoluble product
called fibrin, and a soluble proteid of the globulin class.

Fibrinogen is a globulin. It differs from serum globulin, and may
be separated from it by the fact that half-saturation with sodium
chloride precipitates it. It coagulates by heat at the low temperature
of 56° C. As judged from the yield of fibrin, it is the least abundant
of the proteids of the plasma (see table on upper part of this page).

Serum globulin and serum albwmin.—These substances are con-
sidered in the practical exercises at the head of this lesson: see also

THE BLOOD 87

_ Lesson II. Both serum globulin and serum albumin probably
_ consist of more than one proteid substance (see Lesson XX.).
q Fibrin ferment.—Schmidt’s method of preparing it is to take serum
. and add excess of alcohol. This precipitates all the proteids, fibrin
_ ferment included. After some weeks the alcohol is poured off; the
serum globulin and serum albumin have been by this means rendered
_ insoluble in water; an aqueous extract is, however, found to contain
fibrin ferment, which is not so easily coagulated by alcohol as the
_ other proteids are. A simpler method of preparing fibrin ferment
_ in an impure but efficient form is given in the footnote on p. 81.
E B. Extractives.—These are non-nitrogenous and nitrogenous. The
_ non-nitrogenous are sugar (0°12 per cent.), fats, soaps, cholesterin ; and
| the nitrogenous are urea (0°02 to 0-04 per cent.), and still smaller
, quantities of uric acid, creatine, creatinine, xanthine, and hypoxanthine.
O C. Salts—The most abundant salt is sodium chloride: it consti-
' tutes between 60 and 90 per cent. of the total mineral matter.
Potassium chloride is present in much smaller amount. It consti-
' tutes about 4 per cent. of the total ash. The other salts are phos-
_ phates and sulphates.

Schmidt gives the following table :—

1,000 parts of plasma yield—
Mineral matter . ; ; ; i : 8550
Chlorine F ‘ : E ‘ - 3 3°640
SRE RP fot he og 5 SG ED NG
Se ee. ge AOE
Potassium . : P : : ; 3 0°323
Sodium . : é “ “ : é ~ 3°341
Calcium phosphate - ; 5 ; : 0-311
Magnesium phosphate . ; ‘ : ; 0-222

THE WHITE BLOOD CORPUSCLES

These corpuscles are typical animal cells. Their nucleus consists
of nuclein, their cell-protoplasm yields proteids belonging to the

-nucleo-proteid and globulin groups. The nucleo-proteid obtained

from them is the zymogen of the fibrin ferment, the addition of a

_ealcium salt converting it into the ferment. The protoplasm of these
cells often contains small quantities of fat and glycogen.

THE RED BLOOD CORPUSCLES

The red blood corpuscles are much more numerous than the white,
averaging in man 5,000,000 per cubic millimetre, or 400 to 500 red to
_ each white corpuscle. The method of enumeration of the corpuscles
is described in the Appendix.

88 ESSENTIALS OF CHEMICAL PHYSIOLOGY

They vary in size and structure in different groups of vertebrates.
In mammals they are biconcave (except in the camel tribe, where
they are biconvex) non-nucleated discs, in man averaging 35/55 inch
in diameter; during foetal life nucleated red corpuscles are, however,
found. In birds, reptiles, amphibians, and fishes they are biconvex
oval discs with a nucleus: they are largest in the amphibia.

Water causes the corpuscles to swell up, and dissolves out the
red pigment (oxyhemoglobin), leaving a globular colourless stroma.
Salt solution causes the corpuscles to
shrink : they become crenated or wrinkled.
The action of water and salt solution
suggests the existence of a membrane on
the surface of the corpuscles through which
muieen : osmosis takes place, but the existence of

. 29,—a-e, successive effects of

water ona red blood corpuscle; guch a membrane is still a matter of dis-

Jf,a red corpuscle crenated by salt .

pebaton g, action of tannin on cussion. If there is no actual membrane,

the outer denser portion of the stroma plays

the réle of one during osmotic phenomena. Dilute alkalis (0-2 per
cent. potash) dissolve the corpuscles. Dzlute acids (1 per cent.
acetic acid) act like water, and in nucleated corpuscles render the
nucleus distinct. Tannic acid causes a discharge of hemoglobin
from the stroma, but this is immediately altered and precipitated. It
remains adherent to the stroma as a brown globule, consisting
probably of hematin. Boric acid acts similarly, but in nucleated
red corpuscles the pigment collects chiefly round the nucleus, which
may then be extruded from the corpuscles.

Composition.—1,000 parts of red corpuscles contain—

Water ; ; : : . 688 parts
‘ { organic ; } : . 803°88
Solids < O'S ‘
vnedt inorganic . ; ; : Sis...

100 parts of dried corpuscles contain—

Proteid : : . 6 to 12 parts
Hemoglobin : . : . 86 ;,. 94:
Lecithin. : ‘ ; ms i
Cholesterin . ‘ ; ‘ ea he part

The proteid present appears to be identical with the nucleo-
proteid of white corpuscles. The mineral matter consists chiefly of
chlorides of potassium and sodium, and phosphates of calcium and
magnesium. In man potassium chloride is more abundant than
sodium chloride ; this, however, does not hold good for all animals.

_ guinea-pig, or dog; with difficulty

_ also be made by Stein’s method, which
consists in using Canada balsam in-
__ stead of water in the above experiment.

may be obtained by mixing the blood

tere er
aed “is

THE BLOOD 89

Oxygen is contained in combination with the hemoglobin to form
oxyhemoglobin. The corpuscles also contain a certain amount of
carbonic acid (see RESPIRATION, at the end of this lesson).

The pigment of the red corpuscles.—The pigment is by far the
most abundant and important of the constituents of the red cor-

' puscles. It is a substance which gives the reactions of a proteid, but

differs from most other proteids in containing the element iron; it
is also readily crystallisable.

It exists in the blood in two conditions: in arterial blood it is
combined loosely with oxygen, is of a bright red colour, and is called
oxyhzmoglobin ; the other condition is the deoxygenated or reduced
hemoglobin (better called simply hemoglobin). This is found in the
blood after asphyxia. It also occurs in all venous blood—that is,
blood which is returning to the heart after it has supplied the tissues
with oxygen. » Venous blood, however, always contains a considerable
quantity of oxyhemoglobin also. Hemoglobin is the oxygen-carrier
of the body, and it may be called a respiratory pigment.

Crystals of oxyhemoglobin may be obtained with readiness from
the blood of such animals as the rat,

from other animals, such as man, ape,
and most of the common mammals.
The following methods are the best :—

1. Mix a drop of defibrinated
blood of the rat on a slide with a
drop of water; put on a Gover glass;
in a few minutes the corpuscles are
rendered colourless, and then the
oxyhemoglobin crystallises out from
the solution so formed.

2. Microscopical preparations may

3. On a larger scale the crystals jig. 30—oxyhemogiobin crystals magni-

fied : 1, from human blood: 2, from the
guinea-pig ; 3, squirrel; 4, hamster.
with one-sixteenth of its volume of

_ ether; the corpuscles dissolve and the blood assumes a laky appear-
|) ance. After a period, varying from a few minutes to days, abundant
| etystals are deposited.

The accompanying figures represent the form of the crystals so

_ obtained.

90 ESSENTIALS OF CHEMICAL PHYSIOLOGY

In nearly all animals the crystals are rhombic prisms; but in
the guinea-pig they are rhombic tetrahedra (four-sided pyramids) ;
in the squirrel, hexagonal plates; and in the hamster, rhombohedra
and hexagonal plates.

The crystals also contain a varying amount of water of crystallisa- ~
tion: this may in part explain their different crystalline forms
and solubilities.

Different observers have analysed hemoglobin. They find carbon,
hydrogen, nitrogen, oxygen, sulphur, and iron. The percentage of
iron is 0-4. The amounts of the other elements are variously given,
but, roughly, they are the same as in the proteids. We know at
present as little of the chemical structure of hemoglobin as of the
proteids generally.

Oxyhzemoglobin may be estimated in the blood (1) by the amount
of iron in the ash, or (2) by certain colorimetric methods which are
described in the Appendix.

On adding an acid or alkali to hemoglobin, it is broken up into
two parts, a proteid called globin, ani a brown pigment called
hematin, which contains all the iron of the original substance.

Globin is a somewhat unique proteid. It is coagulable by heat,
soluble in dilute acids, and precipitable from such solutions by
ammonia. It closely resembles a substance previously separated
from red corpuscles by Kossel and termed by him histone (Schulz).

Hematin is not crystallisable ; according to Hoppe-Seyler its
formula is C3,H3;,N,FeO;; other observers give different for-
mule. It presents different spectroscopic appearances in acid and
alkaline solutions, which we shall study more fully in the advanced
céurse. It also yields several products under the influence of certain
reagents, which we shall also again consider in the advanced course.
For the present, we will mention only two of these, hemin and
hematoporphyrin.

Hemin is of great importance, as the obtaining of this substance
in a crystalline form is the best chemical test for blood. Hesmin
crystals, sometimes called, after their discoverer, Teichmann’s
crystals, are composed of the hydrochloride of hematin. They
may be prepared for microscopic examination by boiling a frag-
ment of dried blood with a drop of glacial acetic acid on a slide;
on cooling, dark brown plates and prisms belonging to the triclinic
system, often in star-shaped clusters and with rounded angles
(fig. 31), separate out.

In the case of an old blood-stain it is necessary to add a crystal

THE BLOOD 91

of sodium chloride in addition. Fresh blood contains sufficient
sodium chloride in itself.

The action of the acetic acid is (1) to split the hemoglobin into
_ hematin and globin; and (2) to evolve hydrochloric acid from the

sodium chloride. The hematin unites with the hydrochloric acid,

and thus hemin is formed. The formula for hemin is C;,;H,,N,FeClO,
(Moérner).

Hematoporphyrin is iron-free hematin; it may be prepared by
mixing blood with strong sul-

phuric acid: the iron is taken ~ are 7 i *%
out as ferrous sulphate. This wi 7H oe
substance is also found sometimes fa 4% > oh

sc hlUuN
in nature: it occurs in certain ae HWS, > *

invertebrate pigments, and may > =

also be found in certain forms of ,-4 al Way =“ ms
pathological urine. It shows well- rr y J “A ae
marked spectroscopic bands, and Yo at i
so is not identical with the iron. 89 + ' g . $°/

free derivative of hxmoglobin —— 4 7 &*

called hzematoidin whichis formed ric. 31.Hemin crystals magnified. (Preyer.)
in extravasations of blood in the
body (see p. 70). The two substances are possibly isomeric.

COMPOUNDS OF HHZMOGLOBIN WITH GASES

Hemoglobin forms at least four compounds with gases :— _
(1. Oxyhamoglobin.

(2. Methzemoglobin.

With carbonic oxide. 3. Carbonic oxide hemoglobin.
With nitric oxide. 4, Nitric oxide hemoglobin.

With oxygen

These compounds have similar crystalline forms: each probably
consists of a molecule of hemoglobin combined with one of the
gas in question. They part with the cSmbined gas somewhat
readily, and are arranged in order of stability in the above list, the
least stable first.

Oxyhemoglobin is the compound that exists in arterial blood.

Many of its properties have been already mentioned. The oxygen
_ linked to the hemoglobin, which is removed by the tissues through
_ which the blood circulates, may be called the respiratory oxygen of
hemoglobin. The processes that occur in the lungs and tissues,
_ resulting in the oxygenation and deoxygenation respectively of the
hzmoglobin, may be imitated outside the body, using either blood or

92 ESSENTIALS OF CHEMICAL PHYSIOLOGY

pure solutions of hemoglobin. The respiratory oxygen can be
removed, for example, in the Torricellian vacuum of ‘a mercurial air-
pump, or by passing a neutral gas like hydrogen through the blood,
or by the use of reducing agents like ammonium sulphide or Stokes’s
reagent. One gramme of hemoglobin will combine with 1°34 c.c.
of oxygen.

If any of these methods for reducing oxyhemoglobin is used, the
bright red (arterial) colour of oxyhemoglobin changes to the purplish
(venous) tint of hemoglobin. On once more allowing oxygen to
come-into contact with the hemoglobin, as by shaking the solution
withthe air, the bright arterial colour returns.

These colour-changes may be more accurately studied with the
spectroscope, and the constant position of the absorption bands seen
constitutes an important test for blood pigment. It will be first
necessary to describe briefly the instrument used.

The Spectroscope.— When a ray of white light is passed through
a prism, it is refracted or bent at each surface of the prism; the
whole ray is, however, not equally bent, but it is split into its con-
stituent colours, which may be allowed to fall on a sereen. The
band of colours beginning with the red, passing through orange,
yellow, green, blue, and ending with violet, is called a spectrum: this
is seen in nature in the rainbow. It may be obtained artificially by
the glass prism or prisms of a spectroscope.

The spectrum of sunlight is interrupted by numerous ack lines
crossing it vertically, called Fraunhofer’s lines. These are perfectly
constant in position, and serve as landmarks in the spectrum.
The more prominent are A, B, and C, in the red; D, in the yellow;
H, 6, and F, in the green; G and H, in the violet. These lines are
due to certain] volatile substances in the solar atmosphere. If the
light"from burning sodium or its compounds is examined spectro-
scopically, it will be found to give a bright yellow line, or rather —
two bright yellow lines very close together. Potassium gives two
bright red lines and one* violet line; and the other elements, when
incandescent, give characteristic lines, but none so simple as sodium.
If now the flame of a lamp be examined, it will be found to give a
continuous spectrum like that of sunlight in the arrangement of its
colours, but unlike it in the absence of dark lines; but if the light
from the lamp be made to pass through sodium vapour before it
reaches the spectroscope, the bright yellow light will be found

' Stokes’s reagent must always be freshly prepared : it is a solution of ferrous
sulphate to which a little tartaric acid has been added, and then ammonia till the
reaction is alkaline.

THE BLOOD 93

absent, and in its place a dark line, or rather two dark lines very
close together, occupying the same position as the two bright lines
of the sodium spectrum. The sodium vapour thus absorbs the same

Fie. 32.—Diagram of spectroscope.

rays as those which it itself produces at a higher temperature. Thus
the D line, as we term it, in the solar spectrum is due to the presence

Fic. 33.—Spectroscope : A, collimator with adjustable slit at one (left) end and collimating lens at
the other (right) end ; B, telescope moving on graduated are divided into degrees ; C, prism or
combination of prisms; D, tube for scale; E, mirror for illuminating scale; F, vessel with
parallel glass sides for holding fiuid, shown with the fiat side towards the reader ; I, long spectro-
scope bottle for examining a deep layer of fluid; H, Argand burner; G, cond for ¢
trating the light from H on the slit. (From a photograph taken by Dr. MacMunn for
McKendrick’s ‘ Physiology.’)

of sodium vapour in the solar atmosphere. The other dark lines are
similarly accounted for by other elements.

The large form of spectroscope (fig. 32) consists of a tube A, called
the collimator, with a slit at the end S, and a convex lens at the end L.
The latter makes the rays of light passing through the slit from the

94 ESSENTIALS OF CHEMICAL PHYSIOLOGY

source of light parallel: they fall on the prism P, and then the

spectrum so formed is focussed by the telescope T.

The third tube, D, seen in the next figure (fig. 33), carries a small
transparent scale of wave lengths, as in accurate observations the
position of any point in the spectrum is given in the terms of the
corresponding wave-lengths.

If we now interpose betwéen the source of light and the slit S
a piece of coloured glass (H in fig. 32), or a solution of a coloured
substance contained in a vessel with parallel sides (the hamatoscope
of Hermann, F in fig. 33), the spectrum is found to be no longer

continuous, but is interrupted by a number of dark shadows, or

absorption bands, corresponding to the light absorbed by the coloured
medium. Thus a solution of oxyhemoglobin of a certain strength
gives two bands between the D and E lines ; hemoglobin gives only
one ; and other red solutions, though to the naked eye similar to.
oxyhzmoglobin, will give characteristic bands in other positions.

Crownglas Crownglass Crowngelass

Flintglass

Fig. 34.—Arrangement of prisms in direct-vision spectroscope.

A convenient form of small spectroscope is the direct-vision
spectroscope, in which, by an arrangement of alternating prisms
of crown and flint glass, placed as in fig. 34, the spectrum is
observed by the eye in the same line as the tube furnished with
the slit—indeed slit and prisms are both contained in the same
tube.

Such small spectroscopes may be used for class purposes, and
may for convenience be mounted on a stand provided with a gas-
burner and a receptacle for the test-tube (see fig. 35).

In the examination of the spectrum of small coloured objects, a.
combination of the microscope and direct-vision spectroscope, called
the micro-spectroscope, is used.

Fig. 36 illustrates a method of representing absorption spectra.
diagrammatically. The solution was examined in a layer 1 cen-
timetre thick. The base line has on it at the proper distances
the chief Fraunhofer lines, and along the right-hand edges are

etiam

——

THE BLOOD 95

percentages of the amount of oxyhemoglobin present in I, of
hemoglobin in II. The width of the shadings at each level represents

Fic. 35.—Stand for direct-vision spectroscope : S, spectroscope: T, test-tube for coloured
substance under investigation.

the position and amount of absorption corresponding to the per-
centages.

U |

Eb F G ABC D Eb F

I II

Fic. 36.—Graphic representations of the amount of absorption of light by solution (I) of oxyhemo-

globin, (II) of hemoglobin, of different strengths. The shading indicates the amount of absorp-
tion of the spectrum ; the figures on the right border express percentages. (Rollett.)

The characteristic spectrum of oxyhemoglobin, as it actually
appears through the spectroscope, is seen in the nexé figure (fig. 37,

96 ESSENTIALS OF CHEMICAL PHYSIOLOGY

spectrum 2). There are two distinct absorption bands between the
D and E lines; the one nearest to D (the a band) is narrower,
darker, and has better defined edges than the other (the 6 band).
As will be seen on looking at fig. 36, a solution of oxyhsmoglobin of
concentration greater than 0°65 per cent. and less than 0°85 per cent.

(examined in a cell of the usual thickness of 1 centimetre) gives one —

thick band overlapping both D'and EH, and a stronger solution only
lets the red light through between C and D. A solution which gives
the two characteristic bands must therefore be a very dilute one.
The one band (y band) of hemoglobin (fig. 37, spectrum 3) is not so

Cc D Eé F G
is aks > S 2
1 2) iB. 3 8 R

Fic. 37.—1, Solar spectrum ; 2, spectrum of oxyhamoglobin (0°37 per cent. solution) ; 3, spectrum of
hemoglobin; 4, spectrum of CO-hemoglobin} 5, spectrum of methemoglobin (concentrated
solution).

well defined as the a or B bands. On dilution it fades rapidly, so
that in a solution of such strength that both bands of oxyhemoglobin
would be quite distinct the single band of hemoglobin has dis-
appeared from view. The oxyhemoglobin bands can be distinguished
in a solution which contains only one part of the pigment to 10,000
of water, and even in more dilute solutions which seem to be colourless
the a band is still visible.

Methemoglobin.—This may be produced artificially by adding
such reagents as potassium ferricyanide or amyl nitrite to a solution
of oxyhemoglobin ; it may also occur in certain diseased conditions in
the urine ; it is therefore of considerable practical importance. It
can be crystallised, and is found to contain the same amount of

THE BLOOD 97

oxygen as oxyhemoglobin, only combined differently. The oxygen
is not removable by the air-pump, nor by a stream of a neutral gas
like hydrogen. It can, however, by reducing agents like ammonium
_ sulphide, be made to yield hemoglobin. Methemoglobin is of a
_brownish-red colour, and gives a characteristic absorption band in
_ the red between the C and D lines (fig. 37, spectrum 5).

The ferricyanide of potassium or sodium not only causes the
conversion of oxyhemoglobin into methemoglobin, but if the reagent
_ is added to blood which has been previously laked by the addition of
twice its volume of water there is an evolution of oxygen. Ifa small
amount of sodium carbonate or ammonia is added as well to prevent
the evolution of any carbonic acid, and the oxygen is collected and
measured, it is found that all the oxygen previously combined in
oxyhemoglobin is discharged. This is at first sight puzzling, because,
as just stated, methemoglobin contains the same amount of oxygen
that is present in oxyhemoglobin. What occurs is that after the
oxygen is discharged from oxyhemoglobin, an equal quantity of
oxygen takes its place from the reagents added. The oxygen atoms
of the methemoglobin must be attached to a different part of the
hematin group from the oxygen atoms of the oxyhemoglobin, sc
that the hematin group when thus altered loses its power of com-
bining with oxygen and carbonic oxide to form compounds which
are dissociable in a vacuum.

Dr. Haldane, to whom we owe these interesting results, gives the
following provisional equation to represent what occurs :—

HbO, +4Na,;Cy,Fe+4NaHCO;,;=HbO,+4Na,Cy,Fe.

[oxyhzemo- [sodium ferri- {sodium bicar- [methzemo- [sodium ferro-
globin cyanide] bonate] globin] cyanide]
+4C0,+2H,0+03,,

[carbonic [water] [oxygen]
acid]

Carbonic Oxide Hemoglobin may be readily prepared by passing
a stream of carbonic oxide or coal gas through blood or through a
solution of oxyhemoglobin. It has a peculiar cherry-red colour. Its
absorption spectrum is very like that of oxyhemoglobin, but the two
bands are slightly nearer the violet end of the spectrum (fig. 37,
‘Spectrum 4). Reducing agents, like ammonium sulphide, do not
change it; the gas is more firmly combined than the oxygen in
oxyhzemoglobin. CO-hemoglobin forms crystals like those of
oxyhemoglobin : it resists putrefaction for a very long time,
Carbonic oxide is given off during the imperfect’ combustion of
carbon such as occurs in charcoal stoves: this acts as a powerful
H

98 ESSENTIALS OF CHEMICAL PHYSIOLOGY

_ poison by combining with the hemoglobin of the blood, and thus
interfering with normal respiratory processes. The colour of the
blood and its resistance to reducing agents are in such cases charac-
teristic.

Nitric Oxide Hemoglobin.— When ammonia is added to blood, and
then a stream of nitric oxide passed through it, this compound is
formed. It may be obtained in erystals isomorphous with oxy- and
CO-hemoglobin. It also has a similar spectrum. It is even more

stable than CO-hemoglobin ; it has little practical interest, but is of

theoretical importance as completing the series.

Bohr has advanced a theory that hemoglobin forms a compound with
carbonic dioxide, and that there are numerous oxyhemoglobins containing
different amounts of oxygen, but his views have not been accepted.

Very dilute solutions of hemoglobin and its derivatives show an absorption
band in the ultra-violet region in addition to those just described in the visible
regions of the spectrum (see more fully Advanced Course).

CHEMISTRY OF RESPIRATION

The consideration of the blood, and especially of its pigment, is so
closely associated with respiration thata brief account of that process
follows conveniently here.

The lungs consist essentially of numerous little hollow sacs in the
walls of which is a close plexus of capillary blood vessels. These air
sacs, or alveoli, communicate with the external air by the trachea,
bronchi, and bronchial tubes. Inspiration is due to a muscular effort
that enlarges the thorax, the closed cavity in which the lungs are
situated. Owing to the atmospheric pressure the lungs become dis-
tended. The atmospheric air does not, however, actually penetrate
beyond the bronchial tubes; the gases which get into the smallest
tubes and air sacs do so mainly by diffusion. Expiration is ordinarily
brought about by the elastic rebound of the lungs and chest walls, and
is only a muscular effort when forced: but even the most vigorous.

expiratory effort is unable to expel the alveolar air. This air and the.

blood in the capillaries are only separated by the thin capillary and
alveolar walls. The blood parts with its excess of carbonic acid
and watery vapour to the alveolar air; the blood at the same time
receives from the alveolar air the oxygen which renders it arterial.
The intake of oxygen is the commencement, and the output of
carbonic acid the end, of the series of changes known as respiration.
The intermediate steps take place all over the body, and constitute
what is known as internal or tissue respiration. The exchange of
gases which,occurs in Re hE sometimes called in contradistinc-

Pes
ee

THE BLOOD 99

tion external respiration. We have already seen that oxyhemo-
globin is only a loose compound, and in the tissues it parts with
its oxygen. The oxygen does not necessarily undergo immediate
union with carbon to form carbonic acid, and with hydrogen to form
water, but in most cases, as in muscle, is held in reserve by the tissue
itself. Ultimately, however, these two oxides are formed: they are
the chief products of combustion. Certain other products which
represent the combustion of nitrogenous material (urea, urie acid, &c.)
ultimately leave the body by the urine. All these substances pass into
the venous blood, and the gaseous products, carbonic acid, and a
portion of the water find an outlet by the lungs.

Inspired and Expired Air.—The composition of the inspired and
the expired air may be compared in the following table :-—

— Inspired or atmospheric Air Expired Air

| eee

po Oxygen . = . | 20°96 vols. per cent. 16-03 vols. per cent.
_ Nitrogen . ‘ kh ae = a 79 pa m=

| Carbonic acid . iG Des | A. £< |< ',, A
Watery vapour. ; variable saturated

| Temperature . - = that of body (37° C.)

The nitrogen remains unchanged. The recently discovered gases,
argon, erypton, &c., are in the above table reckoned in with the
nitrogen. They are, however, only present in minute quantities.
The chief change is in the proportion of oxygen and carbonic acid.
The loss of oxygen is about 5, the gain in carbonic acid 44. If the
inspired and expired airs are carefully measured at the same tempera-
ture and barometric pressure, the volume of expired air is thus rather
less than that of the inspired. The conversion of oxygen into carbonic
acid would not cause any change in the volume of the gas, for a
molecule of oxygen (O,) would give rise to a molecule of carbonic acid
(CO,), which would occupy the same volume (Avogadro's law). It
must, however, be remembered that carbon is not the only element
which is oxidised. Fats contain a number of atoms of hydrogen
which during metabolism are oxidised to form water; a certain small
amount of oxygen is also used in the formation of urea. Carbo-
hydrates contain sufficient oxygen in their own molecules to oxidise
their hydrogen ; hence the apparent loss of oxygen is least when a
vegetable diet (that is, one consisting largely of starch and other
carbohydrates) is taken, and greatest when much fat and proteid are

eaten. The quotient CO, given off i. called the respiratory quotient.

‘ Oz-absorbed ARTVENT
Of

Sete. ed Se re Ak eB

100 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Normally it is He =0°9, but this varies considerably with diet, as

just stated. It varies also with muscular exercise, when the output
of carbonic acid is much increased both absolutely and relatively to
the amount of oxygen used up.

Gases of the Blood.— From 100 volumes of blood about 60 volumes
of gas can be removed by the mercurial air-pump (see Appendix).
The average composition of this gas from dog’s blood is—

— Arterial Blood | Venous Blood |

Oxygen ; : =| 20 8 to 12 |
Nitrogen. ; A | 1 to 2 | 1 to 2
40 46

Carbonic acid

The nitrogen in the blood is simply dissolved from the air just
as water would dissolve it: it has no physiological importance. The
other two gases are present in much greater amount than can be
explained by simple solution ; they are, in fact, chiefly present in loose
chemical combinations. Less than one volume of the oxygen and about
two of carbonic acid are present in simple solution in the plasma.

Oxygen in the Blood.—The amount of gas dissolved in a liquid
varies with the pressure of the gas; double the pressure andthe amount
of gas dissolved is doubled. Now this does not occur in the ease of
oxygen and blood; very nearly the same amount of oxygen is dissolved
whatever be the pressure. We thus have a proof that oxygen is not
merely dissolved in the blood, but is in chemical union : and the fact
that the oxygen of oxyhemoglobin can be replaced by equivalent
quantities of other gases, like carbonic oxide, is a further proof of the
same statement. The tension or partial pressure of oxygen in the air
of the alveoli is less than that in the atmosphere, but greater than that
in venous blood; hence oxygen passes from the alveolar air into
the blood; the oxygen immediately combines with the hemoglobin,
and thus leaves the plasma free to absorb more oxygen; and this goes
on until the hemoglobin is entirely, or almost entirely, saturated with
oxygen. The reverse change occurs in the tissues where the partial
pressure of oxygen is lower than in the plasma, or in the lymph that
bathes the tissue elements; the plasma parts with its oxygen to the
lymph, the lymph to the tissues; the oxyhemoglobin then undergoes
dissociation to supply more oxygen to the plasma and lymph, and this
in turn to the tissues once more. This goes on until the oxyhemo-
globin loses a great portion of its store of oxygen, but even in asphyxia
it does not lose all. ;

THE BLOOD 101

The following values are given by Fredericq for the tension of
oxygen in percentages of an atmosphere. His experiments were made
on dogs :— c

Alveolar air . - ; 3 = : 18 y
Arterial blood * ‘ : : 14

The arrow shows the direction in which the gas passes.

The methods of obtaining the gases of the blood and analysing
them are described in the Appendix. When the gases are being
pumped off from the blood, very little oxygen comes off until the
pressure is greatly reduced, and then, at a certain point, itis suddenly
disengaged. This shows it is not in simple solution, but is united
chemically to the hemoglobin as oxyhemoglobin, which is dissociated
when the pressure is extremely low.

The avidity of the tissues for oxygen is shown by Ehrlich’s experi-
ments with methylene blue and similar pigments. Methylene blue is
lation of a living animal, and the animal killed a few minutes later,
the blood is found dark blue, but the organs colourless. On ex-
posure to oxygen the organs become blue. In other words the tissues
have removed the oxygen from methylene blue to form a colourless
reduction product; on exposure to the air this once more unites
with oxygen to form methylene blue.

Carbonic Acid in the Blood.— What has been said for oxygen holds
good in the reverse direction for carbonic acid. Compounds are
formed in the tissues where the tension of the gas is high : these pass
into the lymph, then into the blood, and in the lungs the compounds
undergo dissociation, carbonic acid passing into the alveolar air where
the tension of the gas is comparatively low, though it is greater here
than in the expired air.

The relations of this gas and the compounds it forms are more
complex than in the case of oxygen. If blood is divided into plasma
and corpuscles, it will be found that both yield carbonic acid, but the
yield from the plasma is the greater. If we place blood in a vacuum
it bubbles, and gives out all its gases; addition of a weak acid
causes no further liberation of carbonic acid. If plasma or serum is
similarly treated the gas comes off, but from 10 to 18 per cent. of the
carbonic acid is fixed—that is, the addition of some stronger acid,
like phosphoric acid, is necessary to displace it. Fresh red cor-
puscles will, however, take the place of the phosphoric acid, and thus

102 ESSENTIALS OF CHEMICAL PHYSIOLOGY

it has been surmised that oxyhzemoglobin has the properties of an
acid.

One hundred volumes of venous blood contain forty-six volumes
of carbonic acid. Whether this is in solution or in chemical
combination is determined by ascertaining the tension of the gas in
the blood. One hundred volumes of blood plasma would dissolve
more than an equal volume of the gas at atmospheric pressure, if its
solubility in plasma were equal to that in water.' If, then, the
carbonic acid were in a state of solution, its tension would be very
high, but it proves to be only equal to 5 per cent. of an atmosphere.
This means that when venous blood is brought into an atmosphere
containing 5 per cent. of carbonic acid, the blood neither gives off
any carbonic acid nor takes up any from that atmosphere. Hence
the remainder of the gas, 95 per cent., is in a condition of chemical
combination. The chief compound appears to be sodium _bicar-
bonate. ‘

The carbonic acid and phosphoric acid of the blood are in a state
of constant struggle for the possession of the sodium. The salts
formed by these two acids depend on their relative masses. If
carbonic acid is in excess, we get sodium carbonate (Na ,CO;), and
mono-sodium phosphate (NaH,PO,); but if the carbonie acid is
diminished, the phosphoric acid obtains the greater share of sodium
to form disodium phosphate (Na,.HPO,). In this way, as soon as
the amount of free carbonic acid diminishes, as in the lungs, the
amount of carbonic acid in combination also decreases; whereas in
the tissues, where the tension of the gas is highest, a large amount is
taken up into the blood, where it forms sodium bicarbonate.

The tension of the carbonic acid in the tissues is high, but one
cannot give exact figures ; we can measure the tension of the gas in
certain secretions; in the urine it is 9, in the bile 7 per cent. The
tension in the cells themselves must be higher still.

The following figures (from Fredericq) give the tension of carbonic
dioxide in percentages of an atmosphere :-—

Tissues . ; , : 4 5 to9

Venous blood . . , . B88 to 4} in dog. }
Alveolar air .. ‘ : 5 28

External air . : : p 0:04

The arrow indicates the direction in which the gas passes, namely,
in the direction of pressure from the tissues to the atmosphere.

' To be exact, the solubility of carbon dioxide in plasma is a little less than
in pure water.

THE BLOOD 103

In some other experiments, also on dogs, the following are the
_ figures given :— |

Arterial blood . ¢ y x 3 ‘ 28

Venous blood . 2 : é : 3 5-4
b Alveolar air. : 3 c . ‘ 3°56
| Expired air 4 : 4 : ‘ . 2-8

It will be seen from these figures that the tension of carbonic acid
in the venous blood (5:4) is higher than in the alveolar air (3-56) ; its
| passage into the alveolar air is therefore intelligible by the laws of
| diffusion. Diffusion, however, should cease when the tension of the gas

in the blood and alveolar air are equal. But the transference goes
beyond the establishment of such an equilibrium, for the tension of the
gas in the blood continues to sink until it is, when the blood is arterial,
ultimately less (2°8) than in the alveolar air.

The whole question is beset with great difficulties and contradic-
tions. Analyses by different observers have given very different
results, but if such figures as those just quoted are ultimately found to
be correct, we can only explain this apparent reversal of a law of nature
by supposing with Bohr that the alveolar epithelium possesses the
power of excreting carbonic acid, just as the cells of secreting glands
are able to select certain materials from the blood and reject others.
Recent work by Bohr and Haldane has also shown that in all
probability the same explanation—epithelial activity—must be called
in to account for the absorption of oxygen. Haldane, in fact, states that
the tension of oxygen in the blood is greater than in the atmosphere,
In the swim-bladder of fishes (which is analogous to the lungs of
mammals) the oxygen is certainly far in excess of anything that can be
explained by mere diffusion. The storage of oxygen, moreover, ceases
when the vagus nerves which supply the swim-bladder are divided.

Some continental observers have stated that certain noxious
substances are ordinarily contained in expired air which are much
more poisonous than carbonic acid, but researches in this country
have entirely failed to substantiate this. If precautions be taken by
absolute cleanliness to prevent admixture of the air with exhalations
from skin, teeth, and clothes, the expired air only contains one noxious
substance, and that is carbonic acid.

Tissue-Respiration.—Before the processes of respiration were fully
understood the lungs were looked upon as the seat of combustion ; they
were regarded as the stove for the rest of the body where effete
material was brought by the venous blood to be burnt up. When it
was shown that the venous blood going to the lungs already contained

304. >*. ESSENTIALS OF CHEMICAL PHYSIOLOGY

carbonic acid, and that the temperature of the lungs is not greater
than that of the rest of the body, this explanation had of necessity to
be dropped.

Physiologists next transferred the seat of the combustion to the
blood ; but since then innumerable facts and experiments have shown
that itis in the tissues themselves, and notin the blood, that combustion
occurs. The methylene-blue experiments already described (p. 101)
show this ; and the following experiment is also quite conclusive. A
frog can be kept alive for some time after salt solution is substituted
for its blood. The metabolism goes on actively if the animal is kept
in pure oxygen. The taking up of oxygen and giving out of carbonic
acid must therefore occur in the tissues, as the animal has no blood.

Ss ie

105

LESSON X
URINE

1. Test the reaction of urine to litmus paper.

2. Determine its specific gravity by the urinometer.

3. Test for the following inorganic salts :

(a) Chlorides.—Acidulate with nitric acid and add silver nitrate; a white
precipitate of silver chloride, soluble in ammonia, is produced. The object
of acidulating with nitric acid is to prevent phosphates being precipitated by
the nitrate of silver.

(6) Sulphates.— Acidulate with hydrochloric acid and add barium
chloride. A white precipitate of barium sulphate is produced. Hydro-
chloric acid is again added first, to prevent precipitation of phosphates.

(c) Phosphates—i. Add ammonia; a white crystalline precipitate of
earthy (that is, calcium and magnesium) phosphates is produced. This
becomes more apparent on standing. The alkalime (that is, sodium and
potassium) phosphates remain in solution.

ii. Mix another portion of urine with half its volume of nitric acid; add
ammonium molybdate, and boil. A yellow crystalline precipitate falls.
This test is given by both kinds of phosphates.

4. Urea.—Take some urea crystals. Observe that they are readily soluble
in water, and that effervescence occurs when fuming nitric acid (i.e. nitric
acid containing nitrous acid in solution) is added to the solution. The
effervescence is due to the breaking up of the urea. Carbonic acid and
nitrogen come off. A similar bubbling, due to evolution of nitrogen, occurs
when an alkaline solution of sodium hypobromite is added to another
portion of the solution. {

5. Heat some urea crystals in a dry test-tube. Biuret is formed, and
ammonia comes off. Add a drop of copper-sulphate solution and a few drops
of 20-per-cent. potash. A rose-red colour is produced.

6. Quantitative estimation of urea. ‘

For this purpose Dupré’s apparatus (fig. 38) is the most convenient. It
consists of a bottle united to a measuring tube by indiarubber tubing. The
measuring tube (an inverted burette will do very well) is placed within a
eylinder of water, and can be raised and lowered at will. Measure 25 c.c. of
alkaline solution of sodium hypobromite (made by mixing 2 c.c. of bromine
with 23 c.c. of a 40-per-cent. solution of caustic soda) into the bottle.
Measure 5 c.c. of urine into a small tube, and lower it carefully, so that no
urine spills, into the bottle. Close the bottle securely with a stopper per-
forated by a glass tube; this glass tube! is connected to the measuring tube by
indiarubber tubing and a T-piece. The third limb of the T-piece is closed
by a piece of indiarubber tubing and a pinch-cock, seen at the top of the

* The efficiency of the apparatus is increased by having a glass bulb blown on

this tube to prevent froth passing into the rest of the apparatus. This is not
shown in the figure.

106 ESSENTIALS OF CHEMICAL PHYSIOLOGY

figure. Open the pinch-cock and lower the measuring tube until the surface
of the water with which the outer cylinder is filled is at the zero point of the
Sraduation. Close the pinch-cock, and raise the measuring tube to ascertain
if the apparatus is air-tight. Then
lower it again. Tilt the bottle so as
to upset the urine, and shake well for
a minute or so. During this time
there is an evolution of gas. Then
immerse the bottle in a large beaker
containing water of the same tempera-
ture as that in the cylinder. After
two or three minutes raise the measur-
ing tube until the surfaces of the
water inside and outside it are at the
same level. Read off the amount of
gas evolved. This is nitrogen. The
carbonic acid resulting from the de-
composition of urea has been absorbed
by the excess of soda in the bottle.
35'4 cc. of nitrogen are yielded by
0'1 gramme of urea. From this the
quantity of urea in the 5 c.c. of urine
and the percentage of urea can be
calculated. If the total urea passed
in the twenty-four hours is to be
ascertained, the twenty-four hours’
urine must be carefully measured and
thoroughly mixed. A sample is then
taken from the total for analysis; and
then, by a simple sum in proportion,
the total amount of urea is ascertained.
Sometimes the measuring tubes sup-
plied with this apparatus are graduated
in divisions corresponding to percent-
ages of urea.

7. Creatinine.—This substance may
be detected by adding a little sodium
nitro-prusside and caustic soda to the
urine. A red colour develops which
Fie, 38.—Dupré’s urea apparatus. fades on boiling.

The kidney is a compound tubular gland, the tubules of which it
is composed differing much in the character of the epithelium that
lines them in various parts of their course. The true secreting part
of the kidney is the glandular epithelium that lines the convoluted
portions of the tubules; there is in addition to this what is usually
termed the filtering apparatus: tufts of capillary blood vessels called
the Malpighian glomeruli are supplied with afferent vessels from the
renal artery; the efferent vessels that leave these have a smaller
calibre, and thus there is high pressure in the Malpighian capillaries.
Certain constituents of the blood, especially water and salts, pass

ee Ee eee

URINE - 107

through the thin walls of these vessels into the surrounding Bow-
man’s capsule which forms the commencement of each renal tubule.

- Bowman’s capsule is lined by a flattened epithelium, which is reflected

over the capillary tuft. Though the process which occurs here is
generally spoken of as a filtration, yet it is no purely mechanical
process, but the cells exercise a selective influence, and prevent the
albuminous constituents of the blood from escaping. During the
passage of the water which leaves the blood at the glomerulus through
the rest of the renal tubule, it gains the constituents urea, urates,
&c., which are poured into it by the secreting cells of the convoluted
tubules.

The term excretion is better than secretion as applied to the
kidney, for the constituents of the urine are not actually formed in
the kidney itself (as, for instance, the bile is formed in the liver), but
they are formed elsewhere; the kidney is simply the place where
they are picked out from the blood and eliminated from the body.

GENERAL CHARACTERS OF URINE

Quantity A man of average weight and height passes from 1,400
to 1,600 c.c., or about 50 oz. daily. This contains about 50 grammes
(13 0z.) of solids. The urine should be collected in a tall glass vessel —
capable of holding 3,000 c.c., which should have a smooth-edged neck
accurately covered by a ground-glass plate to exclude dust and avoid
evaporation. The vessel, moreover, should be graduated so that the
amount may be easily read off. From the total quantity thus
collected in the twenty-four hours, samples should be drawn off for
examination.

Colour.—This is some shade of yellow which varies considerably
in health with the concentration of the urine. It appears to be due
to a mixture of pigments: of these wrobilin is the one of which we
have the most accurate knowledge. Urobilin has a reddish tint and
is ultimately derived from the blood pigment, and like bile pigment
is an iron-free derivative of hemoglobin. The bile pigment (and
possibly also the hematin of the food) is in the intestines converted
into stercobilin; most of the stercobilin leaves the body with the
feces ; but some is reabsorbed and is excreted with the urine as
urobilin. Urobilin is very like the artificial reduction product of
bilirubin called hydrobilirubin (see p. 73). Normal urine, however,
contains very little urobilin. The actual body present is a chromogen
or mother substance called urobilinogen, which by oxidation (such as
occurs when the urine stands exposed to the air) is converted into

108 ESSENTIALS OF CHEMICAL PHYSIOLOGY

the pigment proper. In certain diseased conditions the amount of
urobilin is considerably increased.

The most abundant urinary pigment is a yeliow’6 one called
urochrome. It shows no absorption bands. It is probably an oxi-
dation product of urobilin (Riva, A. E. Garrod). (See Lesson XXVI.)

Reaction.—The reaction of normal urine is acid. This is not due
to free acid, as the uric and hippuric acids in the urine are combined
as urates and hippurates respectively. The acidity is due to acid salts,
especially acid sodium phosphate. Under certain circumstances the
urine becomes less acid and even alkaline; the most important of
these are as follows :—

1. During digestion. Here there is a formation of free acid in
the stomach, and a corresponding liberation of bases in the blood

Fie. 40.—Crystals of urea : a, four-sided

prisms ; 6, indefinite crystals, such

Fic. 39.—Urinometer floating in urine as are ustally formed from alcohol
in a testing glass. solutions.

which passing into the urine diminish its acidity, or even render it
alkaline. This is called the alkaline tide ; the opposite condition, the
acid tide, occurs after a fast, for instance, before breakfast.

2. In herbivorous animals and vegetarians. The food here con-
tains excess of alkaline salts of acids like tartaric, citric, malic, &c.
These acids are oxidised into carbonates, which passing into the urine
give it an alkaline reaction.

Specific Gravity —This should be taken in a spl of the twenty-
four hours’ urine with a good urinometer (see fig. 39)

The specific gravity varies inversely as the quantity of urine
passed under normal conditions from 1015 to 1025. A specific
gravity below 1010 should excite suspicion of hydruria; one over
1030 of a febrile condition, or diabetes, a disease in which it may rise

URINE Poe ae

to 1050. The specific gravity has, however, been known to sink as
low as 1002 (after large potations, wrina potus), or to rise as high as
1035 (after great sweating) in perfectly healthy persons.

Composition.—The following table gives the average amounts of
the urinary constituents passed by a man in the twenty-four
hours :—

Water 1500-00 grammes
Total solids 72-00 a
Urea . 33°18 a
Uric acid 0°55 13
Hippuric acid 0-40 pi
Creatinine ; 0-91 z
Pigment and ‘other organic » substances ‘ 10-00 ri
Sulphuric acid : : 2°01 -
Phosphoric acid : r F 2 3°16 te.
Chlorine : : 3 : ‘ 7-50 ‘a
Ammonia { “ 0-77 bs
Potassium 2-50 pe
Sodium 11-09 a
Calcium 0-26 Ms
Magnesium 0-21 a

The most abundant constituents of the urine are water, urea, and
sodium chloride. In the foregoing table the student must not be
misled by seeing the names of the acids and metals separated. The
acids and the bases are combined to form salts :—urates, chlorides,
sulphates, phosphates, &c.

UREA

Urea, or Carbamide, CO(NH.,)s, is isomeric (that is, has the same
empirical, but not the same structural formula) with ammonium
cyanate (NH,)CNO, from which it was first prepared synthetically by
Wohler in 1828. Since then it has been prepared synthetically in
other ways. Wohler’s observation derives interest from the fact that
this was the first organic substance which was prepared synthetically
by chemists.

It may be crystallised out from the urine, and it is then found to
be readily soluble both in water and in alcohol: it has a saltish taste,
and is neutral to litmus paper. The form of its crystals is shown in

fig. 40.

wb treated with nitric acid, nitrate of urea (CON,H,.HNO,) is
formed; this crystallises in octahedra, lozenge-shaped tablets, or
hexagons (fig. 41, a). When treated with oxalic acid, flat or pris-
matic crystals of urea oxalate (CON,H,.H.C,0,+H,O) are formed
(fig. 41, 5).

110 ESSENTIALS OF CHEMICAL PHYSIOLOGY

These crystals may be readily obtained in an impure form by
adding excess of the respective acids to urine which has been con-
centrated to a third or a quarter of its bulk."

Under the influence of an organised ferment, the torula or micro-
coceus ure, which grows readily in stale urine, urea takes up water,
and is converted into ammonium carbonate [CON,H,+2H,0=
(NH,).CO;]. Hence the ammoniacal odour of putrid urine.

By means of nitrous acid, urea is broken up into carbonic acid,
water, and nitrogen, CON,H,+2HNO,=CO,+3H,0+2N,. This.
may be used as a test for urea. Add fuming nitric acid (i.e. nitric acid
containing nitrous acid in solution) to a solution of urea, or to urine ;
an abundant evolution of gas bubbles takes place.

Fic, 41.—a, nitrate; b, oxalate of urea.

Hypobromite of soda decomposes urea in the following way :—
CON,H, + 3NaBrO = CO, a No 4+ 2H,O + 3NaBr

{urea] {sodium] {earbonie [nitrogen] [water] {sodium
hypobromite] acid] bromide]

This reaction is important, for on it one of the readiest methods for
estimating urea depends. There have been various pieces of appa-
ratus invented for rendering the analysis easy ; but the one described
in the practical exercise at the head of this lesson appears to be the
best. If the experiment is performed as directed, nitrogen is the
only gas that comes off, the carbonic acid being absorbed by excess
of soda. The amount of nitrogen is a measure of the amount of
urea.

The quantity of urea excreted is somewhat variable, the chief cause
of variation being the amount of proteid food ingested. Inamanina

1 The preparation of urea nitrate and urea oxalate is postponed to the next,
lesson, when other microscopic crystals will also be under examination.

URINE .

state of equilibrium the quantity of urea excreted daily averages 33
grammes (500 grains). The normal percentage in human urine is
2 per cent.; but this also varies, because the concentration of the
urine varies considerably in health. In dogs it may be 10 per cent.
The excretion of urea is usually at a maximum three hours after a
meal, especially after a meal rich in proteids. The urea does not
come, however, direct from the food; the food must be first assimi-
lated, and become part of the body before it can break down to form
urea. An exception to this rule is to be found in the case of the amido-
acids, especially leucine and arginine, which are formed in the intes-
tinal canal from proteids duringdigestion. These substancesare carried
to the liver and converted into urea ; but only a very small fraction of
the urea in the urine is formed in this way. Food increases the elimi-
nation of urea because it stimulates the tissues to increased activity ;
their waste nitrogenous products are converted into urea, which,
passing into the blood, is directly excreted by the kidneys. The
greater the amount of proteid food given, the more waste producis do
the tissues discharge from their protoplasm, in order to make room
for the new proteid which is built into its substance.

Muscular exercise has little immediate effect on the amount of
urea discharged. During intense muscular work there is a slight im-
mediate increase of urea, but this is quite insignificant when compared
with the increase of work. This is strikingly different from what occurs
in the case of carbonic acid; the more the muscles work, the more
carbonic acid do they send into the venous blood, which is rapidly
discharged by the expired air. Careful research has, however, shown
that an increase of nitrogenous waste does occur on muscular exertion,
but appears as urea in the urine to only a slight extent on the day of
the work ; the greater part is excreted during the next day.

Where is Urea formed ?—The older authors considered that it
was formed in the kidneys, just as they also erroneously thought that
carbonic acid was formed in the lungs. Prévost and Dumas were the
first to show that after complete extirpation of the kidneys the forma-
tion of urea goes on, and that it accumulates in the blood and tissues.
Similarly, in those cases of disease in which the kidneys cease work,
urea is still formed and accumulates. This condition is called wremia
(or urea in the blood), and unless the urea be discharged from the
body the patient dies. There is no doubt, however, thatit is not urea
but some antecedent of urea that acts most poisonously, and is the
cause of death, for considerable quantities of urea can be injected into
the circulation without untoward results.

Where, then, is the seat of urea formation? Nitrogenous waste

112 ESSENTIALS OF CHEMICAL PHYSIOLOGY

occurs in all the living tissues, and the principal final result of this
proteid metabolism is urea. It may not be that the formation of urea
is perfected in each tissue, for if we look to the most abundant tissue,
the muscular tissue, very little urea is to be found. Yet there can be
no doubt that the chief place from which urea ultimately comes is the
muscular tissue. Some intermediate step occurs in the muscles; the
final steps occur elsewhere.

In muscles we find a substance called creatine in fairly large quan-
tities. If creatine is injected into the blood it is discharged as crea-
tinine. But there is very little creatinine in normal urine; what
little there is can be nearly all accounted for by the creatine in the
food ; if the muscular creatine and creatinine are discharged as urea,
they must undergo some further change before they leave the
muscle. !

Similarly, other cellular organs, spleen, lymphatic glands, secreting
glands, participate in the formation of urea; but the most important
appears to be the liver: at any rate this is the organ where the final
changes take place. The urea is then carried by the blood to the
kidney, and is there excreted.

The facts of experiment and of pathology point very strongly in
support of the theory that urea is formed in the liver. The principal
are the following :—

1. After removal of the liver in such animals as frogs, urea forma-
tion almost ceases, and ammonia is found in the urine instead.

2. In mammals, the extirpation of the liver is such a serious
operation that the animalsdie. But the liver of mammals can be very
largely thrown out of gear by the operation known as Hck’s fistula,
which consists in connecting the portal vein directly to theinferior vena
cava. Under these circumstances the liver receives blood only by the
hepatic artery. The amount of urea is lessened, and its place is taken
by ammonia.

3. When degenerative changes occur in the liver, asin cirrhosis of
that organ, the urea formed is much lessened, and its place is taken
by ammoma. In acute yellow atrophy urea is almost absent in the
urine, and, again, there is considerable increase in the ammonia. In
this disease leucine and tyrosine are also found in the urine; undue
stress should not be laid upon this latter fact, for the small amounts
of leucine and tyrosine found doubtless originate in the intestine, and,
escaping further decomposition in the degenerated liver, pass as such
into the urine.

We have to consider next the intermediate stages between proteid
and urea. A few years ago Drechsel succeeded in artificially pro-

eatin tein i i ca lel

URINE a

ducing urea from casein. More recent work has shown that this is
true for other proteids also. If a proteid is decomposed by hydro-
chloric acid, a little stannous chloride being added to prevent oxida-
tion, a number of products are obtained, such as ammonium salts,
leucine, tyrosine, aspartic and glutaminic acids. This was known
before, so the chief interest centres round two new substances,
precipitable by phosphotungstic acid. One of these is called lysine
(C,H,,N.O., probably di-amido-caproic acid); the other was first
ealled lysatinine. Hedin then showed that lysatinine is a mixture of
lysine with another base called arginine (C;H,,N,O.); it is from the
arginine that the urea comes in the experiment to be next described.
Arguing from some resemblances between this substance and creatine,
Drechsel expected to be able to obtain urea from it, and his expectation
was confirmed by experiment. He took a silver compound of the
base, boiled it with barium carbonate, and after twenty-five minutes’
boiling obtained urea. Drechsel’s comparison of arginine to creatine
has turned out to be correct; on decomposition it breaks up into
di-amido-valerianic acid and cyanamide (CN.NH,) from which the
urea originates. (Schulze and Winterstein.) Lysine and arginine
are two of the hexone bases (see p. 32).

It is, however, extremely doubtful whether the chemical decom-
positions produced in laboratory experiments on proteids are com-
parable with those occurring in the body. Many physiologists consider
that the amido-acids are intermediate stages in the metabolic pro-
cesses that lead to the formation of urea from proteids. We have
already alluded to this question in relation to the creatine of muscle,
and we are confronted with the difficulty that injection of creatine
into the blood leads to an increase not of urea, but of creatinine in
the urine. If creatine is an intermediate step, it must undergo some
further change before it leaves the muscle. Other amido-acids, such
as glycocine (amido-acetic acid) and leucine (amido-caproic acid) and
arginine, are to be included in the same category. The facts upon
which such a theory depends are (1) that the introduction of glycocine
or leucine into the bowel, or into the circulation, leads to an increase of
urea in the urine; there is, however, no evidence that tyrosine acts in
this way; and (2) that amido-acids appear in the urine of patients
suffering from acute yellow atrophy of the liver. Then again it is
perfectly true that, in the laboratory, urea can be obtained from
creatine, and also from uric acid, but such experiments do not prove
that creatine or uric acid is a normally intermediate product of urea
formation in the body. Still, if we admit for the sake of argument

114 ESSENTIALS OF CHEMICAL PHYSIOLOGY

that amido-acids are normally intermediate stages in preted meta-
bolism, and glance at their formule—

Glycocine, C,.H;NO, Creatine, C,H,N,O,
Leucine, C,H,;NO, - Arginine, C,H,,N,0,

—we see that the carbon atoms are more numerous than the nitro-
gen atoms. In urea, CON.H,, the reverse is the case. The amido-
acids must therefore be split into simpler compounds, which unite
with one another to form urea. Urea formation is thus in part
synthetic. There have been various theories advanced as to what
these simpler compounds are. Some have considered that cyanate,
others that carbamate, and others still that carbonate of ammonium
is formed. Schréder’s work proves that ammonium carbonate is one
of the urea precursors, if not the principal one. The equation which
represents the reaction is as follows :—
(NH,),CO,—2H,O=CON,H,

{ammonium [water] [urea]
carbonate]

Schréder’s principal experiment was this: a mixture of defibrinated
blood and ammonium carbonate was injected into the liver by the
portal vein ; the blood leaving the liver by the hepatic vein was found
to contain urea in great abundance. This does not occur when thesame
experiment is performed with any other organ of the body, so that
Schréder’s experiments also prove the great importance of the liver
in urea formation. Similar results were obtained by Nencki with
ammonium carbamate.

There is, however, no necessity to suppose that the formation of
amido-acids is a necessary preliminary to urea formation. The con-
version of the leucine and arginine formed in the intestine into
ammonium salts and then into urea does certainly occur, but this
only accounts for quite an insignificant fraction of the urea in the
urine. If this also occurs in tissue metabolism, we ought to find
considerable quantities of leucine, glycocine, creatine, arginine, and
such substances in the blood leaving the various tissues and entering
the liver; but we do not. We do, however, constantly find ammonia,
which, after passing into the blood or lymph, has united with car-
bonic acid to form either carbonate or carbamate of ammonium. It
is quite probable that the nitrogenous waste that leaves the muscles
and other tissues is split off from them as ammonia, and not in the
shape of large molecules of amido-acid which are subsequently con-
verted into ammonia. The experiments outside the body which most
closely imitate those occurring within the body are those of Drechsel,

———

URINE 115

in which he passed strong alternating currents through solutions of
proteid-like materials. Such alternating currents are certainly
absent in the body, but their effect, which is a rapidly changing series
of small oxidations and reductions, is analogous to metabolic pro-
cesses; under such circumstances the carbon atoms are burnt off as
carbon dioxide, the nitrogen being split off in the form of ammonia,
and by the union of these two substances ammonium carbonate is
formed.

The following structural formule show the relationship between
ammonium carbonate, ammonium carbamate, and urea.

NH NH
O=C<O NE O=C<ONH, O=C<yH,

[ammonium pare {ammonium carbamate] {urea or carbamide]

The loss of one molecule of water from ammonium carbonate pro-
duces ammonium carbamate; the loss of a second molecule of water
produces urea.

THE INORGANIC CONSTITUENTS OF URINE

The inorganic or mineral constituents of urine are chiefly chlorides,
phosphates, sulphates, and carbonates ; the metals with which these
are in combination are sodium, potassium, ammonium, calcium, and
magnesium. The total amount of these salts varies from 19 to 25
grammes daily. The most abundant is sodium chloride, which
averages in amount 10 to 13 grammes per diem. These substances
are derived from two sources—first from the food, and secondly as the
result of metabolic processes. The chlorides and most of the phos-
phates come from the food ; the sulphates and some of the phosphates
are a result of metabolism. The salts of the blood and of the urine
are much the same, with the important exception that, whereas the
blood contains only traces of sulphates, the urine contains abundance
of these salts. The sulphates are derived from the changes that occur
in the proteids of the body ; the nitrogen of proteids leaves the body
as urea and uric acid ; the sulphur of the proteids is oxidised to form
sulphuric acid, which passes into the urine in the form of sulphates.
The execretion of sulphates, moreover, runs parallel to that of urea.
The chief tests for the various salts have been given in the practical
exercises at the head of this lesson.

Chlorides.—The chief chlorideis that of sodium. The ingestion of
sodium chloride is followed by its appearance in the urine, some on the
same day, some on the next day. Some is decomposed to form the
hydrochloric acid of the gastric juice. The salt, in passing through

116. ESSENTIALS OF CHEMICAL PHYSIOLOGY

the body, fulfils the useful office of stimulating metabolism and
excretion.

Sulphates.—The sulphates in the urine are principally those of
potassium and sodium. They-are derived from the metabolism of
proteids in the body. Only the smallest trace enters the body with the
food. Sulphates have an unpleasant bitter taste (for instance, Epsom
salts) ; hence we do not take food that contains them. The sulphates
vary in amount from 1°5 to 3 grammes daily.

In addition to these sulphates there is a small quantity of
sulphuric acid comprising about one-tenth of the total which
is combined with organic radicles; the compounds are known as
ethereal sulphates, and they originate from putrefactive processes
occurring in the intestine. The most important of these ethereal
sulphates are phenyl sulphate of potassium and indoxyl sulphate
of potassium. The latter originates from the indole formed in the
intestine, and as it yields indigo when treated with certain reagents
it is sometimes called indican. It is very important to remember
that the indican of urine is not the same thing as the indican of
plants. Both yield indigo, but there the resemblance ceases.

The equation representing the formation of potassium phenyl-
sulphate is as follows :--

C,H,0H+80,<0F =80,<0Cs4s 4 1,0

{phenol} {potassium {potassium {water ]
hydrogen sulphate] phenyl-sulphate]

The formation of potassium indoxyl-sulphate may be represented
as follows:— Indole, C,H, eae .CH
C,H, eee .OH.CH

, on absorption is converted

into indoxy] :

Indoxyl then interacts with potassium hydrogen sulphate as
follows :—

- CH,NO + 80, OF —g0, <OCssN 4,0
{indoxy!] [potassium {potassium ’
hydrogen sulphate] todoxyieniphate) { water]

‘The formation of such sulphates is important ; the aromatic sub-
stances liberated by putrefactive processes in the intestine are
poisonous, but their conversion into’ethereal sulphates renders them
innocuous.

Carbonates.—Carbonates and bicarbonates of sodium, calcium,
magnesium, and ammonium are only present in alkaline urine. They
arise from the carbonates of the food, or from vegetable acids (malic,

URINE | 117

tartaric, &c.) in the food. They are, therefore, found in the urine of
herbivora and vegetarians, whose urine is thus rendered alkaline.
Urine containing carbonates becomes, like saliva, cloudy on standing,
the precipitate consisting of calcium carbonate, and also phosphates.

Phosphates—Two classes of phosphates occur in normal
urine :-—

(1) Alkaline phosphates—that is, phosphates of sodium (abundant)
and potassium (scanty).

(2) Earthy phosphates—that is, phosphates of calcium (abundant)
and magnesium (scanty).

The composition of the phosphates in urine is liable to variation.
In acid urine the acidity is due to the acid salts. These are chiefly
sodium dihydrogen phosphate, NaH,PO,, and calcium dihydrogen
phosphate, Ca(H,PO,)>.

In neutral urine, in addition, disodium hydrogen phosphate
(Na,HPO,), calcium hydrogen phosphate, CaHPO,, and magnesium
hydrogen phosphate, MgHPO,, are found. In alkaline urine there
may be instead of, or in addition to the above, the normal phosphates
of sodium, calcium, and magnesium [Na;PO,, Ca3(PO,)2, Mg3(POx,)2]-

The earthy phosphates are precipitated by rendering the urine
alkaline by ammonia. In decomposing urine ammonia is formed
from the urea: this also precipitates the earthy phosphates. The

phosphates most frequently found in the white creamy precipitate
_ which occurs in decomposing urine are—

(1) Triple phosphate or ammonio-magnesium
phosphate (NH,MgPO,+6H,0). This crystallises
in ‘ coffin-lid ’ crystals (see fig. 42) or feathery stars.

(2) Stellar phosphate, or calcium phosphate,
which crystallises in star-like clusters of prisms.

As a rule normal urine gives no precipitate when
it is boiled; but sometimes neutral, alkaline, and
occasionally faintly acid urines give a precipitate of
calcium phosphate when boiled; this precipitate is
amorphous, and is liable to be mistaken for albumin.
It may be distinguished readily from albumin, as it 6. ate aeiad
is soluble in a few drops of acetic acid, whereas _ phosphate.
coagulated proteid does not dissolve.

The phosphoric acid in the urine chiefly originates from the phos-
phates of the food, but is partly a decomposition product of the phos-
phorised organic materials in the body, such as lecithin and nuclein.
The amount of P.O; in the twenty-four hours’ urine varies from 2°5
to 3°5 grammes, of which the earthy phosphates contain about half
(1 to 1°5 gr.).

118 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON XI
URINE (continued)

1. Urea Nitrate——Evaporate some urine in a capsule to a quarter of its
bulk. Pour the concentrated urine into a watch-glass ; let it cool, and add a
few drops of strong, but not fuming, nitric acid. Crystals of urea nitrate
separate out. Examine these microscopically.

2. Urea Oxalate.—Concentrate the urine as in the last exercise, and add
oxalic acid; crystals of urea oxalate separate out. Examine these micro-
scopically.

3. Urie Acid —Examine microscopically the crystals of uric acid in some
urine, to which 5 per cent. of hydrochloric acid has been added twenty-four
hours previously. Note that they are deeply tinged with pigment, and to the
naked eye look like granules of cayenne pepper.

When microscopically examined, the crystals are seen to be large bundles,
principally in the shape of barrels, with spicules projecting from the ends,
and whetstones. If oxalic acid is used instead of hydrochloric acid in this
experiment, the crystals are smaller, and more closely resemble those observed
in pathological urine in cases of uric acid gravel (see fig. 48).

Dissolve the crystals in caustic potash and then carefully add excess of
hydrochloric acid. Small crystals of uric acid again form.

Murexide Test.—Place a little uric acid, or a urate (for instance, serpent’s
urine), in a capsule ; add a little dilute nitric acid and evaporate to dryness.
A yellowish-red residue is left. Adda little ammonia carefully. The residue
turns to violet. This is due to the formation of murexide or purpurate of
ammonia. On the addition of potash the colour becomes bluer.

Schiff’s Test.—Dissolve some uric acid in sodium carbonate solution. Put
a drop of this on blotting paper, add a drop of silver nitrate, and warm gently;
the black colour of reduced silver is seen on the paper.

4, Deposit of Urates or Lithates (Lateritious Deposit).—The specimen of
urine from the hospital contains excess of urates, which have become deposited
on the urine becoming cool. They are tinged with pigment (uroerythrin),
and have a pinkish colour, like brickdust; hence the term ‘ lateritious.’
Examine microscopically. The deposit is usually amorphous—that is, non-
crystalline. Sometimes crystals of calcium oxalate (envelope crystals—
octahedra) are seen also; these are colourless.

The deposit of urates dissolves on heating the urine. }

5. Deposit of Phosphates.—Another specimen of pathological urine contains
excess of phosphates, which have formed a white deposit on the urine be-
coming alkaline. This precipitate does not dissolve on heating; it may be
increased. It is, however, soluble in acetic acid. Examine microscopically
for coffin-lid crystals of triple phosphate (ammonio-magnesium phosphate),
for crystals of stellar (calcium) phosphate, and for mucus. Mucusis flocculent
to the naked eye, amorphous to the microscope.

N.B.—On boiling neutral, alkaline, or even faintly acid urine it may be-
come turbid from deposition of phosphates. The solubility of this deposit in

i i

URINE 119

a few drops of acetic acid distinguishes it from albumin, for which it is
liable to be mistaken.

Some of the facts described in the foregoing exercises have been already
dwelt upon in the preceding lesson. They are, however, conveniently grouped
nee here, as all involve the use of the microscope.

We have now studied urea, the principal nitrogenous constituent
of urine, at some length. There are still left for our consideration a
number of other nitrogenous constituents, the most important of which
are uric acid, hippuric acid, and creatinine.

URIC ACID

Uric acid (C;N,H,O,) is in mammals the medium by which only a
small quantity of nitrogen is excreted from the body. It is, however,
in birds and reptiles the principal nitrogenous constituent of their
urine. It is not present in the free state, but is combined with bases
to form urates.

It may be obtained from human urine by adding 5 c.c. of hydro-
chloric acid to 100 c.c. of the urine, and allowing the mixture to stand
for twelve to twenty-four hours. The crystals which form are deeply
tinged with urinary pigment, and though by repeated solution in-
caustic soda or potash, and reprecipitation
by hydrochloric acid, they may be obtained Dae
fairly free from pigment, pure uric acid is pe
more readily obtained from the solid urineof “7 Loe Si es
a serpent or bird, which consists principally BS a a
of the acid ammonium urate. This is dis- g =
solved in soda, and then the addition of BS 8 e S
hydrochloric acid produces as before the B JZ
erystallisation of uric acid from the solution. § mG

The pure acid crystallises in colourless s)

rectangular plates or prisms. _ In striking ci
contrast to urea it is a most insoluble sub- 3 & A&R
stance, requiring for its solution 1,900 parts ;
of hot and 15,000 parts of cold water. The *™ *—°"e eit crystals.
forms which uric acid assumes when precipitated from human urine,
either by the addition of hydrochloric acid or in certain pathological
processes, are very various, the most frequent being the whetstone
shape ; there are also bundles of crystals resembling sheaves, barrels,
and dumb-bells (see fig. 43).

The murexide test which has just been described among the

120 ESSENTIALS OF CHEMICAL PHYSIOLOGY

practical exercises is the principal test for uric acid. The test has
received the name on account of the resemblance of the colour to the
purple of the ancients, which was obtained from certain snails of the
genus Mure.

Another reaction that uric acid undergoes (though it is not appli-
cable as a test) is that on treatment with certain oxidising reagents
urea and oxalic acid can be obtained fromit. Itis, however, doubtful
whether a similar oxidation occurs in the normal metabolic processes
of the body (see p. 113).

Uric acid is dibasic, and thus there are two classes of urates—the
normal urates and the acid urates. A normal urate is one in which
two atoms of the hydrogen are replaced by two of a monad metal like
sodium; an acid urate is one in which only one atom of hydrogen is
thus replaced. The formule would be—

C;H,N,O,=urie acid
C;H,;NaN,O,=acid sodium urate
C;H,Na,.N,O,;=normal sodium urate

The acid sodium urate is the chief constituent of the pinkish deposit
of urates, which, as we have already stated, is called the lateritious
deposit.

The quantity of uric acid excreted by an adult varies from 7 to
10 grains (0°5 to 0°75 gramme) daily.

The best method for determining the quantity of uric acid in the
urine is that of Hopkins. Ammonium chloride in crystals is added
to the urine until no more will dissolve. This saturation completely
precipitates all the uric acid in the form of ammonium urate. After
standing for two hours the precipitate is collected on a filter, washed
with saturated solution of ammonium chloride, and then dissolved in
weak alkali. From this solution the uric acid is precipitated by
neutralising with hydrochloric acid.. The precipitate of uric acid is
collected on a weighed filter, dried and weighed, or titration may be
performed with potassium permanganate (see Advanced Course).

Origin of Uric Acid.—Uric acid is not made by the kidneys.
When the kidneys are removed uric acid continues to be formed and
accumulates in the organs, especially in the liver and spleen. The
liver has been removed from birds, and uric acid is then hardly formed
at all, its place being taken by ammonia and lactic acid. It is there-
fore probable that ammonia and lactic acid are normally synthesised
in the liver to form uric acid.

The principal conditions which lead to an increase of uric acid in
the urine are—

PE —_—

URINE as

1. Increase of meat diet and diminution of oxidation processes,
such as occur in people with sedentary habits.

2. Increase of white corpuscles in the blood, especially in the
disease known as leucocythemia. This latter fact is of great interest,
as leucocytes contain large quantities of nuclein. Nuclein yields
nitrogenous bases which are closely related to uric acid.

Purine Substances.—The bases just alluded to are sometimes
called the xanthine bases, because xanthine is a prominent member
of the group. Sometimes they are termed the nuclein bases, because
of their origin from nuclein. Another name they have received is
the alloxuric bases, because they contain in combination two radicles,
one of alloxan, and the other of urea. Emil Fischer’s recent work,
however, has shown that they are to be rather regarded as deriva-
tives of a substance he has named purine ; hence the name by which
they are generally called now, the purine bases. The empirical
formule for purine, the purine bases, and uric acid are as follows :—

Purine . . C,H,N,

Hypoxanthine. C;H,N,O Monoxypurine

Xanthine . - C,;H,N,0O, Dioxypurine Purine
Adenine . . C3H,N,NH Amino-purine bases
Guanine . . C;H,N,O.NH Amino-oxypurine

Uric acid . - ©,H,N,O,; Trioxy-purine

There are a vast number of purine derivatives, but only a few of
them have at present any physiological importance. Others in addition
to those already enumerated are -theophylline (dimethyl-xanthine),
theobromine (also a dimethyl-xanthine), caffeine (trimethyl-xanthine) ;
these are of interest, as they occur in tea, cocoa, and coffee. A few
words more may be added in respect to those in our list.

Purine itself-has never been discovered in the body. It has the
following structural formula :

N=C—H

es
H—C C—NH

1 »o-#
N-—-C—N

The purine nucleus is depicted in the next formula, and its atoms

have been empirically numbered as shown for convenience of
description :—

122 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Hypoxanthine or Sarcine is found in the body tissues and fluids,
and in the urine. It is derived from some nucleins, especially those
from fishes’ spermatozoa. It may be termed 6-oxypurine, as the
oxygen is attached to the atom numbered 6 in the purine nucleus.

Xanthine is found with hypoxanthine in the body, and has been
obtained from a number of nucleins (from spermatozoa, thymus,
pancreas, &c.). It is 2, 6-dioxypurine, its oxygen atoms being
attached to the atoms numbered 2 and 6 in the purine nucleus.

NH —C=O NH —-C=0
| |
H—- C — NH oe O an é —NEX
Il I = II C—H
se At ine NH—C— N74
([hypoxanthine] {xanthine}

Adenine is found in the tissues, blood, and urine. It is obtained
from several nucleins, but especially from the nuclein derived from
the thymus. It is 6-amino-purine.

Guanine is also a decomposition product of nucleins, especially
of that obtained from the pancreas. Combined with calcium it
gives the brilliancy to the scales of fishes, and is also found in the
bright tapetum of the eyes in these animals. It is a constituent of
guano, and here is probably derived from the fish eaten by marine
birds. It is 2-amino-6-oxypurine.

N=C—NH, Nokes.
byt |
H-—-C C—NH H,N—C C— NH
Yo YC - H
alee: Vs Il Il po
N-C— N N —C- WN
{adenine] (guanine]

Uric Acid is 2, 6, 8-trioxypurine.

NH —C=O0O

| |
O=O ae NHL

NH O— a
{uric acid]

We thus see the close relationship between uric acid and the
nuclein bases. Leaving aside other possible ways in which uric
acid is undoubtedly formed in the organism, we have here a way in
which uric acid may arise by oxidation from the nuclein bases and
thus ultimately from the nuclei of cells. Certain forms of diet
inerease uric acid formation by leading to an increase of leucocytes

URINE 123

and consequently increase in the metabolism of their nuclei; some
investigators think, however, that the increase is chiefly due to
nuclein in the food. The question is
not yet settled.

HIPPURIC ACID

Hippuric acid (C,H,NO;), com-
bined with bases to form hippurates,
is present in small quantities in
human urine, but in large quantities
in the urine of herbivora. This is
due to the food of herbivora con-
taining substances belonging to the <\
aromatic group—the benzoic acid i. At’ Tigiaiin sid crystal
series. If benzoic acid is given to
a man, it unites with glycocine with the elimination of a molecule
of water, and is excreted as hippuric acid—

CH,.NH, CH,NH.CO.C,H;

C,H;.COOH + | = | +H,0O

COOH COOH
[benzoic acid] [glycocine} {hippuric acid] (water)

This is a well-marked instance of synthesis carried out in the
animal body, and experimental investigation shows that it is ac-
complished by the living célls of the kidney itself ; for if a mixture of
glycocine, benzoic acid, and blood is injected through the kidney (or
mixed with a minced kidney just removed from the body of an
animal), their place is found to have been taken by hippuric acid.

CREATININE

The creatinine in the urine is nearly all derived from the creatine
contained in the meat of the food. There is, however, a small
amount in the urine even during starvation: this represents a small
percentage of creatine from the muscles.

Sarcosine (C;H;NO,) united to cyanamide (CN.NH,) yields
creatine (C,H,N;0.). Cyanamide plus water yields urea (CON.H,).

The formation of creatinine from creatine is represented in the
following equation :—

C,H,N,;0,—H,O=C,H;N;0

[creatine] [water] [ereatinine]
Creatine and creatinine are of considerable chemical interest, because
urea can be obtained from them as one of their decomposition products

124 ESSENTIALS OF CHEMICAL PHYSIOLOGY

in the laboratory ; the equation which represents the formation of urea
from creatine is as follows :—
C,H,N;0,+H,O=C;H,NO,+CON.H,
[ereatine] [water] [sarcosine] {urea]
The second substance formed is sarcosine. Sarcosine is methyl-glyco-
cine—that is, amido-acetic acid in which one H is replaced by methyl
(CH;)

on SE

It is, however, doubtful whether decompositions of this kind occur
in the body (see p. 118).

With sodium nitro-prusside creatinine gives a colour reaction
described on p. 106.

Creatinine with zine chloride gives a characteristic crystalline
precipitate (groups of fine needles) with composition C,H,N3;0.Zn(Cl,.

According to the recent researches of G. 8. Johnson, urinary
creatinine, though isomeric with the creatinine obtained artificially

Fic, 45.—Creatine crystals. Fie. 46.—Creatinine crystals.

from the creatine of flesh, differs from it in some of its properties,
such as reducing power, solubility, and character of its gold salts.
The reducing action of urinary creatinine has led to some confusion,
for some physiologists have supposed that the reducing action on
Febling’s solution and picric acid of normal urine is due to sugar,
whereas it is really chiefly due_to creatinine. The readiest way of
separating creatinine from urine is the following :—To the urine a
twentieth of its volume of a saturated solution of sodium acetate is
added, and then one-fourth of its volume of a saturated solution of
mercuric chloride: this produces an immediate abundant precipitate

URINE ~ ~ 9S5

of urates, sulphates, and phosphates, which is removed by filtration ;
the filtrate is then allowed to stand for twenty-four hours, when the
precipitation of a mercury salt of creatinine (C,H;HgN;OHCI)
3HgCl,+2H,0 occurs in the form of minute spheres, quite typical
on microscopic examination. This compound lends itself very well
to quantitative analysis. It may be collected, dried, and weighed,
and one-fifth of the weight found is creatinine.' Creatinine may be
obtained from it by suspending it in water, decomposing it with
sulphuretted hydrogen, and filtering. The filtrate deposits creatinine
hydrochloride, from which lead hydrate, Pb(OH),, liberates creatinine.
An important point in Johnson’s process is that all the operations are
carried out in the cold ; if heat is applied one obtains the creatinine
of former writers, which has no reducing power.

URINARY DEPOSITS

The different substances that may occur in urinary deposits are
formed elements and chemical substances.

The formed or anatomical elements may consist of blood
corpuscles, pus, mucus, epithelium cells, spermatozoa, casts of the
urinary tubules, fungi, and entozoa. All of these, with the exception
of a small quantity of mucus, which forms a flocculent cloud in the
urine, are pathological, and the microscope is chiefly employed in
their detection.

The chemical substances are uric acid, urates, calcium oxalate,
calcium carbonate, and phosphates. Rarer forms are leucine, tyro-
sine, xanthine, and cystin. We shall, however, here only consider
the commoner deposits, and for their identification the microscope
and chemical tests must both be employed.

Deposit of Uric Acid —This is a sandy reddish deposit resembling
cayenne pepper. It may be recognised by its crystalline form (fig.
43, p. 119) and by the murexide reaction. The presence of these
erystals generally indicates an increased formation of uric acid, and,
if excessive, may lead to the formation of stones or calculi in the
bladder. The way in which uric acid is split off from the urates is
of great interest to pathologists, and will be found fully discussed in
Sir W. Roberts’s Croonian Lectures.”

Deposit of Urates—This is much commoner, and may, if the
urine is concentrated, occur in normal urine when it cools. Ié is

1 If only a quantitative analysis is required, the process may be considerably
accelerated by boiling the first filtrate for ten minutes instead of letting it stand

for twenty-four hours.
? Published by Smith, Elder, & Co., London, 1892.

126 ESSENTIALS OF CHEMICAL PHYSIOLOGY

generally found in the concentrated urine of fevers; and there
appears to be a kind of fermentation, called the acid fermentation,
which occurs in the urine after it has been passed, and which leads
to the same result. The chief constituent of the deposit is the acid

ae,
de

&
‘ne

Fic. 47.—Acid sodium urate. Fia, 48,—Aeid ammonium urate,
sodium urate, the formation of which from the normal sodium urate
of the urine may be represented by the equation—

2C;,H,Na,.N,O; ote H,O oe Co, a 2C;H;NaN,O, a Na,CO,
{normal sodium {water] [carbonic {acid sodium (sodium

urate] acid] urate] carbonate]

This deposit may be recognised as follows :—
1, It has a pinkish colour ; the pigment called wro-erythrin is one

0 0
OSsSe

Fig. 49,—Envelope crystals Fic, 50.—Cystin erystals,
of calcium oxalate.

aa =

of the pigments of the urine, but its relationship to the other urinary
pigments is not known (see further Lesson XXVI.).

2. It dissolves upon warming the urine.

3. Microscopically it is usually amorphous, but crystalline forms
similar to those depicted in figs. 47 and 48 may occur.

Crystals of calcium oxalate may be mixed with this deposit (see
fig. 49).

Deposit of Calcium Oxalate—This occurs in envelope crystals
(octahedra) or dumb-bells.

It is insoluble in ammonia, and in acetie acid. It is soluble with
difficulty in hydrochloric acid.

a alae

URINE 127

Deposit of Cystin—Cystin (C;H,.N.S,0,) is recognised by its
colourless six-sided crystals (fig. 50). These are rare: they oceur
only in acid urine, and they may form concretions or calculi. Cystin- |
uria (cystin in the urine) is hereditary. Mod

Deposit of Phosphates—These occur in alkaline urine. The
urine may be alkaline when passed, due to fermentative changes
occurring in the bladder. All urine, however, if exposed to the air
(unless the air is perfectly pure, as on the top of a snow mountain),
will in time become alkaline owing to the growth of the mtcrococcus
ure@. This forms ammonium carbonate from the urea.

CON.H, + 2H,O = (NH,).CO;
[urea] (water] {ammonium
carbonate]

The ammonia renders the urine alkaline and precipitates the
earthy phosphates. The chief forms of phosphates that occur in
urinary deposits are—

1. Calcium phosphate, Ca;(PO,),; amorphous.

Fi6, 51.—Triple phosphate crystals. Fig. 52.—Crystals of phosphate of lime
(stellar phosphate).

2. Triple or ammonio-magnesium phosphate, MgNH,PO, ; coffin-
lids and feathery stars (fig. 51).

3. Crystalline phosphate of calcium, CaHPO,, in rosettes of
prisms, in spherules, or in dumb-bells (fig. 52).

4. Magnesium phosphate, Mg,(PO,),+22H.O, occurs oceasion-
ally, and crystallises in long plates.

All these phosphates are dissolved by acids, such as acetic acid,
without effervescence.

’ They do not dissolve on heating the urine; in fact, the amount

of precipitate may be increased by heating. Very often neutral or

128

ESSENTIALS OF CHEMICAL PHYSIOLOGY

alkaline urine will become cloudy when boiled: this may be due to

albumin or to phosphates.

It is very important to distinguish be-
tween these two, as albuminuria is a serious condition.

They may

be distinguished by the use of acetic acid, which dissolves phosphates

but not albumin.

A solution of ammonium carbonate (1l-in-5) eats magnesium
phosphate away from the edges; it has no effect on the triple phos-

phate.
be deposited in acid urine.

A phosphate of calcium (CaHPO,+2H,O) may occasionally
Pus in urine is apt to be mistaken for

phosphates, but can be distinguished by the microscope.
Deposit of calcium carbonate, CaCO;, appears but rarely as

whitish balls or biscuit-shaped bodies.

It is commoner in the

urine of herbivora (see p. 116). It dissolves in acetic or hydrochloric

acid, with effervescence.

The following is a summary of the chemical sediments that may

occur in urine :—

CHEMICAL SEDIMENTS IN URINE

In Actp URINE

Uric Acid.—Whetstone, dumb-
bell, or sheaf-like aggregations of
crystals deeply tinged by pigment
(fig. 48).

Urates.—Generally amorphous.
The acid urate of sodium (fig. 47) and
of ammonium (fig. 48) may some-
times occur in star-shaped clusters of
needles or spheroidal clumps with
projecting spines. Tinged brick-red.
Soluble on warming.

Calcium Oxalate. — Octahedra,
so-called envelope crystals (fig. 49).
Insoluble in acetic acid.

Cystin.—Hexagonal plates (fig.
53). Rare.

Leucine and Tyrosine.—Rare.

Caleciwm Phosphate.

CaHPO, + 2H,0.—Rare.

In ALKALINE URINE

Phosphates.—Calcium phosphate,
Ca,(PO,),. Amorphous.

Triple phosphate,
MgNH,PO,+6H,0. Coffin-lids or
feathery stars (figs. 42 and 51).

Calcium hydrogen phosphate,
CaHPO,. Rosettes, spherules, or
dumb-bells (fig. 52).

Magnesium phosphate,
Mg,(PO,), + 22H,0. Long plates.

All soluble in acetic acid without
effervescence.

Calctwm Carbonate, CaCO,.—
Biscuit-shaped crystals. Soluble in
acetic acid with eifervescence.

Ammonium Urate,
C,H,(NH,)..N,0,. — ‘Thorn-apple ’
spherules.

Leucineand Tyrosine.—Very rare.

EE en

129

LESSON XII
PATHOLOGICAL URINE

1. Urine A is pathological urine containing albumin. It gives the usual
proteid tests. e two following are most frequently used in practice.

(a) Boil the top of a long column of urine in a test-tube. If the urine is
acid, the albumin is coagulated. If the quantity of albumin is small, the
cloudiness produced is readily seen, as the unboiled urine below it is clear.
This is insoluble in a few drops of acetic acid, and so may be distinguished
from phosphates. If the urine is alkaline, it should be first rendered acid with
a little dilute acetic acid.

(b) Heller’s Nitric-Acid Test.—Pour some of the urine gently on to the
surface of some nitric acid in a test-tube. A ring of white precipitate occurs
at the junction of the two liquids. This test is used for small quantities of
albumin.

If the urine is cloudy, it should be filtered before applying these tests.

2. Estimation of Albumin by Esbach’s Albuminometer.—Esbach’s reagent
for precipitating the albumin is made by dissolving 10 grammes of picric acid
and 20 grammes of citric acid in 800 or 900 c.c. of boiling water, and then
adding sufficient water to make up to a litre (1,000 ¢.c.).

Fic. 53.—Albuminometer of Esbach.

Pour the urine into the tube up to the mark U ; then the reagent up to
the mark R. Close the tube with a cork, and to ensure complete mixture
tilt it to and fro a dozen times without shaking. Allow the corked tube to
stand upright twenty-four hours; then read off on the scale the height of the
coagulum. The figures indicate grammes of dried albumin ina litre of urine.
The percentage is obtained by dividing by 10. Thus, if the coagulum stands
at 3, the amount of albumin is 3 grammes per litre, or 0°3 gr. in 100 ec. If
the sediment falls between any two figures, the distance +, 3, or 3 from the
upper or lower figure can be read off with sufficient accuracy. Thus the
surface of the sediment being midway between 3 and 4 would be read as 3°5.
When the albumin is so abundant that the sediment is above 4, a more
accurate result is obtained by first diluting the urine with one or two volumes
of water, and then multiplying the resulting figure by 2 or 3, as the case may
be. If the amount of albumin is less than 0-05 per cent., it cannot be accu-
rately estimated by this method.

3. Urine B is diabetic urine. Ithas a high specific gravity. The presence
of sugar is shown by the reduction (yellow precipitate of cuprous oxide) that
occurs on boiling with Fehling’s solution. Fehling’s solution is an alkaline
solution of copper sulphate to which Rochelle salt has been added. The
Rochelle salt (double tartrate of potash and soda) holds the cupric hydrate in

180 ESSENTIALS OF CHEMICAL PHYSIOLOGY

solution. Fehling’s solution should always be freshly prepared, as, on stand-
ing, racemic acid is formed from the tartaric acid, and this substance itself
reduces the cupric to cuprous oxide. Fehling’s solution should, therefore,
always be tested by boiling before it is used. If it remains unaltered by
boiling, it is in good condition.

4, Quantitative Determination of Sugar in Urine.—Fehling’s solution is pre-
pared as follows :—34°639 grammes of copper sulphate are dissolved in about
200 c.c. of distilled water; 173 grammes of Rochelle
salt are dissolved in 600 c.c. of a 14-per-cent. solution
of caustic soda. The two solutions are mixed and
diluted to a litre. Ten c.c. of this solution are
equivalent to 0°05 gramme of dextrose. Dilute 10 c.c.
of this solution with about 40 c.c. of water, and boil it
in a white porcelain dish. Run into this from the
burette (see fig. 54) the urine (which should be
previously diluted with nine times its volume of
distilled water) until the blue colour of the copper
solution disappears—that is, till all the cupric hydrate
is reduced. The mixture in the basin should be boiled
after every addition.1 The- quantity of diluted urine
used from the burette contains 0:05 gramme of sugar.
Calculate the percentage from this, remembering that
urine has been diluted to ten times its original
volume.

The following formula will be found useful in con-
verting grammes into grains :—

«x =number of grains of sugar in the 24 hours.

a=number of ounces of urine in the 24 hours.

1 ounce = 28°396 c.c.

b=number of ¢.c. of urine used from the burette
to decompose 10 c.c. of Fehling’s solution (equivalent
to 0°05 gramme = 0°77 grain of sugar).

Then

ek bes Sew eccabioe se a= % x 28396 x 077 = % x 21-865.
stand. (Sutton.) E b

Pavy’s modification of Fehling’s solution is some-
times used. Here ammonia holds the copper in solution, and no precipitate
forms on boiling with sugar, as ammonia holds the cuprous oxide in solution,
The reduction is complete when the blue colour disappears; 10 c.c. of Pavy’s
solution=1 ¢.c. of Fehling’s solution =0-005 gramme of dextrose. In some
cases of diabetic urine where there is excess of ammonio-magnesic phosphate,
the full reduction is not obtained with Fehling’s solution,. and when the
quantity of sugar is small it may be missed. In such a case excess of soda
or potash should be first added; the precipitated phosphates filtered off, and
the filtrate after it has been well boiled may then be titrated with Fehling’s
or Pavy’s solution.

5. Picric Acid Test.—The work of Sir George Johnson and G. S. Johnson
has shown the value of this reagent in detecting both albumin and sugar
in the urine. The same reagent may be employed for the detection of
both substances. The method of testing for albumin has been already studied
with Esbach’s tubes. To test for sugar do the following experiment. Take
a drachm (about 4 ¢.c.) of diabetic urine; add to it an equal volume of
saturated aqueous solution of picrie acid, and half the volume (i.e. 2 c.c.) of
the liquor potasse of the British Pharmacopeia. Boil the mixture for about

feo hm ener e's Meee

' On cooling the blue colour reappears, owing to re-oxidation.

PATHOLOGICAL URINE ~) ee

a minute, and it becomes so intensely dark red as to be opaque. Now do
the same experiment with normal urine. An orange-red colour appears
even in the cold, and is deepened by boiling, but it never becomes opaque,
and so the urine for clinical purposes may be considered free from sugar.
This reduction of picric acid by normal urine is due to creatinine (see p. 123).

The full significance and causes of pathological urine cannot be
appreciated until a theoretical and practical acquaintance with disease
is obtained, and we shall briefly consider only those abnormal con-
stituents which are most frequently met with.

PROTEIDS IN THE URINE

There is no proteid matter in normal urine, and the most common
cause of the appearance of albumin in the urine is disease of the
kidney (Bright’s disease). The best methods of testing for and esti-
mating the albumin are given in the practical heading to this lesson.
The term ‘albumin’ is the one used by clinical observers. Properly
speaking, it is a mixture of serum albumin and serum globulin.

A condition called ‘ peptonuria,’ or peptone in the urine, is ob-
served in certain pathological states, especially in diseases where
there is a formation of pus, and particularly if the pus is decomposing
owing to the action of a bacterial growth called staphylococcus ; one
of the products of disintegration of pus cells appears to be peptone ;
and this leaves the body by the urine. The term ‘peptone,’ however,
is in the strict sense incorrect; the proteid present is deutero-
proteose. In the disease called ‘ osteomalacia ’ a proteose is usually
found in the urine, which more nearly resembles hetero-proteose in
its characters.

SUGAR IN THE URINE

Normal urine contains no sugar, or so little that for clinical pur-
_ poses it may be considered absent. It occurs in the disease called
_ diabetes mellitus, which can be artificially produced by puncture of
the medulla oblongata, by extirpation of the pancreas, and by the
administration of the drug called phloridzin. The disease as it occurs
in man may be due to disordered metabolism of the liver, to disease
of the pancreas, or to other not fully understood causes. Transitory
glucosuria is found in many diseases.

The methods usually adopted for detecting and estimating the
sugar are given at the head of this lesson. The sugar present is
dextrose. Lactose may occur in the urine of nursing mothers.
Diabetic urine also contains hydroxybutyric acid, and may contain
or yield on distillation acetone and ethyl-diacetic acid.

K 2

132 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Fehling’s test is not absolutely trustworthy. Often a normal urine will
decolorise Fehling’s solution, though seldom a red precipitate is formed.
This is due to excess of urates and creatinine. Another substance called
glycuronic acid (C,H,,0,) is, however, very likely to be confused with
sugar by Fehling’s test; the cause of its appearance is sometimes the
administration of drugs (chloral, camphor, &c.); but sometimes it appears
independently of drug treatment. The cause of this is not known, but the
condition has not the serious meaning one attaches to diabetes; hence, for
life assurance purposes, it is most necessary to confirm the presence of sugar
by other tests.

Then, too, in the rare condition called aleaptonuria, confusion may
similarly arise. Alcapton is a substance which probably originates from
tyrosine by an unusual form of metabolism. It gives the urine a brown tint,
which darkens on exposure to the air. It is an aromatic substance, and the
researches of Baumann and Wolkow! have identified it with homogenti-
sinic acid (C,H,.(OH),CH,.COOH).

The best confirmatory tests for sugar are the phenyl-hydrazine
test (see Lesson XIII.), and the fermentation test, which is performed
as follows :—

Half fill a test-tube with the urine and add a little German yeast.
Fill up the tube with mercury ; invert it in a basin of mercury, and
leave it in a warm place for twenty-four hours. The sugar will
undergo fermentation: carbonic acid gas accumulates in the tube,
and the liquid no longer gives the tests for sugar, or only faintly, but
gives those for alcohol instead. A control experiment should be
made with yeast and water in another test-tube, as a small yield of
carbonic acid is sometimes obtained from impurities in the yeast.

Sir W. Roberts introduced a method for estimating sugar in urine, hy the
diminution in specific gravity which it undergoes on fermentation. Every
degree lost in the specific gravity corresponds to one grain of sugar per fluid
ounce. Four ounces of urine are placed in a bottle, and a piece of German
yeast about the size of a small walnut is added. The bottle is closed with a
cork, through which a small hole is bored to allow the carbonic acid to
escape. This is put in a warm chamber (40° C.), and beside it is placed
another similar bottle containing 4 ounces of the urine without any yeast.
After 18 to 24 hours, fermentation is complete, and the specific gravity of
both is taken: suppose that the specific gravity of the unfermented urine is
1040, and that of the urine which has undergone fermentation is 1030: the
number of degrees lost is ten; i.e. the urine contained 10 grains of sugar per
ounce. The percentage of sugar may be ascertained by multiplying the
degrees of specific gravity lost by 0:22; thus the percentage in the example

just given will be 0°22x10=2:2. The method, however, is too rough for
trustworthy observations to be made, and has dropped out of use.

BILE IN THE URINE

This occurs in jaundice. The urine is dark-brown, greenish, or
in extreme cases almost black in colour. The most readily applied
test is Gmelin’s test for the bile pigments. Pettenkofer’s test for the

1 Zeit. physiol. Chem. xv. 228,

PATHOLOGICAL URINE ~ 133

bile acids seldom succeeds in urine if the test is done in the ordinary
way. The best method is to warm a thin film of urine and cane-
sugar solution in a flat porcelain dish. Then dip a glass rod in
strong sulphuric acid, and draw it across the film. lis track is
marked by a purplish line. Excess of urobilin should not be mis-
taken for bile pigment.

BLOOD AND BLOOD PIGMENT IN THE URINE

When hzmorrhage occurs in any part of the urinary tract, blood
appears in the urine. It is found in the acute stage of Bright's
disease. If a large quantity is present, the urine is deep red.
Microscopic examination then reveals the presence of blood cor-
puscles, and on spectroscopic examination the bands of oxyhemo-
globin are seen.

If only a small quantity of blood is present, the secretion—
especially if acid—has a characteristic reddish-brown colour, which
physicians term ‘ smoky.’

The blood pigment may, under certain circumstances, appear in
the urine without the presence of any blood corpuscles at all. This
is produced by a disintegration of the corpuscles occurring in the
circulation, and the most frequent cause of this is a disease allied to
ague, which is called parorysmal hemoglobinuria. The pigment is
in the condition of methemoglobin mixed with more or less
oxyhemoglobin, and the spectroscope is the means used for identi-
fying these substances (see pp. 96-152).

PUS IN THE URINE

Pus occurs in the urine as the result of suppuration in any part

of the urinary tract. It forms a white sediment resembling that of
phosphates, and, indeed, is always mixed with phosphates. The pus
corpuscles may, however, be seen with the microscope ; their nuclei
» are rendered evident by treatment with 1 per cent. acetic acid, and
the pus corpuscles are seen to resemble white blood corpuscles,
which, in fact, they are in origin.
_ Some of the proteid constituents of the pus cells—and the same
is true for blood—pass into solution in the urine, so that the
urine pipetted off from the surface of the deposit gives the tests for
albumin.

On the addition of liquor potassz to the deposit of pus cells a
ropy gelatinous mass is obtained. This is distinctive. Mucus treated
in the same way is dissolved.

134 ESSENTIALS OF CHEMICAL PHYSIOLOGY

DETECTION OF PHYSIOLOGICAL PROXIMATE PRINCIPLES

Subsequent lessons may be very usefully employed by the class in testing
for the various substances the properties of which they have studied. The
following scheme will form a rough guide to the tests to be employed for
the most important of the proximate principles :—

1. Note reaction, colour, clearness or opalescence, taste, smell. Coloured
liquids suggest blood, bile, urine, &c. Opalescent liquids suggest starch, gly-
cogen, or certain proteids.

2. Add iodine. A colour is produced:

If blue: Starch. Confirm by converting into a reducing sugar by saliva
at 40° C., or boiling with dilute sulphuric acid.

If reddish brown: Glycogen or dextrin. Glycogen forms an opalescent
solution in water, and is readily precipitated by alcohol. It is precipitated by
basic lead acetate. Dextrin forms a clear solution: it is not precipitated
by basic lead acetate unless ammonia is added also. It is not precipitated by
alcohol unless a large excess is added. Both dextrin and glycogen are, like
starch, convertible into a reducing sugar.

3. Add copper sulphate and caustic potash.

(a) Blue solution: boil; yellow or red precipitate. Dextrose, maltose, or
lactose (for distinguishing tests see Lesson XIII.).

(6) Blue solution; no reduction on boiling; boil some of the original
solution with 25 per cent. sulphuric acid, and then boil with copper sulphate
and caustic potash ; abundant yellow or red precipitate : Canesugar. Confirm
by HCl test (see p. 9).

(c) Violet solution: Proteids (albumins, globulins, albuminates). In
presence of magnesium sulphate the potash causes also a white precipitate of
magnesia.

(ad) Pink solution ; biuret reaction. Peptones or albumoses (proteoses).
In presence of ammonium sulphate very large excess of potash is necessary
for this test. Only a trace of copper sulphate must be used.

4. When proteids are present proceed as follows: Boil the original solution
(after adding a trace of 2-per-cent. acetic acid).

(@) Precipitate produced: Albumins or globulins.

(0) No precipitate : Albuminates, proteoses, or peptones.

5. If albumin, or globulin, or both, are present, saturate a fresh portion with
magnesium sulphate or half saturate with ammonium sulphate; filter; the
precipitate contains the globulin, the filtrate the albumin. Test temperature
of heat coagulation.

DETECTION OF PROXIMATE PRINCIPLES 135

6. If albumin or globulin is absent :—

(a) Neutralisation causes a precipitate soluble in excess of weak acid or
alkali. Acid albumin or alkali albumin, according as the reaction of the
original liquid is acid or alkaline respectively. If the original liquid is
neutral, acid albumin and alkali albumin must be both absent.

(6) Neutralisation produces no such precipitate : Proteose or peptone.

7. If proteose, or peptone, or both, are present, saturate a fresh portion
with ammonium sulphate:

(a) Precipitate : Proteose. (6) No precipitate: Feptone.

If both are present, the precipitate contains the proteose, and the filtrate
the peptone.

8. To a fresh portion add nitric acid (proteids having been proved to be
present).

(a) No precipitate, even though excess of sodium chloride be also added:
Peptone.

(6) No precipitate, until excess of sodium chloride is added: Deutero-
proteose.

(c) Precipitate which disappears on heating and reappears on cooling:
Proteoses. This is the distinctive test of all the proteoses or albumoses, and
is given by all of them. For one of them, however (deutero-proteose), excess
of sodium chloride must be added also.

(@) Precipitate little altered by heating: Albumin or globulin.

In all four cases nitric acid plus heat causes a yellow colour, turned orange
by ammonia.

9. Confirmatory tests for proteids :—

(a) Millon’s test.

(6) Ferrocyanide of potassium and acetic acid causes a precipitate (except
in the case of peptones and some proteoses).

(ce) To test for fibrinogen :-—

i. It coagulates by heat at 56° C.

ii. It is changed into fibrin by fibrin ferment and calcium chloride.

(d) To test for caseinogen :—

i. It is not coagulated by heat.

ii. It is changed into casein by rennet and calcium chloride.

' 10. If blood is suspected—
(a) Examine spectroscopically, diluting if necessary.
i. Oxyhemoglobin shows two bands between D and E.
ii. Add ammonium sulphide; one band only appears.

iii. Carbonic oxide hemoglobin shows two bands also, but will not reduce
with ammonium sulphide. y
iv. Methemoglobin gives a typical band in the red between C and D.

v. Hematin &c. show special spectra (see Advanced Course).
(6) Dry: boil with glacial acetic acid and a crystal of sodium chloride on
a glass slide under a cover glass. Hemin crystals are obtained.

186 ESSENTIALS OF CHEMICAL PHYSIOLOGY

i. Try heemin test.

ii, Dissolve it in potash; add ammonium sulphide, and examine for
spectrum of hemochromogen.

11. If bile is suspected.
(a) Try Gmelin’s test for bile pigments. This is the test for bile in urine,
(6) Try Pettenkofer’s test for bile salts. ;

12, Miscellaneous substances :

(a) Mucin. Precipitated by acetic acid or by alcohol. The precipitate is
soluble in lime water. By collecting the precipitate and boiling it with 25-
per-cent. sulphuric acid, a reducing sugar is obtained. Mucin gives the
proteid colour tests.

(6) Nucleo-proteid.— Precipitated by acetic acid or by alcohol. The pre-
cipitate is often viscous. It is soluble in dilute alkalis like 1 per cent. sodium
carbonate. This solution causes intra-vascular clotting. If the precipitate
is collected and subjected to gastric digestion, an insoluble deposit of nuclein
is left, which is rich in phosphorus. Nucleo-proteid gives the proteid colour
tests.

(c) Gelatin. This also gives the proteid colour tests. It is not coagulated,
but dissolved in hot water. The solution gelatinises when cold.

(d) Urea. Very soluble in water. The solution effervesces when sodium
hypobromite or fuming nitric acid is added. Concentrate a fresh portion,
add nitric acid, and examine for crystals of urea nitrate. Solid urea heated
in a dry test-tube gives off ammonia, and the residue is called biuret. This
gives a rose-red colour with copper sulphate and caustic potash.

(e) Urie acid. Very insoluble in water ; soluble in potash, and precipitated
from this solution in crystals by hydrochloric acid. Uric acid erystals from
human urine are deeply pigmented red. Try murexide test.

(f) Cholesterin. Characteristic flat crystalline plates. Play of colours
with iodine and concentrated sulphuric acid.

13. Urine. Normal constituents :

(a) Chlorides. Acidulate with nitric acid; add silver nitrate; white
precipitate.

: (0) Sulphates. Acidulate with nitric or hydrochloric acid: add barium
chloride; white precipitate. }

(c) Phosphates. Acidulate with nitric acid; add ammonium molybdate ;
boil; and a yellow crystalline precipitate forms. To another portion add
ammonia; earthy (i.e. calcium and magnesium) phosphates are precipitated.

(d) Urea (see above).

(e) Uric acid. To 100 c.c. of urine add 5 ¢.c. of hydrochloric acid; leave
for twenty-four hours, and pigmented crystals of uric acid are formed. For
tests see above.

(f) Hippuric acid. Evaporate the urine with nitric acid, and heat the
residue in a dry test-tube. A smell of oil of bitter almonds is given off.

(g) Creatinine. Take 100 c.c, of urine: add 5 c.c. of a saturated solution

;

|
;
;

. a

: DETECTION OF PROXIMATE PRINCIPLES 137

of sodium acetate and 20 c.c. of a saturated solution of mercuric chloride.

Filter. Set the filtrate aside for twenty-four hours, and the spherical mercury

compound of creatinine crystallises out. Examine this with the microscope.
For colour test with sodium nitro-prusside see p. 106. ‘

14. Urine. Abnormal constituents. :
(a) Blood. Microscope (blood corpuscles). Spectroscope (for oxyhxmo-

globin or methemoglobin). Hzmin test.

(6) Blood pigment may be present without blood corpuscles. Spectro-
Scope.

(d) Pus. White deposit. Microscope (pus cells). Add potash ; it becomes
stringy.

(e) Albumin. (i.) Precipitated, if acid, by boiling; precipitate insoluble
in acetic acid, so distinguishing it from phosphates. (ii.) Precipitated by
nitric acid in the cold. (iii.) Precipitated by picric acid.

(f) Sugar. (i.) Brown colour with potash and heat (Moore’s test). (ii.)
Ferments with yeast. (iii.) Reduces Fehling’s solution. (iv.) Urine has a
high specific gravity. (v.) Add picric acid, potash, and boil; the urine
becomes a dark opaque red; the similar ae colouration in sonal urine

due to creatinine.

(g) Mucus. Flocculent cloud; may be increased by acetic acid; soluble
in alkalis. A little mucus in urine is not abnormal.

(h) Deposits.

i. Examine microscopically for blood corpuscles, pus cells, crystals, &c.

ii. Phosphates. White deposit often mixed with mucus or pus. In-
soluble on heating; soluble in acetic acid. Urine generally alkaline
Examine microscopically for coffin-lids of triple phosphate and star-like
clusters of stellar (calcium) phosphate.

iii. Uric acid. Like cayenne pepper. Whetstone &c. crystals. Very
insoluble in water. Soluble in potash. Murexide test. Urine acid.

iv. Urates. Pink deposit, usually amorphous; may be mixed with
envelope crystals of calcium oxalate. Deposit soluble on heating urine.
Murexide test.

Sheer Lg) =
ce SRE id

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Brae:

aa
Se Fan)

ADVANCED COURSE

INTRODUCTION

Ir will be presupposed that students who take the following lessons have

already been through the elementary course. The order in which the
subjects are treated is the same as that already adopted. The instructions
given will be mainly practical; theoretical matter on which they depend,
or-to which they lead, is, as a rule, too lengthy to be discussed in a short
manual like the present volume. The Appendix contains a description of
various instruments which are not generally contained in sufficient numbers
in a physiological laboratory to admit of each student being able to use them
in a class. It also contains a description of certain methods of research
which should always be shown in demonstrations, though there may be
practical difficulties in allowing each member of the class to perform the
experiments. The description of the phenomena of polarised light and their
application has been considerably extended, and a section added on osmosis,
a subject which is becoming of more and more importance to the student of
physiology.

~The few experiments in which living animals are employed will also
necessarily be of the nature of demonstrations.

140 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON XIII
CARBOHYDRATES

1. Glycogen—A rabbit which has been fed five or six hours previously
on carrots is killed by bleeding. The chest and abdomen are opened quickly
and a cannula inserted into the portal vein, and another into the vena cava
inferior. A stream of salt solution is then allowed to pass through the liver -
until it is uniformly pale. The washings are collected in three beakers
labelled a, b, and c.

The liver is cut out quickly, chopped into small pieces, and thrown into
boiling water acidulated with acetic acid. The acidulated water extracts a
small quantity of glyco-
gen. The pieces of scalded
liver are then ground up
in a mortar with hot
water, and thoroughly
extracted with boiling
water. Filter. A strong
solution of glycogen is
thus obtained.

Test the solution when
cold with iodine.

To separate the glyco-
gen! evaporate the solu-
tion to a small bulk on the
water-bath and add excess
of alcohol; the glycogen
is precipitated as a floccu-
lent powder, which is col-
lected on a filter and dried
in an oven at the tempe-
rature of 100° (see fig. 55).

If the experiment is to be a quantitative one, the piece of liver taken and
the glycogen obtained must be weighed.

Fic. 55,—Hot-air oven with gas regulator (G). (Gscheidlen.)

' This method of preparation of glycogen has the advantage that only traces
of proteid are mixed with it. In Kiilz’s method (extraction with dilute potash)
there is more proteid. This is precipitated by the alternate addition of hydro-
chloric acid and potassio-mercuric iodide.

Fig. 56.—Plate of osazone crystals highly magnified.
A, phenyl-glucosazone. B, phenyl-maitosazone. CO, phenyl-lactosazone.

CARBOHYDRATES 141

2. Examine the washings of the liver in the beakers a, b, and ec for sugar.
This may be done in a rough quantitative manner as follows :—Take equal
quantities of a, 6, and ¢ in three test-tubes; to each add an equal amount of

Fehling’s solution, and boil: a will give a heavy precipitate, 6 one not so

heavy, and ¢ least of all, or none at all.

3. Micro-chemical detection of Glycogen.—A thin piece of the same liver is
hardened in 90 per cent. alcohol. Sections are cut by the free hand, or after
embedding in paraffin. If paraffin is used, this is got rid of by means of
turpentine; and the sections prepared by either method are treated with
chloroform in which iodine is dissolved, and mounted in chloroform balsam
containing some iodine. The glycogen is stained brown, and is most abundant
in the cells around the radicles of the hepatic vein.

4, Phenyl-Hydrazine Test for Sugars.—To 5 c.c. of the suspected fiuid (e.g.
diabetic urine) add 1 decigramme of phenyl-hydrazine hydrochloride, 2 deci-
grammes of sodium acetate, and heat on the water-bath for half an hour.
On cooling, if not before, a crystalline or amorphous precipitate separates out.
If amorphous, dissolve it in hot alcohol ; dilute the solution with water, and
boil to expel the alcohol, whereupon the osazone separates out in yellow
erystals. Examine the crystals with the microscope (see accompanying
plate).

Dextrose gives a precipitate of phenyl-glucosazone (C,,.H,.N,0,), which
erystallises in yellow needles (melting-point 205° C.).

Levulose yields an osazone identical with this. :

Galactose yields a very similar osazone (phenyl-galactosazone). It differs
from phenyl-glucosazone by melting at 190—-193°, and in being optically in-
active when dissolved in glacial acetic acid.

Cane sugar does not form a compound with phenyl-hydrazine.

Lactose yields phenyl-lactosazone (C,,H.,,N,0,). It crystallises readily
in needles, usually in clusters (melting-point 200° C.). It is soluble in
80-90 parts of boiling water. Lactose in urine does not give this test
readily.

Maltose yields phenyl-maltosazone (C,,H,.N,0,). It erystallises in yellow
needles much wider than those yielded by glucose or lactose (melting-point
206° C.). Unlike phenyl-glucosazone it dissolves in 75 parts of boiling water,
and is still more soluble in hot alcohol.

Isomaltose is a sugar formed at the same time as maltose by the action of
diastase, ptyalin, and amylopsin on starch. It is also an intermediate
product in the formation of dextrose by mineral acids from starch. An
amylolytic ferment in blood serum, capable of forming dextrose from starch,
aéts similarly. It is readily soluble in water, is very sweet, and ferments
very slowly with yeast. Its formula and general chargoting are like
those of maltose, but its osazone forms fine yellow needles, which melt at
150° C.

The chemistry of the phenyl-hydrazine reaction is represented in the
following equations, dextrose being taken as an example of the sugar

142 ESSENTIALS OF CHEMICAL PHYSIOLOGY

I. CH,OH [CH(OH)],CH(OH)COH + H,N.NH(C,H,)
[dextrose] [phenyl-hydrazine]
COH,OH [CH(OH)],CH(OH)OH:

= Il * H,0

N-NH(C,H,)
[hydrazone] [water]

II. CH,OH [CH(OH)}],CH(OH)CH
Hl + C,H,.NH—NH,

N-—NH(C,H,)
[hydrazone] [phenyl-hydrazine]
CH,OH[CH(OH)],C—CH
= # ot +H, + H,O
C,H,.NH-N N-~NH.O,H,
[osazone] [hydrogen] [water]

5, Barfoed’s Reagent.—Dissolve 1 part of cupric acetate in 15 parts of
water ; to 200 c.c. of this solution add 5 ¢.c. of acetic acid containing 38 per
cent. of glacial acetic acid. Dextrose reduces this reagent on boiling; mal-
tose and lactose do not. This test is not very delicate.

6. The Polarimeter.—Estimate the strength of a solution of dextrose by
means of the polarimeter (see Appendix).

—

* 129th 5 nl

148

LESSON XIV
CARBOHYDRATES: ACTION OF MALT UPON STARCH

1. Prepare a 0°5-per-cent. solution of starch.

2. Prepare some malt extract by digesting 10 grammes of powdered malt
with 50 c.c. of water at 50° C. for three hours, and subsequently straining.
This extract contains the diastatic or malting ferment.

Solutions 1 and 2 may be conveniently prepared beforehand by the
demonstrator.

8. To the starch solution add one-tenth of its volume of malt extract, and
place the mixture in a water-bath at 40° C, From time to time test portions
of the liquid by mixing a drop with a drop of iodine solution on a testing
slab. The blue colour at first seen is soon replaced by a violet (mixture of
blue and red), and then by a red reaction (due to erythrodextrin) which
gradually vanishes. Alcohol added to the liquid when all starch and erythro-
dextrin have gone still causes a precipitate of a dextrin, which, as it gives no
colour with iodine, is called achrod-dextrin. The liquid also contains a
reducing sugar, maltose.

4. Treat the solution of starch as before with one-tenth of its volume of
malt extract, and keep it in the warm chamber (50° C.) for three hours.
This may be conveniently done beforehand by the demonstrator.

5. Take 50 c.c. of the product (which is a solution of maltose and isomal-
tose, the reducing action of which is the same as that of maltose), and
determine how much of it is necessary to reduce 10 c.c. of Fehling’s solution.
The manner of carrying out Fehling’s quantitative method is given in
Lesson XII.

6. Take another 50 c.c. and boil it with 1 ¢.c. of strong sulphuric acid for
half an hour in a flask. This converts it into dextrose. After cooling bring
the liquid to its original volume (50 c.c.) by adding water, and again determine
its increased reducing power with Fehling’s solution. If x=c.c. of maltose

solution necessary to reduce 10 c.c. of Fehling’s solution, then = cc. of

dextrose solution necessary for the same purpose. The strength of the
maltose solution can be calculated from the fact that 10 ¢.c. of Fehling’s
solution corresponds to 0°05 gramme of dextrose.

144 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON XV
CRYSTALLISATION OF EGG ALBUMIN

Fresh egg-white is mixed with an equal bulk of fully saturated, filtered,
neutral ammonium sulphate solution. 100 c.c. of the former are measured
into a porcelain basin or strong beaker, and 100 ¢.c. of ammonium sulphate
solution are added in successive quantities of 10 or 15 ¢.c., the mixture being
thoroughly churned with an egg whisk after each addition. The whole should
be finally so thoroughly beaten up as to form a large proportion of light froth,
After the greater part of the froth has broken down the mixture is thrown on
a folded filter-paper, moderately rapid filtration being obtained without the
use of a filter-pump. The filtrate is strongly alkaline to litmus, and smells of
ammonia. ‘To the filtrate in a flask, or to as much of it as can be obtained in
a convenient time of filtration, further ammonium sulphate solution is very
cautiously added (best, drop by drop from a burette) until a slight permanent
precipitate remains, and this precipitate is afterwards just redissolved by the
equally cautious addition of water. Dilate acetic acid (10 per cent.) from a
burette is now added drop by drop until such a stage of reaction is reached that
a precipitate forms and only just redissolves. Finally one or two drops (not
more) of acid are added in excess of this, whereupon a bulky white precipitate
falls. The flask is now corked and allowed to stand. In 24 hours or less the
precipitate, which will have increased in quantity, will be found to consist
entirely of acicular crystals. Small portions should be examined under a
4th objective, avoiding pressure on the cover slip (F. G. Hopkins).

: 145

a ee eS ——. e

LESSON XVI
COAGULATION OF MILK

1. Prepare a solution of rennet by extracting the fourth stomach of the
sucking calf with glycerin. Clark’s rennet, which is sold for making junkets,
will do equally well.

2. Prepare a solution of pure caseinogen in the following way :—Saturate
milk with magnesium sulphate by shaking it with excess of the powdered
salt; or the caseinogen may be more readily precipitated by half saturation
with ammonium sulphate—that is, by adding to the milk an equal volume
of saturated solution of ammonium sulphate. Filter. The caseinogen and
fat remain together on the filter. Save the filtrate, andlabel it A. Wash
the precipitate on the filter with saturated solution of magnesium sulphate
or half-saturated ammonium sulphate solution, as the case may be, until the
washings contain no albumin. Add water to the precipitate. The caseinogen
dissolves, the fat being insoluble. In this way a solution of caseinogen in
weak magnesium or ammonium sulphate is obtained. So far the operations
should be performed beforehand by the demonstrator.

8. To this solution add acetic acid. The caseinogen is precipitated ;
collect it on a filter; wash the acid away with distilled water. Dissolve the
precipitate in lime water by grinding it up in a mortar with the lime water;
filter, and an opalescent solution of caseinogen is obtained.

4. Toa portion of this solution add a few drops of rennet extract. Put
it in the water-bath at 40° C., and if the caseinogen has been thoroughly
washed to free it from calcium salts no coagulation will occur. The calcium
contained in the lime water subsequently added is not effective in promoting
coagulation.

5. Treat another portion in the same way, adding, however, a few drops
of 0°5-per-cent. phosphoric acid as well as the rennet. Warm to 40° C.
Coagulation—that is, formation of casein from caseinogen—usually occurs in
afew minutes. The addition of the phosphoric acid causes the formation of
calcium phosphate, which is effective in promoting coagulation.

6. Examine the filtrate A (see above). Saturate a portion with sodium
chloride. A small amount of precipitate of a proteid comes down. This is
the so-called lacto-globulin. This contains only a trace of true globulin: it
is mostly caseinogen previously left in solution, together with calcium
sulphate.

7. Heat another portion of A to 77°, acidifying faintly with a few drops
of 2-per-cent. acetic acid. Lactalbumin is coagulated at this temperature.

146 ESSENTIALS OF CHEMICAL PHYSIOLOGY

8. Ringer’s method of showing the conversion of caseinogen into casein :—
Milk is strongly acidified with acetic acid. This precipitates the caseinogen
and entangled fat. The precipitate is collected on a filter, thoroughly washed
with distilled water, and ground up in a mortar with calcium carbonate.
The mixture is thrown into excess of distilled water. The fat rises to the
top: the excess of calcium carbonate falls to the bottom. The intermediate
fluid contains the caseinogen in solution ; it is usually very opalescent. Take
some of this solution and divide it into three parts, A, B, and C.

To A add rennet.

To B add a few drops of 2-per-cent. solution of calcium chloride.

To C add both rennet and calcium chloride.

Put all three in the water-bath at 40° C. A clot of casein forms in C, but
not in A and B.

2. The formation of casein from caseinogen is a double process; the first
action is that of the ferment, which converts the caseinogen into what may
be called soluble casein ; the second action is that of the calcium salt, which
precipitates the casein in an insoluble form, or curd. This is probably a
caseate of lime. This may be shown by taking some of Ringer’s caseinogen
solution and adding rennet. Warm to 40° C.; no visible change occurs, but
nevertheless soluble casein and not caseinogen is now present. Then boil
this mixture to destroy the rennet, cool, and add caleciumchloride. A forma-
tion of insoluble curd now occurs.

; me 7)

LESSON XVIL
THE ALBUMOSES

1. Witte’s peptone contains very little true peptone, but consists chiefly
of albumoses, which are soluble like peptone, in neutral saline solutions.

2. Make a solution of this substance in 10-per-cent. sodium chloride
solution, and filter. Very little residue is left on the filter. This consists of
dysalbumose, an insoluble form of hetero-albumose, formed during the process
of preparing the substance. If hot saline solution is used instead of cold as
a solvent, this amount of insoluble residue is increased, hetero-albumose
being to a slight extent precipitated by heat.

3. The solution gives the following tests :—

(a) Biuret reaction (due both to peptone and albumoses).

(6) A drop of nitric acid, best added by a glass rod, gives a precipitate
which dissolves upon heating and reappears on cooling. (This is due to the
albumoses present.)

Otherwise it gives the ordinary proteid reactions.

4. For the separation of the albumoses and peptone proceed as follows :—

(a) Saturate the solution with ammonium sulphate, and filter. The
filtrate contains the peptone and the precipitate the albumoses. The peptone
is not precipitated by nitric acid, nor by most of the reagents that precipitate
other proteids. It is precipitated completely by alcohol, tannin, and potassio-
mercuric iodide ; imperfectly by phospho-tungstic and phospho-molybdie acid.

It gives the biuret reaction, but in the presence of ammonium sulphate a
large excess of caustic potash is necessary.

(6) Dialyse another portion of the solution; hetero-albumose is pre-
cipitated.

(c) Saturate another portion of the solution with sodium chloride (or half
saturate with ammonium sulphate) after faintly acidulating with acetic acid.
Proto-albumose and hetero-albumose are precipitated. Filter. The filtrate
contains the deutero-albumose and peptone.

The proto- and hetero-albumose may be redissolved by adding distilled
water, and may be separated from each other by dialysis (see 5).

Deutero-albumose may be separated from the peptone by saturation with
ammonium sulphate, or by the addition of a crystal of phosphoric acid.
These reagents precipitate the deutero-albumose, but not the peptone.

Deutero-albumose gives the nitric acid reaction (see 3, 6) characteristic of
the albumoses only in the presence of excess of salt. If the salt is removed
by dialysis, nitric acid then causes no precipitate.

nu 2

148 ESSENTIALS OF CHEMICAL PHYSIOLOGY

5. Among the important reactions of proteids is Piotrowski’s reaction—
that is, the coloration produced by copper sulphate and a caustic alkali; the
term ‘ biuret reaction ’ is applied to the rose-red colour which proteoses and
peptones give with these reagents, because biuret (a derivative of urea) gives
a similar colour. It does not, however, prove that biuret is contained in the
proteid molecule. Biuret and proteid both contain some radicle to which
the colour is due. Gnezda' thought it was cyanogen, and that the cyanogen
was differently combined in the peptones and native proteids (albumins and.
globulins) respectively ; hence the rose-red given by one group and the violet
by the other. More recent work by Pickering,’ however, points to a CONH
group rather than cyanogen. Gnezda found that if a dilute solution of
nickel sulphate is used instead of copper sulphate, the native proteids give
different colours from the peptones and proteoses, and Pickering has found
the same with cobalt. Their results may be given in the following
table :—

Copper sul- } Copper sul- | Nickel sul- | Nickel sul- | Cobalt sul- | Cobalt sul-
Proteid phate and | phate and | phate and | phate and | phate and | phate and
ammonia potash ammonia potash ammonia potash

Albumins and : : ‘ Heliotrope-
Peeiae } Blue Violet Nil Yellow Nil corte
2 |
Sees } Rose-red | Rose-red | Yellow Orange Nil Red-brown

6. Another delicate test introduced by McWilliam may here be mentioned :
Salicyl-sulphonic acid precipitates albumins and globulins: on heating the
precipitate is coagulated. The same reagent precipitates proteoses. On
heating the precipitate dissolves and reappears on cooling. It does not
precipitate peptones.

7. The use of trichloracetic acid for the separation of various proteids
may be illustrated by the following experiment. Take some blood and add
to it some solution of Witte’s peptone (i.e. proteoses and peptone). Add to
this mixture an equal volume of a 10-per-cent. solution of trichloracetic acid.
There is an abundant precipitate. Boil rapidly and filter hot, The filtrate
contains the proteoses and peptone, all the other proteids being contained in
the precipitate. On cooling, the filtrate deposits some of the proteose. The
proteose and peptone may be detected in the usual way.

1 Proc. Roy. Society, vol. xlvii. p. 202.
2 Journal of Physiology, vol. xiv. Most of the other colour reactions of pro-
teids depend on the aromatic radicle they contain.

149

LESSON XVIII
DIGESTION

1. Activity of pepsin solutions. Examine the comparative digestive
power of the glycerin extracts of two stomachs. Take in two test-tubes an
equal small weighed quantity of fibrin stained with carmine. Add to each
10 c.c. of 0-2-per-cent. hydrochloric acid. Add to one a measured quantity of
one glycerin extract, and to the other an equal quantity of the other glycerin
extract. As the fibrin is digested the carmine is set free, and colours the
liquid ; that which is more deeply stained is that which contains the more
active preparation of pepsin.

This exercise illustrates the principle of Griitzner’s method of comparing
the digestive powers of solutions. In the original method the amount of
earmine set free is estimated by an artificial scale consisting of ten solutions
of carmine of different known strengths.

The carmine solution for staining the fibrin is prepared by dissolving
1 gramme of carmine in about 1 c.c. of ammonia; to this 400 c.c. of water
are added, and the mixture is kept in a loosely stoppered bottle till the smell
of ammonia has become faint.

The fibrin is stained by taking it perfectly fresh and clean. Itis chopped
finely and placed in the carmine solution for twenty-four hours. The fluid is
strained off and the fibrin washed in water till the washings are colourless. It
is kept in a stoppered bottle with just enough ether to cover it.

2. Tests for Free Hydrochloric Acid. (a) Gunsberg’s reagent consists of 2
parts of phloroglucinol, 1 part of vanillin, and 30 parts of rectified spirit. A
drop of filtered gastric juice is evaporated with an equal quantity of the
reagent. Red crystals form, or if much peptone is present, there will be ared
paste. The reaction takes place with 1 part of hydrochloric acid in 10,000.
The organic acids do not give the reaction.

(6) Tropzolin test. Drops of a saturated solution of tropzolin-00 in 94
per cent. methylated spirit are allowed to dry on a porcelain slab at 40° C.
A drop of the fluid to be tested is placed on the tropzolin drop, still at 40° C. ;
and if hydrochloric acid is present, a violet spot is left when the fluid has
evaporated. A drop of 0°006 per cent. hydrochloric acid leaves a distinct
mark.

(For a very complete account of these and other colour reactions see
“ Diseases of the Stomach,’ chap. v. By Sidney Martin, F.R.S. 1895.)

3. Pancreatic Digestion. A finely divided ox-pancreas has been allowed
to digest at 40° C. for twenty-four to thirty-six hours in a litre of 1 per cent.

150 ESSENTIALS OF CHEMICAL PHYSIOLOGY

solution of sodium carbonate to which the white of an egg has been added
every ten hours. Note the odour due to putrefaction. Another preparation

has been similarly made, except that thymol has been added to prevent de-.

composition. These should be got ready by the demonstrator.

Filter some of the extract and examine for leucine and tyrosine as.

follows:

(a) To some of the liquid add Millon’s reagent and filter off the pre-

cipitated proteid. Boil the filtrate. The presence of tyrosine is indicated
by a red colour. If tyrosine is abundant the red colour appears without.
boiling.

(6) Faintly acidify another portion of the filtered extract with 2-per-cent..
acetic acid and boil ; filter off the proteid thus coagulated; reduce the filtrate
to a small bulk by evaporation on the water-bath at the boiling temperature.
Examine a drop microscopically for crystals of leucine and tyrosine. Treat
the remainder with excess of alcohol, to precipitate the albumoses and
peptones, and again filter. Concentrate the filtrate on the water-bath till it

becomes sticky from the presence of leucine. Examine some of the concen-.

trated fluid with the microscope; leucine will be found in crystalline
spheroidal clumps.

Examine microscopic specimens of leucine and tyrosine which have been
prepared by the demonstrator.!

4. Zymogen granules——Examine microscopically, mounting in aqueous.

humour or serum (or in glycerin after treatment with osmic acid vapour),
small pieces of the pancreas, parotid and submaxillary glands in a normal
guinea-pig” and also in one in which profuse secretion had been produced by
the administration of pilocarpine.

Note that zymogen granules are abundant in the former, and scarce in:
the latter, being situated chiefly at the free border of the cells.

Extremely good, though not permanent, microscopic specimens may be-

obtained by teasing in a 33-per-cent. solution of caustic potash.

* The deposit often found in rather old specimens of Benger’s liquor pancrea-
ticus will be found to consist of leucine and tyrosine crystals.

? The guinea-pigs should be killed by bleeding, and the blood collected and
defibrinated, and utilised for the preparation of oxyhemoglobin crystals. This will
give students an opportunity of seeing the exceptional form (tetrahedra) in which
the blood-pigment of this animal crystallises.

The three methods of obtaining crystals described on p. 89 all give good results...
If amy] nitrite is used instead of ether in the third method, crystals of methwmo-
globin are obtained.

RR ar ea

SSS a a .

151 |

LESSON XIX
HZMOGLOBIN AND ITS DERIVATIVES

Defibrinated ox-blood suitably diluted may be used in the following
experiments as in those described in Lesson IX.

1. Place some in a hematoscope (see fig. 33, p. 93) in front of the large
spectroscope. Note the position of the two characteristic bands of oxyhemo-
globin ; these are replaced by the single band of hemoglobin after reduction
by the addition of Stokes’s reagent (see footnote, p. 92) or ammonium sulphide.
By means of a small rectangular prism a comparison spectrum showing
the bright sodium line (in the position of the dark line named D in the solar
spectrum) may be obtained, and focussed with the absorption spectrum.

2. Obtain similar comparison spectra by the use of the microspectroscope.
For this purpose a cell containing a small quantity of oxyhemoglobin
solution may be placed on the microscope stage, and a test-tube containing
carbonic oxide hemoglobin in front of the slit in the side of the instrument.
Notice that the two bands of carbonic oxide hemoglobin are very like those
of oxyhemoglobin, but are a little nearer to the violet end of the spectrum.

Carbonic oxide hemoglobin may be readily prepared by passing a stream
of coal gas through the diluted blood. It has a cherry-red colour, and is not
reduced by the addition of ammonium sulphide (fig. 57, spectrum 4).

3. Methemoglobin.—Add a few drops of ferricyanide of potassium to
dilute blood and warm gently. The colour changes to mahogany-brown.
Place the test-tube in front of the small direct vision spectroscope. Note
the characteristic band in the red (fig. 57, spectrum 5). On dilution other
bands appear (fig. 57, spectrum 6). Treat with ammonium sulphide, and
the band of hemoglobin appears.

4, Acid Hematin.—Add a few drops of glacial acetic acid to dilute blood;
the colour changes to brown. Examine with the spectroscope. Compare
the position of the absorption band in the red with that of methemoglobin ;
that of acid hematin is further from the D line (fig. 57, spectrum 7).

Take some undiluted blood and add glacial acetic acid as before. Extract
this with ether by gently agitating it with that fluid. The ethereal extract
should then be poured off and examined. The band in the red is seen, and
on further diluting with ether three additional bands appear.

5. Alkaline Hematin.—Add to diluted blood a small quantity of strong
caustic potash and warm. The colour changes to brown, and with the spectro-
seope a faint shading on the left side of the D line is seen (fig. 57, spectrum 8).

6. Hemochromogen.—Add ammonium sulphide to a solution of alkaline

152 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Cc D ES F G

3 > S S S
; : Ss yy
1 RI “4 3 me 4 Si

;

Fig. 57,—1, Solar spectrum. 2, Spectrum of oxyhsemoglobin (0°37 p.c. solution). First band, A 589-
564 ; second band, A 555-517. 3,Spectrum ot hemoglobin. Band, A 597-535. 4,Spectrum of CO-
hemoglobin. First band, A 583-564; second band, A 547-521. 5, Spectrum of methemoglobin
(concentrated solution). 6, Spectrum of methemoglobin (dilute solution). First band, A 647-
622; second band, A 587-571; third band, A 552-532; fourth band, A 514-490. 7, Spectrum of
acid hematin (ethereal solution). First band, A 656-615 ; second band, A 597-577 ; third band, A
557-529 ; fourth band, A 517-488, 8, Spectrum of alkaline hematin. Band from A 630-581.
9, Spectrum of hwmochromogen (reduced hematin). First band, A 569-542; second band, A
535-504, 10, Spectrum of acid heamatoporphyrin, First band, A 607-593; second band, A 585-
536, 11, Spectrum of alkaline hematoporphyrin. First band, A 633-612; second band, A 589-564;
third band, A 549-529; fourth band, A 518-488, The above measurements (after MacMunn) are
in millionths of a millimetre. The liquid was examined in a layer 1 centimetre thick. The edges
of ill-defined bands vary a good deal with the concentration of the solutions,

H2MOGLOBIN 153

hematin ; the other changes’to red, and two bands are seen, one between D
and E, and the other nearly coinciding with E and 6 (fig. 57, spectrum 9).
The spectrum of alkaline hematin reappears for a short time after vigorous
shaking with air. ;

7. Hematoporphyrin.—To some strong sulphuric acid in a test-tube add a
few drops of undiluted blood. and observe the spectrum of acid hemato-

G i HKLM N

Solar spectrum,

Hbo

Fic. 58,—The photographic spectrum of hemoglobin and oxyhemoglobin. (Gamgee.)

Oxyhemoglobin,

Methemoglobin.

Pr6, 59.—The photographic spectrum of oxyhzmoglobin and methemoglobin. (Gamgee.)

porphyrin (iron-free hematin) (fig. 57, spectrum 10).- Map out all the spectra
you see on a chart.

8, The Photographic Spectrum.—Hzmoglobin and its compounds also
show absorption bands in the ultra-violet portion of the spectrum. This
portion of the spectrum is not visible to the eye, but can be rendered visible

154 ESSENTIALS OF CHEMICAL PHYSIOLOGY

by allowing the spectrum to fall on a fluorescent screen, or on a sensitive
photographic plate. In order to show absorption bands in this part of the
spectrum very dilute solutions of the pigment must be used.

In order to demonstrate these bands, the telescope of a large spectroscope
is removed, and a beam of sunlight or of light from the positive pole of an
are lamp is allowed to fall on the slit of the collimator. The spectrum is
focussed on a fluorescent screen. The slit is then opened very widely, and
the coloured solution is interposed on the path of the beam falling on the
slit.

Oxyhzemoglobin shows a band (Soret’s band) between the lines G and H.
In hemoglobin, carbonic oxide hemoglobin, and nitric oxide hemoglobin,
this band is rather nearer G. Methzmoglobin and hematoporphyrin show
similar bands.

The two preceding figures show the ‘ photographic spectra’ of hzmo-
globin, oxyhemoglobin, and methemoglobin, and will serve as examples of
the results obtained. I am greatly indebted to Prof. Gamgee, to whom we
owe most of our knowledge on this subject, for permission to reproduce these
two specimens of his numerous photographs.

9. Preparation of Pure Oxyhemoglobin.—The following method is described
in Stirling’s ‘ Practical Physiology’ (8rd edit. p. 65). Centrifugalise dog’s
defibrinated blood and pour off the serum. Centrifugalise again with physio-
logical saline solution repeatedly until the supernatant fluid contains only
traces of proteid. Mix the magma of corpuscles with two or three volumes of
water saturated with acid-free ether; the solution becomes clear. Then add
a few drops of 1-per-cent. solution of acid sodium sulphate till the mixture
looks tinted like fresh blood, owing to the precipitation of the stromata.
These can be separated by centrifugalising. (I have found that they aggre-
gate together and can be easily removed by filtration.) Pour off the clear red
fluid: cool it to 0° C., add one-fourth of its volume of absolute aleohol pre-
viously cooled to 0° C. Shake well, and then let the mixture stand at 5°-15° C.
for 24 hours. As arule the whole passes into a glittering crystalline mass.
Filter at 0° C. and wash with ice-cold 25-per-cent. alcohol. Redissolve the
crystals in a small quantity of water, and recrystallise as before. The
crystals may then be spread on plates of porous porcelain, and dried in a
vacuum over sulphuric acid.

1 Fluorescent screens, similar to those in common use in observations sinte
with Réntgen rays, may be made by coating white cardboard with barium pletino-
cyanide.

a ie we

Te NR ee te

155

LESSON XX
SERUM

1. The following methods of precipitating serum globulin (paraglobulin
should be performed :—

(a) Panum’s Method.—Dilute serum with fifteen times its bulk of water.
It becomes cloudy owing to partial precipitation of the serum globulin. Add
a few drops of 2-per-cent. acetic acid; the precipitate becomes more abun-
dant, and it dissolves in excess of the acid. It was formerly called ‘serum
casein.”

(6) Alexander Schmidt's Method.—Dilute serum with twenty times its bulk
of water and pass a stream of carbonic acid through it. A fairly abundant
precipitate of serum globulin falls. Let it settle, and an additional precipitate
ean be obtained from the decanted liquid by treating it with a trace of acetic
acid (the ‘serum casein’ mentioned above). Repeat the carbonic acid
method without dilution ; no precipitate forms.

(c) By Dialysis.—Put some serum in a dialyser with distilled water in the
outer vessel. The water must be frequently changed. In order to prevent
decomposition afew crystals of thymol are added. In a day or two the salts
have passed out; the proteids remain behind: of these the serum albumin
is still in solution ; the serum globulin is in part precipitated, as it requires
a small quantity of salt to hold it in solution.

(d) By Addition of Salts :—

(i.) Schmidt’s method. Saturate some serum with sodium chloride. A
precipitate of serum globulin is produced.

(ii.) Hammarsten’s method. Use magnesium sulphate instead of sodium
chloride. A more abundant precipitate is produced, because this salt is a-
more perfect precipitant of serum globulin than sodium chloride. In order
to obtain complete saturation with these salts it is necessary to shake the
mixture of salt and serum for some hours.'

(iii.) Kauder’s method. Half saturate serum with ammonium sulphate..
This is done by adding to the serum an equal volume of saturated solution
of ammonium sulphate. This precipitates the globulin. Complete satura-
tion with the salt precipitates the albumin also.

2. Heat Coagulation.—Saturate serum with magnesium sulphate and
filter off the precipitate ; preserve the filtrate and label it‘B.’ Wash the
precipitate on the filter with saturated solution of magnesium sulphate until
the washings do not give the tests for albumin,* then dissolve the precipitate

? This may be conveniently done by a shaking machine before the class meets,

? On account of the prolonged nature of these operations they must necessarily
be performed by the demonstrator beforehand.

156 ESSENTIALS OF CHEMICAL PHYSIOLOGY

by adding distilled water, It readily dissolves, owing to the salt adherent
to it. The solution is opalescent. Label it ‘ A.’

Render A faintly acid with a drop of 2-per-cent. acetic acid, and heat ina
test-tube. The temperature of the test-tube may be raised by placing it in
.a flask of water gradually heated over a flame. A thermometer is placed in
the test-tube, and should be kept moving so as to ensure that all parts of the
liquid are at the same temperature. The quantity of liquid in the test-tube
should be just sufficient to cover the bulb of the thermometer. A flocculent
precipitate of coagulated serum globulin separates out at about 75°.

Now take the filtrate B. This contains the serum albumin. Dilute it
with an equal volume of water; render it faintly acid as before, testing the
reaction with litmus paper. Heat. A flocculent precipitate (a) falls at about
‘73° C; filter this off; note that the filtrate is less acid than that from
which the precipitate has separated, or it may even be alkaline. Ifso, make
it faintly acid again, and heat ; a precipitate falls at 77-79° C. (8). A third
precipitate is similarly obtained at 84-86° C. (y). In the serum of the ox,
sheep, and horse the a precipitate is absent: in cold-blooded animals, the B
and y varieties are absent.

3. Take a fresh portion of B, and saturate it with sodium sulphate. The
serum albumin is precipitated (completely after a long shaking). This is
due to the formation of sodio-magnesium sulphate. B was already saturated
with magnesium sulphate (MgSO,+7H,O); on adding sodium sulphate a
double salt (MgSO,.Na,SO,+6H,O) is formed. Shake some serum with
sodium sulphate alone. A small precipitate of globulin is produced. Saturate
another portion of the serum with sodio-magnesium sulphate ; both globulin
and albumin are precipitated.

Of the methods used for precipitating serum globulin practically only two
are used now. These are Hammarsten’s and Kauder’s. The other methods
only precipitate the globulin incompletely. Kauder’s method is rapid and
efficacious : if the globulin is filtered off, the albumin may be precipitated in
the filtrate by complete saturation with the same salt, ammonium sulphate.
This method avoids the trouble of using two salts as described under 3. This
last method is instructive, but not nearly so quick as Kauder’s.

With regard to the separation of serum albumin into a, y, and 8 varieties
by the use of the method of fractional heat coagulation, it must be men-
tioned that at present no further difference has been shown to exist between
them, and the opinion has been very freely expressed that the results
obtained are not trustworthy. I am convinced that the method is a good one,
especially as in other cases (see Muscie) the proteids so separated can be
shown to possess other differences. In the case of serum, however—and
the same is true for egg albumin—the matter must still be considered sub
judice.

Recent research has shown that serum globulin is not a single proteid.
We have already seen that the precipitation that occurs by means of
dialysis is incomplete. It has now been shown that serum globulin as
‘ salted out’ by means of the sulphate of magnesium or ammonium really
consists of two proteids; one of these (eu-globulin) is precipitable by
dialysis: the other (pseudo-globulin) is not.

TIN II TOE ON TIES LOLS ELE LEE i

157

LESSON XXI
COAGULATION OF BLOOD

1. Salted plasma and oxalate plasma are the two kinds of blood-plasma.
which are most easily prepared (Lesson IX.). The separation of the plasmx
and corpuscles may most readily be carried out by the centrifugal machine
(one form of this is represented in the next figure) ; the corpuscles settle and
the supernatant plasma is pipetted off.

Fic. 60.—Centrifugal machine as made by Runne of Heidelberg. Glass vessels containing the sub--
stances to be centrifugalised are placed within the six metallic tubes which hang vertically while
the dise is at rest ; when the machinery is set going they fly out into the horizontal position. A.
water motor or gas engine may be used to work the instrument. A small but effective hand-
centrifuge is made by Watson, Laidlaw, & Co., Glasgow.

The principal properties of these two forms of plasma have already been
described in Lesson IX.; the following additional experiments may now be
performed.

2. Heat a portion of the salted plasma to 60° C. The fibrinogen is
precipitated (coagulated by heat) at 56°C. Filter. Dilute the filtrate as in
Lesson IX. 3, and add fibrin ferment. No coagulation occurs.

3. Oxalate plasma or decalcified plasma coagulates when a little calcium

158 ESSENTIALS OF CHEMICAL PHYSIOLOGY

-chloride is added, but not if the oxalate plasma has been previously heated to
-60° C. and filtered, because the fibrinogen has been thus removed.

4, Hydrocele Fluid.—This does not clot spontaneously, or only very slowly:
Divide it into four parts—A, B, C, and D.

To A add an equal volume of serum.

To B add a few drops of fibrin-ferment solution.

To C add a piece of buffy coat.

Put them into the warm bath, and coagulation takes place in each. The
serum or the buffy coat supplies the missing fibrin ferment. The serum
does not produce its effect in virtue of the serum globulin it contains;
hydrocele fluid contains both fibrinogen and serum globulin, as the following
experiment shows :—

Take the portion D and half saturate it with sodium chloride by adding
to it an equal bulk of saturated solution of sodium chloride. Fibrinogen is
precipitated. The precipitate is a small one, and on standing aggregates
together, and so becomes more apparent. Filter, and saturate the filtrate
with sodium chloride, or, better, magnesium sulphate; serum globulin is
precipitated.

5. Intravascular coagulation.—A solution of nucleo-proteid from the
thymus, testis, lymphatic glands, or kidney has been prepared beforehand by
the demonstrator. It may be prepared in one of two ways.

(a) Wooldridge’s Method.—The gland is cut up small and extracted with
water for twenty-four hours. Weak acetic acid (0°5 ¢.c. of the acetic acid of
the ‘ Pharmacopeeia’ diluted with twice its volume of water for every 100 c.c.
of extract) is then added to the decanted liquid. After some hours the pre-
cipitated nucleo-proteid (called tissue-fibrinogen by Wooldridge) falls to the
bottom of the vessel. This is collected and dissolved in 1-per-cent. sodium
carbonate solution.

(b) The Sodiwm Chloride Method.—The finely divided gland is ground
up in a mortar with about an equal volume of sodium chloride. The re-
sulting viscous mass is poured into excess of distilled water. The nucleo-
proteid rises to the surface of the water, where it may be collected and
dissolved as before.

A rabbit is anesthetised, and a cannula inserted into the external jugular
vein. The solution is injected into the circulation through this. The
animal soon dies from cessation of respiration; the eyeballs protrude and
the pupils are widely dilated. On opening the animal the heart will be found
still beating, and its cavities (especially on the right side) distended with
clotted blood. The vessels, especially the veins, also are full of clot.

a ee oe

159

LESSON XXII
MUSCLE AND NERVOUS TISSUES

1. A rabbit has been killed and its muscles washed free from blood by a
stream of salt solution injected through the aorta. The muscles have been
quickly removed, chopped up small, and extracted with 5-per-cent. solution
of magnesium sulphate. This extract is given out.

2. The extract will probably be faintly acid. The acid is lactic acid. It
may be identified by the following reaction :—

A solution of dilute ferric chloride and carbolic acid is made as follows :—

10 c.c. of a 4-per-cent. solution of carbolie acid.

20 c.c. of distilled water.

1 drop of the liquor ferri perchloridi of the British Pharmacopeia.

On mixing a solution containing a mere trace (up to 1 part in 10,000) of
lactic acid with this violet solution, it is instantly turned yellow. Larger
percentages of other acids (for instance, more than 0-2 per cent. of hydro-
chloric acid) are necessary to decolorise the test solution.

3. The coagulation of muscle is very like that of blood. This may be
shown with the salted muscle plasma (the extract given out) as follows:
Dilute some of it with four times its volume of water; divide it into two
parts ; keep one at 40° C. and the other at the ordinary temperature. Coagu-
lation, that is, formation of a clot of myosin, occurs in both, but earliest in
that at 40° C.

4. Add a few drops of 2-per-cent. acetic acid to some of the extract; a
stringy precipitate of myosinogen is produced.

5%Remove the clot of myosin from 3; observe it is readily soluble in
10-per-cent. sodium chloride, and also in 0-2-per-cent. hydrochloric acid,
forming syntonin.

6. Perform fractional heat coagulation—

(a) With the original extract. Coagula are obtained at 47°, 56°, 63°,
73° C.

(6) With the liquid (salted muscle serum) in 3, after separation of the
clot. Coagula are obtained at 63° and 73° C.

(c) With muscle extract which has been saturated with magnesium
sulphate and filtered. The globulins are thus separated. Coagulation now
occurs at 73° C., but the amount of coagulum is small.

160 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The following table represents these facts concisely :

| Coagula- Is it glo-
Name of proteid tion tem- Action of MgSO, bulin or Fate
perature albumin ?
1. Massulin or Le ce P These go to form!
paramyosinogen ' 47° C. precipitated | globulin 1 chy asviuals
2. Myosinogen | 56°C. ‘% 3 clot (myosin)
8. Myo-globulin | 68°C. ‘ i la esgacienae
4, Myo-albumin §_ 73° C. | not precipitated | albumin antag

7. Pigments of Muscle :—
(a) Notice the difference between the red and pale muscles of the rabbit.
(b) Examine a piece of red muscle (e.g. the diaphragm) spectroscopically
for oxyhemoglobin (or it may be more convenient to make an aqueous.
extract of the muscle and examine that).

| |
+P. D Ee OF G a
sisi |8 S| § $ g §
J
2

Fx. 61.—1, Absorption spectrum of myohzmatin, as seen in muscle rendered transparent by
glycerin. 2, Absorption spectrum of modified myohzmatin,

(c) A piece of the pectoral muscle of a pigeon has been soaked in glycerin.
Press a small piece between two glass sides and place it in front of the spec-
troscope. Observe and map out the bands of myohematin.

(d) Pieces of the same muscles have been placed in ether for twenty-four:
hours. The ether dissolves out a yellow lipochrome from the adherent fat..
A watery fluid below contains modified myohematin. Filter it ; compare
its spectrum with that of hemochromogen. The myohematin bands are
rather nearer the violet end of the spectrum (fig. 61, spectrum 2) than those.
of hemochromogen (fig. 57, spectrum 9),

8. Creatine :—

(a) Take some of the red fluid described in 7, d, and let it evaporate to-
dryness in a desiccator over sulphuric acid (fig. 62).

' This proteid appears to be absent in many forms of involuntary muscle.

MUSCLE AND NERVOUS TISSUES “265

In a day or two crystals of creatine tinged with myohematin separate out.

(6) Take an aqueous extract of muscle, like Liebig’s extract or beef-tea ;
add baryta water to precipitate the phosphates, and filter. Remove excess
of baryta by a stream of carbonic acid; filter off the barium carbonate and
evaporate the filtrate on the water-bath toa thick syrup. Set it aside to cool,
and in a few days crystalline deposits of creatine will be found at the bottom
of the vessel. These are washed with alcohol and dissolved in hot water.
On concentrating the aqueous solution crystals once more separate out,
which may be still further purified by recrystallisation.

Creatinine can be obtained from meat extracts by Johnson’s method (see
pp. 124, 125): mdeed, Johnson states it is usually more abundant than crea-
tine, and that the same is true for fresh meat.

Note.—The exercises on muscle plasma described in this lesson have
been in the main derived from my own investigations on the subject

S=
=
=a

Fic. 62.—A desiccator. (Gscheidlen.)

(‘ Journ. of Physiol.,’ viii. p. 133, ‘ Chemical Physiology and Pathol.,’ Chap.
XX.). The same subject has been recently taken up by v. Fiirth (‘ Arch.
Exp. Path. u. Pharm. 1895,’ vol. xxxvi. p. 231). His nomenclature of the
proteids is somewhat different from mine, but on main questions we are
in substantial agreement—viz. that in the muscle plasma there are two
principal proteids; these become changed, and so lead to the formation of
the muscle clot, or myosin-fibrin as he terms it. He uses physiological saline
solution to extract the muscle plasma, and this coagulates spontaneously on
standing ; paramyosinogen (v. Fiirth’s myosin) passes directly into the con-
dition of myosin-fibrin ; but myosinogen (v. Fiirth’s myogen) first passes into
a soluble condition (coagulable by heat at the remarkably low temperature of
40° C.) before the myogen-fibrin separates out. The myo-albumin which
occurs in minute amount is probably serum albumin from adherent blood or
lymph. The main points of difference between us are (1) he looks upon
myosinogen as not being a true globulin, though like a globulin in some of its
characters ; (2) myo-globulin is not a separate proteid, but only some myosino-
gen which has escaped coagulation. (3) The phenomenon I have termed re-
coagulation of muscle plasma he looks upon as being merely a reprecipitation

162 ESSENTIALS OF CHEMICAL PHYSIOLOGY

of the globulins, and not a true coagulation process. He does not appear to
have investigated the question of a specific myosin ferment.

When a muscle is gradually heated, at a certain temperature it contracts
permanently and loses its irritability. This phenomenon is known as heat-
rigor, and is due to the coagulation of the proteids in the muscle. Ifa
tracing is taken of the shortening, it is found that the first shortening occurs
at the coagulation temperature of paramyosinogen (47°-50° C.), and if the
heating is continued a second shortening occurs at 56° C., the coagulation
temperature of myosinogen. If frog’s muscles are used there are three
shortenings—namely, at 40°, 47°, and 56° C.; frog’s muscle thus contains
an additional proteid which coagulates at 40° C. (T. G. Brodie). Von Fiirth
regards this additional proteid as the soluble myosin alluded to above, some
of which in frog’s muscle is present before rigor mortis occurs.

In addition to all the proteids previously enumerated, there is a small
quantity of nucleo-proteid.

NERVOUS TISSUES

The chemical investigation of nervous tissues does not lend itself very
well to class exercises; still it may not be uninteresting to state briefly the

principal known facts in relation to this subject. The most important points

which any table of analyses will show are: (1) the large percentage of water,
especially in the grey matter; (2) the large percentage of proteid. In grey
matter, where the cells are prominent structures, this is most marked, and
of the solids, proteid material here comprises more than half of the total.
The following are some analyses which give the mean of a number of
observations on the nervous tissues of human beings, monkeys, dogs, and
cats :

| Percentage of
pi Water Solids Proteids in
Solids
Cerebral grey matter . ; : 83°5 16°5 51
is white = (5; 55 : ant 69:9 8071 33
Cerebellum . : : ; as 79°8 20:2 42,
Spinal cord as a whole emai 716 28°4 31
Cervical cord . 3 : ol 72°5 27°5 31
Dorsal cord. : ; ; sat 69°8 80°2 28
Lumbar cord : : . ee 72°6 'D7°4 33
Sciatie nerves. : ‘ a 651 34:9 | 29

The most abundant proteid is nwcleo-proteid; there is also a certain
amount of globulin, which, like the paramyosinogen of muscle, is coagulated
by heat dt the low temperature of 47° C. A certain small amount of neuro-
keratin (especially abundant in white matter) is included in the above table
with the proteids. The granules in nerve cells (Nissl’s bodies), which stain
readily with methylene blue, are nucleo-proteid in nature. The next most
abundant substances are of a fatty nature; the most prominent of these is
the phosphorised fat called lecithin (see p. 18). In the nervous tissues
lecithin is combined with cerebrin to form a complex substance called
protagon, which erystallises out on cooling a hot alcoholic extract of brain or

——

MUSCLE AND NERVOUS TISSUES 163

«ther nervous structures. Cerebrin is aterm which probably includes
several substances, which are nitrogenous glucosides; they yield on hydrolysis
_ the sugar called galactose (see p. 12). They are sometimes called
_ cerebrosides. The crystalline monatomic alcohol cholesterin (see p. 73) is
_ also a fairly abundant constituent of nervous structures, especially of the
white substance of Schwann. Finally there are smaller quantities of other
extractives, and a small proportion of mineral salts (about 1 per cent. of the
solids).
i nervous tissues are alkaline, but, like most other living structures,
_ they turn acid after death. The change is particularly rapid in grey matter.
_ The acidity is due to lactic acid.

Little or nothing is known of the chemical changes nervous tissues
undergo during activity. We know that oxygen is very essential, especially
for the activity of grey matter; cerebral anemia is rapidly followed by loss

' of consciousness and death. Waller has suggested that small quantities of
| earbonic acid are produced during activity, because the increase in the action
current (detected by the galvanometer) which occurs after a nerve has been
repeatedly excited is very like the increase also noted on the application of small
quantities of this gas. Waller’s statement that the action current is increased
by small amounts of carbonic acid has, however, received other explanations.
Certainly large quantities of carbonic acid act like an anesthetic, abolishing
nervous activity. Of all parts of the nervous system, the cells in the grey
matter are those which most readily manifest fatigue ; the next most sensitive
region is the termination of nerves in such endings as the end plates. Fatigue
_ in amedaullated nerve trunk has never yet been experimentally demonstrated ;
| Waller’s view that this is due to inter-nutritional changes between the axis
i cylinder and the investing medullary sheath can hardly be considered proved,
| for it is just as difficult to demonstrate fatigue in non-medullated nerves.
Chemistry of nerve-degeneration.—_Mott and I have shown that in the
' disease General Paralysis of the Insane, the marked degeneration that oecurs
__ in the brain is accompanied by the passing of the products of degeneration
_ into the cerebro-spinal fluid. Of these, nucleo-proteid and choline—a decom-
position product of the lecithin (see p. 18)—are those which can be most readily
detected. Choline can also be found in the blood. But this is not peculiar
to the disease just mentioned, for in various other degenerative nervous
diseases (combined sclerosis, disseminated! sclerosis, meningitis, alcoholic
neuritis, beri-beri) choline can also be detected in these situations. The
tests employed to detect choline are mainly two: (1) a chemical test, namely
the obtaining of the characteristic octahedral crystals of the platinum double
salt from the alcoholic extract of the cerebro-spinal fluid or blood;* (2) a

1 This test is performed as follows: the fiuid is diluted with about five times
its volume of alcohol and the precipitated proteids are filtered off. The filtrate is
evaporated to dryness at 40° C. and the residue dissolved in absolute alcohol and
filtered ; the filtrate from this is again evaporated to dryness, and again dissolved
in absolute alcohol, and this should be again repeated. To the final alcoholic solu-
tion, an alcoholic solution of platinum chloride is added, and the precipitate so
formed is allowed to settle and washed with absolute alcohol by decantation ; the
precipitate is then dissolved in 15-per-cent. alcohol, filtered, and the filtrate is
allowed to slowly evaporate in a watch-glass at 40° C. The crystals can then be
seen with the microscope. They are recognised not only by their yellow colour

u 2

164 ESSENTIALS OF CHEMICAL PHYSIOLOGY

physiological test, namely the lowering of arterial blood-pressure (partly
cardiac in origin, and partly due to dilatation of peripheral vessels) which a.
saline solution of the residue of the alcoholic extract produces: this fall is
abolished, or even replaced by a rise of arterial pressure, if the animal has.
been atropinised. It is possible that such tests may be of diagnostic value
in the distinction between organic and so-called functional diseases of the
nervous system. The chemical test can frequently be obtained with 10 c.c.
of blood.

A similar condition can be produced artificially in animals by a division
of large nerve trunks; and is most marked in those animals in which the
degenerative process is at its height as tested histologically by the Marchi
reaction.! A chemical analysis of the nerves themselves was also made. A
series of cats was taken, both sciatic nerves divided, and the animals subse-
quently killed at intervals varying from 1 to 106 days. The nerves remain
practically normal as long as they remain irritable, that is up to 8 days after
the operation. They then show a progressive increase in the percentage of
water, and a progressive decrease in the percentage of phosphorus until
degeneration is complete. When regeneration occurs, the nerves return
approximately to their previous chemical condition. The chemical explana-
tion of the Marchi reaction appears to be the replacement of phosphorised by
non-phosphorised fat. When the Marchi reaction disappears in the later
stages of degeneration, the non-phosphorised fat has been absorbed. This
absorption oceurs earlier in the peripheral nerves than in the central nervous
system.

Further, it has been found that in human spinal cords in which a unilateral
degeneration of the pyramidal tract has been produced by a lesion in the
opposite hemisphere, and which gives the Marchi reaction, there is a similar
increase of water and diminution of phosphorus in the degenerated side.

The table on next page shows these main results in the experiments on cats
just described.

Cerebro-spinal ffuid.—This functions as the lymph of the central nervous
system, but differs considerably from all other forms of lymph. It is a very
watery fluid, containing, besides some inorganic salts similar to those of the
blood, a trace of proteid matter (globulin) and a small amount of a reducing
substance, the nature of which is uncertain. It is, however, not sugar, but is
possibly an aromatic substance allied to pyrocatechin. It contains the
merest trace of choline ; but this is not devoid of significance, for this fact
taken in conjunction with another—namely, that physiological saline solution
will extract from perfectly fresh nervous matter a small quantity of choline—
shows us that protagon is not a stable substance, but is constantly breaking
down and building itself up afresh; in fact, undergoing the process called
metabolism. This is most marked in the most active region of the brain—
viz., the grey matter.
and octahedral form, and by their solubility in water and 15-per-cent. alcohol, but

also by the fact that on incineration they yield 31 per cent. of platinum and give
off the odour of trimethylamine.

' The Marchi reaction is the black staining that the medullary sheath of
degenerated nerve fibres shows when, after being hardened in Miiller’s fluid, they are
treated with Marchi’s reagent, a mixture of Miiller’s fluid and osmicacid. Healthy
nerve fibres are not affected by the reagent, but degenerated myelin is blackened
like the fat of normal adipose tissue.

165,

Condition of
nerves
| |
=
Minimal Nerves irritable
Normal 651 | 349 1 Sesh
1 to 3 days after section 645 35°55 og {ta clei {and i logically
Choline more | { Ititability lost ;
4 to 6 Pa }eo3 | 307 | . 09 | Spundant [aeeemectioe ho-
.
8 | 6s2 318 05 n well
10 es | 70-7 | 293 | 03 { Choline abun- | shown by Marchi
13 ” | 1-3 | 287 | 0-2 xan ‘wae
Marchi reac
|
25-27 : | 721 | 279 | traces | { Choline much {sit seen, but ab-
1 29 < ” } 725 27°5 | 0 } } generated fat has
. \ set in
. |
Absorption of fat
44 2 | 726 | 97-4 | 0 ( Choline al- {practical com-
f | | + most disap- plete
100t0 106, 62) 38 | o9 | (peared { Return of funetion,
:

: The foregoing figures relate to the peripheral portions of the nerves. Noll
__ has also shown that the phosphorised material protagon diminishes some-
_ what in the central ends of cut nerves due to ‘ disuse atrophy.’

166° ESSENTIALS OF CHEMICAL PHYSIOLOG ¢

LESSON XXIII
UREA AND CHLORIDES IN URINE

ESTIMATION OF UREA

If albumin is present it must be first separated by boiling after acidulation
with acetic acid if necessary, and filtering off the flakes of coagulated proteid.

The three chief methods of estimating urea are—

(a) The mercuric nitrate or Liebig’s method.

(6) The hypobromite, or Hiifner’s method.

a. Liebig’s Method.—The combination between urea and mercury has.

the formula (CON,H,).Hg(NO,),(HgO),. It forms a white precipitate, in-

soluble in water and weak alkaline solutions. It is therefore necessary to.

prepare a standard solution of mercuric nitrate, and to have an indicator by
which to detect the point when all the urea has entered into combination

with the mercury, and the latter slightly predominates. This indicator is.

sodium carbonate, which gives a yellow colour with the excess of mercury,
owing to the formation of hydrated mercuric oxide.

Theoretically, 100 parts of urea should require 720 parts of mercuric
oxide, but practically 772 of the latter are necessary to remove all the
urea, and at the same time show the yellow colour with alkali; consequently

the solution of mercuric nitrate must be of empirical strength in order to.

give accurate results.

The following solutions must be prepared—

i. Standard mercuric nitrate solution. Dissolve 77°2 grammes of red oxide
of mercury (weighed after it has been dried over a water-bath), or 71°5 gr.
of the metal itself, in dilute nitric acid. Expel excess of acid by evapo-
rating the liquid to a syrupy consistence. Make up to 1,000 c.c. with distilled
water, adding the water gradually. This solution is of such a strength that

19 c.c. will precipitate 10 c.c. of a 2-per-cent urea solution. Add 52°6 c.c. of

water to the litre of the mercuric nitrate solution and shake well; then 20 c.c.
(instead of 19) = 10 c.c. 2-per-cent. urea solution, ie. 1 ¢.c. = ‘01 gr. urea.

ii. Baryta mixture. This is a mixture of two volumes of solution of

barium hydrate with one of solution of barium nitrate, both saturated in the
cold.
Analysis.—Take 40 ¢.c. of urine. Add to this 20 c.c. baryta mixture and

filter off the precipitate of barium salts (phosphates and sulphates). Take-

15 c.c. of the filtrate (this corresponds to 10 ¢.c. of urine) in a beaker. Run
into it the mercuric nitrate solution from a burette, until it is found that, on
mixing a drop of the mixture with a drop of a saturated solution of sodium

NES

UREA AND CHLORIDES IN URINE See (oe:

carbonate on a white tile, a pale lemon colour is produced. Then read the
amount used from the burette, and calculate thence the percentage of urea.

Corrections.—This method only approaches accuracy when the quantity
of urea present is about 2 per cent., which is about the normal percentage of
urea in urine. The chlorine m the urine must also be estimated, and the
quantity of urea indicated reduced by the subtraction of 1 gramme of urea
for every 1°3 gramme of sodium chloride found. If the urine contains less
than 2 per cent. of urea, 0°1 c.c. of mercuric nitrate solution must be deducted
for every 4 c.c. used; if more than 2 per cent. of urea, a second titration
must be performed with the urine diluted with half as much water as has
been needed of the mercurial solution above 20 ¢.c. Suppose that 28 c.c.
have been used in the first titration, the excess is 8 c.c.; therefore 4 c.c. of
water must be added to the urine before the second titration ismade. When
ammonium carbonate is present, first estimate the urea in one portion of
urine, and the ammonia by titration with normal sulphuric acid in another ;
0017 gramme of ammonia = 0°030 of urea. The equivalent of ammonia
must be added to the urea found in the first portion of urine.

b. The Hypobromite Method.—This is a far easier method. It consists in
decomposing urea into water, carbonic acid, and nitrogen by means of an
alkaline solution of hypobromite of soda; the carbonic acid combines with
the soda, and the nitrogen which is evolved is measured, and the quantity of
urea calculated from this. There are many kinds of apparatus for per-
forming this operation, but the best is that of Dupré (see Lesson X.).

Reactions and Corrections.—The reaction by which urea is decomposed
in this proceeding may be denoted by the following formula :—

CON,H, + 3NaBrO = CO, + N, + 2H,O + 3NaBr.

From 1 gramme of urea 0°46 gramme of nitrogen = 372°7 c.c. is obtained.

In practice, however, it is found that only 354°3 ¢.c. are obtained,’ except
in diabetic urine, in which the urea yields nearly the normal amount of
nitrogen. Moreover, urine contains small quantities of creatinine and
urates, which yield some of their nitrogen when acted on by sodium hypo-
bromite. When great exactitude is required these must be removed—
creatinine by an alcoholic solution of zine chloride, and the urates by acetate
of lead followed by sodium phosphate (Yvon).

5 c.c. of a 2-per-cent. solution of urea in urine yield 35:4 ¢.c. of nitrogen.
This quantity is taken as representing 2 per cent. of urea, and serves as a
basis for the graduations of the tubes, which are marked in percentages.

When exactitude is required, the quantity of nitrogen must be measured
in cubie centimetres, and the volume obtained corrected for temperature,
pressure, and tension of aqueous vapour by the formula below.”

' The cause of this loss of nitrogen has been investigated by Luther, Zeit.
physiol. Chem. xiii. p. 500. He finds part is combined as a nitrate, and part in an
unknown organic compound which gives off ammonia when distilled with alkali.

2 V’'= correct volume; V = vol observed; B = barometric pressure corrected
for temperature ; ¢ = temp. in degrees Centigrade ; T = tension of aqueous vapour
in millimetres of mercury at ¢° (see table, p. 8). Then .

Vv x (B - T)

V'= 760 x (1 + 0-003665t)

168 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Of the two methods just described, Liebig’s is so cumbrous and inexact
that it has almost passed outof use. The hypobromite method holds its own,
as it is easy and sufficiently exact for clinical purposes. When absolute
accuracy is necessary, one of the numerous recently introduced methods
must be employed, and of these the method of Mérner and Sjéqvist appears
to be best.

i, A saturated solution of barium chloride containing 5 per cent. of
barium hydrate.

ii. A mixture of ether and alcohol in proportion 1 : 2.

iii. The apparatus, &c., necessary for carrying out Kjeldahl’s method of
estimating nitrogen (see p. 199).

Analysis.—Five c.c. of urine are mixed with 5 ¢.c. of the barium mixture
and 100 ¢.c. of the mixture of ether andaleohol. By this means all nitrogenous
substances except urea are precipitated. Twenty-four hours later this is filtered
off, and the precipitate is washed with 50 c.c. of the ether-alcohol mixture,
the filter-pump being used to accelerate the process. The washings are
added to the filtrate; a little magnesia is added to this to drive off ammonia,
The alcohol and ether is then driven off at a temperature of 55° C. and
evaporation is continued at this temperature until the volume of the residue
is 10-15 c.c. The nitrogen in this is estimated by Kjeldahl’s method. The
nitrogen found is multiplied by 2°148, and the result is the amount of urea,

PREPARATION OF UREA FROM URINE

(1) Evaporate the urine to a small bulk. Add strong pure nitric acid in
excess, keeping the mixture cool during the addition of the acid. Pour off
the excess of fiuid from the crystals of urea nitrate which are formed ; strain
through muslin and press between filter paper. Add to the dry product
barium carbonate in large excess. This forms barium nitrate and sets the
urea free. Mix thoroughly with sufficient methylated spirit to form a paste.
Dry on a water-bath and extract with alcohol; filter; evaporate the filtrate
on a water-bath and set aside. The urea crystallises out, and may be de-
colorised by animal charcoal and purified by recrystallisation.

(2) The following method is well adapted for the preparation of micro-
scopic specimens of urea and urea nitrate: Take 20 c.c. of ure; add

aryta mixture’ (two volumes of barium hydrate solution and one volume
of barium nitrate solution, both saturated in the cold) until no further preci-
pitate is produced; filter, evaporate the filtrate to a thick syrup on the
water-bath, and extract with alcohol; pour off and filter the alcoholic
extract; evaporate it to dryness on the water-bath and take up the residue
with water. Place a drop of the aqueous solution on a slide and allow it to
crystallise ; crystals of urea separate out. Place another drop on another
slide and add a drop of nitric acid ; crystals of urea nitrate separate out.

ESTIMATION OF CHLORIDES

The chlorides in the urine consist of those of sodium and potassium, the
latter only in small quantities. The method adopted for the determination

UREA AND CHLORIDES IN URINE ~ 169

of the total chlorides consists in their precipitation by a standard solution
of silver nitrate or mercuric nitrate.

Mohr’s Method.—Precipitation by silver nitrate.

The following solutions must be prepared :

Standard silver nitrate solution. Dissolve 29°075 grammes of fused
nitrate of silver in a litre (1,000 c.c.) of distilled water; 1 ¢.c. = 0°01 gramme
of sodium chloride.

(a) Saturated solution of neutral potassium chromate.

Analysis.—Take 10 c.c. of urine; dilute with 100 c.c. of distilled water.

Add to this a few drops of the potassium chromate solution.

Drop into this mixture from a-burette the standard silver nitrate solu-
tion ; the chlorine combines with the silver to form silver chloride, a white
precipitate. When all the chlorides are so precipitated, silver chromate (red
in colour) goes down, but not while any chloride remains in solution. The
silver nitrate must therefore be added until the precipitate has a pink tinge.

From the amount of standard solution used, the quantity of sodium
chloride in 10 c.c. of urine, and thence the percentage, may be calculated.

Sources of Error and Corrections.—A high-coloured urine may give rise
to difficulty in seeing the pink tinge of the silver chromate: this is overcome
by diluting the urine more than stated in the preceding paragraph.

1 c.c. should always be subtracted from the total number of c.c. of the
silver nitrate solution used, as the urine contains small quantities of certain
compounds more easily precipitable than the chromate.

_ (6) To obviate such sources of error the following modification of the
test, as described by Sutton,’ may be used: 10 c.c. of urine are measured
into a thin porcelain capsule and 1 gramme of pure ammonium nitrate
added ; the whole is then evaporated to dryness, and gradually heated over
asmall spirit lamp to low redness till all vapours are dissipated and the
residue becomes white. It is then dissolved in a small quantity of water,
and the carbonates produced by the combustion of the organic matter
neutralised by dilute acetic acid ; a few grains of pure calcium carbonate to
remove all free acid are then added, and one or two drops of potassium
chromate. The mixture is then titrated with decinormal silver solution
(16-966 gr. of silver nitrate per litre) until the end reaction, a pink colour,
appears. Each c.c. of silver solution represents 0°005837 gr. of salt ; con-
sequently, if 12-5 c.c. have been used, the weight of salt in the 10 c.c. of

_ urine is 0-07296 gr., or 07296 per cent. If 5-9 c.c. of urine are taken for

titration, the number of c.c. of silver solution used will represent the number

of parts of salt per 1,000 parts of urine.

The following solutions must be first prepared :

i. Standard mercuric nitrate solution: Dissolve 20 grammes of pure
mercury in boiling nitric acid; then dilute to nearly a litre. To dilute this
to the right strength, preliminary experiments must be performed with a
standard solution of pure sodium chloride, 20 grammes to the litre. Take
10 c.c. of the standard sodium chloride solution, add to this 2 ¢.c. of a 4-per-
cent. solution of urea and 5 c.c. of a saturated solution of sodium sulphate.

! Volumetric Analysis, p. 309.

170 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Into this mixture allow the mercuric nitrate solution to flow from a burette,
stirring the mixture the while. A precipitate forms, which redissolves on
stirring; add the mercuric nitrate solution till a permanent precipitate (not
an opalescence) forms; the reaction is then complete. The strength of the
mercurial solution is thus determined, and it is then diluted so that 20 c.c.
= 02 gramme of sodium chloride = 10 ¢.c. of the standard sodium chloride
solution ; 1 c.c., therefore, corresponds to 0°01 gramme of sodium chloride,
or 0:006059 gramme of chlorine.

ii. Baryta mixture, made by adding two volumes of barium hydrate
solution to one of barium nitrate solution, both saturated in the cold.

iii. Dilute nitric acid (1 in 20).

Analysis.—Take 40 c.c. of urine.

Add 20 c.c. of baryta mixture. Filter off the precipitate which forms,
which consists of sulphate and phosphate of barium.

Take 15 c.c. of the filtrate: this corresponds to 10 c.c. of the original
urine.

Render this slightly acid with dilute nitric acid.

Run in the standard mercuric nitrate solution from a burette, stirring the
mixture well until a permanent precipitate appears.

Read off the number of ¢.c. used; multiply by 0°01. This gives the
amount of chloride as sodium chloride contained in 10 c.c. urine.

Explanation and Corrections.—This test depends on the fact that when
mercuric nitrate and sodium chloride in solution are mixed, sodium nitrate
and mercuric chloride, which are both soluble in water, are formed. It is
not till all the chloride in the urine is so decomposed that mercuric nitrate
begins to combine with the urea present to form a permanent white pre-
cipitate. Hence the necessity of estimating the chlorides when using
Liebig’s method for the determination of urea.

In order to obtain the exact point at which the precipitate becomes a
permanent one, the process must be repeated in another specimen. The
advantage of this process is its simplicity: its disadvantage is that the end
point is rather obscure.

If the urine used is albuminous, the albumin must be first removed by
boiling, after the addition of a few drops of acetic acid, and filtering off the
precipitated proteid.

Fl a ECO ee he

171

LESSON XXIV
ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE

ESTIMATION OF PHOSPHATES

The phosphoric acid in the urine is combined with soda, potash, lime, and
magnesia.

(a) Estimation of the total phosphates.

For this purpose the following reagents are necessary :

i. A standard solution of uranium nitrate. The uranium nitrate solution
contains 35°5 grammes in a litre of water ; 1 c.c. corresponds to 0005 gramme
of phosphoric acid (P,0O.).

ii. Acid solution of sodium acetate. Dissolve 100 grammes of sodium
acetate in 900 c.c. of water; add to this 100 c.c. of glacial acetic acid.

iii. Solution of potassium ferrocyanide.

Method.—Take 50 c.c. of urine. Add 5 c.c. of the acid solution of sodium
acetate. Heat the mixture to 80° C.

Run into it while hot the standard uranium nitrate solution from a
burette until a drop of the mixture gives a distinct brown colour with a drop
of potassium ferrocyanide placed on a porcelain slab. Read off the quantity
of solution used and calculate therefrom the percentage amount of phosphoric
acid in the urine.

(6) Estimation of the phosphoric acid combined with lime and magnesia
(alkaline earths).

Take 200 c.c. urine. Render it alkaline withammonia. Lay the mixture
aside for twelve hours. Collect the precipitated earthy phosphates on a filter ;
wash with dilute ammonia (1 in 3). Wash the precipitate off the filter with
water acidified by a few drops of acetic acid. Dissolve with the aid of heat,
adding a little more acetic acid if necessary. Add 5 c.c. of the acid solution
of sodium acetate. Bring the volume up to 50 c.c., and estimate the phos-
phates in this volumetrically by the standard uranium nitrate as before.
Subtract the phosphoric acid combined with the alkaline earths thus obtained
from the total quantity of phosphoric acid, and the difference is the amount
of acid combined with the alkalis soda and potash. ‘

(c) Instead of uranium nitrate a standard solution of uranium acetate
may be used. The directions for the making of these standard solutions will
be found in Sutton’s ‘ Volumetric Analysis.’ As a rule, it is less troublesome,
and not much more expensive, to purchase standard solutions ready-made.

In using uranium nitrate it is imperative that sodium acetate should

accompany the titration in order to avoid the possible occurrence of free nitric acid
in the solution. If uranium acetate is used, it may be omitted.

172 ESSENTIALS OF CHEMICAL PHYSIOLOGY

ESTIMATION OF SULPHATES

The sulphates in the urine are of two kinds: the pre-formed sulphates—
viz. those of soda and potash, and the combined or ethereal sulphates.

(a) For the determination of the total amount of sulphuric acid (SO,) (i.e.
pre-formed and combined sulphuric acid together) in the urine, one of two
methods is adopted :

1. Volumetric method.

2. Gravimetric method. te

Both methods will be given here; the former is, however, better suited
for class experiments.

1. Volumetric Determination.—This process consists in adding to a
given volume of the urine a standard solution of chloride of barium so long
as a precipitate of barium sulphate is formed.

The following solutions are necessary :—

i. Standard barium chloride solution: 80°5 grammes of crystallised
chloride of barium in a litre of distilled water; 1 ¢.c. of this solution corre-
sponds to 0°01 gramme of sulphuric acid (SO,).

ii. Solution of sulphate of potash: 20 per cent.

iii. Pure hydrochloric acid.

Method.—100 c.c. of urine are taken in a flask. This is rendered acid by
5 ec. of hydrochloric acid, and boiled. The combined sulphates are thus
converted into ordinary sulphates, and give a precipitate like them with
barium chloride. The chloride of barium solution is allowed to drop into
this mixture as long as any precipitate occurs, the mixture being heated before
every addition of barium chloride to it. After adding 5 to 8 c.c. of the
standard solution, allow the precipitate to settle; pipette off a few drops of
the clear supernatant fluid into a watch-glass; add to it a few drops of the
standard barium chloride solution. If any precipitate occurs, return the
whole to the flask and add more barium chloride ; again allow the precipitate
to settle, and test as before; go on in this way until no more barium sulphate
is formed on the addition of barium chloride.

Excess of barium chloride must also be avoided; when only a trace of
excess is present a drop of the clear fluid removed from the precipitate gives
a cloudiness with a drop of the potassium sulphate solution placed on a
glass plate over a black ground. If more than a cloudiness appears, too
large a quantity of barium chloride has been added, and the operation must
be repeated. From the quantity of barium chloride solution used, the per-
centage of sulphuric acid in the urine is calculated.

2. Gravimetric Determination (i.e. by weight).—This method consists in
weighing the precipitate of barium sulphate obtained by adding barium
chloride to a known volume of urine; 100 parts of sulphate of barium
correspond to 34°33 parts of sulphuric acid (SO,).

Method (Salkowski).—100 ¢.c. of urine are taken in a beaker. This is
boiled with 5 c.c. of hydrochloric acid as before.

Chloride of barium is added till no more precipitate occurs.

The precipitate is collected on a small filter of known ash, and washed
with hot distilled water till no more barium chloride occurs in the filtrate—

PDI et ER OR BE EN mn

EE RT: AO os

RB. .) @h ye
* ~~

ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE 173

i.e. until the filtrate remains clear after the addition of a few drops of hydric
sulphate. Then wash with hot alcohol, and afterwards with ether. Remove
the filter, and place it with its contents in a platinum crucible. Heat to
redness. Cool over sulphuric acid in a desiccator; weigh, and deduct the
weight of the crucible and filter ash ; the remainder is the weight of barium
sulphate formed.

Error.—When the experiment is carried out as above there is a slight
error from the formation of a small quantity of sulphide of barium. This
may be corrected as follows: After the platinum crucible has become cool
add a few drops of pure sulphuric acid (H,SO,). The sulphide is converted
into sulphate. Heat again to redness to drive off excess of sulphuric acid.

(6) The following is Salkowski’s* method of estimating combined
sulphuric acid—that is, the amount of SO, in ethereal sulphates :—100 c.c.
of urine are mixed with 100 c.c. of alkaline hein chloride solution, which is.
a mixture of two volumes of solution of barium hydrate with one of barium
chloride, both saturated in the cold. The mixture is stirred, and after a *)¥
minutes filtered : 100 c.c. of the filtrate (=50 c.c. of urine) are acidified with
10 c.c. of hydrochloric acid, boiled, kept at 100° C. on the water-bath for an
hour, and then allowed to stand till the precipitate has completely settled ; if
possible, it should be left in this way for twenty-four hours. The further
treatment of this precipitate (= combined sulphates) is then carried out as in
the last case.

Calculation.—233 parts of barium sulphate correspond to 98 parts of
H,SO,, or 80 parts of SO, or 32 ope of S. To calculate the H,SO,, multiply

the weight of barium sulphate by 5 = = 0-4206 ; to calculate the SO, multiply

by on 0°34335 ; to calculate the S multiply by = = 013734. This method

of calculation applies to the gravimetric estimation both of total sulphates
and of combined sulphates.

amount of combined SO, from the total amount of SQ,. The difference is
the pre-formed SO,.

Example: 100 c.c. of urine gave 0°5 gramme of total barium sulphate.
This multiplied by Pa =0°171 gr. =total SO,. Another 100c.c. of the same
urine gave 0°05 gr. of barium sulphate from ethereal sulphates; this multi-
plied by a = 0-017 gr. of combined SO, Total SO,—combined SO,
= 0171 —0°017 = 0°154 gr. of pre-formed SO,.

} Zeit. physiol. Chem. x. p. 346. This method is a modification of Baumann’s
original method, ibid. i. p. 7L.

174 ESSENTIALS OF CHEMICAL PHYSIOLOGY

LESSON XXV
URIC ACID AND CREATININE

1. Prepawation of Pure Uric Acid.—If one wishes to prepare pure uric acid
the solid urine of a reptile or bird, which consists principally of the acid
ammonium salt, should be selected; one has not then to separate any pig-
ment. It is boiled with 10 per cent. caustic soda or ammonia, diluted, and
then allowed to stand. The clear fluid is decanted and poured into a large
excess of water to which 10 per cent. of hydrochloric acid has been added ;
after twenty-four hours, crystals of uric acid are deposited. These may be
purified by washing, re-solution in soda, and re-precipitation by acid.

2. Estimation of Urie Acid (Hopkins’s method),—The following reagents
are required :

Pure chloride of ammonium, finely powdered.

A wash bottle containing a filtered saturated solution of the same salt.

A twentieth normal solution of potassium permanganate made by
dissolving 1581 grammes of permanganate in a litre of water.

Separation of wric acid from the wrine.—Measure 100 c.c. of urine into
a beaker of about 150 c.c. capacity.

Add to this 25 grammes (approximately weighed) of ammonium chloride,
stirring briskly till all the salt is dissolved. Now add 2 e.c. of strong
ammonium hydrate, and allow the mixture to stand until the precipitate of
ammonium urate, which rapidly forms, has wholly settled to the bottom of
the beaker ; its subsidence is promoted by occasional brisk stirring.

Adjust a small filter paper (7 cm. diam.) in a funnel of such size that only
a small margin of glass projects above the edge of the folded paper, and
transfer to this the ammonium urate precipitate.

Filtration should not be commenced until the precipitate has settled
satisfactorily, and the supernatant liquid is clear. The latter should be first
poured on to the filter, the precipitate being so far as possible retained in the
beaker until the greater part of the clear liquid has filtered through; finally
transfer the whole to the filter with the help of a wash bottle containing
saturated ammonium chloride solution. After the filter has thoroughly
drained, wash the precipitate twice again with the same solution.

While the last washings are running through the paper, distilled water
should be heated to boiling in a wash bottle provided with a jine jet. The
funnel containing the filter is now held horizontally over a small porcelain
basin (of about 50 ¢.c. capacity) and the precipitate washed into the latter
with a jet of hot water, the filter itself being afterwards opened out over the
basin in order that any urate adhering to fs folds can be washed off. Not

0 EEE

URIC ACID AND CREATININE ~ 195

more than 20-30 c.c. of water need be employed in this transference : if much

more has been used the liquid should be concentrated over the water bath at
this stage. A little strong HCl (1 c.c.) is next added to the-contents of the
basin, and the whole is then heated over a burner until it just reaches
the boiling point. It is then set aside for the uric acid to crystallise out.

Titration of the wric acid.—If the mixture is artificially cooled all the
uric acid will separate out in two hours, otherwise it is best allowed to stand
over night or longer.

The crystals are filtered off through a very small filter paper (4 cm.
diam.); the filtrate is received into a graduated cylinder so that the
amount of mother liquor may be noted (see below). The uric acid is next
washed with cold distilled water until free from chlorides. It is unnecessary
to transfer the whole to the filter ; the greater part may be washed by decanta-
tion. Such of the crystals as are upon the filter are now washed back into
the basin (best by the aid of hot water) and the whole-quantity is dissolved
by heating to boiling with 1 ¢.c. of 10-per-cent. sodium carbonate solution
and as much distilled water as the basin will safely hold.

The solution is transferred to a }-litre Erlenmeyer flask, which should be
marked roughly at 100 c.c. The solution is made up to this mark with dis-
tilled water, and cooled to the temperature of the room.

The standard permanganate solution being ready in a burette, 20 c.c. of
strong sulphuric acid are added to the contents of the flask, and the mixture
shaken and titrated.

During the addition of the standard solution the liquid in the flask should
be kept in vigorous movement. It will be found that at first the disappear-
ance of the pink colour is so rapid that each drop as it is added is decolorised
before it has time to diffuse through the whole liquid. The first instan-
taneous appearance of a diffused flush throughout the solution indicates the
end point of the reaction. This colour rapidly disappears, but it will be found
that the effect of adding further quantities of permanganate after the end
point has been passed is quite different from the effect before the end point
was reached; each drop is now able to diffuse throughout the fluid.

For each c¢.c. of the solution necessary to produce the end point just
described 0:00375 gramme of uric acid is present. To the value so obtained
1 mgm. must be added for each 15 c.c. of the mother liquor from which the
crystals separated.

Thus the uric acid from 100 c.c. of a sample of urine used up 18°5 e.c.
of the standard permanganate solution. The mother liquor filtered from the
crystals measured 25 c.c.

18°5 x ‘00375 = -0694 gr. nearly
Oo1x 2 O17 _—C=~
15

Total = -0711

The urine contained 71 mgms. uric acid per 100 e.c.

8. Estimation of Creatinine.—The crystalline compound which creatinine
forms with zine chloride is employed in estimating the quantity of creatinine
in urine; 100 parts of the compound correspond to 62°42 of creatinine.

Method.—Take 250 c.c. of urine. Add milk of lime and calcium chloride

176 ESSENTIALS OF CHEMICAL PHYSIOLOGY

in excess to precipitate the phosphates. Filter, and evaporate the filtrate to
a small bulk; to this add 50 ¢.c. absolute alcohol, and let the mixture stand
for six hours. Then add 10 or 15 drops of an alcoholic solution of zine

chloride; the crystals form, and after two or three days’ standing in a

dark place may be collected on a weighed filter.

Wash with 90 per cent. alcohol, dry and weigh, and thence calculate the
percentage of creatinine.

4, Estimation of Creatinine. Johnson’s Method.—Take 100 c.c. of urine;
add to it 5 ¢.c. of a saturated solution of sodium acetate, and then 20 c.c. of a
saturated solution of mercuric chloride. This produces an abundant precipi-
tate of urates, sulphates, and phosphates. Filter. Set the filtrate aside for
twenty-four hours, and the mercury compound of creatinine crystallises out.
Examine this deposit with the microscope: note it is composed of spherules.
For quantitative purposes, this is collected, washed, dried, and weighed in
the usual way. One-fifth of the weight obtained is creatinine. Throughout
the processes no heat is used, otherwise the characteristic properties of urinary
creatinine are altered; but if only a quantitative analysis is wanted the
method may be hastened by boiling the first filtrate for ten minutes, instead
of letting it stand twenty-four hours.

In order to separate the base itself (see p. 125), much larger volumes of
urine must be employed, for there is considerable loss in the later stages
of the process. Johnson himself used some hundreds of litres.

.™

EE ee Se

177

LESSON XXVI
THE PIGMENTS OF THE URINE

The urinary pigments are numerous, and have from time to time been
described under different names by various observers.

1. Urochrome.—This is the essential yellow pigment of the urine. The
word was originally introduced by Thudichum, and the substance he obtained
is now recognised to have been a mixture of several pigments, of which,
however, the essential yellow pigment formed a large proportion. Garrod’s
method of separating it from the urine is as follows :—

The urine is saturated with ammonium sulphate and filtered. The
filtrate contains the pigment; this is shaken with alcohol. The alcohol
separates readily from the saline mixture, and as it does so dissolves out
much of the urochrome. By repeated extraction all the pigment passes into
solution in the alcohol. The alcoholic solution is diluted with water, and
the mixture again saturated with ammonium sulphate. The alcohol con-
taining the pigment in solution again separates out. The second alcoholic
solution is made faintly alkaline with ammonia and evaporated to dryness.
The residue is extracted once or twice with acetic ether, and then again
dissolved in strong alcohol. Finally the alcohol is concentrated till it is
deep orange in tint, and poured into an equal volume of ether. The pure
pigment is by this means precipitated as an amorphous brown powder.

Urochrome shows no absorption bands. As already stated (p. 108), it is
probably an oxidation product of urobilin.

2. Urobilin.—Urobilin is a derivative of the blood-pigment, and is identical
with stercobilin (see pp. 73,107). Probably both reduction and hydration
occur in its formation. It is very like the substance named hydrobilirubin
by Maly, which he obtained by the action of sodium amalgam on bilirubin.
The following formule’ show the relationship between these allied pig-
ments :—

Hematin , ‘ . ; , . C,,H,.N,0,Fe
Bilirubin , ‘ . ‘ <u ep (Oebtaeae
Hydrobilirubin . : , : 4 Cette D.

Urobilin is probably a further stage in reduction.
Normal urine contains but little urobilin; what is present is chiefly in
the form of a colourless chromogen, which by oxidation is converted into

urobilin. In numerous pathological conditions urobilin is abundant. The

' The formule given are those of Nencki and Sieber. They differ from hose
previously given (p. 90) by Hoppe-Seyler.

178 ESSENTIALS OF CHEMICAL PHYSIOLOGY

so-called ‘pathological urobilin’ described by previous writers is ordinary
urobilin incompletely separated from various impurities.

The following are the methods introduced by Garrod and Hopkins for its
separation from the urine :—

(a) The urine is first saturated with ammonium chloride, and the urate
so precipitated is filtered off. The filtrate is then acidified with sulphuric
acid and saturated with ammonium sulphate. This causes a precipitate of
urobilin, which may be collected and dissolved in water. The aqueous
solution is again saturated with ammonium sulphate, and the pigment
is thus precipitated in a state of purity.

(6) The urates are first removed, then the urine is acidified and saturated
with ammonium sulphate as before. The urobilin is then extracted from the
mixture by shaking it with a mixture of chloroform and ether (1:2) in a
large separating funnel. The ether-chloroform extract is then rendered
faintly alkaline and shaken with distilled water, and the urobilin passes into
solution in the water. The aqueous solution is now once more saturated
with ammonium sulphate and slightly acidified; it then once more yields its
pigment to ether chloroform.

By means of either of these methods urobilin is obtained in a pure con-
dition; even normal urine will give some, for the chromogen is partly con-
verted into the pigment by the acid employed.

Urobilin dissolved in alcohol exhibits a green fluorescence, which is
greatly increased by the addition of zinc chloride and ammonia. It shows a
well-marked absorption band between d and F, slightly overlapping the latter
(fig. 63, spectrum 4).

Urobilin, like most animal pigments, shows acidic tendencies and forms
compounds with bases; it is liberated from such combinations by the
addition of an acid.

If urobilin is dissolved in caustic potash or soda, and sufficient sulphuric
or hydrochloric acid is added to render the liquid faintly acid, a turbidity is
produced. This turbid liquid shows an additional band in the region of the
E line (fig. 63, spectrum 6), which is probably due to the special light absorp-
tion exercised by fine particles of urobilin in suspension. It wholly dis-
appears when the precipitate is filtered off, and when it is re-dissolved the
ordinary band alone is visible.

8. Uroerythrin.—This is the colouring matter of pink urate sediments,
It may be separated from the sediment as follows :—The deposit is washed
with ice-cold water, dried, and placed in absolute alcohol. The alcohol,
though a solvent for uroerythrin, does not extract it from the urates. The
alcohol is poured off, and the deposit dissolved in warm water. From this
solution the pigment is easily extracted by amylic alcohol.

Uroerythrin has a great affinity for urates, with which it appears to form
a loosecompound. Its solutions are rapidly decolorised by light. Spectro-
scopically it shows two rather ill-defined bands (fig. 63, spectrum 7). It
gives a green colour with caustic potash, and red or pink with mineral acids,
Uroerythrin appears to be a small but constant constituent of urine. Its
origin and relationship to other pigments are unknown.

4, Hematoporphyrin.—This also occurs in small quantities in normal

THE PIGMENTS OF THE URINE 179

_ urine. In some pathological conditions, especially after the administration

of certain drugs (e.g. sulphonal), its amount is increased. Its amount is

Stated to increase when the urine stands; this points to the existence of a

colourless chromogen. It may be separated from the urine as follows :—

Caustic alkali is added to the urine; this causes a precipitate of phos-
B O D Eb F G

“eae |
B oO D E »b F G
Fic. 63.—Chart of absorption spectra: 1, acid hematoporphyrin ; 2, alkaline hematoporphyrin ;
3, hematoporphyrin as found sometimes in urate sediments; 4, acid urobilin, concentrated ;
5, acid urobilin, dilute; 6, the E band spectrum of urobilin ; 7, uroerythrin; 8, urorosein con-
centrated—on dilution the band shrinks rapidly from redward end. (After F. G. Hopkins.)
phates, which carries down the pigment with it: the pigment may be dis-
solved out with chloroform. The chloroform is evaporated, the residue
washed with alcohol, and finally dissolved in acidified aleohol. Urines rich
in the pigment yield it easily to acetic ether or to amylic alcohol.
When the urine is sufficiently rich in the pigment, the bands shown are
n2

180 ESSENTIALS OF CHEMICAL PHYSIOLOGY

those of alkaline heematoporphyrin (fig. 63, spectrum 2). On adding sulphuric
acid the spectrum of acid hematoporphyrin is seen (fig. 63, spectrum 1).
Occasionally urate sediments are pigmented with a form of the pigment which
shows a two-banded spectrum, very like that of oxyhemoglobin (fig. 63.
spectrum 38); by treatment with dilute mineral acids this changes immedi-

ately to the spectrum of acid hematoporphyrin.

5. Chromogens in urine.—In addition to the chromogens of urobilin and
hematoporphyrin alluded to in the foregoing paragraphs there are others of
which the following may be mentioned :—(a) Indoxyl.—The origin of this
substance from indole is mentioned on p. 116. It is easily oxidised to
indigo-blue or indigo-red.

0.0H fe) CO
20H.<eiy > OH + 0, = OH, hyp >O:C cg >OsH, + 28,0.
{indoxyl] [indigo blue]

Indigo red is isomeric with indigo-blue, its structural formula being
CO C.OH. ‘ :

C,H C Tt f tuall

<nA> <o.H, >N. is very rare for the urine to be actually

pigmented with indigo, for the urinary indoxyl is excreted as a conjugated
sulphate which resists oxidation. When the urine is mixed with an equal
volume of hydrochloric acid, indoxyl is liberated from the sulphate. A
solution of a hypochlorite is then added drop by drop, when indigo-blue is
formed, and on shaking the mixture with chloroform the indigo-blue passes
into the chloroform. (Jaffé.) (6) Skatoxyl.—When skatoxy]l is given by the
mouth it passes into the urine, and yields skatoxyl-red on oxidation. (c)
Urorosewm is distinct from indigo-red. It is produced from its chromogen
by the action of mineral acids. It frequently appears when urine is treated
with strong hydrochloric acid and allowed to stand, but it appears more
readily when an oxidising agent is added as well. It is readily soluble in
amylic alcohol, but not in ether. The chromogen is precipitated by satura-
tion with ammonium sulphate. The colour is destroyed by alkalis. It
shows an absorption band between the D and E lines (fig. 63, spectrum 8).

6. Pathological pigments.—The most frequently appearing of abnormal
pigments are those of blood and bile. The urine may contain accidental
pigments due to the use of drugs (rhubarb, senna, logwood, santonin); in
carbolic acid poisoning pyrocatechin and hydrochinon are chiefly responsible
for the brown colour of the urine, which increases on exposure to the air.
The black or dark brown pigment called melanin may pass into the urine
in cases of melanotic sarcoma. For alcaptonuria see p. 182.

ES aS ee ee

APPENDIX

HEMACYTOMETERS

Gowers’s Hemacytometer.—The enumeration of the blood corpuscles is
readily effected by the hemacytometer of Gowers. This instrument consists
of a glass slide (fig. 64, C), the centre of which is ruled into 4, millimetre
squares and surrounded by a glass rim 4 millimetre thick. It is provided
with measuring pipettes (A and B), a vessel (D) for mixing the blood with a
saline solution (sulphate of soda of specific gravity 1015), a glass stirrer (E),
and a guarded needle (F).

Fie. 64.—Heemacytometer of Sir W. Gowers.

Nine hundred and ninety-five cubic millimetres of the saline solution are
measured out by means of A, and then placed in the mixing jar; 5 cubic
millimetres of blood are then drawn from a puncture in the finger by means
of the pipette B, and blown into the solution. The two fluids are well
mixed by the stirrer, and a small drop of this diluted mixture placed in the

182 ESSENTIALS OF CHEMICAL PHYSIOLOGY

centre of the slide C, a cover glass is gently laid on (so as to touch the drop
which thus forms a layer 4 millimetre thick between the slide and cover
glass), and pressed down by two brass springs. In a few minutes the
corpuscles have sunk to the bottom of the layer of fluid, and rest on the
squares. The number on ten squares is then counted, and this multiplied
by 10,000 gives the number in a cubic millimetre of blood. The average
number of red corpuscles in each square ought therefore in normal human
blood to be 45-50.

Oliver’s Hemacytometer.—The following method devised by Dr. George
Oliver is a ready way of determining the total number of corpuscles. It is,
however, not possible to determine the relative proportion of red and white
corpuscles by this means.

Fia, 65,—Oliver’s heemacytometer,

APPENDIX . - 3283

The finger is pricked, and the blood allowed to flow into the small
capillary pipette (fig. 65, a) until it is full. This is washed out by the
dropping tube 6 into a graduated flattened test-tube, c, with Hayem’s fluid.’
The graduations of the tube are so adjusted that with normal blood con-
taining 5,000,000 coloured corpuscles per cubic millimetre, the light of a
small wax candle placed at a distance of 9 feet from the eye in a dark room
is just transmitted as a fine bright line when looked at through the tube held
edgeways between the fingers (d) and filled up to the 100 mark of the gradua-
tion. If the number of corpuscles is less than normal, less of the diluting
solution is required for the light to be transmitted ; if above the normal, more
of the Hayem’s fluid must be added. The tube is graduated, so as to indicate
in percentages the decrease or increase of corpuscles per cubic millimetre as
compared with the normal standard of 100 per cent.

HEMOGLOBINOMETERS

Gowers’s Hemoglobinometer.—The apparatus consists of two glass tubes,
C and D of the same size. D contains glycerin jelly tinted with carmine to
a standard colour—viz., that of normal blood diluted 100 times with distilled
water. The finger is pricked and 20 cubic millimetres of blood are measured
out by the capillary pipette, B. This is blown out into the tube C, and diluted

seats i

lure

Fic. 66.—Hzemoglobinometer of Sir W. Gowers.

with distilled water, added drop by drop from the pipette stopper of the
bottle, A, until the tint of the diluted blood reaches the standard colour. The
tube C is graduated into 100 parts. If the tint of the diluted blood is the
same as the standard when the tube is filled up to the graduation 100, the
quantity of oxyhemoglobin in the blood is normal. If it has to be diluted

_' Sodium sulphate 5 grammes, sodium chloride 1 grm., mercuric chloride
0-5 grm., distilled water 200 c.c.

184 ESSENTIALS OF CHEMICAL PHYSIOLOGY

more largely, the oxyhemoglobin is in excess; if to a smaller extent, it is
less than normal. If the blood has, for instance, to be diluted up to the
graduation 50, the amount of hemoglobin is only half what it ought to be—
50 per cent. of the normal—and so for other percentages.

The instrument only yields approximate results, but is extremely useful
in clinical observations.

Von Fleischl’s Hemometer.—The apparatus (fig. 67) consists of a stand
-bearing a white reflecting surface (S) and a platform. Under the platform is
a slot carrying a glass wedge stained red (K) and moved by a wheel (R).
On the platform is a small cylindrical vessel divided vertically into two com-
partments, a and a’.

Fill with a pipette the compartment a’ over the wedge with distilled
water. Fill about a quarter of the other compartment (a) with distilled
water.

Fia. 67.—Fleischl’s heemometer.

Prick the finger and fill the short capillary pipette provided with the
instrument with blood. Dissolve this in the water in compartment a, and
fill it up with distilled water.

Having arranged the reflector (S) to throw artificial light vertically
through both compartments, look down through them, and move the wedge
of glass by the milled head (T) until the colour in the two is identical.

Read off the scale which is so constructed as to give the percentage of
hemoglobin.

Dr. Oliver’s Hemoglobinometer.—This method consists in comparing a
specimen of blood suitably diluted with water in a shallow white palette,
with a number of standard tests very carefully prepared by the use of
Lovibond’s coloured glasses. The capillary pipette ¢ (fig. 68) is first filled
with blood obtained by pricking the finger. This is washed with water

APPENDIX 185

by the mixing pipette d into the blood cell e; the cell is then just filled with
water, and the blood and water thoroughly mixed by the handle of ¢ being

Fic. 68.—Oliver’s hreemoglobinometer : a, standard gradations ; b, lancet; ¢, capillary
measuring pipette ; d, mixing pipette ; ¢. blood cell and cover glass.

used as a stirrer. The cover glass is then adjusted, when a small bubble
should form a clear sign that the cell has not been overfilled. The cell is

186 ESSENTIALS OF CHEMICAL PHYSIOLOGY

then placed by the side of the standard gradations, and the eye quickly
recognises its approximate position on the scale. The camera tube provided
with the instrument will more accurately define it. Artificial light should
be used.

If it is proved that the blood solution is matched in depth of colour by
one of the standard grades, the observation is at-an end; but if the tint is
higher than one grade, but lower than another, the blood cell is placed
opposite to the former, and riders (not shown in the illustration) are added
to complete the observation. The standard gradations are marked in per-
centages, 100 per cent. being taken as normal.

‘ Worth’ of the Corpuscles.—If the percentage of hemoglobin is 100, and the

percentage number of corpuscles is 100 also, then the quotient 7°) = 1 is

taken as the normal. This varies in health from 0°95 to 1:05 in men, and
from 0°9 to lin women. This quotient has been termed the worth of the
corpuscle.

Specific Gravity of Blood.—Of the numerous methods introduced for taking
the specific gravity of fresh blood, that of Hammerschlag is the simplest. A
drop of blood from the finger is placed in a mixture of chloroform and
benzene. If the drop falls, add chloroform till it just begins to rise ; if the drop
rises, add benzene till it just begins to fall. The fluid will then be of the
same specific gravity as the blood. Take the specific gravity of the mixture
in the usual way with an accurate hydrometer.

Schmalz’s capillary picnometer is more accurate.

POLARISATION OF LIGHT

If an object, such as a black dot on a piece of white paper, be looked at
through a crystal of Iceland spar, two black dots will be seen; and if the
crystal be rotated, one black dot will move round the other, which remains
stationary. That is, rays of light entering such a crystal are split into two
rays, which travel through the crystal with different velocities, and conse-
quently one is more refracted than the other. One ray travels just as it
would through glass; this is the ordinary ray, the ray which gives the
stationary image ; the other ray gives the moveable image when the erystal
is rotated; the ordinary laws of refraction do not apply to it, and it is called
the extraordinary ray. Both rays are of equal brilliancy. In one direction,
however, that of the optic axis of the crystal, a ray of light is transmitted
without double refraction.

Ordinary light, according to the wave theory, is due to vibrations occur-
ring in all planes transversely to the direction of the propagation of the wave.
Light is said to be plane polarised when the vibrations take place all in one
plane. The two rays produced by double refraction are both polarised, one
in one plane, the other in a plane at right angles to thisone. Doubly refract-
ing bodies are called anisotropous; singly refracting bodies, isotropous.
The effect of polarisation may be very roughly illustrated by a model.

If a string be stretched as in the figure, and then touched with the finger,
it can be made to vibrate, and the vibrations will be free to occur from above
down, or from side to side, or in. any intermediate position. If, however, a

SS ae

APPENDIX - = ie

dise with a vertical slit be placed on the course of the string, the vibrations
will be all obliged to take place in a vertical plane, any side to side movement
being stopped by the edges of the slit* (fig. 69).

Light can be polarised not only by the action of crystals, but by reflection
from a surface at an angle which varies for different substances (glass

rH | =

| aa

| be

Fic. 69.

54° 35’, water 52° 45’, diamond 68°, quartz 57° 32’, &c.). It is also found
that certain non-crystalline substances, like muscle, cilia, &e., are doubly
refracting.

The Nicol’s Prism is the polariser usually employed in polariscopes ; it
consists of a rhombohedron of Iceland spar divided into two by a section
through its obtuse angles. The cut surfaces are polished and cemented

together in their former position with canada balsam. By this means the

ordinary ray is totally reflected through the canada balsam; the extra-
ordinary ray passes on and emerges in a direction parallel to the entering
ray. In this polarised ray there is nothing to render its peculiar condition
visible to the naked eye; but if the eye is aided by a second nicol’s prism,
which is called the analyser, it is possible to detect the fact that it is
polarised.

This may be again illustrated by reference to our model (fig. 70)

Fie. 70.

Suppose that the string is made to vibrate, and that the waves travel in
the direction of the arrow. From the fixed point ¢ to the disc a, the string
is theoretically free to vibrate in any plane;* but after passing through the
vertical slit in a, the vibrations must all be vertical also ; if a second similar
dise 6 be placed further on, the vibrations will also pass on freely to the other
extremity of the string d, if as in the figure (fig. 70) the slit in 6 be also placed

' Such a model is, of course, imperfect ; it does not, for instance, represent the
splitting of the ray into two, and moreover the polarisation takes place on each
side of the slit; whereas, in regard to light, it is only the rays on one side of a
polariser, viz. those that have passed through it, which are polarised.

? The imperfection of the model has been explained i in preceding footnote.

188 ESSENTIALS OF CHEMICAL PHYSIOLOGY

vertically. If, however, b be so placed that its slit is horizontal (fig. 71), the
vibrations will be extinguished on reaching 0, and the string between b andd
will be motionless.

Fig. 71.

c here represents a source of light, and the vibrations of the string the
undulations which by the nicol’s prism @ are polarised so as to occur in one
plane only; if the second nicol or the analyser 6 is parallel to the first, the
vibrations will pass on to the eye, whichis represented by d; but if the planes
of the two nicols are at right angles, the vibrations allowed to pass through
the first are extinguished by the second, and so no light reaches the eye. In
intermediate positions, 6 will allow only some of the light to pass through it.
It must be clearly understood that a nicol’s prism contains no actual slits,
but the arrangement of its molecules is such that their action on the particles
of «ther may be compared to the action of slits in a diaphragm to vibrations
of more tangible materials than xther.

The Polarising Microscope consists of an ordinary microscope with certain
additions; below the stage is the polarising nicol; in the eye-piece is the
analysing nicol ; the eye-piece is so arranged that it can be rotated; thus the
directions of the two nicols can be made parallel, and then the field is bright;
or crossed, and then the field is dark. The stage of the microscope is arranged
so that it also can be rotated.

The polarising microscope is used to detect doubly refracting substances.
Let the two nicols be crossed, so that the field is dark; interpose between
the two, that is, place upon the stage of the microscope, a doubly refracting
plate of which the principal plane is parallel to the first prism or polariser

the ray from the first prism is unaffected by the plate, but will be extinguished ~

by the second; the field therefore still remains dark. If the plate is parallel
to the second nicol the field is also dark; but in any intermediate position,
the light will be transmitted by the second nicol. In other words, if between
two crossed nicols, which consequently appear dark, a substance be interposed
which in certain positions causes the darkness to give place to illumination,
that substance is doubly refractive. How this takes place may be explained
as follows :— “

Let N,N, (fig. 72) represent the direction of the principal plane of the
first nicol, and N,N, that of the second. They are at right angles, and so
the ray transmitted by the first will be extinguished by the second. Let
PP represent the principal plane of the interposed doubly refractive plate.
The extraordinary ray transmitted by N,N, vibrates in the plane N,N,, and
falls obliquely on the plate PP; it is by this plate itself split into two rays,
an ordinary and an extraordinary one, at right angles to one another, one

I ee

a ie oe led

APPENDIX ~ 189

vibrating in the plane PP, the other in the plane P'P’. These two rays
meet the second nicol, which can only transmit vibrations in the plane N,N,.
The vibrations in PP can be resolved

into a vibration in N,N, and a N,
vibration in N,N,; the former is
extinguished, the latter transmitted. is
Similarly the vibration in P'P’ can

be resolved into two sub-rays in

N,N, and N,N, respectively, the

latter only being transmitted. The yy ap
illumination is thus due to two ~ ex.
sub-rays, one of the vibrations in
PP, the other of those in P!P' which

have been made to vibrate in N,N,. S.
Now, although these two sub- Pp Pp
rays vibrate in the same plane,
they are of different velocities ; N,
hence the phases of the vibrations Fic. 72.

do not coincide, and thus the

phenomena of interference are obtained. If we have two sets of vibrations
fused, the crest of one wave may coincide with the crest of the other,
in this case the wave will be higher; or the crest of one may coincide
with the hollow of the other, that is, the undulation would be extinguished ;
in other intermediate cases, the movement would be interfered with, either
helped or hindered more or less. Interference in the case of many kinds
of doubly refracting substances (Iceland spar is in this an exception) shows
itself in the extinction of certain rays of the white light, and the light seen
through the second nicol is white light minus the extinguished rays; those
extinguished and those transmitted will together form white light,.and are
thus complementary. Moreover, the rays extinguished in one position of
the plate will be transmitted in one at right angles and vice versa; thus a
erystal showing these phenomena of pleochromatism, as it is termed, will
transmit one colour in one position, and the complementary colour in a
position at right angles to the first; blue and yellow, and red and green, are
the pairs of colours most frequently seen in this way.

Rotation of the plane of Polarisation.—Certain crystals such as those of
quartz, and certain fluids such as the essence of turpentine, aniseed, &c.,
and solutions of certain substances like sugar, and albumin, have the power
of rotating the plane of polarised light to the right or left. The polarisation
of light that is produced by a quartz crystal is different from that produced
by a rhombohedron of Iceland spar. The light that passes through the
latter is plane polarised ; the light that passes through the former (quartz)
is circularly polarised, ie. the two sub-rays are made up of vibrations
which occur not ina plane, but are curved. The two rays are circularly
polarised in opposite directions, one describing circles to the left, the other
to the right; these unite on issuing from the quartz plate; and the net
result is a plane polarised ray with the plane rotated to right or left
according as the right circularly polarised ray or the left proceeded through

190 ESSENTIALS OF CHEMICAL PHYSIOLOGY

the quartz with the greater velocity. There are two kinds of quartz, one
which rotates the plane to the right (dextrorotatory), the other to the left
(levorotatory).

Gordon explains this by the following mechanical illustration. Ordinary
light may be represented by a wheel travelling in the direction of its axle,
and the vibrations composing it executed along any or all of its spokes (a),
If the vibrations all take place in the same direction, i.e. along one spoke,

and the spoke opposite to it (0), the light is said to be plane polarised. The
two spokes as they travel along in the direction of the arrow will trace out
a plane (see fig. 73) between b and b’. If this polarised beam be made to
travel now through a solution of sugar, the net result is that the plane so
traced out is twisted or rotated; the two spokes, as in 6b’, do not trace out
a plane, but we must consider that they rotate as they travel along, as
though guided by a spiral or screw thread cut on the axis, so that after a
certain distance the vibrations take place as in b’’; later in b’’’, and so on.
This effect on polarised light is due to the molecules in solution, and the
amount of rotation will depend on the strength of the solution, and on the
length of the column of the solution through which the light passes or in
the case of a quartz plate on its thickness.

If a plate of quartz be interposed between two nicols, the light will not be
extinguished in any position of the prisms, but will pass through various
colours as rotation is continued. The rotation produced for different kinds
of light being different, white light is split into its various constituent colours ;
and the angle of rotation that causes each colour to disappear is constant for
a given thickness of quartz plate, being least for the red and greatest for the
violet. These facts are made use of in the construction of polarimeters.
Polarimeters are instruments for determining the strength of solutions of
sugar, albumin, &c., by the direction and amount of rotation they produce
on the plane of polarised light. They are often called saccharimeters, as
they are specially useful in the estimation of sugar.

POLARIMETERS

Soleil’s Saccharimeter.—This instrument (see fig. 74) consists of a nicol’s
prism, d, called the polariser: this polarises the light entering it, and the
polarised beam then passes through a quartz plate (b in fig. 74), 8°75 mm.
thick, one half of which (d in fig. 75) is made out of dextrorotatory, the other
half (g in fig. 75) of levorotatory quartz.

The light then passes through the tube containing the solution in the
position of the dotted line in fig. 74, then through a quartz plate cut per-

cy OR a ar arte

APPENDIX is

pendicularly to its axis (q in fig. 75), then through an arrangement called

a compensator (r in fig. 75), then through a second nicol called the analyser,
and lastly through a telescope (L in fig. 75).

Fig. 74.—Soleil’s saccharimeter.

The compensator consists of two quartz prisms (RR, fig. 75) cut perpen-
dicularly to the axis, but of contrary rotation to the plate just in front of
them. These are wedge-shaped, and slide over each other, the sharp end of
one being over the blunt end of the other. By ascrew the wedges may be
moved from each other, and this diminishes the thickness of quartz inter-
posed; if moved towards each other the amount of quartz interposed is
increased.

The effect of the quartz plate (d, g) next to the polariser (i in fig. 75)
is to give the polarised light a violet tint when the two nicols are parallel to
each other. But if the nicols are not parallel, or if the plane of the polarised
light has been rotated by a solution in the tube, one half the field will change
in colour to the red end, the other to the violet end of the spectrum, because
the two halves of the quartz act in the opposite way.

Ei a5

Fig. 75.—Diagram of optical arrangements in Soleil’s saccharimeter.

The instrument is first adjusted with the compensator at zero, and the
nicols parallel, so that the whole field is of one colour. The tube containing
the solution is then interposed ; and if the solution is optically inactive the
field is still uniformly violet. But if the solution is dextrorotatory the two
halves will have different tints; a certain thickness of the compensating
quartz plate which is levorotatory must be interposed to make the tint of
the two halves of the field equal again; the thickness so interposed can be
read off in amounts corresponding to degrees of a circle by means of a vernier
and scale (E in fig. 75) worked by the screw which moves the compensator.

If the solution is levorotatory, the screw must be turned in the opposite

direction.

192 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Zeiss’s polarimeter is in principle much the same as Soleil’s; the chief
difference is that the rotation produced by the solution is corrected not by a
quartz compensator but by actually rotating the analyser in the same
direction, the amount of rotation being directly read off in degrees of a
circle,

Laurent’s polarimeter is a more valuable instrument. Instead of using
daylight, or the light of a lamp, monochromatic light (a sodium flame pro-

Fic. 76.—Laurent’s polarimeter,

duced by volatilising common salt in a colourless gas flame) is employed ;
the amount of rotation varies for different colours; and observations are
recorded as having been taken with light corresponding to the D or sodium
line of the spectrum. The essentials of the instrument are, as before, a
polariser, a tube for the solution, and an analyser. The polarised light
before passing into the solution traverses a quartz plate, which, however,
covers only half the field, and retards the rays passing through it by half a
wave-length. In the 0° position the two halves of the field appear equally
illuminated: in any other position, or if rotation has been produced by the

solution when the nicols have been set at zero, the two halves appear un-’

APPENDIX ' 198

equally illuminated. This is corrected by means of a rotation of the analyser
that can be measured in degrees by a scale attached to it.

Specific rotatory power of any substance is the amount of rotation in
degrees of a circle of the plane of polarised light produced by 1 gramme of
the substance dissolved in 1 ¢.c. of liquid examined: in a column 1 deci-

metre long.
Ifa@ = rotation observed.
w = weight in grammes of the substance per cubic centimetre.
l = length of tube in decimetres.

(@)» = specific rotation for light with wave-length corresponding to
the D line (sodium fiame).
a
Then (a)p = + or
In this formula + indicates that the substance is dextrorotatory, — thatit
is levorotatory.
If, on the other hand, (a)p is known, and we wish to find the value of
w, then
43 a
1 (eee

THE SPECTRO-POLARIMETER

This instrument is one in which a spectroscope and polarising apparatus
are combined for the purpose of determining the concentration of substances
which rotate the plane of polarised light. It was invented by E. v. Fleischl

Fic. 77.—Spectro-polarimeter of v. Fleischl.

for the estimation of sugar in diabetic urine. Its chief advantage is that no
_ difficulty arises of forming a judgment as to the identity of two coloured
4 surfaces, as in Soleil’s saccharimeter, or of two shades of the same colour, as
in Laurent’s instrument. The light enters at the right-hand end of the
Oo

194 ESSENTIALS OF CHEMICAL PHYSIOLOGY

instrument, is polarised by the nicol’s prism 0b, and then passes through two.
quartz plates, ce, placed horizontally over each other. One of these plates is
dextro-, the other levorotatory, and they are of such a thickness (7°75 mm.)
that the green rays between the E and 0 lines of the spectrum are circularly
polarised through an angle of 90°, the one set passing off through the upper
quartz to the left, the other through the lower to the right. The light then
continues through a long tube, ff, which contains 15 ¢.c. of the solution under
examination. It then passes through an analysing nicol d, and finally
through a direct-vision spectroscope, e. On looking through the instrument,
the tube # being empty or filled with water or some other optically inert
substance, two spectra are seen, one over the other, but each shows a dark
band between E and 6 owing to the extinction of these rays by the circular
polarisation, produced by the quartz. The analyser can be rotated: -a.
vernier, g, is attached to, and moves with it, round a circular dise (seen in
section at h) graduated in degrees. The two bands in the spectra coincide
when the zeros of vernier and scale correspond. If now the tube f is filled
with an optically active substance like sugar, the bands are shifted, one to the
right, the other to the left, according to the direction of rotation of the sub-
stance in f. The rotation is corrected by rotating the analyser into such a
position that the two bands exactly coincide once more as to vertical position.
The number of degrees through which it is thus necessary to move the
analyser measures the amount of rotation produced by the substance in f,
and is a measure of the concentration of the solution. The degrees marked
on the circular scale are not degrees of a circle, but an arbitrary degree of
such a length that each corresponds to 1 per cent. of sugar in the given
length of the column of fluid in ff (177'°2 mm.).

RELATION BETWEEN CIRCULAR POLARISATION AND CHEMICAL
CONSTITUTION

The first work in this direction was performed by Pasteur, and it was his
publications on this subject that brought him into prominence. He found
that racemic acid, which is optically inactive, can be decomposed into two
isomerides, one of which is common tartaric acid which is dextrorotatory, and
the other tartaric acid differing from the common variety in being levo-
rotatory. The salts of tartaric acid usually exhibit hemihedral faces, while
those of racemic acid are holohedral. Pasteur found that, although all the
tartrate crystals were hemihedral, the hemihedral faces were situated on
some crystals to the right, and on others to the left hand of the observer, so
that one formed, as it were, the reflected image of the other. These crystals
were separated, purified by recrystallisation, and those which exhibited
dextro-hemihedry possessed dextrorotatory power, whilst the others were
levorotatory. Pasteur further showed thatif the mould Penicilliwm glaucwm
is grown in a solution of racemic acid, dextro-tartaric acid first disappears,
and the levo-acid alone remains. The subject remained in this condition for
many years; it was, however, conjectured that probably there is some mole-
cular condition corresponding to the naked-eye crystalline appearances which
produces the opposite optical effects of various substances. What this mole-

APPENDIX ~ 195

cular structure was, was pointed out independently by two observers—Le
Bel in Paris, and Van’t Hoff in Holland—who published their researches
within a few days of each other. They pointed out that all optically active
bodies contain one or more asymmetric carbon atoms, i.e. one or more atoms
of carbon connected with four dissimilar groups of atoms, as in the following
examples :— :

C.H, CO0.0H
|
H—-(C)—CH, H—(C)—O0H
| :
CH,.0H CH,—C0.0H
Amyl alcohol Malie acid

The question, however, remained—do all substances containing such
' atoms rotate the plane of polarised light? It was found that they do not;
this is explained by Le Bel by supposing that these, like racemic acid, are
compounds of two molecules—one dextro-, the other levo-rotatory ; that this

A A
D c c D
Fic. 78.

was the case was demonstrated by growing moulds, the fermenting action of
which is to separate the two molecules in question. Then the other question
—how is it that two isomerides, which in chemical characteristics, in graphic
as well as empirical formule, are precisely alike, differ in optical properties ?
—is explained ingeniously by Van’t Hoff. He compares the carbon atom to
a tetrahedron with its four dissimilar groups, A, B, C, D, at the four corners.
The two tetrahedra represented in fig. 78 appear at first sight precisely alike ;
but if one be superimposed on the other, C in one and D in the other could
never be made to coincide. This difference cannot be represented in any
other graphic manner, and probably represents the difference in the way the
atoms are grouped in the molecule of right- and left-handed substances respec-
tively.

MERCURIAL AIR PUMPS

Pfliiger’s Pump.—/ is a large glass bulb filled with mercury ; from its lower
end a straight glass tube, m, about 3 feet long, extends, which is connected
by an indiarubber tube, m, with a reservoir of mercury, 0, which can be
raised or lowered as required, by a simple mechanical arrangement. From
the upper end of the bulb, /, a vertical tube passes; above the stopcock, k,
_ this has a horizontal branch, which can be closed by the stopcock, f. The

_ vertical part is continued into the bent tube, which dips under mercury in
the trough, h. A stopcock, j, is placed on the course of this tube. Beyond f
the horizontal tube leads into a large double glass bulb, a 6; a mercurial
‘gauge, e, and a drying-tube, d, filled with pieces of pumice-stone moistened.

_ with sulphuric acid, are interposed. a is called the blood-bulb, and the
blood is brought into it by the tube c; the gases, as they come off, cause the
02

196 ESSENTIALS OF CHEMICAL PHYSIOLOGY

blood to froth, and the bulb, 8, is called the froth-chamber, as it intercepts
the froth, preventing it from passing into the rest of the apparatus.

The pump is used in the following way: / is filled with mercury, the
level in 7 and o being the same; & is closed; o is then lowered, and when it
is 30 inches lower than the stopcock, k, the mercury in / falls also, leaving

te | ate al

Fic. 79.—Diagram of Pfitiger’s pump. ’

that bulb empty; 7 being closed and f open, & is then opened, and the air in
a, b, d, &e. rushes into the Torricellian vacuum in 1; f is closed andj opened ;
the reservoir, 0, is raised; the mercury in / rises also, pushing the air before
it, and it bubbles out into the atmosphere through the mercury (the tube, h,
is not-at this stage in position), When 2 is full of mercury, % and j are once
more closed and o is again lowered; when 2 is thus rendered once more a

APPENDIX 197

vacuum, & and f are opened and more of the air remaining in a, b, d, &c.
rushes into the vacuum ; f is closed, 7 is opened, and this air is expelled as
before. The process is repeated as often as is necessary to make a, 3, d, &e.
as complete a vacuum, as indicated by the mercury in the gauge, @, as is
obtainable.

a being now empty and the stopcock, f, closed, blood is introduced by the
tube c; it froths and gives off all its gases, especially if heated to 40-45° C.
In the case of serum, acid has to be added to disengage the more firmly
combined carbonic acid.t The bulb, J, is once more rendered a vacuum, and
k and f are opened, j being closed. The gas from @ and 6 rushes into the
bulb 7, being dried as it passes through d; f is then closed and j opened ; the
reservoir o is raised, and as the mercury in / rises simultaneously, it pushes
the gases into the cylinder, h, which is filled with mercury and inverted over
the end of the bent tube. This gas can be subsequently analysed. By
alternately raising and lowering o, and regulating the stopcocks in the manner
already described, all the gas from the
quantity of blood used can be ultimately a a
expelled into h. =

A good grease for the stopcocks is a BB. eo
mixture of two parts of vaseline to one of =
white wax.

Alvergniat’s pump has the advantage i
over Pfliiger’s of fewer connections, and hk
all of these are surrounded by mercury, Ree
which effectually prevents leakage; it
has the disadvantage of a rather small b
bulb in place of J, and thus it is more
labour to obtain a vacuum. S

cs=3

q
e

Leonard Hill’s Pump.—This is a simpler
instrument, and is sufficient for most B.B:
purposes. It consists of three glass bulbs
(B.B. in fig. 80), which we will call the
blood bulb; this is closed above by a g
piece of tubing and a clip, a; this is
connected by good indiarubber tubing to Fic. 80.—L, Hill’s air-pump.
another bulb, d. Above d, however,
_ there is a stopcock with two ways cut through it: one by means of
which B.B. and d may be connected, as in the figure; and another seen
in section, which unites d to the tube e, when the stopcock is turned through
aright angle. In intermediate positions the stopcock cuts off all communi-
cation from d to all parts of the apparatus above it; d is connected by tubing
to a receiver, R, which can be raised or lowered at will. At first the whole
apparatus is filled with mercury, R being raised. Then a being closed, R is
lowered, and when it is more than the height of; the barometer (30 inches)
below the top of B.B. the mercury falls and leaves the blood bulb empty ; by
lowering R still further, d can also be rendered a vacuum. A few drops of
mercury should be left behind in B.B. B.B. is then detached from the rest

! Phosphoric acid is usually employed.

198 ESSENTIALS OF CHEMICAL PHYSIOLOGY

of the apparatus and weighed, the clips, a and b, being tightly closed. Blood
is then introduced into it by connecting the tube with the clip a on it toa
cannula filled with blood inserted in an artery or vein of a living animal.
Enough blood is withdrawn to fill about half of one of the bulbs. This is
defibrinated by shaking it with the few drops of mercury left in the bulb.
It is then weighed again; the increase of weight gives the amount of blood
which is being investigated. B.B. is then once more attached to the rest of
the apparatus, hanging downwards, ‘as in the side drawing in fig. 80, and
the blood gases boiled off ; these pass into
d, which has been made a vacuum; and
then, by raising R again, the mercury
rises in d, pushing the gases in front of
it through the tube e (the stopcock being
turned in the proper direction) into the
| eudiometer, E, which has been filled with,

’ and placed over, mercury. The gas can
then be measured and analysed.

ANALYSIS OF GASES

Waller’s modification of Zuntz’s more
complete apparatus will be found very
useful in performing gas analysis, say of
the expired air or blood gases: a 10Uc.c.
measuring-tube graduated in tenths of a
cubic centimetre between 75 and 100; a
filling bulb and two gas pipettes are con-
nected up as in the diagram.

It is first charged with acidulated
water up to the zero mark by raising
the filling bulb A, tap 1 being open. It
is then filled with 100 ¢.c. of expired
air, the filling bulb being lowered till
the fluid in the tube has fallen to the
100 mark. Tap 1 is now closed. The
amount of carbonic acid in the expired

Fia. cg igin pote Lipo gas forgas gir is next ascertained; tap 2 is opened,

and the air is expelled into the gas pipette
B containing strong caustic potash solution by raising the filling bulb until the
fluid has risen to the zero mark of the measuring tube. Tap 2 is closed,
and the air left in the gas pipette for a few minutes, during which the
carbonie acid is absorbed by the potash. Tap 2 is then opened and the air
drawn back into the measuring tube by lowering the filling bulb. The
volume of air (minus the carbonic acid) is read, the filling bulb being
adjusted so that its contents are at the same level as the fluid in the
measuring tube. The amount of oxygen is next ascertained in a precisely
similar manner by sending the air into the other gas pipette, which contains
sticks of phosphorus in water, and measuring the loss of volume (due to
absorption fot or aghe in ‘the air when drawn back into the tube. The
remaining gas is nitrogen.

te oe! ee ee Foe

APPENDIX * 199

KJELDAHL’S METHOD OF ESTIMATING NITROGEN

This simple and accurate method has very largely replaced the older
complicated processes.

I take the following account of the method with the modifications pro-
posed by Warington from Sutton’s ‘ Volumetric Analysis.’

From 0-1 to 1 gramme of the dry powdered substance is put into a hitting
flask holding about 100-120 ¢.c. The acid used for the destruction of the
organic material is made by mixing 200 c.c. pure oil of vitriol with 50 c.c.
Nordhausen oil of vitriol, and 2 grammes of phosphoric acid in sticks; all these
must, of course, be free from ammonia: 10-20 c.c. of this mixture are poured
over the substance in the flask and heated on wire gauze over a small Bunsen
flame. The temperature must be kept below boiling ; with prolonged heating
the organic matter is gradually destroyed, and the liquid becomes clear and

Boiling flash (3 nO.

En 50, >. bus ib
Fic. 82.—Kjeldahl’s method. (Waller, after Argutinsky.)

quiet. The nitrogen originally present is thus converted into ammonia, and
this may be hastened by adding to the liquid very minute pinches of pure
potassium-permanganate. A violent commotion takes place with every
addition, but there is no fear of any ammonia being lost. The operation is
ended when the mixture becomes permanently greenish (from one to two
hours), and moderate heat is continued fora few minutes more. The flask
is cooled, some water added, and the contents washed out into a large flask
of 700 ¢.c. capacity with as little water as possible. It is then made alkaline
with excess of either pure caustic soda or potash solution (sp. gr. 1°3). A little
metallic zinc is added to prevent bumping during the subsequent distillation.
‘The fiask is then rapidly closed with a perforated caoutchouc stopper, through
which passes an upright tube with two bulbs about an inch in diameter
blown upon it: these arrest and carry back any spray of soda from the liquid.
“The tube above the bulbs is bent over and connected to a condenser, and the
delivery end of the condenser leads into a flask, containing a measured excess
-of standard acid. The mixture in the flask is then distilled, and the ammonia
passes over into the acid. Distillation is continued from forty-five to sixty
minutes. The amount of acidity is then determined in the distillate by

titration with standard potash or soda, methy} DEP ARAVER TY the
Of

Cow a DR Oy, See ee |

200 ESSENTIALS OF CHEMICAL PHYSIOLOGY

‘indicafor of the end of the reaction. (This gives a pink colour with acid,.
yellow with alkali.)

EHxample.—Suppose 0°15 gramme of a nitrogenous substance is taken,,.
treated with acid, neutralised, and the ammonia distilled over and received by
100 c.c. of a decinormal solution of hydrochloric acid (= 10 ¢.c. normal acid).
The distillate is then titrated with decinormal soda, and it is found that the
neutral point is reached when 60 c.c. of the decinormal soda have been added.
The other 40 c.c. must therefore have been neutralised by the ammonia
derived from the nitrogenous substance under investigation. This 40 c.c. of
decinormal acid = 4 ¢.c. of normal acid = 4 ¢.c. of normal ammonia = 4

x 0°017 = 0:068 gramme of ammonia; 0°15 gramme of the substance
therefore yields 0:068 gramme of ammonia, and this amount contains 0-056.
_ gramme of nitrogen: 100 grammes of the substance will therefore contain
100 x 0°056 .

9-15 = 873 grammes of nitrogen.

Fig. 82 represents the apparatus as modified by Willfarth. In this,
oxidation is assisted by adding a small quantity of metallic mereury (about.
zy ¢.c.). To avoid bumping during distillation tale is added instead of zine.
12 c.c. of strong potassium sulphate solution are also added. Decinormal
sulphuric acid is used as the standard acid, and this is contained in the bulbs.
shown in the figure.

When this method is used for determining the total nitrogen in urine, 5 ¢.c.
of urine and 20 c.c. of the mixed acids are boiled for half an hour in a flask
of 300 ¢.c. capacity. After cooling, this is distilled with soda, and the process.
completed as already described.

SOLUTIONS. DIFFUSION. DIALYSIS. OSMOSIS

The investigations of physical chemists during recent years have given us.
new conceptions of the meaning of the words that stand at the head of this:
article. I propose to state what these new conceptions are, and briefly to
indicate the bearing they have on the elucidation of physiological problems.

Solutions.—Water is the fluid in which soluble materials are usually
dissolved, and our new ideas have'been principally worked out in connection
with this fluid. Water at ordinary temperatures is a fluid the molecules of
which are in constant movement; the hotter the water the more active are
the movements of its molecules, until, when at last it is converted into.
steam, the molecular movements become much more energetic. Perfectly
pure water consists of molecules with the formula H,O, and these molecules.
undergo practically no dissociation into their constituent atoms, and it is.
for this reason that pure water is not a conductor of electricity.

If a substance like sugar is dissolved in the water, the solution still
remains incapable of conducting an electrical current. The sugar molecules.
in solution are still sugar molecules; they do not undergo dissociation.

But if a substance like salt is dissolved in the water, the solution is then
capable of conducting electrical currents, and the same is true for most acids,
bases, and salts. These substances do undergo dissociation, and the simpler
materials into which they are broken up in the water are called ions. Thus
if sodium chloride is dissolved in water a certain number of its molecules:

APPENDIX - 201

become dissociated into sodium ions, which are charged positively with elec-.
tricity, and chlorine ions, which are charged negatively with electricity.
Similarly a solution of hydrochloric acid in water contains free hydrogen
ions and free chlorine ions. Sulphuric acid is decomposed into hydrogen
ions and ions of SO,. The term ion is thus not equivalent to atom, for an
ion may be a group of atoms, like SO,, in the example just given.
Further, in the case of hydrochloric acid, the negative charge of the
chlorine ion is equal to the positive charge of the hydrogen ion; but in the
ease of the sulphuric acid, the negative charge of the SO, ion is equal to the
positive charge of two hydrogen ions. We can_thus speak of monovalent,
divalent, trivalent &c. ions.
Ions positively charged are called kat-ions because they move towards.
the kathode or negative pole; those which are negatively charged are called
an-ions because they move towards the anode or positive pole. The following
are some examples of each class.
Kat-ions. Monovalent :—H, Na, K, NH,, &e.
Divalent :—Ca, Ba, Fe (in ferrous salts), &e.
Trivalent :—Al, Bi, Sb, Fe (in ferric salts), &c.

An-ions. Monovalent:—Cl, Br, I, OH, NO,, &e.
Divalent :—S, Se, SO,, &e.

Roughly speaking, the greater the dilution the more nearly complete is.
the dissociation, and in a very dilute solution of such a substance as sodium
chloride we may consider that the number of ions is double the number of
molecules of the salt present.

The ions liberated by the act of dissociation are, as we have seen, charged
with electricity, and when an electrical current is led into such a solution,
it is conducted through the solution by the movement of the ions. Sub-
stances which exhibit the property of dissociation are known as electrolytes.

The liquids of the body contain electrolytes in solution, and it is owing to-
this fact that they are able to conduct electrical currents.

The conception of electrolytes which we owe to Arrhenius is extremely
important in view of the question of osmotic pressure which we shall be con-
sidering immediately; because the act of dissociation increases the number
of particles moving in the solution and so increases the osmotic pressure, for
infthis relation an ion plays the same part as a molecule.

Another physiological aspect of the subject is seen in a study of the
actions of mineral salts in solution on living organisms and parts of organisms.
Many years ago Dr. Ringer showed that contractile tissues (heart, cilia, &c.}
continue to manifest their activity in certain saline solutions. Indeed, as
Howell puts it, the cause of such rhythmical action is the presence of these
inorganic substances in the blood or lymph which usually bathes them. In
the case of the heart, the sinus, or venous end of the heart is peculiarly
susceptible to the stimulus of the imorganic salts, and the rhythmical
peristaltic waves so started travel thence over the rest of the heart muscle.

Loeb and his fellow workers have confirmed these statements but interpret
them now as ionic action. Contractile tissues will not contract in pure
solutions of non-electrolytes (like sugar, urea, albumin). But different con-
tractile tissues differ in the nature of the ions which are most favourable

202 ESSENTIALS OF CHEMICAL PHYSIOLOGY

stimuli. Thus cardiac muscle, cilia, amceboid movement, karyokinesis, cell
division are all alike in requiring a proper adjustment of ions in their sur-
roundings if they are to continue to act, but the proportions must be different
in individual cases. Ions affecting the rhythmical contractions may be
divided into three classes: (1) Those which produce such contractions; of
these the most efficacious is Na. (2) Those which retard or inhibit
rhythmical contractions ; for instance, Ca and K. (8) Those which act cata-
lytically, that is, they accelerate the action of Na, though they do not them-
selves produce rhythmical contractions directly: for instance, H and OH.
In spite of the antagonistic effect of Ca, a certain minimal amount of it must
bepresent if contractions are to continue for any length of time. Ions produce
rhythmical contraction only because they affect either the physical condition
of the colloidal substances (proteid &c.) in protoplasm, or the rapidity of
chemical processes.

Loeb has even gone so far as to consider that the process of fertilisation is
mainly ionic action. He denies that the nuclein in the head of the sperma-
tozoon is essential, but asserts that all the spermatozoon does is to act as the
stimulus in the due adjustment of the proportions of the surrounding ions.
He supports this view by numerous experiments on ova, in which, without the
presence of spermatozoa, he has produced larve (generally imperfect ones,
it is true) by merely altering the saline constituents of the fluid that bathes
them. Whether such a sweeping and almost revolutionary notion will stand
the test of further verification must be left to the future.

Gramme-molecular Solutions.—From the point of view of osmotic pressure
a convenient unit is the gramme-molecule. A gramme-molecule of any
substance is the quantity in grammes of that substance equal to its molecular
weight. A gramme-molecular solution is one which contains a gramme-
molecule of the substance per litre. Thus a gramme-molecular solution of
sodium chloride is one which contains 58°5 grammes of sodium chloride
(Na = 23°05 ; Cl= 35-45) in a litre. A gramme-molecular solution of grape
sugar (C,H,,0,) is one which contains 180 grammes of grape sugar in a
litre. A gramme-molecule of hydrogen (H,) is 2 grammes by weight of
hydrogen, and if this was compressed to the volume of a litre, it would be
comparable to a gramme-molecular solution. It therefore follows that a
litre containing 2 grammes of hydrogen contains the same number of
molecules of hydrogen in it as a litre ofa solution containing 58°5 grammes
of sodium chloride, or one containing 180 grammes of grape sugar, has in
it of salt or sugar molecules respectively. To put it another way, the heavier
the weight of a molecule of any substance the more of that substance must
be dissolved in the litre to obtain its gramme-molecular solution. Or still
another way: if solutions of various substances are made all of the same
strength per cent., the solutions of the materials of small molecular weight
will contain more molecules of those materials than the solutions of the
materials which have heavy molecules. We shall see that the calculation of
osmotic pressure depends upon these facts.

Diffusion, Dialysis, Osmosis—If two gases are brought together within a
closed space, a homogeneous mixture of the two is soon obtained. This is
due to the movements of the gaseous molecules within the confining space,

__ be a solution of salt of half the original strength of |— —-_~| —~ ==

APPENDIX $08

and the process is called diffusion. In a similar way diffusion will effect in
time a homogeneous mixture of two liquids or solutions. If water is carefully
poured on to the surface of a solution of salt, the salt or its ions will soon be
equally distributed throughout the whole. If a solution of albumin er any
other colloidal substance is used instead of salt in the experiment, diffusion
will be found to occur much more slowly. If, instead of pouring the water
on to the surface of a solution of salt or sugar, the two are separated by a
_ membrane made of such a material as parchment paper, a similar diffusion
_ will occur, though more slowly than in cases where the membrane is absent.
In time, the water on each side of the membrane will contain the same
quantity of sugar or salt. Substances which pass through such membranes
are called crystalloids. Substances which have such heavy molecules
(starch, proteid &c.) that they will not pass through such membranes are
called colloids. Diffusion of substances in solution in which we have to deal
with an intervening membrane is usually called dialysis. The process of
filtration (i.e. the passage of materials through the pores of a membrane
under the influence of mechanical pressure) may be excluded in such experi-
ments by placing the membrane (™) vertically as shown in the diagram
(fig. 83), and the two fluids a and 8 on each side of it. Diffusion through a
membrane is not limited to the molecules of water, but

it may occur also in the molecules of certain substances NM
dissolved in the water. But very few or no membranes
are equally permeable to water and to molecules of the
substances dissolved in the water. If in the accompany- A B
ing diagram the compartment 4 is filled with pure water, (E===> ===
and B with a sodium chloride solution, the liquidsin [—=—
the two compartments will ultimately be found to be |—- - |—-—
equal in bulk as they were at the start, and each will }|—-— .| ——=-

that in the compartment 8. But at first the volume [———_/_ ==
of the liquid in compartment B increases, because more |——_— —
water molecules pass into it from a than salt mole- L——=
cules pass from B into a. The term osmosis is generally
limited to the stream of water molecules passing Fic. 88.
through a membrane, while the term dialysis is applied
to the passage of the molecules in solution in the water. The osmotic
_ Stream of water is especially important, and in connection with this it is
next necessary to define the term osmotic pressure. At first, then, osmosis
(the diffusion of water) is more rapid than the dialysis (the diffusion of the
salt molecules or ions). The older explanation of this was that salt attracted
the water, but we now express the fact differently by saying that the salt in
solution exerts a certain osmotic pressure : the result of the osmotic pressure
is that more water fiows from the water side to the side of the solution than
in the contrary direction. The osmotic pressure varies with the amount of
substance in solution, and is also altered by variations of temperature
occurring more rapidly at high than at low temperatures.

If we imagine two masses of water separated by a permeable membrane,
as many water molecules will pass through from one side as from the other,

204 ESSENTIALS OF CHEMICAL PHYSIOLOGY

and so the volumes of the two masses of water will remain unchanged. If
now we imagine the membrane M is not permeable except to water, and the
compartment A contains water, and the compartment 8 contains a solution
of salt or sugar; under these circumstances water will pass through into B,
and the volume of B will increase in proportion to the osmotic pressure of
the sugar or salt in solution in B, but no molecules of sugar or salt can get
through into a from B, so the volume of fluid in a will continue to decrease,
until at last a limit is reached. The determination of this limit, as measured
by the height of a column of fluid or mercury which it will support, will give
us a measurement of the osmotic pressure. Membranes of this nature are
called semi-permeable. One of the best kinds of semi-permeable membrane is
ferrocyanide of copper. This may be made by taking a cell of porous
earthenware and washing it out first with copper sulphate and then with
potassium ferrocyanide. An insoluble precipitate of copper ferrocyanide is
thus deposited in the pores of the earthenware. If such a cell is filled with
a 1-per-cent. solution of sodium chloride, water diffuses in till the pressure
registered by a manometer connected to it registers the enormous height of
5,000 millimetres of mercury. Theoretically it is possible to measure osmotic
pressure by a manometer in this way, but practically it is never done, and
some of the indirect methods of measurement described later are used
instead. The reason for this is that it has been found impossible to construct
a membrane which is absolutely semi-permeable ; they are all permeable in
some degree to the molecules of the dissolved crystalloid. In course of time
therefore the dissolved crystalloid will be equally distributed on both sides of
the membrane, and osmosis of water will cease to be apparent, since it will
be equal in both directions.

Many explanations of the nature of osmotic pressure have been brought
forward, but none is perfectly satisfactory. The following simple explanation
is perhaps the best, and may be rendered most intelligible by an illustration.
Suppose we have a solution of sugar separated by a semi-permeable mem-
brane from water, that is, the membrane is permeable to water molecules,
but not to sugar molecules. The streams of water from the two sides will
then be unequal; on one side we have water molecules striking against the
membrane in what we may call normal numbers, while on the other side
both water molecules and sugar molecules are striking against it. On this
side, therefore, the sugar molecules take up a certain amount of room, and
do not allow the water molecules to get to the membrane; the membrane is,
as it were, screened against the water by the sugar, therefore fewer water
molecules will get through from the screened to the unscreened side than
vice versa. This comes to the same thing as saying that the osmotic stream
of water is greater from the unscreened water side to the screened sugar side
than it is in the reverse direction. The more sugar molecules that are
present, the greater will be their screening action, and thus we see that the
osmotic pressure is proportional to the number of sugar molecules in the
solution, that is, to the concentration of the solution.

Osmotic pressure is, in fact, equal to that which the dissolved substance
would exert it it occupied the same space in the form of a gas (Van’t Hoff’s
hypothesis). The nature of the substance makes no difference ; it is only the

APPENDIX ~ 205

‘number of molecules which causes osmotic pressure to vary. The osmotic
pressure, however, of substances like sodium chloride, which are electrolytes, is
greater than what one would expect from the number of molecules present.
‘This is because the molecules in solution are split into their constituent ions,
and an ion plays the same part as a molecule, in questions of osmotic pressure.
In dilute solutions of sodium chloride ionisation is more complete, and as the
total number of ions is then nearly double the number of original molecules,
| the osmotic pressure is nearly double what would have been calculated from
the number of molecules.
. The analogy between osmotic pressure and the partial pressure of gases
| is very complete, as may be seen from the following statements.
1. At a constant temperature osmotie pressure is proportional to the
' concentration of the solution (Boyle-Mariotte’s law for gases).
2. With constant concentration, the osmotic pressure rises with and is
proportional to the temperature (Gay-Lussac’s law for gases).
3. The osmotic pressure of a solution of different substances is equal to
the sum of the pressures which the individual substances would exert if they
_ were alone in the solution (Henry-Dalton law for partial pressure of gases).
; 4. The osmotic pressure is independent of the nature of the substance in
solution and depends only on the number of molecules or ions in solution
_ (Avogadro’s law for gases).
Calculation of Osmotic Pressure.— We may best illustrate this by an example,
and to simplify matters we will take an example in the case of a non-electro-
_ lytelike sugar. We shall then not have to take into account any electrolytic
_ dissociation of the molecules into ions. We will suppose we want to caleu-
late the osmotic pressure of a 1-per-cent. solution of cane sugar.
One gramme of hydrogen at atmospheric pressure and 0° C. occupies a
- yolume of 11°19 litres; two grammes of hydrogen will therefore occupy“a
_ volume of 22°38 litres. A gramme-molecule of hydrogen—that is, 2 grammes of
_ hydrogen—when brought to the volume of 1 litre will exert a gas pressure
equal to that of 22°38 litres compressed to 1 litre—that is, a pressure of 22°38
_ atmospheres. A gramme-molecular solution of cane sugar, since it contains the
_ same number of molecules in a litre, must therefore exert an osmotic pressure
_ of 22°38 atmospheres also. A gramme-molecular solution of cane sugar
(C,,H,,0,,) contains 342 grammes of cane sugar in a litre. A 1-per-cent.
solution of cane sugar contains only 10 grammes of cane sugar in a litre ; hence
_ the osmotic pressure of a 1-per-cent. solution of cane sugar is Ba x 22:38
atmospheres or 0°65 of an atmosphere, which in terms of a coher of mercury
= 760 x 0°65 = 494 mm.
It would not be possible to make such a calculation in the case of an
electrolyte, because we should not know how many molecules had been
ionised. In the liquids of the body, both electrolytes and non-electrolytes
_ are present, and so a calculation is here also impossible.
We have seen that for such liquids the osmotic pressure cannot be
_ directly measured by a manometer, because there are no perfect semi-
_ permeable membranes; we now see that mere arithmetic often fails us; and
_ so we come to the question to which we have been so long leading up, viz.
_ how osmotic pressure is actually determined.

206 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Determination of Osmotic Pressure by means of the Freezing-point.—This is
the method which is almost universally employed. A very simple apparatus
(Beckmann’s differential thermometer) is all that.is necessary. The principle
on which the method depends is the following :—The freezing point of any
substance in solution in water is lower than that of water; the lowering of
the freezing-point is proportional to the molecular concentration of the dis-
solved substance, and that, as we have seen, is proportional to the osmotic
pressure.

When a gramme-molecule of any substance is dissolved in a litre of water,
the freezing-point is lowered by 1°87°C., and the osmotic pressure is, as we have
seen, equal to 22°38 atmospheres, that is, 22°38 x 760 = 17,008 mm. of mercury.

We can, therefore, calculate the osmotic pressure of any solution if we
know the lowering of its freezing-point in degrees Centigrade; the lowering
of the freezing-point is usually expressed by the Greek letter A.

A
Osmotic pressure = 787 * 17,008

For example, a 1-per-cent. solution of sugar would freeze at —0°052° C.;
052 x 17,008
ae Lomas
mately equal to that we obtained by calculation.

Mammalian blood serum gives A=0°56°C. A 0°9-per-cent. solution of so-
dium chloride has the same A ; hence serum and a 0°9-per-cent. solution of com-
mon salt have the same osmotic pressure, or are isotonic. The osmotic pressure
of blood serum is ee. =4,987 mm. of mercury, or a pressure of
nearly 7 atmospheres.

The osmoti¢e pressure of solutions may also be compared by observing
their effect on red corpuscles, or on vegetable cells such as those in T'rades-
cantia. If the solution is hypertonic, i.e. has a greater osmotic pressure
than the cell contents, the protoplasm shrinks and loses water, or if red cor-
puscles are used, they become crenated. If the solution is hypotonic, e.g. has
a smaller osmotic pressure than the material within the cell-wall, no shrink-
ing of the protoplasm in the vegetable cell occurs, and if red corpuscles are
used they swell and liberate their pigment. Jsotonic solutions produce
neither of these effects, because they have the same molecular concentration
and osmotic pressure as the material within the cell-wall.

Physiological Applications.—It will at once be seen how important all these
considerations are from the physiological standpoint. In the body we have
aqueous solutions of various substances separated from one another by
membranes. Thus we have the endothelial walls of the capillaries separating
the blood from the lymph; we have the epithelial walls of the kidney tubules
separating the blood and lymph from the urine; we have similar epithelium
in all secreting glands; and we have the wall of the alimentary canal
separating the digested food from the blood-vessels and lacteals. In such
important problems, then, as lymph-formation, the formation of urine and
other excretions and secretions, and absorption of food, we have to take into
account the laws which regulate the movements both of water and of sub-
stances which are held in solution by the water. In the body osmosis is not

its osmotic pressure is therefore = 473 mm., a number approxi-

APPENDIX . 9OT

the only force at work, but we have also to consider filtration, that is, the
forcible passage of materials through membranes, due to differences of
- mechanical pressure. Further complicating these two processes we have to
take into account another force, namely, the secretory or selective activity of
the living cells of which the membranes in question are composed. This is
sometimes called by the name vital action, which is an unsatisfactory and
unscientific expression. The term is merely a confession of ignorance ;
what we cannot understand we may call vital, but it is no explanation of any
matter to say we cannot understand it. The laws which regulate filtration,
imbibition, and osmosis are fairly well known and can be experimentally
verified. But we have undoubtedly some other force, or some other mani-
festation of force, in the case of living membranes. It probably is some
physical or chemical property of living matter which has not yet been
brought into line with the known chemical and physical forces which
operate in the inorganic world. We cannot deny its existence, for it some-
times operates so as to neutralise the known forces of osmosis and filtration.
It is, however, contrary to scientific usage and a bar to all progress in
knowledge to sit quietly down and merely label it as vital; we must look
forward to the time when workers will succeed in unravelling its so-called
mysteries, and show what it really consists in.

_ The more one studies the question of lymph-formation, the more con-
vinced one becomes that mere osmosis and filtration will not explain it entirely.
The basis of the action is no doubt physical, but the living cells do not
behave like the dead membranes of a dialyser; they bave a selective action,
picking out some substances and passing them through to the lymph, while
they reject others.

The question of gaseous interchanges in the lungs has been another
battlefield of a similar kind. Some maintain that all can be explained
by the laws of diffusion of gases; others assert that the action is wholly
vital. Probably those are most correct who admit a certain amount of truth
in both views ; the main facts are explicable on a physical basis, but there
are also some puzzling data that show that the pulmonary epithelium is able
to exercise some other force as well which interferes to some extent with the
known physical process. Take again the case of absorption. The object of
digestion is to render the food soluble and diffusible ; it can hardly be sup-
posed that this is useless; the readily diffusible substances will pass more
easily through into the blood and lymph: but still, as Waymouth Reid has
» shown, if the living epithelium of the intestine is removed, absorption comes

very nearly to a standstiJl, although from the purely physical standpoint
removal of the thick columnar epithelium would increase the facilities for
osmosis and filtration.

The osmotic pressure exerted by crystalloids is very considerable, but
their ready diffusibility limits their influence on the flow of water in the
body. ‘Thus if a strong solution of salt is injected into the blood, the first
effect will be the setting up of an osmotic stream from the tissues to the
blood. The salt, however, would soon diffuse out into the tissues, and would
now exert osmotic pressure in the opposite direction. Moreover both effects
will be but temporary, because excess of salt is soon got rid of by the excreting

‘208 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Osmotic Pressure of Proteids.—It has been generally assumed that proteids,
the most abundant and important constituents of the blood, exert little or no
osmotic pressure. Starling, however, has claimed that they have a small
osmotic pressure ; if this is so, it is of importance, for proteids, unlike salt, do
not diffuse readily, and their effect therefore remains as an almost permanent
factor in the blood. Starling gives the osmotic pressure of the proteids of
the blood-plasma as equal to 30mm. of mercury. By others thisis attributed
to the inorganic salts with which proteids are always closely associated.
Moore, for instance, finds that the purer a proteid is, the less is its osmotic
pressure; the same is true for other colloidal substances. It really does not
matter much, if the osmotic force exists, whether it is due to the proteid
itself, or to the saline constituents which are almost an integral part of a
proteid. It is merely interesting from the theoretical point of view. We
should from the theoretical standpoint find it difficult to imagine that a pure
proteid can exert more than a minimal osmotic pressure. It is made up of
such huge molecules that, even when the proteids are present to the extent
of 7 or 8 per cent., as they are in blood-plasma, there are comparatively few
proteid molecules in solution. Still, by means of this weak but constant
pressure it is possible to explain the fact that an isotonic or even a hypertonic
sdlution of a diffusible crystalloid may be completely absorbed from the
peritoneal cavity into the blood. b

The functional activity of the tissue elements is accompanied by the
breaking down of their proteid constituents into such simple materials as urea
(and its precursors), sulphates, and phosphates. These materials pass into
the lymph, and increase its molecular concentration and its osmotic pressure ;
thus water is attracted (to use the older way of putting it) from the blood to
the lymph, and so the volume of the lymph rises and its flow increases. On
the other hand, as these substances accumulate in the lymph they will in
time attain there a greater concentration than in the blood, and so they will
diffuse towards the blood, by which they are carried to the organs of
excretion.

But, again, we have a difficulty with the proteids ; they are most important
for the nutrition of the tissues, but they are practically indiffusible. We

must provisionally assume that their presence in the lymph is due to filtration

from the blood. The plasma in the capillaries is under a somewhat higher
pressure than the lymph in the tissues, and this tends to squeeze he
constituents of the blood, including the proteids, through the capillary walls,
I have, however, already indicated that the question of lymph-formation is
one of the many physiological problems which await solution by the
physiologists of the future.

The whole subject of osmosis and its applications to physiology is attracting
considerable attention at the present time. I have not attempted to discuss
the question at all fully, but merely to indicate the lines research has followed
and will have to follow in the future. In the preparation of this short sketch
I have had of necessity to consult much of the current literature, particularly
the writings of Waymouth Reid and Starling. I am also indebted for much
help to the very excellent article on the subject in Howell’s ‘ Text-Book of
Physiology,’ and Dr. Kosppe’s ‘ Physikalische Chemie.’

——OoO

__ Acid sodium phosphate, 108

INDEX

In cases where there are several figures after any subject, the one in heavy type indicates where
the Lacie matter in relation to the subject is to be found.

Axssorption, 76; of carbohydrates, 77 ;
of proteids, 77 ; of fats, 78

Absorption bands, 96, 152, 179

Absorption spectra of hemoglobin and
its derivatives, 96, 152; of myohex-
a 160; of urinary pigments,

Accessories of food, 43

Acetic acid, 11, 17

Achroé-dextrin, 15, 47, 143
Acid-albumin, 20, 21, 27, 52, 58, 135

absorption |
Acid, lactic, 88, 56; test for, 159

a
ey
3

Acid tide, 108

Acids, vegetable, 108

Acute yellow atrophy of liver, 112, 113
Adenine, 30, 121, 122

Adipose tissue, 2, 4, 16
Aérobic micro-organisms, 51

Air, expired and inspired, 99

» Air pumps, mercurial, 195
Alanine, 67

Albumin, acid, 21, 27, 52, 58, 135
a" action of acids and alkalis on, |

Albumin, alkali, 20, 27, 60, 62, 135
Albuminates, 27

Albumin in urine, estimation of, 129
Albumin in urine, tests for, 129
Albumins, 4, 22, 26, 27, 59
Albuminoids, 3, 4, 31, 62
Albuminometer of Esbach, 129
Albumose, 19, 20, 50, 58, 77, 134, 147
yeaa and peptone, separation of, |

Albumoses, tests for, 52, 147

Aleapton, 132

Alcaptonuria, 132

Alcohol, action of, on proteids, 25

Alcoholic fermentation, 13, 14, 51; of
milk, 39

Alcohols, 3, 4, 17, 43

Aldehyde, 11, 17

Aleurone grains, 24

Alexander Schmidt’s method of precipi-
tating serum globulin, 155

Alexis St. Martin, case of, 54

Alkali-albumin, 20, 27, 62, 135

Alkaline hematin, 151 -

Alkaline hematoporphyrin, absorption
bands of, 179

Alkaline tide, 108

| Alkaloids, 43

Alloxuric bases, 30, 121

| Alvergniat’s pump, 197
| Amides, 4

Amido-acetic acid, 66, 113
Amido-acids, 22, 66, 113

_ Amido-caproic acid, 66, 113

Amido-ethyl-sulphonie acid, 72

| Amido-iso-butyl-acetic acid, 67

Amido-propionie acid, 67

| Amido-pyrotartaric acid, 62
| Amido-succinic acid, 62
| Amido-valerianic acid, 32

Amidulin, 15

Amines, 4, 22

Amino-oxy-purine, 121

Amino-purine, 121

Ammonia, 4, 22

Ammoniacal odour of putrid urine, 110

Ammonium carbonate and carbamate
as urea precursors, 115

Ammonium cyanate, 109

_ Ammonium sulphate, action of, on pro-

teids, 19, 25, 27, 134, 144, 145
Ameeboid movement, 2
Y

210

Amylolytic ferments, 47, 49; in blood
serum, 141

Amylopsin, 14, 49, 60, 61, 63

Amyloses, 10, 11, 50

Anaérobic micro-organisms, 51

Analysis of gases, 198

Animal gum, 28

Animal starch, 10

Anions, 201

Anisotropous bodies, 186

Anti-peptone, 62, 63

Antiseptics, 48

Apparatus necessary for practical work, 4

Aqueous vapour, tension of, 8

Arginine, 22, 32, 62, 63, 113

Aromatic compounds, 4, 12, 24

Arterial blood, gases of, 100

Artificial gastric juice, 54

Aspartic acid, 62, 112

Assimilation, 2

Atmosphere, 99

Atomic weights, 8

Attraction sphere, 2

Avogadro’s law, 205

Bactuu1, 49

Bacteria, 49, 76

Bacterial action, 65

Barfoed’s reagent, 142

Bath, warm, 20

Baumann and Wolkow on alcapton, 132

Beaumont, Dr., 54

Beef, 34, 39

Beef-tea, 42

Beetroot, 13

Benger’s liquor pancreaticus, 60, 150;
liquor pepticus, 52

Benzene, derivatives of, 4

Benzoie acid, 123

Bernard, Claude, on glycogen, 77

Bile, 64, 69-80; amount secreted, 70;
circulation, 71, 72; characters of,
71; constituents of, 70, 71; mucin
of, 71; pigments, 72; salts of, 72;
uses of, 74; in urine, 132

Biliary fistula, 69, 177

Bilirubin, 70, 72, 107 ; of meconium, 76

Biliverdin, 70, 72; of meconium, 76

¢ Biuret, 24, 105

Biuret reaction, 19, 24, 52, 58, 60, 148

Blood, 81-104; coagulation of, 82, 85,
157; corpuscles, 87 ; detection of, 135 ;
gases of, 100; tablets, 83; pigment,
87-98, 151-156 ; plasma and serum
of, 3, 81, 155; in urine, 133

Bohr on absorption-of oxygen, 103; on
hemoglobin, 98; on tension of
carbonic dioxide, 102

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Bone, composition of, 31; marrow, 16

Bowman’s capsule, 107

Bran, 40

Bread, 34, 41

Bright’s disease, 131

Brodie on muscle proteid, 162

Brown and Morris on digestion of
starch, 47

Buffy coat, 83

Bunge on hematogens, 29; on milk, 36

Bush tea, 43

Butter, 33, 38

Butyric acid, 14, 17, 65

Butyrin, 38

CAFFEINE, 43, 121

Calcium, 3,8; phosphate, 4

Calcium oxalate in urine, 118, 126

Calcium salts, importance of, in coagu-
lation of blood, 81, 85, 157; of milk,
33, 37, 145

Cane sugar, 9, 10, 12, 77,141; in urine,
13; tests for, 9, 13

Caproic acid, 17

Caproin, 38

Caprylin, 38

Carbamide, 109,115. See also Urea

Carbohydrates, 3, 4, 9-16, 140; absorp-
tion of, 77; classification of, 10;
definition of, 10; tests for, 10-16,
140-144

Carbolic acid poisoning and urine, 180

Carbon, 3, 8

Carbonates in blood, 102; in urine, 116

Carbonic acid in air, 99; in blood, 100,
101

Carbonic oxide hemoglobin, 97, 151

Cardiac glands, 53, 54

Carmine-stained fibrin, 149

Carnic acid, 63

Cartilage, hyaline, 31

Casein, 27, 33, 37, 145, 146

Caseinogen, 33, 37, 145 ; preparation of,
145, 146

Cell, definition of, 2; diagram of, 29

Cells, differentiation of, 1

Cellulose, 10, 14, 15, 50

| Centigrade scale, 8

Central cells of cardiac glands, 54

Centrosome, 2

Cerebrin, 162, 163

Cerebrosides 163

Cerebrospinal fluid, 163 ;
164

Centrifugal machine, 157

Cheese, 38

Chemical physiology, 1

Chemical sediments of urine, 128

functions of,

Chemical structure of protoplasm, 2
istry of respiration, 98
Chitin, 31
Chlorides of urine, 115 ; estimation of,
168 ; tests for, 105
Chlorine, 3, 8
- Chlorophyll, 75
Cholalic acid, 72, 75
Cholesterin, 3, 4, 18, 69, 70, 73, 88, 163;
_ ~ tests for, 74, 136
_ Choletelin, 73 -
Choline, 18, 163, 165 ; tests for, 163, 164
Chondrin, 31

Chromogens, 107 ; of urine, 180
Chyle, 78

Chyme, 70

Ciliary movement, 2

stitution, 194

Circulation of bile, 71

Cirrhosis of liver, 112

Citric acid, 108

Clark’s essence of rennet, 43

Claude Bernard on glycogen, 77

Clotting of blood, 82

Coagulated proteids, 27

Coagulation, 25

Coagulation of blood, 82, 157 ; of hydro-
cele fluid, 158; of milk, 37, 145; of
muscle, 159 ; of proteids, 22

Coagulative ferments, 50

Cobalt sulphate, actions on proteids,
148

Cocaine, 43

Cocoa, 43, 121

Coffee, 43, 121

Coffin-lid crystals, 117

Cola nut, 43

Collagen, 21, 31, 62

Collimator, 93, 154

Colloids, 23, 203

Colostrum, 36

Colostrum corpuscles, 36

_ Commercial peptone, 84, 147

-_ Compound proteids, 28

Compounds found in the body, 3

Compounds of carbon, 3

Condiments, 42

INDEX

211

| Crystallisable proteids, 22, 89, 144

Crystallisation of egg albumin, 144
Crystalloids, 23, 204

| Crystals from blood, 82, 89, 150

Curd, 146

_ Curds and whey, 37

Cyclopterine, 32

| Cyanamide, 123
_ Cystin, 127 ; crystals of, 126

DecatcrrreD blood, 81, 157
Decalcified milk, 33, 145, 146
Decomposition, products of, 18

|. Density of water, 8
| Dentine, composition of, 31
_ Deposits in urine, 117-127

Circular polarisation and chemical con- —

Cooking of foods, 41
Copper, 3, 8

Cream, 33

Creatine, 4, 22, 42; crystals, 124; in |
muscle, 160; as a urea precursor, 112

Creatinine, 42, 112, 123, 131; crystals
of, 121 ; detection of, 106; estimation
of, 176 ; preparation of, 124

Crypts of Lieberkiihn, 64

Desiccator, 161

Deutero-albumose, 57, 147

Deutero-proteose, 59, 62, 131

Dextrin, 9,10, 15, 34, 134,143 ; tests for,
10, 47

Dextro-rotatory, 10-15

Dextrose, 9, 10, 11, 33, 77, 141, 142;
crystals, +f - in blood, 11; inurine, 130

Diabetes mellitus, 11, 67, 131

Diabetic urine, 129

Diacetin, 17

Dialyser, 23

Dialysis, 23, 200, 202;
147 ; of serum, 155

Diastase, 49

of albumoses,

| Diastatic ferments, 14, 47, 143

Diet, 34
Diet tables, 35

Dietary, 35

Diffusion, 23, 200, 202

Digestion, 149; gastric, 52-59; intes-
tinal, 64; pancreatic, 60-64 ; salivary
45

: Dimethylxanthine, 121

Dioxy-purine, 121

Direct vision spectroscope, 94, 95
Disaccharides, 10, 11, 12

‘ Disuse atrophy,’ 165

Dough, 33, 40

Drechsel on urea formation, 112
Dripping, 42

Dropsical effusions, 29

| Dulcite, 11

Dupré’s urea apparatus, 105, 106
Dysalbumose, 147

Ecx’s fistula, 112
Egg-albumin, 19, 26, 39; crystallisation
of, 24, 144; in urine, 77
P2

212

Egg-globulin, 19, 26, 39

Eggs, 34, 39

Egg white, 19, 20, 22, 39

Egg yolk, 39

Ehrlich’s experiments with methylene
blue, 101

Elastic fibres, 31

Elastin, 31, 62

Elastoses, 58

Elements found in the body, 3
and atomic weights of, 8

Emulsification, 18

Emulsion, 18, 63

Enamel, composition of, 31

English system of weights and measures,

; symbols |

Envelope crystals, 118, 126

Enzymes, 47, 49, 51

Epidermis, 2

Epithelium of intestine, 78

Epsom salt, 116

Erlenmeyer flask, 175

Erythro-dextrin, 15, 47, 143

Esbach’s albuminometer, 129; reagent,
5; tube, 129

Estimation of chlorides, 168; of creati- |
nine, 175; of dextrose, 130; of lactose, |
13; of maltose, 14, 143; of nitrogen, |
199; of phosphates, 171 ; of sulphates, —
172; of urea, 105, 166; of uric acid,
174

Ethereal sulphates
estimation of, 172

Ethyl alcohol, 17

Ethyl-diacetic acid, 131

Eu-globulin, 156

Expired air, 99

External respiration, 99

Extirpation of pancreas, 67

Extractives of blood, 87; of muscle, 42,
159, 161

Extraordinary ray, 186

in urine, 116;

Faces, 75

Fahrenheit scale, 7

Fats, 4, 16-18, 38

Fats, absorption of, 78-80 ; constitution
of, 16; decomposition products of,
18; melting-point of, 16; tests for,

, 16

Fatty acids, 17

Fehling’s solution, 9, 11, 129

Fehling’s test, 9, 13, 14, 129

Ferment coagulation, 25

Ferment, invert, 64

Fermentation, 47

Fermentation test, 11, 132

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Ferments, 47; of gastric juice, 55; of
pancreatic juice, 61; of saliva, 49;
of succus entericus, 64; organised,
49; unorganised, 49

Ferments, classification of, 49

Fibre, elastic; 31

Fibrin, 27, 84, 85

Fibrin ferment, 50, 84, 85, 158; pre-
paration of, 81, 87

Fibrin filaments, 83

Fibrinogen, 26, 84, 85, 86

Filtration, 203

Fistula, gastric, 54

Fleischl’s hsmometer, 184;
polarimeter, 193

Flour, 33, 40

Fluorescent sereens, 154

Fluorine, 3

spectro-

| Food, 38, 84; cooking of, 41

Formic acid, 17

Fractional heat coagulation of muscle
proteids, 159; of serum proteids, 156

Frauenhofer’s lines, 92, 152, 153

_ Fredericq on oxygen tension, 101; on
tension of carbon dioxide, 102

Free hydrochloric acid in gastric juice,
tests for, 149

Firth, v., on muscle plasma, 161

GauacrosE, 10, 11, 12, 13, 141

Gall stones, 73

Gamgee on secretion of hydrochloric
acid, 55; on photographic spectra,
153

Garrod on urochrome, 108

Garrod and Hopkins on urobilin and
stercobilin, 177

Garrod’s methods of separating pig-
ments from urine, 178

Gas analysis, 198

Gaseous interchange in lungs, 207

Gases of blood, 100

Gastric fistula, 54

Gastric juice, 52; action of, 50, 57;
composition of, 56; glands, 53; pro-
perties of, 57; secretion of, 52

Gay-Lussac’s law, 205

Gelatin, 4, 21, 31, 43; tests for, 136

Gelatinisation, 21

Gelatoses, 58

Germ theory of disease, 48

Globin, 90

Globulin, 22,26, 58, 162

Globulose, 58

Glossopharyngeal nerve, 44

Gluco-proteid, 28

Glucose, 10, 11, 77, 141, 143

INDEX —

_ Glucosuria, transitory, 131
_ Glutaminie acid, 62, 113

' Glycocholate of soda, 70
_ Glycocholic acid, 72
Glycocine, 22, 66, 72, 75, 113, 123

_ Glycogen, 10, 15, 22, 49, 134; micro-

scopical detection of, 141; prepara-
tion of, 140; tests for, 15, 134
Glycosuria, 131
Glycuronic acid, 132
Glyoxylic acid, 5, 19
Gmelin’s tcst, 69, 73
Gnezda on biuret reaction, 148

213

Haldane on methemoglobin, 97; on
absorption of oxygen, 103

| Halogens, action on proteid, 28
_ Hammarsten on blood coagulation, 84 ;

_ Heat coagulation of proteids, 22;

Gowers’s hemacytometer, 181; hzmo-

globinometer, 183
Graham, Thomas,
erystalloids, 22

on colloids and

_ Gram-molecular solutions, 202

Granulose, 14

Grape sugar, see Dextrose

Ground substance, 21, 28

Griitzner’s method of comparing diges-
tive power of solutions of pepsin,
149

- Guanine, 30, 121, 122
_ Guarana, 43

Gums, 10
Gunsberg’s reagent, 149
_ Giirber on serum albumin crystals, 24

Hzmacytometer of Sir William Gowers,
181; of Oliver, 182

Hematin, 75, 90, 177; acid, 151;
a ine, 151 ; iron free, 151 ; of food,

. Hematogen, 29

Hematoidin, 70, 91; crystals, 70

Hematoporphyrin, 91, 153,178 ; absorp-
tion bands of, 153, 179

Hematoscope of Hermann, 94

oa 90,91; crystals, preparation of,

ka , 151

Hemoglobin, 23, 23, '28, 82, 151’; composi-
ny of, 90; derivatives of, 151, 152,

3
binometer of Sir William

Gowers, 183; of Oliver, 184

q Hemoglobinuria, paroxysmal, 133
Hemometer of von Fleischl, 184

method of precipitating serum glo-
bulin, 155

Hammerschlag’s method of estimating

_. the specifie gravity of blood, 186

Hayem’s fiuid, 183

of
serum globulin and serum albumin,
155, 156

Heat rigor, 162

Heidenhain on secretion of gastric juice,
55; on pressure in bile duct, 70

Heller’s nitric acid test, 129

Hemi-peptone, 62

Henry-Dalton law, 205

Herrmann, hematoscope of, 94

Hetero-albumose, 58, 59, 147 ; proteose,
62, 131

Hexatomic alcohols, 11

Hexone bases, 32, 63, 113

| Hill’s air-pump, 187

Hill, Croft, on ferments, 50

Hippuric acid, 123, 136; crystals of,
123

Histidine, 32, 63

Histone of Kossel, 90

Hofmeister on crystallisation of egg-
albumin, 24

Homogentisinic acid, 132

| Hopkins’s method of estimating uric

acid, 120, 174; on crystallisation of
egg-albumin, 24, 144

_ Hoppe-Seyler on hematin, 90; on pro-

teids, 21

Hot-air oven, 140

Hiifner’s method of estimating urea,
167

Hyaline cartilage, 31

Hydrazone, 142

Hydrobilirubin, 73, 107, 177

Hydrocarbon, 17

Hydrocele fiuid, 85, 158

Hydrochinon, 180

Hydrochloric acid, 4; of gastric juice,
55; tests for, 149; test for cane
sugar, 9

Hydrochloride of hematin, 90

Hydrogen, 3, 8

Hydrolysis, 13, 58

Hydrolytic ferments, 50

Hydrometer, 36, 186

Hydroxybutyric acid, 131

Hydroxyl, 17

Hypertonic solutions, 206

bromite of soda, action of, cn urea,

105, 110, 167

Hypotonic solutions, 206

Hypoxanthine, 30, 121

214

InpDIcAN, 116

Indiffusibility of proteids, 23, 208
Indigo, 116; blue, 180; red, 180
Indole, 65, 75, 180

Indoxyl, 116, 180

Indoxy! sulphate of potassium, 116, 180
Infection, 48, 49
Tnorganic compounds,
Tnosite, 11, 12;
Inspired air, 99
Internal respiration, 98

Internal secretion, 67, 68
Interstitial substance, 21, 28
Intestinal juice, 13; digestion, 64
Intravascular coagulation, 158
Inversion, 12, 13

Inversion of cane sugar, 50
Inversive ferments, 50

Invert ferments, 12

Invertin, 50, 64

Todine, 3, 8

Todine test, 10, 15, 33, 134, 143
Ions, 201

Tron, 3, 8; in milk, 36

Tron-free hematin, 90, 153
Iso-cholesterin, 74

Iso-maltose, 14, 141

Isotonic solutions, 206

Isotropous bodies, 186

3; salts, 3
erystals of, 12

Jarré on test for indoxyl, 180
Jaundice, 132

Jelly, Lieberkiihn’s, 28

Jelly, Whartonian, 21

Johnson, G. S., on creatinine, 176; on
' sugar in urine, 130

Johnson, Sir G., on picric-acid test, 130
Juice, intestinal, 12, 13, 64

Junkets, 43

K
Kar-tons, 201

Liver, function of, in relation to ibe:

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Kiihne, on peptone, 62; on precipita-
tion of pepsin, 57

Kiilz’s method of extracting glycogen,
140

Kutscher on antipeptone, 63

LactTaLBumEn, 26, 33, 37, 145; properties
of, 37

Lactic acid, 11, 14, 38, 42, 56; tests for,
159

Lactic acid fermentation, 14, 65; in milk,
38; in muscle, 42, 159; organisms,
13

Lactometer, 33

Lactose, 10, 18, 33, 38, 141; in urine,
131 ; tests for, 141

Laky blood, 89

Lanoline, 74

Lard, 10

Lateritious deposit, 118

Laurent’s polarimeter, 192

Lead, 3, 8

Lecithin, 3, 4,18, 39, 65,71, 88, 162, 163

Leech extract, 84

Leucine, 22, 32, 61, 63, 65, 66; as a
urea precursor, 113; in urine, 128;
tests for, 150

Leucine crystals, 66 ; preparation of, 150

Levo-rotatory, 10, 12, 24

Levulose, 10, 11, 12, 141; in blood, 12;
in muscle, 12; in urine, 12; reactions
of, 12

Lieberkiihn’s crypts, 64; jelly, 28

Liebig’s extract, 161; method of esti-
mating chlorides, 169; urea, 166

Lime water, 21

Lipochrome, 38, 39; in muscle, 160

Lipolytie ferments, 50

Liquor pancreaticus, 60; pepticus, 52

Lithates, see Urates

Lithium, 3, 8

Litre, standard of capacity, fi

111; uric acid, 120

| Living test-tube experiment, 85
_ Loeb on ionic action, 201 ; on fertilisa-

Kauder’s method of precipitating serum —

globulin, 155

Keratin, 2, 4, 31, 75

Ketone, 11

Kidney, 106

Kidneys, removal of part of, 67

Kjeldahl’s method of estimating nitro-
gen, 199

Kossel on protamines, 26, 32; on his-
tone, 90

Koumiss, 39

tion, 202
Lungs, 98
Luther on nitrogen loss in hypobromite
method of estimating urea, 167
Lysatinine, 113
Lysine, 32, 62, 113

McoKenprick on cholesterin, 74
MacMunn on stercobilin, 76

INDEX

MeWilliam’s test for proteids, 148
_ Magnesium, 3, 8

215

| Moore on osmotic pressure of proteids,

sulphate, action of, on pro- |

Magnesium
teids, 19, 26, 156
Malic acid, 108
Mallow, 13
ighian capillaries, 106
Malting ferment, 143
Maltase, 50
Maltose, 10, 14, 47, 77, 141, 143
Malt upon starch, action of,
diastase, 143
Maly on the formation of hydrochloric

143 ;

Marsh gas, 64

Measures of capacity, 7; of length, 6
Meat, 34, 39; constituents of, 40
Meconium, 76

Moore’s test for sugar, 11 -

Mérner and Sjéqvist’s method of esti-
mating urea, 168

Mérner on hemin, 91

_ Morris and Brown on starch digestion,

- Mucic acid, 12
Mucin, 2, 21, 28, 75; in bile, 29, 69; -in

Medulla oblongata, diabetic puncture |

of, 131
Melanin, 180
Mercurial air-pumps, 195

Mercuric nitrate, method of estimating |

chlorides, 168 ; urea, 166

Mercury compound of creatinine, 125,
176

Metabolism, 2, 67

Methzmoglobin, 96, 151; crystals of,
150 ; in urine, 133

Methane, 65

Methylene blue sagan 101

Metric system, 7

Microchemical detection of eon
141

Micrococcus urea, 110, 127

Microspectroscope, 94, 151

Milk, 33, 34, 36; alcoholic fermenta-
tion of, 39; coagulation of, 37, 57,
145 ; composition of, 37 ; fats of, 38 ;

saliva, 45 ; in urine, 125 ; tests for, ‘91,
44, 137

Mucinogen, 45

Mucoids, 28

Mucous glands, structure of, 46

Mucous membrane of frog’s intestine,
78

Mucous salivary glands, 28

Munk on fat absorption, 79

Murexide test, 118

Muscle, 39, 156; clot, 161; pigments of,
160; plasma of, 156; extractives of,
160, 161

Muscle sugar, 12

Muscular movement, 2; exercise and
urea, 111 ; exercise and carbonic acid,

Myo-albumin, 160, 161

Myogen, 161; myogen-fibrin, 161

Myo-globulin, 160, 161

Myo-hematin, absorption spectrum of,
160

Myosin, 4, 27, 39, 58, 161; ferment, 50 ;
fibrin, 161

_ Myosinogen, 26, 159, 160, 161, 162

proteids of, 37; salts of, 39; souring |
38

of,

Milk-curdling ferment of pancreas, 61,
64 ; of stomach, 37, 57 x

- Milk-sugar, 10, 13, 33, 38, 77; erystals,
13°

rete reagent, 5; test, 19, 24, 33,

Mineral compounds, 3

Mohr’s method of estimating chlorides,
169

Moleschott’s diet, 35

Monatomic alcohols, 17

Monoacetin, 17

Monochromatic light, 192

Monosaccharides, 10, 11

Monoxypurine, 121

Myxcedema, 67

Nencxi and Sieber, on pigments, 177 _
Nencki’s experiments on urea, 114
Nerve degeneration, chemistry of, 163
Nervous tissues, 162

Neurokeratin, 31, 162

_ Neutral salts, action of, on proteids, 19,

- 25, 26, 33, 37, 82, 134, 147, 155
Nickel sulphate, action of, on proteids,
148
Nissl’s bodies, 162
Nicol’s prism, 187
Nitrate of urea, 110, 118
Nitrie oxide hemoglobin, 91, 98, 154
Nitrogen, 3; estimation of, 199
Nitrogenous food, 39 ; glueosides, 163°
Nitrous acid, action of, on urea, 110
Nucleic acid, 30 ; decomposition of, 30

Moore and Rockwood on fat absorption, Nocisin, 3, 29, 38, 117, 121; bases from,
20 -

216

Nucleo-proteid, 3, 29, 30, 158, 163; in
bile, 71; decomposition of, 30;
muscle, 162; tests for, 136

Nucleus, functions of, 2

OueErc acid, 17

Olein, 16, 17, 38

Olfactory nerve, 44

Oliver’s hemacytometer, 182; hemo-
globinometer, 184

Organic compounds, 3

Ordinary ray, 186

Organic ferments, 49

Osazones, 141, 142

Osmosis, 23,200, 202

Osmotic pressure, 203, 204, 207; calcu-
lation of, 205, 206; of proteids, 208 ;
physiological application of, 206

Ossein, 31

Osteomalacia, 131

Ovarian cyst fluid, 29

Ovo-mucoid, 28, 39

Oxalate of calcium in milk, 33 ; plasma,
157; in urine, 118, 126

Oxalate of urea, 110, 118

Oxidases, 50

Oxygen, 3, 8; in blood, 100

Oxyhemoglobin, 62, 91, 151; crystals,
82, 89, 150; in muscle, 160

Oxyhemoglobin, pure, preparation of,
154

Oxyntic cells of cardiac glands, 54

Oxyphenyl-amido-propionic acid, 167

Pate muscle, 160

Palmitic acid, 17

Palmitin, 16, 17, 38

Pancreas, extirpation of, 67, 131; graft-
ing the, 67; structure of, 61

Pancreatic digestion, 60, 149

Pancreatic juice, 14; action of, 62;
composition of, 61; secretion of, 60

Panum’s method of precipitating serum
globulin, 155

Paraglobulin, see Serum Globulin

Para-mucin, 29

Paramyosinogen, 160, 161, 162

Parapeptone, 52, 58

Parietal cells of ‘cardiac glands, 54, 55

Parotid gland, 44, 46

Paroxysmal hemoglobinuria, 133

Partial pressure of gases, 100-103

Pasteur on circular polarisation, 194

Pathological urine, 129; pigments of,
178; pigments, 180

in |

ESSENTIALS OF CHEMICAL PHYSIOLOGY

| Pavy on composition of proteid, 28 ; on

glycogenic function of liver, 77; on

estimation of sugar, 130

_ Pawlow on secretory nerve-fibres to the
a glands, 56; to the pancreas,

| Pepsin, 50, 52, 55, 57; solutions,
abe! of, 149 ; hydrochloric acid,
5

Pepsinogen, 55
Peptic digestion, 52
Peptone, 19, 22, 52, 58, 59, 77, 134;
precipitation of, 58, 147; in urine,
131; tests for, 59, 147
Peptonuria, 131
Perfect foods, 34
Pericardial fluids, 85
Pettenkofer’s test for bile salts, 69, 72,
132
_ Pfliiger’s mercurial air-pump, 195, 196
| Phenol, 65, 75
| Phenyl sulphate of potassium, 116
| Phenyl-hydrazine test for sugars, 141
Phloridzin, 131
Phosphates of urine, 117, 127, 128;
> estimation of, 171; tests for, 105
Phosphates, stellar, 127
Phosphates, triple, 127
Phosphorus, 3, 8
Photographic spectra, 153
Physiological chemistry, 1
Physiological proximate
detection of, 134

principles,

Pickering on colour reactions of

teids, 148

Picramie acid, 11

Picric acid, test for sugar, 11, 130

Pigment of red corpuscles, 89; of
muscle, 160; of urine, 177

Pigments, 3; pathological, 180
Piotrowski’s reaction, 19, 24, 148

| Plasma, constituents of, 86; gases of,

86; proteids of, 86

| Plasma of blood, 81, 85, 157; of
muscle, 159

Pleochromatism, 189

Poisonous alkaloids, 65

Polarimeters, 190; of Laurent, 192; of
Soleil, 190; of Zeiss, 192

Polarisation of light, 186

Polariscopes, 187 ¥

Polarised light, 189; action of carbo-
hydrates on, 10-14; of proteids, 24

Polarising microscope, 188

Polysaccharides, 10, 11, 14

Pork, 39

Portal vein, 69

Potash, method of extracting glycogen,
140 ; of showing zymogen granules,
150

| Potassium, 3, 8

”

Potassium ferrocyanide in the estima-
tion of phosphates, 171

of uric acid, 175
Potassium sulphocyanide in saliva, 44,
45

Precipitants of proteids, 24, 25
Precipitation, 24

Prevost and Dumas on formation of |

urea, 111
Primary albumoses, 58; proteoses, 59
Principal cells of stomach, 54

De ee fa dares sieion apickrosaone, 94

Propeptone, 52, 58
Propionic acid, 17
Protagon, 162

Protamines, 26, 32

Proteids, 2, 3, 19-32, 37, 39-41; absorp- |

tion of, 1: classification of, 25, 59;

27; coagulation of, 22,
27; colour reactions of, 24; composi-
tion of, 21; compound, 26, 28; con-
jugated, 26; ~ crystallisation of, 23,
24; definition of, 21; digestion of
by gastric j juice, 57-60; digestion of,
by pancreatic juice, 62, ‘63; molecule,
21; of blood plasma, 86 ; of milk, 37:
of ‘muscle, 160; of serum, 86; os-
motie pressure of, 208; in urine, 131;
precipitants of, 24; simple, 26; solu-
bilities of, 22, 26; tests for, 19, 22,

Proteid sparing food, 31
Proteolytic ferments, 50, 52; enzymes,

62
Proteoses, 19, 26, 27, 58, 59, 62, 77,
131

; 85
Sco albene 58, 59, 62, 147
Protones, 32
Protoplasm, chemical structure of, 3;
properties of, 2

217
Q

QuaNTITATIVE estimation of albumin,
129 ; of chlorides, 168-170 ; of creati-
nine, 175; of dextrose, 130; of
glycogen, 140; of lactose, 13, 130; of
maltose, 14, 130, 143; of nitrogen,
199; of phosphates, 171 ; of sulphates,
172; of sugar, 130; of urea, 105, 166,
167; of uric acid, 120, 174

Racemic acid, 130
Ranke’s diet, 35
Reagents necessary for practical work, 4

| Réaumur’s seale, 7

Red blood corpuscles, 87,88 ; com-
position of, 88; pigment of, 89

Red muscle, 160

Refiex action, 44

Rennet, 33, 50, 145

Rennin, 55

Reproduction, 2

Respiration, chemistry of, 98

Respiratory pigments, 89

iratory quotient, 99

Rigor mortis, 42

Ringer on caseinogen, 146; on con-
tractile tissue, 201

Riva on urochrome, 108

Rockwood and Moore on fat absorption,
80

Saccuanic acid, 12

| Saccharimeters, 190
| Saecharoses, 10, 12
| Salicyl-sulphonic acid, action of, on

Proximate principles, classification of, |

4; of food, 34; scheme for detecting,

134-137
Pseudo-globulin, 156
Pseudo-mucin, 29
oe 30

Ptomaines,
Ptyalin, nth 4, 47, 49
Pulses, 41

Pumps, mercurial air, 195-198
Purine bases, 30, 121

Purpurate of ammonia, 118 2
Pus in urine, 133; tests for, 133
Putrefaction, 48, 49

Pyloric glands, 53

Pyrocatechin, 164, 180

proteids, 148

Saliva, 21, 44; action of, 45; composi-
tion of, 45; secretion of, 44

Salivary ‘corpuscles, 44; glands, 44

Salkowski’s reaction for cholesterin, 74
method of estimating sulphates, 173

Salmine, 32

Salted whey, 33

Salting-out of proteids, 25

Saponification, 63

Sarcine, 122

Sarcolemma, 31

Sarcosine, 123, 124

Schafer on internal secretion, 68 ; on fat
absorption, 79

Schiff on bile circulation, 71, 75 ; test for
uric acid, 118

218

Schizomycetes, forms of, 48

Schmalz’s capillary picnometer, 186

Schmidt on precipitating serum globulin,
155; on salts of plasma, 87; on pre-
paration of fibrin-ferment, 87

Schréder’s work on urea, 114

Schwann, white substance of, 73

Scombrine, 32

Sebum, 74

Secretion, internal, 67; of bile, 69

Sediments in urine, 125-128

Semipermeable membranes, 204

Serous glands, 46

Serum, 83, 155; albumin, 26, 81, 86;
crystallisation of, 24; heat Coagula-
tion of, 155

Serum casein, 155

Serum globulin, 26, 81,
coagulation of, 155, 156

Serum lutein, 81 ~

Serum of blood, 23, 81, 85; proteids of,
86 ; of muscle, 159

Serum proteids, 86; separation of, 82

Sheep’s-wool fat, 74
Shell of eggs, 39

' Siegfried on antipeptone, 63

Silicon, 3, 8

Silver nitrate method of estimating
chlorides, 169

Sir William Gowers’s hemacytometer,
181; hemoglobinometer, 182

Sir William Roberts on estimation of
sugar in urine, 132

Skatole, 65, 75

Skatoxyl, 180; red, 180

Skimmed milk, 33, 36

Smoky urine, 133

Snake venom, 49

Soap, 18, 63, 80, 87

Sodio-magnesium sulphate, action of,
on proteids, 156

Sodium, 3, 8

Sodium bicarbonate in blood, 101, 102

Sodium chloride, 3; action on proteids,
26, 155 ; on nucleo-proteids, 29, 158

86; heat

Sodium hypobromite method of estimat- _

ing urea, 105, 167
pairs phosphate, 21; acid, 21, 102,
108

Sodium sulphate plasma, 81, 157

Solar spectrum, 96, 152

Soleil’s saccharimeter, 190, 191

Soluble starch, 15

Solutions, 200

Sorbite, 11

Soret’s band, 154

Soup, 43

Specific gravity of blood, 186; of milk,
33, 36; of urine, 105, 108

Specific rotatory power, 193

Spectra, 96; of hemoglobin and its |

ESSENTIALS OF CHEMICAL PHYSIOLOGY

derivatives, 152; photographic, of
methemoglobin, oxyhemoglobin, and
hemoglobin, 153; of myohsmatin,
160; of urinary pigments, 108, 179
Spectro-polarimeter, 193
Spectroscope, 92, 93, 94
Spectrum, 92
Spermatozoa, 30
Starch, 4, 9,10, 14, 41, 47, 134; soluble,
15; actionof malt on, 143; digestion
of, 44, 49, 63; test for, 9, 14
Starling on osmotic pressure of proteids,
208
Steapsin, 18, 50, 61; action of, 60
Stearic acid, 17
Stearin, 16, 17, 38
Steatolytic ferments, 50
Stellar phosphates, 127
Stercobilin, 73, 107, 177
Stewart, G. N., dietary, 35
Stirling on preparation of pure oxyhe-
moglobin, 154
Stokes’ fiuid, 92
Stomach, glands of, 53, 54
turine, 32
ublingual gland, 44, 45
Submaxillary gland, 44, 45
Succus entericus, 13, 64, 77
Sucroses, 10, 11
Sugar, 4, 50; cane, 13; in blood, 87;
in urine, 130, 181; muscle, 12; tests
for, 9-14, 141, 142
Sugar, maple, 13
Sulphates of urine, 116; estimation of,
172; tests for, 105
Sulphonal poisoning, 179
Sulphur, 3, 8
Suprarénal gland, removal of, 67
Sutton’s modification of Mohr’s method
of estimating chlorides, 169
Swim-bladder of fishes, 103
Symbols and atomic weights, 8
Syntonin, 21, 27, 52, 58, 159

Tannin, 43

Tartaric acid, 108

Taurine, 72, 75

Tauro-carbamic acid, 75

Taurocholate of soda, 70, 72

Taurocholic acid, 72

Tea, 121

Teichmann’s crystals, 90, 91

Tendon, 21

Tension of aqueous vapour, 8 ; of gases,
102, 103

Testis, removal of, 67

Theine, 43

——

INDEX

Theobromine, 43, 121
Theophylline, 121
Thermometric scales, 7
Thrombin, 84, 85
Thudichum on urochrome, 177
Thyroid gland, removal of, 67
Tissue-fibrinogens, 29, 84, 158
Tissue respiration, 98, 103
Tomes on enamel, 31
Tonsils, 44
Torricellian vacuum, 92, 196
Torula urex, 110
Torulz, 47, 48, 49
Transitory glucosuria, 131
Triacetin, 17
Triatomic alcohol, 17
Trichloracetic acid as a precipitant of
proteids, 148

Trimethyl-xanthine, 121
Triolein, 17
Trioxy-purine, 121
Tripalmitin, 17
Triple phosphate, 117, 118, 127
Tristearin, 17
Trommer’s test, 9, 11-15, 33, 44
Tropzolin test, 149

in, 50; action of, 60, 62

Tyrosine, 22, 32, 61, 62, 63, 65, 66, 113,
132; crystals, 66; in urine, 113,
128; tests for, 150

Umeriicat cord, 21

Uncerystallisable sugar, 12

Unorganised ferments, 46, 49

Uremia, 111

Uranium acetate in estimation of phos-
phates, 171

Uranium nitrate in estimation of phos-
phates 171

Urate, acid ammonium, deposit of, 126 ;
acid sodium, deposit of, 126

Urates, 118, 120, 125, 126

Urea, 3, 4, 22, 47, 71, 87, 105, 109;
composition and compounds, 109;
crystals of, 108; decomposition of,
110; estimation of, 105, 166; mode
and site of formation, 111; prepara-
tion of, 168; quantity excreted, 111;
tests for, 105, 136; where formed,
111, 112

eer? nitrate, 109 ; preparation of, 109,

8
Urea oxalate, 109, 118

219

Urie acid, 22, 87, 119, 121, 122;
erystals of, 119; estimation of, 120,
174; preparation of, 118, 174 ; origin
of, 120; tests for, 118, 136_

Urina potus, 109

Urinary deposits, 125

Urine, 105; composition of, 109; inor-
ganic constituents of, 115; pigments
of, 177; tests for abnormal constitu-
ents of, 129; tests for constituents of,
105, 136

Urinometer, 108

Urobilin, 73, 107, 108, 177, 178 ; absorp-
tion bands of, 179

Urobilinogen, 107

Urochrome, 108, 177

ro-erythrin, 126, 178 ; absorption bands
of, 179

Urorosein, 180; absorption bands of,

179

Vaueric acid, 17, 65

Van’t Hoff on polarisation of light, 195 ;
hypothesis, 204

Vegetable acids, 4; foods, composition
of, 41; parchment, 23

Vegetables, composition of, 41, 43;
green, 43

Venous blood, gases of, 100

Villus, section of, 78

Vital action, 207

Vitellin, 24, 29, 30, 39

Vitellose, 58

Vitreous humour, 21

WwW

Watier’s modification of Zuntz’s gas
apparatus, 198

Warm bath, 20

Water in protoplasm, 2, 4; density
Of, 7:

Wave theory of light, 186

Weights and measures, 6

Whartonian jelly, 21

Whey, 33, 37; salted, 33, 36

Whey-proteid, 37

White blood corpuscles, 2, 87

White of egg, 2, 19, 20, 39, 52, 59

Whole flour, 40

Witte’s peptone, 147, 148

Wohler, preparation of urea, 109

Wooldridge on tissue-fibrinogen, 29, 84,
158

‘ Worth’ of corpuscles, 186

220 ESSENTIALS OF CHEMICAL PHYSIOLOGY

xX Yvon on removal of creatinine and

XANTHINE, 30, 61, 87
Xanthine group, 30,

Xanthoproteic test, 19, 44

urates, 167
, 121, 122
121

¥ Zxuiss’ polarimeter, 192

Yxast, action of, 11,
in bread-making,
sugar, 11, 132

Zine chloride creatinine, 124, 175
12, 13; cells,47; | Zuntz’s gas apparatus 198
41; in testing for | Zymogen, 55,87 granules, 150

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