MEMCAL tSCMOOL

^^<M>^ (ju-iCf^^

CHEMICAL PHYSIOLOGY

H IB
1907

PEEFACE

TO

THE SIXTH EDITION

I HAVE again subjected the book to a thorough revision, and the
changes which are now introduced into the practical exercises are
those which experience has shown to be advisable. In the large
text it has been necessary to rewrite a good many parts, mainly on
account of our increased knowledge of the proteins and of the way
they are utilised in the body. The sections relating to blood coagula-
tion and to respiration have been much amplified in order to include
many facts which are the result of recent research.

In my endeavour to bring the work abreast of advances in
science, and at the same time to keep it within moderate limits,
I have to acknowledge help and valuable suggestions from Mr. J.
Barcroft, M.A. (especially in connection with Eespiration), from
Professor T. G. Brodie, F.E.S., and from my two colleagues at King's
College, Dr. Lyle and Dr. O. Eosenheim ; both of these have been
of great assistance to me in reading the proof-sheets, and Dr. Lyle
is again responsible for the Index.

W. D. Hallibubton.
King's College, 1907.

J 1089

Digitized by the Internet Archive

in 2007 with funding from

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CONTENTS

PAGE

Introduction 1

ELEMENTARY COURSE

Lessox

I. The Elements contained in Physiological Compounds ... 9

II. The Carbohydrates . . .13

III. The Fats - 22

IV. The Proteins 27

V. The Proteins (continued) 29

VI. Foods 49

VII. The Digestive Juices — Saliva and Gastric Digestion ... 62

VIII. The Digestive Juices (continued) — Pancreatic Digestion and Bile 78

IX. The Blood and Eespiration 101

X. Urine 141

XI. Urine (continued) 156

XII. Pathological Urine . . . . ^ 166

Scheme for Detecting Physiological Proximate Principles . .171

ADVANCED COURSE

Introduction 175

Lesson

XIII. Carbohydrates 176

XIV. Action of Malt upon Starch 179

XV. Crystallisation of Egg Albumin 180

XVI. Milk 181

XVII. The Proteoses ...... ... 182

VIU ESSENTIALS OF CHEMICAL PHYSIOLOGY

Lesson page

XVIII. Digestion 184

XIX. HAEMOGLOBIN AND ITS DERIVATIVES 188

XX. Serum 192

XXI. Coagulation of Blood 194

XXII. Muscle and Nebvous Tissue 197

XXIII. Urea and Chlorides in Urine 204

XXIV. Phosphates and Sulphates in Urine 207

XXV. Uric Acid and Creatinine ........ 210

XXVI. The Pigments of the Urine 213

APPENDIX

HiEMACYTOMETERS * .* 217

h.ajmoglobinometebb . . . . . . * . .219

Polarisation of Light 222

polarimeters 226

The Spectro-polarimeter '.'.'. . . 220

Eelation between Circular Polarisation and Chemical Constitution . 230

Mercurial Air-pumps 231

Analysis of Gases 234

Kjeldahl's Method of Estimating Nitrogen . ." . . . . 235

Solutions, Diffusion, Dialysis, Osmosis 236

INDEX 245

LIST OF ILLUSTRATIONS

FIG- - PAGE

1. Dextrose Crystals . Frey 17

2. Inosite Crystals . . . . . . . Frey 18

3. Milk-sugar Crystals Frey 19

4. Section of Pea, showing Starch Grains . , Yeo, after Sachs 20

5. Fat Cells Schdfer 23

6. Simple Warm Bath 29

7. Dialyser 37

8. Dialyser 37

9. Diagram of a Cell Schafer 45

10. Milk Yeo 5S

11. Colostrum Corpuscles HeidenJiain 53

12. Yeast Cells Yeo' s Physiology 63

13. Schizomycetes After Zopf 64

14. Bacillus Anthracis Koch 65

15. Alveoli of Serous Gland Langley 69

16. Mucous Cells Langley 69

17. Submaxillary Gland Heidenhain 69

18. Fundus Gland Klein 71

19. Pyloric Gland Ehstein 71

20. Fundus Gland Langley 72

21. Alveolus of Pancreas Kuhne and Lea 80

22. Leucine Crystals KUhne 86

23. Tyrosine Crystals ^rey 86

24. H^MATOiDiN Crystals . , Frey 89

25. Cholesterin Crystals Frey 93

X ESSENTIALS OF CHEMICAL PHYSIOLOGY

FIG. PAGR

26. Villus of Eat Killed during Fat Absorption . . . Schafer 98

27. Mucous Membrane of Frog's Intestine during Fat

Absorption ScJuifer 99

28. Fibrin Filaments and Blood Platelets .... Schafer 103

29. Action of Reagents on Blood Corpuscles .... Schafer 111

30. Oxyh.i:moglobin Crystals Quairi's Anatomy 112

31. H;emin Crystals ... Preyer 113

32. Diagram of Spectroscope . . . 116

33. Figure of Spectroscope and Accessories . . . McKendrick 116

34. Arrangement of Prisms in Direct-vision Spectroscope GscJieidlen 117

35. Stand for Direct- vision Spectroscope 118

36. Absorption Spectra . " Rollett 118

37. Absorption Spectra 119

38. Loewy's Aerotonometer 132

39. Dissociation Curves of Blood and Hemoglobin . . Bohr 133

40. Barcroft's Blood Gas Apparatus 135

41. Dupre's Urea Apparatus Gamgee 142

42. XJrinometer McKendrick 144

43. Urea Crystals Fi-ey 144

44. Urea Nitrate and Oxalate Frey 146

45. Triple Phosphate Crystals ....... Frey 155

46. Uric Acid Crystals Frey 157

47. Acid Sodium Urate Frey 163

48. Acid Ammonium Urate Frey 163

49. Envelope Crystals of Calcium Oxalate .... Frey 164

50. Cystin Crystals Frey 164

51. Triple Phosphate Crystals Bryant's Surgery 164

52. Calcium Phosphate Crystals .... Bryant's Surgery 164

63. Albuminometeb of Esbach 166

f)4. Two Burettes on Stand . . . . ; . . Sutton 167

55. Hot-air Oven with Gas Regulator .... Gscheidlen 176

56. OsAzoNE Crystals Coloured x>lcde to face 177

57. Absorption Spectra op Hemoglobin, &c 189

58. Photographic Spectrum of Hemoglobin and Oxyhemoglobin Gamgee 190

LIST OF ILLUSTRATIONS XI

FIG. PAGB

59. Photographic Spectrum of Oxyhjemoglobin and Methjemo-

GLOBiN Oamgee 190

60. Centrifugal Machine 195

61. Absorption Spectra of Mtoh-ematin 199

62. A Desiccator Gscheidlen 199

63. Absorption Spectra of Urinary Pigments . . . After Hopkins 215

64. Gowers' Hemacytometer 217

65. Oliver's H.ismacytometer 218

66. Gowers' Hemoglobinometer 219

67. Von Fleischl's Hemometer 220

68. Oliver's Hjemoglobinometer 221

69. Model to Illustrate Polarised Light 223

70. Model to Illustrate Polarised Light 223

71. Model to Illustrate Polarised Light 224

72. Diagram to Explain Polarisation of Light 225

73. Diagram to Explain Polarisation of Light 226

74. Soleil's Saccharimeter 227

75. Diagram of Optical Arrangements in Soleil's Saccharimeter . 227

76. Laurent's Polarimeter 228

77. Spectro-polarimeter of von Fleischl 229

78. Diagram of Asymmetric Carbon Atoms 231

79. Diagram of Pfluger's Pump 232

80. L. Hill's Air-pump 233

81. Waller's Apparatus for Gas Analysis .... Wall^ 234

82. Kjeldahl's Method, Distilling Apparatus 235

83. Diagram to Illustrate Osmosis 239

ESSENTIALS

OF

CHEMICAL PHYSIOLOGY

INTEODUCTION

Chemical Physiology or Physiological Chemistry deals with the

chemical composition of the body and with the chemical changes it
undergoes ; it also deals with the composition of the food which
enters, and the excretions which leave, the body.

When a chemist examines living things he is placed at a dis-
advantage when compared with an anatomist; for the latter can
with the microscope examine cells, organisms, and structures in the
living condition. The chemist, on the other hand, cannot at present
state anything positive about the chemical structure of living matter,
because the reagents he uses will destroy the life of the tissue he
is examining. There is, however, no such disadvantage when he
examines non-living matter, like food and urine, and it is therefore
in the analysis of such substances that chemical physiology has
made 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 alteration in the shape of the cells, by deposition of inter-
cellular matter between the cells, and by chemical changes in the
living matter of the cells themselves. Thus in some situations the
cells are grouped into the various epithehal linings; in others the
n B

2 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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

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

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

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 specially 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) irritability — that is, the property of responding by some
change when subjected to the influence of an external agent or
stimulus : the most obvious of these changes is movement (amoeboid
movement, ciliary movement, muscular movement, &c.); (2) its power
of assimilation — that is, it is able to convert into protoplasm the
nutrient material or food which is ingested ; (3) its power of growth
— this is a natural consequence of its power of assimilation ; (4) its
power of reproduction — this is a variety of growth ; and (5) its power
to excrete, to give out waste materials, the products of its other
activities.

Of all the signs of life, those numbered 2 and 5 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

INTRODUCTION 3

point of view are the complex nitrogenous compounds called Proteins,^
So far as is at present known, protein material is never absent from
living substance, and is never present in anything else thaii that
which is alive or has been formed by the agency of living cells. It
may therefore be stated that Protein 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-iluid, and at least three-quarters of
its weight, often more, are due to water. (2) Proteins. These are
the most constant and abundant of the solids. A protein or albu-
minous substance consists of carbon, hydrogen, nitrogen, oxygen,
with sulphur and phosphorus in small quantities only. In nuclein,
a protein-like substance obtained from the nuclei of cells, phosphorus
is more abundant. The protein obtained in greatest abundance from
the cell-protoplasm is nucleo -protein : that is, a compound of protein
with varying amounts of nucleiji. White of egg is a familiar instance
of an albuminous substance or protein, 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 proteins 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 ; chole-
sterin, a monatomic alcohol : and inorganic salts, especially phos-
phaucs 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.

The substances out of which the body is built consist of
chemical elements and of chemical compounds, or unions of these
elements.

The elements found in the body are carbon, hydrogen, nitrogen,
oxygen, sulphur, phosphorus, fluorine, chlorine, iodine, silicon,
sodium, potassium, calcium, magnesium, lithium, iron, and occa-
sionally manganese, copper, and lead.

Of these very few occur in the free state. Oxygen and nitrogen (to
a small extent) are found dissolved in the blood-plasma ; hydrogen is

' In most English text-books these substances have hitherto been called
Proteids. The change to Protein brings English, American, and German usage
into harmony.

b2

4 ESSENTIALS OF CHEMICAL PHYSIOLOGY

formed by putrefaction in the alimentary canaL With some few ex-
ceptions such as these, the elements enumerated above are found
combined with one another to form compounds.

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

(1) Mineral or inorganic compounds.

(2) Organic compounds, or compounds of carbon.

A convenient practical method of grouping these proximate
principles of the body and of food is the following : —

( Water.

Inorganic . . . . j Salts — e.,<7.chlorides and phosphates of sodium

( and calcium.

/ j Proteins — e.g. albumin, myosin, gelatin.

Nitrogenous \ Simpler nitrogenous bodies — e.g. lecithin,

i creatine, urea.

Organic ■< ( Fats — e.g. butter, fats of adipose tissue.

^, ., J Carbohydrates — e.g sugar, starch.

JNon-mtrogenous j simple organic bodies— e.g. alcohol, chole-

V V sterin, vegetable acids and salts, lactic acid.

Many of the substances enumerated above only occur in small
quantities. The most important are the inorganic substances, water
and salts ; and the organic substances, proteins, 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 proteins,
fats, and carbohydrates forms the starting point, the A B C, as one
might say, of chemical physiology.

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

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.

„ „ 01 per cent.

Nitric acid, concentrated.
Fuming nitric acid.
Hydrochloric acid, concentrated.
„ „ 0-2 per cent.^

Acetic acid, glacial.
„ „ 20 per cent.

?> " . ^ "
Glyoxylic acid.
Formaldehyde.

' Made by adding 994 c.c. of water to C c.c. of the concentrated hydrochloric
acid of the British Pharmacopoeia.

INTRODUCTION 5

Caustic potash, 20 per cent.

» 0-1
Ammonia.

Sodimn 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.

„ sodium phosphate.

„ iodine in potassium iodide.

„ ferrous sulphate.

„ ferric chloride.

„ sodium nitroprusside.

Alcohol.
Ether.

Esbach's reagent.^

Solution of copper sulphate, 1 per cent.
Fehling's solution.
Lime water.

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.
Baryta mixture.^
Sodium acetate solution.'*
Phosphoric acid, 0'5 per cent.

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

Solid sodium chloride.

„ magnesium sulphate.

„ ammonium sulphate-

„ sodio-magnesium sulphate.
Standard solution of uranium acetate or nitrate for estimating
phosphates.^

' Mercury is dissolved in its own weight of strong nitric acid. The solution so
obtained is diluted with twice its volume of water. The decanted clear liquid is
Millon's reagent.

2 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.

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

6 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Standard solution of mercuric nitrate for estimating urea.

„ „ silver „ „ chlorides.

Caustic soda, 40 per cent.
Bromine.

Solution of potassium bichromate.
Phenyl hydrazine hydrochloride.
Solid sodium acetate.
Phospho-tungstic acid.
Glacial phosphoric acid.
Dry cupric oxide.
Soda-Ume.

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-oz. 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. Apparatus and reagents for carrying out the Kjeldahl
process are also necessary.

WEIGHTS AND MEASUKES

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

Weights

(English System)

1 grain = 0*0648 gramme

1 ounce = 437'5 grains = 28*3595 grammes

1 lb. = 16 oz. = 7,000 grains = 453*5925

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

1 milligramme -
1 centigramme =
1 decigramme =
1 gramme
1 decagramme = 10
1 hectogramme = 100
1 kilogramme = 1,000

INTEODUCTION

(Metric System)

0*001 gramme
0-01 „
0-1

grammes

0-015432 grain
0-154323 „
1-543235 ,;
15-43235 grains
154-3235
1543-235
= 15432-35
= 2 lb. 3 oz. 119-8 „

Measures of Length

(English System)

1 inch = 25-4 millimetres

1 foot = 12 inches = 304-8 millimetres

(Metric System)

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

1 millimetre = 0-001 metre

1 centimetre = 0-01 „

1 decimetre = 0-1 „

1 metre

0-03937 inch
0-3937 „
3-93707 inches
39-37079 „

1 minim

1 fluid drachm = 60 minims

1 fluid ounce = 8 fluid drachms

1 pint = 20 fluid ounces

1 gallon = 8 pints

Measures of Capacity
(English System)

= 0*59 cubic centimetre
3-549 cubic centimetres
28-398
567-936

4-54837 litres

(Metric System)

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

1 cubic centimetre (generally written c.c.) = 16-931 minims.

1 litre = 1,000 c.c. - 1 pint 15 oz. 2 drs. 11 m. - 35-2154 fluid ounces.

1 cubic inch = 16-365 c.c.

THEKMOMETEIC SCALES

The scale most frequently used in this country is the Fahrenheit scale ;
in this the freezing-point of water is 32°, and the boiling point 212°. On the
Continent the Reaumur scale is largely employed, in which the freezing-
point is 0°, and the boiling-point 80°. In scientific work the Centigrade

' 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
a 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

scale has almost completely taken the place of these ; in this system the
freezing-point is 0° and the boiUng-point 100°.

To convert degrees Fahrenheit into degrees Centigrade, subtract 32 and
multiply by f , or C = (F — 32) f . Conversely, degrees Centigrade may be
converted into degrees Fahrenheit by the following formula : F = §C + 32.

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

10°. 9-126
11°. 9-751
12°. 10-421
13^ 11-130

14°. 11-882

15°. 12-677

16°. 13-519

17". 14-409

18°. 15-351
19^ 16-345
20°. 17-396
21°. 18-505

22°. 19-675
23^ 20-909
24°. 22-211
25°. 23-582

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

0°. 0-99988
1°. 0-99993
2°. 0-99997
3°. 0-99999
4°. 1-00000
6°. 0-99999
6°. 0-99997
7°. 0-99994

8°- 0-99988
9°. 0-99982
10°. 0-99974
11°. 0-99965
12°. 0-99955
13°. 0-99942
14°. 0-99930
15°. 0-99915

16°. 0-99900
17°. 0-99884
18°. 0-99866
19°. 0-99847
20°. 0-99827
21°. 0-99806
22°. 0-99785
23°. 0-99762

24°. 0-99738
25°. 0-99714
26°. 0-99689
27 \ 0-99662
28°. 0-99635
29^ 0-99607
30°. 0-99579

SYMBOLS AND ATOMIC WEIGHTS OF THE PRINCIPAL
ELEMENTS '

Aluminiun

1 Al

27-1

Fluorine

F

19-0

Oxygen

0

16-0

Antimony

Sb 120-2

Gold

Au

197-2

Phosphorus

i P

31-0

Arsenic

As

75-0

Hydrogen

H

1-0

Platinum

Pt

194-8

Barium

Ba

137-4

Iodine

I

126-97

Potassium

K

39-15

Bismuth

Bi

208-5

Iron

Fe

55-9

Silver

Ag

107-93

Boron

B

11-0

Lead

Pb

206-9

Silicon

Si

28-4

Bromine

Br

79-96

Magnesium

Mg

24-36

Sodium

Na

23-05

Cadmium

Cd

112-4

Manganese

Mn

55-0

Strontium

Sr

87-6

Calcium

Ca

40-1

Mercury

Hg

200-0

Sulphur

S

32-06

Carbon

C

12-0

Nickel

Ni

58-7

Tin

Sn

119-0

Chlorine

CI

35-45

Nitrogen

N

1404

Tungsten

W

184-0

Copper

Cu

63-6

Osmium

Os

191-0

Zinc

Zn

65-4

' The above

atomic

weights are

taken

on the

basis that 0 = 16;

that of

hydrogen will then be 1-008.

ELEMENTAEY COUESE

LESSON I
THE ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS

1. Take a fragment of meat about the size of a pea and place it in a porcelain
crucible over a Bunsen flame. Note that it chars, showing the presence of
carbon, and that it gives off the unpleasant odour of burning flesh, which is
due to the fact that it contains the nitrogenous substances called proteins. In
course of time the organic material is completely burnt up, and a small
amount of white ash or inorganic material is left behind.

2. Kepeat the experiment with a pure organic substance like sugar.
Note that no ash is left. Charring, as before, indicates the presence of carbon,
but there is no characteristic smell of burning nitrogenous substances (absence
of nitrogen).

_ 3. The tests for carbon depend on the fact that when this element is
oxidised it gives rise to carbon dioxide ; the test for hydrogen depends on
the fact that when this element is oxidised it gives rise to water. If all the
carbon dioxide and water formed by oxidation from a weighed amount of
any organic substance under examination are collected and estimated, the
amount of carbon and hydrogen respectively which it contains can be easily
calculated. The following exercises, however, deal only with the qualitative
detection of these elements.

4. Tests for Carbon. — The following tests can be carried out with sugar.
(a) When burnt in the air it chars and subsequently the carbon entirely

disappears, passing off in combination with oxygen as carbon dioxide (carbonic
acid gas).

(h) Mix some of the powdered sugar in a dry mortar with about ten times
the quantity of cupric oxide (which has been freed from water by previous
heating) ; place the mixture in a dry test-tube provided with a rubber cork
perforated by a bent glass tube which dips into either lime water or baryta
water. Heat the tube over a Bunsen flame, and as the carbon of the sugar
becomes oxidised carbon dioxide comes off and causes a white precipitate of
calcium or barium carbonate, as the case may be.

5. Test for Hydrogen. — In the experiment just described (4 h) note that
drops of water due to oxidation of hydrogen condense in the colder parts of
the test-tube.

6. Tests for Nitrogen. — The greater number of tests for this element are
due to the circumstance that on the breaking up of organic substances which
contain it, it is given off as ammonia. If the ammonia is all collected and
estimated, the amount of nitrogen can be easily calculated. Kjeldahl's
method for carrying out this quantitative analysis is described in the Appendix.
The following exercises, however, are qualitative only.

10 ESSENTIALS OF CHEMICAL PHYSIOLOGY

(a) The characteristic odour of burning flesh, horn, hair, feathers, &c., has
been already noted, and, though only a rough test, is very trustworthy.

(6) Take a little dried albumin and mix it thoroughly in a mortar with
about twenty times the amount of soda-lime and heat in a test-tube over a
Bunsen flame. Ammonia comes off in the vapours produced, and may be
recognised by (i.) its odour ; (ii.) it turns moistened red litmus paper (held over
the mouth of the tube) blue ; (iii.) it gives off white fumes with a glass rod
(held over the mouth of the tube) which has been dipped in hydrochloric acid.

(c) Mix some dried albumin with about ten times its weight of a mixture
of equal parts of magnesium powder and anhydrous sodium carbonate. A
small quantity of the mixture — such as would lie on the end of a penknife —
is then carefully heated in a dry test-tube and finally heated more strongly
for about half a minute to red heat. Dip the tube while still glowing into
a beaker containing a few c.c. of distilled water ; the tube will break and its
contents mix with the water. Filter and label the filtrate A ; add to this
filtrate a little strong solution of potash, one or two drops of cold saturated
solution of ferrous sulphate and a drop of ferric chloride solution. Bring
the mixture to boiling point, then cool and acidify with hydrochloric acid.
The fluid becomes bluish green, and gradually a precipitate of Prussian blue
separates out. This test is due to the fact that some of the nitrogen is
fixed as sodium cyanide, and this gives the Prussian blue reaction with the
reagents added.

7. Tests for SuljjJmr. — (a) In the foregoing test (6 c) the sulphur of the
albumin combines with the sodium to form sodium sulphide. This may be
detected by taking some of the filtrate A and adding freshly prepared solution
of sodium nitro-prusside ; a reddish violet colour forms.

(6) Test for loosely combined Sulphur. — Add two drops of a neutral
lead acetate solution to a few c.c. of caustic soda solution. The precipitate
of lead hydroxide which is first formed soon dissolves. Heat a small portion
of the albumin with this alkaline solution. The mixture turns black in con-
sequence of the formation of lead sulphide, part of the sulphur present in
albumin in the unoxidised form having been split oft" from it by the caustic
soda as sodium sulphide.

(c) Take some dried albumin and fase with a mixture of potash and
potassium nitrate. Cool ; dissolve in water and filter. The filtrate will give
the following tests for sulphates : — Acidulate with hydrochloric acid and add
barium chloride ; a white precipitate of barium sulphate is produced.

{d) Take some solution of albumin and heat in an open dish in a fume
cupboard for at least an hour with large excess of fuming nitric acid, renewing
the acid from time to time as necessary. The resulting fluid will give the
test for sulphate as in c.

8. Test for Phospliorus. — The two tests just described (7 c and d) maybe
repeated with some substance (such as caseinogen, nucleoprotein, or lecithin)
which contains phosphorus in organic combination ; or the organic matter
may be more conveniently destroyed by Neumann's method, which consists
in heating it with a mixture of sulphuric and nitric acids. The resulting
fluid in each case gives the following test for phosphates : — Mix it with half
its volume of nitric acid ; add ammonium molybdate in excess and boil ; a
yellow crystalline precipitate falls.

The reactions described in the foregoing exercises show how the
processes of pure chemistry may be employed for the detection of
some of the most important elements that occur in substances of
physiological importance, and thus form a fitting introduction to a
study of physiological chemistry.

ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS H

They show, in the first instance, how the substances with which
we have to deal fall under the two main categories of organic and
inorganic. In some of the tissues of the body, like bone and tooth,
the inorganic or mineral material is in excess, but in the softer
portions of the organism the organic compounds are in great pre-
ponderance.

Organic chemistry is sometimes defined as the chemistry of the
carbon compounds ; carbon is in all cases present, and is usually the
most abundant element.

The most important of the nitrogenous substances are the
proteins, as already explained in the introductory chapter, and the
detection and estimation of nitrogen are thus exercises of the highest
interest.

All the proteins contain a small amount of sulphur ; keratin, or
horny material, contains more than most of them do.

Phosphorus is another element of considerable importance, being
present in nuclein and nucleo-proteins, and also in certain complex
fats, of which lecithin may be taken as a type. Iodine occurs united
to protein material in the colloid substance of the thyroid gland ; iron
in the pigment of the blood called haemoglobin ; sodium, calcium,
potassium, and other metals in the inorganic substances of the body.
It would, however, lead us too far into the regions of pure chemistry
to undertake exercises for the detection of these and other elements
which might be mentioned, and have been already commented upon.
The teacher of physiological chemistry is bound to assume that the
students who come before him have already passed through a course
of ordinary chemistry.

The main interest of the exercises selected as types lies in their
physiological application. As a rule an element is detected by
breaking up or oxidising the more or less complex molecule in which
it occurs into substances of simpler nature, and then performing tests
for these simpler products. Thus carbon is identified by the forma-
tion of carbon dioxide, nitrogen by the formation of ammonia, and so
forth.

A great many reactions which can be performed in the test-tube
imitate those which are performed in the body. Eeactions in vitro
and in vivo, to use the technical phrases, often, though not always,
run parallel. Life, from one point of view, is a process of com-
bustion or oxidation ; the fuel is supplied by the food ; this becomes
assimilated, and so forms an integral part of the living substance of
the body ; it is then burnt up by the oxygen brought to it by the
blood-stream, giving rise to animal heat and other manifestations of

12 ESSENTIALS OF CHEMICAL PHYSIOLOGY

energy ; and finally the simple products of oxidation or chemical
breakdown are carried to the organs of excretion (lung, skin, kidney,
&c.), where they are discharged from the body.

A candle consists principally of carbon and hydrogen ; when it is
burnt the products are carbonic acid gas and water ; the former may
be detected by means of lime water, the latter, by holding a dry
beaker upside down for a few moments over the burning candle, when
the moisture will condense on the cold glass.

The body is more complex than a candle, but so far as its carbon
and hydrogen are concerned the main products of combustion are
the same. The carbon dioxide is discharged by the expired air, as
may be proved by blowing it into lime water. The water finds an
outlet by several channels, lungs, skin, and kidneys. The presence of
nitrogen in the body is perhaps the most striking chemical distinction
between it and a candle, and here again the process of metabolism
runs a course analogous in some degree to our experiments in vitro.
Here again the most important and abundant substance which
contains the waste nitrogen is the simple material ammonia, but
ammonia is only discharged as such to a very small extent in health.
It unites with carbon and oxygen to form the body called urea
(CON2H4), which finds its way out of the body via the urine. The
urine also contains the sulphates, due to the oxidation of the sulphur
of the proteins, and the phosphates due to the similar oxidation of
the phosphorus of such substances as lecithin and nuclein. Some
of the salts of the urine, however, in particular the chlorides, come
directly from the food. This we shall discuss at the proper place
when we come to the study of the urine.

LESSON II
THE CARBOHYDRATES

1. Note the general appearance of the specimens of grape sugar or dextrose,
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. — Put a few drops of copper sulphate solution 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 boil-
ing this a yellow or red precipitate (cuprous hydrate or oxide) forms.

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

5. Moore's test. — Add to the dextrose solution about half its volume of
20-per-cent. potash and heat. The solution becomes yellowish brown. Add
to this some sulphuric acid (25 per cent.) and the odour of caramel becomes
apparent.

6. Fermentation test. — Add a fragment of dried yeast to the dextrose
solution in a test-tube ; fill the test-tui)e up with mercury, and invert it over
mercury in a trough. Place it in an incubator at body temperature for 24
hours. The sugar is broken up into alcohol and carbon dioxide ; the latter
gas collects in the upper part of the test-tube.

7. Ca7ie 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 solutionis formed. Dex-
trose, lactose, and maltose do not give this test.

8. Starch. — (a) Examine microscopically the scrapings from the surface
of a freshly cut potato. Note the appearance of the starch grains with their
concentric markings.

(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.

14 ESSENTIALS OF CHEMICAL PHYSIOLOGY

{(J) Conversion into dextrin and dextrose. To some starch solution in a
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.

9. Dextrin. —Add iodine solution to solution of dextrin ; a reddish-brown
colour is produced. The colour disappears on heating and reappears on
coohng.

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

(6) With iodine solution 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.

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.

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. But this definition is
only a rough one, and if pushed too far would include many substances,
like acetic acid, lactic acid, and inosite, which are not carbohydrates.
Kesearch has shown that the chemical constitution of the simplest
carbohydrates is that of an aldehyde, or a ketone, and that the more
complex carbohydrates are condensation products of the simple ones.
In order, therefore, that we may understand the constitution of these
substances, it is first necessary that we should understand what is
meant by the terms aldehyde and ketone.

A primary alcohol is one in which the hydroxyl (OH) is attached
to the last carbon atom of the chain ; its end group is CH2OH. Thus
the formula for common alcohol (primary ethyl alcohol) is

CH3.CH2OH.

The formula for the next alcohol of the same series (primary

propyl alcohol) is

0H3.CH,.CH2OH.

If a primary alcohol is oxidised, the first oxidation product is
called an aldehyde ; thus ethyl alcohol yields acetic aldehyde : —

CH3.CH2OH -f 0=CH3.CH0 + H2O.

[ethj'l alcohol] [acetic aldehyde]

The typical end-group CHO of the aldehyde is not stable, but is
easily oxidisable to form the group COOH, and the compound so
formed is called an acid ; in this way acetic aldehyde forms acetic
acid : —

THE CARBOHYDEATES 15

CH3.CHO + 0=CH3.C00H.

[acetic aldehyde] [acetic acid]

The majority of the simple sugars are aldehydes of more complex
alcohols than this : they are spoken of as aldoses. The readiness with
which aldehydes are oxidisable renders them powerful reducing agents,
and this furnishes us with some of the tests for the sugars.

Let us now turn to the case of the ketones. A secondary alcohol
is one in which the OH group is attached to a central carbon atom ;
thus secondary propyl alcohol has the formula

CH3.CHOH.GH3.

Its typical group is therefore CHOH. When this is oxidised, the
first oxidation product is called a ketone, thus : —

CH3.CHOH.CH3 + 0=CH3.CO.CH3 + H2O.

[secondary propyl alcohol] [propyl ketone]

It therefore contains the group CO in the middle of the chain.
Some of the sugars are ketones of more complex alcohols : these are
called ketoses. The only one of these which is of physiological interest
is levulose.

The alcohols of which we have already spoken are called monatomic,
because they contain only one OH group. Those which contain two
OH groups (like glycol) are called diatomic ; those which contain
three OH groups (like glycerin) are called triatomic ; and so on. The
hexatomic alcohols are those which contain six OH groups. Three
of these hexatomic alcohols with the formula C6H8(OH)(j are of
physiological interest ; they are isomerides, and their names are
sorbite, mannite, and dulcite. By careful oxidation their aldehydes
and ketones can be obtained ; these are the simple sugars ; thus,
dextrose is the aldehyde of sorbite ; mannose is the aldehyde of mannite ;
levulose is the ketone of mannite ; and galactose is the aldehyde of
dulcite. The sugars all have the empirical formula C^HigOe. The
constitutional formula for dextrose is : —

H H H H H H

I I I I I I

H C C C C C C

I I I II I

OH OH OH OH OH O

By further oxidation, the sugars yield acids with various names.
If we take such a sugar as a typical specimen, we see that their general
formula is

C„H2,hO„i

16

ESSENTIALS OF CHEMICAL PHYSIOLOGY

and as a general rule n—m\ that is, the number of oxygen and carbon
atoms is equal. This number in the case of the sugars already
mentioned is six. Hence they are called hexoses.

Sugars are known to chemists, in which this number is 3, 4, 5, 7, &c., and
these are called trioses, tetroses, pentoses, heptoses, &c. The majority of
these have no physiological interest. It should, however, be mentioned that
a pentose has been obtained from the nucleoprotein of the pancreas, of the
liver, and of yeast. If the pentoses that are found in various plants are given
to an animal, they are excreted in great measure unchanged in the urine.

The hexoses are of great physiological importance. The prin-
cipal ones are dextrose, levulose, and galactose. These are called
monosaccharides.

Another important group of sugars is that of the disaccharides :
these are formed by the combination of two molecules of monosac-
charide together with the loss of a molecule of water, thus : —

C5Hi20o + C6Hi206 = Ci2H220ii-f-H20.

The principal members of the disaccharide group are cane sugar,
lactose, and maltose.

If more than two molecules of the monosaccharide group undergo
a corresponding condensation, we get what are called polysaccharides.

The polysaccharides are starch, glycogen, various dextrins, cellu-
lose, gums, &c. We may therefore arrange the important carbo-
hydrates of the hexose family in a tabular form as follows : —

1. Monosaccharides or Glucoses,

2. Disaccharides, Sueroses,
or Saccharoses, CiaHjaO,,

3. Polysaccharides or Amyloses

+ Dextrose.
— Levulose.
+ Galactose.

+ Cane sugar.
+ Lactose.
+ Maltose.

+ Starch.
+ Glycogen.
+ Dextrin.
Cellulose.

The signs + and — in the above list indicate that the substances

to which they are prefixed are dextro- and levo-rotatory respectively

as regards polarised light. ^ The formulae given above are merely

empirical; the quantity w in the starch gi'oup is variable and often

large. The following are the chief facts in relation to each of the

principal carbohydrates.

' 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.

THE CAKBOHYDRATES 17

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
large quantities in the blood and urine
in the disease known as diabetes.

Dextrose is soluble in hot and cold
water and in alcohol. It is crystalline
(see fig. 1), but not so sweet as'cane sugar.
When heated with strong potash certain
complex acids are formed which have a
yellow or brown colour. This constitutes

Moore's test ior SUgSiT. In alkaline SOlu- Fig. l. -Dextrose crystals.

tions dextrose reduces salts of silver,

bismuth, mercury, and copper. The reduction of cupric hydrate to
cuprous hydrate or 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 reduc-
tion of the picric to picramic acid is produced. Another important
property of grape sugar is that under the influence of yeast it is con-
verted into alcohol and carbonic acid (C6Hi206=2C2H60 + 2C02).

Dextrose may be estimated by the fermentation test, by the
polarimeter, and by the use of Fehling's solution. The last method
is the most important : it rests on the same principles as Trommer's
test, and we shall study it and other methods of estimating sugar in
connection with diabetic urine (see Lesson XII.).

Levulose. — When cane sugar is treated with dilute mineral acids
it undergoes a process known as inversion — i.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 levo-
rotator}^ the levo -rotatory power of the levulose being greater than
the dextro-rotatory power of the dextrose formed. Hence the term
inversio7i. The same hydrolytic change is produced by certain
ferments, such as the invert ferment of the intestinal juice, and of
yeast.

Pure levulose can be crystallised, but so great is the difiSculty of
obtaining crystals of it that one of its names was ' uncrystallisable
sugar.' Small quantities of levulose have been found in blood, urine,
and muscle. It has been recommended as an article of diet in
diabetes in place of ordinary sugar; in this disease it does not

c

18 ESSENTIALS OF CHEMICAL PHYSIOLOGY

appear to have the harmful effect that other sugars produce. Levu-

lose 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 (CgHioOg),
which is only sparingly soluble in
water. Dextrose when treated in this
way yields an isomeric acid — i.e. an
acid with the same empirical formula,
called saccharic acid, which is readily
2.-inosite crystals. soluble in Water.

Inosite, formerly called 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,.Hj.^O,;) 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.

DISACCHAEIDES

Cane Sugar. — This sugar is generally distributed throughout the
vegetable kingdom in the juices of plants and fruits, especially the
sugar cane, beetroot, mallow, and sugar maple. It is a substance of
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 — tlcfkt 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 : —

Cj2H220ii+H20 = C6H,206 + C(,H,206

[caoe sugar] [dextrose] [levulose]

THE CARBOHYDRATES 19

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

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

It crystallises in rhombic prisms (see fig. 3). It is much less
soluble in water than cane sugar or dextrose,
and has only a slightly sweet taste. It is
insoluble in alcohol and ether; aqueous
solutions are dextro-rotatory.

Solutions of lactose give Trommer's
test, but when the reducing power is tested
quantitatively by Fehling's solution it is
found to be a less powerful reducing agent ^ /

than dextrose. If it required seven parts fig. 3.— Miik-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 enumerated in connection with cane sugar. The
glucoses formed are dextrose and galactose,

C,2H2,OH + H2O=C6Hi2Oe + 0eH,A

[lactose] [dextrose] [galactose]

With yeast it is first inverted, and then alcohol is formed. Thi^,
however, occurs slowly.

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,,B.,,0,,-\-IL,0=:iG,R,0,

[lactose] [lactic acid]

(2) 4C3He03=2C4H802-H4C02 + 4H2

[lactic acid] [butyric acid]

Maltose is the chief end product of the action of malt diastase on
starch, and is also formed as an intermediate product in the action
of dilute sulphuric acid on the same substance. It is also the chief
sugar formed from starch by the diastatic ferments contained in the
saliva (ptyalin) and pancreatic juice (atnylopsin). It can be obtained

c3

20

ESSENTIALS OF CHEMICAL PHYSIOLOGY

in 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.

0i2H22Oh+H2O = C6H.2O6 + C6Hp,O6

[maltose] [dextrose] [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 (I'0 : 071 : 0-63), by their
rotatory power, or by the phenyl-hydrazine test described in Lesson
XIII.

POLYSACCHARIDES

Starch, is widely diffused through the vegetable kingdom. It
occurs in nature in the form of microscopic grains, varying in size
and appearance, according to their source. Each consists of a
central spot (hilum) round which more or less concentric envelopes

of starch proper or granulose alternate
with layers of cellulose. Cellulose has
very little digestive value, but starch is
a most important food.

Starch is insoluble in cold water : it
forms an opalescent solution in boiling
water, which if concentrated gelatinises
on cooling. Its most characteristic re-
^\mmmsr^smB^^mmm^<^ action is the blue colour it gives with
i^^BSPf^P^H^ iodine solution.

•'^^^^^^^^^^^ On heating starch with dilute mineral

acids dextrose is formed. By the action
of diastatic ferments, maltose is the chief
end product. In both cases dextrin is
an intermediate stage in the process.
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 solution.
Although the molecular weight of starch is unknown, the formula
for soluble starch is probably (CgH, 005)200- Equations that repre-
sent the formation of sugars and dextrin s from this are very complex,
and are at present hypothetical.

Fig. 4.- Section of pea showing starch
aud aleurone grains embedded in the
protoplasm of the cells : a, aleurone
grains ; s(, starch grains ; i, inter-
cellular spaces. (Yeo, after Sachs.)

THE CAKBOHYDRATES 21

Dextrin is the name given to the intermediate products in the
hydrolysis of starch, and two chief varieties are distinguished —
erythro-dextrin, v^hich gives a reddish-brown colour with iodine solu-
tion ; and achroo-dextrin, which does not. v

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

Cellulose. — This is the colourless material of which the cell-walls
and woody fibres of plants are composed. By treatment with
strong mineral acids, it is like starch, converted into glucose, but
with much greater difficulty. The various digestive ferments have
little or no action on cellulose ; hence the necessity of boiling starch
before it is taken as food. Boiling bursts the cellulose envelope of
the starch grains, and so allows the digestive juice to get at the
starch proper. Cellulose is found in a few animals, as in the test or
outer investment of the Tunicates.

Salting out of the Colloid Carbohydrates. — By saturating solutions of
the colloid carbohydrates (starch, soluble starch, glycogen, and some varieties
of dextrin) with such neutral salts as magnesium sulphate or ammonium
sulphate the carbohydrate is thrown out of solution in the form of a white
precipitate. The remaining carbohydrates (sugars and some of the smaller
moleculed dextrins like achroo-dextrin) are not precipitated by this means.
We shall find in connection with the proteins that this method, known as
* salting out,' is one largely employed there for precipitating and distinguish-
ing between classes of proteins. The student is therefore warned that a
precipitate obtained under such circumstances will not necessarily indicate
the presence of protein.

Further information regarding the carbohydrates is given in I^esson XIII,

LESSON III

THE FATS

Lard and olive oil are given round as examples of fats.

1. They are insoluble in water.

2. They dissolve readily in ether. On pouring some of the ethereal solu-
tion on to a piece of blotting paper, a greasy stain is left after the ether has
evaporated.

3. Saponification. — By boiling with potash, fat yields a solution of soap.
On adding some sulphuric acid to this and heating, the fatty acid collects in
a layer on the surface of the fluid. This experiment may conveniently be
performed in the following way : —Melt some lard in an evaporating basin
and pour it into a solution of potash in alcohol - contained in a small flask
and heated carefully on a water-hath nearly to boiling-point. Continue to
boil and saponification is soon completed. When the process is completed
drop some of the solution into a test-tube containing about 10 c.c. of water ;
the solution of soap will be clear, and no oil globules should separate out.
If there is any separation of oil globules continue the boiling.

Then drop the soap solution into some 25-per-cent. sulphuric acid con-
tained in a small beaker and heated nearly to boiling ; the fatty acid soon
separates out and floats on the surface.

4. Beaction of Fatty Acids. — (a) They produce a greasy stain on paper.
(b) Wash the fatty acid obtained in experiment 3 repeatedly with water,

rmtil the wash water is no longer acid, and divide it into two portions.
Dissolve one portion in ether ; this solution reacts acid to phenolphthalein ;
to show this, place a few drops of phenolphthalein in 5 c.c. of water contain-
ing a drop of 20-per-cent. potash. If this red solution is dropped into the
solution of fatty acid, the colour is discharged. Place the second portion of
fatty acid in some half-saturated solution of sodium carbonate and warm ; a
solution of sodium soap is obtained and carbon dioxide comes ofl".

5. Separation of Neutral Fats from Fatty Acids. — In most fats some
free fatty acid is present. They may be separated by the fact that the latter
only dissolves in a solution of sodium carbonate to form soap. The resulting
mass is shaken with water and then with ether ; the two fluids separate on
standing ; the ether contains the neutral fat and the water the soap.

6. Test for Glycerin. — The most important reaction for glycerin, the
other constituent of a fat, is the acrolein test, which is performed in the
following way : — Place some lard in a dry test-tube, add a crystal of potassium
sulphate and heat. Acrolein is given off, which is recognised by its charac-
teristic unpleasant odour, and by the fact that it blackens a piece of filter
paper previously moistened with ammoniacal silver nitrate solution.

7. Osmic Acid Test. — Fat, if it contains olein or oleic acid, is blackened
by osmic acid. Try this with both the lard and the olive oil.

' 30 grammes of potash are dissolved in 20 c.c. of water, and 200 c.c. of 90-per-
cent, alcohol added.

THE FATS

23

8. To determine melting-point of a fat or fatty acid, place a small
quantity in a very narrow test-tube strapped on to a thermometer with an
india-rubber band. Place this in a water-bath which is gradually heated,
and note the temperature at which it melts.

9. Emulsijication. — (a) Take two test-tubes and label them A and B.
Place water in A and soap solution in B. To each add a few drops of olive
oil and shake. In B an emulsion is formed, but not in A.

(6) Shake a few drops of rancid oil with a dilute solution of potash ; an
emulsion is formed because the potash and free fatty acid unite to form a soap.
Divide this into two parts, and to one of them add a little solution of gum or
egg albumin ; the emulsion is much more permanent in this specimen.
These experiments illustrate the favourable action of soap and of a suspend-
ing medium like mucilage upon the formation of an emulsion.

Fat is found in small quantities in many animal tissues. It is,
hov^ever, found in large quantities in three situations, viz. bone

Fia. 5.— A few cells from the margin of a fat lobule ; /. g., fat globule distending fat cell ; ??, nucleus ;
m, membranous envelope of tlie cell ; cr., bunch of crystals within a fat cell ; c, capillary vessel
V, venule ; c. t., connective tissue cell. The fibres of the connective tissue are not represented.

marrow, adipose tissue, and milk. The consideration of the fat in
milk is postponed to Lesson VI.

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 53-65° C. Thus, it is olein v^hich holds the other tv^o dissolved at

24 ESSENTIALS OF CHEMICAL PHYSIOLOGY

the body temperature. Fats are all soluble in hot alcohol, ether, and
chloroform, but insoluble in water.

Chemical Constitution of the Fats. — The fats are compounds of
fatty acids with glycerin, and may be termed glycerides or glyceric
ethers.

The fatty acids form a series of acids derived from the mona-
tomic alcohols by oxidation. Thus, to take ordinary ethyl alcohol,
C2H5HO, the first stage in oxidation is the removal of two atoms
of hydrogen to form aldehyde, CH3.COH : on further oxidation an
atom of oxygen is added to form acetic acid, CH3.COOH (see also
p.U).

A similar acid can be obtained from all the other alcohols,
thus:

From methyl alcohol CH3.HO, formic acid H.COOH is obtained
ethyl

CH3.HO, formic acid H.COOH is

C2H5.HO, acetic „ CH3.COOH

C3H7.HO, propionic,, C^H^.COOH

butyl „ C4H.J.HO, butyric „ C3H7.COOH

amyl „ CsHn-HO, valeric „ C4Hc).C00H

hexyl „ C6H,3.HO,caproic „ GsHn-COOH

propyl
butyl

and so on.

Or in general terms : —

From the alcohol with formula C,iH2„+i.H0 the acid with formula
C„_iH2„_i.C0.0H is obtained. The sixteenth term of this series has
the formula Ci5H3,.C0.0H, and is called palmitic acid; the
eighteenth has the formula Ci;H35.CO.OH, and is called stearic acid.
Each acid, as will be seen, consists of a radical, C„_,H2„_iC0, 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„_]H2„__3COOH.
It is the eighteenth term of the series, and its formula is C17H33.CO.OH.

The first member of the group of alcohols from which this acrylic series of
acids is obtained is called allyl alcohol (CH2 : CH.CHoOH) ; the aldehyde of
this is acrolein (CH2 : CH.CHO), and the formula for the acid (acrylic acid) is
CH2 : CH.COOH. It will be noticed that two of the carbon atoms are united
by two valencies, and these bodies are therefore unsaturated ; they are un-
stable and are prone to undergo by uniting with another element a conversion
into bodies in which the carbon atoms are united by one bond only. This
accounts for their reducing action, and it is owing to this construction that
the colour reactions with osmic acid and Sudan III. are due. Fat which
contains any member of the acrylic series like oleic acid blackens osmic acid,
by reducing it to a lower (black) oxide. Fats like palmitin and stearin do
not give this reaction.

THE FATS 25

Glycerin or Glycerol is a triatomic alcohol, C3H.5(OH)3 — i.e. three
atoms of hydroxyl united to a radical glyceryl (C3H5). The hydrogen
in the hydroxyl atoms is replaceable by other organic radicals. As
an example take the radical of acetic acid called acetyl (CH3.CO).
The following formulae represent the derivatives that can be obtained
by replacing one, two, or all three hydroxyl hydrogen atoms in this
way :—

(OH (OH (OH (O.CH3.CO

C3H5 OH C3H5 -I OH C3H5 O.CH3.CO C3H5 O.CH3.CO

tOH iO.CH3.CO lO.CH3.CO lo.CH3.CO

[glycerin] [monoacetin] [cliacetin] [triacetin]

Triacetin is a type of a neutral fat ; stearin, palmitin, and olein
ought more properly to be called tristearin, tripalmitin, and triolein
respectively. Each consists of glycerin in which the three atoms of
hydrogen in the hydroxyls are replaced by radicals of the fatty acid.
This is represented in the following formulae : —

Acid Radical Fat

Palmitic acid Ci^Hgi.COOH Palmityl Ci,H3,.C0 Palmitin CgH^COCi^Hgi.CO).,
Stearic acid Ci^Hg^.COOH Stearyl Ci.Hgg.CO Stearin C3H,(OCi,H3,.CO)3
Oleic acid Ci.Hgg.COOH Oleyl Cj-Hgg.CO Olein C^B..^{OC,^'R^^.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 : —

C3H5(O.C,5H3iCO)3 + 3H20=C3H5(OH)3 + 3C,5H3iCO.OH

[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 —

C3H5(O.Ci5H3iCO)3 + 3KHO=C3H5(OH)3 + 3C,5H3iCO.OK

[palmitin— a fat] [glycerin] [potassium palmitate— a soap]

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

Lecithin (C42H84NPO9).— This is a very complex fat, which

26 ESSENTIALS OF CHEMICAL PHYSIOLOGY

yields on decomposition not only glycerin and a fatty acid,
but phosphoric acid, and an alkaloid [N.(CH3)3C2H602] 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 (C27H45.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.

' See further under Nervous Tissues, Lesson XXII.

LESSON IV
THE PROTEINS

1. Tests for Proteins. — 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.)

(a) Heat Coagulation. — Faintly acidulate with a few drops of 2-per-cent.
acetic acid and boil. The protein is rendered insoluble (coagulated protein).

(6) Precipitation ivitJi Nitric Acid. — The addition of strong nitric acid to
the original solution also produces a white precipitate.

(c) XantJioproteic Reaction. — On boiling the white precipitate produced
by nitric acid it turns yellow ; after cooling add ammonia ; the yellow becomes
orange.

[d) Millon's Test. — Millon's reagent (which is a mixture of the nitrates
of mercury containing excess of nitric acid ; see p. 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.

(/) Rose's or PiotroiusM's Test. — Add one drop of a 1-per-cent. solution
of cupric sulphate to the original solution and then caustic potash, and a
violet solution is obtained.

Eepeat experiment (/) with a solution of commercial peptone, and note
that a rose-red solution is obtained. This is called the biuret reaction.

(g) Rosenheim's Formaldehyde Reactio?!. — Add to the solution of com-
mercial peptone a very dilute solution of formaldehyde (1 : 2,500), and then
aboiit one third of the volume of strong sulphuric acid containing (as most
commercial specimens of the acid do) a trace of an oxidising agent such as
ferric chloride or nitrous acid. A purple ring develops at the surface of contact.
This reaction probably plays a part in the original

(h) Adamliiewicz Reaction^ in which glacial acetic acid was used instead
of the formaldehyde. Most commercial specimens of glacial acetic acid con-
tain hydrogen peroxide as an impurity ; the oxidising action of this on the
acetic acid leads to the formation of traces of glyoxylic acid and formalde-
hyde ; the necessary factors for the occurrence of the formaldehyde reaction
are thus present. (According to Hopkins glyoxylic acid itself with pure
sulphuric acid gives the test with proteins.)

The same reactions {g and h) are given by the solution of egg-white, but
not so markedly.

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.

(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

28 ESSENTIALS OF CHEMICAL PHYSIOLOaY

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 of both the globulin and albumin. Filter. The filtrate contains no
protein.

(d) Repeat the last experiment (c) with a solution of commercial peptone.
A precipitate is produced of the proteoses it contains. Filter. The filtrate
contains the true peptone. This gives the biuret reaction (see above), but
large excess of strong potash must be added on account of the presence of
ammonium sulphate. Ammonium sulphate added to saturation preci;pitates
all proteins except peptone.

LESSON V

THE PROTEINS {continued)

1. — Action of Acids and Alkalis 07i 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*l-per-cent. solution of caustic potash.

To B add the same amount of 0'1-per-cent. solution of caustic potash.

To C add a rather large amount of 0*l-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 protein is no
longer coagulated by heat, having been converted into alkali- albumin. After

Fig. 6.— Simple warm batli, as described iu footnote.

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 a few
drops of sodium phosphate, colour with litmus, and neutralise as before.

' 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.

30 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Note that the alkali-albumin now requires more acid for its precipitation
than in A, the acid which is first added converting the sodium phosphate into
acid sodium phosphate.

Now remove C from the bath. Boil it. Again there is no coagulation,
the proteins having been converted into acid-alhumin, or syntonin. After
cooling, colour with litmus and neutralise with O'l-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 protein tests with it
enumerated on p. 27. 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 daj'S 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 mucoid 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 mucoid is obtained.

The Proteins are the most important substances that occur in
animal and vegetable organisms, and protein metabolism is, as already
noted, the most characteristic sign of life.

They 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, how^ever, great differences in their chemical and
physical properties.

The proteins in the food form the source of the proteins in the
body tissues, but the latter are usually different in composition from
the former. The food proteins are in the process of digestion
broken up into simpler substances, usually called cleavage products,
and it is from these that the body cells reconstruct the proteins
peculiar to themselves. As a result of katabolic processes in the
body, the proteins are finally again broken down, carbonic acid,
water, sulphuric acid (combined as sulphates), urea, and creatinine
being the principal final products which are discharged in the urine and
other excretions. The intermediate substances between the proteins
and such final katabolites as urea will be discussed under Urine.

The following figures will convince the student how different the
proteins are in elementary composition ; Hoppe-Seyler many years
ago gave the variations in percentage composition as follows : —

a H N 3 0

From 51-5 69 15-2 0-3 20-9
To 5i'5 7-3 17-0 2-0 23*5

THE PROTEINS 31

and recent research has since shown that the variations are even
greater than those just stated.

The same fact is brought home more vividly when the cleavage
products are separated and estimated. These differ both in kind
and in amount, but nearly all of them are substances which are
termed amino-acids. Emil Fischer, to whom we owe so much of our
knowledge in this direction, considers that the proteins are linkages
of a greater or lesser number of these amino-acids, and there is great
hope that in the future his work will result in an actual synthesis of
the protein molecule, and with that will come an accurate knowledge
of its constitution.

When the protein molecule is broken down in the laboratory
by processes similar to those brought about by the digestive
ferments which occur in the alimentary canal, the essential
change is due to what is called hydrolysis : that is, the molecule
unites with water and then breaks up into smaller molecules. The
first cleavage products, which are called proteoses, retain many of
the characters of the original protein ; and the same is true, though
to a less degree, of the peptones, which come next in order of for-
mation. The peptones in their turn are decomposed into short link-
ages of amino-acids which are called polypeptides, and finally the
individual amino-acids are obtained separated from each other.

What we have already learnt about the fatty acids will help us in
understanding what is meant by an amino-acid.

If we take acetic acid, which is one of the simplest of the fatty
acids, we see that its formula is

CH3.COOH.

If one of the three hydrogen atoms in the CH3 group is replaced
by NH2 we get a substance which has the formula

CH2.NH2.COOH.

The combination NH2 which has stepped in is called the amino-
group, and the new substance now formed is called amino-acetic acid ;
it is also termed glycine or glycocoll.

We may take another example from another fatty acid. Propionic
acid is C2H5.COOH ; if we replace an atom of hydrogen by the
amino-group as before, we obtain C2H4.NH2.COOH, which is amino-
propionic acid or alanine. Going a little higher in the scale, and taking
caproic acid, OsHn^COOH, we obtain from it, in an exactly similar
way, CsHio.NHg.COOH, which is amino-caproic acid or leucine.

All the three amino-acids mentioned— glycine, alanine, and leucine

32 ESSENTIALS OF CHEMICAL PHYSIOLOGY

— are found among the final cleavage products of most proteins; but
there are a good many more in addition, for instance : —

Serine (aminO-oxypropionic acid, CHaOH.CHNHo.COOH) ;

Amino-valeric acid ;

Amino-succinamic acid (asparagine) ;

Amino-succinic acid (aspartic acid) ;

Amino-pyrotartaric acid (glutamic acid),
some of which are derived from fatty acids of a different series from
those first enumerated.

But in all these cases there is only one replacement of an atom
of hydrogen by NH2 ; hence they are called monoamino-acids.

Passing to the next stage in complexity, we come to another
group of amino-acids which are called diamino-acids : that is, fatty
acids in which two hydrogen atoms are replaced by NH^ groups. Of
these we may mention lysine, ornithine, arginine, and histidine.

Lysine is diamino-caproic acid.

Caproic acid is C5H11.GOOH.

Mono-amino-caproic acid, or leucine, we have already learnt is
C5H10.NH2.COOH.

Lysine or diamino-caproic acid is C5H,j(NH2)2COOH.

OrnitMne is di-amino-valeric acid, and the following formulae will
show its relationship to its parent fatty acid : —
C4H9.COOH is valeric acid.
C4H7(NH2)2.COOH is diamino-valeric acid or ornithine.

Arginine is a somewhat more complex substance, which contains
the ornithine radical. It belongs to the same group of substances as
creatine, another important cleavage product of the protein molecule.

Creatine is methyl-guanidine acetic acid, and has the formula

HN\ ;

C ; -N(CH3)0H2.COOH

On boiling it with baryta water, it takes up water (H2O) and splits
at the dotted line into urea [CO (NH)2] and sarcosine, as shown

below.

H,N\

C=0 NH.CH3.CH2.COOH
H.N/

[urea] [surcosiiie or inethyl-glyciue]

Arginine decomposes in a similar way, urea being split off on
the left, and ornithine instead of sarcosine on the right. Arginine is
therefore a compound of ornithine with a urea group.

Histidine, the last member of this class, has the formula

I

THE PEOTEINS 33

CfjHgNsOs is probably also a diamino-acid, but its exact constitution
has not yet been made out with certainty.

These substances we have hitherto described as acids, but they
may also play the part of bases, the introduction of a second amino-
group into the fatty-acid molecules conferring upon them basic pro-
perties. The three substances

Lysine (CeHnNsOs)
Arginine (C6H14N4O2)
Histidine (OeHgNgOs)
are in fact often called the hexone bases because each of them contains
six atoms of carbon, as the above empirical formulae show.

But there is still an important group of amino-acids to be con-
sidered, and these are termed the aromatic amino-acids : that is, amino-
acids united to the benzene ring ; and of these we shall mention
three : namely, phenyl-alanine, tyrosine, and a nearly related sub-
stance called tryptophane.

Phenyl-alanine is alanine or amino-propionic acid in which an
atom of hydrogen is replaced by phenyl (C6H5).

Propionic a.cid has the formula C2H5.COOH.
Alanine (amino-propionic acid) is C2H4.NH2.COOH.
Phenyl-alanine is C2H3.GGH5.NH2.COOH.
The formula of phenyl-alanine may also be written another way.
The graphic formula of benzene (C^H^) is :

H

I
C

^ \

H— C C— H

I '^^ t II
. H— C "^i C— H

If the H placed lowermost in the above formula is replaced by
CH2.CH.NH2.COOH we obtain the formula of phenyl-alanine : —

CH2.CHNH0COOH

the remainder of the benzene ring which is unaltered being repre-
sented, as usual, by a simple hexagon.

D

34 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Tyrosine is a little more complicated : it is oxyphenyl-alanine :
that is, instead of phenyl (CeH.^) in the formula of phenyl-alanine we
have now oxyphenyl (CgHj.OH). This gives us C2H3.(OoH4.0H)
NH2.COOH as the formula for tyrosine written one way, or

OH

CH2.CH.NH2.COOH

when written in the other way.

Tryptophane is more complex still ; it is indole-amino-propionic
acid : that is, amino-propionic acid united to another ringed derivative
called indole. Tryptophane is the portion of the protein molecule
which is the parent substance of two evil-smelling products of protein

decomposition called indole CGHiZ-^r-rr"

and scatole or methyl indole. Tryptophane is also the radical in the
protein molecule which is responsible for the colour test called the
Adamkiewicz reaction.

We may sum up what we have learnt up to this point by enume-
rating the principal members of these three groups of amino-
acids : —

1. The mono-amino-acids : glycine, alanine, leucine, amino-
valeric acid, asparagine, aspartic acid, glutamic acid.

2. The di-amino -acids : lysine, ornithine, arginine, and histidine.

3. The ringed amino -acids : phenyl-alanine, tyrosine, and
tryptophane.

But this does not bring us to the end of the list of the cleavage
products of proteins, for we have still left several other groups most
of which are still more complex, and which we will therefore be
content with merely mentioning : namely —

4. Pyrimidine bases such as uracil, thymine and cytosine.

5. Pyrrolidine derivatives.

6. Cystine, a complex amino- acid in which sulphur is present, and
in which the greater part of the sulphur of the protein molecule is
combined.

7. Ammonia.

Our list now represents the principal groups of chemical nuclei
united together in the protein molecule, and its length makes one

THE PROTEINS

35

realise the complicated nature of that molecule and the difficulties
which beset its investigation.

The workers in Fischer's laboratory are steadily working through
the various known proteins, taking them to pieces, and identifying
and estimating the fragments. I do not intend to burden the readers
of this book with anything more than a sample of their results, and
will therefore only give in a brief table the results obtained with
some of the cleavage products of a few proteins. The numbers
given are percentages.

Edes.

irl

Egg-al-
bumin

Serum
globu-
lin

Casei-
nogen

Gelatin

Keratin
from horse-
hair

tin, a
globu-
lin from
hemp

Zein
from
maize

Glia.

din

from

wheat

1 Glycine .

0

0

8-5

0

16-5

seed

0-9

0-8

3.8

+

' Leucine .

20-0

6-1

18-7

10-5

2-1

7-1

15-5

11-2

6-0

Glutamic acid .

7-7

8-0

8-5

11-0

0-8

3-7

17-2

11-8

31-5

Tyrosine .

2-1

1-1

2-5

4-5

0

3-2

2-1

10-1

2-4

Arginine .

4-8

7-6

11.7

1-8

2-75

Tryptophane .

+

-f

+

1-5

0

more

-f

! Cystine .

2-3

0-2

0*7

0-06

than 10

0-2

Such numbers of course are not to be committed to memory, but
they are sufficient to convey to the reader the differences between the
proteins. There are several blanks left on account of no accurate
estimations having yet been made. Where the sign -f occurs, the
substance in question has been proved to be present, but not yet
determined quantitatively. Among the more striking points brought
out are : —

1. The absence of- glycine from albumins.

2. The high percentage of glycine in gelatin.

3. The absence of tyrosine and tryptophane in gelatin.

4. The high percentage of the sulphur containing substance
(cystin) in keratin.

5. The high percentage of glutamic acid in vegetable proteins.
Emil Fischer in his work has sought to make such a list complete,

and month by month the details are being filled in. He has next
tried to discover the way in which the amino-acids are linked to-
gether into groups ; and the culmination of his work will be the
discovery of the way in which such groups are linked together to
form the protein molecule. The last stage he has not yet reached,
but it will be interesting to see what progress he has -made in
ascertaining how the amino-acids are linked together into groups.

D 2

36 ESSENTIALS OF CHEMICAL PHYSIOLOGY

These groups he terms peptides or polypeptides : many of these
have been made synthetically in his laboratory, and so the synthesis
of the protein molecule is foreshadowed.

We may take as our examples of these peptides some of the
simplest, and may write the formulae of a few amino-acids as

follows : —

NH2.CH2.COOH glycine

NH2.CH4.COOH alanine

NH2.C5H10.COOH leucine

or in general terms

HNH.E.COOH

Two amino-acids are linked together as shown in the following
formula : —

HNH.K.CO OH.H NH.R.COOH

What happens is that thehydroxyl (OH) of the carboxyl (COOH)
group of one acid unites with one atom of the hydrogen of the next
amino (HNH) group, and water is thus formed, as shown within the
dotted lines : this is eliminated and the rest of the chain closes up.
In this way we get a dipeptide. The names glycyl, alanyl, leucyl,
&c. are given by Fischer to the NH2.R.CO groups which replace
the hydrogen of the next NH2 group. Thus glycyl-glycine,
glycyl-leucine, leucyl-alanine, alanyl-leucine, and numerous
other combinations and permutations are obtained. If the same
operation is repeated we obtain tripeptides (leucyl- glycyl-alanine.
alanyl-leucyl-tyrosine &c.) ; then come the tetrapeptides and so on.
In the end, by coupling the chains sufficiently often and in appro-
priate order, Fischer has already obtained substances which give some
of the reactions of peptones.

TESTS FOR PROTEINS

Solubilities. — The proteins as a class are insoluble in alcohol and
in ether. Some are soluble in water, others insoluble. Many of the
latter are soluble in weak saline solutions. Some are soluble, others
insoluble in concentrated saUne solutions. It is very largely on
these varying solubilities that proteins are separated into classes,
and from each other.

When, however, one speaks of the solution of a protein, the kind
of solution obtained is usually what physical chemists call colloidal
solution : the condition here is something intermediate between true
solution and a suspension. Many of the properties of proteins, such

THE PEOTEINS

37

as the tendency to coagulate or solidify and the readiness with
which they are ' salted out,' are shared in common with other colloids
some of which aie of inorganic nature.

All proteins are soluble with the aid of heat in concentrated
mineral acids and alkalis. Such treatment, however, decomposes as
well as dissolves the protein. Proteins are also soluble in gastric and
pancreatic juices ; but there, again, they undergo a change, being
converted by hydrolysis into proteins of smaller molecular
weight called peptones. The intermediate substances formed in this
process are called proteoses. Commercial peptone contains a mixture
of proteoses and true peptone.

Heat Coagulation. — Many of the proteins which are soluble in
water or saline solution are rendered insoluble when those solutions

Fig. 7.--Dialyser. The lower opening of the bell jar
suspended in water is tightly covered with parchment
paper. The fluid to be dialysed is placed within this
vessel; the crystalloids pass out into the distilled
water outside through the parchment paper.

Fig, 8. — In this form of dialyser
the substance to be dialysed
is placed within the piece
of tubing suspended in the
larger vessel of water. The
tubing is made of parchment
paper.

are heated. This is true for most of the proteins 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-
teins : thus myosinogen and fibrinogen coagulate at about 56° C. ;
serum albumin and serum globulin at about 75° C.

The proteins which are coagulated by heating their solution come
for the most part into two classes — the alhimiins and the globulins.
The full distinction between these we shall see immediately. We
may, however, state here that the albumins are soluble in distilled

38 ESSENTIALS OF CHEMICAL PHYSIOLOGY

water : the true globulins are not, but require salts to hold them in
solution.

Indiffusibility. — The proteins (peptones excepted) belong to the
class of substances called colloids by Thomas Graham : 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).

Proteins 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 proteins, albumin and globulin,
remain inside. The globulin is, however, precipitated, as the salts
which previously kept it in solution have been removed.

The terms ' diffusion ' and ' 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 albimiin. The
phenomenon is called diffusion. If the solutions are separated by a mem-
brane the term ' dialysis ' is employed. 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 more fully article Osmosis in
Appendix.

Crystallisation. — Haemoglobin, the red pigment of the blood, is a
protein substance, and is crystallisable (for further details, see The
Blood). Like other proteins, it has an enormously large molecule ;
though crystalline, it is not crystalloid in Graham's sense of
that term, although it probably forms a true solution with water.
Blood pigment is not the only crystallisable protein. Long ago
crystals of protein (globulin or vitellin) were observed in the aleurone
grains of many seeds, and in the similar protein occurring in
the egg-yolk of some fishes and amphibians. By appropriate
methods these have been separated and recrystallised. Further,
egg-albumin itself has been crystallised. If a 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

THE PROTEINS 39

sulphuric acid is added (Hopkins). Serum albumin (from come
animals) has also been similarly crystallised (Giirber).

Action on Polarised Light. — All the proteins are levo-rotatory,
but the amount of rotation the} produce varies with the kind of
protein. See Appendix. Several of the compound proteins (for
instance, haemoglobin and nucleo-proteins) are dextro-rotatory,
though their protein components are levo-rotatory (Gam gee).

Colour Reactions. — The principal colour reactions have been
already described in the heading of this lesson.

(1) The xantho-proteic reaction depends on the conversion of the
aromatic group of the protein molecule into nitro-derivatives.

(2) Millon's reaction is due to the presence of the tyrosine group,
and is given by all benzene derivatives which contain a hydroxyl
group (OH) replacing hydrogen.

(3) The formaldehyde reaction (and the Adamkiewicz reaction,
which is probably the same thing) is due to the presence of the trypto-
phan radical (indole-amino-propionic acid).

The presence, absence, or intensity of these colour tests in various
proteins depends respectively on the presence, absence, or amount of
the groups to which they are due.

(4) In the copper sulphate test the proteoses and peptones
behave differently from the native proteins ; the latter give a violet
and the former a rose-red colour, which is called the biuret reaction,
because the same tint is also given by the substance called biuret.^
The name does not imply that biuret is present in protein, but both
biuret and protein give the reaction because they possess the same
group or groups which are probably two GONH2 groups linked either
to a carbon atom, or to a nitrogen a,tom, or directly to one another
(Schiff).

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

Solutions of the proteins are precipitated by —

1. Strong acids, like nitric acid.

2. Picric acid.

3. Acetic acid and potassium ferrocyanide.

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

' Biuret is obtained by heating solid urea ; ammonia is given off and leaves
biuret thus : —

2C0N,H,= CANgHs +NH3

[urea] [biuret] [ammouia]

40 ESSENTIALS OF CHEMICAL PHYSIOLOaY

5. Salts of the heavy metals, like copper sulphate, mercuric
chloride, lead acetate, silver nitrate, &c.

6. Tannin.

7. Alcohol.

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

It is necessary that the words coagulation and precipitation should,
in connection with the proteins, be carefully distinguished. The
term coagulation is used when an insoluble protein (coagulated
protein) is formed from a soluble one. This may occur —

1. When the protein 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 an addition of
certain reagents (nitric acid, picric acid, tannin, &c.).

There are, however, other precipitants of proteins in which the
precipitate formed is readily soluble in suitable reagents, like saline
solution, and the protein continues to show its typical reactions.
This precipitation is not coagulation. Such a precipitate is produced
by saturation with ammonium sulphate. Certain proteins, 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 ammoniun sulphate precipitates all
proteins but peptone. The globulins are precipitated by certain salts
like sodium chloride and magnesium sulphate, which do not precipi-
tate the albumins. The precipitation of proteins by salts in this way
is conveniently termed ' salting out.'

The precipitate produced by alcohol is peculiar in that after
a time it becomes a coagulum. Protein 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 ren-
dered insoluble by this method ; proteoses and peptones are appa-
rently never rendered insoluble by the action of alcohol. This fact
is of value in the separation of these proteins from others.

THE PROTEINS 41

CLASSIFICATION OF PROTEINS

The knowledge of the chemistry of the proteins which is slowly
progressing under Emil Fischer's leadership will no doubt in time
enable us to give a classification of the substances on a strictly
chemical basis. But until that time arrives we must be content very
largely with the artificial classification (on the basis of solubility and
so forth) which has hitherto prevailed. The following classification
must therefore be regarded as a provisional one, which, while it retains
the old familiar names as far as possible, yet attempts also to
incorporate some of the new ideas. The classes of proteins, then,
beginning with the simplest, are as follow : —

1. Protamines.

2. Histones.

3. Albumins.

4. Globulins.

5. Sclero-proteins.

6. Phospho-proteins.

7. Conjugated proteins.

(a) Gluco-proteins.

(b) Nucleo-proteins.

(c) Chromo-proteins.
We shall take these classes one by one.

1. The Protamines

These substances are obtainable from the heads of the spermatozoa
of certain fishes, where they occur in combination with nuclein.
Kossel's view that they are the simplest proteins in nature has met
with general acceptance, and they give such typical protein reactions
as the copper sulphate test (Eose's or Piotrowski's reaction). On
hydrolytic decomposition they first yield substances of smaller
molecular weight analogous to the peptones which are called protones,
and then they split up into amino-acids. The number of resulting
amino-acids is small as compared with other proteins ; hence the
hypothesis that they are simple proteins is confirmed. Notable
among their decomposition products are the diamino-acids or hexone
bases, which have the following names and formulae : —

Lysine (OeHnNaOa)

Arginine (C6H.,N402)

Histidine (CgHgNaOs)
The protamines differ in their composition according to their source,
and yield these products in different proportions.

42 ESSENTIALS OF CHEMICAL PHYSIOLOGY

Salmine (from the salmon roe) and clupeine (from the herring roe)
appear to be identical, and have the empirical formula C30H57N17O6 :
its principal decomposition products are arginine, amino- valeric acid,
and a small quantity of an unknown residue. Sturinc (from the
sturgeon) yields the same products v^rith lysine and histidine in
addition. With one exception, the protamines yield .no aromatic
amino-acids, of w^hich tyrosine is a familiar instance ; the exception
is cyclopterine (from Cycloptertos lumpus) ; this substance is thus an
important chemical link between the other protamines, and the more
complex members of the protein family.

2. The Histones

These are substances which have been separated from blood
corpuscles ; glohin, the protein constituent of heemoglobin, is a well-
marked instance. They yield a larger number of amino-compounds
than do the protamines. They are coagulable by heat, soluble in
dilute acids, and precipitable from such solutions by ammonia. The
precipitability by ammonia is a property possessed by no other
protein group.

3. The Albumins

These are typical proteins, and yield the majority of the cleavage
products enumerated on pp. 31-35.

They enter into colloidal solution in water in dilute saline solutions,
and in saturated solutions of sodium chloride and magnesium
sulphate. They are, however, precipitated by saturating their
solutions with ammonium sulphate. Their solutions are coagulated
by heat usually at 70°-73° C. Serum albumin, egg-albumin, and
lact-albumin are instances.

4. The Globulins

The globulins give the same general tests as the albumins : they
are coagulated by heat, but differ from the albumins mainly in their
solubiHties. This difference in solubility may be stated in tabular form
as follows : —

Reagent Albumin

Globulin

Water I soluble insoluble

Dilute saline solution . . . soluble | soluble

Saturated solution of magnesium

sulphate or sodium chloride . ^ soluble insoluble

Half- saturated solution of ammo-
nium sulphate ....

Saturated solution of ammonium

sulphate i insoluble insoluble

soluble insoluble

THE PROTEINS 43

In general terms globulins are more readily salted out than
albumins ; they may therefore be precipitated and thus separated
from the albumins by saturation with such salts as sodium chloride,
or, better, magnesium sulphate, or by half saturation with ammo-
nium sulphate.

The typical globulins are also insoluble in water, and so may be
precipitated by removing the salt which keeps them in solution.
This may be accomplished by dialysis (see p. 38).

Their temperature of heat coagulation varies considerably. The
following are the commoner globulins : — fibrinogen and serum
globulin in blood ; egg-globulin in white of egg, myosinogen in
muscle, and crystallin in the crystalline lens. We must also include
under the same heading certain proteins which are the result of
ferment coagulation on globulins, such as fibrin (see Blood) and
myosin (see Muscle).

The most striking and real distinction between globulins and
albumins is that the latter on hydrolysis yield no glycine, whereas
the globulins do.

5. The Sclero-proteins

These substances form a heterogeneous group of substances,
which are frequently termed albu7ninoids. The prefix sclero- indicates
the skeletal origin and often insoluble nature of the members of the
group. The principal proteins under this head 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. On digestion it is like ordinary
proteins converted into peptone-like substances and is readily

' 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, lacunae, and canaliculi,
and other proteins and nuclein from the bone corpuscles. There is also a small
quantity of fat even after removal of all the marrow. Dentine is like bone chemi-
cally, but the proportion of earthy matter is rather greater. E?iamel 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.

44 ESSENTIALS OF CHEMICAL PHYSIOLOGY

absorbed. Though it will replace in diet a certain quantity of such
proteins and thus acts as a * protein-sparing ' food, it cannot
altogether take their place as a food. Animals whose sole nitro-
genous food is gelatin waste rapidly. The reason for this is that
gelatin contains neither the tyrosine nor the tryptophane radicals, and
so it gives neither Millon's nor the Adamkiewicz reaction. Animals
which receive in their food gelatin to which tyrosine and tryptophane
are added thrive well.i

4. Chondrin. — This is the name given to the mixture of gelatin
and mucoid which is obtained by boiling cartilage.

5. 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.

6. 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 most proteins in its high
percentage of sulphur. A similar substance, called neurokeratin,
is found in neuroglia and nerve fibres. In this connection it is
interesting to note that the epidermis and the nervous system
are both formed from the same layer of the embryo — the epiblast.

6. The Phospho-proteins

Vitellin (from egg-yolk), caseinogen, the principal protein of milk
and casein, the result of the action of the rennet-ferment on
caseinogen (see Milk), are the principal members of this group. Among
their decomposition products is a considerable quantity of phosphoric
acid. They have been frequently confused with the nucleo-proteins
we shall be studying immediately, and the prefix nucleo- so often
applied to them is entirely misleading, since they do not yield the
products (purine bases) which are characteristic of nucleo-compounds.

7. The Conjugated Proteins

These very complex substances are compounds in which the
protein molecule is united to other organic materials, which are, as
a rule, also of complex nature. This second constituent of the
compound is usually termed a prosthetic group. They may be
divided into the following sub-classes.

i. Chromo-proteins. — These are compounds of proteins with a
pigment, which usually contains iron. They are typified by
haemoglobin and its allies, which will be fully considered under Blood

THE PKOTEINS 45

ii. Gluco-proteins. — These are compounds of protein with a carbo-
hydrate group. This class includes the mucins and the mucoids.

The mucins are widely distributed and may occur in epithelial
cells, or be shed out by these cells (mucus, mucous glands, goblet
cells). The mucin obtained from different sources varies in com-
position and reactions, but they all agree in the following points : —

(a) Physical character. Viscid and tenacious.

(b) They are soluble in dilute alkalis, such as lime water, and are
precipitable from solution by acetic acid.

The mucoids generally resemble the mucins, but differ from them
in minor details. The term is applied to the mucin-like substances
which form the chief constituent of the ground substance of con-
nective tissues (tendo-mucoid, chondro-mucoid, &c.). Another, ovo-
mucoid is found in white of egg, and others (pseudo- mucin and
para-mucin) are occasionally found in dropsical effusions, and in the
fluid of ovarian cysts.

It is probable that the differences between the mucins and the
mucoids are due either to the nature of the carbohydrate group or,
more probably, to the nature of the protein to which it is united.
The carbohydrate substance in the majority of cases is not sugar,
but a nitrogenous substance which has a similar reducing power to
sugar, and which is called glucosamine (CgH, 1O5NH2) .' that is, glucose
in which HO is replaced by NHg.

Pavy and others have shown that a small quantity of the same
carbohydrate derivative can be split off from various other proteins
which we have already placed among the
albumins and globulins. It is, however,
probable that this must not be considered
a prosthetic group, but irS more intimately
united within the protein molecule.

iii. Nucleo-proteins. These are com-
pounds of protein with a complex organic
acid called nucleic acid which contains
phosphorus. They are found both in
the nuclei and cell-protoplasm of cells. piasm iompSed of%on|i'op]iasm
In physical characters they often simu- fatraSff n'?t^vvork'o?X^l^-

lofn mnm'n *^° °'' ^"cleiu ; and «', nucleolus

late mucin. (ScMfer.)

Nuclein is the name given to the
chief constituent of cell-nuclei. It is identical with the chromatin of
histologists (see fig. 9).

On decomposition it 'yields an organic acid called nucleic acid
together with a variable but usually small amount of- protein. It

46 ESSENTIALS OF CHEMICAL PHYSIOLOGY

contains a high percentage (10-11) of phosphorus. The nuclein
obtained from the nuclei or heads of the spermatozoa consists of
nucleic acid without any protein admixture. In fishes' spermatozoa,
however, there is an exception to this rule, for there it is, as we have
ah'eady seen, united to protamine.

The nucleo-proteins of cell protoplasm are compounds of nucleic
acid with a much larger quantity of protein, so that they usually
contain only 1 per cent, or less of phosphorus. Some also contain
iron, and it is probable that the normal supply of iron to the body is
contained in the nucleo-proteins or hcematogens (Bunge) of plant or
animal cells.

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

1. Wooldridges method. The organ is minced and soaked in
water for twenty-four hours. Dilute acetic acid added to the aqueous
extract precipitates the nucleo-protein.

2. Sodimn chloride method. The minced organ is ground up in a
mortar with solid sodium chloride; the resulting viscous mass is
poured into excess of water, and the nucleo-protein rises in strings to
the top of the water.

The solvent usually employed for a nucleo-protein, by whichever
method it is prepared, is a 1-per-cent. solution of sodium carbonate.
The relationship of nucleo-proteins to the coagulation of the blood is
described under that heading.

Nucleic acid on decomposition yields phosphoric acid, various
bases of the xanthine group, and bases also of the pyrimidine group
(cytosine, uracil, &c.). In some cases a carbohydrate radical is also
obtained ; thus a pentose is obtained from the nucleic acid of the
pancreas, the liver, and yeast cells. There appear to be several
nucleic acids, which vary in the relative amount they yield of their
decomposition products, especially of the members of the xanthine
family, which are sometimes called alloxuric, or, more usually, _2?2mw-e
bases. The purine bases are closely allied chemically to uric acid,
and we shall have to study them again in relation to that substance.
The following diagrammatic way of representing the decomposition
of nucleo-protein will assist the student in remembering the relation-
ships of these substances : —

THE PROTEINS

47

NUCLEO-PROTEIN

subjected to gastric digestion yields

Protein converted into
peptone, which goes
into solution.

Protein converted into
acid albumin in solu-
tion.

Nuclein, which remains as an insoluble
residue. If this is dissolved in alkali
and then hydrochloric acid added it
yields

I

I. .

A precipitate consisting of nucleic acid.
If this is heated in a sealed tube with
hydrochloric acid, it yields a number
of substances. But the best known
and constant products of its decom-
position are

Phosphoric acid.

I

Purine bases, viz.
Adenine
Hypoxanthine
Guanine
Xanthine.

I

Pyrimidine
Uracil

Cytosine
Thymine.

, VIZ.

Protein-hydrolysis

When protein material is subjected to hydrolysis, as it is when
heated with mineral acid, or superheated steam, or to the action of
such ferments as pepsin or trypsin in the alimentary canal, it is
finally resolved into the numerous amino-acids of which it is built.
But before this ultimate stage is reached, it is split into substances of
progressively diminishing molecular size, which still retain many of
the protein characters. These may be classified in order of formation
as follows : —

1. Infra-proteins.

2. Proteoses.

3. Peptones.

4. Polypeptides.

The polypeptides are linkages of two or more amino-acids as already
explained. They do not give the biuret reaction. Although most
of the polypeptides at present known are products of laboratory
synthesis, some have been definitely separated from the digestion of

48 ESSENTIALS OF CHEMICAL PHYSIOLOGY

proteins, and so they must appear in our classification. The proteoses
and peptones give the biuret reaction ; the peptones, however, cannot
be salted out of solution like the proteoses ; their molecules are
smaller than those of the proteoses. We shall study them more
fully under digestion. Tt is, however, necessary to add here a brief
description of the infra-proteins, since some of the practical exercises
at the head of this lesson deal. with them.

They are obtained as the first stage of hydrolysis, and also by
the action of dilute acids or alkalis on either albumins or globulins.
The general properties of the acid-albumin or syntonin and the alkali-
alhumin, which are thereby respectively formed are as follows : —
they are insoluble in pure water, but are soluble in either acid or
alkali, and are precipitated by neutralisation unless certain disturbing
influences like sodium phosphate are present. They are precipitated
as globulins are by saturation with such neutral salts as sodium
chloride or magnesium sulphate. They are not coagulated by heat
if in solution. In alkali-albumin some of the sulphur in the original
protein is removed.

The name albuminate used to be applied to these substances ; but this is
an objectionable term, for these first degradation products of protein hydro-
lysis are not salts, as the termination -ate would imply. Moreover, they are
obtainable from both albumins and globulins. The prefix ' infra- " (or pos-
sibly ' meta-, which some prefer) may be taken as an indication of compara-
tively slight chemical alteration.

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 Lieberhiihn'' s jelly. A similar •
jelly is obtainable by adding strong acetic acid to undiluted egg-white.

The word ' albuminate ' is also used for compounds of protein with mineral
substances. Thus if a solution of copper sulphate is added to a solution of
albumin a precipitate of copper albuminate is formed. Similarly, by the
addition of other salts of the heavy metals, other metallic albuminates are
obtainable. The halogens (chlorine, bromine, iodine) also form albuminates
in this sense, and may be used for the precipitation of proteins.

It should be noted, in conclusion, that the foregoing classification of
proteins is mainly applicable to those of animal origin. The vegetable pro-
teins may roughly be arranged under the same main headings, although it is
doubtful if a real and complete analogy exists in all cases. The cleavage
products of the vegetable proteins are in the main the same as those of the
animal proteins, but the quantity of each yielded is usually different.
Vegetable proteins, for instance, as a rule give a very much higher yield of
glutamic acid than do those of animal origin.

Further, there are certain vegetable proteins which have hitherto been
regarded as peptones, but which do not give the biuret reaction. It seems
impossible at present to bring exceptional substances of this kind into any
general classification, and the same is true for those curious vegetable pro-
teins, such as gliadin from the gluten of wheat, and zein from maize, which
stand apart from all other members of the group in being soluble in alcohol.

LESSON VI
FOODS

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

2. Note the specific gravity of fresh milk with the lactometer ; compare
this with the specific gravity of milk from which the cream has been removed
(skimmed milk). The specific gravity of skimmed milk is higher owing to
the removal of the lightest constituent — the cream.

3. The reaction of fresh milk is neutral or slightly alkaline to litmus.

4. Warm some milk in a test-tube to the temperature of the body, and
add a few drops of rennet. After standing, a curd is formed from the con-
version of caseinogen, the chief protein in milk, into casein. The casein
entangles the fat globules. The liquid residue is iexmediwhey. No curdling
is produced if the rennet solution is previously • boiled, because heat kilfs
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 entanghng
the fat is formed.

7. Filter off this precipitate, and in the filtrate test for lactose or milh
sugar by Trommer's test (see Lesson II.) ; for lact-albumin by boiling, or by
Millon's reagent (see Lesson IV.) ; and for earthy (that is, calcium and
magnesium) ;pliosphates 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. Shake up a little milk with twice its volume of ether ; the opacity of
the milk remains nearly as great as before. Repeat this, but first add to the
milk a few drops of caustic potash before adding the ether and then shake.
The milk which lies beneath the ethereal solution of fat becomes trans-
lucent. As a matter of fact ether dissolves the fat without the addition of
alkali, and the opacity of milk is therefore not due to the fat globules alone,
but largely to their caseinogen envelope. The clearing which takes place
when ether and alkali are added is due to an action of the reagents on the
caseinogen.

10. 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

60 ESSENTIALS OF CHEMICAL PHYSIOLOGY

by heat. The precipitate produced by saturation with salt floats to the
surface with the entangled fat, and the clear salted whey is seen below after
an hour or two.

B. Flour. — Mix some wheat flour with a little water into a stiff dough.
Wrap this up in a piece of muslin and knead it under a tap or in a capsule of
water. The starch grains come through the holes in the muslin (identify by
iodine test), and an elastic sticky mass remains behind. This is a protein
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
colour.

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 breadcruat
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 protein food. Cut up some lean
meat into fine shreds and grind these up with salt solution. Filter and test
for proteins.

THE PRINCIPAL FOOD-STUFFS

We can nov^ proceed to apply the knowledge we have obtained of
the proteins, carbohydrates, and fats to the investigation of some
important foods. The chief proximate principles in food are : —

1. Proteins \

2. Carbohydrates h organic.

3. Fats j

4. Water ).

5. Salts [inorgamc.

In milk and in eggs, which form the exclusive foods of young
animals, all varieties of these proximate principles are present in
suitable proportions. Hence they are spoken of as perfect foods.
Eggs, though a perfect food for the developing bird, contain too
little carbohydrate for a mammal. In most vegetable foods carbo-
hydrates are in excess, while in animal food, like meat, the proteins.
Are predominant. In a suitable diet these should be mixed in
proper proportions, which must vary for herbivorous and carnivorous
animals. We must, however, limit ourselves to the omnivorous
animal, man.

A healthy and suitable diet must possess the following cha-
racters : —

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.

FOODS 51

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 protein than beef and mutton, but are not so nutritious, as
they are less digestible, much passing off in the faeces unused.

The nutritive value of a diet depends mainly on the amount of
carbon and nitrogen it contains in a readily digestible form. A man
doing a moderate amount of work, and taking an ordinary diet,
will eliminate, chiefly from the 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 food, and from the metabolism of the tissues ;
various forms of energy, work and heat being the chief, are simul-
taneously 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 to 1.
The proportion of carbon to nitrogen in protein is, however, 53 to 15,
or 3*5 to 1. The extra supply of carbon comes from non-nitrogenous
foods — viz. fat and carbohydrate.

Voit gives the following daily diet : —

Protein 118 grammes.

Fat 100

Carbohydrate 333

Ranke's diet closely resembles Voit's ; it is —

Protein 100 grammes.

Fat 100

Carbohydrate 250 „

In preparing diet tables, such adequate diets as those just given
should be borne in mind. The following dietary (from G. N. 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.

e2

52

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Food-staflE

Quantity

Grammes of

Nitro-
gen

Car-
bon

Pro-
teins

Fat.

Carbo-
hydrates

Salts

1

Metric

Englisli

1

1

system

weiglits

Lean meat

250 grammes

. 9 oz.

8

33

55

8-5

0

4

Bread .

600

18 „

6

112

40

7-5

245

6-5

Milk

500

f pint

3

35

20

20

25

3-5

Butter .

30

1 oz.

0

20

0

27

0

0-5

Fat with

meat .

30

1 „

0

22

0

30

0

0

Potatoes .

450

16 „

1-5

47

10

0

95

4-5

Oatmeal .

75 „

3 „

1-7

30

10

4

48
413

2
21

20-2

299

135

97

Eecent research has shown that, for a certain time at any rate, a
man will maintain his w^eight and health on diets even scantier than
those of Voit and Ranke. The most convincing of these experiments
have been performed by Chittenden upon himself and others.
Chittenden m^ges that the normal diet should contain only about
half the customary quantity of protein. The body certainly does
not assimilate the larger amount usually taken, for the greater part
of the nitrogenous constituents is converted into amino-acids, which
are rapidly transformed by the liver into urea and cast out of the
body, leaving the non-nitrogenous remainder to be utilised as fats
and carbohydrates are, for the production of heat and energy.
"Whether Chittenden's views will meet with general acceptance is at
present doubtful, although his work will bring home to many people
that temperance is necessary in food as well as in drink. The
majority of well-to-do people cerbainly eat an excess of meat, and so
throw an unnecessary strain upon their digestive and excretory
organs. One should hesitate, however, in accepting Chittenden's
conclusions to the full, for it is doubtful if the ^ninimum is also the
optimum diet. It may be that there is a real need for an excess of
protein beyond the apparent minimum. In diamond mining a large
quantity of earth must be crushed to obtain the precious stones. It
may be that among the many cleavage products of protein the
majority may be compared to this waste earth, and we get rid of
them as quickly as possible in the excretions, but some few may be
unusually precious for protein synthesis in the body, and that, in
order to get an adequate supply of these, a comparatively large
amount of protein must be ingested.

FOODS

53

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 protein and fat. It also contains too little
iron (Bunge) ; lience children weaned late become anaemic.

The microscope reveals that it consists of two parts : a clear fluid
and a number of minute particles that float in it. These consist of
minute oil globules, varymg in diameter from 00015 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

o

OoO
O "

AOqO OAT" oP^if^ o'O 9^o V o^Qn » Uo

0

"qoco

00

^ ^o o -O

Fm. 10. — Microscopic appearance of milk in the
early stage of lactation, showing colostrum
corpuscles (a) in addition to fat globules. (Yeo.)

Pig. 11. — a, b, colostrum
corpuscles with fine
and coarse fat globules
respectively ; c, d, e,
pale cells devoid of
fat. (Heidenhain.)

of globuhn instead. Microscopically, cells from the acini of the
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 amphoteric. This is due to the presence of
both acid and alkaline salts ; the latter are usually in excess. Milk
readily turns acid or sour as the result of fermentative change, part
of its lactose being transformed into lactic acid (see p. 19). The
specific gravity of milk is usually ascertained with the hydrometer.
That of normal cow's milk varies from 1,028 to 1,034. When the milk
is skimmed the specific gravity rises, owing to the removal of the
Hght constituent, the fat, to 1,033 to 1,037. In all cases the specific

54 ESSENTIALS OF CHEMICAL PHYSIOLOGY

gravity of water, with which other substances are compared, is taken
as 1,000.

Composition. — Bunge gives the following table, contrasting the
milk of woman and cow ; —

-

Woman

Cow

Proteins (chiefly caseinogen) .

Butter (fat)

Lactose

Salts

Per cent.

1-7
3-4
6-2
0-2

■

Per cent.

3-5
3-7
4-9
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 Proteins of Milk. — The principal protein in milk is called
caseinoge7i : this is the one which is coagulated by rennet to form
casein. Cheese consists of casein with the entangled fat. The other
protein in milk is an albumin. It is present in small quantities
only ; it differs in some of its properties (specific rotation, coagulation,
temperature, and solubilities) from serum-albumin ; it is called lact-
alhumin.

The Coagulation of Milk. — Eennet is the agent usually employed
for this purpose : it is a ferment secreted by the stomach, especially
by sucking animals, and is generally obtained from the calf.

The curd consists of the casein and entangled fat : the liquid
residue called whey contains the sugar, salts, and albumin of the
milk. There is also a small quantity of a new protein called whey-
inotein, which differs from caseinogen by not being convertible into
casein. It is produced by the decomposition of the caseinogen
molecule during the process of curdling.

The curd formed in human milk is more finely divided than that
in cow's milk : hence it is more digestible. In feeding children and
invalids on cow's milk, the lumpy condition of the curd may be
obviated by the addition of lime water or barley water to the milk.

Considerable discussion has taken place as to whether the
caseinogen of human milk may not be a different protein from that
of cow's milk, especially in relation to the amount and manner of
combination of its phosphorus. The differences, however, appear to
be explicable on the hypothesis that they are due to variations in the
amounts of calcium salts and of citric acid which are present.

Caseinogen itself may be precipitated by acids such as acetic acid,

FOODS 55

or by saturation with neutral salts like sodium chloride. This, how-
ever, is not coagulation, but precipitation. The precipitate may be
collected and dissolved in lime water; the addition of rennet then
produces coagulation in this solution, provided that a sufficient
amount of calcium salts is present.

In milk also, rennet produces coagulation, 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 w^ay,
namely, in the production of fibrin-ferment (see Coagulation of
Blood).

Caseinogen is not coagulable by heat. We have already classed
it with vitellin as a phospho-protein (see p. 44).

Caseinogen, as was originally pointed out by Hammarsten, is a protein
with acid properties : it is quite insoluble in water, but it forms soluble
salts with such metallic bases as potassivim, sodium, and calcium. The
caseinogen as it exists in ^milk is combined with calcium as calcium caseino-
genate. When acetic acid is added to milk,- we therefore get calcium acetate,
and a precipitate of free caseinogen. On ' dissolving ' this caseinogen in an
alkali like soda or potash, we have the formation of sodium caseinogenate or
potassium caseinogenate, as the case may be. The precipitate obtained in
milk by the addition of alcohol, or by ' salting out,' is not free caseinogen,
but calcium caseinogenate. When we add potassium oxalate to milk, we
get the reaction represented in the following equation : — Calcium caseino-
genate + potassium oxalate = calcium oxalate + potassium caseinogenate.
When we add calcium chloride to oxalated milk, the following equation
represents what occurs : — Potassium caseinogenate + calcium chloride = cal-
cium caseinogenate -^ potassium chloride.

Calcium caseinogenate forms an opalescent solution in water, and reacts
with the rennin ferment. The caseinogenates of magnesium, barium, and
strontium have similar characters. The caseinogenates of potassium, sodium,
and ammonium differ from the above by forming a nearly clear solution in
water, and they do not react with the rennin ferment. (W. A. Osborne.)

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 old statement that each fat globule is surrounded
by a membrane of caseinogen is, according to Eamsden's recent
work, correct. Milk also contains small quantities of lecithin, a
phosphorised fat ; of cholesterin, an alcohol which resembles fat in
its solubilities (see Bile) ; and a yellow fatty pigment or lipochrome.

56 ESSENTIALS OF CHEiMICAL PHYSIOLOGY

Milk Sugar or Lactose. — This is a saccharose (CisHaaOn). Its
properties have already been described in Lesson II., p. 19.

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. 19. 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.

Alcoholic Fermentation in Milk.— When yeast is added to milk,
the sugar does not readily undergo the alcoholic fermentation. Other
somewhat similar fungoid growths are, however, able to produce
the change, as in the preparation of koumiss ; the milk sugar
is first inverted, that is, dextrose and galactose are formed from it
(see p. 19), 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.

EGGS

In this country the eggs of hens and ducks are those particularly
selected as foods. The shell is made of calcareous matter, especially
calcium carbonate. The lohite 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 protein in nature.
The proteins are albumin, with smaller quantities of egg-globulin
and ovo-mucoid (p. 45). 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 protein in nature, consisting of the
phospho-protein called vitellin (p. 44). 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 protein
constituents become.

FOODS

57

MEAT

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

Meat is the most concentrated and most easily assimilable of
nitrogenous foods. It is our chief source of nitrogen. Its chief solid
constituent is protein, and the principal protein is myosin. In addi-
tion to the extractives and salts contained in muscle, there is always
a certain percentage of fat, even though all visible adipose tissue is
dissected off. The fat-cells are placed between the muscular fibres,
and the amount of fat so situated varies in different animals. It is
particularly abundant in pork ; hence the indigestibility of this form
of flesh ; the fat prevents the gastric juice from obtaining ready
access to the muscular fibres.

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

Constituents
Water

Ox

Calf

Pig

Horse

Fowl

Pike

76-7

75-6

72-6

74-3

70-8

79-3

Sclids

23-3

24-4

27-4

25-7

29-2

20-7

Proteins, includ-

ing gelatin . .

20-0

19-4

19-9

21-6

22-7

18-8

Fat .

1-5

2-9

6-2

2-5

4-1

0-7

Carbohydrate .

0-6

0-8

0-6

0-6

1-3

0-9

Salts .

1-2

1-3

1-1

1-0

1-1

0-8

The large percentage of water in meat should be particularly
noted ; if a man wished to take his daily quantity of 100 grammes
of protein 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 protein. Whole flour is made from the whole grain
minus the husk, and thus contains, not only the white interior, but
also the harder and browner outer portion of the grain. This outer
region contains a somewhat larger proportion of the proteins of the

58

ESSENTIALS OF CHEMICAL PHYSIOLOGY

grain. Whole flour contains 1 to 2 per cent, more protein than the
best white flour, but it has the disadvantage of being less readily
digested. Brown flour contains a certain amount of bran in addi-
tion ; it is still less digestible, but is useful as a mild laxative, the
insoluble cellulose mechanically irritating the intestinal canal as it
passes along.

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

When mixed with water, wheat flour forms a sticky adhesive
mass called dough. This is due to the formation of gluten, and the
forms of grain poor in gluten cannot be made into dough (oats, rice,
&c.). Gluten does not exist in the flour as such, but is formed on the
addition of water from the pre-existing soluble proteins {e.g. globulins)
in the flour. It is a mixture of several proteins (gliadin, mucedin,
gluten -fibrin, &c.).

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

Constituents

Wheat

Barley

Oats

Rice

Lentils

Peas

Potatoes

Water

13-6

13-8

12-4

13-1

12-5

14-8

76-0

Protein

12-4

11-1

10-4

7-9

24-8

23-7

2-0

Fat ... .

1-4

2-2

5-2

0-9

1-9

1-G

0-2

Starch

67-9

64-9

57-8

76-5

54-8

49-3

20-6

Cellulose .

2-5

5-3

11-2

0-6

3-6

7-5

0-7

Mineral salts .

1-8

2-7

3-0

1-0

24

3-1

1-0

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.

Protein, except in potatoes, is pretty abundant, and especially so
in the pulses (lentils, peas, &c.). The protein 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 w^heat 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

FOODS 59

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 protein, 55 of carbo-
hydrate, 1 of fat, 2 of salts, and the rest water.

COOKING OF FOOD

The cooking of foods is a development of civilisation, and much
relating to this subject is a matter of education and taste rather than
of physiological necessity. Cooking, however, serves many useful
ends :--

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

2. In the case of vegetable foods it breaks up the starch grains,
bursting the cellulose and allowing the digestive juices to come into
contact with 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 7'igor 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 protein on its exterior, which
keeps in the juices to a great extent, letting little else escape than
the dripping (fat). Whereas in boiling, unless bouillon and
bouilli are used, there is considerable waste. Cooking, especially
boiling, renders the proteins 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 it is usual to

60 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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,
hypoxanthine, lactic acid, &c.) of flesh.

Many invalids restricted to a liquid diet get tired of milk, and
imagine that they get sufficient nutriment by taking beef tea instead.
It is very important that this erroneous idea should be corrected. One
of the greatest difficulties that a physician has to deal with in these
cases is the distaste which many adults evince for milk. It is
essential that this should be obviated as far as possible by preparing
the milk in different ways to avoid monotony. Some can take
koumiss; but a less expensive variation may be introduced in the
shape of junkets, which, although well known in the West of
England, are comparatively unknown in other parts. The preparation
of a junket consists of adding to warm milk in a bowl or dish a small
quantity of rennet (Clark's essence is very good for this purpose) and
flavouring material according to taste. The mixture 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
proteins, 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
gelatinise.

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, cojfee, cocoa, and similar drinks. These are stimulants
chiefly to the nervous system ; tea, coffee, mat6 (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 (C8H,oN402) ; cocoa
to the closely related alkaloid, theobromine (C7H8N4O2) ; coca to
cocaine. These alkaloids are all poisonous, and used in excess, even
in the form of infusions of tea and coffee, produce over-excitment,

FOODS 61

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 protein. In cocoa,
as manufactured for the market, the amount of fat is reduced to
30 per cent., and the amount of protein rises proportionally to about
20 per cent.

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 protein, 2 to 4 carbohydrates, and 1 to 1*5 cellulose
per cent. The small amount of nutriment in most green foods
accounts for the large meals made by and the vast capacity of the
alimentary canal of herbivorous animals.

LESSON VII
THE DIGESTIVE JUICES

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 ceils and mucus, and apply the
xanthoproteic or Millon's test to the filtrate ; the presence of protein 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 reJl 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.

GASTRIC 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 protein 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-
fully.

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 or syntonin 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

' Benger's liquor pepticus may be used instead of the glycerin extract of
stomach.

THE DIGESTIVE JUICES 63

reaction) is produced. This should be carefully compared with the violet
tint given by unaltered albumin.

(c) To a third portion of the fluid in test-tube B add a drop of nitric acid
proteoses 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 similar circumstances.

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 (torulse) belonging to the fungus
group of plants.

The souring of milk, the transformation of ,, ,„ ^„ ,,,

" ' Pig. 12.— Cells of the yeast

urea into ammonium carbonate in decomposing plant in process of bud-

. . -IS fluig> between which

urine, and the formation of vinegar (acetic acid) are some bacteria.
from alcohol are produced by the growth of very
similar organisms. The complex series of changes known as putre-
faction, 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-
perature or by certain substances (carbolic acid, mercuric chloride,
&c.) called antiseptics. The organisms produce fermentative 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 protein in
nature. The existence of poisonous proteins is a very remarkable
thing, as no profound chemical differences have yet been shown to
exist between them and those which are not poisonous, but which are

64

ESSENTIALS OF CHEMICAL PHYSIOLOGY

useful as foods. Snake venom is an instance of a very virulent
poison of protein nature.

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

Fig. 13.— Typical forms of Scbizomycetes (after Zopf ) : a, micrococcus;
terium ; d, bacillus ; e, Clostridium ; /, monas Okeuii ; ff, leptotlirix ; h,
I, spiruliua ; m, spiromonas ; n, spirochsete ; o, cladothrix.

macrococcus ; c, bac-
vibrio ; /fc, spirillum ;

substances, the result of the activity of living cells. The change of
starch into sugar by the ptyalin of the saliva is an instance.
Ferments may therefore be divided into two classes : —

1. The organised ferments — torulaB, bacteria, &c.

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

THE DIGESTIVE JUICES

65

unorganised 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)
into sugars. Examples : ptyalin, diastase, amylopsin.

(b) Proteolytic — those which change native proteins 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.

A

Fig. 14. — Bacillus anthracis, the agent that produces anthrax or splenic fever (iloch) : A, bacilli
mingled with blood corpuscles from the blood of guinea-pig, some of the bacilli dividing ; B, the
same after three hours' culture in a drop of aqueous humour. They grow out into long leptothrix-
]ike filaments, which subsequently divide up, and spores are developed in the segments.

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

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

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

(C6Hio05)7i+?iH20=3nC02 + 3wCH4

[cellulose]

[water]

[carbonic
acid]

[methane]

2. Inversion of cane sugar by the unorganised ferment invertin : —

Ci2H220]i,+H20 = G6H,206 + C6Hi206

[cane sugar] [water] [dextrose] [levulose]

66 ESSENTIALS OF CHEMICAL PHYSIOLOGY

It appears also that some enzymes are oxygen carriers and pro-
duce oxidation : they are termed oxidases.

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,
cresol, &c., produced by putrefactive organisms from proteins, 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, converts 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. ' Eeversible
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
activity is stopped, but the ferments are not destroyed, by cold ; it is
stopped and the ferments killed by 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
anaerobic, in contradistinction to those that require oxygen, and
which are therefore called aerobic.

The chemical nature of the enzymes is very difficult to investigate ;
they are substances that to a great extent elude the grasp of the
chemist. So far research has taught us that they are either protein
in nature or are substances closely allied to the proteins.

The distinction between organised ferments and enzymes is, how-
ever, more apparent than real ; for the micro-organisms exert their
action by enzymes that they secrete. This has long been known
in connection with the invertin of yeast and with the enzyme
secreted by the micrococcus ureae which converts urea into ammonium
carbonate. In recent years Buchner by crushing yeast cells succeeded
in obtaining from them an enzyme which produces the alcoholic
fermentation, and there is now no doubt that what is true for yeast
is true for all the organised ferments ; in several cases this has
already been proved experimentally.

The view at present current regarding ferment action is that they

THE DIGESTIVE JUICES 67

are catalysing agents. That is to say, their presence induces a
chemical reaction to occur rapidly which in their absence also occurs,
but so slowly that any action at all is difficult to discover. To use
the technical phrase, their action is to increase the velocity of chemical
reactions. The enzymes are catalysts of a colloidal nature, and
certain properties, in which they differ from most inorganic catalysts
are due to this circumstance.

THE SALIVA

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 suppHes 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 having
been shed out to contribute to the secretion (see fig. 15).

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. 16).

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. 17).

COMPOSITION OF SALIVA

On microscopic examination of mixed saliva a few epithelial
scales from the mouth and salivary corpuscles from the tonsils
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 soHds (2*75 per cent.).

f2

68 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 sohds 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 classified
thus : —

a. Mucin : this may be precipitated by acetic acid.

h. Ptyalin : an amylolytic ferment.

c. Protein : of the nature of a globulin.

d. Potassium sulphocyanide.

e. Sodium chloride : the most abundant salt.
/. Other salts : sodium carbonate ; calcium phosphate

and carbonate ; magnesium phosphate ; potassium
chloride.

Organic

Inorganic

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

The chemical action of saliva is due to its active principle,
ptyalin. This substance belongs to the class of unorganised ferments
or e7izymes, 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).

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 achroo-dextrin, which gives no
colour with iodine. Brown and Morris give the following equa-
tion >

10(C6H,o05). + ^nB.,0

[starch] . [water]

=47iCi2H220n + {Ce^ioO,)n + (CeH.oOs),,

[maltose] [achroo-dextriil] [erythro-dextrin]

Ptyalin acts in a similar way, but more slowly on glycogen : it

THE DIGESTIVE JUICES

69

has no action on cellulose ; hence it is inoperative on uncooked
starch grains, for in these the cellulose layers are intact.

Fig. 15. — Alveoli of serous gland ; A, loaded befoi'e secretion ; B, after a short period of active
secretion ; C, after a prolonged period. (Laugley.)

Fig. 16.— Mucous cells from a fresli submaxillary gland of dog : a, loaded with muciuogen granules
before secretion ; 6, after secretion : the granules are fewer, especially at the attached border of
the cell ; a' and 5' 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
mass of mucin traversed by a network of protoplasmic cell-substance. (Foster, after Langley.)

Fig. 17. — 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.

Ptyalin acts best about the temperature of the body (35-40°),
and in a neutral medium ; a small amount of alkali makes but

70 ESSENTIALS OF CHEMICAL PHYSIOLOaY

little difference ; a very small amount of acid stops its activity.
The conversion of starch into sugar by saliva in the stomach
continues for a considerable time, for the swallowed masses which
fall into the fundus of the stomach are not subjected to peristalsis
and admixture with gastric juice until a later stage in digestion ;
the hydrochloric acid which is poured out by the gastric glands j5rst
neutralises the saliva and combines with the proteins 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.

f THE SECRETION OF GASTRIC JUICE

The nice secreted by the glands in the mucous membrane of the
stomach varies in composition in the different regions, but the mixed
juice is a solution of a proteolytic ferment called pepsin in a saline
solution, which also contains a little free hydrochloric acid.

The gastric juice can be obtained during the life of an animal by
means of a gastric fistula. Gastric fistulas 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 v^ound in the abdomen in 1822.
Observations made on him by Dr. Beaumont formed the startmg-
point for our correct knowledge of the physiology of the stomach
and its secretion.

We now make artificial gastric juice by mixing weak hydrochloric
acid (0*2 to 0*4 per cent.) with a glycerin or aqueous extract of the
stomach of a recently killed animal. This acts like the normal juice.

Three kinds of glands are distinguished in the stomach, w^hich
differ from each other in their position, in the character of their
epithelium, and in their secretion. The cardiac glands are simple
tubular glands quite close to the cardiac orifice. The fundus glands
are those situated in the remainder of 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 corresponding cells in
the pyloric glands. They are called i)rinciijal or central cells.
Between them and the basement membrane of the tubule are other
cells which stain readily with aniline dyes. They are called imrietal
or oxyntic {i.e. acid-forming) cells. The pyloric glands, in the pyloric
half of the stomach, have long ducts and short tubules lined with
cubical cells. There are no parietal cells.

THE DIGESTIVE JUICES

71

Fig. 19. — A pyloric glaud from a
section of the dog's stomach
(Ebstein) : m, month ; n, neck ;
tr, a deep portion of tubule cut
transversely.

Fig. 18.— a fundus glund from the dog's stomach (Klein) : d, duct or mouth of the gland ; b, base
of one of its tubules ; on tlie right the base of a tubule is more highly magnified ; c, central
cell ; p, parietal cell.

72

ESSENTIALS OF CHEMICAL PHYSIOLOaY

The central cells of the fundus glands and the cells of the pyloric
glands are loaded with granules. During secretion they discharge
their granules, those that remain being chiefly situated near the
lumen, leaving in each cell a clear outer zone (see fig. 20). These
are the cells that secrete the pepsin. Like secreting cells generally,
they select certain materials from the lymph that bathes them :
these materials are worked up by the protoplasmic activity of the

cells into the secretion, which is then dis-
charged 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 precursors. The
zymogen in the gastric cells is called pep-
sinogen. The rennet-ferment or rennin that
causes the curdling of milk is distinct from
pepsin,* and is preceded by another zymo-
gen ; it is, however, formed by the same
cells.

The parietal cells are also called oxyntic
cells, because 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 chemical theories advanced as to how
this is done, Maly's is the most satisfactory. He considers that the

' The individuality of rennin has been questioned by Pawlow, who regards its
action as a phase of pepsin activity.

Fig. 20.— a fundus gland of simple
form from the bat's stomach.
Osmic acid preparation (Lang-
ley) : c, columnar epithelium
of the surface ; n, neck of the
gland, with central and parietal
cells ; /, base occupied only
by principal or central cells,
which exhibit the granules
accumulated towards the lumen
of the gland.

THE DIGESTIVE JUICES

73

acid originates by the interaction of sodium chloride and sodium
dihydrogen phosphate, as is shown in the following equation : —

NaH2P04 + NaCl=Na2HP04 + HCl

[sodium di-

[sodium

[disodium

[hydro-

hydrogeu

chloride]

hydrogen

chloric

phosphate]

phosphate]

acid]

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

NasHPO^ + CO2 + H20=NaHC03 + NaH2P04.

Other theories have tried to explain the formation of such a strong
acid as hydrochloric by the law of * mass action.' We know that by
the action of large quantities of carbonic acid on salts of the mineral
acids the latter may be liberated in small quantities. We know,
further, that small quantities of acid ions may be continually formed
in the organism by ionisation. But in every case we can only make
use of these explanations if we assume that the small quantities of
acid are carried away as soon as they are formed, and thus give room
for the formation of fresh acid. Even then it is impossible to
explain the whole process. A specific action of the cells is no doubt
exerted, for these reactions can 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 constituents of the
blood, and the hydrochloric acid, as soon as it is formed, passes
into the secretion of the gland in consequence of its high power of
diffusion.

COMPOSITION OF GASTRIC JUICE

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

Constituents

Human

Dog

Water ....

99-44

97-30

Organic sul
^pepsin)

stanc

es (chiefly

0-32
0-20

1-71
0-50

CaCL. .

0-006

0-06

NaCl .

0-14

0-25

KCl .

0-05

0-11

NH.Cl
Ca3(P0,),
Mg3(P0,),
FePO,

0-01

0-05
0-17
0-02
0-008

1
1

74 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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. 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, however, is
derived by fermentative processes from the food.

Pawlow has shown that in dogs the secretory fibres for the gastric
glands are contained in the vagus nerves.

By an ingenious surgical operation he succeeded in separating off
from the stomach a diverticulum which pours its secretion through an
opening in the abdominal wall. This small stomach was found to
act in every way like the main stomach of the animal. The pure juice
so obtained is clear and colourless : it has a specific gravity of 1003
to 1006. It is feebly dextro -rotary, and gives some of the protein
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 (NHJ2SO4

Carbon . . . 50-73 per cent, j 50*37

Hydrogen . . 7'23 „ ! 6-88

Chlorine. . I'Ol to 1-17 „ ' 0*89

Sulphur . . . 0-98 „ 1-34

Nitrogen . . . not estimated 14*55 to 15*0

Oxygen . . . the remainder I the remainder.

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 with meat will cause it to flow.

The larger the proportion of protein in the diet, the more abundant

THE DiaESTIVE JUICES 75

and active is the juice secreted, provided the animal is hungry : the
psychical element is of great importance.

THE ACTION OF GASTRIC JUICE

The principal actions of the gastric juice have been already prac-
tically studied : the action of pepsin in converting the proteins of the
food into the diffusible peptones is its chief action. The curdling of
milk by rennet will be found described in Lesson VI.

There is 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 juice
that possesses this power.

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 employ :
it includes the albumoses (from albumin), globuloses (from globulin),
vitelloses (from vitellin), &c. Similar substances are also formed
from gelatin (gelatoses) and elastin (elastoises).

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

1. Acid-albumin.

{(a) Proto-albumose f ^^^ P^^^^^-^ albumoses, i.e

2. Proteoses (6) Hetero-albumose those which are formed

1 ^ ^ I first.

\{c) Deutero-albumose

3. Peptone.

1. Acid albumin. — The properties of the infra-proteins which are
the first degradation products in the cleavage of the proteins which
occurs during digestion have been described in Lesson V. (see pp. 29
and 48). We shall find later that, in pancreatic digestion, alkali-
albumin is formed instead of acid-albumin. The theory has been
put forward that a protein is capable of playing the part of a base in

76 ESSENTIALS OF CHEMICAL PHYSIOLOGY

virtue of its NHg groups, and also of an acid in virtue of its
COOH groups.

2. Proteoses. — These are the intermediate products in the
hydrolysis of native proteins 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
heating, and reappearing when the liquid cools. The last is a
distinctive property of proteoses. They are slightly diffusible.

The difference between the different proteoses is mainly one of
solubihty. 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.

3. Peptones. — These are the final products of the action of gastric
juice on native proteins. If the action is very prolonged, polypeptides
and amino-acids are split off from the peptones, but in the usual
short stay of food in the stomach very little of these ultimate cleavage
products is found there.

They are soluble in water, are not coagulated by heat, and are
not precipitated by nitric a^cid, copper sulphate, ammonium sulphate,
and a number of other precipitants of proteins. They are precipi-
tated but not coagulated by alcohol. They are also precipitated by
tannin, picric acid, potassio-mercuric iodide, phospho-molybdic acid,
and phospho-tungstic acid. They give the biuret reaction (rose-red,
with a trace of copper sulphate and caustic potash or soda) and are
readily diffusible through animal membranes.

To sum up : the main action of the gastric juice is upon the pro-
teins of the food, converting them into more soluble and diffusible
products. The protein envelopes of the fat globules are dissolved, and
the solid fats are melted. According to some, gastric juice contains
a fat-spliting ferment in small quantities which acts like the steapsin
of pancreatic juice ; but this action if present is very slight. Starch
is unaffected ; but cane sugar is inverted. The inversion of cane
sugar is due to the hydrochloric acid of the juice, and is frequently
assisted by inverting ferments contained in the vegetable food
swallowed. The stomach does not digest itself, because it forms an
antipepsin ; similarly in the intestine an antitrypsin is formed. The
formation of anti-bodies will be treated under the heading Immunity.
(See Blood.)

THE DIGESTIVE JUICES

77

The following table gives us at a glance the chief characters of
proteoses and peptones in contrast with those of such native proteins
as albumin and globulin.

Variety of
protein

Action of
heat

Action of
alcohol

Action of
nitric acid

Action of

ammonium

sulphate

Action of

copper

sulphate

and caustic
potash

!

DiflEusi-
bility

Ditto

1

Slight
Great

1

Albumin

Globulin

Proteoses
Peptones

Coagulated
Ditto

Not coagu-
lated

Not coagu-
lated

Precipitated,
then coagu-
lated

Ditto -

Precipitated
but not co-
agulated

Precipitated
but not co-
agulated

Precipitated
in the cold ;
not readily
soluble on
heating

Ditto

Precipitated
in the cold ;
readily so-
luble on
heating ;
the precipi-
tate reap-
pears on
cooling ^

Not precipi-
tated

Precipitated
by complete
saturation

Precipitated
by half satu-
ration; also
precipitated
by satura-
tion with
MgSO,

Precipitated
by saturation

Not precipi-
tated

Violet
colour

Ditto

Rose-red
colour
(biuret
reaction)

Rose-red
colour
(biuret
reaction)

' In the case of deutero-albumose this reaction only occurs in the presence of
excess of salt.

LESSON VIII

THE DIGESTIVE JUICES [continued)

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.

3. Put all into the water-bath at 40° C. After half an hour, test A and B
for alkali-albumin by neutralisation, for proteoses by nitric acid, and for
proteoses 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 C 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 fluid (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 Proteins. — Fibrin, albumin, &c. are converted into proteoses and
peptone by the ferment trypsin in an alkaline medium.

ii. On Carholiydrates. — 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.

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

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

THE DIGESTIVE JUICES 79

of a mucinoid substance is obtained. Filter this off and boil the filtrate ; no
protein coagiilable by heat is present.

3. Add a few drops of bile to (a) acid-albumin prepared as described in
Lesson V., 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 protein which leaves the stomach.

4. Petteiiliofer'8 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
capsule. 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. Hay's Test for Bile Salts. — Take two beakers full of water ; to
one add a few drops of bile, or solution of bile salts. Sprinkle a little
flowers of sulphur on the surface of each. It remains floating on the
pure water ; but where bile is present the surface tension of the water is
reduced, and the sulphur consequently rapidly sinks. This test is very
sensitive.

7. Preparation of Glycocholic Acid. — The preparation of the bile acids
is usually a task of some difficulty ; the following exercise is a simple one,
though unfortunately, for reasons which are not explicable, it does not always
succeed. Take a stoppered cylinder, place in it 200 c.c. of ox bile, 10 c.c. of
hydrochloric acid, and 25 c.c. of ether, and shake vigorously. Add a crystal
of glycocholic acid, and allow the mixture to stand in a cool place. In a time
varying from a few minutes to some hours, a mass of crystals of glycocholic
acid separates out. This may be filtered off, washed, and dissolved in a little
boiling water, and filtered hot. On cooling, needle-like crystals of the acid
again separate out.

8. Cholesterin. — (a) Examine crystals of this substance with the micro-
scope. Heat these on a slide with a drop of sulphuric acid and water (5:1);
the edges of the crystals turn red.

(6) Salkowski's reaction. Dissolve some cholesterin in chloroform in a
dry test-tube, and gently shake with an equal amount of concentrated
sulphuric acid ; the solution turns red, and the subjacent acid acquires
a green fluorescence. The chloroformic solution of cholesterin is rendered
colourless by pouring it into a wet test-tube, and the colour is restored by
the addition of sulphuric acid.

(c) Liebermann's reaction. Two or three drops of acetic anhydride are
added to a chloroformic solution of cholesterin and then sulphuric acid drop
by drop. A rose-red colour first develops ; this becomes blue and finally
bluish-green.

THE PANCREAS

The Pancreas is a compound tubulo-racemose gland ; between
the secreting acini are situated little masses of epithelial cells without
ducts called * islets of Langerhans.' Examination of the secreting
cells in different stages of activity reveals changes comparable to
those already described in the case of salivary and gastric cells.

80 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 ad-
ministration 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
before the extract is made, so as to ensure that the transformation
of trypsinogen into trypsin has taken place.

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

Quantitative analysis of human pancreatic juice gives the following
results : —

Water .... 97'6 per cent.
Organic solids ... 1*8 „

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.

(b) A small amount of protein matter, coagulable by heat.

(c) Traces of leucine, tyrosine, xanthine, and soaps.

THE DIGESTIVE JUICES 81

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.

(c) The first degradation product is alkali-albumin in place of the
acid-albumin of gastric digestion.

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

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

(/) Trypsin acts further than pepsin, decomposing peptone into
simpler products, of which the most familiar are leucine and tyrosine.

Besides leucine and tyrosine, other amino-acids such as aspartic
acid, glutamic acid, lysine, arginine, and tryptophan and ammonia
are also formed. A more complete list of the cleavage products with
their chemical constitution is given on pp. 31-35.

We know that the action of proteolytic enzymes is, by the
process of hydrolysis, to spHt the heavy protein molecule into
smaller and smaller molecules ; first we get proteoses, then peptones,
and finally after prolonged action simple substances like leucine and
tyrosine.

This cleavage is more easily performed by the more powerful
tryptic enzyme than by the comparatively feeble agent, pepsin-
hydrochloric acid. Still we have already seen that by the very pro-
longed action of the latter, leucine, tyrosine, and other amino-acids

G

82 ESSENTIALS OF CHEMICAL PHYSIOLOGY

are formed, although the quantity of these in an ordinary gastric
digest is negUgible. The essential difference between the two
enzymes is one of degree only ; trypsin is by far the more powerful
catalytic agent, and increases the velocity of the reaction much more
markedly than does pepsin-hydrochloric acid.

2. Action of Amylopsin. — The conversion of starch into maltose
is the 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-
tio7i). The chemistry of this is described on p. 25.

With regard to the formation of emulsions the following are the
principal facts. If olive oil and water are shaken up together and
the mixture is then allowed to stand, the finely divided oil globules
soon separate from and float on the surface of the water ; but if the
oil is shaken up with a solution of soap, the conditions of surface
tension are such that the oil globules remain as such in the mixture,
and a milky fluid or emulsion is the result. This emulsion is
rendered still more permanent by the presence of colloid or viscous
materials, 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 probably forms a thin layer on the outside of each
oil globule, which prevents them running together again. Pancreatic
fluid possesses all the necessary qualifications for forming an
emulsion. It is alkaline, and it liberates fatty acids from the fat :
these acids form soap with the alkali present ; moreover, it is viscous
from the presence of protein.

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

THE DiaESTlVE JUICES 83

THE SECRETION OF PANCREATIC JUICE

One of the most effective ways of producing a flow of the juice
is to introduce acid into the duodenum, and no doubt the acid of the
gastric juice is the normal stimulus for the pancreatic flow. This flow
still occurs when all the nerves supplying the duodenum and pancreas
are cut, and it was held by Popielski and by Wertheimer and Le
Page that it must be due to a local reflex, the centres being situated
in the scattered ganglia of the pancreas and of the solar plexus.
Starling and Bayliss, however, pointed out that it cannot be a
nervous reflex, since it occurs after extirpation of the solar plexus,
and destruction of all nerves passing to an isolated loop of intestine.
Moreover, atropine does not paralyse the secretory action. It must
therefore be due to direct excitation of the pancreatic cells by a
substance or substances conveyed to the gland from the bowel by the
blood-stream.

The exciting substance is not acid ; injection of 0*4 per cent, of
hydrochloric acid into the blood-stream has no influence on the
pancreas. The substance in question must be produced in the
intestinal mucous membrane under the influence of the acid. This
conclusion was confirmed by experiment. If the mucous membrane
of the duodenum or jejunum is exposed to the action of 04 per cent,
hydrochloric acid, a body is produced which, when injected into the
blood-stream in minimal doses, produces a copious secretion of pan-
creatic juice. This substance is termed secretm. It is associated with
another substance which lowers arterial blood-pressure. The two
substances are not identical, since acid extracts of the lower end of
the ileum produce a lowering of blood-pressure, but have no excitatory
influence on the pancreas.

Secretin is split off from a precursor, prosecretin, which is present
in relatively large amounts in the duodenal mucous membrane, and
gradually diminishes in amount throughout the intestine until it
entirely disappears in the ileum. Pro-secretin can be dissolved
out of the mucous membrane by normal saline solution. It has no
influence on the pancreatic secretion. Secretin can be split off from
it by boiling or by treatment with acid.

What secretin is chemically, we do not yet know. It is soluble in
alcohol and ether. It is not a protein, but probably is an organic
substance of low molecular weight. It is, moreover, the same sub-
stance in all animals, and not specific to different kinds of animals.

Whether there are any secretory nerves for the pancreas is at
present doubtful. Pawlow thought he had discovered them in the

g2

84 ESSENTIALS OF CHEMICAL PHYSIOLOGY

vagus ; but as he did not exclude in his experiments the passage of
the acid chyme from the stomach into the duodenum, it is probable
that the pancreatic secretion he obtained was due to that circum-
stance and the consequent formation of secretin.

Injection of secretin also stimulates the flow of bile.

Secretin is an instance of the chemical messengers or hormones
(Starling) of the body. Evidence is accumulating to show that
hormones are extremely important. It has already, for instance,
been shown that one called gastrin is formed as the result of salivary
digestion, and stimulates the flow of gastric juice. Another is
formed from the foetal tissues, which, passing into the mother's
circulation, stimulates the mammae to enlarge and secrete milk.

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 succus entericus or intestinal juice has no action on fats or
native proteins, but it appears to have to some extent the power of
converting starch into sugar ; its best known action is due to a ferment
it contains called invertin, which inverts saccharoses — that is, con-
verts cane sugar and maltose into glucose. The original use of the
term ' inversion ' has been explained on p. 17. It may be extended
to include the similar hydrolysis of other saccharoses, although there
may be no formation of levo-rotatory substances. There are probably
several inverting ferments in the succus entericus, one of which acts
on cane suger, one on maltose, and one on milk sugar.

A few years ago, however, Pawlow showed that succus entericus
has a still more important action, which is to intensify the proteo-
lytic power of the pancreatic juice. Fresh pancreatic juice has
very little power on proteins, for what it contains is not trypsin,
but its precursor, trypsinogen.

If fresh pancreatic and intestinal juices are mixed together, the
result is a very powerful proteolytic mixture, though neither juice by
itself has any proteolytic activity. The substance in the intestinal
juice that activates trypsinogen or, in other words, liberates trypsin
from trypsinogen has been called by Pawlow a ' ferment of the
ferments,' or enter o-kinase.

Intestinal juice contains another ferment called ercpsin (Otto
Cohnheim), which is capable of breaking up proteoses and peptone
into simple substances (leucine, tyrosine, hexone bases, ammonia,
&c.), and so assisting the action of trypsin.

THE DIGESTIVE JUICES 85

Bacterial Action. — The gastric juice is an antiseptic ; the pan-
creatic juice is not. An alkahne 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 proteins, while others again
break up fats. There are, however, certain actions that are entirely
or mainly 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. 19).
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.

ii. On fats. In 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. Eecent 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 diges-
tion to any appreciable extent.

iii. On proteins. Fatty acids and amino-acids are produced, but
these putrefactive organisms are specially efficacious in liberating the
protein cleavage products which have 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
limits, they are useful, helping the pancreatic juice and, further, pre-
venting the entrance into the body of poisonous products. It is
possible that, in digestion, poisonous alkaloids are formed. Certainly
this 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-
phoric acid, fattj^ acids, 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.

86

ESSENTIALS OF CHEMICAL PHySIGLOGY

LEUCINE AND TYROSINE

These two substances have been frequently mentioned in the
preceding pages, and they are the final cleavage products of proteins
which have been longest known. Leuciiie is usually the most
abundant of all the cleavage products (see table on p. 35).

We have already learnt that they are amino-acids, and that
leucine is amino-caproic acid (see p. 31). There are, however, several
isomeric amino-caproic acids. It was thought until quite recently

Fig. 22. — Leucine crystals.

Fig. 23.— Tyrosine crystals

that leucine was the amino-acid of normal caproic acid, but it has
been shown to be a-amino-iso-butyl-acetic acid. The difference in the
structure of these two compounds may be represented by the following
graphic formulae : —

/CH3

CH.,

CH2 Iso-butyl

CH,

Normal

a-amino-caproic
acid

CH3CH3

2 a-ammo-

CH.NH2 acetic acid

ICOOH

CH
CH,
CH.NH2
COOH

Tyrosine is a little more complicated, as it is not only an amino-acid,
but also contains an aromatic radical (see p. 34). Figs. 22 and 23
represent the crystalline forms of leucine and tyrosine.

THE DIGESTIVE JUICES 87

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 previously 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
by 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
respiratory systems is a well-known instance. Eemoval of the
thyroid gland upsets the whole body, producing widespread changes
known as myxoedema. Eemoval of the testes produces, not only a
loss of the spermatic secretion, but changes the whole growth and
appearance of the animal. Eemoval 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,
which leaves the gland via the lymph or venous blood, and is then
distributed to minister to parts elsewhere. Eemoval of such glands
as the thyroid or suprarenal produces disease or death because this
internal secretion can no longer be formed. In the case of the
pancreas, the external secretion of the pancreas (that is, pancreatic
juice) is formed by the cells lining the acini, and the internal secre-
tion, stoppage of which in some way leads to diabetes, has been
attributed by some to the islets of Langerhans ; but if these islets
are only stages in the formation of acini, as they have been shown
to be, it is difficult to fully accept this view.

In diabetes the oxidative powers of the body cells are lessened, and
the capability of these cells to prepare sugar for oxidation is impaired.
In this process the sugar or its derivative glycuronic acid is split into
smaller molecules, and ultimately into water and carbon dioxide. The

88 ESSENTIALS OF CHEMICAL PHYSIOLOGY

close relationship of sugar and glycuronic acid is shown by the following

formulae : —

COH COH

(CHOH), (CHOH)^

CH^OH COOH

[dextrose] [glycuronic acid]

That is, two hydrogen atoms in the CH2OH group are replaced by one of
oxygen. This oxidation is readily brought about in the body, and glycuronic
acid is usually found in diabetic urine ; but the further oxidation into water
and carbon dioxide is a more difficult task, because it involves the disruption
of the linkage of the carbon atoms. Perhaps it is here that the internal
secretion of the pancreas is effective. This, however, is at present a mere
theory, and certainly Lepine's idea that the ferment of the pancreatic
internal secretion is one which initiates glycolysis or sugar-splitting in the
blood, has been abundantly disproved. It may be that the active principle
of the pancreatic internal secretion stimulates the glycolytic action of the
tissue-cells. It is conceivable that in the other great cause of experimental
glycosuria, namely, injury to nervous structures, as in Bernard's puncture
experiment, the derangement of the nervous system exerts some unknown
influence on the pancreas as well as on the liver.

Many poisons produce temporary glycosuria, but the most interesting
and powerful of these is phloridzin. The diabetes produced is very intense.
Phloridzin is a glucoside, but the sugar passed in the urine is too great to be
accounted for by the small amount of sugar derivable from the drug.
Besides that, phloretin, a derivative of phloridzin, free from sugar, produces
the same results.

Phloridzin produces diabetes in starved animals, or in those in which any
carbohydrate store must have been got rid of by the previous administration
of the same drug. Phloridzin-diabetes is therefore analogous to those intense
forms of diabetes in man in which the sugar must be derived from proto-
plasmic metabolism.

A puzzling feature is the absence of an increase of sugar in the blood ; if
the phloridzin is directly injected into one renal artery, sugar rapidly appears
in the secretion of that kidney ; the sugar is formed within the kidney cells
from some substance in the blood, but whether that substance is protein or
not is uncertain. The action of the kidney cells in forming sugar has been
compared to that of the mammary cells in forming lactose.

Death in diabetic patients is usually preceded by deep coma, or uncon-
sciousness. Some poison must be produced that acts soporifically upon the
brain. The breath and urine of these patients smell strongly of acetone ;
hence the term acetoncBmia. This apple-like smell should always suggest
the possible onset of coma and death, but it is quite certain that acetone
(which can certainly be detected in the urine) is not the true poison ;
ethyl-diacetic acid, which accompanies and is the source of the acetone, was
regarded by some as the actual poison ; but these substances, when intro-
duced into the circulation artificially, do not cause serious symptoms. The
principal poison is /S-hydroxybutyric acid or its amino derivative. The
alkalinity and carbonic acid of the blood are decreased, and the ammonia
of the urine is increased : this indicates an attempt of the body to neutralise
the poisonous acid. The acid is the source of the ethyl-diacetic acid and
of the acetone. Research has shown that /3-hydroxybutyric acid originates
in the body from fat.

THE DIGESTIVE JUICES 89

THE BILE

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. At 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 duodenum ; there i-s 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
that in the area from which the portal
vein collects blood — stomach, intestine, # v ^f

spleen, and pancreas — the arterioles are ^ i^ ^? ^^ ^A

all dilated, and the capillaries are thus M ^m "^ \'' ♦ [^
gorged with blood. Further, the active ^^ ^ 9 ^K ^"J^
peristalsis of the intestine and the , ' M ^ ^^

pumping action of the spleen are ad- ^ ^ ^ K^^'

ditional factors in driving more blood j;^,^^M?

onwards to the hver. ^^HH^^^*^''"

The bile is secreted from the portal ^ _

^ Fig. 24.— HaBoiatoidin crystals.

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 — was at one time attributed to the effect of the digestive
products carried by the blood to the liver stimulating the hepatic
cells to activity: this was supported by the fact that protein 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 facts are now more readily explained by the
circumstance that secretin is a stimulant of the liver as well as of
the pancreas.

The chemical processes by which the constituents of the bile are
formed are obscure. We, however, know that the biliary pigment is

90 ESSENTIALS OF CHEMICAL PHYSIOLOGY

produced by the decomposition of haBmoglobin. Bilirubin is, in fact,
identical with the iron-free derivative of haemoglobin called haemat-
oidin, which is found in the form of crystals in old blood-clots such
as occur in the brain after cerebral haemorrhage (see fig. 24).

An injection of haemoglobin into the portal vein, or of substances
such as water which liberate haemoglobin from the red blood corpuscles,
produces an increase of bile pigment. If the spleen takes any part
in the elaboration of bile pigment, it does not proceed so far as to
hberate haemoglobin from the corpuscles. No free haemoglobin 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).

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,
lecithin, urea, and mineral salts, of which sodium chloride and the
phosphates of iron, calcium, and magnesium are the most im-
portant.

Bile is a yellowish, reddish-brown or green fluid, according to
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
reaction.

The specific gravity of human bile from the gall bladder is
1026 to 1032; that from a fistula, 1010 to 1011. The greater con-
centration 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 gall-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 :—

THE DIGESTIVE JUICES

91

Constituents

Fistula bile

(healtliy woman.

Copeman and

Winston)

Fistula bile (case

of cancer. Yeo and

Herroun)

Normal bile
(Prerichs)

I 9-14
' .1-18

2-98
0-78

14-08
85-92

Sodium g^lycocholate
Sodium taurocholate
Cholesterin, lecithin, fat
Mucinoid material .
Pigment ....
Inorganic salts

Total solids .
Water (by difference)

0-6280 1

0-0990
0-1725
0-0725
0-4510

0-165

0-055
0-038

1 0-148
0-878

1-284
98-716

1-4230
98-5570

100-0000

100-000

100-00

,

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-protein ; in human bile, on the other hand, there is
very little if any nucleo-protein ; the mucinoid material present there
is really mucin. (On the general characters of Mucin and Nucleo-
PROTEiNS see pp. 45 to 47.)

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

Glycocholic acid (C26H43NO6) is by the action of dilute acids and
alkalis, and also in the intestine, hydrolysed and split into glycine or
amino-acetic acid and cholalic acid.

Cor.Hj

'2G

3NO6 + H2O:

:CH2.NH2.COOH+C24H4o05

[glycocholic acid] [glycine] [cholalic acid]

The glycocholate of soda has the formula C26H42NaN06.
Taurocholic acid (O2GH45NO7S) similarly splits into taurine or
amino-ethyl-sulphonic acid and cholalic acid,

C26H45NO7S + H,0=C2H4.NH2.HS03 4- C24H40O5

[taurocholic acid] [taurine] [cholalic acid]

The taurocholate of soda has the formula C26H44NaN07S.
Glycocholic and taurocholic acids have lately been prepared
synthetically from cholalic acid and glycine and taurine respectively.
The colour reaction called Pettenkofer's reaction, described in the

92 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 furfuraldehyde, in addition to other products. It is the
furfuraldehyde which 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 in excess. The bile pigments show no absorption bands with the
spectroscope ; their origin from the blood pigment has already been
stated (p. 90).

Bilirubin has the formula CigHigNgOa: it is thus an iron-free
derivative of haemoglobin. The iron is apparently stored up in the
liver cells, perhaps for future use in the manufacture of new hasmo-
globin. The bile contains only a trace of iron.

Biliverdin has the formula Ci6H,gN204 {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.

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
yellow product is called choletelin, CigHigNsOe.

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, C32H44N4O7. With the
spectroscope it shows a dark absorption band between b and F, and a
fainter band in the region of the D line.

Urobilin. — Hydrobilirubin is interesting because a similar substance
is formed from the bile pigment by reduction processes in the intestine,
and constitutes stercohilin, the pigment of the faeces. Some of this
is absorbed and ultimately leaves the body in the urine as one of its
pigments called urobilin. A small quantity of urobilin is sometimes
found preformed in the bile. The identity of urobilin and stercohilin
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

THE DIGESTIVE JUICES

93

in the region of the E line. Hydrobilirubin differs from urobilin in
containing much more nitrogen in its molecule (9*2 instead of 4*1 per
cent.), and is probably a product of less complete reduction than
urobilin. (See further Lesson XXVI.) Urobilin is also formed, by
the oxidation of hsemopyrrol (see Haemoglobin, p. 114).

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, and in blood plasma as an ester
of oleic and palmitic acids. The cholesterin
of nervous tissues is, however, free. In
bile it is normally present in small quanti-
ties 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, but a monatomic
alcohol, probably of the terpene series. Its formula is C27H45.HO.

From alcohol or ether containing water it crystallises 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 tests described under the practical exercises on p. 79. What the
physiological uses of cholesterin are is entirely unknown.

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.

Fl(i. 25.— Cholesterin crystals.

THE USES OF BILE

Bile is doubtless, to a certain extent, excretory. In some animals
it has a slight action on fats and starch, but it appears to be rather a
coadjutor to the pancreatic juice (especially in the digestion of fat) than
to have any independent digestive activity. Its auxiliary action in
starch digestion has been shown in one of our practical exercises
(p. 78). It has a similar assisting power in the digestion of
proteins.

Bile is said to be a natural antiseptic, lessening the putrefactive
processes in the intestine. This is 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

94 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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
increased, owing to the precipitation of unpeptonised protein. 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
juice to have full play. Bile is a solvent of fatty acids, and assists
the absorption of fat.

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 faeces ; they are only represented to a slight extent in the urine ;
hence it is calculated that seven-eighths of them are reabsorbed from
the intestine. Small quantities of cholalic acid, taurine, and glycine
are found in the faeces ; the greater part of these products of the
decomposition of the bile salts is taken by the portal vein to the
liver, where they are once more synthesised into the bile salts.
Some of the taurine is absorbed and excreted as tauro-carbamic
acid (C2H4NHCO.NH2HSO3) in the urine. Some of the absorbed
glycine may be excreted as urea. The cholesterin and mucus are
found in the faeces ; the pigment is changed into stercobilin, a sub-
stance like hydrobilirubin. Some of the stercobilin is absorbed, and
leaves the body as the urinary pigment, urobilin.

THE FiECES

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

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

2. Undigested food : that is, if food is taken in excess, some escapes

THE DIGESTIVE JUICES 96

the action of the digestive juices. On a moderate diet unaltered
protein 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. ; haematin from haemoglobin ;
insoluble soaps like those of calcium and magnesium.

6. Bacteria of all sorts and debris from the intestinal wall ; cells,
nuclei, mucus, &c. This forms a very large contribution.

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
debris from the intestinal wall. The pigment is 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 oesophagus the
thickness of the epithelium and the quick passage of the food through
these parts reduce absorption to a minimum. Absorption takes place
to a small extent in the stomach ; the small intestine, with its folds
and villi to increase its surface, is, however, the great place for absorp-
tion ; 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 protein being converted re-
spectively into the diffusible materials sugar and amino-acids.

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

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

96 ESSENTIALS OF CHEMICAL PHYSIOLOGY

through which the absorbed subtances 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 important. 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.

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 protein 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 Pavy, who
holds 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 protein ; in support
of this he has shown that proteins contain a carbohydrate radical.
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
hfe.

Absorption of Proteins. — It is possible that under abnormal con-
ditions a certain amount of soluble protein is absorbed unchanged.
Thus, after eating a large number of eggs, egg-albumin has been
stated to be discoverable in the urine. Patients fed per rectum derive
nourishment from protein food, although proteolytic ferments are not
present in this part of the intestine.

Under normal conditions, however, the food-proteins are broken

THE DIGESTIVE JIHCES 97

up into substances with smaller molecules, and the ready diffusibility
of peptones led most physiologists to consider that protein was usually
absorbed as peptone, or as proteose and peptone. But proteose
and peptone are absent from the blood and lymph under all circum-
stances, even from the portal blood during the most active digestion.
It is fortunate that this is so, for proteose and peptone when intro-
duced into the blood produce poisonous effects ; the coagulability of
the blood is lessened, blood pressure falls, secretion ceases, and in
the dog 0"3 gramme of commercial peptone per kilogramme of body-
weight is often sufficient to produce death.

This absence of ' peptone ' (using the word to include the prote-
oses) did not, however, absolutely negative the idea that ' peptone '
is the form in which proteins are absorbed, and the difficulty was met
by supposing that during absorption the products of proteolysis were
reconverted into native proteins (albumins and globulins). This
synthesis was further considered to be accomplished by the epithelial
cells that line the intestine.

This view has now had its day, and the change of opinion that
has relegated it to the past is due (1) to our increased knowledge of
the power of trypsin and erepsin ; (2) to a more careful examination
of the intestinal contents, and of the blood during absorption. We
now know that in the intestine the proteins are, by the two enzymes
trypsin and erepsin, broken down beyond the peptone stage into
their final cleavage products, the amino-acids, and that these pro-
bably pass into the blood as such, for the amount of non-protein
nitrogen in that fluid is increased during absorption. These amino-
acids are partly utilised by the cells of the body to repair their
waste, but partly and to a still greater extent converted by the liver
into the waste substance urea, which is finally excreted by the
kidneys. The view that the absorptive epithelium of the alimentary
tract has any special power in building up proteins from these simple
cleavage products has not been confirmed. If an animal is fed on
the cleavage products obtained from a pancreatic digest nitrogenous
equilibrium is still maintained.

We thus see that the cells of the body possess the power of
rebuilding the proteins peculiar to themselves from the fragments
of the molecules of the food proteins. This accounts for the fact that
the animal tissues retain their chemical individuality in spite of the
great variations in the composition of the diet the animal takes.

If a man wishes to build a new house, and to employ for the
purpose the bricks previously used in the building of another house,
he takes the old house to pieces and uses the bricks and stones most

H

98 ESSENTIALS OF CHEMICAL PHYSIOLOGY

appropriate for his purpose, rearranges them in such a way that the
new house has its own special architectural features, and discards as
waste the bricks and stones which are not suitable. This idea under-
lies the term so often used by German writers, who speak of the
cleavage products of protein as Bausteine (building stones). Each
tissue has special architectural features in its protein molecules, and
these molecules are reconstructed by using the Bausteine that pre-
viously had been used in the building of other protein molecules,
either in another animal or in vegetable structures. The Bausteine
which are in excess or are unsuitable are simply got rid of as waste
substance.

A large number of the Bausteine are never actually built into
protoplasm, but are converted by the liver into urea, and this is dis-
charged from the body via the urine (see Urea formation, p. 148).

Fig. 26.— Section of the villus of a rat killed during fat-absorption (PI A. Schafer) :
ep, epithelium ; str, striated border ; c, lympb cells ; c', lymph cells in the epithelium ;
Z, central lacteal containing disintegrating lymph cells.

One can only conjecture at present which are the ones that on
p. 52 we compared to diamonds, because they are unusually precious
for the synthesis of protein by tissue cells ; but probably phenyl-
alanine and its near relation tyrosine come into this category, for if
they are injected into the blood-stream they do not give rise to any
increase in the urea formed.

THE DIGESTIVE JUICES

99

Absorption of fats. — The fats undergo in the intestine two
changes : one a physical change (emulsification), the other a chemical
change (saponification). The lymphatic vessels are the great channels
for fat-absorption, and their name, lacteals, is derived from the
milk-like appearance of their contents (chyle) during the absorption
of fat.

The way in which the minute fat globules pass from the intestine
into the lacteals has been studied by killing animals at varying periods
after a meal of fat and making osmic acid microscopic preparations
of the villi. Figs. 26 and 27 illustrate the appearances observed.

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 pene-
trate 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

,11111 • •, -ii ,1 Fm.27. — Mucous membrane of frog's intestiue

the blood plasma is quite milky ; the during fat-absorption (E. a. Scliafer) : ep,

fat droplets are so small that they t^^^^A:!'"'''^'^'''''''''' '''''^^''
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 from carbo-
hydrates, and possibly, in the opinion of some physiologists, from
protein as well.

As the fat globules were never seen penetrating the striated
border of the epithelial cells, there was a difficulty in understanding
how they reached the interior of these cells ; the cells will not take
up other particles, and it is 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) ._

Eecent research has solved this difficulty;, ,Li ilhe ^rst-. place
particles may be present in the epithelium" arnd lymphoid cells while
no fat is being absorbed. These parti<?]es;are'5roi6pla2imic in nature,

100 ESSENTIALS OF CHEMICAL PHYSIOLOGY

as they stain with reagents that stain protoplasmic granules ; but as
they also stain darkly with osmic acid, they 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 performed a large number of experiments on the subject,
showed that the splitting of fats into glyceiin and fatty acids occurs
to a much greater extent than was formerly supposed : these sub-
stances, being soluble, pass readily into the epithelium cells, and these
cells perform the synthetic act of building them into fat once more ;
the fat so formed 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 Eockwood
has shown that fat is absorbed entirely as glycerin and either fatty
acid or soap ; and that prehminary emulsification, though advan-
tageous for the formation of these substances, is not essential.

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 faeces.

LESSON IX
THE BLOOD AND RESPIRATION

[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 ai 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.^

A 3. 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 lluid residue of the blood after the separation of the
clot ; it is blood plasma minus the fibrin which it yields. The general ap-
pearance of fibrin obtained by whipping fresh blood will already be familiar
to the student, as he has used it in experiments on digestion.

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

Separation of the serum proteins, —{a) Dilute serum with fifteen times
its volume of water. It becomes cloudy owing to the partial precipitation of

' 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
partial precipitation of globulin takes place, and carries down the ferment with it.
After a few hours pour off the supernatant fiuid and dissolve the precipitate in
half a litre of tap-water to which a few drops of 2-per-cent. solution of calcium
chloride have been added. The solution can be then given round to the class as
fibrin ferment.

102 ESSENTIALS OF CHEMICAL PHYSIOLOGY

serum globulin. Add a few drops of 2-per-cent. acetic acid ; the precipitate
becomes more abundant, but dissolves in excess of the acid.

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

(c) Saturate some serum with magnesium sulphate by adding crystals of
the salt and grinding in a mortar. A precipitate of serum globulin is pro-
duced.

(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 pre-
cipitated.

(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 protein.

Haemoglobin.

6. Direct the spectroscope to the window and carefully focus Fraunhofer'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 81 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 oxyhaemoglobin in the first series, and of (reduced)
haemoglobin in the second series. Notice that in the more dilute specimens
of haemoglobin the bands are no longer seen, whereas those of oxyhaemoglobin
in specimens similarly diluted are still visible.

11. Take a tube which shows the single band of reduced haemoglobin
and shake it with the air ; the bright red colour returns to it and it shows
spectroscopically the two bands of oxyhaemoglobin 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 witli a drop of water,
or mount it in a drop of Canada balsam. Examine the crystals of oxyhaemo-
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 haemin.

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 platelets.

After blood is shed it rapidly becomes viscous and then sets into

THE BLOOD 103

a firm 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 fluid, many radiating from small clumps of
blood platelets. 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 having
previously been removed. It may also be 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

Fig. 28.— Fibrin filaments and blood platelets : A, network of fibrin sbown 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 platelets. B (from Osier), blood corpuscles and blood platelets
within a small vein.

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

Blood

Plasma 11^^^.^,
Corpuscles )

It may be roughly stated that in 100 parts by weight of blood 60-65
parts consist of plasma and 35-40 of corpuscles.

The buffy coat is seen when blood coagulates slowly, as in horse's
blood. The red corpuscles sink more rapidly than the white, and
the upper stratum of the clot (buffy coat) consists mainly of fibrin
and white corpuscles.

Coagulation is hastened by —

1. A temperature a little over that of the body.

2. Contact with foreign matter.

3. Injury to the vessel walls.

4. Agitation.

5. Addition of calcium salts.

6. Injection of nucleo-protein produces intravascular clotting

104 ESSENTIALS OF CHEMICAL PHYSIOLOaY

(positive phase). Very minute doses, however, produce the opposite
effect : namely, delay of coagulation (negative effect).
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. Addition of a soluble oxalate, fluoride or citrate.

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

5. Addition of leech extract to the blood, or injection of leech
extract into the circulation while the animal is alive.

6. Contact with the living vascular walls.

7. Contact with oil.

The cause of the coagulation of the blood may be briefly stated as
follows : —

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

When the blood is shed the fibrinogen molecule is altered in such
a way that it gives rise to the comparatively insoluble material yi6ri?t.
The statement has been made that the fibrinogen molecule is split
into two parts : one part is a globulin (fibrinoglobulin), which remains
in solution ; the other and larger part is the insoluble substance
fibrin. It is, however, doubtful if this really represents what
occurs, for recent work seems to show that the fibrinoglobulin is
not a product of fibrinogen, but exists in the blood plasma before-
hand. At any rate, whether this is so or not, the fact remains
that fibrin is the important product and the only one which need
concern us.

The next question is. What causes the transformation of fibrinogen
into fibrin ? And the answer to that is, that the change is due to the
activity of a special unorganised ferment which is called yi^rm/crmew^
or tkromhin.

This ferment does not exist in healthy blood contained in healthy
blood vessels, but is formed by the disintegration of the blood platelets
and colourless corpuscles which occurs when the blood leaves the
blood vessels or comes into contact with foreign matter. Hence the
blood does not coagulate during life. But, it will be said, disintegra-
tion of the blood corpuscles occurs during life ; why, then, does the
blood not coagulate ? The reason is that although the formed elements
do disintegrate in the living blood, such a phenomenon takes place

THE BLOOD 105

very slowly and gradually, so that there can never in normal cir-
cumstances be any massive liberation of fibrin ferment, and, further,
that there are agencies at work to neutralise the fibrin ferment as it is
formed. The most noteworthy of these neutralising agencies is
the presence in the blood of an antiferment called antithrombin,
analogous to the antipepsin and antitrypsin which, we have seen, are
efiQcacious in preventing the stomach and intestines from undergoing
self-digestion.

Thrombin or fibrin ferment belongs to the class of nucleo-proteins,
and other nucleo-proteins (see pp. 45, 46) obtained from most of the
cellular organs of the body produce intravascular clotting when in-
jected into the circulation of a living animal. In certain diseased
conditions intravascular clotting, or thrombosis, sometimes occurs.
This must be due either to the entrance of nucleo-protein into the
circulation from diseased tissues, or to a failure of the body to pro-
duce sufficient antithrombin to neutralise its effect, or to both of
these conditions together.

Thrombin is believed to originate chiefly from the blood platelets
and in part from the leucocytes. Birds' blood clots very slowly, and
the absence of blood platelets in this variety of blood will in part
account for this. Lymph which contains colourless corpuscles, but
no platelets, also clots in time, so in this case the colourless corpuscles
must be the source of the ferment. One should, however, be careful
in speaking of the disintegration of leucocytes to remember that the
word disintegration does not mean complete breakdown, leading to
disappearance ; the colourless corpuscles do not appreciably diminish
in number when the blood is shed, but what occurs in the surviving
leucocytes is a shedding out of certain products, among which fibrin-
ferment is one.

We have now traced fibrin-formation, the essential cause of blood-
clotting, to the activity of thrombin ; it is next necessary to allude
to what has been discovered in relation to the origin of thrombin.
Like other ferments, it is preceded by a mother substance or
zymogen. This zymogen is called prothrombin, or thrombogen, and
there appear to be two necessary agents concerned in the con-
version of thrombogen into thrombin : one of these is the action
of calcium salts, the other is the presence of an activating agent,
analogous to entero-kinase (see p. 84), and called thrombo-hinase.
The exact role played by each is still a matter of speculation, but
we may learn a good deal by studying a little more in detail
some of the methods already enumerated for preventing the blood
from coagulating.

106 ESSENTIALS OF CHEMICAL PHYSIOLOGY

The part played by calcium salts is well illustrated by the fact
that coagulation is prevented by the decalcification of the blood.
This can be accomplished by the addition of a small amount of
soluble oxalate or fluoride to the blood immediately it is shed. The
calcium of the blood plasma is then immediately precipitated as
insoluble calcium oxalate or fluoride, and is thus not available for
the transformation of thrombogen into thrombin. The addition of
the oxalate or fluoride must be rapidly performed, otherwise time will
be given for the conversion of thrombogen into thrombin, and
thrombin, when formed, will act upon fibrinogen whether the calcium
has been removed or not. In other words, calcium is only necessary
for the formation of fibrin-ferment, and not for the action of fibrin-
ferment on fibrinogen. Fibrin is thus not a compound of calcium
and fibrinogen.

The action of a soluble citrate is also in a certain sense a
decalcifying action, for although calcium citrate is a soluble salt it
does not ionise in solution so as to liberate the free calcium ions
which are essential for thrombin formation.

Oxalated blood (or oxalated plasma) will clot when the calcium is
once more restored by the addition of a small amount of calcium
chloride, but such an addition to fluoride plasma will not induce clot-
ting ; thrombin must be added also. In some way sodium fluoride
interferes with the formation of thrombin, probably by preventing the
liberation of thrombo-kinase from the formed elements of the blood.

The other activating agent, thrombo-kinase, is in part liberated
from the formed elements of the blood, but it is also obtained from
many other tissues. If a haemorrhage takes place under ordinary
circumstances the blood as it flows from the wound passes over the
muscles and skin that have been cut, and rapidly clots owing to the
thrombo-kinase supplied by those tissues. If blood is obtained by
drawing it off through a perfectly clean cannula into a clean vessel
without allowing it to touch the tissues, it remains unclotted for a
long time ; in the case of birds' blood this time may extend to many
days ; but the addition of a small piece of tissue such as muscle, or of
an extract of such a tissue, produces almost immediate clotting. If a
solution of fibrinogen is prepared and calcium added it will not clot ;
if thrombin or a fluid such as serum which contains thrombin is added
also it will clot. It will not clot if birds' plasma obtained as above is
added to it ; nor if tissue extract is added to it ; but if both are added
it will. In other words, the thrombogen of the birds' plasma
plus the thrombo-kinase of the tissue extract have the same effect as
thrombin.

THE BLOOD 107

The next point to consider is why blood obtained after the previous
injection of proteoses (or commercial peptone) into the circulation
should not clot. It certainly contains calcium salts, and probably
both thrombogen and thrombo-kinase, for it can be made to clot with-
out the addition of either ; for instance, by dilution or the passage of
a stream of carbon dioxide through it. There must be something in
peptone blood which antagonises the action of thrombin. This some-
thing is an excess of antithrombin. Peptone will not hinder blood
coagulation, or only very slightly if it is added to the blood after it is
shed. The antithrombin must therefore have been added to the
blood while it was circulating in the body. We can even go further
than this and say what part of the body it is which is concerned in the
production of antithrombin : it is the liver, for if the liver is shut
off from the circulation, peptone is ineffective in its action. The
converse experiment confirms this conclusion, for if a solution of
peptone is artificially perfused through an excised surviving liver, a
substance is formed which has the power of hindering or preventing
the coagulation of shed blood.

We are thus justified in two conclusions : —

(1) That the antithrombin which is normally present in healthy
blood in sufficient quantities to prevent intravascular clotting, is
formed in the liver.

(2) That commercial peptone in virtue of the proteoses it contains
stimulates this action of the liver to such an extraordinary degree,
that the accumulation of antithrombin in the blood becomes suffi-
ciently great to prevent the blood from clotting even after it is shed.

It should be noted, however, that this effect upon the Uver varies
in different animals, and is most marked in the dog.

We shall conclude by considering only one more of the hindrances
to coagulation, and that by no means the least interesting. The
leech lives by sucking the blood of other animals ; from the leech's
point of view^ it is therefore necessary that the blood should flow
freely and not clot. The glands at the head end of the leech, often
spoken of roughly as its salivary glands, secrete something which
hinders the blood from coagulating, and everyone knows by experience
who has been treated by leeches how difficult it is to prevent a leech-
bite from bleeding after the leech has been removed ; complete
cleansing is necessary to wash away the leech's secretion from the
wound. Now if an extract of leeches' heads is made with salt solution
and filtered, that fluid will prevent coagulation, whether it is injected
into the blood-stream or added to shed blood. The substance in the
extract is called hirudin, and this is believed to be antithrombin itself.

108

ESSENTIALS OF CHEMICAL PHYSIOLOGY

We may summarise our present knowledge of the causes of
coagulation in the following tabular way : —

From the platelets,
and to a lesser degree
from the leucocytes, a
nucleo-protein is shed
out called

Thrombogen.

From the formed
elements of the blood,
but also from the tissues
over which the escaping
blood Hows, is shed out
an activating agent
called
Thrombo-kinase.

In the blood plasma
a protein substance ex-
ists caUed

Fibrinogen.

In the presence of calcium salts, thrombo-
kinase activates thrombogen in such a way that
an active ferment is produced, which is called
Thrombin.

Thrombin or fibrin -ferment acts on fibrinogen in such a way that it is
transformed into the insoluble stringy material which is called
Fibrin.

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
a calcium salt ; plasma obtained by the use of a strong solutfon of
salt coagulates when this is diluted by the addition of water, the
addition of fibrin ferment being necessary in most cases ; where
coagulation occurs without the addition of fibrin-ferment, no doubt
some is present from the partial disintegration of the corpuscles which
has already occurred. Pericardial and hydrocele fluids resemble pure
plasma very closely in composition. As a rule, however, they contain
few or no white corpuscles, and do not clot spontaneously, but after
the addition of fibrin ferment or liquids like serum that contain fibrin
ferment they always yield fibrin.

THE BLOOD 109

Pure plasma may be obtained from horse's veins by what is known
as the ' Hving test-tube ' experiment. If the jugular vein is ligatured
in two places, so as to include a quantity of blood within it, then
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 pipette.

The plasma is alkaline, yellowish in tint, and its specific gravity
is about 1,026 to 1,029.

Its chief constituents may be enumerated as follows : —

1,000 parts of plasma contain —

Water . . . . . . . 902-90

Solids . . . . . . 97-10

Proteins : 1, yield of fibrin . . 4-05

2, other proteins . . 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 protein in nature.

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

Proteins of Pldsma \ Proteins of Serum

Fibrinogen Serum globulin

Serum globulin Serum albumin

Serum albumin Fibrin ferment (nucleo- protein)

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

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

A. Proteins. — Fibrinogen. — This is the parent substance of fibrin,
It is a globuHn. It differs from serum globulin, and may be sepa-
rated from it by the fact that half saturation with sodium chloride
precipitates it. It coagulates by heat at the low temperature of
56° 0. As judged from the yield of fibrin, it is the least abundant of
the proteins of the plasma (see table on upper part of this page).

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

110

ESSENTIALS OF CHEMICAL PHYSIOLOaY

Lesson IV. Both serum globulin and serum albumin probably con-
sist of more than one protein substance (see Lesson XX.).

Fibrin ferment. — Schmidt's method of preparing it is to take serum
and add, excess of alcohol. This precipitates all the proteins, 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 proteins are. A simpler method of preparing fibrin
ferment in an impure but efficient form is given in the footnote
on p. 101.

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 O'Od per cent.), and still smaller
quantities of uric acid, creatine, creatinine, xanthine, and hypoxan-
thine.

C. Salts. — The most abundant salt is sodium chloride : it con-
stitutes 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 . . . . . • . 8*550

Chlorine

3-640

SO3 .

0-115

P2O5 . . .

0*191

Potassium

0*323

Sodium .

3*841

Calcium phosphate

0*311

Magnesium phosphate 0*222

THE WHITE

BLO(

)D (

:!ORPTJSC

LES

These corpuscles are typical animal cells. Their nucleus consists
of nuclein ; their cell-protoplasm yields proteins belonging to the
nucleo-protein and globulin groups. 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.

THE BLOOD 111

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 -j^o i^^ch
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 (oxyhaemoglobin), leaving a globular colourless stroma.
Salt solution causes the corpuscles ^ ,

shrink : they become crenated or wrinkled. § f| ^ #^ /^
The action of water and salt solution is 8 © fir ^'^ ^-^
explained by the existence of a membrane

on the surface of the corpuscles through •''-^^ ^C^

which osmosis takes place. Dilute alkalis ^^ V^

. 7 N T 1 - 1 ^^^- 29.— a-^, successive effects of

(0"2 per cent, potash) dissolve the cor- water on a red blood corpuscle :

, -r^.y , -7/1 X J.' /, a red corpuscle crenated by salt

pUSCleS. JJimte acids (1 per cent, acetic solution ; ,<7, action of tannin on

acid) act like water, and in nucleated cor- ^^ corpusc e.
puscles render the nucleus distinct. Tannic acid causes a discharge
of haemoglobin from the stroma, but this is immediately altered and
precipitated. It remains adherent to the stroma as a brown globule,
consisting probably of haematin. 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

Solids 'o>-g^°i«. • ■ • • 303-88 „

(inorganic . . . 8*12 „
100 parts of dried corpuscles contain —

Protein . ~ . . . . . 5 to 12 parts

Haemoglobin . . . . 86 ,, 94 ,,

Lecithin . . . . .1*8 part

Cholesterin . . . . .0*1 ,,

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

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

The pigment of the red corpuscles. — The pigment is by far the

112

ESSENTIALS OF CHEMICAL PHYSIOLOGY

most abundant and important of the constituents of the red cor-
puscles. It differs from most other proteins 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
oxyhgemoglobin ; the other condition is the deoxygenated or reduced
haemoglobin (better called simply haemoglobin). This is found in the
blood after asphyxia. It also occurs in all venous blood — that is,
blood which is returning to the heart after it* has supplied the tissues
with oxygen. Venous blood, however, always contains a considerable
quantity of oxyhaemoglobin also. Haemoglobin is the oxygen-carrier
of the body, and it may be called a respiratory pigment.

Crystals of oxyhaemoglobin may be obtained with readiness from
the blood of such animals as the rat, guinea-pig, or dog; with diffi-
culty from other animals, such as man,
ape, and most of the common mam-
mals. The following methods are the
best : —

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

2. Microscopical preparations may
also be made by Stein's method, which
consists in using Canada balsam in-
stead of water in the above experi-
ment.

3. On a larger scale the crystals
may be obtained by shaking the blood
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
crystals are deposited.

The accompanying figures represent the form of the crystals so
obtained.

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

Fig. 30.— Oxyhaemoglobiu crystals magni-
fied : 1, from human blood ; 2, from the
guinea-pig ; 3, squirrel ; 4, hamster.

THE BLOOD 113

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 haemoglobin. They
find carbon, hydrogen, nitrogen, oxygen, sulphur, and iron. The
percentage of iron is 0'4. Oxyhaemoglobin may be estimated in the
blood (1) by the amount of iron in the ash, or (2) by certain colori-
metric methods w^hich are described in the Appendix.

Haemoglobin is a conjugated protein (see p. 44), and on the addi-
tion of an acid or alkali it is broken up into two parts, a protein called
(jlobin, and a brown pigment called hcematin, which contains all the
iron of the original substance.

Globin is coagulable by heat, soluble in dilute acids, and preci-
pitable from such solutions* by ammonia. It is a member of the
group of proteins called histories (see p. 42).

Haematin is not crytallisable : according to Nencki and Sieber
its formula is C.32H32N404Fe. It presents different spectroscopic
appearances in acid and alkaline solutions, and yields several products
under the influence of certain reagents, which we shall consider in
the advanced course. For the present, we shall mention only three
of these, hsemin, hgematoporphyrin, and hgemopyrrol.

Hsemin is of great importance, as the obtaining of this substance
in a crystalline form is the best chemical test for blood. Haemin
crystals, sometimes called Teich-

mann's crystals, are prepared for SLT^' ^^ ^.

microscopic examination by boiling y^'J^-^/r v, ,^ \

a fragment of dried blood with a f^ ^ ^"^ j ^=<^

drop of glacial acetic acid on a ^ ^^ *'' \c^ ^ \j ^>^

sHde ; on coohng, dark - brown v ^^^ | X " ^ ^

plates and prisms belonging to the S"! ^^^ > /:;l

triclinic system, often in star- , ^'^ ^ r '^w ^ , ]^ji^

shaped clusters and with rounded *^' CiJ "^ /^^ "**

angles (fig. 31), separate out. "^ ^ ^ '^ %

In the case of an old blood- -rlr -| /■ -^^ ''

stain it is necessary to add a Fig. 31.— Hajmiu crystals magulfied. (Preyer.)

crystal of sodium chloride. Fresh

blood contains sufiftcient sodium chloride in itself. The action of the
acetic acid is (1) to split the haemoglobin into haematin and globin ;
and (2) to evolve hydrochloric " acid from the sodium chloride. The
haematin unites with the hydrochloric acid, and thus haemin is formed.
Nencki has further shown that, when prepared in this way, haemin
also contains the acetyl gi'oup.

Haematoporphyrin is iron-free haematin : it may be prepared by

I

114 ESSENTIALS OF CHEMICAL PHYSIOLOaY

mixing blood with strong sulphuric acid ; the iron is taken out as
ferrous sulphate. This substance is also found sometimes in nature ;
it occurs in certain invertebrate pigments, and may also be found in
certain forms of pathological urine. It shows well-marked spectro-
scopic bands, and so is not identical with the iron-free derivative of
haemoglobin called haematoidin which is formed in extravasations of
blood in the body (see p. 90). The two substances are possibly
isomeric.

Haemopyrrol is methyl-propyl-pyrrol, with the formula

H.C-C — CH,.C,H5

II II
H.C G — CH3

N.H

and is obtained by reduction from haematoporphyrin. Ifc is also
similarly obtained from the derivative of chlorophyll called phyllo-
porphyrin, a fact which illustrates the near relationship of the
principal animal and vegetable pigments.

COMPOUNDS OF H.ffiMOGLOBIN WITH GASES

Haemoglobin forms at least four compounds with gases : —

^T-,, ( 1. Oxyhaemoglobin.

With oxygen i r. ^r .^ -. , •

•^^ 12. Methaemoglobm.

With carbonic oxide 3. Carbonic oxide haemoglobin.

With nitric acid 4. Nitric oxide haemoglobin.

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

Oxyhaemoglobiii is the compound that exists in arterial blood. The
oxygen linked to the haemoglobin, which is removed by the tissues
through which the blood circulates, may be called the resjnratory
oxygen of hcevwglobin. The processes that occur in the lungs and
tissues, resulting in the oxygenation and deoxygenation respectively
of the haemoglobin, may be imitated outside the body, using either
blood or pure solutions of haemoglobin. The respiratory oxygen can
be removed, for example, in the Torricellian vacuum of a mercurial
air-pump, or by passing a neutral gas like hydrogen through the
blood or by the use of reducing agents such as ammonium sulphide or

THE BLOOD 115

Stokes's reagent.^ One gramme of haemoglobin will combine with
1-34: c.c. of oxygen.

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

These colour-changes may be more accurately studied with the
spectroscope, and the constant position of the absorption bands seen
constitutes an important test for blood pigment.

The Spectroscope. — "When a ray of white Hght 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 spht into its con-
stituent colours, which may be allowed to fall on a screen. 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.

The spectrum of sunlight is interrupted by numerous dark 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 0, in the red ; D, in the yellow ;

E, b, 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 Hues 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 lainp 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 brighb yellow light will be found

absent, and in its place a dark line, or rather two dark lines very

close together, occupying the same position as the two bright hues

of the sodium spectrum. The sodium vapour thus absorbs the same

rays as those which it itself produces at a higher temperature. Thus

the D line, as we term it, in the solar spectrum is due to the presence

of sodium vapour in the solar atmosphere. The other dark lines are

similarly accounted for by other elements.

* 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.

I 2

116

ESSENTIALS OF CHEMICAL PHYSIOLOGY

The large form of spectroscope (fig. 32) consists of a tube A, called
the collimator, with a slit at the end S, and a convex lens at the end L.
The latter makes the rays of light passing through the slit from the
source of light parallel : they fall on the prism P, and then the
spectrum so formed is focussed by the telescope T.

Fig. 32. — Diagniin of aptctroscope.

The third tube, D, seen in the next figure (fig. 33) carries a small
transparent scale of wave-lengths, so that the position of any
point in the spectrum may be given in terms of the corresponding
wave-lengths.

Fig. 33. — Spectroscope: A, collimator with adinstablc slit at one (left) end and collimating lens at
the other (right) end ; B, telei-c(Ji)e moving on graduated arc divided into degrees ; C, prism or
combination of prisms ; D, tube for scale ; E, miiTor for illuminating scale ; F, vessel with
parallel glass sides for holding lluid, shown with the flat ?ide towards the reader ; I, long
spectroscope bottle for examining a deep layer of fluid ; H, Argaud burner ; G, condenser for
concentrating the light from H on the slit, (From a photograph taken by Dr.MacMunii for
McKendrick's ' Physiologj'.')

If we now interpose between the source of light and the slit S
a piece of coloured glass (H in fig. 32), or a solution of a coloured

THE BLOOD 117

substance contained in a vessel with parallel sides (the haematoscope
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
absoyytion hands, corresponding to the light absorbed by the coloured
medium. Thus a solution of oxyhaemoglobin of a certain strength
gives two bands between the D and E lines ; haemoglobin gives only
one ; and other red solutions, though to the naked eye similar to
oxyhaemoglobin, will give characteristic bands in other positions.

A convenient form of small spectroscope is the direct-vision
sioectroscoye, in which, by an arrangement of alternating prisms
of crown and flint glass (see fig. 34), the spectrum is observed
by the eye in the same line as the tube furnished with the slit.
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 combina-
tion of the microscope and direct-vision spectroscope, called the
micro- spectroscope, is used.

Fig. 36 illustrates a method of representing absorption spectra

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

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
the percentage amounts of oxyhaemoglobin present in I, of haemo-
globin in II. The width of the shadings at each level represents the
position and amount of absorption corresponding to the percentages.
The characteristic spectrum of oxyhaemoglobin, as it actually
appears through the spectroscope, is seen in the next figure (fig. 37^
spectrum 2). There are two distinct absorption bands between the
D and E lines ; the one nearest to D (the a band) is narrower,
darker, and has better defined edges than the other (the /3 band).
As will be seen on looking at fig. 36, a solution of oxyhaemoglobin 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

118

ESSENTIALS OF CHEMICAL PHYSIOLOaY

thick band overlapping both D and E, and a stronger solution only
lets the red light through between C and D. A solution which gives

Fig. 35.— Stand for direct-vision spectroscope : S, spectroscope ; T, test-tnbe for coloured
substance under investigation.

the two characteristic bands must therefore be a very dilute one.
The one band (y band) of haemoglobin (fig. 37, spectrum 3) is not so

Fig. 36.— Graphic representations of the amount of absorption of light by solution (I) of oxyhaemo-
globin, (IT) of haemoglobin, of different strengtlis. The shading indicates the amount of
absorption of the spectrum ; the figures on the right border express percentages. (Rollett.)

well defined as the a and p bands. On dilution it fades rapidly, so
that in a solution of such strength that both bands of oxyhaemoglobin

THE ELOOD

119

would be quite distinct the single band of haemoglobin has dis-
appeared from view. The oxyhaemoglobin bands can be distinguished
in a solution which contains only one part of the pigment to 10,000
of water, and even in more dilute solutions which seem to be colourless
the a band is still visible.

Methaemoglobm. — This may be produced artificially by adding
such reagents as potassium ferricyanide or amyl nitrite to a solution
of oxyhaemoglobin ; it may also occur in certain diseased conditions
in the urine ; it is therefore of considerable practical importance. It
can be crystallised, and is found to contain the same amount of
oxygen as oxyhaemoglobin, only combined differently. The oxygen
is not removable by the air-pump, nor by a stream of a neutral gas
C D E& F G

Fig. 37,-1, Solar spectrum : '2. ^p. Mrrum of oxyhaemoglobin (0-37 per cent, solution) ; 3, spectrum of
hasmoglobin ; 4, spectrum of CO-h«moglobin ; 5, spectrum of methfemoglobin (concentrated
solution).

like hydrogen. It can, however, by reducing agents like ammonium
sulphide, be made to yield haemoglobin. Methaemoglobin is of a
brownish-red colour, and gives a characteristic absorption band in
the red between the C and D lines (fig. 37, spectrum 5).

Tho ferricyanide of potassium or sodium not only causes the
conversion of oxyhaemoglobin into methaemoglobin, but if the reagent
is added to blood which has been previously laked by the addition of
twice its volume of water there is an evolution of oxygen. If a small
amount of sodium carbonate 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
oxyhaemoglobin is discharged. This is at first sight puzzling, because,

120 ESSENTIALS OF CHEMICAL PHYSIOLOOY

as just stated, methacmoglobin contains the same amount of oxygen
that is present in oxyhaBmoglobin. What occurs is that, after the
oxygen is discharged from oxyhaemoglobin, an equal quantity of
oxygen takes its place from the reagents added. The oxygen atoms
of the methsemoglobin must be attached to a different part of the
haematin group from the oxygen atoms of the oxyhaemoglobin, so
that the haematin group when thus altered loses its power of com-
bining with oxygen and carbonic oxide to form compounds which
are dissociable in a vacuum.

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

Hb02 + 4Na3CycFe + 4NaHCO,=Hb02 + 4Na4Cy,Fe

[oxyhaemo- [sodium ferri- [sodium bicar- [metlifemo- [sodium ferro-
globin] cyanide] bnnate] globin] cyanide]

+ 4CO2 + P.H2O + O2.

[carbonic [water] [oxygen]
acid]

Carbonic Oxide Haemoglobin may be readily prepared by passing
a stream of carbonic oxide or coal gas through blood or through a
solution of oxyhaemoglobin. It has a peculiar cherry-red colour. Its
absorption spectrum is very like that of oxyhaemoglobin, but the two
bands are slightly nearer the violet end of the spectrum (fig. 37,
spectrum 4). Keducing agents, such as ammonium sulphide, do not
change it ; the gas is more firmly combined than the oxygen in
oxyhaemoglobin. CO-haemoglobin forms crystals like those of oxy-
haemoglobin : 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 ; it is a powerful poison
combining with the haemoglobin of the blood, and thus interfering
with normal respiratory processes. The colour of the ])lood and its
resistance to reducing agents are in such cases characteristic. .

Nitric Oxide Haemoglobin. — When ammonia is added to blood, and
then a stream of nitric oxide is passed through it, this compound is
formed. It may be obtained in crystals isomorphous with oxy- and
CO-haemoglobin. It also has a similar spectrum. It is even more
stable than CO-haemoglobin ; it has little practical interest, but is of
theoretical importance as completing the series.

TESTS FOR BLOOD

These may be gathered from preceding descriptions. Briefly,
they are microscopic, spectroscopic, and chemical. The best chemical
test is the formation of haemin crystals. The old test with tincture

THE BLOOD 121

of guaiacum and hydrogen peroxide, the blood causing the red tincture
to become green, is not very trustworthy, as it is also given by many
other organic substances ; it is, however, with certain precautions,
used by some.

In medico-legal cases it is often necessary to ascertain whether
a red fluid or stain upon clothing is or is not blood. In any such
case it is advisable not to rely upon one test only, but to try every
means of detection at one's disposal. To discover whether it is blood
or not, is by no means a difficult problem, but to distinguish human
blood from that of the common mammals is possible only by the
* biological ' test described at the end of the next section.

IMMUNITY

The chemical defences of the body against injury and disease
are numerous. The property that the blood possesses of coagu-
lating is a defence against haemorrhage ; the acid of the gastric
juice is a protection against harmful bticteria introduced with food.
Bacterial activity in urine is inhibited by the acidity of that secretion.

Far more important and widespread in its effects than any of the
foregoing is the bactericidal {i.e. bacteria-killing) action of the blood
and lymph ; a study of this question has led to many interesting
results, especially in connection with the important problem of
immunity.

It is a familiar fact that one attack of many of the infective maladies
protects us against another attack of the same disease. The person
is said to be immune, either partially or completely, against that
disease. Vaccination produces in a patient an attack of cowpox or
vaccinia. This disease is either closely related to smallpox, or
may be it is smallpox modified and rendered less malignant by passing
through the body of a calf. At any rate, an attack of vaccinia renders
a person immune to smallpox for a certain number of years. Vac-
cination is an instance of what is called 'protective inoculation, which
is now practised with more or less success in reference to other
diseases, such as plague and typhoid fever. The study of immunity
has also rendered possible what may be called curative hwculation, or
the injection of antitoxic material as a cure for diphtheria, tetanus,
snake poisoning, &c.

The power the blood possesses of slaying bacteria is not limited
to the colourless corpuscles ox phagocytes, but is also a property of the
fluid part of the blood, at any rate in the case of some micro-
organisms. The chemical characters of the substances which kill
the bacteria are not fully known ; but they appear to be protein

122 ESSENTIALS OF CHEMICAL PHYSIOLOaY

in nature. The bactericidal powers of blood are destroyed by
heating it for an hour to 55° C. The balance of evidence appears
to be in favour of the view that the substances in question
originate from the leucocytes, and phagocytosis becomes more
intelligible if this is accepted. The substances, whatever be their
source or their chemical nature, are called hacterio-lysins.

Closely allied to the bactericidal power of blood, or blood-serum,
is its globulicidal power. By this one means that the blood-serum of
one animal has the power of dissolving the red blood-corpuscles of
another species. If the serum of one animal is injected into the
blood-stream of an animal of another species, the result is a destruction
of its red corpuscles, which may be so excessive as to lead to the
passing of the liberated haemoglobin into the urine (haemoglobinuria).
The substances in the serum that possess this property are called
hcemolysins, and though there is some doubt whether bacterio-
lysins and haemolysins are absolutely identical, there is no doubt
that they are closely related substances.

Normal blood thus possesses not only j^hagocytes, which eat up
bacteria, but also a certain amount of chemical substances which are
inimical to the life of our bacterial foes. But suppose a person gets
' run down ' ; everyone knows he is then more liable to ' catch any-
thing.' This coincides with a diminution in the bactericidal power of
his blood. But even a perfectly healthy person has not an unlimited
supply of bacterio-lysins, and if the bacteria are sufficiently numerous
he will fall a victim to the disease they produce. Here, however,
comes in the remarkable part of the defence. In the struggle he
will produce more and more bacterio-lysin, and if he gets well it
means that the bacteria are finally vanquished, and his blood remains
rich in the particular bacterio-lysin he has produced, and so will
render him immune to further attacks from that particular species
of bacterium. Every bacterium seems to cause the development of
a specific anti -substance.

Immunity can more conveniently be produced gradually in animals,
and this applies, not only to the bacteria, but also to the toxins they
form. If, for instance, the bacilli which produce diphtheria are
grown in a suitable medium, they produce the diphtheria poison, or
toxin, much in the same way that yeast-cells will produce alcohol
when grown in a solution of sugar. Diphtheria toxin is associated
with a proteose, as is also the case with the poison of snake venom.
If a certain small dose called a ' lethal dose ' is injected into a guinea-
pig the result is death. But if the guinea-pig receives a smaller
dose it will recover ; a few days after it will stand a rather larger

THE BLOOD 123

dose ; and this maj^ be continued until, after many successive gradually
increasing doses, it will finally stand an amount equal to many lethal
doses without any ill effects. The gradual introduction of the toxin
has called forth the production of an antitoxin. If this is done in
the horse instead of the guinea-pig the production of antitoxin is
still more marked, and the serum obtained from the blood of an
immunised horse maybe used for injecting into human beings suffering
from diphtheria, and it rapidly cures the disease. The two actions of
the blood, antitoxic and antibacterial, are frequently associated, but
may be entirely distinct.

The antitoxin is also a protein probably of the nature of a globulin ;
at any rate it is a protein of larger molecular weight than a proteose.
This suggests a practical point. In the case of snake-poisoning the
poison gets into the blood rapidly owing to the comparative ease with
which it diffuses, and so it is quickly carried all over the body. In
treatment with the antitoxin or antivenin, speed is everything if life
is to be saved ; injection of this material under the skin is not much
good, for the diffusion into the blood is too slow. It should be
injected straight away into a blood-vessel.

There is no doubt that in these cases the antitoxin neutralises the
toxin much in the same way that an acid neutralises an alkali. If
the toxin and antitoxin are mixed in a test-tube, and time allowed
for the interaction to occur, the result is an innocuous mixture. The
toxin, however, is merely neutralised, not destroyed ; for if the mix-
ture in the test-tube is heated to 68° C. the antitoxin is coagulated
and destroyed, and the toxin remains as poisonous as ever.

Immunity is distinguished into active and passive. Active immunity is
produced by the development of protective substances in the body ; passive
immunity by the injection of a protective serum. Of the two the former is
the more permanent.

Bicin, the poisonous protein of castor-oil seeds, and ahriv, that of the
Jequirity bean, also produce when gradually given to animals an immunity,
due to the production of antiricin and antiabrin respectively.

Ehrlich's hypothesis to . explain such facts is usually spoken of as the
Hide-chain theory of imnmnity. He considers that the toxins are capable of
uniting with the protoplasm of the living cells by possessing groups of atoms
like those by which nutritive proteins are united to cells during nonnal
assimilation. He terms these haptoj^hor groups, and the groups to which
these are attached in the cells he terms receptor groups. The introduction
of a toxin stimulates an excessive production of receptors, which are finally
thrown out into the circulation, and the free circulating receptors constitute
the antitoxin. The comparison of the process to assimilation is justified by
the fact that non-toxic substances like milkor egg-white introduced gradually
by successive doses into the blood-stream cause the formation of anti-sub-
stances capable of coagulating them.

Up to this point I have spoken only of the blood, but m.onth by month
workers are bringing forward evidence to show that other cells of the body

124 ESSENTIALS OV CHEMICAL PHYSIOLOaV

may by similar measures be rendered capable of producing a corresponding
protective mechanism.

One further development of the theory I must mention. At least two
different substances are necessary to render a serum bactericidal or globulicidal.
The bacterio-lysin or haemolysin consists of these two substances. One of
these is called the immune body, the other the comjjlement. We may
illustrate the use of these terms by an example. The repeated injection of
the blood of one anhnal {e.g. the goat) into the blood of another animal {e.g.
a sheep) after a time renders the latter animal immune to farther injections,
and at the same time causes the production of a serum which dissolves
readily the red blood-corpuscles of the first animal. The sheep's serum is
thus hemolytic towards goat's blood-corpuscles. This power is destroyed by
heating to 56° C. for half an hour, but returns when fresh serum of any
animal is added. The specific immunising substance formed in the sheep
is called the immune body ; the ferment-like substance destroyed by heat
is the complement. The latter is not specific, since it is furnished by the
blood of non-immunised animals, but it is nevertheless essential for haemo-
lysis. Ehrlich believes that the immune body has two side groups — one
which connects with the receptor of the red corpuscles, and one which unites
with the haptophor group of the complement, and thus renders possible the
ferment-like action of the complement on the red corpuscles. Various
antibacterial serums, which have not been the success in treating disease they
were expected to be. are probably too poor in complement, though they may
contain plenty of the immune bodj'.

To put it another way : the cell-dissolving substances cannot act on their
objects of attack without an intermediate substance to anchor them on the
substance in question. This intermediary substance, known as the immune
body or amboceptor, is specific, and varies with the substance to be attacked
(red corpuscles, bacterium, toxin, &c.). The complement may be compared
to a person who wants to unlock a door ; to do this effectively he must be
provided with the proper key (amboceptor or immune body).

Quite distinct from the bactericidal, globulicidal, and antitoxic
properties of blood is its agglutinating action. This is another result
of infection with many kinds of bacteria or their toxins. The blood
acquires the property of rendering immobile and clumping together
the specific bacteria used in the infection. The test applied to the
blood in cases of typhoid fever, and generally called Widal's reaction,
depends on this fact.

The substances that produce this effect are called agglutinins.
They also are probably protein-like in nature, but are more resistant
to heat than the lysins. Prolonged heating to over 60° C. is necessary
to destroy their activity.

We thus see that the means the body possesses of combating
bacterial invasion are numerous. In some cases the bacteria are
killed by bacterio-lysins, and in other cases they are directly attacked
and devoured by the phagocytes. Bacteria which are destroyed in
this way produce no evil results, whereas those which are not
destroyed are called pathogenic, or disease-producing organisms.
There is still another means of defence, for if the bacteria are not

THE 13L00D 126

'destroyed the poisons or toxins they produce are in certain other
cases ueutrahsed by antitoxins.

Metschnikoff's view, which is very widely accepted by bacterio-
logists, is that the most stress should be laid upon phagocytosis as
the principal factor in tha resistance of the body to bacteria ; and the
recent discovery of opsonins by Sir A. E. Wright not only emphasises
this opinion, but shows how the body fluids co-operate with the
phagocytes in the process. The word * opsonin ' is derived from a
Greek word which means 'to prepare the feast.' Washed bacteria
from a culture are distasteful to leucocytes, and would therefore,
other things being equal, be pathogenic if injected into an animal's
body. But if the bacteria have been previously soaked in serum,
especially if that serum has been obtained from the blood of an
animal previously immunised against that special bacterium, then
the leucocytes devour them eagerly. It was at first supposed that
something had been added to the bacterium to make it tasty, and
that each kind of bacterium requires its own special sauce or
opsonin. It is, however, equally possible that the serum has not
added anything to the bacterium, but removed from it something
that previously made it distasteful. At any rate the ultimate effect
is the same, and the bacterium is rendered non-pathogenic. When a
person is attacked with some invading organism, say the tubercle
bacillus, if that person's blood is naturally rich in the proper kind of
opsonin he will not be troubled with tuberculosis ; but if the opsonic
power of his blood is low the organism will produce the disease.
The modern treatment of tuberculosis aims at increasing the opsonic
power of the blood by improving the general condition of the patient
by good food and pure air, and also by the injection of the appro-
priate opsonin into his -blood.

Lastly, we come to a question which more directly appeals to the
physiologist than the preceding, because experiments in relation to
immunity have furnished us with what has hitherto been lacking,
a means of distinguishing human blood from the blood of other
animals.

The discovery was made by Tchistovitch (1899), and his original
experiment was as follows : — Eabbits, dogs, goats, and guinea-pigs
were inoculated with eel-serum, which is toxic ; he thereby obtained
from these animals an antitoxic serum. But the serum was not only
antitoxic ; it also produced a precipitate when added to eel-serum,
though not when added to the serum of any other animal. In other
w^ords, not only has a specific antitoxin been produced, but also a
specific precipitin. Numerous observers have since found that this is

126

ESSENTIALS OF CHEMICAL PHYSIOLOGY

a general rule throughout the animal kingdom, including man. If, for
instance, a rabbit is treated with human blood, the serum ultimately
obtained from the rabbit contains a specific precipitin for human
blood ; that is to say, a precipitate is formed on adding such a rabbit's
serum to human blood, but not when added to the blood of any other
animal.^ The great value of the test is its delicacy: it will detect
the specific blood when it is greatly diluted, after it has been dried
for weeks, or even when it is mixed with the blcod of other animals.

CHEMISTRY OF RESPIRATION

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

The air in the alveoli of the lungs, and the blood in the pulmonary
capillaries are only separated by the thin capillary and alveolar
walls. The blood parts with its excess of carbonic acid and watery
vapour to the alveolar air ; the blood at the same time receives from
the alveolar air the oxygen which renders it arterial.

The intake of oxygen is the commencement, and the output of
carbonic acid the end, of the series of changes known as respiration.
The intermediate steps take place all over the body, and constitute
what is known as internal or tissue respiration. The exchange of
gases which occurs in the lungs is sometimes called in contradistinc-
tion exterjial respiration. We have already seen that the oxyhaemo-
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 substances pass into the venous
blood, and the carbonic acid and a portion of the water find an outlet
by the lungs.

Inspired and Expired Air. — The composition of the inspired and
expired air may be compared in the following table : —

-

Inspired or atmospheric Air

Expired Air

16-03 vols, per cent.
79
4-4 „

saturated
that of body (37° C.) '

Oxygen .

Nitrogen .
Carbonic acid .
"Watery vapour .
Temperature

20-96 vols, per cent.
79
0-04 „

variable

' There may be a slight reaction with the blood of allied animals ; for instance
with monkey's blood in the case of man.

RESPIEATION 127

The nitrogen remains unchanged. The recently discovered gases,
argon, crypton, &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 4^. If the
inspired and expired airs are carefully measured at the same tempera-
ture and barometric pressure, the volume of expired air is thus rather
less than that of the inspired. The conversion of oxygen into carbonic
acid would not cause any change in the volume of the gas, for a
molecule of oxygen (0^) would give rise to a molecule of carbonic acid
(CO2) 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 protein are

eaten. The quotient ^—v^-^T^-, is called the respiratory quotient.
O2 absorbed

Normally it is =0*9, but this varies considerably with diet, as
o

just stated.

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

20

lto2

40

Venous Blood

Oxygen
Nitrogen .
Carbonic acid .

8 to 12

lto2

46

The nitrogen in the blood is simply dissolved from the air just
as water would dissolve it : it has no physiological importance. The
other two gases are present in much greater amount than can be
explained by simple solution ; they are, in fact, chiefly present in
loose chemical combinations. Less than one volume of the oxygen
and about two of carbonic acid are present in simple solution in the
plasma.

Oxygen in the Blood. — The amount of gas dissolved in a liquid
varies with the pressure of the gas ; double the pressure and the

128 ESSENTIALS OF CHEMICAL PHYSIOLOGY

amount of gas dissolved is doubled. The oxygen in the blood does
not vary directly with oxygen pressure, for the amount of that gas
in simple solution forms only a small fraction of the total present.
This small amount is of coarse doubled by doubling the pressure, but
such an increase is insignificant, the bulk of the gas being in chemical
union w^ith haemoglobin. The oxygen of oxyhtemoglobin can be
replaced by equivalent quantities of other gases, such as carbonic
oxide. 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 haemoglobin, and
thus leaves the plasma free to absorb more oxygen ; and this goes on
until the haemoglobin is entirely, or almost entirely, saturated with
oxygen. The reverse change occurs in the tissues where the partial
pressure of oxygen is lower than in the plasma, or the lymph that
bathes the tissue elements ; the plasma parts with its oxygen to the
lymph, the lymph to the tissues ; the oxyhaemoglobin then undergoes
dissociation to supply more oxygen to the plasma and lymph, and this
in turn to the tissues once more. This goes on until the oxyhaemo-
globin loses a great portion of its store of oxygen, but even in asphyxia
it does not lose all.

The following values are given for the tension of oxygen in per-
centages of an atmosphere : —

External air 20-96

Alveolar air 13-16 ^

Arterial blood ..... 14 |

Tissues . 0

The arrow shows the direction in which the gas passes.

The methods of obtaining the gases of the blood and analysing
them are described in the Appendix. When the gases are being
pumped off from the blood, very little oxygen comes off until the
pressure is greatly reduced, and then, at a certain point, it is suddenly
disengaged. This shows it is not in simple solution, but is united
chemically to the haemoglobin as oxyhaemoglobin, which is dissociated
when the pressure is extremely low.

The ability of the tissues to form reduction products is shown by
Ehrlich's experiments with methylene blue and similar pigments.
Methylene blue is more stable than oxyhaemoglobin ; but if it is injected
into the circulation of a living animal, and the animal killed a few
minutes later, the blood is found dark blue, but the organs colourless.
On exposure to oxygen the organs become blue. In other words
the tissues have formed a colourless reduction product from the

RESPIRATION 129

methylene blue ; on exposure to the air this is oxidised, and the
blue pigment is thus regenerated.

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,
such as phosphoric acid, is necessary to displace it. Fresh red cor-
puscles will, however, take the place of the phosphoric acid, and thus
it has been surmised that oxyhaemoglobin 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 com-
bination 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 solu-
bility 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 about 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. Hence the remainder of the
gas, 95 per cent., is in a condition of chemical combination. The
chief compound appears to be sodium bicarbonate.

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 (NagCOa), and
mono-sodium phosphate (NaH2P04) ; but if the carbonic acid is
diminished, the phosphoric acid obtains the greater share of sodium

' To be exact, the solubility of carbon dioxide in plasma is a little less than
in pure water.

130 ESSENTIALS OF CHEMICAL PHYSIOLOGY

to form disodium phosphate (Nti2HP04). 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 .
Venous blood
Alveolar air
External air

5 to 9 )
3-8 to 5-4 in dog
2-8 j

0-04

The arrow indicates the direction in which the gas passes : namely,
in the direction of pressure from the tissues to the atmosphere.

In some other experiments, also on dogs, the following are the

figures given : —

Arterial blood . . . . . . 2 '8

Venous blood . . ". . . . 5*4 ^

Alveolar air ..... . 3"56 |

Expired air . . . . . . 2*8

It will be seen from these figures that the tension of carbonic acid
in the venous blood (5-4) is higher than in the alveolar air (3-56) ; its
passage into the alveolar air is therefore intelligible by the laws of
diffusion. Diffusion, however, should cease when the tension of the
gas in the blood and in the 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 tliis 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. Eecent 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

EESPIRATION 131

greater than iu the atraosphere. In the swim-bladder of fishes
(which is analogous to the lung 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.

Tension of Gases in Fluids. — It is necessary to understand
thoroughly the expression * tension of gases in fluids ' ; we will
therefore go into the matter a little more fully.

The first question which arises is, In what circumstances will a
gas, dissolved in a fluid, diffuse out of the fluid into the air in contact
with it ? or vice versa, in what circumstances will a gas diffuse out of
the air into the fluid, and at what rate will it do so ?

The answer depends upon the physical constants of the fluid and
of the atmosphere ; and these must be determined experimentally.

As an example the following instance may be taken : — 100 c.c.
of water charged with 80 c.c. of carbonic acid are shaken with pure
air in a closed bottle of 500 c.c. capacity. The carbonic acid will
come out of the water at first, but as the shaking continues the
carbonic acid will come out more and more slowly until it entirely
ceases to do so. Analysis of the air and of the water in the bottle
would show that the water had not parted with all its carbonic acid.
It would be found that the water contained 16 c.c. of carbonic acid
dissolved in it, while 64 c.c. have diffused out into the air.

At the end of this experiment, then, there will be in the atmosphere
64 c.c. of carbonic acid in a space of 400 c.c. If the whole space
(400 c.c.) were filled with carbonic acid its pressure would be 760 mm.
The partial pressure of carbonic acid in the atmosphere of the bottle
is therefore 760 x /q^o mm- of mercury =122 mm. Water, therefore,
containing 16 volumes per cent, of carbonic acid is in equilibrium
with an atmosphere in which the carbonic acid exerts a pressure of
122 mm. This fact is stated shortly by the phrase * when water has
a carbonic acid tension of 122 mm. it contains 16 per cent, of
carbonic acid.'

The amount of carbonic acid which would be contained in any
other fluid when it was in equilibrium with an atmosphere having a

k2

132

ESSENTIALS OF CHEMICAL PHYSIOLOGY

122 mm. pressure of carbonic acid would be different from that
contained in water. For instance, alcohol would contain 50 per cent,
of the gas. In both fluids the tension of carbonic acid is the same
but the quantity of carbonic acid would be as stated, and therefore
different.

The tension of a gas in a liquid, therefore, is the pressure of that
gas in an atmosphere of such a composition, that the liquid would
neither acquire that gas from the atmosphere nor impart that gas to
it, if the liquid and the atmosphere were brought into intimate contact,
as by shaking.

Measurement of Tension in Fluids — Aerotonometer.— The measure-
ment of the tension of gases in fluids is conducted upon the principles
of the example given above. The instrument for
the purpose is called an aerotonometer. The form
used by Loewy consists simply of a closed bottle,
into which the blood and the air can be put, and from
which they can be withdrawn by suitable means.
Through the stopper of the bottle three tubes pass —
A, B, and c— each of which is provided with a piece
of rubber tubing and a clip. The tube A is used
for introducing or expelling the blood (e). c is
used for introducing or expelling the air ; b is con-
nected with a rubber bag containing water inside
Loewy's Agrotouometer. the bottlc, while outside a connection can be made
with a syringe. A little mercury should be put into
the bottle to defibrinate the blood. To determine the carbonic acid
tension in blood several bottles should be filled with gases of known
composition from gasometers before the experiment. Into each bottle
some blood is drawn from the animal. This is done by attaching a to
a cannula in one of its vessels, and then, when water is withdrawn
from the bag d by the syringe, a corresponding amount of blood enters
the aerotonometer. Each bottle is shaken violently for some time.
When equilibrium has been estabhshed the air can be taken from
the air space f by attaching c to an air-analysis apparatus, and forcing
water into the bag d from the syringe attached to b. An example may
illustrate the result which might be obtained.

Determination of carbonic acid tenaion of blood.

Bottle ....

Initial percentage of the gas
Final percentage of gas present

From the above figures it will be seen that the blood has acquired

Fig. 38.

I

II

III

IV

V

6-0

5-5

5-0

4-5

4

5-8

5-4

5-1

4-7

4-8

RESPIRATION

133

carbonic acid from the air in bottles I and II, while the reverse has
taken place in bottles III, IV, and V. The carbonic acid tension of
the blood is therefore between 5'4: and 5*1 per cent.

Relation of Tension to Composition. — The aerotonometer may be
used to determine the relation of the tension of a gas in the blood
(oxygen or carbonic acid) to the quantity contained in that fluid.
The example given above might be extended for this purpose in the
following way. After the air has been w^ithdrawn for analysis, it is
only necessary to attach a to the blood-bulb of a mercurial gas pump,
and, by forcing more water into b, expel a sufficient quantity of
blood for analysis. If an analysis was made for each of the bottles,
the quantity of carbonic acid would be determined corresponding to
the tensions, 5'8, 5"4, 5*1, 4'7, and 4-3 per cent, respectively.

The comparision of the quantity of carbonic acid in the blood
with its tension is not of importance, but the corresponding measure-
ments for oxygen have been carried out with great care by a number
of observers, both for blood and for solutions of haemoglobin.

It is found that the two sets of observations agree very closely,
and this fact forms the most substantial evidence for our belief that
the oxygen in the blood is almost entirely associated with the haemo-
globin.

The relation of the quantity of oxygen in the blood to the tension
which it exerts may be conveniently set forth in a curve, which is
called the dissociation curve of oxyhaemoglobin.

An example may illustrate
the use to which the informa-
tion, given in this curve, may
be put. The amount of blood
which comes, say, from the
pancreas is too small to allow^
of a direct determination of the
tension of the oxygen which it
contains. We can, however,
calculate it from the following
data. (1) The amount of oxygen
which the blood would contain
if saturated (this can be arrived

at by saturating arterial blood and analysing it). (2) The amount of
"oxygen in the venous blood. In an actual experiment (1 ) was 22*0 c.c,

^

^-^ — r"t"1 i !

/

H^

^-"

U M M

/ .

/"

/

/

'\f>

\\\

,.l

/■

"\l

ll-

_.JJ

_i

n^ 20 to 43 so £} 70 tO DO 100 ItJ 120 133 MO

i;. 3!).— Dissociation of oxygen curves for Wood (n)
and Li«;moglobin solution (h) at 38° C. The
figures along the base line are pressures in milli-
metres of mercury, aud those along the ordinate
are percentages of oxygen. (Bohr.)

(2) was 10-7 c.c

By referring to the dissociation

10-7

The saturation then was 100 x ^^ = 49

per cent,
curve w^e see that 49 per cent.

184 ESSENTIALS OF CHEMICAL rHYSTOLOGY

saturation corresponds with an oxygen tension of about 14 mm. of
mercm'y, or 1*8 per cent, of an atmosphere.

The Carbonic Oxide Method of Estimating the Oxygen Tension of Arterial
Blood. — This method was devised by Haldane, and is considered by him and
Lorrain Smith to give more trustworthy results than those obtained by the
aerotonometer. If blood is exposed to a mixture of carbonic oxide and
oxygen, the haemoglobin will become saturated by these gases according to
their relative tensions. If a number of experiments are performed using
different percentages of carbonic oxide, the results may be expressed
graphically as the curve of dissociation of carboxyhaemoglobin in air. When,
in place of such experiments in vitro, an animal is made to breathe air contain-
ing a known percentage of carbonic oxide, the comparison of the saturation
of its blood with the saturation of its blood in vitro, exposed to the same
percentage of carbonic oxide in air (which has an oxygen tension of 20*9 per
cent.) gives us the means of discovering the oxygen tension in the arterial
blood of the lung capillaries : this will be higher or lower than that of the air
according as the saturation by carbonic oxide is correspondingly lower or higher.
A small animal like a mouse is made to breathe air containing a known per-
centage of carbonic oxide. After a sufficient time the animal is killed and
the amount of carboxyhaemoglobin is determined colorimetrically in a drop
of its blood. The data thus obtained are compared with the data previously
expressed in the curve of dissociation of carboxyhaemoglobin in air ; it is
then easy to calculate whether the oxygen tension in the blood is higher or
lower than that of air. The results of the method show generally that the
tension of oxygen in the arterial blood as it leaves the lungs is liigher than
could result from simple diffusion of the oxygen through the alveolar
epithelium ; in other words, the epithelial cells are capable of secreting oxygen
into the blood until an oxygen pressure is reached considerably above that in
the alveolar air.

The results expressed in percentages of an atmosphere are as follows : —
Oxygen tension of arterial blood in man, 38-5; in mouse, 22*6; in dog.
21 ; in cat, 35-3 ; in rabbit, 27*6, and in birds 30 to 50 per cent. The results
in the case of man and larger animals probably require revision, as it is not
certain that the time allowed for the establishment of the balance of carbonic
oxide and oxygen has been sufficient in any of the experiments.

Tissue-Respiration. — Before the processes of respiration were fully
understood the lungs were looked upon as the seat of combustion ;
they were regarded as the stove for the rest of the body where effete
material was brought by the venous blood to be burnt up. When it
was shown that the venous blood going to the lungs already contained
carbonic acid, and that the temperature of the lungs is not greater
than that of the rest of the body, this explanation had of necessity to
be dropped.

Physiologists next transferred the seat of the combustion to the
blood ; but since then innumerable facts and experiments have
shown that it is in the tissues themselves, and not in the blood, that
combustion occurs. The methylene-blue experiment already described
(p. 128) shows this ; and the following experiment is also quite con-
clusive. A frog can be kept alive for some time after salt solution is

IlESPI RATIO X

135

is necessary (1) to
analyse the blood

#0^

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.

The following are the amounts of oxygen used up per minute by
one gramme of certain epithelial and muscular organs respectively : —
Submaxillary gland 004 c.c, pancreas 0-05 c.c, kidney O'OS c.c,
heart (contracting very feebly and slowly) 0-007 c.c, muscles of leg
(with spinal cord destroyed) 0*003 c.c.

In order to obtain data, such as the above, it
analyse the blood going to the organ ; (2) to
emerging from the organ ; (3) to determine the
amount of blood passing through the organ in
one minute.

Analysis of the blood may be performed
by either of two methods, the mercurial air-
pump (see Appendix) or the chemical method of
expelling the oxygen and carbonic acid from the
blood by means of potassium ferricyanide and
phosphoric acid respectively.

Chemical Method of Blood Gas Analysis. —
When a solution of hsemoglobin is treated W'ith
potassium ferricyanide it yields all its oxygen to
the air on shaking, just as urea yields its nitrogen
when treated with sodium hypobromite. The
apparatus used for determining the oxygen in
blood is very similar to a Dupre's urea apparatus
(see p. 142). The blood (5 c.c.) is placed in the
large bottle (f) (fig. 40) underneath a layer of
dilute ammonia solution. The blood is thus pro-
tected from the air, while the apparatus becomes
equal in temperature to the bath in which it is
placed. The blood is shaken with the ammonia
solution which lakes it thoroughly; the ferri-
cyanide solution is then spilt into the laked blood
from the tube g, and the oxj^gen is shaken out of the solution.
When the oxygen has been determined the bottle is opened and
phosphoric acid is placed in the small tube g : this is subsequently
spilt into the mixture of blood, ammonia, and ferricyanide ; it liberates
the carbonic acid, w^hich is also shaken out of the fluid. The carbonic
acid does not come completely out, however, and a con-ection has to be
introduced for the quantity w^hich remains in solution. The gas w^hich

Fio. 40.— Barcroft's appa-
ratus for obtaining tJie
blood gases.

136

ESSENTIALS OF CHEMICAL PHYSIOLOGY

comes off passes into the tube c, which was originally filled up to the
zero mark with water in continuity with that in the reservoir a. This
would depress the column of fluid in c and raise that in the open
tube D, which is graduated in millimetres. In practice, however, it
is convenient to keep the gas always at the same volume : this may
be done by raising the pressure in the open tube (d) by squeezing
some of the water with which the gauge is filled out of a rubber
reservoir b, which forms the base of the gauge ; thus the level of the
water in c is kept at the zero, while that in d rises from h to i.
The actual measurement then is the increase of pressure {i.e. the
height of the column of water hi) which is necessary to keep the
gases in the tube c at the same volume as that which was previously
occupied by the air in that tube. From this the quantity coming off
can be calculated.

The chemical method is not quite so accurate as the vacuum pump,
but it is much more convenient for the study of many problems, as it
requires less blood, and, owing to its simplicity, a great number of
observations can be made upon a single animal.

Changes in Tissue Respiration caused by Activity.— In all organs,
so far as is known, increased activity is associated with increased
tissue respiration. The increase is commonly three- to six-fold. It is

Organ

1 1

'■ Oxygen i
Condition of rest used per Condition of activity
1 minute ,

Oxygen used
per minute

Muscle .

Nerves cut

1
1

0-003 c.c. i Tonic (nerves

nncut)

0-006 c.c.
0-020 c.c.

Heart .

Very slow and
feeble contrac-
tions

0-007 c.c. ! Normal contrac-
tions
Very active

1

0-014
0-054

Submaxillary
gland

Nerves cut ' 0*04 c.c. ' Chorda tympani

stimulated

1

0-12 c.c.
0-20 1

1

Pancreas

i

Not secreting

0-05 c.c. Secretion after in-
jection of secre-
tin

1
Kidney .

Scanty
secretion

0-03 c.c. After injection of
a diuretic

i

007 c.c.

KESPIRATION

137

often more easy to demonstrate the augmented oxygen consumption
than the augmented output of carbonic acid. This is due to several
causes : (1) carbonic acid is soluble in the tissues in which it is pro-
duced; (2) any change in the chemical reaction of the tissues alters
the amount of carbonic acid which they give out to the blood ; if, for
instance, it becomes more alkaline it retains a portion of its carbonic
acid.

The preceding table shows the variations which take place in the
oxygen intake of several organs as the result of activity produced by
widely different forms of stimulus (Barcroft).

The relation of the oxygen taken in to the carbonic acid given out
is well shown in the following experiment performed by Zuntz on the
leg of the dog : —

Blood-vessel

Gases in blood per cent.

Remarks

Femoral vein
1 Carotid artery

1

Oxygeu

1-2
14-4

(Jarbon
dioxide

36-32
4-92

Muscles tonic, nerves
being intact

1

j Exchange

i

1 Femoral vein
Carotid artery

18-20

14-4

2-85
13-30

33-16
23-06

10-1

After section of sciatic
and crural

Exchange

10-45

Two points must be noted in considering the above table : —

(1) That the exchange of gases was decreased on cutting the
nerves. The decrease in metabolism was greater even than the
figures show, for the blood flow through the leg was decreased.

(2) That the oxygen exchange and the carbonic acid exchange

changed in about the same proportions. The ratio of the carbonic

14-4
acid given out to the oxygen taken in was -^-^ with the nerves intact

±o'2

and

lO'l
10-45

with the nerves cut.

Effect of reduced Oxygen Tension on Tissue Respiration. — When
the oxygen tension in the blood is reduced, the tissues still take up the
same quantity of oxygen as before and give out as much, or slightly

138 ESSENTIALS OF CHEMICAL PHYSIOLOGY

more, carbonic acid ; thus, in the case of a dog, when the oxygen
tension in the blood is approximately 18 millimetres of mercury or
one fortieth of an atmosphere, 163 c.c. of oxygen per minute were
used up by the animal, and 172 c.c. of carbonic acid were given out ;
normally by the same animal the oxj^gen consumption was 157 c.c.
and the carbonic acid output 158 c.c. The reason why the tissues
extract oxygen with such readiness from the blood, even when the
oxygen exists in the blood at a low tension, is that there is no free
oxygen in the tissues themselves (and they always thirst for it). This
fact can be demonstrated in more than one way.

(1) No oxygen can be extracted from the tissues by exposing them
to the vacuum of an air-pump.

(2) The tissues possess the power of reducing such substances as
methylene blue (see p. 128).

Respiration in excised Tissues.— Excised frog's muscle retains its
power of contraction for a considerable time. During this time it
gives out carbonic acid. These facts are true whether the muscle be
in air or in nitrogen. In either case the muscle must be regarded as
partially or entirely asphyxiated, for the individual elements of the
muscle are cut off from that ready supply of oxygen which normally
reaches them. During life (and the living condition can be imitated
by placing an excised muscle in an atmosphere of pure oxygen) the
muscular substance breaks down into a number of somewhat simpler
substances : one of these is carbonic acid. The others, however, or
some of them, are at once built up again with the inclusion of oxygen
and some carbon-containing substance, perhaps sugar, into living
material. The muscle, therefore, does not contain any of the by-
products of its own metabolism. In excised muscle, when the oxygen
supply is deficient the by-products accumulate, as a result of which
very striking alterations take place. (1) The reaction of the muscle
becomes acid and the phenomena of fatigue and functional death set
in. (2) The proteins become coagulated, and this is the phj^sical basis
of rigor mortis.

The Chemical Stimulus to Respiration. — Haldane and Priestley
have introduced a new and simple method of obtaining the com-
position of the air in the alveoli. It consists in collecting one
sample of air expelled by a deep expiration at the end of a quiet
inspiration, and another of the air expelled by a deep expiration at
the end of a quiet expiration ; the mean of the two gives the com-
position of alveolar air. This is much simpler than the method
formerly employed by Pfliiger, which consisted in pumping off the
air from an occluded portion of a dog's lung by the means of a fine

RESPIRATION 189

elastic catheter introduced via the trachea and bronchus. It has the
further advantage that it can be appHed to the human subject.

They found that under constant atmospheric pressure in man, the
alveolar air contains a nearly constant percentage of carbon dioxide in
the same person. In different individuals this percentage varies
somewhat, but averages in men o'lG, in women and children 4-77 per
cent, of an atmosphere. With varying atmospheric pressures the
percentage varies inversely as the atmospheric pressure, so that the
pressure or tension of the carbon dioxide remains constant. The
oxygen pressure, however, varies widely under the same con-
ditions.

These observations and the next to be immediately described
furnish the chemical key to the cause of the amount of pulmonary
ventilation, and play an important part ill conjunction with the
respiratory nervous system in the regulation of breathing. For the
respiratory centre is not only affected by the impulses reaching it by
the vagi and other afferent nerves, but it is also very sensitive to any
rise in the tension of carbon dioxide in the blood that supplies it.
The changes in the tension of this gas in the arterial blood are
normally proportional to the changes in the carbon-dioxide pressure
in the alveoli, and thus the changes in the lung alveoli are trans-
mitted to the respiratory centre. They found that a rise of 0"2 per
cent, in the alveolar carbon-dioxide pressure is sufficient to double
the amount of alveolar ventilation during rest. During work the
alveolar carbon-dioxide pressure increases slightly, and the pulmonary
ventilation is consequently increased. Changes in the oxygen pressure
within wide limits have no such influence ; the normal chemical
stimulus to respiration is therefore the presence of an increase of carbon
dioxide, and not diminution of oxygen. If these limits are exceeded,
as when the oxygen pressure falls below 13 per cent, of an atmosphere,
the respiratory centre begins to be excited by want of oxygen, for
the alveolar carbon -dioxide pressure is lower than normal under such
circumstances. ' Apnoea 'is the name given to the cessation of breathing
which temporarily follows excessive ventilation of the lungs, as when
one takes a number of deep breaths in rapid succession. The deep
and rapid breathing clears out the carbon dioxide in the alveoli until
it is so small in quantity that it is insufficient to excite the respiratory
centre via the blood to action, the oxygen pressure at the same time
remaining sufficiently high not to excite the centre either. Hence
breathing ceases. The old idea that apnoea is due to over-oxygena-
tion of the blood has been abundantly disproved, but Head and others
have gone to an extreme in assuming that apncea is purely nervous

140 ESSENTIALS OF CHEMICAL PHYSIOLOGY

in origin. Haldane considers it is unnecessary to assume the
existence of a vagus apncea in man at all under normal circum-
stances. It is, however, probable that in normal breathing the
nervous reflex and the chemical stimulus have both to be reckoned
with, though the relative importance of these two factors is a question
for the future.

LESSON X

URINE

1. Test the reaction of urine on 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.

{h) Suljjhates. — Acidulate with hydrochloric acid and add barium chloride.
x\ white precipitate of barium sulphate is produced. Hydrochloric acid is
again added first, to prevent precipitation of phosphates.

(c) Phospliates. — i. Add ammonia ; a white crystalline precipitate of
earthy (that is, calcium and magnesium) phosphates is produced. This
becomes more apparent on standing. The alJfaline (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 oft'. 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 Dupre's apparatus (fig. 41) 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
cylinder of water, and can be raised and lowered at will. Measure 25 c.c. of
alkaline solution of sodium hypobromite (made by mixing 2 c.c. of bromine
witli 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

* 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.

142

ESSENTIALS OF CHEMICAL PHYSIOLOaY

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
tigure. Open the pinch-cock and lower the measuring tube until tlie surface

of the water with which the outer cylin-
der is filled is at the zero point of the
graduation. Close the pinch- cock, and
raise the measuring tube to ascertain
whether 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 measiu--
ing tube until the surfaces of the
water inside and outside it are at the
same level. Head off the amount of
gas evolved. This is nitrogen. The
carbonic acid resulting from the de-
composition of urea has been absorbed
by the excess of soda in the bottle.
35*4 c.c. of nitrogen are yielded by
0*1 gramme of urea. From this the
(juantity 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.
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
FIG. 41.-Dnpre"s urea apparatus. ^^^^^ 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
portion 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

URINE 143

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 undoubtedly exercise a selective influence, and,
among other things, prevent the albuminous constituents of the blood
from escaping. During the passage of this dilute urine 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.

GENERAL CHARACTERS OF URINE

Quantity. — A man of average weight and height passes from 1,400
to 1,600 c.c, or about 50 oz., daily. This contains about 50 grammes
(1^ oz.) of solids. The urine should be collected in a tall graduated
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. From the total quantity thus
collected in the twenty-four hours samples should be drawn off for
examination.

Colour. — This is some shade of yellow which varies considerably
in health with the concentration of the urine. It appears to be due
to a mixture of pigments : of these urobilin is the one of which we
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 haemoglobin. The bile pigment (and
possibly also the hsematin of the food) is in the intestines converted
into stercobilin ; most of the stercobilin leaves the body with the
faeces ; 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. 92). 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
the pigment proper. In certain diseased conditions the amount of
urobilin is considerably increased.

The most abundant urinary pigment is a yellow one called
urochrome. It shows no absorption bands. It is probably an
oxidation product of urobilin (Eiva, A. E. Garrod). (See Lesson
XXVI.)

Reaction. — The reaction of normal urine is acid. This is not due

144

ESSENTIALS OF CHEMICAL PHYSIOLOGY

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. In certain circumstances the
urine becomes less acid and even alkahne ; the most important of
these are as follov^s : —

1. During digestion. Here there is a formation of free acid in the
stomach, and a corresponding liberation of bases in the blood 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 alkahne salts of acids such as tartaric, citric, malic, &c.

Fici. 42. — Urinometer floating iii urine
in a testing glass.

Fig. 43.— Crystals of urea : a, four-sidai
prisms ; fc, indefinite crystals, such
as are usually formed from alcohol
solutions.

These acids are oxidised into carbonates, which passing into the urine
give it an alkaline reaction.

Specific Gravity. — This should be taken in a sample of the twenty-
four hours' urine with a good urinometer (see fig. 42).

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
to 1050. The specific gravity has, however, been known to sink as
low as 1002 (after large potations, urina 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 (taking an ordinary

IFliINK

145

diet containing about 100 grammes of protein) in the twenty-four
hours : —

Water

Total solids .

Urea .

Uric acid

Hippuric acid

Creatinine

Pigment and other organic substances

Sulphuric acid

Phosphoric acid

Chlorine

Ammonia

Potassium

Sodium

Calcium

Magnesium .

1500'00 grammes
72-00
33-18

0-55

0-40

0-91
10-00

201

3-16

7-50

0-77

2-50
11-09

0-26

0-21

The most abundant constituents of the urine are water, urea, and
sodium chloride. In the foregoing table the student must not be
misled by seeing the names of the acids and metals separated. The
acids and the bases are combined to form salts : — urates, chlorides,
sulphates, phosphates, &c.

UREA

Urea or Carbamide, CO(NH.2)2, is isomeric (that is, has the same
empirical, but not the same structural formula) with ammonium
cyanate (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 syntheti-
cally by chemists.

When separated from the urine, it is 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. 43.

When treated with nitric acid, nitrate of urea (CON2H4.HNO3)
is formed ; this crystallises in octahedra, lozenge-shaped tablets, or
hexagons (fig. 44, a). . When treated with oxalic acid, prismatic
crystals of urea oxalate (CON2H4.H2C2O4 + H2O) are formed (fig.
44, b).

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 certain organised ferments, such as the
micrococcus ureae, which grows readily in stale urine, urea takes up

' The preparation of urea nitrate and urea oxalate is postponed to the next
lesson, when other microscopic crystals will also be under examination.

146

ESSENTIALS OF CHEMICAL PIIYSIOLOCrY

water, and is converted into ammonium carbonate [CON2H4 + 2H2O
= (NH4)2C03]. Hence the ammoniacal odour of putrid urine.

By means of nitrous acid, urea is broken up into carbonic acid,
water, and nitrogen, CON2H4 + 2HN02=C02 + 3H20 + 2N2. This
may be used as a test for urea. Add fuming nitric acid {i.e. nitric acid
containing nitrous acid in solution) to a solution of urea, or to urine ;
an abundant evolution of gas bubbles takes place.

Hypobromite of soda decomposes urea in the following way : —

CON2H4 + 3NaBrO = CO2 + N2 + 2H2O + 3NaBr

[urea]

[sodium
liypobromLte]

[carbouic
acid]

[nitro-
gen]

[water] [sodium

bromide]

This reaction is important, for on it one of the readiest methods for
estimating urea depends. There have been various pieces of appa-
ratus invented for rendering the analysis easy ; but the one described

Fig. 44.— a, nitrate ; 5, oxalate of urea.

in the practical exercise at the head of this lesson is 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 protein food ingested. In a man
taking the usual Voit diet containing about 100 grammes of protein
(which will contain about 16 grammes of nitrogen) the quantity of
urea excreted daily averages 33 grammes (500 grains). The per-
centage in human urine would then be 2 per cent. ; but this also
varies, because the concentration of the urine varies considerably in
health. The excretion of urea is usually at a maximum three hours
after a meal, especially after a meal rich in protein.

Muscular exercise has but little effect on the amount of nitrogen

URINE 147

discharged. 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 venous blood, which is rapidly discharged by the
expired air. Muscular energy is derived normally from the combus-
tion of non-nitrogenous material ; this is very largely carbohydrate.
If the muscles, however, are not supplied with the proper amount of
carbohydrate and fat, or if the work done is very excessive, then they
consume some of their more precious protein material.

Where is Urea formed ?— -The older authors considered that it
was formed in the kidneys, just as they also erroneously thought that
carbonic acid was formed in the lungs. Prevost and Dumas were the
first to show that after complete extirpation of the kidney the forma-
tion of urea and other waste products goes on, and these accumulate
in the blood and tissues. Similarly, in those cases of disease in
w^hich the kidneys cease work, urea is still formed and accumulates.
This condition is called urcemia (or urea in the blood), and unless the
waste substances are discharged from the body the patient dies.

UrcBmia. — This term was originally applied on the erroneous supposition
that it is urea or some antecedent of urea which acts as the poison. There
is no doubt that the poison is not any constituent of normal urine ; if the
kidneys of an animal are extirpated the animal dies in a few days, but there
are no uraemic convulsions. In man also, if the kidneys are healthy or
approximately so, and suppression of urine occurs from the simultaneous
blocking of both renal arteries by clot, or of both ureters by stones, again
uraimia does not follow. On the other hand, uraemia may occur even while
a patient with diseased kidneys is passing a considerable amount of urine.
What the poison is that is responsible for the convulsions and coma is
unknown. It is doubtless some abnormal katabolic product, but whether
this is produced by the diseased kidney cells, or in some other part of the
body, is also unknown.

Where, then, is the seat of urea formation ? The facts of experi-
ment and of pathology point very strongly in support of the theory
that urea is formed in the liver. The principal are the following : —

1. After removal of the liver in such animals as frogs, urea forma-
tion almost ceases, and ammonia is found in the urine instead.

2. In mammals, the extirpation of the liver is such a serious
operation that the animals die. But the liver of mammals can be very
largely thrown out of gear by the operation known as Eck's fistula,
which consists in connecting the portal vein directly to the inferior
vena cava. In these circumstances the liver receives blood only
by the hepatic artery. The amount of urea is lessened, and its place
is taken by ammonia.

3. When degenerative changes occur in the liver, as in cirrhosis of
that organ, the urea formed is much lessened, and its place is taken
by ammonia. In ac2ite yelloiv atrophy, urea is almost absent from the

L 2

1 48 ESSENTIALS OF CHEMICAL PHYSTO^,OGY

urine, and, again, there is considerable increase in the ammonia. In
this disease amino-acids such as leucine and tyrosine are also found
in the urine ;. these 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 protein
and urea. In order that the student may grasp the meaning of urea
formation it would be advisable for him to turn again to p. 52 and
read the paragraph there relating to Chittenden's views on diet, and
to pp. 96 to 98, which treat of protein absorption, for the question,
What is a normal diet ? is intimately bound up with the question,
What is a normal urine ? If, for instance, the diet of the future is to
contain only half as much protein as in the past, the urine of the
future will naturally show a nitrogenous output of half of that which
has hitherto been regarded as normal. In people on such a reduced
diet, Folin has shown that the decrease in urinary nitrogen falls
mainly on the urea, but certain other nitrogenous katabolites, parti-
cularly one called creatinine, remain remarkably constant in absolute
amount in spite of the great reduction in the protein ingested.

The laws governing the composition of urine are obviously the
effect of the laws that govern protein katabolism. Many years ago
Voit supposed that the protein ingested was utilised partly in tissue
formation, and partly remained in the circulating fluids as ' circulating
protein ' ; he further considered that the breakdown of the protein in
the tissues was accomplished with much greater difficulty than that
in blood and lymph, and that the small amount of ' tissue-protein '
which disintegrates as the result of the wear and tear of the tissues
was dissolved and added to the ' circulating protein,' in which alone
the formation of final katabolic products such as urea was supposed
to occur. As time went on, it was shown that many facts were
incompatible with this theory, and so it was largely displaced by
Pfliiger's view, in which it was held that the food protein must first
be assimilated, and become part and parcel of living cells, before
katabolism occurs. We now know that neither of these views is
correct, and that nitrogenous katabolism is of two kinds : one kind
varies with the food ; it is therefore variable in amount, and occurs
almost immediately or within a few hours after the food is absorbed ;
the amino-acids absorbed from the intestine are in great measure never
built into living protoplasm at all, and are simply taken to the liver,
where they are converted into urea. This variety of katabolism is
called exogenous. The other kind of metabolism is constant in quantity
and smaller in amount, and is due to the actual breakdown of protein
matter in the body cells and tissues, which had been built into them

URINE 149

previously. This form of metabolism is called endogenous or tissue
metabolism, and the final product is not urea to any great extent, but
the waste nitrogen finds its way out of the body in other substances,
of which creatinine appears to be the most important. This form of
metabolism sets a limit to the lowest level of nitrogenous requirement
attainable ; the protein sufficient to maintain it is indispensable-
Whether the amount of protein which is exogenously metabolised
can be entirely dispensed with is at present questionable, and those
who seek to replace it entirely by non-nitrogenous food are living
dangerously near the margin. A point of considerable importance in
this connection is, that the nitrogen of the protein is split off from it
by hydrolysis without oxidation. There is thus very little loss of
potential energy, the energy of the products being nearly equal to
that of the original protein ; it is, however, the non-nitrogenous
residue which is mainly available for oxidation, and thus for calorific
processes. The fact that muscular work does not normally increase
nitrogenous metabolism becomes intelligible in the light of the con-
sideration that protein katabolism, in so far as its nitrogen is concerned,
is independent of the oxidations which give rise to heat, or to the
energy which is converted into work. Those who in the past have
endeavoured to study the relation of muscular work to nitrogen
excretion have usually estimated the urea. Now that we know urea
is the chief end product of exogenous and not of tissue katabolism,
we see that estimations of urea can give us but little real information
on this point. The substance which ought to be estimated is
creatinine, and it has been found recently that even this is not notably
increased in the urine, provided the muscles receive their normal
supply of fat and carbohydrate. The body is very economical in so
far as protein is concerned, and tissue or endogenous katabolism is
kept at a low^ level.

What is the proportion between exogenous and endogenous
nitrogen katabolism ? It is very difficult to give any exact estimate.
We do know that in ordinary diets, the former is far in excess, and
probably in a man excreting 16 grammes of nitrogen daily (that is,
the amount corresponding to an intake of 100 grammes of protein),
only a quarter of this or even less represents tissue breakdown.

The view we have advanced concerning urea formation, then, is,
that it is mainly the result of the conversion, by the liver, of amino-
acids absorbed from the intestine into that substance. This view
receives confirmation from experiments in which certain amino-acids,
such as glycine, leucine, and arginine, have been injected direct into
the blood-stream. The result is an increased formation of urea. In

150 ESSENTIALS OF CHEMICAL PHYSIOLOGY

the case of arginine the exact chemical decomposition which takes
place is known. We have already seen that arginine is a compound
of a urea radical and a substance called ornithine (di-amino-valeric
acid, see p. 32) ; the liver is able to hydrolyse arginine, and so urea
is liberated. This power is due to the action of a special ferment
called arginase, which, although it is also found in other organs, is
specially abundant in the liver. It is, however, possible that the
ornithine itself may be further broken up, and so an extra quantity of
urea formed. On the other hand there are some amino-acids {e.g.
tyrosine) which on injection do not lead to any increase in urea
formation.

If, however, we glance at the formula of ornithine, we see that it
has one point in common with other amino-acids, such as glycine and
leucine, to take simple examples : —

Ornithine 05lI,2N2O2
Glycine C2H5NO2
Leucine C6H13NO2

This is, that in all cases the carbon atoms are more numerous than
the nitrogen atoms. In urea, CON2H4, the reverse is the case. The
amino-acids must therefore be split into simpler compounds, which
unite with one another to form urea. Urea formation is thus in
part synthetic. These simpler compounds are ammonium salts.
Schroder's work proves that ammonium carbonate is one of the
urea precursors, if not the principal one. The equation which
represents the reaction is as follows : —

(NH4)2C03=CON2H4 + 2H2O

[ammouiam [urea] [water]

carbonate]

Schroder'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 the
same experiment is performed with any other organ of the body, so
that Schroder's experiments also prove the great importance of the
liver in urea formation. Similar results were obtained by Nencki
with ammonium carbamate.

We must further remember that ammonia itself is one of the
products of digestion of protein in the intestine, and it may possibly,
to a small extent, be a result of tissue katabolism. This ammonia
passes into the blood, where it unites with carbonic acid to form either
the carbamate or carbonate of ammonium. Thus ammonia, whether

UKINE 151

formed directly or by the breakdown of amino-acids, is the principal
immediate precursor of urea.

The following structural formulae show the relationship between
ammonium carbonate, ammonium carbamate, and urea : —

o-r/ONH4 o-r/NH2 o-r/NH2

"-'^XO.NH^ ^-^XO.NH^ "-^\NH,

[auimoniuin carbonate] [ammouium 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.

AMMONIA

The urine of man and carnivora contains small quantities of
ammonium salts. The reason that some ammonia always slips
through into the urine is that a part of the ammonia-containing
blood passes through the kidney before reaching organs, such as the
liver, which are capable of synthesising urea. In man the daily
amount of ammonia excreted varies between 0*3 and 1*2 gramme :
the average is 0*7 gramme. The ingestion of ammonium carbonate
does not increase the amount of ammonia in the urine, but increases
the amount of urea, into which substance the ammonium carbonate
is easily converted. But if a more stable salt, like ammonium
chloride, is given, it appears as such in the urine.

Under normal circumstances the amount of ammonia depends on
the adjustment between the production of acid substances in meta-
bolism and the supply of bases in the food. Ammonia formation is
the physiological remedy for deficiency of bases.

When the production of acids is excessive (as in diabetes), or
when mineral acids are given by the mouth or injected into the
blood-stream, the result is an increase of the physiological remedy,
and excess of the ammonia passes over into the urine. Under normal
conditions ammonia is kept at a minimum, being finally converted
into the less toxic substance urea, which the kidneys easily excrete.
The defence of the organism against acids which are very toxic is an
increase of ammonia formation, or, to put it more correctly, less of
the ammonia formed is converted into urea.

Under the opposite conditions — namely, excess of alkali, either in
food or given as such — the ammonia disappears from the urine, all
being converted into urea. Hence the diminution of ammonia in the
urine of man on a vegetable diet, and its absence in the urine of
herbivorous animals.

Not only is this the case but if ammonium chloride is given to a

152 ESSENTIALS OF CHEMICAL PHYSIOLOGY

herbivorous animal like a rabbit, the urinary ammonia is but little
increased. It reacts with sodium carbonate in the tissues, forming
ammonium carbonate (which is excreted as urea) and sodium chloride.
Herbivora also suffer much more from, and are more easily killed by,
acids than carnivora, their organisation not permitting a ready supply
of ammonia to neutralise excess of acids.

CREATININE

Creatinine is one of the substances in the urine which represents
the end-stage of the tissue or endogenous katabolism of protein. It
is the most abundant of such katabolites, and the one concerning
which we know most.

The most abundant of our active tissues is muscle, and the
amount of nitrogenous waste in this tissue which leaves it as urea
is insignificant. The place of urea is taken by another substance
called creatine. As already explained on p. 32, creatine is a com-'
pound of a urea radical with another group, and when it is boiled
with baryta water it takes up water and splits into two substances
— namely, urea and sarcosine or methyl-glycine ; this is shown in
the following equation : —

^'"^ N(CH3)CH2.COOH + H20=H2N/^^

[creatiue] [water] [urea]

+ NH.CH3.CH2.COOH

[sarcosine]

It is, however, extremely doubtful whether this decomposition occurs
in the body, and therefore whether creatine is to any important degree
a precursor of urea. For, when creatine is introduced directly into
the blood-stream, the amount of urea is not increased in the urine.
What is increased is another substance called creatinine, which is
creatine minus wdter, as shown in the following equation : —
C4H9N3O2 =C4H7N30 +U,0

[creatine] [creatinine] [water]

Creatinine, therefore, comes from nitrogenous katabolism in the
tissues, especially muscular tissues, and creatine is an intermediate
stage in its formation. Some of the creatinine, however, has a
different origin — namely, an exogenous one ; that is, from the food
instead of from tissue metabolism ; and the substance in the food
which gives rise to it is the creatine contained in the flesh eaten.

The best method for estimating creatinine and creatine is given in
Lesson XXV.

URINE 168

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 excreted 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 sulphates are derived from the
changes that occur in the proteins ; the nitrogen of proteins leaves the
body chiefly as urea ; the sulphur of the proteins is oxidised to form
sulphuric acid, which passes into the urine in the form of sulphates.
The excretion of sulphates, moreover, runs parallel to that of urea.
Sulphates, like urea, are the result of exogenous protein metabolism ;
endogenous metabolism so far as sulphur is concerned is represented
in the urine partly as ethereal sulphates, but chiefly by less fully
oxidised compounds of sulphur. The chief tests for the various salts
have been given in the practical exercises at the head of this lesson.

Chlorides. — The chief chloride is 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
the body, fulfils the useful office of stimulating metabolism and
excretion.

Sulphates. — The sulphates in the ludne are principally those of
potassium and sodium. They are derived from the metabolism of
proteins 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 radicals ; the compounds are known as
ethereal sulphates, and they originate mainly 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
ill the intestine, and as it yields indigo when treated with certain

154 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 : —

CH^OH + SO^/Of = SO,/0^«H,^jj^O

[phenol] [potassium [potassium [water]

hydrogen sulphate] phenyl-sulphate]

The formation of potassium indoxyl-sulphate may be represented

CH

as follows : — Indole, C6H4/ ^CH on absorption is converted

NH
O.OH

intoindoxyl: CgH/^CH

NH
Indoxyl then interacts with potassium hydrogen sulphate as
follows : —

[indoxyl] [potassium [potassium [water]

hydrogen sulphate] iudoxyl-sulphate]

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. (For tests for indoxyl in urine see Advanced Course,
Lesson XXVI.)

Carbonates. — Carbonates and bicarbonates of sodium, calcium,
magnesium, and ammonium are present in alkaline urine only. They
arise from the carbonates of the food, or from vegetable acids (malic,
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 stand-
ing, the precipitate consisting of calcium carbonate, and also phos-
phates.

Phosphates. — Two classes of phosphates occur in normal
urine : —

(1) Alkaline phosphates — that is, phosphates of sodium (abundant)
and potassium (scanty).

URINE

165

(2) Earthy phosphates — that is, phosphates of calcium (abundant)
and magnesium (scanty).

The composition of the phosphates in urine is hable to variation.
In acid urine the acidity is due to the acid salts. These are chiefly
sodium dihydrogen phosphate, NaH2P04, and calcium dihydrogen
phosphate, Ca(H2P04)2.

In neutral urine, in addition, disodium hydrogen phosphate
(Na2HP04), calcium hydrogen phosphate, CaHP04, and magnesium
hydrogen phosphate, MgHP04, are found. In alkahne urine
there may be instead of, or in addition to the above, the normal
phosphates of sodium, calcium, and magnesium [Na3P04, Ca3(P04)2,

Mg3(P04)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 (NH4MgP04 + 6H20). This crystallises
in ' coffin-lid ' crystals (see fig. 45) 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
is soluble in a few drops of acetic acid, whereas
coagulated protein 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 thephos-
phorised organic materials in the body, such as lecithin and nuclein.
The amount of P2O5 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.).

Fig. 45. — Ammouio-
magnesium or triple
phosphate.

LESSON XI
UBINE {contmued)

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 m-ine as in the last exercise, and add
oxalic acid. Crystals of urea oxalate separate out. Examine these micro-
scopically.

3. Uric 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. 46).

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. Add a little ammonia carefully. The residue
turns to violet. This is due to the formation of nuirexide 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 pax)er, add a drop of silver nitrate, and warm
gently ; the black colour of reduced silver is seen on the paper.

4. Deposits 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 cofiin-lid crystals of triple phosphate (ammonio-magnesium phosphate),
for crystals of stellar (calcium) phosphate, and for mucus. Mucus is flocculent
to the naked eye, amorphous to the microscope.

N.B. — On boiling neutral, alkaline, or even faintly acid urine it may be-
come turbid from deposition of phosphates. The solubility of this deposit in

URINE 157

a few drops of acetic acid distinguishes it from albumin, for which it is liable
to be mistaken.

Some of the facts described in the foregoing exercises have been already-
dwelt upon in the preceding lesson. They are, however, conveniently grouped
together here, as all involve the use of the microscope.

URIC ACID

Uric Acid (C5N4H403)is in mammals the medium by which only a
small quantity of nitrogen is excreted from the body. It is, however,
in birds and reptiles the principal nitrogenous constituent of their
urine. It is not present in the free state, but is combined with bases
to form urates.

It may be obtained from human urine by adding 5 c.c. of hydro-
chloric acid to 100 c.c. of the urine, and allowing the mixture to stand
for twelve to twenty-four hours. The crystals which form are deeply
tinged with urinary pigment, and though by repeated solution in
caustic soda or potash, and reprecipitation ,,^^^ y^^^^

l)y hydrochloric acid, they may be obtained ^^^^^^^^^^^ / — \^^

fairly free from pigment, pure uric acid is /^^ ^

more readily obtained from the solid urine of /^ ^^^^^ rj
a serpent or bird, which consists principally ^ ^ 5^^

of the acid ammonium urate. This is dis- 0 ^-^^

solved in soda, and then the addition of ^^ fl % ^

hydrochloric acid produces as before the % ^ ^p a

crystallisation of uric acid from the solution, f ^ ^ I — .

The pure acid crystallises in colourless 0 w

rectangular plates or prisms. In striking
contrast to urea it is a most insoluble sub-
stance, requiring for its solution 1,900 parts
of hot and 15,000 parts of cold water. The '''''• *'-''''" ''^"' "'"'"^'•
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. 46).

The murexide test which has just been described among the
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 Murex.

Another reaction that uric acid undergoes (though it is not appli-
cable as a test) is that on treatment w4th certain oxidising reagents

158 ESSENTIALS OF CHEMICAL PHYSIOLOGY

urea aud oxalic acid can be obtained from it. It is, however, doubtful
whether a similar oxidation occurs in the normal metabolic processes
of the body.

Uric acid is dibasic, and thus there are two classes of urates — the
normal urates and the acid urates. A normal urate is one in which
two atoms of the hydrogen are replaced by two of a monad metal like
sodium; an acid urate is one in which only one atom of hydrogen.is
thus replaced. The formulae would be —

C5H4N40;j =uric acid
C.5H3NaN403=acid sodium urate
05H2Na9N4O3=normal sodium urate

The acid sodium urate is the chief constituent of the pinkish deposit
of grates, which, as w^e have already stated, is called the lateritious
deposit.

If uric acid is represesented by H^U, the normal urates may be represented
by MoU and the acid urates by MHU. Bence Jones, and later Sir W. Roberts,
considered that the urates which actually occur in urine are quadriurates,
MHU.H^U. There is much doubt whether such compounds actually exist ;
if they do they are readily decomposed into acid urates MHU and free uric
acid, HoU.

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 in these animals ammonia and lactic acid are
normally synthesised in the liver to form uric acid.

This synthetic origin of uric acid, which is so important in birds
and snakes, does not, however, occur in mammals. In mammals
uric acid is the chief end-product of the katabolism of cell nuclei or

UHINK 159

of imclein, the principal constituent of the nuclei. This therefore
leads us next to study : —

Purine Substances. — Emil Fischer has shown that the decom-
position products of nuclein are derivatives of a substance he has
named purine. The empirical formulae for purine, the purine bases,
and uric acid are as follows : —

Purine . . C5H4N4

Hypoxanthine . C5H4N4O Monoxypurine \

Xanthine . . C5H4N4O2 Dioxypurine | Purine

Adenine . . C5H3N4.NH2 Amino-purine I bases.

Guanine . . C5H3N4O.NH2 Amino-oxypurine/

Uric acid . . C-,H4N403 Trioxypurine

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 dimethy] -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

I I

H - C C - NH.

II !l ^C-H

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 : —

IN -«C
I i

3N _ 4C - 9N

/

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 number 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.

1C)0 ESSENTIALS OF CHEMICAL PHYSIOLOaY

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=0 NH-C = 0

II II

H-C C-NH. 0 = 0 C-NH.

II II >C~H I II >C-H

[hypoxanthiue] [xftnthine]

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.

H

N = C - NH.,

1 1

NH-

1

-C = 0

H-C C - NH. H2N -
II II >C-H

N - C - W/

1
-C

II

N -

C- NH.

II >c

- C - N'^

[adenine]

[guanine]

Uric acid is 2, 6, 8-trioxypurine.

NH - C = 0

1 1

0 = C C - NH.

1 II >co

NH-C - NH/

[nric acid]

The close chemical relationship of uric acid to the purine bases is
obvious from a study of the formulae just given. Just as in the case
of urea, uric acid, however, may be exogenously or endogenously
formed. Certain kinds of food increase uric acid because they con-
tain nuclein (for instance, sweetbreads) in abundance, or purine bases
(for instance, hypoxanthiue in meat) ; the uric acid which originates
in this way is termed exogenous. Certain diets, on the other hand,
increase uric acid formation by leading to an increase of leucocytes,
and consequently increase in the metabolism of their nuclei ; in other
cases the leucocytes may increase from other causes, as in the disease
named leucocythaemia. The uric acid that arises from nuclear kata-
bolism is termed e^idogenous. Although special attention has been

URINE 161

directed to the nuclei of leucocytes because they can be readily
examined during life, it must be remembered that nuclein meta-
bolism of all cells may contribute to uric acid formation.

A study of uric acid formation forms a useful occasion on which
to allude to ferment actions in metabolism generally. Ferments of
a digestive kind are not confined to the interior of the alimentary
canal ; but most of the body cells are provided with ferments to
assist them either in utilising the nutrient materials brought to them
by the blood-stream, or in breaking them down previously to expel-
ling them as waste substances. The ferment which enables the liver
cells to turn glycogen into sugar is the one which has been known
longest. The ferment called arginase (see p. 150), which leads to
the hydrolysis of arginine into urea and ornithine, is one of the most
recently discovered. Other examples which may be mentioned are
proteolytic enzymes (tissue erepsin, &c.) found in many organs.

The formation of uric acid from nuclein is perhaps the best
instance of all, for here we have to deal with numerous ferments
acting one after another. The first to come into play is called
nuclease ; this liberates purine bases such as adenine and gua-
nine from the nuclein ; the next ferments that act remove the
amino-group from the purine bases just mentioned ; in this way
adenine (G5H3N4.NH0) is converted into hypoxanthine C5H4N4O ;
and guanine (C5H3N4O.NH2) into xanthine C5H4N4O2. These two
ferments are respectively called adenase and guanase. Finally, oxi-
dising ferments, or oxidases, step in and oxidise hypoxanthine into
xanthine, and xanthine into uric acid. By examining extracts of
various organs, the distribution of these numerous ferments has been
determined, and in general terms the spleen and liver are the organs
where they are most abundant. But the examination of such extracts
has shown in addition that the list of ferments is not yet complete ;
for some extracts in part break up the uric acid, which is formed into
simpler substances ; the uric acid destroying ferment is called the
uricolytic ferment. We therefore learn that the uric acid discharged
in the urine is only the balance left over when the amount destroyed
is deducted from the amount originally formed. In other words, the
body possesses to some extent the power of protecting itself from an
excessive formation of uric acid, and so from the evils which would
result from an accumulation of this substance.

162 ESSENTIALS OF CHEMICAL PHYSIOLOGY

HIPPURIC ACID

Hippuric acid (C9H9NO3), combined with bases to form hip-
purates, is present in small quantities in human urine, but in large
quantities in that of herbivora. This is due to the food of herbivora
containing substances belonging to the aromatic group — the benzoic
acid series. If benzoic acid is given to a man, it unites with glycine
with the elimination of a molecule of water, and is excreted as
hippuric acid —

CH2NH2 CH2NH.CO.C6H5
CeHsCOOH + | = | +H2O

COOH COOH

[benzoic acid] [glycine] [hippuric acid] [water]

This is a well-marked instance of synthesis carried out in the
animal body, and experimental investigation shows that it is accom-
plished by the living cells of the kidney itself ; for if a mixture of
glycine, benzoic acid, and defibrinated 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.

It may be crystallised from horse's urine by evaporating to a syriip and
saturating with HCl. The crystals are dissolved in boiling water, filtered,
and on cooling the acid again crystallises out. It melts at 186° C, and on
further heating gives rise to the odour of bitter almond oil.

URINARY DEPOSITS

The different substances that may occur in urinary deposits are
formed elements and chemical substances.

The formed or histological 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. Earer 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.
46, p. 157) and by the murexide reaction. The presence of these
crystals generally indicates an increased formation of uric acid, and,

URINE 163

if excessive, may lead to the formation of stones or calculi in the
bladder.

Deposit of Urates. — This is much commoner, and may, if the
urine is concentrated, occur in normal urine when it cools. It is
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
sodium urate, the formation of which from the normal sodium urate
of the urine may be represented by the equation —

205H2Na,N4O3 + H2O + C02=2C5H3NaN403 + NagCOa

[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 uro-erythrin is one

Fig. 47. — Acid sodium urate. FiG. 48. — Acid ammonium urate.

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.

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 acetic acid. It is soluble with
difficulty in hydrochloric acid.

Deposit of Cystin.—Gystin (OgHisNiSaOi) is recognised by its
colourless six-sided crystals (fig. 50). These are rare : they occur
only in acid urine, and they may form concretions or calculi. Cystin-
uria (cystin in the urine) is hereditary.

Deposit of Phosphates. — These occur in alkaline urine. The

M 2

164

ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 micrococciis
itrecB. This forms ammonium carbonate from the urea.

[urea] [water] [ammonium

carbonate]

The ammonia renders the urine alkaline, and precipitates the

^

0

0 o

Fiu. 49.— Envelope crjstiils
of calcium oxalate.

Fig. 50.— Oystin crystals.

earthy phosphates. The chief forms of phosphates that occur in
urinary deposits are —

1. Calcium phosphate, Ca3(P04)2 ; amorphous.

2. Triple or ammonio-magnesium phosphate, MgNH4P04 + 6H2O ;
coffin-hds and feathery stars (fig. 51).

Fio. 51.— Triple phosphate crystals.

Fjg. 52.— Crystals of phosphate of lime
(stellar phosphate).

3. Crystalline phosphate of calcium, CaHPO}, in rosettes of
prisms, in spherules, or in dumb-bells (fig. 52).

4. Magnesium phosphate, Mg;3(P04).2 i 2H2O, occurs occasionally,
and crystallises in long plates.

UEINE

165

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.

A solution of ammonium carbonate (l-in-5) eats magnesium
phosphate away from the edges ; it has no effect on the triple phos-
phate. A phosphate of calcium (CaHP04 + 2H20) may occasionally
be deposited in acid urine. Pus in urine is apt to be mistaken for
phosphates, but can be distinguished by the microscope.

Deposit of calcium carbonate, CaCOa, appears but rarely as
whitish balls or biscuit-shaped bodies. It is commoner in the urine
of herbivora (see p. 154). 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 UEINE

In Acid Urine

Uric Acid. — Whetstone, dumb-
bell, or sheaf-like aggregations of
crystals deeply tinged by pigment
(fig. 46).

Urates. — Generally amorphous.
The acid urate of sodium (fig. 47) and
of ammonium (fig. 48) may some-
times occur in star-shaped chisters of
needles or spheroidal clumps with
projecting spines. Tinged brick-red.
Soluble on warming.

Calcmm Oxalate. — Octahedra,
so-called envelope crystals (fig. 49).
Insoluble in acetic acid.

Cystin. — Hexagonal "plates (fig.
50). Bare.

Leucine and Tyrosine. — Kare.

Calcium Phosphate.

CaHP04-f2H,0.— Rare.

In Alkaline Urine

Phosphates. — Calcium phosphate,
Ca3(P04)2. Amorphous.

Triple phosphate,
MgNH,P0, + 6H,0. Coffin-lids or
feathery stars (figs. 45 and 51).

Calcium hydrogen phosphate,
CaHPO^. Rosettes, spherules, or
dumb-bells (fig. 52).

Magnesium phosphate,
Mg3(P04).;j + 2H.,0. Long plates.

All soluble in acetic acid without
effervescence.

Calciinn Carbonate, CaCO^. —
Biscuit-shaped crystals. Soluble in
acetic acid with effervescence.

Ammonium Urate,
C,H,(NH,)o.N,03. — ' Thorn-apple '
spherules.

Leucine and Tyrosine. — Very rare.

LESSON XII
PATHOLOGICAL UBINE

1. Urine A is pathological urine containing albumin. It gives the usual
protein tests. The following two are most frequently used in practice.

(rt) 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.c).

:ir«i:?<i»:4i=<n»>'ifc

FfG. 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
precipitate. The figures indicate grammes of dried albumin in a litre of urine.
The percentage is obtained by dividing by 10. Thus, if the sediment stands
at 3, the amount of albumin is 3 grammes per litre, or 0-3 gr. in 100 c.c. If
the sediment falls between any two figures, the distance }, i, or ^ 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. It has a high specific gravity. The presence
of sugar is shown by the reduction (yellow precipitate of cuprous oxide) that
occurs on boiling with Fehling's solution. Fehling's solution is an alkaline
solution of copper sulphate to which Rochelle salt has been added. The
Rochelle salt (double tartrate of potash and soda) holds the cupric hydrate in

PATHOLOGICAL URINE

167

solution. Fehling's solution should always be freshly prepared, as, on stand-
ing, an isomeride is formed from the tartaric acid, and this substance itself
reduces the cupric to cuprous oxide. Fehling's solution should, therefore,
always be tested by boiling before it is used. If it remains unaltered by
boiling, it is in good condition.

4. Quantitative Determination of Sugar in Urine. — Fehling's solution is pre-
pared as follows : — 34*639 grammes of copper sulphate are dissolved in about
200 c.c. of distilled water ; 173 grammes of Eochelle
salt are dissolved in 600 c.c. of a 14-per-cent. solution
of caustic soda. The two solutions are mixed and
diluted to a litre. Ten c.c. of this solution are
equivalent to 0*05 gramme of dextrose. Dilute 10 c.c.
of this solution with about 40 c.c. of water, and boil it

■in a white porcelain dish. Run into this from the
burette (see fig. 54) the virine (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 hj^drate
is reduced. The mixture in the basin should be boiled
after every addition.^ The quantity of diluted urine
used from the burette contains O'Oo gramme of sugar.
Calculate the percentage from this, remembering that
the urine has been diluted to ten times its original
volume.

Example.— Su-pTpose that 20 c.c. of the diluted
urine are found necessary to reduce the 10 c.c. of
Fehling's solution. This wiU be equivalent to 2 c.c.
of the undiluted urine ; 2 c.c. of the original urine
will therefore contain 0*05 gramme of sugar ; 1 c.c.

will contain ^^ and 100 c.c. will contain ^^ x 100
2 2

= 2'5 grammes of sugar.

Pavy's modification of Fehling's solution is some-
times used. Here ammonia holds the cuprous oxide
in solution, so that no precipitate forms on boiling
Pavy's solution with a reducing sugar. The reduc-
tion is complete when the blue colour disappears : 10 c.c. of Pavy'
tion = 1 c.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 solu-
tion, 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 perform the following experiment.
Take a drachm (about 4 c.c.) of diabetic urine ; add to it an equal volume of
saturated aqueous solution of picric acid, and half the volume {i.e. 2 c.c.) of
the liquor potassae of the British Pharmacopoeia. Boil the mixture for about
a minute, and it becomes so intensely dark red as to be opaque. Now do
the same experiment with normal urine. An orange-red colour appears
even in the cold, and is deepened by boiling, but it never becomes opaque,

I'Ki. 54. — Two burettes on
stand. (Sutton.)

solu-

On cooling the blue colour reappears, owing to re-oxidation.

168 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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.

6. Test for Acetone. — Acetone is often found in diabetic urine. Add to the
urine a dilute solution of sodium nitro-prusside and a little 20-per-cent.
caustic potash. A red colour is produced. Acidify with strong acetic
acid. The colour disappears at once in the absence of acetone, but re-
mains or is intensified in its presence.

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.

PROTEINS IN THE URINE

There is no protein 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 protein 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 the methods
described on pp. 87, 88.

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.

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

PATHOLOGICAL UEINE 169

glycuronic acid (C^HjqO-) 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 (see p. 88).

Then, too, in the rare condition called alcaptonuria, confusion may
similarly arise. Alcapton is a substance which probably originates, from
tyrosine by an unusual form of metabolism. It gives the urine 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 homogentisinic
acid [C,H3.(0H),CH,.C00H].

The best confirmatory tests for sugar are the phenyl-hydrazine
test (see Lesson XIII.), and i\\Q fermentation test (Lesson IL).

Sir W. Eoberts introduced a method for estimating sugar in urine, by the
diminution in specific gravity which it undergoes on fermentation with yeast.
Ever}- degree lost in the specific gravity corresponds to one grain of sugar per
fluid ounce. 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*22 x 10 = 2*2. The method, however, is rough 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
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. Its track is
marked by a purplish line. Hay's sulphur test (p. 79) is very trust-
worthy. Excess of urobilin should not be mistaken for bile pigment.

BLOOD AND BLOOD PIGMENT IN THE URINE

When haemorrhage 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 oxyhaemo-
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

170 ESSENTIALS OF CHEMICAL PHYSIOLOGY

the urine without the presence of any blood corpuscles at all. This
is produced by a disintegration of the corpuscles occurring in the
circulation. The condition so produced is called hcBmoglobiiiuria,
and it occurs in several pathological states, as, for instance, in the
tropical disease known as ' Black-water fever.' The pigment is in
the condition of methaemoglobin mixed with more or less oxyhaemo-
globin, and the spectroscope is the means used for identifying these
substances (see p. 119).

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 protein constituents
of the pus cells — and the same is true for blood — pass into solution,
so that the urine pipetted off from the surface of the deposit gives
the tests for albumin. On the addition of liquor potassae to the
deposit of pus cells a ropy gelatinous mass is obtained. This is
distinctive. Mucus treated in the same way is dissolved.

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 have been previously
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,
glycogen, or certain proteins,

2. Add iodine. A colour is produced :

If blue : Starch. Confirm by converting into a reducing sugar by saHva
at 40° C, or by 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 glj'cogen are, like
starch, convertible into a reducing sugar.

3. Add copper sulphate and caustic potash.

(«) Blue solution : boil ; yellow or red precipitate. Dextrose, levulose,
maltose, lactose, and other reducing sugars (for distinguishing tests see
Lesson XIII.).

(b) 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 : Cane sugar. Confinn
by HCl test (see p. 13).

(c) Violet solution : Proteins (albumins, globulins, infra-proteins). In
presence of magnesium sulphate the potash causes also a white precipitate of
magnesia.

(d) Pink solution ; biuret reaction. Peptones or 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 proteins are present proceed as follows : Boil the original solution
(after adding a trace of 2-per-cent. acetic acid).

(a) Precipitate produced : Albumins or globulins.

(b) No precipitate : Infra-proteins,^ proteoses, or peptones.

5. If albumin, or globulin, or both are present, saturate a fresli portion
with magnesium sulphate or half saturate with ammonium sulphate; filter;
the precipitate contains the globulin, the filtrate the albumin. Test tempera-
ture of heat coagulation.

' The infra-proteins (see pp. 47 and 75) are by some called ' meta-proteins.'

172 ESSENTIALS OF CHEMICAL PHYSIOLOGY

6. If proteins are present, but albumin or globulin 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 :

(rt) Precipitate : Proteose. (&) No precipitate : Peptone.
If both are present, the precipitate contains the proteose, and the filtrate
the peptone.

8. To a fresh portion add nitric acid (proteins 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.

(d) Precipitate little altered by heating: Albumin or globulin.

In all four cases nitric acid^jZi^s heat causes a yellow colour, tinned orange
by ammonia (xantho-proteic reaction).

9. Confirmatory tests for proteins : —
{a) Millon's test (see p. 27).

(6) Adamkiewicz's reaction (see p. 27).

(c) Ferrocyanide of potassium and acetic acid cause a precipitate (except
in the case of peptones and some proteoses).

(d) To test for fibrinogen : —

i. It coagulates by heat at 56° C.

ii. It is changed into fibrin by fibrin ferment and calcium chloride.

(e) To test for caseinogen : —
1. 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. Oxyhaemoglobin shows two bands between D and E.

ii. Add ammonium sulphide ; one band only appears.

iii. Carbonic oxide haemoglobin shows two bands also, but will not reduce
with ammonium sulphide.

iv. Methsemoglobin gives a typical band in the red between C and D.

v. Haematin, &c., show special spectra (see Advanced Course, Lesson XIX.).

(6) Dry : boil with glacial acetic acid and a crystal of sodium chloride on
a glass slide under a cover glass. When cold, haemin crystals are seen.

DETECTION OF PROXIMATE PRINCIPLES 173

(c) If the blood is old and dry, and its haemoglobin converted into
hsematin :

i. Try haemin test.

ii. Dissolve it in potash ; add ammonium sulphide, and examine for
spectrum of haemochromogen.

11. If bile is suspected :

(a) Try Gmelin's test for bile pigments (see p. 79).
(6) Try Pettenkofer's test for bile salts (see p. 79).
(c) Try Hay's sulphur test (see p. 79).

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-like substance is obtained. Mucin
gives the protein colour tests.

(b) Nucleo-protein. — Precipitated by acetic acid or by alcohol. The pre-
cipitate is often viscous. It is soluble in dilute alkalis such as 1-per-cent.
sodium carbonate. This solution causes intravascular clotting. If the pre-
cipitate is collected and subjected to gastric digestion, an insoluble deposit of
nuclein is left, which is rich in phosphorus. Nucleo-protein gives the protein
colour tests.

(c) Gelatin. This also gives some of the proteid colour tests, but not
those of Millon or Adamkiewicz. 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) Uric acid. Very insoluble in water ; soluble in potash, and precipitated
from this solution in crystals by hydrochloric acid. Uric acid crystals from
human urine are deeply pigmented red. Try murexide test (see p. 156).

(/) Cholesterin. Characteristic flat crystalline plates. For various colour
tests see p. 79.

13. Urine. Normal constituents

(a) Chlorides. Acidulate with nitric acid ; add silver nitrate ; white
precipitate.

(6) Sulphates. Acidulate with nitric or hj'drochloric 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.c. of hydrochloric acid ; leave
for twenty-four hours, and pigmented crystals of uric acid are formed. For
tests see above.

(/) 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.

174 ESSENTIALS OF CHEMICAL PHYSIOLOGY

(g) Creatinine. Take 100 c.c. of urine : add 5 c.c. of a saturated solution
of sodium acetate and 20 c.c. of a saturated solution of mercuric chloride-
Filter. Set the filtrate aside for twenty-four hours, and a spherical mercury
compound of creatinine crystallises out. Examine this with a microscope.

For colour test with sodium nitro-prusside see p. 142.

14. TTrine. Abnormal constituents.

{a) Blood. Microscope (blood corpuscles). Spectroscope (for oxyhaemo-
globin or methaemoglobin). Haemin test.

(6) Blood pigment may be present without blood corpuscles. Spectro-
scope.

(c) Bile. Gmelin's test. Hay's sulphur test.

(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.

(/) Sugar, (i.) Brown colour with potash and heat (Moore's test), (ii.)
Ferments with yeast, (iii.) Keduces 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 slight coloration in normal urine is
due to creatinine.

(jjl) Acetone. For colour test see p. 168.

(7i) Mucus. Flocculent cloud ; may be increased by acetic acid ; soluble
in alkalis. A little mucus in urine is not abnormal.

(i) Deposits.

i. Examine microscopically for blood corpuscles, pus cells, crj^stals, &c.

ii. Phosphates, White deposit often mixed with nmcus or pus. Insoluble
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. Urates. Pink deposit, usually amorphous; may be mixed with
envelope crystals of calcium oxalate. Deposit soluble on heating urine.
Murexide test.

iv. Uric acid. Deposit like cayenne pepper. Microscope. Tests as
above.

ADVANCED COUESE

INTKODUCTION

It 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 few experiments in which living animals are employed
will also necessarily be of the nature of demonstrations.

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 then add
excess of alcohol ; the
glycogen is precipitated
as a flocculent powder,

which is collected on a filter and dried in an oven at the temperature 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.^

2. Examine the washings of the liver in the beakers a, b, and c for sugar.
This may be done in a rough quantitative manner as follows : — Take equal

' This method of preparation of glycogen has the advantage that only traces
of protein are mixed with it. In Kiilz's method (extraction with dilute potash)
there is more protein. This is precipitated by the alternate addition of hydro-
chloric acid and potassio-mercuric iodide. Pavy and also Pfliiger recommend
extraction with strong potash, and subsequent precipitation with a certain per-
centage of alcohol ; this method extracts all the glycogen easily.

Fig. 55. — Hut-air oven with gas reguUitor (<;). (Gscheidlen.)

Fig. 56.— Plate of osazone crystals highly magnified.
A, phmyl-glucosazone. B, phenyl-maUosazone. C\ phenyl-lactosazone.

CARBOHYDRATES 177

quantities of a, b, and c in three test-tubes ; to each add an equal amount of
FehHng's sohition, and boil : a will give a heavy precipitate of cuprous
oxide, b one not so heavy, and c 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 m 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 radicals of the hepatic vein.

4. Phenyl-Hydrazine Test for Sugars. — To 5 c.c. of the suspected fluid {e.g.
diabetic urine) add 1 decigramme of phenyl-hydrazine hydrochloride, 2 deci-
grammes of sodium acetate, and heat on the water-bath at 100*" C. for 30 to
60 minutes. 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 crystals. Examine the crystals with the microscope (see
accompanying plate).

Dextrose gives a precipitate of phenyl-glucosazone C,3Hio04(N2H.CgH3)2,
which crystallises 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 190-198°, 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 0,2H.3oOj)(NoH.CyIl5).^. It crystallises
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 (C2|H32N40y). It crystallises in yellow
needles much wider than those yielded by ghicose 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.

The chemistry of the- phenyl-hydrazine reaction is represented in the
following equations, dextrose being taken as an example of the sugar used :-

I. CH20H[CH(0H)] 3CH(0H)C0H -f H,N.NH(C6H5)

[dextrose] [phenyl-hydrazine]

CH.0H[CH(0H)]3CH(0H)CH

II + H,0

N-NH(C6H,)

[hydrazone] . [water]

II. CH20H[CH(OH)]3CH(OH)CH

N - NH(CoH,)

[hydrazone] [phenyl-hydrazine]

CH.OH [CH(OH)] 3C - CH

II II + H2 -f H2O

CeH^.NH-N N-NH.C,H,

[osazone] [hydrogen] [water]

N

178 ESSENTIALS OF CHEMICAL PHYSIOLOaY

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.c. of acetic acid containing 38 per
cent, of glacial acetic acid. Dextrose reduces this reagent on boiling ;
maltose and lactose do not. This test is not very delicate, and the reagent
must be freshly prepared.

6. The Polarimeter. — Estimate the strength of a solution of dextrose by
means of the polarimeter (see Appendix).

7. Formation of Mucic Acid. — Take 1 gramme of lactose and heat it in a
porcelain capsule with 12 c.c. of nitric acid on a water-bath until the fluid is
reduced to one-third of its original volume. A precipitate of mucic acid
separates out. Cane sugar, maltose, dextrose, dextrin, and starch treated in
the same way, yield an isomeric acid called saccharic acid, which, being
soluble, does not separate out. Lactose yields both acids ; galactose mucic
acid only.

8. Pentoses give the ordinary reduction tests for sugar and yield osazones,
but do not ferment with yeast. They give the two following characteristic
tests ; they may be performed with gum arable (which contains arabinose)
or pine- wood shavings (which contain xylose).

(a) Phloroglucin reaction. Warm some distilled water with an equal
vohime of concentrated hydrochloric acid in a test-tube and add phloroglucin
until a little remains undissolved. Add a small quantity of gum arable,
and keep the mixture warm in the water-bath at 100° C. The solution
becomes cherry-red, and a precipitate settles out, which is soluble in amyl
alcohol. This solution gives an absorption band between the D and E lines.

(b) Orcin reaction. Substitute orcin for phloroglucin in the foregoing
experiment. The solution becomes violet on warming, then blue, red, and
finally green. A bluish green precipitate settles out, soluble in amyl alcohol.
This solution gives an absorption band between C and D.

9. Glycuronic acid gives all the above reactions ; it may be distinguished
as follows : —

Take 50 c.c. of glycuronic acid solution in a capsule ; add 1 gramme of
p-bromphenyl-hydrazine and rather more than the same amount of sodium
acetate. Keep the mixture in the water-bath at 100° C. for a quarter of an
hour, when yellow crystals of ^;-bromphenyl-hydrazone separate out. After
cooling filter off the crystals and wash them with absolute alcohol, in which
they are insoluble. Under the same conditions carbohydrates yield ^j-brom-
phenyl-osazones, but these ^re soluble in absolute alcohol. The j)-brom-
phenyl-hydrazone is soluble in absolute alcohol to which pyridine has been
added ; the rotatory power of this solution is greater than that of any of the
osazones.

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 CO. 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.

3. 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 achron -dextrin. The liquid also contains a
reducing sugar, maltose.

4. Take 50 c.c. of a solution of maltose and determine how much of it
is necessary to reduce 10 c.c. of Fehling's solution.

5. Take another 50 c.c. and boil it with 1 c.c. of strong sulphuric acid fo

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 j? = c.c. of maltose

2x
solution necessary to reduce 10 c.c. of Fehling's solution, then ^ - c.c. of

o

dextrose solution necessary for the same purpose. The strength of the

maltose solution can be calculated from the fact that 10 c.c. of Fehling's

solution correspond to 0*05 gramme of dextrose.

N 2

LESSON XV
CBYSTALLISATION 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.c. of ammonium sulphate
solution are added in successive quantities of 10 or 15 c.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. Dilute 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
^th objective, avoiding pressure on the cover slip. (F. G. Hopkins.)

LESSON XVI

MILK

1. Caseinogen in milk exists in the form of a salt (calcium caseinogenate).
Add acetic acid to milk, and this salt is decomposed, and free caseinogen
(with entangled fat) is precipitated. Collect the precipitate so produced from
a pint of milk on a filter, and wash thoroughly with distilled water : grind it
up with calcium carbonate in a mortar, and add about a pint of distilled
water ; allow the mixture to stand for about an hour. 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 if all calcium salts have been successfully washed away, nor in 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 may be shown by
taking some of the 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
calcium chloride. A formation of insoluble curd now occurs.

3. Caseinogen may be precipitated as a salt from milk by the addition of
alcohol. This reagent also precipitates the other milk proteins.

4. The method of salting out described in Lesson VI., Exercise 10 (p. 41),
may also be used. Add to some milk an equal volume of saturated solution
of ammonium sulphate.. Caseinogen as a salt is thus precipitated, and
entangles the fat; with it. Filter off the precipitate and examine the filtrate
as follows : — Saturate it with sodium chloride ; a small amount of precipitate
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. Filter it off; acidify the filtrate with a few
drops of 2-per-cent. acetic acid and heat it in a water-bath gradually. About
77° C. the remaining protein (lactalbumin) is coagulated.

LESSON XVII

THE PROTEOSES

1. Witte's peptone contains very little true peptone, but consists chiefly
of proteoses, 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 pro-
cess 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) It does not coagulate on heating.

(b) Biuret reaction (due both to peptone and proteoses).

(c) A drop of nitric acid, best added hj a glass rod, gives a precipitate
which dissolves upon heating and reappears on cooling. (This is due to the
proteoses present.)

(d) The precipitate produced by the addition of acetic acid and a drop of
potassium ferrocyanide is also soluble on heating, and reappears on cooling.

4. For the separation of the proteoses and peptone proceed as follows : —

(a) Saturate the solution with ammonium sulphate, and filter. The
filtrate contains the peptone, and the precipitate the proteoses. The peptone
is not precipitated by nitric acid, nor by most of the reagents that precipitate
other proteins. It is precipitated completely by alcohol, tannin, and potassio-
mercuric iodide ; imperfectly by phospho-tungstic and phospho-molybdic acid.

It gives the biuret reaction, bid in the jJresence of aminoniiim sulphate a
large excess of caustic potash is necessary.

(b) Dialyse another portion of the solution ; hetero-proteose 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-proteose and hetero-proteose are precipitated. Filter. The filtrate
contains the deutero-proteose and peptone.

The proto- and hetero-proteose may be redissolved by adding distilled
water, and may be separated from each other by dialysis (see b).

Deutero-proteose 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-proteose, but not the peptone.

Deutero-proteose gives the nitric acid reaction (see 3, c) characteristic of
the proteoses only in the presence of excess of salt. If the salt is removed
by dialysis, nitric acid then causes no precipitate.

THE PROTEOSES

183

5. Among the important reactions of proteins is Rose's or 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 (see p. 39). Gnezda found that if a dilute
solution of nickel sulphate is used instead of copper sulphate, the native pro-
teins 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 : —

Proteiu

Copper sul- Copper sul-
phate and phate and
ammonia potash

Nickel sul-
phate and
ammonia

Nil

Nickel sul-
phate and
potash

Cobalt sul-
phate and
ammonia

Cobalt sul-
phate and
potash

Albumins and\
globulins /

Blue ': Violet

Yellow
Orange

Nil

Heliotrope-
purple 1

Proteoses and \
peptones /

Rose-red Rose-red

i

Yellow

Nil

Red browu

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 proteins
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 proteins 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.

LESSON XVIII

DIGESTION

1. Activity of Pepsin Solutions (Griitzner's Method). — Examine the com-
parative 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. • In the original method the amount
of carmine set free is estimated by an artificial scale consisting of ten solutions
of carmine of different known strengths.

The carmine solution for staining the fibrin is prepared by dissolving
1 gramme of carmine in about 1 c.c. of ammonia ; to this 400 c.c. of water
are added, and the mixture is kept in a loosely stoppered bottle till the smell
of ammonia has become faint.

The fibrin is stained by taking it perfectly fresh and clean. It is chopped
finely and placed in the carmine solution for twenty-four hours. The fluid is
strained off and the fibrin washed in water till the washings are colourless.
It is kept in a stoppered bottle with just enough ether to cover it.

2. Mett's Tubes.— A method which is now more generally employed for
estimating the proteolytic activity of a digestive juice is one originally
introduced by Mett. Pieces of capillary glass tubing of known length are
filled with white of egg. This is set into a solid by heating to 95° C. They
are then placed in the digestive fluid at 36° C, and the coagulated egg-white
is digested. After a given time the tubes are removed ; and if the digestive
process has not gone too far, onh' a part of the little column of coagulated
protein will have disappeared ; the length of the remaining column is easily
measured, and the length that has been digested is a measure of the digestive
strength of the fluid.' This forms a very convenient method to use in experi-
ments on velocity of reaction. Schiitz's Law states that the amount of action
is proportional to the square root of the amount of ferment. If this rule
applies (which is doubtful) it applies only to the action of pepsin hydrochloric

' Hamburger has used the same method in investigating the digestive action of
juices on gelatin. The tubes are filled with warm gelatin solution, and this jellies
on cooling. They are placed as before in the digestive mixture, and the length of
the column that disappears can be easily measured. These experiments must, how-
ever, be performed at room temperature, for the usual temperature (.36°-40° C.) at
which artificial digestion is usually carried out would melt the gelatin. He has also
used the same method for estimating araylolytie activity, by filling the tubes with
thick starch paste.

DIGESTION 186

acid. In most cases the rapidity of action »is directly proportional to the
amount of ferment present.

3. The Acid of Gastric Juice. — The digestive powers of the acids are pro-
portional to their dissociation and the number of H ions liberated. The
anions, however, modify this by having different powers of retarding the
action. The greater suitability of hydrochloric over lactic acid, for instance,
in gastric digestion is due to the fact that the former acid more readily
undergoes dissociation.

Hydrochloric acid is absent or diminished in some diseases of the
stomach, especially in cancer ; this is true for cancer in general even when
the stomach is not involved ; the best colour tests for it are the following : —

(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. Ked crystals form, or, if
much peptone is present, there will be a red paste. The reaction takes place
with one part of hydrochloric acid in 10,000. The organic acids do not give
the reaction.

(6) Tropaeolin test. Drops of a saturated solution of tropseolin-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 tropaeolin drop, still at 40° C. ;
and if hydrochloric acid is present a violet spot is left when the fluid has
evaporated. A di'op of 0'006-per-cent. hydrochloric acid leaves a distinct
mark. ,

(c) Topfer's test. A drop of dimethyl-amino-azo-benzol is spread in a
thin film on a white plate. A drop of dilute hydrochloric acid (up to 1 in
10,000) strikes with this in the cold a bright red colour.

Lactic acid is sometimes present in the gastric contents, being derived by
fermentative processes from the food. It is soluble in ether, and is generally
detected by making an ethereal extract of the stomach contents, and evapo-
rating the ether. If lactic acid is present in the residue it may be identified
by Uffelmann's reaction in the following way : —

A solution of dilute ferric chloride and carbolic acid is made as follows : -

10 c.c. of a 4-per-ceut. solution of carbolic acid.

20 c.c. of distilled water.

1 drop of the liquor ferri perchloridi of the British Pharmacopoeia.

On mixing a solution containing a mere trace (up to 1 part in 10,000) of
lactic acid with this violet solution, it is instantly turned yellow. Larger
percentages of other acids— for instance, more than 0*2 per cent, of hydro-
chloric acid — are necessary to decolorise the test solution.

The reaction is not absolutely convincing, since other acids (though in
larger percentages) decolorise the solution, but the characteristic yellow colour
given even by dilute lactic acid is not developed. Note the decolorisation
which occurs when 0*2 hydrochloric acid is added to Uffelmann's reagent.

Hopkins's reaction for Lactic Acid. — Place 3 drops of a 1-per-cent. alco-
holic solution of lactic acid in a clean, dry test-tube, add 5 c.c. of concen-
trated sulphuric acid and 3 drops of a saturated solution of copper sulphate.
Mix thoroughly and place the test-tube in a beaker of boiling water for five
minutes. Then cool thoroughly under the tap, and add 2 drops of a 0*2-per-

186 ESSENTIALS OF CHEMICAI. PHYSIOLOGY

cent, alcoholic solution of thiophene and shake. Replace the tube in the
boiling water ; as the mixture gets warm a cherry-red colour develops.

4. Demonstration of Pancreatic Secretion. — In an anaesthetised dog insert
a cannula into the main pancreatic duct, and collect the juice in a suitable
vessel. Inject some 0-4-per-cent. hydrochloric acid into the duodenum, and
note after some minutes the abundant flow of pancreatic juice. Next
ligature off and remove two or three feet of the upper part of the small
intestine, wash out the contents and slit it open ; scrape off the mucous
membrane with the back of a scalpel ; preserve a small quantity of the
scrapings for future use and label this A. Grind up the remainder in a
mortar with clean sand or powdered glass, and add 0*4 per cent, hydrochloric
acid. Transfer the mixture to a flask, boil thoroughly, and when cool
neutralise with a little caustic soda solution. Filter ; the filtrate contains
secretin, which has been formed by the acid from the ^jro-secretin of the
intestinal epithelium. Inject some of this solution through a cannula into
the external jugular vein of the dog, and an abundant flow of pancreatic
juice is an almjst immediate result.

5. Characters of the juice so obtained : —

{a) It is a clear, colourless fluid, and very strongly alkaline.

(6) Mixed with starch solution and kept at 40° C. dextrin and maltose are
rapidly formed.

(c) Mixed with milk there may be some curdling produced, but the most
marked effect is that the milk rapidly becomes acid, and a smell of fatty
acids is noticeable.

{d) Added to fibrin and kept at 40° protein digestion occurs very slowly;
next day, however, the fibrin will be in large measure digested.

{e) Mix some of the pancreatic juice with the scraping of the intestine
which was preserved and labelled A. Then add fibrin. The fluid is now
strongly proteolytic, and at 40° C. the fibrin rapidly dissolves ; trypsin has
been liberated from the trypsinogen of the juice by the intestinal entero-
Tiinase.

6. Products of Pancreatic Digestion of Proteins. — A pancreatic digest
should be prepared beforehand by the demonstrator. This may be done by
digesting a quantity of protein with artificial pancreatic juice, if the natural
juice prepared by the action of secretin is not available ; in the latter case
the addition of intestinal epithelium (entero-kinase) should not be forgotten.
Unless an antiseptic has been added putrefaction will also occur, and its
odour will be very perceptible after the mixture has been placed in the warm
chamber for some time.

A very good mixture for the purpose will be found to be the following :—

100 grammes of plasmon (caseinogen).
10 grammes of sodium carbonate.

1 litre of water.
25 c.c. of Benger's liquor pancreaticus.

0*5 gramme sodium fluoride.

3 c.c. chloroform.

The last two items on the list are added to prevent putrefaction.

i)iaESTioN 187

After digestion has progressed for one to two days another 10 c.c. of Hquor
pancreaticus may be added.

The products of digestion in one case should be examined, say, after six
hours' digestion, and in another case after thirty-six hours' digestion or more.
The digestive products should then be searched for ; the early products of
digestion (alkali-albumin, deutero-proteose, &c.) will become less abundant
with the length of time that digestion has been allowed to progress, and the
later products (peptone, leucine, tyrosine, tryptophane, &c.) will become more
abundant. The methods for testing most of these substances have been
already given. The following are the tests for tryptophane, leucine, and
tyrosine : —

(a) Try2}to;plian6. — Add a few drops of bromine water ; a violet colour is
produced.

(6) Leucine and Ty7'osine. — i. Examine microscopical specimens of these.
The deposit generally found in rather old specimens of Banger's liquor pan-
creaticus will be a convenient source of these crystals.

ii. To some of the pancreatic digest add Millon's reagent and filter ofi'
the precipitated protein. Boil the filtrate, and the presence of tyrosine is
indicated by a red colour. If tyrosine is abundant the red colour appears
without boiling. Leucine does not give this test.

iii. Faintly acidify another portion of the filtered digest with acetic acid
and boil ; if any protein matter is still undigested it will be thus coagulated
and can be filtered off. Eeduce the filtrate to a small bulk until it begins to
become syrupy. Leave overnight in a cool place, and crystals mainly of
tyrosine will separate out. Filter these off through fine muslin, and
evaporate down the filtrate to the consistency of a thick syrup ; leave this
overnight again, and a second crop of crystals, forming a skin on the surface
and consisting mainly of leucine, will have separated out.

7. 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 guinea-pigs should be killed by bleeding, and the blood collected and
defibrinated, and utilised for the preparation of oxyheemoglobin 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. 112 all give good results.
If amyl nitrite is used instead of ether in the third method, crystals of methasmo-
globin are obtained.

LESSON XIX

HEMOGLOBIN AND ITS DERIVATIVES

Defibrinated ox-blood suitably diluted may be used in the following
experiments as in those described in Lesson IX.

1. Place some in a haematoscope (see fig. 33, p. 116) in front of the large
spectroscope. Note the position of the two characteristic bands of oxyhaemo-
globin ; these are replaced by the single band of hsemoglobin after reduction by
the addition of Stokes's reagent (see footnote, p. 115) 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 oxyhaemoglobin
solution may be placed on the microscope stage, and a test-tube containing
carbonic oxide hgemoglobin in front of the slit in the side of the instrument.
Notice that the two bands of carbonic oxide hsemoglobin are very like those
of oxyhaemoglobin, but are a little nearer to the violet end of the spectrum.

Carbonic oxide haemoglobin may be readily prepared by passing a stream
of coal gas through the diluted blood. It has a cherry-red colour and is not
reduced by the addition of ammonium sulphide (fig. 57, spectrum 4).

3. Methsemoglobin. — 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 haemoglobin appears.

4. Acid Haematin. — (a) Prepare the following mixture : — 150 c.c. of 90-per-
cent, alcohol and 6 c.c. of concentrated sulphuric acid ; take about 5 c.c. of
this mixture and boil it in a test-tube. While still hot drop into it a few
drops of undiluted defibrinated blood, and filter. Note the brown colour of
the filtrate. Compare the position of the absorption band in the red with
that of methaemoglobin ; that of acid haematin is further from the D line
(fig. 57, spectrum 7).

(b) Add some glacial acetic acid to undiluted defibrinated blood. Extract
this with ether by gently agitating it with that fluid. The ethereal extract
should then be poured off and examined spectroscopically. The band in the
red is seen, and on further diluting with ether three additional bands appear.

5. Alkaline Haematin. — {a) Add to diluted blood a small quantity of strong

HEMOGLOBIN

189

Fio. 57.— 1, Solar spectrum. 2, Spectrum of oxyhEemoglobin (0*37 p.c. solution). First band, A 589-
564 : second band, A 555-517. 3, Spectrum of haemoglobin. Band, A 597-535. 4. Spect-um of CO-
haemoglobin. First band, A 583-56 i ; second band. A 547-521. 5, Spectrum of methsemoglobin
(concentrated solution). 6, Speatrum of methaemoglobin (dilute solution). First band, A 647-
622: second band, A 587-571; tliird band, A 552-532; fourth band, A 514-490. 7, Spectrum of
acid lisematin (ethereal solution). First band, A 656-615 ; second band, A 597-577 ; third band, A
557-529; fourth band, A 517-488. 8, Spectrum of alkaline haematin. Band from A 630-581.
9, Spectrum of hfemochromogen (reduced haBmatin). First band, A 569-542 ; second band, A
535-504. 10, Spectriim of acid htematoporphyrin. First band, A 607-593 ; second band, A 585-
536. 11, Spectrum of alkaline hajmatoporphyrin. First baud, A 633-612 ; second band, A 589-564 ;
third band, A 549-529 ; fourth band, A 518-488. The above measurements (after MacMunu) are
in millionths of a millimetre. The 1 quid was examined in a layer 1 centimetre thick. The edges
of ill-defined bands vary a good deal with Dhe concentration of the solutions.

190

ESSENTIALS OF CHEMICAL PHYSIOLOGY

caustic potash and boil. The colour changes to brown, and with the
spectroscope a faint shading on the left side of the D line is seen (fig. 57,
spectrum 8).

(6) The band is much better seen in an alcoholic solution. Prepare the
following mixture : — 150 c.c. of 90-per-cent. alcohol, and 18 c.c. of 50-per-
cent, potash. Take about 5 c.c. of this mixture in a test-tube and boil it.

Fig. 58.— The photographic spectrum of haemoglobin oxyhaemoglobin. (Gamgee.)

G «K LM N O

Fig. 59.— The photographic spectrum of oxyhaemoglobin and methaemoglobin. (Gamgee.)

While still hot drop into it a few drops of undiluted defibrinated blood. The
fluid then shows the spectrum of alkaline hsematin. This may then be used
for the next experiment.

6. Haeinochromogen. — Add ammonium sulphide to the solution of alkaline
hgematin ; the colour changes to red, and two bands are seen, one between D

HiEMOGLOBIN 191

andE, and the other nearly coinciding with E and b (fig. 57, spectrum 9).
The spectrum of alkaline haematin reappears for a short time after vigorous
shaking witli air.

7. Haematoporphyrin. — To some strong sulphuric acid in a test-tube add a
few drops of undiluted blood, and observe the spectrum of acid haemato-
porphyrin (iron-free haematin) (fig. 57, spectrum 10). Map out all the spectra
you see on a chart.

8. The photographic Spectrum. — Haemoglobin 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
by allowing the spectrum to fall on a fluorescent screen, or on a sensitive
photographic plate. In order to show absorption bands in this part of the
spectrum very dilute solutions of the pigment must be used.

In order to demonstrate these bands, the telescope of a large spectroscope
is removed, and a beam of sunlight or of light from the positive pole of an
arc lamp is allowed to fall on the slit of the collimator. The spectrum is
focussed on a fluorescent screen.^ The slit is then opened very widely, and
the coloured solution is interposed on the path of the beam falling on the
slit.

Oxyhaemoglobin shows a band (Soret's band) between the lines G and H.
In haemoglobin, carbonic oxide haemoglobin, and nitric oxide haemoglobin,
this band is rather nearer G. Methaemoglobin and haematoporphyrin show
similar bands.

The two preceding figures show the ' photographic spectra ' of haemo-
globin, oxyhaemoglobin, and methaemoglobin, and will serve as examples of
the results obtained. I am greatly indebted to Prof. Gamgee, to whom we
owe most of our knowledge on this subject, for permission to reproduce these
two specimens of his numerous photographs.

0. Preparation of Pure Oxyhaemoglobin. — The following method is described
in Stirling's ' Practical Physiology.' Centrifugalise dog's defibrinated blood
and pour off the serum. Centrifugalise again with physiological saline
solution repeatedly until the supernatant fluid contains only traces of protein.
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 filtration. Pour off the clear red fluid : cool it to 0° C, add
one-fourth of its volmne of absolute alcohol previously cooled to 0° C. Shake •
well, and then let the mixture stand at 5°-15° C. for 24 hours. As a rule
the whole passes into a glittering crystalline mass. Filter at 0° C. and wash
with ice-cold 25-per-cent. alcohol. Eedissolve the crystals in a small quantity
of water, and recrystallise as before. The crystals may then be spread on
plates of porous porcelain, and dried in a vacuum over sulphuric acid.

' Fluorescent screens, similar to those in common use in observations made
with Eontgen rays, may be made by coating white cardboard with barium platino-
cyanide.

LESSON XX

SERUM

1. The following methods of precipitating serum globulin should be per-
formed : —

(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 abundant
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
can be obtained from the decanted liquid by treating it with a trace of acetic
acid (the ' serum casein ' mentioned above). Repeat the carbonic acid
method without dilution ; no precipitate forms.

(c) By Dialysis. - Put some serum in a dialyser with distilled water in the
outer vessel. The water must be frequently changed. In order to prevent
decomposition a few crystals of thymol are added. In a day or two the salts
have passed out ; the proteins 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. (See also bottom of p. 193.)

(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
by adding distilled water. It readily dissolves, owing to the salt adherent
to it. The solution is opalescent. Label it 'A.'

' This may be conveniently done by a shaking machine before the class meets.
2 On account of the prolonged nature of these operations, they must necessarily
be performed by the demonstrator beforehand.

SERUM 193

Render A faintly acid with a drop of 2-per-cent. acetic acid, and heat in a
test-tube. The temperature of the test-tube may be raised 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 sufi&cient to cover the bulb of the thermometer. A flocculent
precipitate of coagulated serum globulin separates out at about 75°.

Now take the filtrate B. This contains the serum albumin. Dilute it
with an equal volume of water ; render it faintly acid as before, testing the
reaction with litmus paper. Heat. A flocculent precipitate (a) falls at about
73° C. ; filter this off; note that the filtrate is less acid than that from
which the precipitate has separated, or it may even be alkaline. If so, make
it faintly acid again^ and heat; a precipitate falls at 77-79° C. (/3). 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 (3
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,^ + 7H2O) ; on adding sodium sulphate a
double salt (MgSO^.Na.^SO^ + 6H0O) 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. There 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 nitrate 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, jS, and y varieties
by the use of the method of fractional heat coagulation, it must be men-
tioned that at present no further difference has been shown to exist between
them, and the opinion has been very freely expressed that the results
obtained are not trustworthy. I am convinced that the method is a good one,
especially as in other cases (see Muscle) the proteins 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 subjudice.

Recent research has shown that sermn globulin is not a single protein.
We have already seen that the precipitation which occurs by means of
dialysis is incomplete. It has now been shown that serum globulm as
' salted out ' by means of the sulphate of magnesium or ammonium really
consists of two proteins ; one of these (eu-globulin) is precipitable by dialysis :
the other (pseudo-globulin) is not.

* That is at room temperature ; if the temperature is raised to 36° C. sodium
sulphate acts like ammonium sulphate and precipitates all the proteins, except
peptone.

0

LESSON XXT

COAGULATION OF BLOOD

1. Effect of decalcifying Agents in hindering Coagulation. — From an
anaesthetised dog collect samples of blood from the carotid artery, into
which a suitable cannula should have been previously inserted.

(a) Collect the first sample in an equal volume of 0*4 per cent, solution
of potassium oxalate made with physiological salt solution.

(6) Collect the second sample in an equal volume of 0-4 solution of
sodium fluoride.

(c) Collect the third sample in a quarter of its volume of 10 per cent,
solution of sodium citrate.

In all three cases mix thoroughly, and coagulation is hindered owing to
decalcification, as explained on page 106.

The separation of the plasma from the corpuscles may be most readily
carried out by a centrifugal machine, one form of which is shown in the
next figure; the corpuscles settle and the supernatant plasma can be
then pipetted off. Sedimentation is specially rapid in the case of citrate
blood, and a well-marked layer of colourless corpuscles and platelets may
usually be seen on the top of the mass of red corpuscles.

Oxalate plasma and citrate plasma coagulate on the restoration of the
calcium by adding a few drops of calcium chloride solution, as we have
already seen in the elementary course (p. 101). Fluoride plasma does not
coagulate unless fibrin ferment (or some fluid such as serum which contains
fibrin ferment or thrombin) is added as well as the calcium salt. Fluoride
plasma thus forms a convenient test -fluid for fibrin ferment.

If in either case the plasma is previously heated to 60° C. and filtered,
coagulation — that is to say, fibrin formation — can never be produced, because
its mother-substance, fibrinogen, which is coagulated by heat at 56° C, has
been destroyed and removed.

2. Influence of Leech Extract on Coagulation. — The same dog still under the
anaesthetic may be next used for the following experiments : —

(a) Draw off a sample of blood into a clean test-tube, and note the time
it takes to clot.

(6) Draw off a second sample into about half its volume of leech extract,
made by grinding up the heads of about twenty leeches in 20 c.c. of salt
solution, and filtering. This remains unclotted for hours or days.

(c) Inject 10 c.c. of the extract into the jugular vein of the animal, and
draw off samples of blood from time to time, comparing the coagulation
time (which gradually lengthens) with that of specimen a.

{d) Having obtained a specimen which does not clot at all, dilute it with

COAGULATION OF BLOOD

195

salt solution and pass a stream of carbon dioxide through it. Clotting is not
produced as it is in ' peptone ' blood (which see). In order to produce clot-
ting, excess of serum, or some fluid containing thrombin must be added.
The action of leech extract is mainly due to the fact that it contains anti-
thrombin.

(e) The experiments described under d may be repeated with leech
extract plasma, obtained from the blood by centrifugalising.

(/) Instead of leech extract, a solution of its active principle (hirudin)
may be used. This produces no fall of blood pressure, and so contrasts

Fiu. tJO.— Centrifugal machiue as made by Kumie. Glass vessels containing the substances t«j be
centrlfugalised a:e placed within the six metallic tubes whicli hang vertically while the disc is at
rest ; when tlie machinery is set going they fly out into the horizontal position.

with what occurs in ' peptone ' injection. Leech extract produces a very
small fall of arterial pressure.

3. Influence of Commercial Peptones (Proteoses) on Coagulation.— For the
purpose of the following experiments another dog must be employed.

The animal having been anaesthetised a cannula is placed in the
external jugular vein for the injection of the * peptone.'

The carotid artery is connected to a mercurial manometer for the
registration of arterial pressure.

Another convenient artery must be exposed and a cannula inserted into
it for the collection of samples of blood.

{a) First draw off a sample of blood and note its coagulation time.

(5) Draw a second sample into a strong solution of commercial peptone.
The coagulation time is somewhat longer than in a.

(c) Then inject the peptone quickly, so that the animal receives 0*3
gramme per kilo, of body-weight. Note during and for some time after the
injection a great fall in arterial blood pressure. This has been shown by
the oncometer to be due to vascular dilatation.

o 2

196 ESSENTIALS OF CHEMICAL PHYSIOLOGY

(d) After the injection draw off successive samples, and note the great
prolongation of the coagulation time which is soon produced.

(e) Dilute some of the blood which does not clot with twice its volume of
salt solution, and pass a stream of carbonic acid through the mixture ; co-
agulation soon occurs.

(/) The same experiment may be repeated with the same result, if
' peptone ' plasma obtained by centrifugalising is used instead of the whole
blood.

{g) Finally bleed the animal to death, collecting the blood in three
successive glass cylinders. Place them in the ice chest, and examine them
a few days or a week later.

The first lot of blood collected will show sedimentation of corpuscles,
and a slight clot at the junction of the corpuscles and supernatant plasma —
that is, at the place where the white corpuscles and platelets lie.

The last lot of blood collected shows less sedimentation, and will
probably have clotted throughout. This is because the blood removed last
has been diluted by tissue lymph, which has passed into the blood -stream in
an attempt to increase the volume of the blood, which has been lessened
by the previous bleeding ; the clot produced is probably due to the action
of thrombokinase.

The middle sample will show something intermediate between the two
extremes, the usual state of things being clot through the sediment, and the
plasma above it still fluid..

4. Intravascular Coagulation. — A solution of nucleo-protein from the
thymus, testis, lymphatic glands, or kidney has been prepared beforehand by
the demonstrator. It may be prepared in one or 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.c. of the acetic acid of
the ' Pharmacopoeia ' diluted with twice its volume of water for every 100 c.c.
of extract) is then added to the decanted liquid. After some hours the pre-
cipitated nucleo-protem (called tissue-fibrinogen by Wooldridge) falls to the
bottom of the vessel. This is collected and dissolved in 1 -per- cent, sodium
carbonate solution.

(6) The Sodiuin 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-
protein rises to the surface of the water, where it may be collected and
dissolved as before.

A rabbit is angesthetised, and a cannula inserted into the external jugular
vein. The solutionis 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. The blood of
the portal vein is usually clotted most. If a dog is employed instead of a rabbit
in this experiment, coagulation is usually confined to the portal area. This
is related to the greater venosity of the blood in this situation. If venosity
is increased in any other area, as by tetanising the muscles of one leg, clot-
ting will be found also in the veins of this region.

LESSON XXII

MUSCLE AND NERVOUS TISSUE

1. Hopkins's Lactic Acid Test (see p. 185) may be applied as follows. Eemove
one hind limb of a pithed frog. Stimulate the sacral plexus of the other side
for ten minutes with a strong Faradic current. Then amputate the other
hind limb. Skin both legs, and chop up the muscles of the two sides
separately. Pound each in a mortar with clean sand and then with 15 c.c.
of 95-per-cent. alcohol. Transfer the mixture to a beaker, and warm in the
water-bath for a few minutes. Filter, and evaporate the filtrate to dryness
in a water-bath. Extract the residue with about 5 c.c. of cold water, rubbing
it up thoroughly with a glass rod. Filter and boil the filtrate in a test-tube
for about a minute with as much animal charcoal as will lie on a threepenny-
piece. Filter again and evaporate the filtrate to dryness in a water-bath.
Allow the residue to cool, and dissolve it by shaking in 5 c.c. of concentrated
sulphuric acid. Transfer this to a dry test-tube ; add three drops of
saturated solution of copper sulphate, and place the tube in boiling water
for five minutes. Cool and add 2 drops of 0*2-per-cent. solution of thiophene
in alcohol ; replace the tube in the boiling water. A cherry-red colour
develops in the tube containing the extract from tetanised muscle, but not
in the other.

2 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. It will probably be
faintly acid. The acid is sarco-lactic acid. It may be identified by
Ufifelmann's (p. 185) or Hopkins's reaction.

3. The coagulation of musele is very like that of blood. This ma3' 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. Co-
agulation— that is, formation of a clot of myosin — occurs in both, but earliest
in that at 40° C.

4. Kemove the clot of myosin from 3 ; observe it is soluble in 10-per-
cent, sodium chloride, and also in 0-2-per-cent. hydrochloric acid, forming
syntonin.

5. Make an extract of muscle in the same way, using a small quantity
of physiological salt solution instead of the strong solution of magnesium
sulphate employed in the foregoing experiments. Such an extract contains
the two principal muscular proteins, viz. paramyosinogen (v. Fiirth's myosin)

198 ESSENTIALS OF CHEMICAL PHYSIOLOGY

and myosinogen (v. Fiirth's myogen), the two precursors of the musele-clot
or myosin (v. Fiirth's muscle-fibrin). Small quantities of other proteins also
present are mainly due to unavoidable mixture with small amounts of blood
and lymph.

These two proteins diflFer in temperature of heat coagulation. Take the
extract and heat it in a test-tube within a water-bath ; at 4V C. para-
myosinogen is coagulated ; filter this off and heat the filtrate ; at 56° C.
flocculi of the coagulated myosinogen separate out.

6. Paramyosinogen is precipitable by dialysis and is a true globulin.
Myosinogen is what is called an atypical globulin, and corresponds to the
pseudo-globulin of blood serum and egg-white. Though readily salted out of
solution like paramyosinogen it is not precipitable by dialysis.

7. In the process of clotting, such as occurs in rigor mortis, paramyosino-
gen is directly converted into myosin ; whereas myosinogen first passes into
a soluble modification (coagulable by heat at the remarkably low temperature
of 40° C.) before myosin is formed. This is shown in a diagrammatic way
in the following scheme : —

Proteins of the living muscle

Paramyosinogen Myosinogen

"^\ Soluble myosin

Myosin
(the protein of the muscle-clot).

8. When a muscle is gradually heated, at a certain temperature it con-
tracts permanently and loses its irritability. This phenomenon is known as
heat-rigor, and is due to the coagulation of the proteins in the muscle. If a
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 protein which coagulates at 40° C. This additional protein is
the soluble myosin alluded to above, some of which, in the muscle of cold-
blooded animals, is present before rigor mortis occurs.

In addition to the proteins mentioned, there is a small quantity of nuclco-
protein.

9. Involuntary Muscle. — The main facts just described for voluntary are
true also for involuntary muscle. The chief distinction lies in the quantity
of nucleo-protein, which is more abundant in those forms of muscle the
fibres of which are least different from the mesoblastic cells from which all
ultimately originate. This may be readily shown by the following simple
experiment.

MUSCLE AND NERVOUS TISSUE

199

Take equal parts of voluntary muscle, heart muscle, and plain muscle
(say from the stomach wall), and extract each for the same time with equal
amounts of 0'15 per-cent. solution of sodium carbonate. Filter and add to
each filtrate acetic acid, drop by drop. The extract of voluntary muscle

Vy

D

E

F

Fig. 61. — 1, Absorption spectrum of myohEematin, as seen in ninscle rendered transparent by
glycerin. 2, Absorption spectrnni of modified myohaematin.

gives an opalescence only ; in the case of the plain muscle there is an
abundant precipitate ; the heart muscle gives a result intermediate between
the other two.

10. Pigments of Muscle : —

{a) Notice the difference between the red and pale muscles of the rabbit.

Fig. 62.— a desiccator.

(Gscheidlen.) a, glass plate ; b, belljar ; c, dish of sulphuric add ;
d, stand for capsules.

(IS) Examine a piece of red muscle {e.g. the diaphragm) spectroscopically
for oxyhaemoglobin (or it may be more convenient to make an aqueous
extract of the muscle and examine that).

{c) A piece of the pectoral muscle of a pigeon has been soaked in glycerin.

200

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Press a small piece between two glass slides and place it in fi'ont of the spec-
troscope. Observe and map out the bands of myohsematin. This pigment is
doubtless a derivative of haemoglobin.

(d) Pieces of the same muscles have been placed in ether for twenty-four
hours. The ether dissolves out a yellow lipochrome from the adherent fat.
A watery fluid below contains modified myohaematin. Filter it ; compare
its spectrum with that of hEemochromogen. The myohaematin bands are
rather nearer the violet end of the spectrum (fig. 61, spectrum 2) than those
of haemochromogen (fig. 57, spectrum 9).

11. Creatine : —

(a) Take some of the red fluid described in 10, d, and let it evaporate to
dryness in a, desiccator over sulphuric acid (fig. 62).

In a day or two crystals of creatine tinged with myohaematin separate out.

(b) Take an aqueous extract of muscle, like Ijiebig's extract or beef-tea ;
add baryta water to precipitate the phosphates, and filter. Eemove excess
of baryta by a stream of carbonic acid ; filter off the barium carbonate and
evaporate the filtrate on the water-bath to a thick syrup. Set it aside to cool,
and in a few days crystalline deposits of creatine will be found at the bottom
of the vessel. These are washed with alcohol and dissolved in hot water.
On concentrating the aqueous solution crystals once more separate out.
which may be still further purified by recrystallisation.

NERVOUS TISSUES

The chemical investigation of nervous tissues is not well adapted to
class exercises ; still it may not be uninteresting to state briefly the princi-
pal known facts in relation to this subject. The most important points
which any table of analysis will show are : (1) the large percentage of water,
especially in the grey matter ; (2) the large percentage of protein. In grey
matter, where the cells are prominent structures, this is most marked, and
of the solids, protein material here comprises more than half of the total.
The following are some analyses which give the mean of a number or
observations on the nervous tissues of human beings, monkeys, dogs, and
cats : —

Percentage of

Water

Solids

Proteins in

SoUds

Cerebral grey matter .

83-5

16-5

51

„ white „ . . .

69-9

30-1

33

Cerebellum

79-8

20-2

42

Spinal cord as a whole .

71-6

28-4

31

Cervical cord ....

72-5

27-5

31

Dorsal cord

69-8

30-2

28

Lumbar cord ....

72-6

27-4

33

Sciatic nerves ....

65-1

34-9

29

The most important protein is nude o -protein ; there is also a certain
amount of glohvlin, which, like the paramyosinogen of muscle, is coagulated
by heat at the low temperature of 47° C. A certain small amount of neuro-

MUSCLE AND NEKVOUS TISSUE 201

keratin (especially abundant in white matter) is included in the above table
with the proteins. The granules in nerve cells (Nissl's bodies), which stain
readily with methylene blue, are nucleo-protein 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. 25). A complex substance
called protagon, which crystallises out on cooling a hot alcoholic extract
of brain or other nervous structures is of uncertain composition. Cerebrin
is a term which probably includes several substances, which are nitro-
genous glucosides ; they yield on hydrolysis the sugar called galactose
(see p. 18). They are sometimes called cerebrosides. There are other
phosphorised fats as well, of which Tiejphalin is the best known. The crystal-
line monatomic alcohol cliolestcr'm (see p. 93) is also a fairly abundant con-
stituent of nervous structures, especially of the white substance of Schwann.
Finally there are smaller quantities of other extractives, and a small pro-
portion of mineral salts (about 1 per cent, of the solids).

Fresh 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.

Very little is known of the chemical changes nervous tissues under-
go during activity. We know that ox^^gen is very essential, especially
for the activity of grey matter ; cerebral anaemia is rapidly followed by loss
of consciousness and death. Waller has suggested that small quantities of
carbonic 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 suggestion has recently been confirmed by
direct experiments in which the amount of carbon dioxide formed has been
estimated. Large quantities of carbonic acid act like an anaesthetic, 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 a meduUated nerve-trunk has never yet been experimentally demonstrated ;
Waller's view that this is due to inter-nutritional changes between the axis
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 occurs
in the brain is accompanied by the passing of the products of degeneration
into the cerebro-spinal fluid. Of these, nucleo-protein and choline— a decom-
position product of the lecithin (see p. 26) — 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-bei-i, &c.) choline can also be detected in these situations. The
tests employed to detect choline are mainly three : (1) The fluid is diluted
with about five times its volume of alcohol and the precipitated proteins are
filtered off. The filtrate is evaporated to dryness at 40° C. and the residue

202 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 solution, an alcohohc solution
of platinum chloride is added, and the precipitate so formed is allowed to
settle and is 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
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. There is a danger of mistaking
such cr^fstals for those obtained from the chlorides of potassium and am-
monium ; but the presence of such contaminations may be minimised by the
use of alcohol as water-free as possible. (2) The following test, however,
is entirely distinctive of choline and leads to no risk of confusion with other
substances. The final alcoholic solution prepared as above is evaporated to
dryness, and the residue taken up with water, to this is added a strong
solution of iodine (2 grammes of iodine and G grammes of potassium
iodide in 100 c.c. of water). In a few minutes dark-brown prisms of
choline periodide are formed. These look very like haemin crystals. If
the slide is allowed to stand so that the liquid gradually evaporates, the
crystals slowly disappear, and their place is taken by brown oily droplets^
but if a fresh drop of the iodine solution is added the crystals slowly form
once more. (3) A 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 pro-
duces : this fall is abolished, or even replaced by a rise of arterial pressure, if
the animal has been atropinised. Such tests have already been shown to be
of diagnostic value in the distinction between organic and so-called functional
diseases of the nervous system.

A similar condition can be produced artificiall}^ 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 about three
days after the operation. They then show a progressive increase in the per-
centage 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. ■ One chemical feature
of degeneration is the replacement of phosphorised by non-phosphorised fat.
When the Marchi reaction disappears in the later stages of degeneration, the

' 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 osmic acid. Healthy
nerve fibres are but little affected by the reagent, but degenerated myelin is
blackened like the fat of normal adipose tissue.

MUSCLE AND NERVOUS TISSUE

203

non-phosphoriscd fat has been absorbed. This absorption occurs earUer in
tlie peripheral nerves than in the central nervous system. The non-phos-
phorised fat of degenerated myelin is also either richer in olein, or the olein
is more loosely combined than in the healthy medullary sheath; hence
the deeper reaction with osmic acid even in the presence of chromic acid
as in the Marchi test. The following table gives details of these experi-
ments : —

Cat's sciatic nerves

Condition
of blood

( Minimal

i traces of cho-

{ line present

j Choline more
( abundant

f Choline abun-
1 dant

j Choline much
jless

( Choline al-
J most disap-
( peared

Condition of
nerves

■
— Water

Normal 65-1 ^
1 to 3 days after section 64-5

4 to G „ 69-3

8 „ 68-2
10 „ 70-7
13 „ 71-3

25-27 „ 72-1
29 „ 72-5

44 „ 72-6
100 to 106 „ G6-2

1

Solids

34-9
35-5

30-7

31-8
29-3

28-7

27-9
27-5

27-4
33-8

Percentage of

phosphoras in

solids

1-1
0-9

0-9

0-5
0-3

U-2

traces
0

0
0-9

( Nerves irritable

J and histologically

i healthy
Irritability lost :
degeneration be-

, ginning
Degeneration well

■ shown by Marchi
( reaction

Marchi reaction
still seen, but ab-

■ sorption of de-
generated fat has
set in
Absorption of fat

■ practically com-
(plete

j Keturn of function,
t nerves regenerated

The foregoing figures relate to the peripheral portions of the nerves. Noll
has also shown that the phosphorised fat diminishes somewhat in the central
ends of cut nerves due to ' disuse atroph3^'

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 on the degenerated side.

Cerebro-spinal Fluid. — This plays the part of 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 protein matter (globulin) and a small amount
of a reducing substance, the nature of which was for a long time uncertain
but which seems now to have been proved to be sugar. 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 lecithin 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 —
vi7.. the grey matter.

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 protein.
The hypobromite method of estimation (see p. 141) holds its own, as it is easy
and sufficiently exact for clinical purposes. It has entirely replaced the older
method of Liebig (titration with mercuric nitrate), which is now of purely
historical interest.

When absolute accuracy is necessary, one or other of many recently
introduced methods must be employed. We shall be content with describing
two of these.

(a) Folin's Method. — This depends on the fact that urea is decomposed
quantitatively into ammonia and carbonic acid by boiling with magnesium
chloride solution. The ammonia is estimated by distillation into standard
acid and subsequent titration.

Analysis. — Three c.c. of urine, 20 grammes of magnesium chloride and2c.c.
of concentrated hydrochloric acid are boiled in a flask, closed by a cork through
which a glass tube 20 centimetres in height passes. This acts as a reflux
condenser. The boiling is continued for 25 to 30 minutes. After diluting
with water the mixture is then transferred to a litre flask, 7 c.c. of 20-per-
cent, caustic soda are added, and the ammonia is distilled off and estimated
as described in the final operation in Kjeldahl's method (see Appendix, p. 235).
Every c.c. of decinormal ammonia in the distillate corresponds to 3 milli-
grammes of urea. A small correction has to be made for the ammonia
present as such in the original urine.

(h) Method of Mbrner and Sjbqvist. — The following reagents are necessary:

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. 235).

Analysis. — Five c.c, of urine are mixed with 5 c.c. of the barium mixture
and 100 c.c. of the mixture of ether and alcohol. By this means all nitrogenous
substances except urea are precipitated. Twenty-four hours later this is filtered
off, and the precipitate is washed with 50 c.c. of the ether-alcohol mixture,
the filter-pump being used to accelerate the process. The washings are
added to the filtrate ; a little magnesia is added to this to drive off ammonia.

UKEA AND CHLORIDES IN URINE 205

The alcohol and ether are 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*143, and the result is the amount of urea.

PKEPARATION OF UREA FROM URINE

(1) Evaporate the urine to a small bulk. Add strong pure nitric acid in
excess, keeping the mixture cool during the addition of the acid. Pour off
the excess of fluid from the crystals of urea nitrate which are formed ; strain
through muslin and press between filter paper. Add to the dry product
barium carbonate in large excess. This forms barium nitrate and sets the
urea free. Mix thoroughly with sufficient methylated spirit to form a paste.
Dry on a water-bath and extract with alcohol ; filter ; evaporate the filtrate
on a water-bath and set aside. The urea crystallises out, and may be de-
colourised 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 lu-ine ; add
baryta mixture (see footnote 3, p. 5) until no further precipitate 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 dry-
ness 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
of the total chlorides consists in their precipitation by a standard solution
of silver nitrate (Mohr's method).

The following solutions must be prepared : —

Standard silver nitrate solution. Dissolve 29*075 grammes of fused
nitrate of silver in a litre (1,000 c.c.) of distilled water : 1 c.c. =-0'01 gramme
of sodium chloride.

(a) Saturated solution of neutral potassium chromate.

Analysis. — Take 10 c.c. of 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. AVhen 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 Correctio7is. — 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.

One c.c. should always be subtracted from the total number of c.c. of the

206 ESSENTIALS OF CHEMICAL PHYSIOLOaY

silver nitrate solution used, as the urine contains small quantities of certain
compounds more easily precipitable than the chromate.

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 a small
spirit lamp to low redness till all vapours are dissipated and the residue
becomes white. It is then dissolved in a small quantity of water, and the
carbonates produced by the combustion of the organic matter neutralised by
dilute acetic acid ; a few grains of pure calcium carbonate to remove all free
acid are then added, and one or two drops of potassium chromate. The
mixture is then titrated with decinornial 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 ; consequently, if 12*5 c.c. have
been used, the weight of salt in the 10 c.c. of urine is 0-07296 gr., or 0'7296
per cent. If 5*9 c.c. of urine are taken for titration, the number of c.c. of
silver solution used will represent the number of parts of salt per 1,000 parts
of urine.

LESSON XXIV

ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE

ESTIMATION OF PHOSPHATES

The phosphoric acid in the urine is combined with soda, potash, hine, and
magnesia.

(a) Estimation of the total pliosphates.

For this purpose the following reagents are necessary : —

i. A standard solution of uranium nitrate. The uranium nitrate solution
contains 85*5 grammes in a litre of water ; 1 c.c. corresponds to 0*005 gramme
of phosphoric acid (P^O^).

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.

Another indicator which may be used is cochineal tincture, a few drops of
which may be added to the mixture. A change of colour from red to green
is the sign of the end of the reaction.

(6) Estimation of the 'phosphor ic acid combined with lime and magnesia
(alkaline earths).

Take 200 c.c. of urine. Render it alkaline with ammonia. Lay the mixtm-e
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.

' 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 ha omitted.

208 ESSENTIALS OF CHEMICAL PHYSIOLOGY

(c) Instead of uranmm 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.

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.

Both methods will be given here : the former is, however, better suited
for class experiments.

1. Volu7netric 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 : 30*5 grammes of crystallised
chloride of barium in a litre of distilled water ; 1 c.c. of this solution corre-
sponds to O'Ol gramme of sulphuric acid (SOg).

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 c.c. of hydrochloric acid, and boiled. The combined sulphates are thus
converted into ordinary sulphates, and give a precipitate like them with
barium chloride. The chloride of barium solution is allowed to drop into
this mixture as long as any precipitate occurs, the mixture being heated before
every addition of barium chloride to it. After adding 5 to 8 c.c. of the
standard solution, allow the precipitate to settle ; pipette off a few drops of
the clear supernatant fluid into a watch-glass ; add to it a few drops of the
standard barium chloride solution. If any precipitate occurs, return the
whole to the flask and add more barium chloride ; again allow the precipitate
to settle, and test as before ; go on in this waj^ 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 bariinn 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. Oravimetric 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 (SO3).

ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE 209

Method (Salkowski). — 100 c.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 —
i.e. until the filtrate remains clear after the addition of a few drops of sulphuric
acid. 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 (H2SO4). The sulphide is converted
into sulphate. Heat again to redness to drive off excess of sulphuric acid.

(b) The following is Salkowski's method of estimating the coinhined
suljyliuric acid — that is, the amount of SO., in ethereal sulphates : — 100 c.c.
of urine are mixed with 100 c.c. of alkaline barium chloride solution, which is
a mixture of two volumes of solution of barium hydrate with one of barium
chloride, both saturated in the cold. The mixture is stirred, and after a few
minutes filtered : 100 c.c. of the filtrate ( = 50 c.c. of urine) are acidified with
10 c.c. of hydrochloric acid, boiled, kept at 100° C. on the water-bath for an
hour, and then allowed to stand till the precipitate has completely settled ;
if possible, it should be left in this way for twenty-four hours. The further
treatment of this precipitate ( = combined sulphates) is then carried out as in
the last case.

Calculation. — 233 parts of barium sulphate correspond to 98 parts of

HoSO^, or 80 parts of SO3 or 32 parts of S. To calculate the H.^SO^, multiply

98
the weight of barium sulphate by --^ = 0*421 ; to calculate the SO3, multiply

Zoo
on Qo

by ry^= 0-343; to calculate the S multiply bvJ;" =0-137. This method
233 " 233

of calculation applies to the gravimetric estimation both of total sulphates
and of combined sulphates.

(c) To obtain the amount of pre-formed sulphuric acid, subtract the
amount of combined SO., from the total amount of S0.(. The difference is
the pre-formed SO3.

Example : 100 c.c. of urine gave 0-5 gramme of total barium sulphate.

80
This multiplied by ^^" = 0-171 gr. = total SO.,. Another 100 c.c. of the same
2oo

urine gave 0*05 gr. of barium sulphate from ethereal sulphates ; this multi-
plied by ^^ = 0-017 gr. of combined SO3.
=-.0-171-0-017 = 0-154 gr. of pre-formed SO,.

plied by ^^ = 0-017 gr. of combined SO3. Total SO, -combined SO,
2oo

LESSON XXV
URIC ACID AND CREATININE

1. Preparation of Pure Uric Acid. — If one wishes to prepare pure uric acid,
the solid urine of a reptile or bird, which consists principally of the acid
amnionmni 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 Uric 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 dis-
solving 1-581 grammes of permanganate in a litre of water.

Measure 100 c.c. of urine into a beaker of about 150 c.c. capacity.

Add to this 25 grammes (approximately weighed) of ammonium chloride,
stirring briskly till all the salt is dissolved. Now add 2 c.c. of strong
ammonia, 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. The precipitate should be as 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, distiUed water
should be heated to boiling in a wash-bottle provided with a fine jet. The
funnel containing the filter is now held horizontally over a small porcelain
basin (of about 50 c.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 its folds may be washed off. Not
more than 20-30 c.c. of water need be emploj^ed in this transference : if much
more has been used the liquid should be concentrated over the water-bath at

URIC ACID AND CREATININE 211

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 crj'stallise out.

If the mixture is artificially cooled all the uric acid will separate out in
two hours, otherwise it is best allowed to stand overnight 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 liquid may be noted (see below). The uric acid is next
washed with cold distilled water until free from chlorides. It is unnecessary
to transfer the whole to the filter ; the greater part may be washed by decanta-
tion. Such of the crystals as are upon the filter are now washed back into
the basin (best by the aid of hot water) and the whole quantity is dissolved
by heating to boiling with 1 c.c. of 10-per-cent. sodium carbonate solution
and as much distilled water as the basin will safely hold.

The solution is transferred to a {-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.

Twenty c.c. of strong sulphuric acid are added to the contents of the
flask, and the mixture shaken and titrated with the standard permanganate
solution.

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 c.c. of the 'standard permanganate solution. The mother liquor
filtered from crystals measured 25 c.c.

18-5 X -00375 = -0694 gr.

•001 X ^^ = -0017
15

Total = -OTli

The urine contained 71 mgms. uric acid per 100 c.c.

3. Estimation of Creatinine. — The following colorimetric method (Folin's)
is now generally employed for the estimation of creatinine ; and with a
slight modification it may also be used for the estimation of creatine. It is
based on the red colour which Jaffe showed develops when an alkaline solu-
tion of picric acid is added to a solution of creatinine ; this is coiiipared
wdth the colour of a standard solution of potassium bichromate, the tint of
the two fluids being almost identical. If creatine has to be estimated, this is

p3

212 ESSENTIALS OF CHEMICAL PHYSIOLOGY

first transformed into creatinine by boiling with hydrochloric acid. Tlie
apparatus and reagents necessary- are : —

1. A colorimeter consisting of two tubes, the height of the column in
which can be read by graduations in tenths of a millimetre.

2. A half normal solution of potassium bichromate (24*5 grammes per
Htre).

3. A saturated solution of picric acid.

4. 10 per cent, caustic soda.

To perform the analysis one tube of the colorimeter is filled with the
bichromate solution up to the height of 8 mm. Ten c.c. of urine are measured
into a half-litre flask, 15 c.c. of the picric acid solution and 5 c.c. of the
caustic soda solution added, and then water until the total volume of the
mixture is 500 c.c. This solution is poured into the second tube of the colori-
meter to such a height (which is read off) that, on looking down through it
the intensity of the colour is the same as that in the standard tube by its
side. The amount necessary for this corresponds exactly to 8'1 milligi-ammes
of creatinine. From this the total in the sample of urine can be easily
calculated.

LESSON XXVI

THE PIGMENTS OF THE URINE

The urinary pigments are numerous, and have from tnne 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 v/hich,
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. 143), 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. 92, 143). Probably both reduction aud 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 formulae show the relationship between these allied pig-
ments : —

Haematin . . .... Cg^Hg^N^O^Fe

Bilirubin C32H3,N,0,'

Hydrobilirubin C-jaH^oNiO^

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 following are the two methods introduced by Garrod and Hopkins
for its separation from the urine : —

(a) The urine is first saturated with ammonium chloride, and the urate

214 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 maj- 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.

(b) 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
lai'ge 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 b and F, slightly overlapping the latter
(fig. 63, spectrum 4).

Urobilin, like most animal pigments, shows acidic tendencies and forms
compounds with bases ; it is liberated from such combinations by the
addition of an acid.

If urobilin is dissolved in caustic potash or soda, and sufficient sulphuric
or hydrochloric acid is added to render the liquid faintly acid, a turbidity is
produced. This turbid liquid shows an additional 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 whoUy dis-
appears when the precipitate is filtered off, and when it is re-dissolved the
ordinary band alone is visible.

3. Uroerythrin. — This is the colouring matter of j)ink 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 amy lie alcohol.

Uroerythrin has a great affinity for urates, with which it appears to form
a loose compound. Its solutions are rapidly decolorised by light. Bpectro-
scopically it shows two rather ill -defined bands (fig, 63, spectrmii 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. Haematoporphyrin. — This also occurs in small quantities in normal
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-

THE PIGMENTS OF THE URINE

215

phates, which carries down the pigment with it : the pigment may be dis-
solved out with chloroform. The chloroform is evaporated, the residue
washed with alcohol, and finally dissolved in acidified alcohol. Urines rich
in the pigment yield it easily to acetic ether or to amylic alcohol.

i

cEg

I

Fig. 63.— Chart of absorption spectra : 1, acid liaematoporphyriii ; 2, alkaline haBUiatoporphyrin ;
3, haematoporpbyrin 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.)

When the urine is sufficiently rich in the pigment, the bands shown are
those of alkaline htematoporphyrin (fig. 63, spectrum 2). On adding sulphuric
acid the spectrum of acid hyematoporphyrin 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 oxyhgemoglobin (fig. 63,

216 ESSENTIALS OF CHEMICAL PHYSIOLOGY

spectrum 3) ; by treatment with dilute mineral acids this changes immedi-
ately to the spectrum of acid haematoporphyrin.

5. Chromogens in Urine. — In addition to the chromogens of urobilin and
haematoporphyrin alluded to in the foregoing paragraphs there are others, of
which the following may be mentioned : — (a) Indoxyl.—The origin of this
substance from indole is mentioned on p. 154. It is easily oxidised to
indigo-blue or indigo-red.

2C,H,<^2^>CH f 0, = C,3H, <^,^>C:C<^^>C,H, + 2H,0.

[indoxyl] [indigo-blue]

Indigo-red is isomeric with indigo-blue, its structural formula being

CO C OTT

C(,H^<^-g->C:C<p*TT >N. It is very rare for the urine to be actually

pigmented with indigo, for the urinary indoxyl is excreted as a conjugated
sulphate which resists oxidation. When the urine is mixed with an equal
volume of hydrochloric acid, indoxyl is liberated from the sulphate. A
solution of a hypochlorite is then added drop by drop, when indigo-blue is
formed, and on shaking the mixture with chloroform the indigo-blue passes
into the chloroform. (Jaffe.) This test, however, is not a very good one, for
the hypochlorite solution has always to be freshly prepared, and even then a
small excess will cause the colour to disappear owing to' oxidation of the
indigo, and it is difficult to hit off the exact amount to give the reaction. A
better test for indoxyl-sulphuric acid (indican) consists in adding 10 c.c. of
a 20-per-cent. solution of lead acetate to 50 c.c. of urine, and filtering the
mixture. The filtrate is shaken with an equal volume of hydrochloric acid
(containing 0*2 to 0*4 per cent, of ferric chloride) and a few c.c. of chloroform.
The indigo-blue passes into the chloroform (Obermayer.) (6) Shatoxyl. —
When skatoxyl is given by the mouth it passes into the urine, and yields
skatoxyl-red on oxidation, (c) Urorosein is distinct from indigo -red. It is
produced from its chromogen by the action of mineral acids. It frequently
appears when urine is treated with strong hydrochloric acid and allowed to
stand, but it appears more readily when an oxidising agent is added as well.
It is readily soluble in amylic alcohol, but not in ether. The chromogen is
precipitated by saturation with ammonium sulphate. The colour is de-
stroyed 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 greenish-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. 169.

APPENDIX

HEMACYTOMETERS

Gowers's Haemacytometer. — The enumeration of the blood corpuscles is
readily effected bj^ the haemacytometer of Gowers. This instrument consists
of a glass slide (fig. 64, C), the centre of which is ruled into ^ millimetre
squares and surrounded by a glass rim i 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).

Fig. 64.— Ha?macvtometer 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

218

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.

Differential counts to show the relative proportions of the varieties of
leucocytes are made in appropriately stained specimens.

Oliver's Haeniacytoiiieter. — The following method, devised by Dr. George

FiO. G5.— Uliver'.s liiumacytouieter.

APPENDIX

219

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.

The linger is pricked, and the blood allowed to flow into the small
capillary pipette (fig. 65, a) until it is full. This is washed out by the
dropping tube b into a graduated flattened test-tube, c, with Haycm'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.

H^MOGLOBINOMETERS

Gowers's Haemoglobinoineter. — ^The apparatus consists of two glass tubes,
C and D, of the same size. D contains glycerin jelly tinted with carmine to
a standard colour — viz., that of normal blood diluted 100 times with distilled
water. The finger is pricked and 20 cubic millimetres of blood are measured

Fig. 66.— HaRmoglohinometer of Sir W. Gowers.

out by the capillary pipette, B. This is blown out into the tube C, and diluted
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

' Sodium sulphate 5 grammes, sodium chloride 1
0-5 grm., distilled water 200 c.c.

':rm., mercuric chloride

220

ESSENTIALS OF CHEMICAL PHYSlOLOaY

same as the standard when the tube is filled up to the graduation 100, the
quantity of oxyhsemoglobin in the blood is normal. If it has to be diluted
more largely, the oxyhsemoglobin 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 haemoglobin is only half what it ought to be —
50 per cent, of the normal — and so for other percentages.

Haldane's Haemoglobinometer is more frequently used. Instead of tinted
gelatin, the standard of comparison is a sealed tube filled with a solution of
carbonic oxide haemoglobin of known strength. This keeps unchanged for
years. A stream of coal gas is passed through the blood to be examined.
This converts all the haemoglobin present into carboxyhaemoglobin ; this is
then diluted with water to match the standard.

Von Fleischl's Haemometer. — The apparatus (fig. 67) consists of a stand
bearing a white reflecting surface (S) and a platform. Under the platform is

Fig. 67. — Von Fleischl's liiemomete

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 water.

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 hyemoglobin.

Oliver's Haemoglobinometer. — This method consists in compaiing a speci-

APPENDIX

221

men of blood, suitably diluted with water in a shallow white palette,
with a number of standard tests very carefully prepared by the use of

riG.68.-01iver;sh^moglobiuometer: a, standard gradations ; &, lancet ; <• capillary
measuring pipette ; d, mixing pipette ; e, blood cell and cover glass.

Lovibond's coloured glasses. The capillary pipette c (fig. 68) is first filled
with Dlood obtained by .pricking the finger. This is washed with water

222 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 c being
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
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 gr&dations are marked in per-
centages, 100 per cent, being taken as normal.

* "Worth ' of the Corpuscles. — If the percentage of haemoglobin is 100, and the

100
percentage number of corpuscles is 100 also, then the quotient ^ = 1 is

100

taken as the normal. This varies in health from 0*95 to 1'05 in men, and
from 09 to 1 in women. This quotient has been termed the ' worth ' of the
corpuscle.

Specific Gravity of Blood.— Of the numerous methods introduced for taking
the specific gravity of fresh blood, that of Hammerschlag is the simplest. A
drop of blood from the finger is placed in a mixture of chloroform and
benzene. If the drop falls, add chloroform till it just begins to rise ; if the drop
rises, add benzene till it just begins to fall. The fluid will then be of the
same specific gravity as the blood. Take the specific gravity of the mixture
in the usual waj' with an accurate hydrometer.

Schmalz's capillary pycnometer 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, each ray of light entering such a crystal is 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 movable image when the crystal
is rotated ; the ordinary laws of refraction do not apply to it, and it is called
the extraordinary ray. Both rays are of equal brilliancj'. 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 this one. Doubly refract-

APPENDIX

223

ing bodies are called anisotropoiis ; singly refracting bodies, isotrojJOiifi.
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
disc with a vertical slit be placed on the course of the string, the vibrations
will all be 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
54° 35', water 52° 45', diamond 68°, quartz 57° 32', &c.). It is also found
that certain non-crystalline substances, like muscle, cilia, &c., are doubly
refracting.

Nicol's Prism is the polariser usually employed in polariscopes ; it con-
sists 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

Fig. 70.

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).

Suppose that the string is made to vibrate, and that the waves travel in
the direction of the arrow. From the fixed point c to the disc a, the string

' 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.

224

ESSENTIALS OF CHEMICAL PHYSIOLOGY.

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
disc h 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 b be also placed
vertically. If, however, b is so placed that its slit is horizontal (fig. 71) the
vibrations will be extinguished on reaching b, and the string between b and d
will be motionless.

I

Fig. 71.

c here represents a source of light ; the vibrations of the string represent
the undulations which by the Nicol's prism a are polarised so as to occur in
one plane only ; if the second Nicol's prism or the analyser b is parallel to the
first, the vibrations will pass on to the ej^e, which is 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, b 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 aether may be compared to the action of slits in a diaphragm to vibratiojis
of more tangible materials than aether.

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 can also 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^Nj (fig. 72} represent the direction of the principal plane of the
first nicol, and N0N2 that of the second. They are at right angles, and so

» The imperfection of the model has been explained in the preceding footnote.

APPENDIX

225

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 NjNj vibrates in the plane NjN,, 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

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 NgNg.

The vibrations in PP can be resolved

into a vibration in NjNi and a

vibration in N.^N., ; the former is

extinguished, the latter transmitted.

Similarly the vibration in P^P^ can

be resolved into two sub-rays in

NjNj and N2N.J respectively, the

latter only being transmitted. The

illumination is thus due to two

sub-rays, one of the vibrations in

PP, the other of those in P^P^ which

have been made to vibrate in N^N^.

Now, although these two sub-
rays vibrate in the same plane,
they are of different velocities ;
hence the phases of the vibrations
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 ether 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
crystal 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, i.e. the two sub -rays are made up of vibrations

226 ESSENTIALS OF CHEMICAL PHYSIOLOGY

which occur not in a 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
the quartz with the greater velocity. There are two kinds of quartz, one
which rotates the plane to the right (dextro-rotatory), the other to the left
(IsBvo-rotatory).

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 (b), 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 bb', 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 Ught being different, white hght 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 Saccbarimeter. — 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), 3-75 mm.

APPENDIX 227

thick, one half of which {d in fig. 75) is made out of dextro-rotatory, the other
half {g in fig. 75) of laBVO-rotatory 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-
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).

The compensator consists of two quartz prisms (KR', fig. 75) cut perpen-
dicularly to the axis, but of contrary rotation to the plate just in front of

Fig. 74. — Soleil's saccharimeter.

them. These are wedge-shaped, and slide over each other, the sharp end of
one being over the blunt end of the other. By a screw the wedges may be
moved from each other, and this diminishes the thickness of quartz inter-
posed ; if moved towards each other the amount of quartz interposed is
increased.

The effect of the quartz plate {d, g) next to the polariser {i in fig. 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

f 7^

HBO - - B-

E

EiiliiiY^^^

r

11'

Fig. 75.— Diagram of optical arrangements in Soleil's saceharimeter.

in colour to the red end, the other to the violet end of the spectrum, because
the two halves of the quartz act in the opposite way.

The instrument is first adjusted with the compensator at zero, and the
nicols parallel, so that the whole field is of one colour. The tube containing
the solution is then interposed ; and if the solution is optically inactive the
field is still uniformly violet. But if the solution is dextro-rotatory the two
halves will have different tints ; a certain thickness of the compensating
quartz plate which is Isevo-rotatory 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

q2

228

ESSENTIALS OF CHEMICAL PHYSIOLOGY

and scale (E in fig. 76) worked by the screw which moves the compensator.
If the solution is Isevo-rotatory, the screw must be turned in the opposite
direction.

Zeiss' s polarimeter is in principle much the same as Soleil's ; the chief
difference is that the rotation produced by the solution is corrected, not by a
quartz compensator, but by actually rotating the analyser in the same
direction, the amount of rotation being directly read off in degrees of a
circle.

Laurent's polarimeter is a more valuable instrument. Instead of using
daylight, or the light of a lamp, monochromatic light (a sodium flame pro-

FiG. 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 1) or sodium
line of the spectrum. The essentials of the instrument are, as before, a
polariser, a tube for the solution, and an analyser. The polarised light
before passing into the solution traverses a quartz plate, which, however,
covers only half the field, and retards the rays passing through it by half a
wave-length. In the 0° position the two halves of the field appear equally

APPENDIX

2^9

illuminated : in any other position, or if rotation has been produced by the
solution when the nicols have been set at zero, the two halves appear un-
equally illuminated. This is corrected by means of a rotation of the analyser
that can be measured in degrees by a scale attached to it.

The specific rotatory power of any substance is the amount of rotation in
degrees of a circle of the plane of polarised light produced by 1 gramme of
the substance dissolved in 1 c.c. of liquid examined in a column 1 deci-
metre long.

If a = rotation observed.

w = weight in grammes of the substance per cubic centimetre.
I = length of tube in decimetres.

[a]n = specific rotation for light with wave-length corresponding to
the D line (sodium flame).

Then[u]i>

a

In this formula + indicates that the substance is dextro-rotatory, - that it
is laevo-rotatory.

If, on the other hand, [a]^ is known, and we wish to find the value of w,
then "

I a Id X Z

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

Fig. 77.— Spectro-polarimeter of v. Fleischl.

which rotate the plane of polarised light, It was invented by E. v. Fleischl
for the estimation of sugar in diabetic urine. Its chief advantage is that no
difficulty arises in forming a judgment as to the identity of two coloured

230 ESSENTIALS OF CHEMICAL PHYSIOLOGY

surfaces, as in Soleil's saccharimeter, or of two shades of the same colour, as
in Laurent's instrument. The light enters at the right-hand end of the
instrument, is polarised by the Nicol's prism b, and then passes through two
quartz plates, cc, placed horizontally over each other. One of these plates is
dextro-, the other laevo-rotatory, and they are of such a thickness (7*75 mm.)
that the green rays between the E and b lines of the spectrum are circularly
polarised through an angle of 90°, the one set passing off through the upper
quartz to the left, the other through the lower to the right. The light then
continues through a long tube,//, which contains 15 c.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 b owing to the extinction of these rays by the circular
polarisation, produced by the quartz. The analyser can be rotated : a
vernier, g, is attached to, and moves with it, round a circular disc (seen in
section at h) graduated in degrees. The two bands in the spectra coincide
when the zeros of vernier and scale correspond. If now the tube / is filled
with an optically active substance like sugar, the bands are shifted, one to the
right, the other to the left, according to the direction of rotation of the sub-
stance in /. The rotation is corrected by rotating the 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 /,
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 // (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 foimd
that racemic acid, which is optically inactive, can be decomposed into two
isomerides, one of which is common tartaric acid which is dextro-rotatory, and
the other tartaric acid differing from the common variety in being laevo-
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 dextro-rotatory power, while the others were
lavo-rotatory. Pasteur further showed that if the mould Pe7iicillium glaucum
is grown in a solution of racemic acid, dextro-tartaric acid first disappears,
and the laevo-acid alone remains. The subject remained in this condition for
many years ; it was, however, conjectured that proba.bly there is some mole-
cular condition corresponding to the naked-eye crystalline appearances which

APPENDIX 231

produces the opposite optical effects of various substances. What this mole-
cular structure is, was pointed out independently by two observers — Le
Bel in Paris, and Van 't HofT 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.Ha CO.OH

^ I

H-(<^)— CH3 H— ©_0H

CH^.OH CH— CO.OH

[Amyl alcohol] [Malic 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 laevo-rotatory ; that this

/4

Fig. 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 formulae, 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
respectively.

MERCURIAL AIR-PUMPS

Pfluger's Pump. — I is a large glass bulb filled with mercury ; from its lower
end a straight glass tube, 7n, about 3 feet long, extends, which is connected
by an india-rubber tube, n, with a reservoir of mercury, o, which can be
raised or lowered as required, by a simple mechanical arrangement. From
the upper end of the bulb, Z, a vertical tube passes ; above the stopcock, 7c,
this has a horizontal branch, which can be closed by the stopcock, /. The
vertical part is continued into the bent tube, which dips under mercury in
the trough, h. A stopcock, j, is placed on the course of this tube. Beyond/
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

232

ESSENTIALS OF CHEMICAL PHYSIOLOGY

blood is brought into it by the tube c ; the gases, as they come ofif, cause the
blood to froth, and the bulb, 6, is called the froth-chamber, as it intercepts
the froth, preventing it from passing into the rest of the apparatus.

The pump is used in the following way : I is filled with mercury, the
level in I and o being the same ; k is closed ; o is then lowered, and when it

Fio. 79.— Diagram of PflUger's pump.

is 30 inches lower than the stopcock, li, the mercury in I falls also, leaving
that bulb empty : j being closed and / open, Ti is then opened, and the air in
a, 6, d, &c., rushes into the Torricellianvacuum in Z ; / is closed and j opened ;
the reservoir, o, is raised ; the mercury in I rises also, pushing the air before
it, and it bubbles out into the atmosphere through the mercury (the tube, h,
is not at this stage in position). When I is full of mercury, h andy are once

APPENDIX

233

more closed and o is again lowered ; when I is thus rendered once more a
vacuum, h and / are opened and more of the air remaining in a, 6, d, &c.
rushes into the vacuum ; / is closed, j is opened, and this air is expelled as
before. The process is repeated as often as is necessary to make a, 6, d, &c.
as complete a vacuum, as indicated by the mercury in the gauge, e, as is
obtainable.

a being now empty, and the stopcock, /, closed, blood is introduced by the
tube c ; it froths and gives forth 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.^ The bulb, I, is once more rendered a vacuum, and
k and / are opened, j being closed. The gas from a and h rushes into the
bulb Z, being dried as it passes through d; f'm then closed and j opened ; the
reservoir o is raised, and as the mercury in I rises simultaneously, it pushes
the gases into the cylinder, 7i, 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
expelled into h.

A good grease for the stopcocks is a
mixture of two parts of vaseline to one of
white wax.

Alvergniat's pump has the advantage
over Pfliiger's of fewer connections, and
all of these are surrounded by mercury,
which effectually prevents leakage ; it has
the disadvantage of a rather small bulb
in place of I, and thus it is more labour to
obtain a vacuum.

Leonard Hill's pump.— This is a simpler
instrument, and is sufiicient for most
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
piece of tubing and a clip, a ; this is
connected by good india-rubber tubing to
another bulb, d. Above d^ however,

there is a stopcock with two ways cut through it : one by means of
which B.B. and d may be connected, as in the figure ; and another seen
in section, which unites d to the tube e, when the stopcock is turned through
a right angle. In intermediate positions the stopcock cuts off all communi-
cation from d to all parts of the apparatus above it ; d is connected by tubing
to a receiver, E, which can be raised or lowered at will. At first the whole
apparatus is filled with mercury, E being raised. Then, a being closed, E 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 E still further, d can also be rendered a vacuum. A few drops of

' Phosphoric acid is usually employed.

^^3^

B.B

Fig. 80. — L. Hill's air-pump.

234

ESSENTIALS OF CHEMICAL PHYSIOLOGY

mercury should be left behind in B.B. B.B. is then detached from the rest
of the apparatus and weighed, the clips, a and 6, being tightly closed. Blood
is then introduced into it by connecting the tube with the clip a on it to ^
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 a 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 K 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 100 c.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.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
air 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 filHng 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
carbonic 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

Fig. 81.— Waller's apparatus for gas
analysis.

APPENDIX 235

absorption of oxygen) in the air when drawn back into the tube. The
remaining gas is nitrogen.

KJELDAHL'S METHOD OF ESTIMATING NITROGEN

This simple method can be used in connection with most substances of
physiological importance. Briefly, it consists in converting all the nitrogen
present into ammonia by means of sulphuric acid ; then rendering alkaline
with soda, and distilling over the ammonia into standard acid, the diminu-
tion in acidity of which measures the amount of ammonia present.

The following modification of the original method is used in this
laboratory.

About 1 gramme of the substance under investigation (or in the case of
urine when one wishes to make an estimation of total nitrogen, 5 or 10 c.c.
of that fluid) is placed in a round bottomed Jena flask of about 250 c.c.
capacity, and 20 c.c. of pure sulphuric acid added. Six grammes of
potassium sulphate and about half a gramme of copper sulphate are also
added. The flask should be provided with a loose balloon stopper, and
arranged in a sloping direction over a small flame. The mixture is heated
slowly until it boils. In about twenty minutes the fluid becomes nearly
colourless ; boiling is continued for another forty-five minutes. By this
time all the nitrogen will be in combination as ammonia.

After cooling, the fluid is washed into a litre flask of Jena glass (fig. 82, A)
and water added until the total volume of the fluid is about 400 c.c. Add
then an excess of 40 per cent, caustic soda solution,
a few pieces of granulated zinc to avoid bumping in the
subsequent distillation, and immediately fit the glass
tube B into the neck of the flask by means of a well-
fitting rubber stopper. The other end of B leads into
the flask C which contains a measured amount (50 or
100 c.c.) of standard sulphuric acid ; ^ normal acid is a
convenient strength to use. The bulb D shown in the
figure guards against regurgitation, and the end of the
tube should dip just below the surface of the acid in C. fig. 82. — Kjeidabi's
The mixture in the flask is now boiled for about half an ^ppamtus*^''*'"'"^

hour when all the anmionia will have distilled over ;
the use of a condenser around the tube B is unnecessary. The acidity of the
standard acid is then determined by titrating with standard alkali, a few
drops of methyl orange being added to act as the indicator of the end of the
reaction (this gives a pink colour with acid, yellow with alkali).

Example. — Suppose 1 gramme of a nitrogenous substance is taken, and
the ammonia distilled over into 100 c.c. of ^ normal sulphuric acid ( = 20
c.c. normal acid). This is then titrated with a corresponding solution of
soda, and it is found that the neutral point is reached when 60 c.c. of the
soda solution have been added. The other 40 c.c. must therefore have been
neutralised by the ammonia derived from the substance under investigation.
This 40 c.c. of acid = 8 c.c. of normal acid = 8 c.c. of normal ammonia =
8 X 0'017 = 0*136 gramme of ammonia. One gramme of the substance ana-
lysed, therefore, yields 0-136 gramme of ammonia, and this contains 0"112

236 ESSENTIALS OF CHEMICAL PHYSIOLOGY

gramme of nitrogen ; 100 grammes will therefore contain 11-2 grammes of
nitrogen. If the strength of the acid is that just recommended, each c.c.
corresponds to 0'0028092 of nitrogen.

SOLUTIONS. DIFFUSION. DIALYSIS. OSMOSIS.

The investigations of physical chemists dming 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 at ordinary temperatures it 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 H2O, 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
become dissociated into sodium ions, which are charged with positive elec-
tricity, and chlorine ions, which are charged with negative 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 SOj^. The term ion is thus not equivalent to atom, for an
ion may be a group of atoms, like SO4, 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
case of the sulphuric acid, the negative charge of the SO^ ion is equal to the
positive charge of tv/o hydrogen ions. We can thus speak of monovalent,
divalent, trivalent, &c. ions.

Ions charged with positive electricity are called Teat-ions because they
move towards the kathode or negative pole ; those which are charged with
negative electricity 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„ &c.

Divalent : Ca, Ba, Fe (in ferrous salts), &c.

Trivalent: Al, Bi, Sb, Fe (in ferric salts), &c.
An-ions. Monovalent : CI, Br, I, OH, NO3, &c.

Divalent : — S, Se, So^, &c.

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

APPENDIX 237

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 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
in this relation the ion plays the same part as a molecule.

The liquids of the body contain electrolytes in solution, and it is owing
to this fact that they are able to conduct electrical currents.

Another physiological aspect of the subject is seen in a study of the
action of mineral salts in solution on living organisms and parts of organisms.
Many years ago Einger 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 inorganic 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 piu-e
solutions of non-electrolytes (like sugar, urea, albumen). But different con-
tractile tissues differ in the nature of the ions which are most favourable
stimuli. Thus cardiac muscle, cilia, amoeboid 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 rhythmical con-
tractions ; for instance, Ca and K. (3) Those which act catalytically, that
is, they accelerate the action of Na, though they do not themselves produce
rhythmical contractions directly : for instance, H and OH. In spite of the
antagonistic effect of Ca, a certain amount of it must be present if con-
tractions are to continue for any length of time. Ions produce rhythmical
contraction only because they affect either the physical condition of the
colloidal substances (protein, &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 larvae (generally imperfect
ones, it is true) by merely altering the saline constituents of the fluid that
bathes them. Whether such a notion will stand the test of further verifica-

238 ESSENTIALS OF CHEMICAL PHYSIOLOGY

tion must be left to the future. So also must the equally important idea
that the basis of a nervous impulse is electrolytic action, though it receives
support from Macdonald's recent investigations.

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 ; CI = 35"45) in a litre. A gramme-molecular solution of grape
sugar (C^jHioO^j) 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 were 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 of a 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 on these facts.

Diifusion, 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
and the process is called diffusion. In a similar way diffusion will effect in
time a homogeneous mixture of two liquids or solutions. If v/ater 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 or 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, protein, &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 (M) vertically as shown in the diagram
(fig. 83), and the two fluids A and B on each side of it. Diffusion through a
membrane is not limited to the molecules of water, but it may occur also in

APPENDIX

239

H

A

B

:=rz^-

Fig. 83.

the molecules of certain substances 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-
ing diagram the compartment A is filled with pure water,
and B with a sodium chloride solution, the liquids in
the two compartments will ultimately be found to be
equal in bulk as they were at the start, and each will
be a solution of salt of half the original strength of
that in the compartment jB, But at first the volume
of the liquid in compartment B increases, because more
water molecules pass into it from A than salt mole-
cules pass from B into^. The term osmosis is generally
limited to the stream of water molecules passing
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 necessary
to explain 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 flows 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,
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 B contains a solution
of salt or sugar; in 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 5, 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 fiUed 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 seldom done, and
some of the indirect methods of measurement described later are used

240 ESSENTIALS OF CHEMICAL PHYSIOLOGY

instead. The reason for this is that it has been found difficult to construct
a membrane which is absohitely semi-permeable.

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 if it occupied the same space in the form of a gas (Van 't Hofif's
hypothesis). The nature of the substance makes no difference ; it is only the
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 complete, as may be seen from the following statements : —

1. At a constant temperature osmotic 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's 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-
lyte like sugar. We shall then not have to take into account any electrolytic

APPENDIX 241

dissociation of the molecules into ions. We will suppose we want to calcu-
late the osmotic pressure of a 1 -par-cent, solution of cane sugar.

One gramme of hydrogen at atmospheric pressure and 0" C. occupies a
volume of 11'2 litres; two grammes of hydrogen will therefore occupy a
volume of 22*4 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*4 litres compressed to 1 litre — that is, a pressure of 22*4
atmospheres. A gramme-molecular solution of cane sugar, since it contains
the same number of molecules in a litre, must therefore exert an osmotic pres-
sure of 22*4 atmospheres also. A gramme-molecular solution of cane sugar
(CiaH^.jOn) 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 of
water ; hence the osmotic pressure of a 1-per-cent. solution of cane sugar is

- X 22*4 atmospheres, or 0*65 of an atmosphere, which in terms of a

column 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 the difficulty of directly measuring osmotic pressure by a
manometer ; we now see that mere arithmetic often fails us ; and so we come
to the question to which we have been leading up, viz. how osmotic pressure
is actually determined.

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*4 atmospheres : that is, 22*4 x 760 = 17,024 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.

Osmotic pressure = ,~- x 17,024.
1-87

For example, a 1-per-cent. solution of sugar would freeze at -0-052° C. ;

. ,, o -052x17,024 .^o ,

its osmotic pressure is therefore t'-^^ — =473 mm., a number approxi-

1'87

mately equal to that we obtained by calculation.

Mammalian blood serum gives A = 0*56° C. A 0'9-per-cent. solution of
sodium chloride has the same A ; hence serum and a 0-9-per-cent. solution
of common salt have the same osmotic pressure, or are isotonic. The osmotic

242 ESSENTIALS OF CHEMICAL PHYSIOLOGY

'56 X 17 024

pressure of blood serum is - _' — =5,000 mm. of mercury approxi-

1*87

mately, or a pressure of nearly 7 atmospheres.

The osmotic pressure of solutions may also be compared by observing
their effect on red corpuscles, or on vegetable cells such as those in Trades-
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
corpuscles 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
shrinking of the protoplasm in the vegetable cell occurs, and if red corpuscles
are used they swell and liberate their pigment. Isotonic 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 waUs 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
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 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 manifestation of force, in
the case of Uving 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 sometimes operates so as to neutralise
the known forces of osmosis and filtration.

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 have 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

APPENDIX 248

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 which 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 Keid has
shown, if the living epithelium of the intestine is removed, absorption comes
very nearly to a standstill, although from the purely physical standpoint
removal of the thick columnar epithelium would increase the faciUties 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
excretions.

Osmotic Pressure of Proteins. — It has been generally assumed that proteins,
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 proteins, 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 proteins of
the blood-plasma as equal to 30 mm. of mercury. By others this is attributed
to the inorganic salts with which proteins are always closely associated.
Moore, for instance, finds that the purer a protein 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 protein
itself, or to the saline constituents which are almost an integral part of a
protein. It is merely interesting from the theoretical point of view. We
should from the theoretical standpoint find it difficult to imagine that a pure
protein can exert more than a minimal osmotic pressure. It is made up of
such huge molecules that, even when the proteins are present to the extent
of 7 or 8 per cent., as they are in blood-plasma, there are comparatively few
protein molecules in solution, and probably none in true solution. Still, by
means of this weak but constant pressure it is possible to explain the fact that
an isotonic or even a hypertonic solution of a diffusible crystalloid may be
completely absorbed from the peritoneal cavity into the blood.

The functional activity of the tissue elements is accompanied by the
breaking down of their protein constituents into such simple materials as uvea
(and its precursors), sulphates, and phosphates. These materials pass into
the lymph, and increase its molecular concentration and its osmotic pressure ;
thus water is attra.cted (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

r2

244 ESSENTIALS OF CHEMICAL PHYSIOLOGY

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 proteins ; 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 the
constituents of the blood, including the proteins, 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.

INDEX

In cases where several references are made to any subject, the figure in heavy type indicates
where the principal matter in relation to the subject is to be found.

A

Abkin, 123

Absorption, 95, 243 ; of carbohydrates,
96 ; of fats, 99 ; of proteins, 96

Absorption bands, 117, 118, 119

Absorption spectra of haemoglobin and
its derivatives, 119, 189 ; of myohae-
matin, 199 ; of urinary pigments,
215

Accessories of food, 60

Acetic acid, 24, 25, 63

Acetompmia, 88

Acetone, 88, 168 ; test for, 168

Acetyl, 25

Achroo-dextrin, 21, 68, 179

Acid-albumin, 29, 30, 48, 62, 75, 79, 81,
172

Acid haematin, 188

Acid haematoporphyrin, 189, 215 ; ab-
sorption bands of, 189, 215

Acid, lactic, see Lactic acid

Acid sodium phosphate, 144

Acid tide, 144

Acids, vegetable, 4, 144

Acrolein, 22, 24

Acute yellow atrophy of liver, 147

Adamkiewicz's reaction, 27^ 34, 39

Adenase, 161

Adenine, 46, 159, 160, 161

Adipose tissue, 2, 23

Aerobic micro-organisms, 66

Aerotonometer, 132

Agglutinating action, 124 ; agglutinins,
124

Air, expired and inspired, 126 ; pumps,
mercurial, 231

Alanine, 31, 33

Alanyl, 36

Albumin, 4, 237 ; action of acids and
alkalis on, 29, 30

Albumin, acid-, 29, 30 ; alkali-, 29

Albumin in urine, 166 ; estimation of,
166 ; tests for, 156

Albuminate, 48

Albuminoids, 43

Albuminometer of Esbach, 166

Albumins, 37, 41, 42, 77, 171

Albumose, 75

Albumoses and peptone, separation of,
182 ; tests for, 182

Alcapton, 169

Alcaptonuria, 169

Alcohol, action of, on proteins, 182

Alcoholic fermentation, 56 ; of milk,
56

Alcohols, 4, 14, 24, 25

Aldehyde, 14, 24

Aldoses, 15

Aleurone grains, 38

AlkaU-albumin, 29, 30, 48, 81, 172,
187

Alkaline hiematin, 188

Alkaline haematoporphyrin, 215 ; ab-
sorption bands of, 189, 215

Alkaline tide, 144

Alkaloids, 60

AUoxuric bases, 46

Allyl alcohol, 24, 231

Aluminium, 8

Alvergniat's pump, 233

Amboceptor, 124

Amidulin, 20

Amino-acetic acid, see Glycine

Amino-acids, 31, 81, 85, 86, 97

Amino-caproic acid, 31, 86

Amino- ethyl-sulphonic acid, 91

Amino-iso-butyl-acetic acid, 86

Amino-oxy-purine, 159

Amino-propionic acid, 31

Amino-purine, 159

Amino-pyrotartaric acid, 32

Amino-succinamic acid, 32

Amino-succinic acid, 32

246

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Ammo- valeric acid, 32

Ammonia, 34, 151

Ammoniacal odour of putrid urine,

146
Ammonio-magnesium phosphate, 156
Ammonium carbonate and carbamate

as urea precursors, 150
Ammonium cyanate, 145
Ammonium sulphate, action of, on

proteins, 27, 28, 40, 165, 182
Ammonium urate, 165
Amoeboid movements, 237
Amylolytic ferments, 65, 68, 184
Amylopsin, 19, 65, 78, 80 ; action of,

82
Amyloses, 16, 65
Anaerobic micro-organisms, 66
Analyses of gases, 234
Animal starch, 21
Anions, 236

Anisotropous bodies, 223
Anti-bodies, 77
Antimony, 8
Antipepsin, 77, 105
Antiseptics, 63
Antithrombin, 105, 107
Antitoxic material, 121
Antitoxin, 123
Antitrypsin, 77, 105
Antivenin, 123
Apparatus necessary for practical

work, 4, 5, 6
Appendix, 217

Aqueous vapour, tension of, 8
Arabinose, 178
Arginase, 150, 161
Arginine, 32, 33, 35, 41, 81, 150, 161
Aromatic amino-acids, 33
Aromatic series, 18
Arsenic, 8

Arterial blood, gases of, 127
Artificial gastric juice, 62
Asparagine, 32
Aspartic acid, 32, 81
Asphyxia, 128
Atmosphere, 126
Atomic weights, 8
Attraction sphere, 2
Atypical globulin, 198
Avogadro's law, 127, 240

B

Bacilli, 65

Bacteria, 65

Bacterial action, 85

Bacterio-lysin, 122

Barcroft's apparatus for obtaining

blood gases, 135
Barfoed's reagent, 178
Barium, 8

Bath, warm, 29

Baumann and Wolkow on alcapton,
169

Bausteine, 98

Beaumont, Dr., on gastric secretion,
70

Beef, 57 ; beef-tea, 59, 200

Beetroot, 18

Benger's liquor pancreaticus, 78, 186 ;
liquor pepticus, 62

Benzene, 33

Benzoic acid, 162

Bernard, Claude, on glycogen of liver,
96 ; diabetic puncture, 88

Bile, 78, 89, 100 ; amount secreted, 90
secretion of, 89 ; circulation, 90
characters of, 90 ; constituents, 90
mucin of, 91 ; pigments, 79, 90, 95
salts of, 79, 91, 94 ; uses of, 93
actions of, 94 ; in urine, 169

Biliary fistula, 89

Bilirubin, 90, 91, 92, 93, 95, 213; of
meconium, 95

Biliverdin, 91, 92, 95

Bismuth, 8

Biuret, 39, 77, 141; reaction, 27, 39,
183

Blood, 3, 101-126 ; coagulation of, 102,
104, 194 ; corpuscles, 103 ; detection
of, 120, 121; gases of, 127; specific
gravity of, 222 ; tests for, 120, 121 ;
pigment. 111 ; biological test for,
121 ; plasma and serum of, 101, 108;
in urine, 169; platelets, 103, 104,
108, 196

Blood bulb, 234

Blood clot, 103

Bohr on absorption of oxygen, 130 ;
on tension of carbonic dioxide, 130

Bone, composition of, 43 ; marrow, 23

Boron, 8

Bowman's capsule, 143

Boyle-Mariotte's law for gases, 240

Bran, 58

Bread, 50, 52, 58; composition of, 59

Bright 's disease, 168

Bromine, 8

Brown and Morris on digestion of starch,
68

Buchner on ferments, 66

Buffy coat, 103

Bunge on hsematogens, 46 ; on milk, 53

Bush tea, 60

Butter, 49, 52

Butyric acid, 19, 24, 85

Butyrin, 55

Cadmium, 8

Caffeine, 60, 159

Calcium, 5, 8 ; phosphate, 49

INDEX

247

Calcium caseinogenate, 55, 181
Calcium oxalate in urine, 156, 162, 165 ;

crystals, 164
Calcium salts, 3, 105 ; importance of, in

coagulation of blood, 104, 105, 194 ;

of milk, 55, 181
Cane sugar, 13, 16, 18, 74, 96, 171, 177,

178 ; urine, 18 ; tests for, 13, 18
Caproic acid, 24, 32
Caproin, 55
Carbamate, 150

Carbamide, 151. See also Urea
Carbohydrates, 4, 13-21, 176-179; ab-
sorption of, 96 ; classification of, 16 ;

definition of, 14 ; tests for, 13-21, 171,

176, 177, 178, 179
Carbolic acid poisoning and urine,

216
Carbon, 3, 8 ; tests for, 3, 9
Carbonates in blood, 129, 130 ; in urine,

154
Carbonic acid in air, 126 ; in blood, 129,

130
Carbonic oxide hssmoglobin, 114, 120,

172, 188
Cardiac glands, 70 ; muscle, 237
Carmine-stained fibrin, 184
Cartilage, 44
Casein, 49, 54, 60, 172
Caseinogen, 10, 35, 49, 64, 65, 172, 181,

186
Caseinogenate of calcium, of sodium, of

potassium, 55
Catalysing agents, 67, 82
Catalysts, 67

Cell, definition of, 2 ; diagram of, 45
Cells, differentiation of, 1
Cellulose, 16, 21, 85, 95
Centigrade scale, 7
Central cells of fundus glands, 70, 71,

72
Centrifugal machine, 195 -
Centrosome, 2
Cerebrin, 201
Cerebrosides, 20]
Cerebrospinal fluid, 203 ; functions of,

203
Chemical physiology, 1
Chemical sediments of urine, 165
Chemical structure of protoplasm, 3
Chemistry of respiration, 126; of nerve

degeneration, 201
Chittenden's views on diet, 52, 148
Chlorides of urine, 141, 153 ; estimation

of, 205 ; tests for, 141
Chlorine, 3, 8
Chlorophyll, 95
Cholalic acid, 91, 94
Cholesterin, 3, 26, 56, 79, 90, 91, 93, 94,
95, 111, 201 ; crystals of, 93, 173 ;
tests for, 93

Choletelin, 92

ChoHne, 26, 85, 201 ; tests for, 201, 202
Chondrin, 44
Chorda tympani, 68
Chromatin, 45
Chromogens in urine, 216
Chromo-proteins, 41, 44
Chyle, 99
Chyme, 89, 94
Cilia, 237

Ciliary movement, 237
Circular polarisation and chemical con-
stitution, 230
' Circulating protein,' 148
Circulation of bile, 90
Cirrhosis of liver, 147
Citric acid, 54, 144
Clark's essence of rennet, 60
Cleavage, 30 ; products of digestion, 81,

97,98
Clotting of blood, 102-108, 194-196
Clupeine, 42
Coagulated proteins, 43
Coagulation, 40
Coagulation of blood, 102-108, 194-196 ;

of milk, 49, 54, 181 ; of muscle, 197-

198 ; of proteins, 27
Coagulative ferments, 65
Cobalt sulphate, actions on proteins,

183
Cocaine, 60
Cocoa, 60, 159
Coffee, 60, 159
Coffin-lid crystals, 155, 157
Cola nut, 60
Collagen, 43, 81
Collimator, 116
Colloid carbohydrates, salting out of,

21
Colloidal solution, 36 ; substances, 237,

238
Colloids, 37, 38, 243
Colostrum, 4, 53
Colostrum corpuscles, 53
Commercial peptone, 37, 107
Complement, 124
Compounds found in the body, 4
Condiments, 60
Conjugated proteins, 41, 44
Cooking of food, 59
Copper, 3, 8
Cream, 49, 53
Creatine, 32, 60, 110, 152, 200; in

muscle, 200
Creatinine, 30, 110, 142, 145, 148, 149,

152, 168, 174, 210 ; detection of, 142 ;

estimation of, 211
Cresol, 66

Croft Hill on ferments, 66
Crypts of Lieberkiihn, 84
Crystallin, 43

248

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Crystalline lens, 43

Crystallisable proteins, 38, 112, 113, 187,

191
Crystallisation of egg albumin, 38, 180
Crystalloids, 38, 238
Crystals from blood, 112, 113, 187, 191
Curative inoculation, 121
Curd, 54, 181
Curds and whey, 54
Cyclopterine, 42
Cystin, 34, 35, 162, 163, 165 ; crystals

of, 164
Cystinuria, 164
Cytosine, 34, 46

Decalcified blood, 101, 104, 194

Decalcified milk, 49, 181

Decomposition products of fats, 25

Density of water, 8

Dentine, composition of, 43

Deposits in urine, 162-165

Desiccator, 199

Deutero-albumose, 75, 77

Deutero-proteose, 81, 168, 172, 187

Dextrin, 13, 16, 21, 68, 171, 178; tests
for, 21

Dextro-rotatory, 17 ; quartz, 226

Dextrose, 13, 16, 17, 88, 110, 168, 171,
177,178; crystals, 17; in blood, 17,
110 ; in urine, 166, 168

Diabetes mellitus, 17, 87

Diabetic coma, 88

Diabetic urine, 88, 166, 168

Diacetin, 25

Dialyser, 37

Dialysis, 37, 38, 192, 236, 238, 239;
of proteoses, 182 ; of serum, 192

Di-amino acids, 32, 34

Di-amino-caproic acid, 32

Di-amino-valeric acid, 32, 150

Diastase, 65

Diastatic ferments, 19, 68, 179

Diet, 51

Diet tables, 51

Dietary, 51

Diffusion, 130, 236, 238

Digestion, 184; gastric, 62, 70-77; in-
testinal, 84-86 ; pancreatic, 78-84 ;
salivary, 62-70

Diraethylxanthine, 159

Dioxy-purine, 159

Dipeptide, 36

Diphtheria-toxin, 122

Direct-vision spectroscope, 117

Disaccharides, 16, 18, 66

Dissociation, 236, 237 ; of oxygen, 133

Disuse atrophy, 203

Divalent elements, 236
Dough, 58
Dripping, 59
Dropsical effusions, 45
Dulcite, 15

Dumb-bell crystals, 157
Dupr6's urea apparatus, 141
Dysalbumose, 182

E

149

Eck's fistula,

Edestin, 35

Egg-albumin, 35, 43, 96 ; crystallisation
of, 180

Egg-globulin, 27, 28

Eggs, 50, 56

Egg-white, 27, 28, 62, 78

Egg yolk, 44

Ehrlich's experiments with methylene
blue, 128 ; hypothesis, 123

Elastic fibres, 44

Elastin, 44, 75, 81, 95

Elastoses, 75

Electrolytes, 237

Electrolytic action, 238

Elements found in the body, 3 ; sym-
bols and atomic weights of, 8

Emulsification, 23, 25, 99

Emulsion, 82

Enamel, composition of, 43

Endogenous metabolism, 149 ; uric
acid, 160

English system of weights and measures,
6,7

Entero-kinase, 84, 105, 186

Envelope crystals, 156

Enzymes, 63, 66, 67, 68

Epidermis, 2

Epithelium of intestine, 98, 99

Epsom salts, 153

Erepsin, 97, 161

Erlenraeyer flask, 2] 1

Erythro-dextrin, 21, 68, 179

Esbach's albuminometer, 5, 166 : re-
agent, 5 ; tube, 166

Estimation of chlorides, 205 ; of crea-
tinine, 211 ; of dextrose, 166, 167 ; of
lactose, 19, 179 ; of maltose, 20, 179 ;
of nitrogen, 235 ; of phosphates, 207 ;
of sulphates, 208 ; of urea, 204 ; of uric
acid, 210

Ethereal sulphates in urine, 153, 154 ;
estimation of, 208

Ethyl alcohol, 24

Ethyl-diacetic acid, 88, 168

Eu-globulin, 193

Exogenous katabohsm, 148

Exogenous uric acid, 160

INDEX

249

Expired air, 126
External respiration, 126
Extirpation of pancreas, 87
Extractives of blood, 110; of muscle,

60
Extraordinary ray, 222

F^CES, 94
Fahrenheit scale, 7
Fatigue, 201

Fat, 2, 96 ; absorption of, 99 ; constitu-
tion, 24 ; decomposition products of,

25 ; melting point of, 23 ; tests for,

22 ; digestion of, 100
Fat-splitting ferment, 80
Fats, 4, 22-26, 78, 93 ; of milk, 55
Fatty acids, 22, 94, 100
Fehling's test, 33, 17, 19, 168 ; solution,

166
Ferment coagulation, 40
Ferment, invert, 84
Ferment of ferments, 84
Fermentation, 63
Fermentation test, 13, 169
Ferments, 63-67 ; of gastric juice, 65 ;

of pancreatic juice, 65 ; of saliva, 65 ;

of succus entericus, 65 ; organised,

64 ; unorganised, 65
Ferments, classification of, 64 ; dias-

tatic, 19, 68, 179
Fertilisation, 237
Fibres, elastic, 44
Fibrin, 78, 101, 103, 106, 108, 184
Fibrin ferment, 40, 65, 101, 103, 104,

105, 106, 108, 109, 110 ; preparation

of, 101
Fibrin filaments, 103
Fibrinogen, 43, 104, 106, 108, 109, 172
Fibrino-globulin, 104
Filtration, 23B, 237
Fischer on proteins, 31
Fistula, gastric, 70
Fleischl's haBmometer, 220 ; spectro-

polariraeter, 229
Flour, 50, 57, 58
Fluorescent screens, 191
Fluorine, 3

Folin's method of estimating urea, 204
Foods, 49 ; cooking of, 59 ; accessories

of, 60
Formaldehyde, 4 ; reaction, 27, 39
Formic acid, 24
Fractional heat coagulation of muscle

proteins, 198 ; of serum proteins, 192,

193
Fraunhofer's lines, 102, 115, 119
Fredericq on tension of carbon dioxide,

130

Free hydrochloric acid in gastric juice,

tests for, 185
Fundus glands, 70, 71, 72
Fiirth on muscle plasma, 195

Galactose, 15, 16, 18, 178, 201
Gallstones, 93

Gamgee on photographic spectra, 190
Garrod and Hopkins on urobilin and

stercobihn, 213, 214
Garrod on urochrome, 213
Garrod's method of separating pigments

from urine, 213
Gas, analysis of, 234
Gaseous interchange in lungs, 128, 129,

130
Gases in blood, 109, 127
Gastric digestion, 62
Gastric fistula, 70
Gastric juice, 70, 85, 185 ; action of, 75;

composition of, 73 ; glands, 70, 71 ;

properties of, 74 ; secretion of, 70
Gastrin, 84

Gay-Lussac's law, 240
Gelatin, 30, 35, 48, 44, 75, 173; tests

for, 30
Gelatinisation, 30
Gelatoses, 75

Germ theory of disease, 63
Gliadin, 35, 48, 58
Globin, 42, 113
Globulicidal power, 122
Globulin, 37, 41, 42, 75, 77, 123, 171, 200
Globulose, 75

Glossopharyngeal nerve, 67
Gluco-proteins, 41, 45
Glucosamine, 45
Gluccse, 16, 65, 96, 177, 178
Glutamic-acid, 32, 35, 81
Gluten, 48, 50, 58
Gluten- fibrin, 58
Glyceric ethers, 24
Glycerides, 24

Glycerin, 25, 85, 100 ; tests for, 22
Glycerol, 25

Glycine, 31, 35, 41, 91, 94, 150
Glycocholate of soda, 90, 91
Glycocholic acid, 91 ; preparation of, 79
Glycocine, see Glycine
Glycogen, 14, 21, 65, 96, 161, 171, 176,

177 ; microscopical detection of, 177 ;

preparation of, 176 ; tests for, 14, 21
Glycol, 15
Glycolysis, 88
Glycosuria, 88

Glycuronic acid, 4, 87, 88, 169, 178
Glycyl, 36

250

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Glyoxylic acid, 4. 27

Gmelin's test, 79, 92, 169

Gnezda on biuret reaction, 183

Goblet cells, 45

Gold, 8

Gowers, Sir William, his hgemacyto-
meter, 217 ; hsemoglobinometer, 219

Graham, Thomas, on colloids and crys-
talloids, 38

Gram-molecular solutions, 238

Granulose, 20

Grape-sugar, see Dextrose

Green vegetables, 61

Ground substance, 30

Griitzner's method of comparing diges-
tive power of solutions of pepsin, 184

Guanase, 161

Guanine, 47, 159, 160, 161

Guarana, 60

Gums, 95

Gunsberg's reagent, 185

Giirber on serum albumin crystals, 39

H

Ha:macytometer of Sir William Gowers,

217 ; of Oliver, 218
Heematin, 95, 113, 213; acid, 188;

alkaline, 188 ; iron free, 191 ; of food,

143
Haematogen, 46
Haematoidin, 114 ; crystals, 89
Haematoporphyrin, 113, 191, 214, 215,

216 ; absorption bands of, 189, 215
Haematoscope of Hermann, 117
Haemin, 101, 113 ; crystals, 113 ; pre-
parations of. 102, 113
Haemochromogen, 190, 200
Haemoglobin, 11, 38, 92, 95, 102, 112,

114, 188 ; composition of, 113 ; deri-
vatives of, 188
Haemoglobinometer of Sir William

Gowers, 219 ; Haldane, 220 ; of Oliver,

220
Haemoglobinuria, 122, 170
Haemopyrrol, 93, 113, 114
Hasmolysins, 122
Haemometer of von Fleischl, 220
Haldane on metheemoglobin, 120 ; on

absorption of oxygen, 130 ; on oxygen

tension, 134
Haldane's haemoglobinometer, 220
Hamburger on digestive action of juices,

184
Hammarsten's method of precipitating

serum globulin, 192
Hammerschlag's method of estimating

the specific gravity of blood, 222
Haptophor group, 123
Hayem's fluid, 219

Hay's test for bile salts, 79, 169

Heart muscle, 199

Heat coagulation of proteins, 37, 192,

193, 198 ; of serum globulin and

serum albumin, 192, 198
Heat rigor, 198
Heidenhain on secretion of gastric juice,

72 ; on pressure in bile duct, 89
Heller's nitric acid test, 166
Henry-Dalton Law, 240
Heptoses, 16

Hermann, haematoscope of, 117
Hetero-albumose, 75, 76, 182 ; proteose,

81
Hexone bases, 33, 84, 150
Hexoses, 16
Hill's air-pump, 233
Hill, Croft, on ferments, 66
Hippuric acid, 162, 173 ; tests for, 162
Hirudin, 107, 195
Histidine, 32, 33, 41
Histone of Kossel, 41, 42, 113
Hofmeister on crystallisation of egg

albumin, 38
Homogentisinic acid, 169
Hopkins's method of estimating uric

acid, 158, 210 ; on Adamkiewicz

reaction, 27 ; on crystallisation of egg

albumin, 39, 180 ; reaction for lactic

acid, 185
Hoppe-Seyler on proteins, 30
Hot-air oven, 176
Howell on rhythmical action, 237
Htifner's method of estimating urea,

141
Hyaline cartilage, 44
Hydrazone, 177
Hydrobilirubin, 92, 93, 94, 143
Hydrocele fluid, 108
Hydrochinon, 216
Hydrochloric acid, 70

of gastric juice, 72, 73

tests for cane sugar, 13
Hydrogen, 3, 8 ; tests for,
Hydrolysis, 19, 75
Hydrolytic ferments, 65
Hydrometer, 49, 53, 141
Hydroxybutyric acid, 88,
Hydroxyl, 24
Hypertonic solutions, 242
Hypobromite of soda, action of, on

urea, 141, 146
Hypotonic solutions, 242
Hypoxanthine, 47, 60, 159, 160, 161

Immune body, 124

Immunity, 121, 123 ; side chain, theory
of, 123

formation of, 71
tests for, 185

9

168

INDEX

251

Indican, 154, 216

Indiffusibility of proteins, 38, 244

Indigo, 154 ; blue, 216 ; red, 216

Indole, 85, 95, 154

Indole-amino-propionic acid, 39

Indoxyl, 154, 216

Indoxyl sulphate of potassium, 154, 216

Infection, 122, 123

Infra-proteins, 47, 48, 171

Inoculation, curative, and protective,

121-126
Inorganic compounds, 4 ; salts, 4
Inosite, 14. 18 ; crystals, 18
Inspired air, 126
Internal respn-ation, 126
Internal secretion of pancreas, 88
Interstitial substance, 30, 45
Intestinal juice, 18, 84 ; digestion, 84
Intravascular coagulation, 103, 105, 196
Inversion, 17, 18, 84
Inversion of cane sugar, 18, 65
Inversive ferments, 65, 66
Invertin, 65, 84
Involuntary muscle, 198
Iodine, 3, 8, 11 .
Iodine test, 13, 14, 176, 179
Ionic action, 237
lonisation, 73
Ions, 73, 236, 240
Iron, 3, 8, 112, 113 ; in milk, 53
Iron-free hsematin, 191
Islets of Langerhans, 79, 87
Iso-cholesterin, 93
Isotonic solutions, 241
Isotropous bodies, 223

Jaffe on test for indoxyl, 216

Jaundice, 169

Jelly, Whartonian, 30

Johnson, G. S., on creatinine, 152 ; on

sugar in urine, 167
Juice, intestinal, 18, 84
Junkets, 60

Karyokinesis, 237

Katabolites, 30, 148

Rations, 236

Kauder's method of precipitating serum

globulin, 192
Kephalin, 201
Keratin, 2, 11, 35, 44, 95
Ketone, 14
Ketoses, 15
Kidney, 142
Kidneys removal of part of, 87 ; removal

of both, 147

Kjeldahl's method of estimating nitro-
gen, 235

Kossel on protamines, 41 ; on histone,
113

Koumiss, 56, 60

Kiihue on precipitation of pepsin, 74

Kiilz's method of extracting glycogen,
176

Lactalbumin, 42, 64, 181 ; properties of,

54
Lacteals, 95, 99
Lactic acid, 4, 53, 56, 60, 95, 158, 185 ;

Hopkins's reaction for, 185, 197 ; tests

for, 185
Lactic acid fermentation, 19 ; in milk,

56 ; in muscle, 197 ; organisms, 19
Lacto-globulin, 181
Lactometer, 49
Lactose, 13, 16, 19, 49, 56, 65, 171, 177,

178; in urine, 168; tests for, 19,

177
Laky blood, 112
Lanoline, 93
Lard, 22

Lateritious deposit, 156
Laurent's polarimeter, 228
Lead, 3, 8
Lecithin, 3, 4, 10, 25, 26, 56, 85, 91, 93,

111, 155, 201
Leech extract, 107, 194
Lethal dose, 122
Leucine, 31, 80, 81, 84, 85, 86, 148, 165,

187 ; as a urea precursor, 150 ; tests

for, 187
Leucine crystals, 86, 150 ; preparation

of, 187
Leucocytes, 160, 161
Leucocythoemia, 160
Leucyl, 36

Levo-rotatory, 17 ; quartz, 226
Levulose, 16, 17, 171, 177 ; in blood, 17 ;

in muscle, 17 ; in urine, 17 ; reactions

of, 17
Lieberkiihn's crypts, 84 ; jelly, 48
Liebermann's reaction, 79
Liebig's extract, 200
Lime water, 30
Lipochrome, 55, 56, 200 ; in muscle,

200
Lipolytic ferments, 65
Liquor pancreaticus, 78, 187 ; pepticus,

62
Lithates, see Urates, 156
Lithium, 3, 8

Litre, standard of capacity, 7
Liver, function of, in relation to urea,

147 ; uric acid, 158
Living test-tube experiment, 109

252

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Loeb on ionic action, 237 ; on fertilisa-
tion, 237
Loewy's aerotonometer, 132
Lungs, 126

Lymph formation, 242
Lysine, 32, 33, 81

M

McMuNN on stercobilin, 95

McWilliam's test for proteins, 183

Magnesium, 3, 8

Magnesium phosphate, 165

Magnesium sulphate, action of, on pro-
teins, 27, 40, 49

Malic acid, 231

Mallow, 18

Malpighian capillaries, 142

Malt upon starch, action of, 179 ; dias-
tase, 179

Maltase, 65

Malting ferment, 179

Maltose, 13, 16, 19, 65, 66, 68, 78, 171,
177, 178, 179

Maly on the formation of hydrochloric
acid, 72, 73

Mammary glands, 84, 88

Manganese, 3, 8

Mannite, 15

Mannose, 15

Marchi reaction, 202

Marsh gas, 65

' Mass Action,' 73

Measures of capacity, 7 ; of length, 7

Meat, 50, 52, 67 ; constituents of, 57

Meconium, 95

Melanin, 216

Mercurial air-pumps, 231

Mercury, 8

Metabolism, 2

Meta-proteins, see Infra-proteins

Methffimoglobin, 114, 119, 170, 172,
191 ; in urine, 170

Methane, 65, 85

Methyl-indole, 34

Methyl-glycine, 32

Methylene blue experiments, 128

Methyl-guanidine acetic acid, 32

Methyl-orange, 235

Metric system, 7

Mett's tubes, 184

Microchemical detection of glycogen,
177

Micrococcus urea, 66

Microspectroscope, 117

Milk, 23, 49, 50, 52, 53, 181 ; alcoholic
fermentation of, 56 ; coagulation of,
54, 181 ; composition of, 54 ; fats of.

54 ; proteins of, 54 ; salts of, 56 ;

souring of, 56 ; sugar, 56
Milk-curdling, ferment of pancreas, 80,

82 ; of stomach, 49, 54, 55
Milk-sugar, 19, 49, 56 ; crystals, 19, 96
Millon's reagent, 5, 27 ; test, 27 ; re-
action, 39
Mineral compounds, 3
Mohr's method of estimating chlorides,

205
Monatomic alcohols, 24
Monoacetin, 25
Monoamino-acids, 32, 34
Monoamino-caproic acid, 32
Monochromatic light, 228
Monosaccharides, 16, 17, 66
Monovalent, 236
Monoxypurine, 159
Moore and Rockwood on fat absorption,

100
Moore on osmotic pressure of proteins,

243
Moore's test for sugar, 13, 17
Morner and Sjoqvist's method of esti-
mating urea, 204
Morris and Brown on starch digestion,

68
Mucedin, 58
Mucic acid, 18, 178
Mucin, 2, 30, 45, 67, 68, 91, 94, 95,

173 ; in bile, 91 ; in saliva, 62, 67 ;

in urine, 162 ; tests for, 62
Mucinogen, 67
Mucoids, 45

Mucous glands, structm-e of, 45, 69
Mucous membrane of frog's intestine,

99
Mucous salivary glands, 69
Mucus, 170

Munk on fat absorption, 100
Murex, 157
Murexide test, 156
Muscle, 199 ; clot, 198 ; pigments of,

199 ; plasma of, 197 ; extractives of,

200
Muscle, cardiac, 237
Muscle fibrin, 198
Muscle sugar, 18
Muscle tissue, 197
Muscles, pale and red, 199 ; involuntary,

198
Muscular movement, 2 ; exercise and

carbonic acid, 136
Mutton, 57

Myogen, 198 ; myogen fibrin, 198
Myo-haematin, 199, 200 ; absorption

spectrum of, 200
Myosin, 4, 57, 75, 197, 198; fibrin,

198
Myosinogen, 37, 198
Myxoedema, 87

INDEX

253

N

Negative effect, 104

Nencki and Sieber on pigments, 113

Nencki's experiment on urea, 150

Nerve degeneration, chemistry of, 201

Nervous impulse, 238

Nervous tissue, 197, 200; composition
of, 200

Neurokeratin, 44

Neutral fats, 23

Neutral salts, action of, on carbo-
hydrates, 21 ; on proteins, 27, 28, 37,
43, 49, 77, 101, 102, 182, 192, 193

Nickel, 8

Nickel sulphate, action of, on proteins,
183

Nicol's prism, 223

Nissl's bodies, 201

Nitrate of urea, 146, 156

Nitric oxide haemoglobin, 114, 120

Nitrogen, 3, 8 ; estimation of, 235 ;
tests for, 9, 10

Nitrogenous food, 50, 51 ; glucosides,
201

Nitrous acid, action of, on urea, 146

Non-electrolytes, 237

Nuclease, 161

Nucleic acid, 45, 46 ; decomposition of,
47

Nuclein, 3, 45, 155, 160, 161

Nucleo-protein, 3, 10, 41, 45, 46, 91,
103, 105, 109, 173, 196, 200; in
bile, 79, 91 ; decomposition of, 47 ; in
muscle, 198 ; tests for, 79, 173

Nucleus, functions of, 2

0

Oatmeal, 52

Oleic acid, 24

Olein, 23, 55, 203

Olfactory nerve, 67

Oliver's hamacytometer, 218 ; ha?mo-
globinometer, 220

Opsonins, 125

Orcin reaction, 178

Ordinary ray, 222

Organic compounds, 4

Organised ferments, 64

Ornithine, 32, 150, 161

Osazones, 177

Osborne, W. A., on coagulation of milk,
55

Osmic acid test, 22, 24

Osmium, 8

Osmosis, 38, 236, 239

Osmotic pressure, 238, 239, 243 ; cal-
culation of, 240 ; of proteins, 243 ;
determination of, by freezing point,
241 ; physiological application, 242

Ossein, 43

Osteomalacia, 168

Ovarian cyst fluid, 45

Ovo-mucoid, 56

Oxalate of calcium, in milk, 47, 55;
plasma, 106 ; in urine, 156, 164

Oxalate of urea, 146, 156

Oxidases, 66, 161

Oxygen, 3, 8 ; in blood, 128 ; tension,
137

Oxyhsemoglobin, 101, 102, 112, 113,
114, 169, 170, 172, 188, 191 ; crystals,
102, 112, 187; in muscle, 200; pre-
paration of, 191

Oxyhtemoglobin pure, preparation of,
191

Oxyntic cells of fundus glands, 70, 72,
73

Oxyphenyl-alanine, 34

Pale muscle, 199

Palmitic acid, 24

Palmitin, 23, 55

Pancreas, extirpation of, 87 ; grafting
the, 87 ; structure of, 79, 80

Pancreatic digestion, 78, 186, 187 ; se-
cretion, 186

Pancreatic juice, 81, 85 ; action of, 78,
81 ; composition of, 80 ; secretion of,

. 83

Panum's method of precipitating
serum globulin, 192

Paraglobulin, see Serum globulin

Para-mucin, 45

Paramyosinogen, 43, 197, 198

Parietal cells of fundus glands, 70, 71

Parotid gland, 67

Partial pressure of gases, 126-140

Pasteur on circular polarisation, 230

Pathological urine, 166 ; pigments of,
216 ; pigments, 216

Pavy on glycogenic function of liver 96,
176 ; an estimation of sugar, 167

Pavy's solution, 167

Pawlow on secretory nerve-fibres to the
gastric glands, 74 ; to the pancreas, 83

Peas, 58

Pentoses, 16, 178

Pepsin, 65, 70, 72, 74, 81 ; solutions,
activity of, 184 ; hydrochloric acid,
70, 74, 82

Pepsinogen, 72

Peptic digestion, 75, 185

Peptides, 36

Peptone, 28, 31, 37, 47, 65, 75, 76, 77,
85, 96, 107, 171, 187, 195 ; precipita-
tion of, 76 ; in urine, 168 ; tests lor, 27,
76

254

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Peptone blood, 195

Peptonuria, 168

Perfect foods, 50, 53

Pericardial fluids, 108

Pettenkofer's test for bile salts, 79, 91,
92, 169

Pfliiger's mercurial air-pump, 231, 232

Phagocytes, 121, 122

Phenol, 66, 85, 95

Phenolphthalein, 22 ; phenyl-alanine,
33, 34, 98

Phenyl sulphate of potassium, 154

Phenyl-hydrazine test for sugar, 177

Phloretin, 88

Phloridzin, 88 ; diabetes, 88

Phloroglucin reaction, 178

Phosphates of urine, 141, 154, 156, 163 ;
estimation of, 207 ; tests for, 141, 156,
163, 165

Phosphates, stellar, 155, 156, 164, 165

Phosphates, triple, 155, 156, 164, 165

Phospho-molybdic acid, 182

Phospho-proteins, 41, 44

Phosphorised fat, 201

Phosphorus, 3, 8, 11 ; tests for, 10

Phospho-tungstic acid, 182

Photographic spectra, 191

Physiological chemistry, 1

Physiological proximate principles, de-
tection of, 171

Pickering on colour reactions of proteins,
183

Picramic acid, 17

Picric acid, 17 ; tests for sugar, 17, 167

Pigment of red corpuscles, 110-114 ; of
muscle, 199 ; of urine, 213, 215

Pigments, pathological, 215

Pilocarpine, administration of, 80, 187

Piotrowski's reaction, 27, 41, 183

Plain muscle, 198, 199

Plasma, constituents of, 109 ; gases of,
109 ; proteins of, 109

Plasma of blood, 101, 103, 108, 109,
194 ; of muscle, 197

Plasmon, 186

Platinum, 8

Pleochromatism, 225

Poisonous alkaloids, 85

Polarimeters, 178, 226 ; of Laurent, 228 ;
of Soleil, 227 ; of Zeiss, 228

Polarisation, circular, and chemical con-
stitution, 230

Polarisation of light, 222

Polariscopes, 226

Polarised light, 222 ; action of carbo-
hydrates on, 16, 17, 18, 19 ; of pro-
teins, 39

Polarising microscope, 224

Polypeptides, 31, 36, 47

Polysaccharides, 16, 20

Pork, 57

Portal vein, 89

Positive phase, 104

Potash, method of extracting glycogen,
176 ; of showing zymogen granules,
187

Potassio-mercuric iodide, 182

Potassium, 3, 8

Patassium caseinogenate, 55

Potassium ferricyanide in preparation
of methaemoglobin, 188

Potassium ferrocyanide in the estimation
of phosphate, 207

Potassium permanganate in estimation
of uric acid, 210, 211

Potassium sulphocyanide in saliva, 62,
68

Potatoes, 52

Precipitants of proteins, 39, 40, 182,
183

Precipitation, 40

Precipitin, 125

Prevost and Dumas on formation of
urea, 147

Primary albumoses, 75; proteoses, 75

Principal cells of stomach, 70, 71

Prisms in direct-vision spectroscope,
117

Propionic acid, 24, 31, 33

Prosecretin, 83, 186

Prosthetic group, 44

Protagon, 201

Protamines, 41, 42

Protective inoculation, 121

Protein-hydrolysis, 47

Protein metabolism, 3

Proteins, 3, 9, 11, 27-48, 57, 68-, 81, 85,
93, 96, 98, 123, 171, 238, 244; absorp-
tion of, 96, 97 ; classification of, 41 ;
coagulated, 37 ; coagulation of, 37 ;
colour reactions of, 27, 39 ; composi-
tion of, 3, 30 ; conjugated, 44 ; crys-
tallisation of, 38 ; definition of, 30 ;
digestion of, by gastric juice, 75 ; diges-
tion of, by pancreatic juice, 81 ; heat
coagulation of, 37 ; of blood plasma,
109; of milk, 54 ; of muscle, 197, 198 ;
of serum, 109 ; osmotic pressure of,
243 ; in urine, 168 ; precipitants of,
27, 39, 40 ; sclero-, 43 ; simple solu-
bilities of, 36 ; tests for, 14, 36, 182,
183

Protein-sparing food, 44

Proteolytic ferments, 74j enzymes, 74,
81, 161

Proteoses, 31, 37, 47, 65, 75, 76, 77, 79, 97,
107, 171, 172, 182, 195 ; in urine, 168

Prothrombin, 105

Proto-albumoses, 75, 76, 182

Protones, 41

Protoplasm, chemical structure of, 3 ;
properties of, 2

INDEX

255

Proximate principles, classification of, 4 ;

of food, 50 ; scheme for detecting,

171-174
Pseudo-globulin, 193, 198
Pseudo-mucin, 45
Ptomaines, 63
Ptyalin, 19, 05, 68, 69
Pulses, 58

Pumps, mercurial air-, 231-234
Purine bases, 44, 46, 47, 159, 161
Purpurate of ammonia, 156
Pus in urine, 170; tests for, 170
Putrefaction, 19, 66, 85
Pyloric glands, 70, 71
Pyrimidine bases, 34
Pyrocatechin, 216
Pyrrolidine derivatives, 34

Q

QUADRIURATES, 158

Quantitative estimation of albumin, 166 ;
of chlorides, 205 ; of creatinine, 211 ;
of dextrose, 167 ; of glycogen, 176,
177 ; of lactose, 19, 179 ; of maltose,
20, 179; of nitrogen, 235; of phos-
phates, 209 ; of sulphates, 210 ; of
sugar, 167 ; of urea, 205 ; of uric acid,
210

R

Kanke's diet, 51

Reagents necessary for practical work,

4, 5, 6
Reaumur's scale, 7
Receptor group, 123
Red blood corpuscles, 110 ; composition

of, 110 ; pigment of, 110
Red muscle, 199
Reflex action, 67

Reid, Wavmouth, on absorption, 243
Rennet, 40, 60, 65, 72
Rennin, 60, 72, 82
Reproduction, 2
Resins, 95
Respiration, chemical stimulus to, 138 ;

chemistry of, 126 ; in excised tissues,

138
Respiratory oxygen of haemoglobin, 114
Respiratory pigments, 112
Respiratory quotient, 127
Reversible action, 66
Rhythmical contraction, 237
Ricin, 123
Rigor mortis, 198
Ringed amino acids, 34
Ringer on contractile tissue, 237
Riva on urochroiij^ 143

Roberts, Sir William, on estimation of

sugar in urine, 169
Rock wood and Moore on fat absorption,

100
Rose's test for protein, 27, 41, 183
Rosenheim's formaldehyde reaction, 27

Saccharic acid, 18

Saccharimeters, 230

Saccharoses, 16, 65, 84

St. Martin, Alexis, case of, 70

Salicyl-sulphonic Acid, action of, on
proteins, 183

Saliva, 30, 62, 67 ; action of, 68, 70 ;
composition of, 67 ; secretion of, 67

Salivary corpuscles, 67 ; glands, 2, 67,
69, 107

Salkowski's reaction for cholesterin, 79,
93 ; method of estimating sulphates,
209

Salmine, 42

Salted plasma, 101

Salted whey, 54

Salting-out of colloid carbohydrates, 21
of proteins, 40, 55, 193

Salts of milk, 56

Saponification, 22, 82, 99

Sarcine, 159

Sarcolemma, 44

Sarco-lactic acid, 197

Sarcosine, 32

Schafer on fat absorption, 98, C9

Schili on bile circulation, 90, 94 test
for uric acid, 156

Schizomycetes, forms of, 64

Schmalz's capillary picnometer, 222

Schmidt, Alexander, on precipitating
serum globulin, 192 ; on salts of
plasma, 110 ; on preparation of fibrin-
ferment, 110

Schroder's work on urea, 150

Schiitz's law, 184

Schwann, white substance of, 93

Sclero-proteins, 41, 43

Sebum, 93

Secretion, 83, 84, 89, 186

Secretion, internal, 87

Sediments in urine, 165

Semipermeable membranes, 239

Serine, 32

Serous glands, 69

Serum, 101, 103, 108, 192; albumin,
35, 37, 42, 101, 109, 168 ; crystallisa-
tion of, 38 ; gases of, 109 ; heat
coagulation, 37, 42, 193

Serum, bactericidal, globulicidal, 124

Serum casein, 192

256

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Serum globulin, 35, 37, 42, 43, 101, 102,

109, 168 ; heat coagulation, 192, 193
Serum lutein, 101
Serum of blood, 101, 102 ; proteins of,

101, 102
Serum proteins, 109 ; separation of, 101
Sheep's wool-fat, 93
Shell of eggs, 56
Side-chain theory, 123
Silicon, 3, 8
Silver, 8
Silver nitrate method of estimating

chlorides, 205
Skatole, 34, 85, 95
Skatoxyl, 216 ; red, 216
Skimmed milk, 49, 53
Smoky urine, 169
Snake venom, 64, 121, 122
Soap, 22, 25, 82, 90, 100
Sodium, 3, 8

Sodium bicarbonate in blood, 129, 130
Sodium chloride, 3 ; action on proteins,

109, 182 ; on nucleo-proteins, 46,

196
Sodium hypobromite, method of esti-
mating urea, 141, 142
Sodium phosphate, 29, 30 ; acid, 144
Sodium sulphate plasma, 101, 104
Solar spectrum, 119, 189
Soleil's saccharimeter, 227
Soluble starch, 20
Solutions, 286
Sorbite, 15
Soret's band, 191
Soup, 60

Souring of milk, 56, 63
Specific gravity of blood, 222 ; of milk,

49, 53 ; of urine, 141, 144
Specific rotatory power, 229
Spectra, 189 ; of haemoglobin and its

derivatives, 189; photographic, of

metheemoglobin, 190, 191 ; oxyheemo-

globin, and haemoglobin, 190, 191 ; of

urinary pigments, 215
Spectro-polarimeter, 229
Spectroscope, 115, 116, 117
Spectrum, 119, 189, 215
Spermatozoa, 41
Starch, 4, 13, 20, 65, 93, 95, 238;

soluble, 21 ; action of malt on, 179 ;

digestion of, 68, 70; test for, 20,

171
Starling and Bayliss on pancreatic

secretion, 83
Starling on osmotic pressure of proteins,

243
Steapsin, 25, 65, 78, 80, 82, 85 ; action

of, 78, 82
Stearic acid, 24
Stearin, 23, 55
Steatolytic ferments, 65

Stein's method of making oxyheemo-

globin crystals, 112
Stellar phosphates, 156
Stercobihn, 92, 94, 95, 143
Stewart, G. N., dietary, 51, 52
Stirling on preparation of pure oxyhsemo-

globin, 191
Stokes's fluid, 115, 188
Stomach, glands of, 71, 72
Strontium, 8
Sturine, 42
Sublingual gland, 67
Submaxillary gland, 67, 68, 69
Succus entericus, 84
Sucroses, 16
Sudan III. stain, 24
Sugar, 4, 9, 13, 17-20, 87, 237; cane, 13,

18, 79 ; in blood, 110 ; in urine, 167,

168 ; in muscle, 18 ; tests for, 166,

174
Sugar-maple, 18
Sulphates of urine, 141, 153 ; estimation

of, 208 ; tests for, 141
Sulphonal poisoning, 214
Sulphur, 3, 8 ; tests for, 10
Suprarenal gland, removal of, 87
Suspending medium, 23
Sweetbreads, 160
Swim-bladder of fishes, 131
Symbols and atomic weights, 8
Syntonin, 30, 48, 62, 75

Tannin, 61, 182

Tapetum, 160

Tartaric acid, 144

Taurine, 91, 94

Tauro-carbamic acid, 94

Taurocholate of soda, 90, 91

Taurocholic acid, 91

Tchistovitch's experiment, 125

Tea, 60, 61, 159

Teichmann's crystals, 113

Tendo-mucoid, 45

Tendon, 30

Tension in fluids, measurement of, 132

Tension of aqueous vapour, 8 ; of gases,

128, 130, 131
Tension, relation of, to composition,

132
Testis, 196 ; removal of, 87
Tetrapeptides, 36
Tetroses, 16
Theine, 60

Theobromine, 60, 159 '
TheophyUine, 159
Thermometric scales, 7
Thrombin, 104, 105, 106, 108
Thrombogen, 105, 1^, 107, 108

INDEX

257

Thrombo-kinase, 105, 106, 107, 108,

196
Thrombosis, 105
Thudichum on urochrome, 213
Thymine, 34
Thymus, 159, 196
Thyroid gland, 87 ; removal of, 87
Tin, 8

Tissue-fibrinogens, 196
Tissue metabolism, 149
Tissue respiration, 126, 134, 136
Tomes on enamel, 43
Tooth, 43
Topfer's test, 185
Torricellian vacuum, 114
Torula ureae, 145
Torulffi, 63, 145
Toxins, 122, 123
Transitory giucosuria, 167
Triacetin, 25
Triatomic alcohol, 25
Trichloracetic acid as a precipitant of

proteins, 183
Trimethyl-xanthine, 159
Triolein, 25
Trioses, 16
Trioxy-purine, 159
Tripalmitin, 25
Tripeptides, 36
Triple phosphate, 156, 165
Tristearin, 25
Trivalent elements, 236
Trommer's test, 13, 17, 18, 19, 20
Tropffiolin test, 185
Trypsin, 65, 80, 81, 82, 84, 97 ; action

of, 81
Trypsinogen, 80, 84, 186
Tryptophan, 33, 34, 35, 81, 187; test

for, 187
Tungsten, 8
Tunicates, 21
Tyrosine, 33, 34, 35, 42, 80, 81, 84, 86,

165, 187 ; crystals,-86 ; in urine, 165 :

tests for, 187 D"»f,

tu

XJffelmann's reaction, 185

Umbilical cord, 30

Uncrystallisable sugar, 17

Unorganised ferments, 64, 66, 68

Uracil, 46

Uraemia, 147

Uranium acetate in estimation of phos-
phates, 207

Uranium nitrate in estimation of phos-
phates, 207

Urate, acid ammonium, deposit of, 163 ;
acid sodium, deposit of, 163

Urates, 156, 163, 165, 168, 214

Urea, 4, 12, 30, 32, 87, 94, 97, 98, 110,
141, 145-151, 161, 173, 237; com-
position and compounds, 146 ;
crystals of, 145 : decomposition of,
66 ; estimation of, 204 ; mode and
site of formation, 147 ; preparation
of, 205 ; quantity excreted, 143 ; tests
for, 141, 145 ; where formed, 147

Urea nitrate, 146, 156 ; preparation of,
156

Urea oxalate, 146, 156 ; preparation of,
156

Uric acid, 110, 145, 156, 157-161, 159,
160, 165, 173 ; crystals of, 157 ; esti-
mation of, 158, 210 ; origin of, 158 ;
preparation of, 157, 210 ; tests for,
156, 157

Uricolytic ferment, 161

Urina potus, 144

Urinary deposits, 162

Urine, 141 ; composition of, 144, 145,
173 ; inorganic constituents of, 153,
173 ; pigments of, 143, 213 ; tests for
abnormal constituents of, 174 ; tests
for constituents, 142, 156

Urinometer, 141, 144

Urobilin, 92, 93, 143, 169, 213, 214, 216 ;
absorption bands of, 215

Urobilinogen, 143, 213

Urochrome, 143, 213 ; uro-erythrin,
156, 214 ; absorption bands of, 215
j Urorosein, 216 ; absorption bands of,
i 215

Valeric acid, 24, 32, 85

Van 't Hoff on polarisation of light, 231 ;
hypothesis, 240

Vegetable acids, 4, 144 ; food, composi-
tion of, 58 ; parchment, 37

Vegetables, composition of, 58 ; green,
61

Velocity of chemical reactions, 67, 82

Venous blood, gases of, 127

Villus, section of, 98, 99

Vinegar, 63

Vital action, 242

Vitellin, 38, 44, 56, 58, 75

Vitellose, 75

Vitreous humour, 30

Voit's diet, 51, 146

Voluntary muscle, 199

W

Waller's modification of Zuntz's gas

apparatus, 234
Warm bath, 29
Water in protoplasm, 3, 4 ; density of, 8

S

258

ESSENTIALS OF CHEMICAL PHYSIOLOGY

Wave theory of light, 222

Weights and measures, 6, 7

Whartonian jelly, 30

Whetstones, 156

Whey, 49, 54 ; salted, 49

Whey protein, 54

White blood corpuscles, 110, 196

White of egg, 3, 27, 29, 37, 38, 43, 45,

62, 78, 180
Whole flour, 57, 58
Widal's reaction, 124
Witte's peptone, 182
Wohler, preparation of urea, 145
Wooldridge on tissue-fibrinogen, 46, 196
' Worth' of corpuscles, 222

Xanthine, 47, 80, 110, 169-161
Xanthine group, 46, 159

Xanthoproteic test, 27, 39
Xylose, 178

Yeast, action of, 17, 19, 66 ; cells, 63 ;

in bread-making, 58 ; in testing for

sugar, 169
Yellow lipochrome, 200
Yolk of eggs, 56

Zein, 35, 48
Zeiss's polarimeter, 228
Zinc, 8

Zuntz's gas apparatus, 234
Zymogen, 70, 72, 80, 105, 187 ; granules,
187

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MESSRS. LONGMANS' WORKS ON MEDICINE, SURGERY, ETC. 9

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10 MESSRS. LONGMANS' WORKS ON MEDICINE, SURGERY, ETC.

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16 MESSRS. LONGMANS' WORKS ON MEDICINE, SURGERY, ETC.

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