Monographs on Biochemistry
UC-NRLF
NATURAL BASES
to Mo.\W Lib* U- 2^ ^ feO
MONOGRAPHS ON BIOCHEMISTRY
EDITED BY
R. H. A. PLIMMER, D.Sc.
AND
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
GENERAL PREFACE.
THE subject of Physiological Chemistry, or Biochemistry, is
enlarging its borders to such an extent at the present time,
that no single text-book upon the subject, without being
cumbrous, can adequately deal with it as a whole, so as to
give both a general and a detailed account of its present
position. It is, moreover, difficult, in the case of the larger
text-books, to keep abreast of so rapidly growing a science
by means of new editions, and such volumes are therefore
issued when much of their contents has become obsolete.
For this reason, an attempt is being made to place this
branch of science in a more accessible position by issuing
a series of monographs upon the various chapters of the
subject, each independent of and yet dependent upon the
others, so that from time to time, as new material and
the demand therefor necessitate, a new edition of each mono-
graph can be issued without re-issuing the whole series. In
this way, both the expenses of publication and the expense
to the purchaser will be diminished, and by a moderate
outlay it will be possible to obtain a full account of any
particular subject as nearly current as possible.
The editors of these monographs have kept two objects
in view : firstly, that each author should be himself working
at the subject with which he deals ; and, secondly, that a
Bibliography, as complete as possible, should be included,
in order to avoid cross references, which are apt to be
wrongly cited, and in order that each monograph may yield
full and independent information of the work which has been
done upon the subject.
It has been decided as a general scheme that the volumes
first issued shall deal with the pure chemistry of physiological
products and with certain general aspects of the subject.
Subsequent monographs will be devoted to such questions
as the chemistry of special tissues and particular aspects of
metabolism. So the series, if continued, will proceed from
physiological chemistry to what may be now more properly
termed chemical physiology. This will depend upon the
success which the first series achieves, and upon the divisions
of the subject which may be of interest at the time.
R. H. A. P.
F. G. H.
MONOGRAPHS ON BIOCHEMISTRY
EDITED BY
R. H. A. PLIMMER, D.Sc.
AND
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
ROYAL 8vo.
THE NATURE OF ENZYME ACTION. By
W. M. BAYLISS, D.Sc., F.R.S. Third Edition.
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THE CHEMICAL CONSTITUTION OF THE
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(Two Parts.)
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THE POLYSACCHARIDES. By ARTHUR R. LING,
F.I.C.
COLLOIDS. By W. B. HARDY, M.A., F.R.S.
RESPIRATORY EXCHANGE IN ANIMALS. By
A. KROGH, Ph.D.
NUCLEIC ACIDS. THEIR CHEMICAL PRO-
PERTIES AND PHYSIOLOGICAL CON-
DUCT. By WALTER JONES, Ph.D.
PROTAMINES AND HISTONES. By A. KOSSEL,
Ph.D.
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MACLEAN, M.D., D.Sc.
ORGANIC COMPOUNDS OF ARSENIC AND ANTI-
MONY. By GILBERT T. MORGAN, D.Sc., F.I.C.
LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, BOMBAY AND CALCUTTA.
THE
SIMPLER NATURAL BASES
BV
GEORGE BARGER, M.A., D.Sc.
FORMERLY FELLOW OF KING'S COLLEGE, CAMBRIDGE
PROFESSOR OF CHEMISTRY IN THE ROYAL HOLLOWAY COLLEGE, UNIVERSITY OF LONDON
LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON
NEW YORK, BOMBAY AND CALCUTTA
1914
0? CALIFORNIA
Y
TO
H. H. D.
AGR1C, DEPT,
PREFACE.
IN the following pages I have endeavoured to give an account
of those basic substances of animals and plants which are of
general biological interest, either because of their wide distri-
bution, or on account of their close relationship to the proteins
and phosphatides. In contradistinction to the typical vegetable
alkaloids, these bases have a simple chemical constitution.
By a more or less arbitrary delimitation of the subject matter,
involving for instance the total exclusion of purine bases, I
have aimed at giving, in the space at my disposal, a somewhat
detailed account of the chemistry of the bases dealt with, and
of their derivatives. Some, like the amines and adrenaline,
are remarkable on account of their physiological action, and in
each case, therefore, a brief description of this action has been
added. In this way I have endeavoured to make the mono-
graph also of interest to those who are concerned with the
biological rather than with the chemical aspect of the subject.
A brief chapter on the practical methods used in the
isolation of the simple bases has been added, and special
attention has been given to the bibliography which extends
to the autumn of 1913.
It is a pleasant duty to express my great indebtedness to
Dr. H. H. Dale, without whose advice and criticism much of
the pharmacological sections would have remained unwritten.
G. B.
ENQLEFIELD GREEN,
SURREY,
November, 1913.
CONTENTS.
CHAPTER III.
BETAINES
PAGE
INTRODUCTION AND SCOPE - i
CHAPTER I.
AMINES DERIVED FROM PROTEIN ----- .7
SECTION
1. The Putrefactive Decomposition of Amino-acids - 7
2. Methylamine, Ethylamine, Dimethylamine - n
3. Trimethylamine - n
4. Isobutylamine - - - - - - - -12
5. Isoamylamine - - - - 13
6. Pyrrolidine - - - 13
7. Amino-ethyl Disulphide - 13
8. Putrescine and Cadaverine - - 14
9. Agmatine ... - - - 16
10. Phenyl-ethylamine - 16
11. p-Hydroxy-phenyl-ethylamine - 18
12. Hordenine - ... 20
13. Indolethylamine (3-/3-Amino-ethylindole) - - 21
14. y8-Iminazolyl-ethylamine - 22
15. Physiological Properties of the Amines derived from Amino-
acids - ... 25
CHAPTER II.
u>- AMINO-ACIDS AND OTHER BASES CONTAINING A CARBOXYL GROUP - 33
SECTION
1. /?-Alanine (/3-Amino-propionic Acid) - 34
2. y-Amino-n-butyric Acid - - 34
3. 8-Amino-n-valeric Acid - .35
4. e-Amino-caproic Acid - - 35
5. /3-Iminazolyl-propionic Acid - - 35
6. Carnosine (Ignotine) - 36
7. Urocanic Acid (Iminazolyl-acrylic Acid) - - 36
8. Kynurenic Acid - 37
39
SECTION
1. Betaine (Trimethyl-glycine) - - 40
2. Physiological Properties and Importance of Betaine 42
3. Stachydrine (Dimethyl-proline) - 43
4. Betonicine and Turicine (Dimethyl-oxyproline) 44
CONTENTS vii
SECTION
5. Trimethyl-histidine . . 45
6. Ergothioneine (Thiolhistidine Betaine) - - - - 46
7. Hypaphorine (Trimethyl-tryptophane) - - - 47
8. Trigonelline (Methylnicotinic Acid) - . . 47
9. Other Pyridine Bases - - - - 48
10. y-n-Butyrobetaine - 49
11. Carnitine (Novaine, a-Hydroxy-y-butyrobetaine) - - - 50
12. Myokynine - 52
CHAPTER IV.
CHOLINE AND ALLIED SUBSTANCES - - - - - 53
SECTION
1. Choline- . 54
2. Ammo-ethyl Alcohol (Colamine) and the Origin of Choline ;
the possible Presence of other Bases in Phosphatides 58
3. Neurine- 60
4. Physiological Action of Choline and of Neurine - - 61
5. Natural and Synthetic Muscarines and their Physiological
Action 64
6. Trimethylamine Oxide - -67
7. Neosine- ......... 55
CHAPTER V.
CREATINE, CREATININE, GLYCOCYAMINE AND GUANIDINES - - 69
SECTION
1. Creatine and Creatinine - - 69
2. Physiology of Creatine and Creatinine - 71
a. Distribution - 71
b. Metabolism - - - 73
c. Possible Precursors of Creatine - .77
3. Glycocyamine and Glycocyamidine - - 78
4. Guanidine - - 79
5. Methylguanidine - - 79
6. as-Dimethylguanidine - 80
CHAPTER VI.
ADRENALINE - 81
SECTION
1. Historical - 81
2. Nomenclature and Synonyms - 83
3. Preparation and Purification of Natural Adrenaline - 84
4. Syntheses of Adrenaline - 85
5. Adrenaline Substitutes - 87
6. Physical and Chemical Properties of Adrenaline. Salts and
Derivatives. Constitution - 87
7. Colour Reactions of Adrenaline and Colorimetric Estimation 89
8. Amount of Adrenaline in the Suprarenal Gland ; Yield ;
Distribution in other Organs ; Origin - 92
viii CONTENTS
SECTION PAGE
9. Physiological Action of Adrenaline - - 96
a. Action on the Circulatory System - - 96
b. Action on other Organs containing Involuntary Muscle,
and on Glands - - 97
c. Action on Carbohydrate Metabolism - - 99
d. Toxic Action - - 100
10. The Physiological Action of Dextro- and of Racemic Adrena-
line - - 100
11. Physiological Methods of Estimating Adrenaline - 101
CHAPTER VII.
BASES OF UNKNOWN CONSTITUTION - 106
SECTION
1. Spermine - 106
2. Bases from Muscle - 107
3. Bases from Urine - 107
4. Putrefaction Bases - 108
5. The Active Principle of the Pituitary Body - 108
6. Vitamine, Oryzanin, Torulin - 1 1 1
7. Sepsine - - 113
8. Secretine - 114
CHAPTER VIII. (APPENDIX.)
PRACTICAL CHEMICAL METHODS AND DETAILS - 116
A. General Methods for the Separation and Isolation of Bases - 1 16
B. Special Methods. Properties of Individual Bases and of their
Salts - - 124
Bases of Chapter I. - 124
Bases of Chapter II. - 135
Bases of Chapter III. - 141
Bases of Chapter IV. - - 150
Bases of Chapter V. - 1 5 7
BIBLIOGRAPHY - 167
INDEX -213
INTRODUCTION AND SCOPE.
THE substances described in this monograph do not constitute a
homogeneous group, like the proteins or carbohydrates, and the choice
of a title was therefore difficult. Many are derived in various ways
from the amino-acids of protein, a few are constituents of phosphatides ;
some are of bio-chemical interest on account of their wide distribution
in animals and in plants, others are important because of their phy-
siological action.
It is common to nearly all the simpler natural bases, however, that
they are insoluble in ether and chloroform and readily soluble in
water, so that their isolation is generally more difficult than that of
the complex vegetable alkaloids, which can be extracted by making
the aqueous solutions of their salts alkaline and then shaking with a
solvent immiscible with water. The separation of the simpler bases
from each other and from non-basic substances like peptones must be
carried out by means of suitable precipitants and crystalline derivatives.
The special technique required for this purpose constitutes the chief
bond between the bases with which we are here concerned. This
technique was first elaborated in a systematic manner by Brieger, who
employed mercuric chloride in the isolation of putrefaction bases.
The introduction of phosphotungstic acid, by Drechsel, as a general
precipitant for basic substances and its use for preparative purposes
marked a great advance ; later Kossel added the silver method for the
separation of imino-bases, such as arginine and histidine. Since then
the details of technique have been chiefly elaborated in three centres.
Schulze at Zurich, in a long series of researches on plant bases,
discovered phenylalanine and arginine and more lately extended our
knowledge of betaines. Kutscher and his pupils, in Germany, have
isolated bases from a variety of sources, and Gulewitsch, at Moscow,
has studied exhaustively the bases in meat-extract.
The history of the simpler natural bases has been greatly influenced
by the need of special methods for their isolation. Another influence,
adverse to their study, was the presence of alkaloids in drugs and
stimulants, which directed attention to these complex bases having
obvious physiological actions rather than to simpler bases of more
I
2 THE SIMPLER NATURAL BASES
general biological importance. Thus the basic nature of morphine was
recognised as long ago as 1806, and in 1820 quite half a dozen of the
most important vegetable alkaloids were known, but our knowledge of
animal bases is of a much later date. Pettenkofer prepared creatinine
from urine in 1844 and Strecker first obtained choline from pig's bile
in 1849, but for a long time hardly any other animal bases were known,
and betaine, which is now known to occur in many plants and some
animals, was not discovered until 1863. The more volatile amines,
trimethylamine and amylamine, were obtained as putrefaction pro-
ducts in 1855 and 1857 respectively, and about the year 1866 it
became generally recognised that bases are formed in putrefaction, but
for a long time these bases were regarded as similar to the vegetable
alkaloids, and their isolation was attempted by similar methods. For
this there were two reasons. In the first place the poisonous properties
of putrid material were considered analogous to those of plant alkaloids,
and secondly the medico-legal examination of corpses in murder trials
revealed the presence of bases (called ptomaines by Selmi) which gave
reactions like those of coniine, nicotine, atropine, etc. In no single
instance did these early investigations result in the preparation of a
pure substance, so that they do not concern us further.
The chemistry of putrefaction bases may be said to begin in 1876
when Nencki correctly analysed a base C8HnN, obtained from putrid
gelatin ; he afterwards identified it as phenylethylamine. It seems
highly probable that this amine, perhaps mixed with diamines, was
the " animal coniine " of earlier investigators.
The next great advance was due to Brieger who, breaking away
from the methods used for plant alkaloids, and relying chiefly on
mercuric chloride, platinic chloride and similar reagents, discovered
putrescine, cadaverine, and many putrefaction bases which had been
overlooked by his predecessors. Gradually it became evident that pto-
maines, or putrefaction bases, are the products of bacterial action on
protein and phosphatides, and since then our knowledge of these bases
has become more and more intimately associated with what we know
of the amino-acids from which protein is built up. Two examples of
this association may be given. The discovery of phenylalanine by
Schulze and Barbieri in 1881 enabled Nencki to surmise the constitu-
tion of his base C8HnN referred to above ; it is derived from the amino-
acid by loss of carbon dioxide. Later Ellinger proved that Brieger's
diamines were similarly derived from the amino-acids ornithine and
lysine.
Since then the amines corresponding to nearly all the known
INTRODUCTION AND SCOPE 3
amino-acids have been found to occur as putrefaction products. These
amines are described in Chapter I and include substances with inter-
esting physiological actions. Another group of bases, likewise derived
from protein, is described in Chapter II. The members of this group
still retain a carboxyl group of the amino-acid, so that they are but
feebly basic, and without marked physiological action. They include
the <y-amino-acids, formed by putrefaction, and urocanic and kynurenic
acids, two substances occurring in dog's urine and derived from
histidine and tryptophane respectively. A third group of simple bases
related to the amino-acids of protein is dealt with in Chapter III,
namely that of the betaines, derived from amino-acids by methylation.
Several new examples of this class have been discovered during the
last few years, both in animals and in plants.
The first three chapters deal therefore with bases which are de-
rived by slight modifications from the constituent units of protein.
These modifications are irreversible. As long as protein is not
broken down beyond the amino-acid stage, its fragments are still
available for synthesis. Thus when an amino-acid is set free in the
germinating seed by the action of proteoclastic enzymes, it may
re-enter a protein molecule in a cell of the growing point. If the de-
gradation of protein proceeds farther, if the amino-acid is de-aminized
or decarboxylated and also probably if it is methylated, it is no
longer available for protein synthesis in animals and in the higher
plants ; it no longer constitutes a food, except for bacteria and some
fungi. To these degradation products of protein which have passed
out of the metabolic circulation, Ackermann and Kutscher [1910, 2]
have applied the term aporrhegmata. They include under this denomina-
tion not only bases, but also acidic products, such as succinic acid,
which is derived from aspartic acid by the loss of an amino-group
during putrefaction.
In addition to the proteins, lecithin and other phosphatides con-
stitute a source of bases in the organism. Here there is less variety,
for only two primary fission products of basic character are known with
certainty, choline and ammo-ethyl alcohol. Neurine and trimethyl-
amine are secondary decomposition products of choline and there are
also a few closely related bases, like muscarine. All these bases are
described in Chapter IV (with the exception of trimethylamine, which
is included in Chapter I as it may also be formed from sources other
than choline).
Of the bases dealt with in the first four chapters some are found in
animals, some in plants, and many in both ; the remaining chapters
4 THE SIMPLER NATURAL BASES
are devoted entirely to animal bases, Chapter V dealing with creatine,
creatinine, and other guanidine derivatives, and Chapter VI with
adrenaline, one of the most interesting of simple bases.
Twenty years ago it could hardly have been imagined that the
suprarenal gland constantly secretes into the blood minute quantities
of a base having an intense physiological action, and that this base
has a simple chemical constitution and can be synthesised. At first
adrenaline stood entirely by itself; later some of the putrefactive
amines of Chapter I were found to have considerable physiological
activity, and one of them, p-hydroxy-phenyl-ethylamine, which
resembles adrenaline chemically, was found to have an essentially
similar, although weaker, action on the animal organism. There are
moreover indications that other internal secretions owe their activity to
bases of comparatively small molecular weight. This appears to be
the case with the highly active principle of the pituitary body which
is possibly a histidine derivative, and shows some analogies to fi-
iminazolyl-ethylamine described in Chapter I. Unfortunately hardly
anything is known with regard to the chemistry of the pituitary active
principle, so that it is only included in Chapter VII (bases of unknown
constitution) on account of its physiological importance. Secretine,
the substance which when introduced into the blood stream, causes
secretion of pancreatic juice, is probably also a base — and like the active
principles of the adrenal gland and of the pituitary body, it is moder-
ately stable in boiling aqueous solution.
The case of the bacterial toxins and antitoxins, which are rapidly
destroyed below the temperature of boiling water, is very different.
After working on the products of putrefactive bacteria, Brieger in-
vestigated the bases produced in cultures of pathogenic organisms,
such as the typhoid and the tetanus bacillus, but the simple bases
which he obtained could not be regarded as the principal cause of
disease, and his further work on tetanus toxin showed this substance
to be extremely active and apparently also extremely complex. We
may say " apparently " for the following reason. When a minute
quantity of an active principle accompanies large quantities of proteins
and other colloids it may remain adsorbed on these in such a way as
to make a separation impossible, even when the active principle has a
comparatively small molecular weight. The difficulties are particularly
great when the active principle is very soluble in water but hardly at
all in alcohol, as is often the case with bases of the animal body. A
good deal of optimism is required for the belief that our present
methods will ever suffice for the isolation of bacterial toxins in a state
INTRODUCTION AND SCOPE 5
of purity, and here we are likely to learn more from colloidal than
from organic chemistry. Recent work on anaphylaxis seems to indicate
that this phenomenon is primarily concerned with a basic part of the
protein molecule which is resolved by hydrolysis into diamino-acids.
We are almost as ignorant of the more interesting toxic products
of putrefaction as we are of pathogenic toxins. Very little is known
about the poisonous substances in food, popularly called ptomaines.
Many cases of so-called ptomaine poisoning are in reality bacterial
infections, but others are purely chemical intoxications. Perhaps the
best known of these is due to Bacillus botulinus which, without obvious
signs of putrefaction, produces in meat or even in vegetable nitrogenous
substances (beans) an excessively poisonous toxin, readily destroyed
at 80° and capable of yielding an antitoxin (Van Ermengem [1907,
1912; Ch. I]; Ornstein [1913; Ch. I]). The poisonous properties
occasionally exhibited by boiled mussels are on the other hand due to
a thermostable base [Brieger, 1886, I, p. 65 ; Ch. I]. The physi-
ological actions of the most active amines described in Chapter I do
not account satisfactorily for such intoxications ; other substances must
be present, and one of these is sepsine, a base of simple constitution
obtained by Faust from putrid yeast. The experimental difficulties
of the subject are illustrated by the fact that 100 kilos, of yeast did not
yield enough of the pure substance for quite satisfactory analysis.
Against this difficulty, that many of the bases described in the follow-
ing chapters are only obtainable in minute quantity from natural
sources, we may, however, set the advantage of a simple constitution,
so that when the latter has once been fully established, a synthesis on
a large scale may be possible, which in some cases has greatly increased
our knowledge of the chemical and physiological properties of the base.
Without an exact knowledge of the properties, the identification is
often very difficult and for this reason detailed descriptions have as far
as possible been given in the appendix. Many bases which have been
insufficiently characterised have not been mentioned, except where it
was possible to suggest identity with better known ones.
In conclusion we may discuss the meaning of the following terms.
Base. — Many substances of physiological importance are at the
same time acids and bases ; those in which the basic character predomin-
ates have been included in this monograph ; others, like the tf-amino-
acids of protein are not generally regarded as bases, although glycine, for
instance, yields a hydrochloride. The predominance of the basic char-
acter may be deduced from a comparison of the (basic and acidic) affinity
constants (see the beginning of Chapter II). For our purposes a better
6 THE SIMPLER NATURAL BASES
practical definition is to describe a base as a substance which is pre-
cipitated by phosphotungstic acid. Adopting this criterion we consider
creatinine to be a base but creatine not.
Alkaloid. — Some writers have used this term to include all natural
bases, but the objections to this are evident from what has been said
above, and the word is best restricted to complex vegetable heterocyclic
bases derived from pyridine, quinoline, etc.1 There is no doubt as to
what is generally meant by an alkaloid, but nevertheless a rigid definition
is almost impossible. On the one hand narceine, for instance, is a
typical alkaloid from opium, but the nitrogen atom does not form part
of a ring ; narceine is an amine. On the other hand histidine and its
derivatives are not classed as alkaloids, although they contain the
heterocyclic glyoxaline ring, which is also present in pilocarpine. The
latter substance is an undoubted alkaloid In a few cases the inclusion
of bases in this monograph is arbitrary ; thus hordenine, which is usually
called an alkaloid, has been included on account of its relationship to
tyrosine ; ephedrine, which is isomeric with hordenine, has been ex-
cluded. All betaines have been included, for no typical alkaloid shows
a betaine structure. One further point should be noted. The typical
alkaloids are generally found only in one or a few closely related
species, but the simpler natural bases, in accordance with their close
connection with proteins and phosphatides, have generally a much
wider distribution.
Ptomaine was originally applied by Selmi to bases from corpses
and afterwards became identical with putrefaction base (Brieger).
Some writers have restricted the term to poisonous bases. Lately it
has fallen into disuse.
Leucomaine was a term used by Gautier for animal bases such as
creatinine, which are not formed by putrefaction ; this term is now
quite obsolete.
Toxins are poisonous bacterial products which when injected cause
the production of anti-bodies, neutralising their poisonous properties ;
an example is diphtheria toxin. Gautier has applied the word, in a
different sense, to simple poisonous putrefaction bases.
1 Winterstein and Trier define plant alkaloids as nitrogenous substances which can no
longer be utilised for building up protein. Thus they would call betaine an alkaloid.
CHAPTER I.
AMINES DERIVED FROM PROTEIN.
The Putrefactive Decomposition of Amino-acids.
BOTH animals and plants decompose proteins into their constituent
amino-acids ; the hydrolysis by trypsin and by erepsin in animals is
similar to the formation of amino-acids in germinating seeds, which
has been studied especially by Schulze and his pupils. The hydrolysis
of proteins into their constituent amino-acids is also the first stage of
putrefaction, but bacteria (and other fungi) are peculiar in being able
to break down the amino-acids themselves into bases and acids which
in general have not been demonstrated as products of the metabolism
of animals and the higher plants.
This degradation may take place in two ways : either an amino-
group may be eliminated (deaminization) or a carboxyl-group may be
removed (decarboxylation) ; various modifications and combinations of
these two processes are possible. Little is known about the conditions
determining which process takes place; generally the two go on
simultaneously and deaminization preponderates. Ackermann who
has carried out a number of experiments on the bacterial decarboxy-
lation of pure amino-acids,- finds that this process is favoured by the
addition of peptone which serves as a source of nitrogen and in this
way lessens deaminization. An organism which decarboxylates histi-
dine has been isolated by Mellanby and Twort [1912]. Berthelot
and Bertrand [1912, 1,2; 1913, 1,2; Bertrand and Berthelot, 1913]
have described a similar organism from the human intestine, Bacillus
aminophilus intestinalis, which decarboxylates histidine, tyrosine, tryp-
tophane, etc.
The various amines dealt with in the present chapter are all deriv-
able from monobasic amino-acids by decarboxylation, and it is therefore
with this process that we are more particularly concerned. Decar-
boxylation may take place by the simple removal of carbon dioxide :
R R
CHNH3 = CHa . NHa + CO2.
I co° | H
L», -i.
8 THE SIMPLER NATURAL BASES
or the carboxyl-group may be eliminated as formic acid, in which case
reduction must take place : —
R R
CHNH3 + H = CH2 . NHa + H . COOH.
| COOH H |
Neubauer [1911] considers that decarboxylation generally takes
place in both these ways, since carbon dioxide and formic acid are
among the regular products of putrefaction. In either case a primary
amine results.
The same process, applied to dibasic monamino-acids, results in
the formation of oi-amino-acids, which are feebly basic putrefaction
products and are described in the next chapter. a>- Ammo-acids are also
formed by the deaminization of diamino-acids ; the deaminization of
monamino-acid yields non-nitrogenous acids such as isocaproic (from
leucine) and succinic (from aspartic acid). Deaminization is accom-
panied by reduction, since hydroxy-acids and un saturated acids ap-
parently do not occur in putrefaction : —
R R
CHNH2 + 2H = CH2 + NH3.
COOH COOH
By a combination of the two processes of decarboxylation and
deaminization, methane may be formed from glycine and «-butyric
acid from glutamic acid (Neuberg and Rosenberg [1907]). A putre-
factive process involving only reduction is the conversion of proline into
8-aminovaleric acid.
The importance of reduction in the above bacterial actions is ex-
pressed by the fact that they chiefly take place under anaerobic con-
ditions. Bienstock [1899, 1901], one of the chief workers in this field
on the bacteriological side, concludes that putrefaction, in the ordinary
sense, cannot take place without an obligate anaerobe, such as Bacillus
putrificus. B. coli hinders the action of B. putrificus and B. tetani has
no action on fibrin. Rettger [1906, 1907 ; Rettger and Newell, 1912]
shares the view that putrefaction is the work of strict anaerobes.
The access of oxygen induces further changes ; p-hydroxy-phenyl-
propionic acid (formed by the deaminization of tyrosine) is oxidised,
according to Baumann and Nencki, to p-hydroxy-phenyl-acetic acid,
which is successively converted into p-cresol and phenol, and simi-
larly indole-propionic acid (from tryptophane) yields indole-acetic acid,
skatole, and indole. Oxidation also accounts for the shortening of
AMINES DERIVED FROM PROTEIN 9
the carbon chain in the production of succinic acid from glutamic acid
by putrefaction.
Some putrefaction bases are formed from substances other than
proteins ; thus lecithin is broken down to choline, neurine, trimethyl-
amine, monomethylamine, and ammonia; creatine yields monomethyl-
guanidine and perhaps also dimethylguanidine ; the trimethylamine of
stale urine is derived from more complex betaines ; purine and pyrimid-
ine bases probably also contribute to the formation of putrefaction bases.
When an entire tissue or organ, and to a less extent when a single
protein is putrefied, as in the experiments of Nencki, Gautier, Brieger,
Salkowski, Emmerling, Barger and Walpole, and the earlier experi-
ments of Ackermann, a complex mixture of bases is obtained from
various parent substances. A better insight into the chemistry of
putrefaction is possible when a simple substance, such as a single
amino-acid, is subjected to bacterial action. This method depends on
a knowledge of the constituents of protein, and was first applied to the
study of bases by Ellinger, who showed that putrescine and cadaverine
are derived from ornithine and lysine respectively. Further work in
this direction has been carried out principally by Ackermann and by
Neuberg. (The products of the action of bacteria on indole-propionic
acid (Nencki) and of yeast on proteins (F. Ehrlich) are not bases, and
they are therefore not included in this monograph.) It is generally
much more difficult to grow bacteria in a solution of a pure amino-acid
than on protein, and Ackermann therefore adds 0^25 per cent. Witte pep-
tone to the solution, together with 0*5 percent, glucose and a few drops
of sodium phosphate and magnesium sulphate ; calcium carbonate is
sometimes added to prevent the solution becoming acid, but a faint
alkaline reaction is secured more certainly by adding sodium carbonate
from time to time. Although Neuberg [1911, l] has pointed out the
theoretical objections to the addition of peptone he yet agrees with
Ackermann that in many cases this addition is desirable. For the
decomposition of histidine Mellanby and Twort [1912] used a culture
medium containing only ammonium tartrate and inorganic salts (see
p. 133). A similar medium was used by Berthelot and Bertrand
[1912, I].
Of late years nearly all the putrefaction products, which might be
expected to result from the known amino-acids, have been obtained
by bacterial action. Exceptions are e-amino-caproic acid which might
be formed from lysine, guanidino-valeric acid (from arginine), pyrroli-
dine (from proline), oxypyrrolidine (from oxyproline) and the amines
from cystine and serine.
10 THE SIMPLER NATURAL BASES
The decarboxylation of amino-acids is not necessarily accompanied
by any obvious sign of bacterial action such as putrefactive odour ;
some of these amines occur in cheese and they have repeatedly been
obtained in fermentation experiments supposed to be sterile (Langstein,
Emerson, Lawrow ; see the section on putrescine and cadaverine).
The difficulties of ensuring sterility, particularly in autolysis, have
often been underestimated and have been emphasised by Schumm
[1905-6], Rothmann [1908], Kikkoji [1909], Salkowski [1909], Ohta
[1910], Harden and Maclean [1911], Beker [1913]. Chloroform
should not be used in conjunction with toluene, which dissolves the
chloroform from the aqueous layer. It is best, according to Schumm
and Kikkoji, to use water saturated with chloroform, or chloroform in
excess and to ensure continued saturation by means of stoppered bottles
and frequent shaking. Sterility tests should be made by smear.
In the absence of bacteria, decarboxylation of amino-acids does not
occur; at least the corresponding primary amines are not found.
(Kutscher and Lohmann [1905], Schumm [1905-6], Bissegger and
Stegmann [1908], Schulze [1906], Kiesel [1911].) The occur-
rence of methylated bases such as tetramethyl putrescine and hordenine
in the higher plants perhaps implies the intermediate formation of
primary amines. Apart from putrefaction, putrescine and cadaverine
occur in cystinuric urine, agmatine in herring spawn and p-hydroxy-
phenyl-ethylamine in the salivary gland of Cephalopoda. It has further
been established that fresh fungi may contain amines resulting from the
decarboxylation of amino-acids or at any rate these amines are formed
by autolysis independently of bacterial action. The close relationship
between the fungi proper and bacteria makes this less surprising.
Ergot, which has been examined more thoroughly than any other
fungus, contains p-hydroxy-phenyl-ethylamine, /3-iminazolyl-ethyl-
amine, putrescine, cadaverine, agmatine, and probably isoamylamine, and
owes much of its physiological action to the first two of these bases. It
is almost certain that they are to some extent present in fresh ergot, but
the amount is increased after death, probably by autolysis. Reuter
[1912] recently found putrescine in fresh specimens of Boletus edulis
and when this fungus was autolysed under sterile conditions, isoamyl-
amine, phenyl-ethylamine, probably p-hydroxy-phenyl-ethylamine and
possibly iminazolyl-ethylamine were formed in addition. Schenck
[1905, I] had previously obtained putrescine from autolysed yeast.
Reuter's experiments are of particular interest ; sterility tests showed
that bacteria were absent, and he concludes that fungi possess ferments
capable of decarboxylating amino-acids.
AMINES DERIVED FROM PROTEIN it
Methylamine, Ethylamine, Dimethylamine.
Methylamine occurs according to Trier [1912, 3; p. 8] in species of
Mercurialis and the root of Acorus Calamus and has been frequently
met with as a product of bacterial action (see P. Rona, Biochemisches
Handlexicon, Band IV, p. 801). It is perhaps formed from glycine, by
decarboxylation, but so far it has not been possible to demonstrate this
experimentally. The source of methylamine is in most cases more
probably trimethylamine (from choline). Thus Hasebroek [1887]
obtained this amine along with ammonia by the anaerobic putrefaction
of choline, and Morner [1896] found amines present in a peculiar
Swedish food (" surfisk "). This fish is pickled with a little salt and
allowed to ferment anaerobically ; it probably contains monomethyl-
amine, and certainly dimethylamine and choline, but not putrescine or
cadaverine. Ackermann and Schiitze [1910, 1911] also found that a
little methylamine, together with trimethylamine, is formed by the action
of Bacterium prodigiosum on choline. Emmerling [1897] obtained
mono- and trimethylamine by the action of Streptococci on fibrin, but
here also the amines appear to be derived from admixed lecithin.
Ethylamine was said more than fifty years ago to be produced in
the putrefaction of yeast and of wheat flour, but these observations re-
quire confirmation. It might result by the decarboxylation of alanine,
from which it is indeed formed on destructive distillation.
Dimethylamine was stated by Bocklisch [1885] and by Morner
[1896] to occur in putrid fish, and by Ehrenberg [1887] in cultures
from a bacillus isolated from poisonous sausages. In the latter case
at least a confusion with putrescine was not unlikely, since the platini-
chlorides of the two bases have nearly the same composition. If
dimethylamine is formed at all it would be most probably derived from
choline and trimethylamine, although it could also result from the
decarboxylation of sarcosine (from creatine).
Trimethylamine, N(CH3)3.
Trimethylamine occurs in the leaves of Chenopodium Vulvaria (the
Stinking Goosefoot) where it is readily detected by the odour on bruis-
ing the leaves ; it is also present in hawthorn flowers (Cratagus
Oxyacantha] and in ergot. Unlike the other amines dealt with in
this chapter, trimethylamine is not formed from an amino-acid, but is
a decomposition product of choline and allied quaternary bases ; it is
therefore of common occurrence in putrefaction. Thus it is present in
herring brine, the first natural source to be discovered by Winckler in
12 THE SIMPLER NATURAL BASES
1855. On an industrial scale it is formed by the destructive distilla-
tion of beet sugar molasses ; here the parent substance is betaine.
Examples of the production of trimethylamine by pure cultures
are the action of Proteus vulgaris on wheat gluten and on meat, of
Bacillus liquefaciens on commercial gelatin and of Bacteriumprodigiosum
on choline and on lecithin. Ackermann and Schutze [1910, 1911]
found that the last-named organism does not produce trimethylamine
from betaine, and that B. vulgatus does not decompose choline.
The alleged occurrence of trimethylamine in urine has been the
subject of several investigations. Long ago Dessaignes [1856] ob-
tained it by distillation of urine with caustic soda (37 grm. of the free
base from 65 litres of human urine). He, however, left open the
question whether trimethylamine is present as such or is formed by the
decomposition of some other compound by the alkali. This question
was likewise left unanswered by de Filippi [1906] who worked out
a process for the estimation of urinary trimethylamine (see appendix).
Takeda [1909] used magnesium oxide instead of caustic soda,
and distilled under reduced pressure ; he found no trimethylamine in
the urine of horses and of dogs and only doubtful traces in human
urine ; it is however formed in putrefaction. Kinoshita [1910, l], using
Herzig and Meyer's method for the estimation of N-methyl groups,
found only traces, and Erdmann [1910] has also arrived at the con-
clusion that " fresh normal urine does not contain trimethylamine ".
According to Kutscher the trimethylamine in urine is formed from
such bases as novaine and reducto-novaine.
Isobutylamine, 3cH . CH2. NH
This base was obtained by the putrefaction of racemic a-amino-
isovaleric acid (d.l. valine) by Neuberg and Karczag [1909]. A
solution of 10 grams of the amino-acid in 450 c.c. of water, with a
little KC1, Na2HPO4 and MgSO4 was rendered alkaline with sodium
carbonate and yielded after inoculation and four weeks' incubation at
37° 0-424 grm. of a platinichloride (C4HnN)2H2PtCl6, mp. 226-227°,
in all probability that of isobutylamine.
A butylamine has also been obtained by Gautier from cod liver oil
prepared by the old putrefactive process.
In Fagara xanthoxyloides isobutylamine occurs in combination with
piperonylacrylic acid as an amide, fagaramide (Thorns and Thumen,
Isobutylamine is the lowest amine causing any appreciable rise of
blood pressure when injected intravenously.
AMINES DERIVED FROM PROTEIN 13
Isoamylamine, > CH . CH2 . CH2 . NH2.
CH/
An amylamine has been obtained from putrid yeast (Muller
[1857]), from cod liver oil (Gautier and Mourgues [1888]), from
putrid horse meat (Barger and Walpole [1909, i]), putrid
placenta (Rosenheim [1909]), from Boletus edulis on sterile autolysis
(Reuter [1912]), and probably from fresh ergot (Barger and Dale
[1909]).
In all these cases isoamylamine (derived from leucine) was pro-
bably mixed with the isomeride 2-methylamino-butane (derived from
isoleucine), and possibly with normal amylamine. from norleucine. Iso-
amylamine is further formed from leucine on rapid heating, and in the
dry distillation of bones and horn. Ciamician and Ravenna (quoted
by Trier [1912, 3]) found isoamylamine in tobacco. The oxalate of
isoamylamine was obtained in an impure form from putrid meat by
Abelous, Ribaut, Soulie and Toujan [1906, I, 2]; Abelous and Ribaut
[1908] deduced the erroneous formula CGHUON for the base, and
were the first to observe its power of raising the blood pressure when
injected intravenously. Extracts of putrid meat were shown by Barger
and Walpole to owe their pressor action principally to isoamylamine
and to p-hydroxy-phenyl-ethylamine.
Pyrrolidine, C4H9N.
This base should result from the amino-acid proline by decarboxy-
lation, but has never been isolated as a putrefaction product, probably
because putrefactive bacteria rupture the pyrrolidine ring by reduction
(see Chapter V).
Pyrrolidine has, however, been isolated in minute quantity from
carrot leaves (Daucus Carota] by Pictet and Court [1907]- They
also found pyrrolidine and N-methylpyrroline in minute quantities in
tobacco, and have termed these bases proto-alkaloids.
Amino-ethyl Disulphide, S2(CH2 . CH2 . NH2)2.
Neuberg and Ascher [1907] obtained this amine in small quantity
by the dry distillation of cystine, from which it is derived by loss of
carbon dioxide. 'Ite pi crate melts at 197°. The amine has no pro-
nounced physiological activity, and has so far not been obtained by
bacterial action.
I4 THE SIMPLER NATURAL BASES
Putrescine and Cadaverine, C4H12N2 and C5HUN2.
These two homologous diamines have similar properties and
generally accompany each other, so that they may be most conveniently
considered together. They were discovered by Brieger [1885, 1, 2] by
his new method of investigating putrefaction bases ; cadaverine was
soon afterwards shown by Ladenburg [1886] to be identical with
the pentamethylene-diamine previously obtained by reduction of tri-
methylene dicyanide, and later Udranszky and Baumann [1888, 2]
proved the identity of putrescine with tetramethylene-diamine.
Putrescine and cadaverine are among the commonest of all putre-
faction bases. They probably escaped the notice of earlier investigators
on account of their sparing solubility in ether and in chloroform, but
Brieger obtained them repeatedly from various sources and they have
been isolated many times since. The possibility of the formation of
cadaverine from lysine by loss of CO2 was already considered by
Udranszky and Baumann and the origin of both amines was definitely
established by Ellinger [1900] who obtained putrescine by the
action of putrefactive bacteria on ornithine :
NH2 . CH2 . CH2 . CH2 . CH(NH2) . COOH = NH2 . CH2 . CH2 . CH2 . CH2 . NH2 + CO2 ;
and similarly cadaverine from lysine :
NH2 . CH2 . CH2 . CH2 . CH2 . CH(NH2) . COOH =
NH2 . CH2 . CH2 . CH2 . CH2 . CH2 . NH2 + CO2.
These important results furnished the first examples of the bacterial
decarboxylation of amino-acids. With access of air Ellinger ob-
tained a 1 2 per cent, yield of putrescine and under anaerobic conditions
a 50-60 per cent, yield (three days at 37°) ; with cadaverine the yield
was 36 per cent. Ackermann [1909, I], who more recently repeated
Ellinger's experiments, was at first unable to obtain putrescine and
cadaverine from the pure amino-acids but succeeded in the case of
the products of the hydrolysis of caseinogen by acids. He showed
that putrescine but not cadaverine is formed in the putrefaction of
gliadin [1909, 2], which does not contain lysine, and ultimately he
[1910, 3] found that the addition of 0*25 per cent. Witte peptone and
0-5 per cent, glucose to the culture medium greatly facilitated decar-
boxylation. In the earlier experiments only traces of inorganic salts
had been added. When once formed, cadaverine and putrescine are
apparently very resistant to the action of micro-organisms, for Brieger
and others isolated the bases in considerable quantity after putrefac-
tion had been going on for months.
Apart from such bacterial formation of putrescine and cadaverine,
AMINES DERIVED FROM PROTEIN 15
both bases have been isolated from ergot by Rielander [1908] and
putrescine has been found in autolysed yeast by Schenck [1905, i], in
fresh specimens of Boletus edulis by Reuter [1912] and in Datura
(a Phanerogam) by Ciamician and Ravenna (Trier [1912, 3]).
The diamines further occur in some cases of cystinuria (Udranszky
and Baumann [1889], Cammidge and Garrod [1900], Loewy and
Neuberg [1904], Garrod and Hurtley [1906]; the last-named paper
should be consulted for the literature of other cases). In some
cases of cystinuria the diamines are only excreted occasionally, or not
at all, in Loewy and Neuberg's case only when arginine and lysine
were given by the mouth. On the other hand the diamines do not
pass into the urine when given by the mouth to a normal animal
(Udranszky and Baumann [1890]). Garrod's impression [1909] is
"that the likelihood that diamines will be detected in any given
specimen of cystin urine is comparatively small, but that if in any case
the examination be continued over sufficiently long periods they are
likely to be found eventually". Lately Ackermann and Kutscher
[1911] have found a minute quantity of lysine in cystinuric urine.
The excretion of diamines in the urine indicates a peculiarity of meta-
bolism, probably not intimately connected with the excretion of
cystine.
Cadaverine was also found by Roos [1892] in the urine in two
cases of malaria, but this may have been the result of bacterial action.
Other cases of the alleged fermentative formation of the two diamines
may safely be ascribed to this cause. Thus Lawrow [1901] ob-
tained both bases in the autolysis of pig's stomach, Langstein
[1901, 1902] isolated cadaverine after digesting egg white with pepsin
for more than a year, Steyrer (referred to by Emerson [1901]) ob-
tained the same base from a pancreatic digest and Werigo [1892]
from pancreas macerated with chloroform water. In some of Werigo's
experiments incipient putrefaction was indeed noticed, and we may
well attach more weight to the experiments of Kutscher and Lohmann
[1905] and of Schumm [1905-6], who could not isolate either
putrescine or cadaverine when pancreas was autolysed under sterile
conditions, and to those of Bissegger and Stegmann [1908] who
likewise could not obtain the diamines by the tryptic or peptic diges-
tion of caseinogen. Schulze showed [1906] that putrescine and
cadaverine, unlike their parent substances, are absent from germinating
seedlings.
Among the cases where putrescine and cadaverine are formed
by bacterial action we may further mention that both bases have
been obtained from putrid Soy beans (Yoshimura [1910]) and from
16 THE SIMPLER NATURAL BASES
Emmenthaler cheese (Winterstein and Thony [1902]). Van Slyke
and Hart [1903] found a little putrescine in ordinary Cheddar
cheese, but none in a sterile chloroform cheese.
According to Garcia [1892-3, 2, 3] the -%\, Tof the diamines from
putrid horse meat and from pancreas is dimimsheol by the addition of
carbohydrates (compare p. 25); four-fifths is already formed in the
first twenty-four hours of incubation and the maximum is reached
after three days. Once formed, putrescine and cadaverine appear to be
very resistant to bacterial action. Gulewitsch [ 1 894] obtained cadaverine
from horse meat kept four months at 15°.
Hyoscyamus muticus contains tetramethyl-putrescine (see appendix).
Agmatine, C5HUN4.
Agmatine, or guanidino-butylamine, was obtained by Kossel
[1910, i] from herring spawn after heating with dilute sulphuric acid
(5 per cent, by volume) in an autoclave at 4 atmospheres pressure.
The base differs from arginine by CO.2, the chief amino-acid in herring
spawn, so that it may be considered as being derived from arginine by
decarboxylation : —
NH2 . C( : NH) . NH . CH2 . CH2 . CH2 . CH2 . NH3 agmatine.
NH3 . C( : NH) . NH . CH3 . CH2 . CH2 . CH(NH3) . COOH arginine.
Agmatine has also been isolated from ergot by Engeland and Kutscher
[1910, I, 2] who obtained from their base on oxidation guanidine and
guanidino-butyric acid,
NH2 . C( : NH) . NH . CH2 . CH2 . CHa . COOH.
Kossel [1910, 2] synthesised agmatine from cyanamide and tetra-
methylene diamine,
NH2 . CN + NH2(CH2)4NH3=NH2 . C( : NH) . NH . (CHQ)4 . NH2 .
Phenyl-ethylamine, C6H5 . CH2 . CH2 . NH2.
/3-Phenyl-ethylamine is of some interest, since it was the first putre-
faction base of which the composition was determined. Nencki
[1876] obtained the base from a mixture of 200 grams of ox pancreas
and 600 grams of gelatin dissolved in 10 litres of water, which was
putrefied at 40° for five days.
Nencki, like Selmi and other early investigators of putrefaction
bases, was most impressed by their analogy to vegetable alkaloids such
as coniine and nicotine, and he at first considered his base to be a pyri-
dine homologue, dimethylpyridine or collidine. Finding later that his
hydrochloride, unlike that of collidine, yielded on destructive distilla-
AMINES DERIVED FROM PROTEIN 17
tion a substance resembling xylene in odour and other properties, he
concluded [1882] that the base obtained from gelatin was an aro-
matic amine, probably a-phenyl-ethylamine, C6H5. CH(NH2). CH3.
Still later he regarded enylalanine, which Schulze and Barbieri had
discovered in etiolated lupin seedlings, as the parent substance of his
putrefaction base, which he [1889] therefore considered to be /3-
phenyl-ethylamine, formed according to the equation : —
C6H5 . CHa . CH(NH2) . COOH = C6H6 . CHa . CH2 . NH2 + CO2.
Nencki was thus also the first to invoke the decarboxylation of an
amino-acid in explanation of the origin of a putrefaction base.
Nencki's "collidine" was further obtained from putrefied egg white
by his1 pupil Jeanneret [1877]. The identity of the base from
putrid gelatin with /3-phenyl-ethylamine was first rendered absolutely
certain by Spiro [1901]. Putrefaction bases of the formula C8HUN
or of a similar formula, with properties somewhat resembling those
of phenyl-ethylamine, have at various times been obtained by other
investigators and one is tempted to regard all these bases as
identical with that first isolated by Nencki. In some cases this is
indeed almost or quite certain. Thus by the action of a Strepto-
coccus on fibrin, Emmerling [1897] obtained a base of the formula
C8HnN of which the picrate melted at the same temperature as
that of synthetic /3-phenyl-ethylamine ; the only discrepancy is that
the platinichloride is described as readily soluble in water. Similarly
a base obtained from putrid horse meat by Barger and Walpole
[1909, 1], and having the boiling point and physiological properties of
$-phenyl-ethylamine, was doubtless identical with this amine.
It is much more difficult to draw the same conclusion with regard
to certain bases described as pyridine derivatives and isolated by
Gautier and Etard [1882, 1883] and by Oechsnerde Coninck,[ 1886-91].
The former investigators obtained from putrid mackerel a base, boiling
at 210°, d0 = 1*0296, which was analysed as platinichloride. The
formula deduced was C8H13N and the base was named dihydrocollidine,
but the analyses are in better, although not good, agreement with the
formula C8HUN. No evidence of its being a pyridine derivative was
adduced and Nencki [1882] at first regarded Gautier and Etard's
hydrocollidine as identical with phenyl-ethylamine, but subsequently
[1889], after a visit to Gautier, he gave up this view. Oechsner de
Coninck obtained a base of the formula C8HnN from putrid cuttle-
fish ; on oxidation it yielded nicotinic acid ; it was examined much
more closely than Gautier and Etard's " hydrocollidine " and in this
2
1 8 THE SIMPLER NATURAL BASES
case at least, a confusion with phenyl-ethylamine seems completely ex-
cluded. Compare further the section on p. 48.
Phenyl-ethylamine does not accompany phenyl-alanine in seedlings
(Schulze [1906]), but with regard to the higher plants it should be
mentioned that Le Prince [1907] has isolated a volatile base C8HnN
from the European mistletoe (Viscum album} and that Crawford
[1911] attributes the pressor action of the U.S.P. extract of the
American mistletoe (Phoradendron flavescens) to the presence of abase,
C7HUN or C8HUN, which he thinks is perhaps identical with phenyl-
ethylamine. This base requires further investigation ; the presence of
phenyl-ethylamine may possibly depend on the fact that the mistletoe
is a semi-parasite. Although phenyl-ethylamine has not been found in
any fresh fungus, Reuter [1912] obtained it from Boletus edulis by
aseptic autolysis. Derivatives of phenyl-ethylamine have been found
in various essential oils ; thus phenyl-ethyl-alcohol
C6H5.CH2 .CH2OH
occurs in rose oil and is also produced from phenyl-ethylamine by
yeast (Ehrlich); phenyl-acetonitrile, C6H5 . CH2 . CN, was found by Hof-
mann [1874] in the essential oil of Nasturtium officinale^ and phenyl-
ethyl-z'jtf-thiocyanate is present in the oil from the root of Reseda
according to Bertram and Walbaum [1894], and yields phenyl-ethyl-
amine on hydrolysis. Possibly phenyl-ethylamine is an intermediate
stage in the formation of all three substances from phenyl-alanine.
p-Hydroxy-phenyl-ethylamine, OH . C6H, . CH2 . CH2 . NH2.
This amine was first obtained by Schmitt and Nasse [1865] by
heating tyrosine, when the following change occurs : —
HO/ \CH2 . CH (NH2) COOH = HO/'" ~\CH2 . CH2 . NH2 + CO2.
p-Hydroxy-phenyl-ethylamine was subsequently isolated from auto-
lysed pancreas by Emerson [1901] and from a prolonged peptic
digestion of egg-albumin by Langstein [1901, 1902]. It seems pretty
certain that in these experiments bacterial action was not completely
excluded (see p. 10). Gautier and Mourgues [1888] isolated the base
from the mother liquors obtained in the putrefaction of cod-livers
(in the old process of making cod-liver oil). Gautier also obtained in
small quantity a lower homologue C7H7NO and a higher one
C9HUNO and named the three bases " tyrosamines ". The last two
do not, however, appear to have been sufficiently well characterised.
p-Hydroxy-phenyl-ethylamine is fairly abundant in various kinds
of cheese. It was found by Van Slyke and Hart [1903] in Cheddar
AMINES DERIVED FROM PROTEIN 19
cheese prepared in the usual manner, but not in a cheese prepared
with chloroform milk, so as to ensure sterility. The normal cheese
was found to give off considerable quantities of carbon dioxide during
ripening and Van Slyke and Hart consider that the carbon dioxide
arose from the decarboxylation of amino-acids. The chloroformed
cheese produced only traces of carbon dioxide and when finally
analysed yielded a considerable quantity of arginine, while the
normal cheese contained only traces of arginine, but instead of it
guanidine and putrescine were present. The cavities in Emmenthaler
("Gruyere") cheese are mostly filled with carbon dioxide, and
p-hydroxy-phenyl-ethylamine was isolated from this kind of cheese by
Winterstein and Kiing [1909].
It is further almost certain that one of Brieger's ptomaines, my dine
[1886, i, p. 26], was identical with p-hydroxy-phenyl-ethylamine. The
base had the composition C8HnNO, yielded a soluble platinichloride,
and a picrate crystallising in broad prisms melting at 190°. It was ob-
tained from putrid human viscera, and was non-poisonous ; ferric and
gold salts were reduced by it. (The picrate of the synthetic amine
crystallises in " short prisms " melting at 200° ; the other properties
are identical with those described for mydine by Brieger.)
The physiological action of p-hydroxy-phenyl-ethylamine was first
brought to light by its identification, by Barger and Walpole [1909, i],
as the chief pressor constituent in extracts of putrid meat. The blood
pressure raising property of such extracts had already been observed
by Abelous, Ribaut, Soulie, and Toujan [1906, I, 2]. Dixon and
Taylor [1907] had also noticed that extracts of human placenta raised
the blood pressure on intravenous injection and caused, in addition,
contraction of the pregnant uterus. Rosenheim [1909] showed that
this effect was mot produced by extracts of perfectly fresh placenta,
and after Barger and Walpole' s identification of the pressor con-
stituent of putrid meat, he was further able to show that the active
constituent in Dixon and Taylor's placental extracts was also p-
hydroxy-phenyl-ethylamine. Finally this amine is the chief pressor
constituent of certain extracts of ergot, as shown by Barger and Dale
[1909]. A certain quantity is apparently present in perfectly fresh
ergot, where it has also been found by Engeland and Kutscher [1910, 2]
and by Burmann [1912]. p-Hydroxy-phenyl-ethylamine is pro-
bably also present in autolysed Boletus edulis (Reuter [1912]). That
tyrosine is indeed the parent substance of p-hydroxy-phenyl-ethylamine
was shown by Barger and Walpole [1909, i]; the yield in putrefaction
was minute (less than I per cent, of the tyrosine present). Ackermann
20 THE SIMPLER NATURAL BASES
[1909, I] also isolated the base after putrefying the mixture of ammo-
acids obtained by boiling caseinogen with sulphuric acid.
Henze [1913] has made the most interesting observation that
p-hydroxyphenyl-ethylamine occurs in the salivary gland of Cephalo-
poda and has a paralysing action on crabs, which are the chief food of
these Molluscs.
Syntheses.
Larger quantities of p-hydroxy-phenyl-ethylamine are obtained
by synthesis, most conveniently by the reduction of p-hydroxy-phenyl-
acetonitrile with sodium and alcohol (Barger [1909, i]), according to
the equation : —
OH . C6H4. CH2. CN + 4H = OH . C6H4 . CH2. CH2. NH2.
Two other syntheses of this amine were described by Barger and
Walpole [1909, 2]; according to one of these benzoyl-phenyl-ethyl-
amine is nitrated and the p-nitro-derivative is reduced, diazotised, and
hydrolysed : —
C6H5 . CH2 . CH2 . NH . CO . C6H5->NO2 . C6H4 . CH2 . CH2 . NH . CO . C6H5
-»NH2 . C6H4 . CH2 . CH2 . NH . CO . C6H5-»OH . C6H4 . CH2 . CH2 . NH . CO . C6H6
-»OH-. C6H4 . CH2 . CH2 . NH2.
The other synthesis starts from anisaldehyde which is successively
converted into p-methoxy-phenyl-acrylic acid, p-methoxy-phenyl-
propionic acid, and its amide, p-methoxy-phenyl-ethylamine and p-
hydroxy-phenyl-ethylamine : —
CH3O . C6H4 . CHO->CHSO . C6H4 . CH : CH . COOH->CH3O . C6H4 . CH3 . CH2 . COOH
_»CH3O . C6H4 . CH2 . CH2 . CO . NH2-»CH3O . C6H4 . CH2 . CH2 . NH 2
-5.0H . C6H4 . CH2 . CH2 . NH3 .
The yield by the last synthesis is poor ; the p-methoxy-phenyl-
ethylamine is better prepared by Rosenmund's method [1909], by
the reduction of the condensation product of anisaldehyde with nitro-
methane : —
CH30 . C8H4 . CHO + CH3 . NO2 = CH3O . C6H4 . CH : CH . NO2
-»CH80 . C6H4 . CH2 . CH : NOH-»CH3O . C6H4 . CH2 . CH2 . NH2.
Rosenmund then boils the latter compound with colourless hydriodic
acid and obtains p-hydroxy-phenyl-ethylamirie.
Hordenine, OH . C6H, . CH2 . CH2 . N(CH3)2 .
An infusion of barley germs, a by-product obtained in the pre-
paration of malt, had been employed in the South of France against
dysentery. This led to the isolation by Leger [1906, l] of an
"alkaloid" from barley germs, which he named hordenine. The
base was found by Leger [1906, 2,3, I9O7] and independently also
by Gaebel [1906] to be p-hydroxy-phenyl-ethyl-dimethylamine
~2 . CHa . N (CH3)2
AMINES DERIVED FROM PROTEIN 21
The constitution of hordenine was deduced by Leger from the oxi-
dation of acetyl-hordenine to acetyl-p-hydroxy-benzoic acid and the
distillation of the ammonium base from hordenine methiodide
methyl-ether, which yielded trimethylamine and p-vinylanisole,
CH3O.C6H4.CH:CH2.
Gaebel, on methylating and oxidising, obtained anisic acid from
hordenine.
The synthesis of hordenine was first carried out by Barger [ 1 909, 2]
from phenyl-ethyl -alcohol, a commercial product, as follows :
C6H5 . CH2 . CH2 . OH-»C6H5 . CH2 . CH2 . C1-»C6H6 . CH2 . CH3 . N(CH3)2
I
HO.C6H4.CH2.CH3.N(CH3)2<-NH2.C6H4.CH3.CH2.N(CH3)2«-N02.C6H4.CH3.CH3.N(CH3)2
Closely related to this is the synthesis from tyrosol, by Ehrlich
[1912]:-
OH . C6H4 . CH3 . CH2OH->OH . C6H4 . CH2 . CH2C1->OH . C6H4 . CH2 . CH2 . N(CH3)2
The attempted conversion of p-hydroxy-phenyl-ethylamine into horde-
nine by methyl-iodide resulted only in the formation of the quaternary
iodide, but Rosenmund [1910] has succeeded in methylating p-methoxy-
phenyl-ethylamine to the tertiary base, hordenine methyl-ether, from
which hordenine was obtained by boiling with hydriodic acid. Other
syntheses are by reduction of p-hydroxy-phenyl-dimethyl-amino-methyl-
ketone
HO . C6H4 . CO . CH2 . N(CH3),£
(Voswinckel [1912]) and by distillation in a vacuum of the quaternary
hordenine methiodide (prepared from p-hydroxy-phenyl-ethylamine)
according to D.R.P. 233069 of Farbenfabriken vorm. F. Bayer &
Co.:—
OH . C6H4 . CR, . CH2 . N(CH3)3I = OH . C6H4 . CHa . CH a. N(CH3)2 + CH3I .
Hordenine has only a transitory existence during the germination of
barley. According to Torquato Torquati [1910] it is not present in the
ungerminated seed and is most abundant after four days, when the
rootlets contain 0-4 - 0*45 per cent. It then gradually diminishes
and has disappeared after twenty-five days. It is absent in germinating
wheat, peas and lupins.
Indolethylamine (3-/3-Amino-ethylindole), C10H12N2.
3-/3-Amino-ethylindole is the amine derived from tryptophane by
decarboxylation. It was obtained by Ewins and Laidlaw [1910, 2]
both synthetically and by the action of putrefactive bacteria on the
amino-acid.
The synthesis, subsequently described by Ewins [1911], is the
22 THE SIMPLER NATURAL BASES
most convenient method for obtaining the base in quantity ; ry-amino-
butyrylacetal is heated with phenyl-hydrazine and zinc chloride.
CH2 . CH2 . CH2 . NH3
,'NH . NH2 CH (OC2H5)2
(C.CH2.CH2.NH2
CH + NH3 + 2C2H6OH
NH
From the concentrated solution of the crude hydrochloride (obtained
by washing the reaction mixture with ether and removing the zinc as
sulphide) the free base is precipitated by sodium hydroxide as an oil,
which on keeping crystallises to a mass of fine needles.
Laidlaw [1911] dissolved 0*5 grm. tryptophane in 250 c.c. of tap
water, together with 0*5 grm. peptone, 2 grm. glucose, traces of sodium
phosphate and magnesium sulphate and added 5 grm. of calcium car-
bonate ; this is the culture medium employed by Ackermann in the
decarboxylation of histidine (p. 132). After infection with a subculture
from putrid pancreas and incubation for a fortnight the mixture was
boiled with charcoal and concentrated. Picric acid then precipitated
the deep orange red picrate of indolethylamine. Yield after purifica-
tion = 0-14 grm. = 14 per cent, of the theoretical.
The decarboxylation of tryptophane cannot be effected by heat.
The author's experiments in this direction were carried out under a
pressure of I mm. ; the only substance which could be isolated from
the sublimate was a small quantity of unchanged tryptophane.
/5-Iminazolyl-ethylamine, C5H9N3.
/2-Iminazolyl-ethylamine (4-/3-amino-ethyl-glyoxaline) is the amine
derived from histidine by decarboxylation ; it is of considerable in-
terest on account of its great physiological activity. The base was
first obtained by Windaus and Vogt [1907] who prepared it by
Curtius's method from iminazolyl-propionic acid, which can be
made by synthesis as well as from histidine. A few years later
Ackermann [1910, I] submitted pure histidine hydrochloride to the
action of putrefactive bacteria and obtained a relatively large yield of
iminazolyl-ethylamine (together with a small quantity of iminazolyl-
propionic acid). The physiological activity of the amine, however,
remained unknown until the latter was identified as one of the active
principles of ergot by Barger and Dale [1910, 2-4]. The same active
principle was simultaneously isolated from ergot by Kutscher [1910, l]
who at first regarded it as closely related to iminazolyl-ethylamine, but
AMINES DERIVED FROM PROTEIN 23
not identical with it, on account of a supposed difference in the physio-
logical action of the two bases. Iminazolyl-ethylamine has also been
obtained from the intestinal mucosa by Barger and Dale [1911];
it is therefore present in crude solutions of secretine, to which it gives
a depressent action. Its formation in the intestinal wall is probably
due to bacilli, isolated by Mellanby and Twort [1912] and by Berthelot
and Bertrand [1912, I, 2]. The base has further been isolated from
putrid Soy beans by Yoshimura1 [1910]; it probably also occurs in
commercial extracts of meat, of yeast, etc.
The yield from almost all the above sources is very small ; larger
quantities may be prepared from histidine, as well as by direct syn-
thesis. The decarboxylation of histidine has been carried out indi-
rectly by Windaus and Vogt [1907] as mentioned above.
The reactions involved are the transformation of histidine (I)
CH-NH^ CH-NH, CH-NH.
CH3 . CH (NH2) . COOH CH2 . CHC1 . COOH CHa . CHa . COOH
I II III
CH—NH. CH— NHv CH—
<- C -- N 4- C __
CH2 . CH2 . NH3 CH2 . CH2 . CONH . NH,, CH2 . CH. . COOCaH5
VI V IV
into a-chloro-/2-iminazolyl-propionic acid (II) (by sodium nitrite and
hydrochloric acid) ; the reduction of this substance to /3-iminazolyl-
propionic acid (III), which can also be synthesised from glyoxyl-propi-
onic acid ; the successive 'conversion of this acid into the ester (IV)
and the hydrazide (V) ; finally the conversion of the latter into the
azide and urethane (in alcoholic solution by amylnitrite and hydrogen
chloride) and the hydrolysis of the urethane by concentrated hydro-
chloric acid, which gives the hydrochloride of the desired amine (VI).
The direct decarboxylation of histidine can be carried out more
conveniently by bacterial action and is applied industrially, according
to patents by Hoffmann, La Roche & Co. [1912], and by Farben-
fabriken vorm. F. Bayer & Co. (D.R.P. 250110). Details of the
method are given in the appendix.
An attempt to decarboxylate histidine by heat alone results only
1 Yoshimura [1909] probably obtained iminazolylethylamine by putrefaction before
Ackermann, but he did not identify it. He found that the Japanese beverage Tamari-
Shoyu, prepared from Soy beans, contains per litre o'7 grm. of a base C6H9N3, which he
surmised was derived from histidine.
24 THE SIMPLER NATURAL BASES
in the formation of traces of the amine, and Ackermann, by heating
histidine with lime, could only obtain glyoxaline. Ewins and Pyman
[1911], however, obtained a 10-20 per cent, yield by heating benzoyl
histidine in a vacuum to 240° and subsequent hydrolysis, and a 24
per cent, yield by heating histidine hydrochloride with 20 per cent,
sulphuric acid to 265-270°. The most convenient method of prepar-
ing iminazolyl-ethylamine is, however, by the synthetical method of
Pyman [1911]. Diaminoacetone dihydrochloride (I) (obtained from
citric acid) is heated with one molecular proportion of potassium
sulphocyanide ; the thiolglyoxaline (II), thus formed by
CHa.NH3.HCl CH.NH, CH . NHX
* i° * f-^CSH -> f-
CH2.NHa.HCl JH..NH, CH2OH
I II III
I
CH.NHv CH.NH, CH . NH
11 X~H II >XH II
C N ^ C N< +_ C
CH2 . CH2 . NH2 CH2 . CN CH2C1
VI V IV
Gabriel's general method, is oxidised with nitric acid ; the nitrous acid
formed in the reaction further attacks the amino-group so that a
glyoxaline alcohol (III) results. This is successively converted into
the chloro-compound (IV) and the cyano-compound (V) ; the latter
yields on reduction the desired amine (VI).
The lower homologue, iminazolylmethylamine, has been prepared
by Windaus and Opitz [191 1].
PHYSIOLOGICAL PROPERTIES OF THE AMINES
DERIVED FROM AMINO-ACIDS.
The chief interest attached to the amines described in this chapter
is due to their physiological action and to the possibility of their forma-
tion in the organism, wherever proteins or amino-acids are exposed to
bacterial action as, for instance, in the intestine. By far the most
active amines are those containing a ring, namely those derived from
phenyl-alanine, tyrosine, tryptophane, and histidine. Their formation
does not take place in acid solution, and would, therefore, appear to
be prevented or lessened by the sour-milk treatment recommended by
MetchnikofT. Berthelot and Bertrand [1913, l] find, however, that
their Bacillus aminophihis even produces /3-iminazolylethylamine in
O'3 per cent, lactic acid, unless much glucose is present, when the sugar
alone is attacked. The same investigators [1913, 2] find that rats, fed
on a milk diet, are not affected by either Proteus vulgaris or B. amino-
philus intestinalis when given separately, but that if the two organisms
are given simultaneously, the rats may develop a fatal diarrhoea in
from 4-8 days. Normally these putrefactive amines appear to be de-
stroyed in the liver; Ewins and Laidlaw [1910, 3; 1913] have shown
that p-hydroxy-phenyl-ethylamine and indole-ethylamine are trans-
formed by perfusion through a surviving liver into p-hydroxy-phenyl-
acetic acid and indole-acetic acid respectively. Oehme [1913] states
that 0'6 mg. may kill a rabbit when given intravenously, but that the
lethal dose is much higher when injected into the portal circulation.
Rabbits will even stand 0*5 grm. by the mouth. Nevertheless the
amines may perhaps play a part in certain diseases ; thus p-hydroxy-
phenyl-ethylamine may be connected with a persistent high blood
pressure, and Mellanby [1911] has attempted to connect /3-iminazolyl-
ethyl-amine with cyclic vomiting. Pharmacologically these bases are
important on account of their presence in ergot.
Ehrlich and Pistschimuka [1912] have shown that they are
transformed by yeast into the corresponding alcohols, and according
to Czapek [1903] the amines with 3-7 carbon atoms are a good
source of nitrogen for Aspergillus.
The action of many synthetic amines has been examined ; it seems
25
26 THE SIMPLER NATURAL BASES
that the most active are cyclic ones with a side chain of two carbon
atoms like the last four naturally occurring ones described in this
chapter. This conclusion with regard to the side chain was deduced
for aromatic amines by Barger and Dale [1910, I] ; it is further sup-
ported by toxicity determinations of several iminazole derivatives by
Friedberger and Moreschi [1912]. Von Braun and Deutsch [1912]
have found, however, that when the side chain of hordenine is
lengthened the pressor action is diminished and the toxicity is in-
creased. With four and five carbon atoms in the side chain the
toxicity is ten times as great as with three carbon atoms.
The natural amines described in this chapter may be arranged in
two groups, of monamines and of diamines, and physiological action
is more or less of the same type within each group. The monamines
(see p. 29) produce effects similar to those caused by stimulation of
the sympathetic nervous system. They may be termed sympatho-
mimetic (see p. 98). The most powerful sympathomimetic base is
adrenaline (see Chapter VI). Of the bases already described
the most powerful is p-hydroxy-phenyl-ethylamine : the others in
descending order of activity are phenyl-ethylamine, isoamylamine,
isobutylamine.
One of the most marked of sympathomimetic actions is the raising
of the blood pressure on intravenous injection and isobutylamine is the
lowest amine which has any marked pressor action. 10-20 mg. of
isoamylamine, injected intravenously as the hydrochloride, produce a
marked rise of blood pressure in the cat (Dale and Dixon [1909]).
The effect of other aliphatic monamines is very similar. Normal
amylamine has a slightly greater activity than its isomeride, and hexyl-
amine is still more active, but in ascending the series beyond this point
the activity again declines, heptylamine being less active than hexyl-
amine and octylamine much less so (Barger and Dale [1910, l]).
The introduction of a benzene ring in phenyl-ethylamine greatly
increases the activity and this base is at least five times as active as
any aliphatic amine. Thus 2 mg. of the base may raise the blood
pressure of a cat from 30 to 180 mm. Phenyl-ethylamine has the
same carbon skeleton as adrenaline.
p-Hydroxy~phenyl-ethylamine has an activity something like ^ of
that of adrenaline, and has been studied by Dale and Dixon
[1909].
Doses of 1-2 mg., injected intravenously, cause a sudden and
pronounced rise of arterial blood pressure, which is somewhat less
transitory than that caused by adrenaline. As with the latter sub-
AMINES DERIVED FROM PROTEIN 27
stance, the output of the heart is increased, the non-pregnant cat's
uterus relaxes, the pregnant cat's uterus contracts, the salivary gland
is stimulated to secretion.
p-Hydroxy-phenyl-ethylamine differs from adrenaline in causing
little vase-constriction when applied locally to a mucous surface, and
in being hardly toxic. Thus 100 mg. given hypodermically to a cat,
produced all the symptoms of intense stimulation of sympathetic
nerves, but no after-effects and no glycosuria.
Since p-hydroxy-phenyl-ethylamine is formed from tyrosine by the
action of faecal bacteria, it doubtless occurs in the alimentary canal and
might therefore perhaps play a part in certain pathological states in
which a high blood-pressure is the most prominent symptom. A
pressor substance has been found in the urine by Abelous and termed
urohypertensine (perhaps identical with isoamylamine) and Bain
[1909, 1910] obtained from normal urine a pressor base, giving
Millon's reaction ; the latter base was not isolated in a state of purity
and its identity with p-hydroxyphenylethylamine, suggested by Bain,
is very doubtful. Bain found that the amount of this base was
diminished in the urine from gouty patients and particularly in that
from patients with a high blood pressure ; on the other hand it did
not disappear from normal urine during a milk diet or when medicinal
doses of antiseptics were administered.
On account of the possible clinical significance of p-hydroxy-phenyl-
ethylamine, as indicated above, Ewins and Laidlaw [1910, 3] have in-
vestigated the fate of this amine in the organism. They found that
when given by the mouth to dogs, something like one-half the amount
is excreted in the urine as p-hydroxy-phenylacetic acid ; the other
half remains unaccounted for. The conversion of the amine into the
acid readily takes place in the perfused rabbit's liver, and also to some
extent in the perfused isolated uterus, but in the isolated heart the
amine, when perfused, was completely destroyed and no p-hydroxy-
phenylacetic acid could be isolated.
Other papers of clinical interest are those by Harvey [1911],
who induced renal disease and vascular sclerosis in rabbits by pro-
longed intravenous and oral administration of p-hydroxy-phenyl-
ethylamine, by Clark [1910] and by Findlay [1911] who examined
the effect of this amine on man. Clark found that large doses (30-
200 mg.) given by the mouth generally gave a slight rise of blood
pressure lasting for several hours, and that 20-60 mg., given sub-
cutaneously, produced in the healthy subject a considerable rise of
blood pressure, lasting for about twenty minutes. The suggestion by
28 THE SIMPLER NATURAL BASES
Burmann [1912] and Heimann [1912] that p-hydroxy-phenyl-ethyl-
amine can replace ergot, or even that it is the most important con-
stituent of this drug, is erroneous (see especially a paper by
Guggenheim [1912]). The action of the base has also been studied
lately by Frohlich and Pick [1912], by Handovsky and Pick [1913]
and by Bickel and Pawlow [1912].
According to Engel [1912] p-hydroxy-phenyl-ethylamine has
no necrotising effect on tumours, although this effect is produced by
phenyl-ethylamine, which has only one-fifth of the pressor activity of
the first-named base. The effect is also shown by hordenine and by
adrenaline.
p-Hydroxyphenyl-ethylamine has a paralytic action on Crustacea
and occurs in the salivary gland of Cephalopoda which feed on crabs
[Henze, 1913].
Hordenine, which is the N-dimethyl-derivative of the last-named
base, has a much weaker action, and has been studied by Camus
[1906]. The minimal lethal dose of the sulphate is 0*3 grm. per
kilo, for dogs, injected intravenously, and 2 grm. per kilo, for guinea-
pigs injected subcutaneously, so that the toxicity is very slight. The
base has a feeble pressor action. Its methiodide, however, causes a
very rapid and evanescent rise of blood pressure in cats, when in-
jected intravenously in doses of I mg. The effect superficially
resembles that of adrenaline but is in reality of the nicotine type
(Barger and Dale [1910, I]). Von Braun and Deutsch [1912] have
prepared homologues of hordenine, having the formula
OH.C(iH4.(CH2)n.N(CH3).2
with ti = 3, 4 and 5. In these the pressor action of hordenine is
diminished. The lethal dose for rabbits is respectively cri grm., croi
grm.,o*O2 grm., as compared with 0-3 grm. for hordenine. Comp.
von Braun, Ber. deutsch. chem. Ges., 1914,47, 492.
The physiological action of indolethylamine has been studied by
Laidlaw [1911]. Doses of 10-20 mg. of the hydrochloride given
intravenously to rabbits and cats, produce a transient stimulant effect
upon the central nervous system, causing clonic and tonic convulsions,
tremors of limbs, and vaso-constriction. In the spinal cat 2 mg.
causes a large rise of blood pressure due to vaso-constriction and in-
creased cardiac activity. In this respect the amine resembles p-hydroxy-
phenyl-ethylamine. Indolethylamine has further a direct stimulant
action on plain muscle, which is most marked in the arterioles, the
iris, and the uterus. This action of the amine from tryptophane is on
the whole much less than that of the amine from histidine. Speaking
AMINES DERIVED FROM PROTEIN 29
very broadly, indolethylamine (with two nitrogen atoms of which only
one is basic) has a physiological action intermediate between that of
the sympathomimetic monamines such as p-hydroxy-phenyl-ethylamine,
and the diamines, like iminazolyl-ethylamine.
Ewins and Laidlaw [1913] have more recently studied the fate
of indolethylamine in the organism ; in the perfused liver the base is
converted into indole-acetic acid, a change quite comparable to the
transformation of p-hydroxy-phenyl-ethylamine into p-hydroxy-
phenyl-acetic acid (see p. 27). In dogs the indole-acetic acid is
however excreted in the urine in combination with glycine as indole-
aceturic acid C8H6N . CH2 . CO . NH . CH2 . COOH, mp. 94°, forming
an orange red picrate which melts at 145°.
Among diamines /3-iminazolyl-ethylamine is the only one having a
cyclic structure, and it is by far the most active. Putrescine and
cadaverine have at most a very slight toxicity ; on intravenous injection
in the cat they lower the blood pressure. Agmatine has according to
Engeland and Kutscher [1910, l] a powerful action on the isolated
uterus, causing contraction, but Dale and Laidlaw [1911, p. 194]
state that agmatine does not make any significant contribution to
the activity of ergot and is only feebly active as compared with
/3-iminazolyl-ethylamine, also present in ergot. Thus 5 mgs. of
agmatine produced a much smaller effect on the cat's uterus than
O'l mg. of the latter base.
The physiological action of ^-iminazolyl-ethylamine has been
investigated by Ackermann and Kutscher [1910, I] and more fully by
Dale and Laidlaw [1910, 191 1].1 According to the latter authors the
fundamental and characteristic feature of the action is a direct stimulant
effect on plain muscle, producing exaggerated rhythm or tonic con-
traction, according to the dose. The most sensitive plain muscle is
the non-pregnant uterus of some species and it is this reaction which
led to the identification of the base in ergot. A marked contraction
of the isolated uterus is produced by adding to the bath of Ringer's
solution sufficient of the base to give a concentration of I : 25,000,000
and the effect of I : 250,000,000 is often quite definite (compare
also Frohlich and Pick [1912] and Sugimoto [1913]). The muscular
coats of the bronchioles are also highly sensitive to the action of
/3-iminazolyl-ethylamine, especially in rodents, but not in the ox
(Trendelenburg [1912]). Baehr and Pick [1913, I] have studied the
effect on the musculature of the surviving guinea-pig's lung. Here
1 Many scattered observations on its action occur in the pharmacological literature of the
last few years.
30 THE SIMPLER NATURAL BASES
the contraction due to /3-iminazolylethylamine is permanently abolished
by adrenaline, which is not so in the intact animal. Large guinea-
pigs are killed in a few minutes by an intravenous injection of
O'5 mg., owing to asphyxia resulting from the constriction of
the bronchioles ; post-mortem the lungs are found to be permanently
distended. This corresponds closely to the effects of poisoning
by Witte's peptone and the toxic effects of serum or other protein
in the sensitised guinea-pig, known as anaphylactic shock. Unlike
peptone, iminazolyl-ethylamine does not, however, possess in any
marked degree the power of rendering the blood incoagulable. Ac-
cording to Popielski the physiological effect of peptone is produced by
a hypothetical substance " vasodilatin," and he [1910, 2] has suggested
that iminazolyl-ethylamine acts by liberation of vasodilatin, when
injected intravenously, a supposition rejected by Dale and Laidlaw
[1911]. Attention may also be drawn to a possible connection
between iminazolyl-ethylamine and the " depressor substances " of
various observers, such as the urohypotensine of Abelous and Bardier
[1909]; the depressent action of Bayliss and Starling's secretine is
indeed explained by the isolation from it of iminazolyl-ethylamine
by Barger and Dale [1911].
The resemblance of the symptoms of poisoning with iminazolyl-ethyl-
amine to those of anaphylactic shock is indeed very striking (Dale
and Laidlaw [1910, 1911], Pfeiffer [1911], Biedl and Kraus [1912],
Schittenhelm and Weichardt [1912], Aronson [1912], Friedberger
and Moreschi [1912]); not only does it extend to the bronchial
constriction in guinea-pigs, mentioned above, but also to a fall of body
temperature, which is one of the characteristics of the milder degree
of the " shock ". Thus the intraperitoneal injection of 3 mgs. of
iminazolyl-ethylamine was found by Dale and Laidlaw to lower the
rectal temperature of a guinea-pig gradually from 38*5° to 28*5° in the
course of two hours ; next day it was again 38°. Extremely minute
doses of serum may, on the other hand, cause a rise of body temperature
in an anaphylactic animal, and the same applies to iminazolyl-ethylamine
when given in sufficiently small doses to a (normal) guinea-pig, as
has been shown by Pfeiffer [191 1]. The correspondence is also illus-
trated by the relatively great resistance of dogs, both to anaphylactic
shock and to the amine. In this connection we may refer to a paper
by Engeland [1908, 3] in which evidence is adduced that histidine
derivatives are more readily broken down by carnivora than by
herbivora. No data are available to fix the lethal dose of /3-iminazolyl-
ethylamine in man, but a Macacus monkey of 1*25 kilo, was killed by
AMINES DERIVED FROM PROTEIN
an intravenous injection of 0*065 grm. of the hydrochloride [Berthelot
and Bertrand, 1912, 3],
Lately the close similarity between the symptoms of poisoning by
/9-iminazolyl-ethylamine and those of anaphylactic shock have been
emphasised anew by Oehme [1913]. He and Loewit [1913 ; Ch. V,
methyl guanidine] both criticise the conclusion of Heyde [1912 ; Ch.
V, methylguanidine] that methylguanidine rather than iminazolyl-
ethylamine is of importance in this respect.
The supposed connection between /3-iminazolyl-ethylamine and ana-
phylactic shock has even led to the statement (by Aronson [1912])
that the amine is formed by incubating histidine with normal guinea-pigs'
serum, but this has been disproved by Friedberger and Moreschi
[1912] and Modrakowski [1912] denies that the amine is the cause of
anaphylactic shock since it does not render the blood incoagulable.
In recording the fact, "as a point of interest and possible signifi-
cance," that the immediate symptoms with which an animal responds
to an injection of a normally inert protein, to which it has been
previously sensitised, are to a large extent those of poisoning by
/3-iminazolyl-ethylamine, Dale and Laidlaw consider that " the corre-
spondence cannot yet be regarded as sufficient basis for theoretical
speculation ". Pfeiffer thinks that /3-iminazolyl-ethylamine will cer-
tainly be of significance for the solution of the problem of anaphylaxis.
The effect of iminazolyl-ethylamine on the vascular system is
complex and varies in different species, as well as in the same species
under different conditions. In rodents a rise of blood-pressure occurs,
owing to constriction of the arterioles, but may be masked by embar-
rassed respiration. It was the different behaviour of rabbits to the
base from histidine and that from ergot, which led Kutscher [1910, I]
to regard the two bases as different. Barger and Dale [1910, 3] have
however shown that both kinds of physiological effect are obtainable
with the base from either source, so that the identity cannot be doubted.
In carnivora, in the fowl, in the monkey (and probably therefore in
man) iminazolyl-ethylamine causes vasodilatation and a fall of systemic
blood pressure. The following table (Barbour [1913]) gives the effects
of the amine, compared with those of adrenaline and p-hydroxy-phenyl-
ethylamine : —
Blood
Pressure.
Peripheral
Vessels.
Coronary
Vessels (Ox).
Non-pregnant
Uterus.
Epinephrin (adrenaline) ....
+
+
_
_
Tyramin (p-hydroxy-phenyl-ethylamine)
Histamin (/3-iminazolyl-ethylamine)
+
+
+
+
+
-r-
+ means rise of blood pressure or constriction, - the opposite ; the last-named amine
may have a pressor effect in some animals.
32 THE SIMPLER NATURAL BASES
The pulmonary arterioles, however, are constricted and the pulmon-
ary blood pressure is raised. This combination of a vasodilator fall of
systemic blood pressure with a vasoconstrictor rise of pulmonary pres-
sure has been described as characteristic of the action of ergot (Bradford
and Dean [1894]), and is doubtless due to the iminazolyl-ethylamine
present in the drug. For the effect of the base on the pulmonary
vessels consult Baehr and Pick [1913, 2], and on the frog's blood
vessels, Handovsky and Pick [1913].
Finally it should be mentioned that iminazolyl-ethylamine has a
weak stimulant action on the salivary glands and on the pancreas,
qualitatively resembling that of pilocarpine, which alkaloid also contains
a glyoxaline ring. The action on the pancreas is not at all like that
of secretine, being abolished by a small dose of atropine.
CHAPTER II.
o> AMINO-ACIDS AND OTHER BASES DERIVED FROM PROTEIN CONTAINING
A CARBOXYL-GROUP (UROCANIC AND KYNURENIC ACIDS).
IN the monamino-acids, formed by the hydrolysis of proteins, the
acidic properties of the carboxyl-group are neutralised more or less
completely by an adjoining amino-group in the a-position, and only
the diamino-acids histidine, lysine, and arginine are bases. When the
amino-group is not in the a-position the basic character is more pro-
nounced, and the so-called w-amino-acids are feeble bases, being pre-
cipitated by phosphotungstic acid ; several of them are formed from
protein fission products by putrefaction, and these are described in this
chapter.
The influence of the position of the amino-group on the acid dis-
sociation constant Ka and on the basic dissociation constant K6 is
evident from the following table (Ley [1909, p. 358]) : —
Ka
K&
Glycine
o-amino-propionic acid
j8-amino-propionic acid
7-amino-butyric acid .
180 x 10 - 12
230 x 10 - ia
71 x io~12
37 x 10 _ 12
2*7 X IQ-12
3-1 x to -12
51 x io~12
170 x 10 ~12
An ammo-acid may also be rendered basic by complete methylation
of the nitrogen atom, as in the betaines described in Chapter III.
o>-Amino-acids are produced by putrefaction in three ways ; —
1. By partial deaminization of a diamino-acid, as in the formation
of S-amino-valeric acid from ornithine : —
NH2. CH2CH2CHaCH(NH2)COOH + 2H = NH2. CH2CH2CH2CH2COOH + NH3.
2. By the partial decarboxylation of a dibasic amino-acid, e.g.
the production of 7-amino-butyric from glutamic acid : —
COOH . CH(NH2) . CH2 . CH2 . COOH = NH3 . CH2 . CHa . CH2 . COOH + CO2.
3. By the reduction of a cyclic amino-acid. Ackermann [191 1, 2]
and Neuberg [191 1, l] have recently shown that a-pyrrolidine car-
boxylic acid (proline) yields S-amino-valeric acid in putrefaction :—
33 3
34 THE SIMPLER NATURAL BASES
CH2— CH2
CH2 CH . COOH + 2H = NH2 . CH2 . CH2 . CH2 . CH2 . COOH.
NH
The (o-amino-acids differ from a-amino-acids in being precipitated by
phosphotungstic acid, even in dilute solutions ; they yield platini-
chlorides soluble in alcohol (Ackermann). The 7-, 8-, and e-amino-
acids are so weakly acidic that they do not form blue copper salts on
boiling with cupric oxide, or on addition of cupric acetate, this pro-
perty belonging only to a- and /3-amino-acids (Fischer and Zemplen
[1909, p. 4883]). On heating 7-amino-butyric and S-amino-valeric
acids are transformed into their anhydrides, pyrrolidone and piperidone.
/9-Alanine, /3-amino-propionic Acid, NH2. CH2. CH2. COOH.
This substance, long known synthetically, was first isolated from
Liebig's extract of meat by Engeland [1908, I] ; Micko [1905] had
previously obtained an alanine from the same source and assumed that
it was the a-amino-acid.
/3-Alanine is formed from the meat base carnosine by hydrolysis
(see next section), and since Engeland's process of isolation involved
evaporation in hydrochloric acid solution, Gulewitsch [1911; see
under carnosine] questions whether /8-alanine is present as such in
muscle.
It was to be expected that /?-alanine could also be formed from
aspartic acid by putrefaction, according to the second general method
given in the preceding section, and after some failures Ackermann
[1911, I] has succeeded in demonstrating this.
One hundred grm. of aspartic acid in a culture medium similar
to that used for preparing ^-iminazolyl-ethylamine yielded 2 grm. of
yQ-alanine hydrochloride.
ft- Alanine is broken down to urea in the dog (Abderhalden and
Schittenhelm [1907]).
7-Amino-n-butyric Acid, NH2 . CH2 . CH2 . CH2 . COOH.
This acid is formed in putrefaction from glutamic acid by the
second general process (p. 33).
Ackermann [1910, 3] obtained 2'i grm. of 7-amino-butyric acid
aurichloride from 50 grm. of glutamic acid. Abderhalden and Kautzsch
[1912] lately failed to repeat Ackermann's experiment, but afterwards
Abderhalden, Fromme and Hirsch [1913] obtained 0*3 grm, of the
platinichloride of 7-amino-butyric acid from 25 grm. of glutamic acid.
o>-AMINO-ACIDS 35
S-Amino-n-valeric Acid, NH2 . CH2 . CH2 . CH2 . CH2 . COOH.
This, the first known example of a natural w-amino-acid, was ob-
tained by E. and H. Salkowski [1883] from putrefied fibrin and
muscle, and later by H. Salkowski [1898] from putrefied gelatin.
Ackermann [1907, 2] isolated it from putrid pancreas (and at first called
it putridine, because he failed to identify it). The substance was pre-
pared synthetically by Schotten [1884] by the oxidation of benzoyl-
piperidine with potassium permanganate.
S-Amino-valeric acid is derived in putrefaction from both arginine
(ornithine) and proline. Ackermann [1910, 3] submitted 56 grm. of
arginine carbonate to putrefaction in the same way as aspartic acid
and glutamic acid (preceding sections), and obtained putrescine,
ornithine, S-amino-valeric acid (about 1 5 grm. of the aurichloride) but
not agmatine. The arginine is no doubt first broken down to ornithine,
and the latter by the first general process (p. 33) yields S-amino-valeric
acid.
The putrefactive formation of S-amino-valeric acid from proline
(a-pyrrolidine carboxylic acid) has been observed more recently by
both Ackermann and Neuberg ; two hydrogen atoms are added and
the ring is opened.
e-Amino-caproic Acid, NH2 . (CH2)5 . COOH.
This substance should be obtainable from lysine by putrefactive
deaminization ; an attempt to prove this was made by Ackermann
[1910, 3] with 98 grm. of lysine chloride. He obtained a large
quantity of cadaverine and a small quantity of a platinichloride fairly
readily soluble in alcohol and in water ; the analysis of this salt did
not agree with the composition required for the platinichloride of the
desired amino-caproic acid.
/Mminazolyl-propionic Acid,
CH = C— CH2.CH2.COOH
N NH
V
CH
This acid was first obtained from histidine by chemical means and
was also prepared synthetically by Knoop and Windaus [1906] (see
Plimmer's " Chemical Constitution of the Proteins," Part I, p. 1 26). Ac-
kermann [1910, i] then showed that it is also formed by putrefaction
from pure histidine hydrochloride ; the principal product was imin-
3*
36 THE SIMPLER NATURAL BASES
azolyl-ethylamine (described in Chapter I, p. 22), but in addition a
small quantity of iminazolyl-propionic acid was obtained.
Carnosine (Ignotine), C9H14O3N4.
This substance is described in this chapter as it is a derivative of
y8-alanine. Carnosine is, after creatine, the most abundant base in
meat extract. It was discovered by Gulewitsch and Amiradz'ibi
[1900, 1,2]; Krim berg [1906, I] obtained 0*13 per cent, from fresh
ox meat. Ignotine, subsequently isolated by Kutscher [1905]
from meat extract and regarded by him as an isomeride, was shown
by Gulewitsch [1906], by direct comparison, to be identical with
carnosine, and the identity has been admitted by Kutscher after pro-
longed controversy. Carnosine has also been obtained from horse
meat, to the extent of 1-82 grm. per kilo. (Smorodinzew [1913]) and
from fish, crabs, oysters and wild rabbits.
On heating with baryta to 140°, carnosine is hydro lysed to histi-
dine and /3-alanine in equimolecular proportions (Gulewitsch [1907,
191 1 ]) according to the equation : —
C9H14O3N4 + HaO = C6H9O2N3 + C3H7O2N.
It is, therefore, similar to a dipeptide and must be either histidyl-/3-
alanine or /3-alanyl-histidine ; it gives the red coloration with sodium
p-diazobenzene sulphonate, characteristic of histidine, and yields on boil-
ing with cupric carbonate a copper salt similar to that of /3-alanine.
Perhaps, therefore, histidyl-yS-alanine is the more likely constitution : —
CH = C— CH2 . CH . CO . NH . CHa . CH2 . COOH
II 'I
N NH NH2
Urocanic Acid, Iminazolyl-acrylic Acid,
CH = C— CH = CH . COOH
N NH
v
CH
This acid contains two hydrogen atoms less than iminazolyl-pro-
pionic acid described above and may be considered to be derived from
histidine by loss of ammonia, without reduction. It was discovered
by Jarfe" [1874, 1875] in the urine of a dog; after a few days
the dog ran away, and, to Jaffe's great disappointment, it was never
recaptured. The substance, was not observed again until Siegfried
a>-AMINO-AClDS 37
[1898] found it once more in dog's urine. In both cases the substance
was constantly present ; no other case of its occurrence in urine has
been observed and it would appear that the two dogs presented a rare
anomaly of metabolism. Recently Hunter [1912], although unable to
find a dog secreting urocanic acid, obtained the same substance by
prolonged tryptic digestion of caseinogen and was able to identify it
by comparison with a specimen of iminazolylacrylic acid which Barger
and Ewins [1911] had obtained as a degradation product of ergo-
thioneine and had also synthesised.
Among closely related substances from human urine we may men-
tion histidine itself, a base yielding a picrolonate C5H7O2N3, C10H8O5N4
melting at 244°, and a base giving an aurichloride C15H36O13N8, HAuCl4
very soluble in water and blackening at 100°. These bases were ob-
tained by Engeland [1908, 3] who regards the second as amino-imin-
azolylacetic acid, a lower homologue of histidine, and the third as
probably & polypeptide of histidine. According to Engeland histidine
is broken down more readily by carnivora than by herbivora ; the urine
of rabbits and horses gives a stronger reaction with p-diazobenzene
sulphonic acid than that of the cat or dog.
OH
Kynurenic Acid,
v
N
Long ago Liebig [1853] discovered an acid which occasionally
separated from dog's urine in minute quantity. The substance was
further investigated by Schmiedeberg and Schultzen [1872] and by
Kretschy [1881-84] who showed that the product formed by heating
the acid above its melting point, the so-called kynurine, C9H7ON, was an
oxyquinoline, and that kynurenic acid was therefore an oxyquinoline
carboxylic acid. Heated with zinc dust kynurine was reduced to
quinoline, and on oxidation of kynurenic Kretschy obtained oxalyl-
anthranilic acid,
COOH
\NH . CO . COOH.
Hence, when Wenzel [1894] had shown by synthesis that kynurine
is 4-hydroxy-quinoline, kynurenic acid was found to be either 4-hydroxy-
3 -quinoline carboxylic acid, or 4-hydroxy-2-quinoline carboxylic acid.
OH OH
^\/\ COOH or ^\/\
I II I I I! I
I /"*/-\/"\TT
>x /\ A L/UUri.
N N
38 THE SIMPLER NATURAL BASES
Camps [1901, I, 2] prepared both acids and wrongly concluded that
the former was identical with the acid from dog's urine, but Miss
Homer [1913] has shown, by the mixed melting point, that kynurenic
acid has the latter constitution.
Liebig [1853], Kretschy [1881] and others had already found
that kynurenic acid only makes its appearance, or is most abundant,
in the urine of dogs fed on large quantities of meat. Many fruitless
investigations were undertaken to find the precursor of the acid, until
finally its formation was shown to depend on a product of tryptic
digestion of protein (Glaessner and Langstein [1902]). This Ellinger
[1904, I, 2] identified as tryptophane (see Plimmer's " Chemical
Constitution of the Proteins," Part I, p. 137). Abderhalden, London,
and Pincussohn [1909] have shown that the transformation of trypto-
phane into kynurenic acid does not take place in the liver.
Kynurenic acid, taken by the mouth, is not excreted in the urine
in man and in the rabbit (Hauser [1895], Solonin [1897]); the
reason is probably that the acid is an intermediate product of metabol-
ism which is not destroyed so rapidly in the dog as in man.
CHAPTER III.
BETAINES.
THE betaines are amino-acids in which the nitrogen atom is completely
methylated. In addition to trimethyl-glycine, which has been known
for a long time and occurs both in plants and in animals, fully methyl-
ated derivatives of proline, oxyproline, histidine, and tryptophane have
so far been obtained from plants, and corresponding derivatives of y-
amino-butyric and of y-amino-hydroxy-butyric acid from animals.
Except in the case of trigonelline, which occurs in many plants but is
not related to any known decomposition product of protein, the betaine
grouping does not occur in the typical vegetable alkaloids ; the two
cases of its alleged occurrence, in damascenine and in chrysanthemine,
have lately been disproved (respectively by Ewins [1912] and
Yoshimura and Trier [1912, section on stachydrine]).
The betaines therefore form a fairly natural group comprising
feeble bases of simple constitution ; the a-betaines are devoid of marked
physiological activity, but the two y-betaines (being presumably stronger
bases) have a distinct action. A comprehensive study of the chemical
behaviour of betaines has been made by Willstatter [1902, l] whose
nomenclature is here employed. He points out that a-betaines and
the isomeric esters of dimethyl-amino-acids are interconvertible : —
/CH3 /CH,
COOCH3 COO CH3
In the case of the betaines of $-and-y-amino-acids the above change
only proceeds from left to right, but not in the reverse direction. From
the methyl ester of /3-dimethyl-amino-propionic acid £-propio-betaine
is thus obtainable ; when y-dimethyl-amino-butyrate is heated, the y-
butyro-betaine which no doubt first results, is unstable and yields
trimethylamine and y-butyro-lactone. Further details concerning the
interconversion in the case of trimethyl-glycine are given in the next
section.
The a-betaines differ greatly in the ease with which they split off
39
40 THE SIMPLER NATURAL BASES
trimethylamine. Some are so unstable that they cannot be formed by
the ordinary process of methylation. Thus aspartic acid, when treated
with methyl iodide and alkali, breaks up into trimethylamine and
fumaric acid. The same applies to tyrosine and it is noteworthy that
the betaines of tyrosine and of phenylalanine have never been found
in nature, whereas the corresponding unsaturated acids (p-cumaric and
cinnamic acids) are often met with in plants. The betaine of tryptophane
is somewhat more stable, and ergothioneine requires heating with
concentrated alkali to decompose it into trimethylamine and the un-
saturated acid.
The free betaines when dried above 1 00° have a composition
corresponding to a cyclic anhydride (the second of the above formulae).
Salts are formed by direct addition of an acid, when the ring is broken
down. Most betaines crystallise with one molecule of water and in
this condition their constitution is probably illustrated by the for-
mula : —
/OH
(CH3)3 : N/
\CH2.COOH.
The main physiological interest of betaines is derived from the question
whether they may re-enter the metabolism of plants or whether they
are merely waste products ; this question is further discussed in the
next section. Pharmacologically the a-betaines are inert, but y-butyro-
betaine is toxic to higher animals.
Betaine, Trimethylglycine, (CH0)3 : N/
XCH
While searching for alkaloids in Solanacece^ Husemann and Marme
[1863, 1864] isolated a base from Lycium barbarum, which was
found to have the composition C5HnO2N and was named by them
lycine. Three years later Scheibler [1866] obtained from the sap
of the sugar beet (Beta vulgaris) and from beet molasses a " soluble
alkaloid" which he described in detail later [1869] and called
betaine. Soon afterwards Scheibler [1870] and Liebreich [1870]
showed the identity of betaine with oxyneurine, a base prepared by
Liebreich [1869, 2] by the oxidation of " bilineurine " ( = choline) and
also synthetically by the action of trimethylamine on mono-chloracetic
acid. Griess [1875] prepared betaine according to his general method,
by methylating glycine and Husemann [1875] proved the identity
of lycine with betaine ; the second (and later) name for this base has,
however, passed into general use.
BETAINES 41
Betaine is of rather widespread occurrence in plants and has also
been found repeatedly in animals, but it is by no means so common as
choline. Stanek and Domin [1910] have given a list of plants con-
taining betaine ; it was found in all species of Chenopodiacece examined ;
this natural order includes the sugar beet and also Chenopodium
Vulvaria which gives off trimethylamine during life. In the closely
related order of Amarantacece betaine was found by Stanek and Domin
in some genera only ; in other orders it only occurs sporadically and
in small amount. The dry leaves of A triplex canescens (N.O. Cheno-
podiaceae) contain as much as 378 per cent, of betaine, but in rye the
amount is only 0*3 per cent, of the dry weight. Young sugar beets
contain 2-5 per cent, old ones I per cent, of betaine (Scheibler).1
Various authors have at different times expressed the view that
betaine may replace choline in lecithin. According to Trier [1912, 3,
p. 83 ; Ch. IV, choline] they were misled on account of the difficulty
of purifying the phosphatide.
In the manufacture of beet sugar most of the betaine remains in
the molasses, but crude beet sugar may contain 0375 per cent, of
betaine (Waller and Plimmer [1903]). When the molasses are
desaccharified by means of strontium, the final liquor (" Schlempe ")
is very rich in betaine (i 15 grm. per kilo., Andrlik [1903-4]).
Syntheses of betaine by Liebreich [1869, 2] and by Griess [1875]
have been referred to above ; it is also formed by isomeric change
from the methyl ester of dimethylamino-acetic acid in sealed tubes at
200° (see below). The estimation of betaine and its separation from
choline by Schulze's method [1909 ; Ch. IV, choline] and by Stanek's
method [1906, I, 2; Ch. IV, choline] are described on pp. 150-152.
1 Other sources of betaine are : Lycium barbarum (Husemann and Marme [1863]),
the press cake of cotton seeds (Ritthausen and Weger [1884]), malt and wheat germs
(Schulzeand Frankfurt [1893 ; Ch. IV, choline]) ; (Yoshimura [1910, Ch. IV, choline] recently
found 0-06 per cent, of betaine in air dry malt germs) ; sunflower seeds (Schulze and Castoro
[1904]), tubers of Helianthus tuberosus (Schulze [1910]), seeds of Avena sativa (Schulze
and Pfenninger [1911; Ch. IV, choline]), Kola nuts (Polstorff [1909, 2; Ch. IV, choline]),
bamboo shoots (Totani [1910,2; Ch. IV, choline]), green tobacco leaves (Deleano and
Trier [1912]), ergot (Kraft [1906, Ch. IV, choline], Rielander [1908, Ch. I]) and com-
mercial mushroom extract (Kutscher [1910, 4 ; Ch. IV, choline]).
For a long time the only recorded instance of the occurrence of betaine in animals
was Brieger's discovery of the base in mussels (Mytilus edulis ; [1886, 1, pp. 77-79; Ch. I)].
Later a number of other animal sources have become known : in commercial shrimp
extract (Ackermann and Kutscher [1907, 3]), in the muscles of Acanthias vulgaris, 2 per
cent, in embryos, 0*07 per cent, in adults (Suwa [1909, i], Kutscher [1910, 3]), in the crayfish,
Astacus fluviatilis (Kutscher [1910, 2]), in a cuttle-fish (Octopus) (Henze [1910]). A sub-
stance from the Japanese cuttle-fish Ommastrephcs identified by Suzuki and Yoshimura
[1909] as 5-amino-valeric acid is, according to Kutscher [1909], betaine. Betaine is also
present in mammalia; Bebeschin [1911] isolated 0*05 per cent, of betaine from ox-kidneys.
42 THE SIMPLER NATURAL BASES
Physiological Properties and Importance of Betaine.
The question as to whether betaine can be utilised by the animal
organism as a source of nitrogen is of some interest on account of the
increasing use of molasses as a cattle food. In the dog after intra-
venous injection nearly the whole of the betaine is rapidly excreted
in the urine, but when given by the mouth only about one quarter is
so excreted (Andrlik, Velich and Stanek [1902-3], Voltz [1907]).
Ruminants are more able to decompose betaine ; a cow accustomed
to molasses excreted no betaine in its urine, and a sheep only during
the first few days of feeding on molasses. Nevertheless, according to
Voltz, the whole of the betaine nitrogen is excreted in sheep even
when there is a deficiency of nitrogen in the food, and the organism
only retains the non-nitrogenous part of the betaine.
Although betaine is therefore not a food, it appears to be quite
harmless. Andrlik, Velich and Stanek for instance gave a rat intra-
venously betaine representing 0*24 per cent, of its body weight without
any appreciable effect.
Riesser [1913 ; Ch. V, creatine] injected betaine into rabbits and
thereby increased their muscular creatine content by 6-3-11-3 per cent.
He thinks that betaine may condense with an equimolecular proportion
of urea to form creatine and methyl alcohol. When betaine chloride
is melted with an excess of urea, methyl alcohol is given off. See
also pp. 77-78.
Waller and Sowton [1903 ; Ch. IV, choline] have described a toxic
action of betaine in the excised frog's heart and on isolated nerves, and
Waller and Plimmer [1903] on intravenous injection. According to
Velich [1904-5] the effects observed were due to hydrochloric acid,
owing to insufficient neutralisation of the betaine chloride injected.
Further experiments (unpublished) by Waller and Plimmer showed
that the injection of the betaine produced a slight lowering of the
blood pressure, which allowed some of the magnesium sulphate solution,
contained in the cannulae, to enter the circulation and exert a toxic
action. A slight effect on the frog's heart has also been noted by
Kohlrausch [1909, 1911].
With regard to the physiological importance of betaine in plants,
Stanek [1911, I] has recently attempted to prove that the base is not
a waste product. He has shown that more betaine is present in the
leaves than in the seeds from which the plant has been grown ; the
sugar beet may contain as much as I -2 per cent, of its dry weight as
betaine. Schulze and Trier [1912, I] have similarly found that betaine
BETAINES 43
is formed during germination in Vicia sativa and trigonelline in Pisum
sativum. In a later paper Stanek [1911, 2] has concluded that there
is more betaine in the dry substance of the young leaves than in that of
the old, that betaine is formed during the germination of the seeds and
that it travels from the roots to the leaves during the sprouting ; the
base collects in the etiolated leaves and on ripening of the organs it
disappears, probably because it travels back into the root. This latter
conclusion is not shared by Schulze and Trier [1910, I] who consider
betaine to be a waste product which no longer takes part in metabolism
(see also Trier [1912, 3, pp. 83-7 ; Ch. IV, choline]). These authors
point out that yeast cannot utilise betaine as a source of nitrogen
(Stanek and Miskovsky [1907]) and that betaines pass unchanged
through the animal organism. Some other fungi do utilise betaine,
however. Ehrlich and Lange [1913] have shown that, in contra-
distinction to ordinary cultivated yeasts, some wild yeasts like Willia
anomala transform betaine to glycollic acid : —
(CH3)3N . CH2 . COO + H2O = CH2(OH) . COOH + N(CH3)3
This is analogous to the change of primary amines, described on page
25. In any case it seems justifiable to draw the conclusion from
Stanek's experiments that betaine occurs most abundantly in those
parts of the plant where the vegetative processes are most active, and
Schulze and Trier consider that betaines collect in young leaves be-
cause they are formed there. Young orange leaves also contain a
greater proportion of stachydrine than the old ones.
Stachydrine, C7H13O2N.
Von Planta [1890] discovered a base in the edible tubers of
Stachys tuberifera. The base closely resembled betaine but yielded
an aurichloride with a smaller gold content ; it was further investigated
by von Planta and Schulze [1893, i, 2] who found it had the compo-
sition C7H13O2N, and Jahns [1896] isolated the same base from the
leaves of the orange tree (Citrus vulgaris] and proved the presence of a
carboxyl-group. Stachydrine is also present in the flowers of Chry-
santhemum cinerariczfolium and in Galeopsis ochroleuca (Yoshimura and
Trier [1912]) and (with betonicine) in Betonica officinalis (Schulze and
Trier [1912, I, section on betaine]). Stachydrine gives off dimethyl-
amine on heating with potassium hydroxide, and since it contains
two hydrogen atoms less than is required for a homologue of betaine,
Jahns considered it to be dimethylamino-angelic acid. The base is,
however, stable to potassium permanganate, and the deficiency of two
44 THE SIMPLER NATURAL BASES
hydrogen atoms is not due to unsaturation but to ring formation ; on
heating, vapours are formed which give the pyrrole reaction with pine
wood, and these facts led Schulze and Trier [1909, 2; 1910, 2] to re-
gard the base as a derivative of a-pyrrolidine carboxylic acid (proline)
which had meanwhile been recognised as a common fission product
of proteins. They suggested for stachydrine the formula I, which was
I
t
H2C CH,
H2C CH3 H2C
I
/•• • '•' -
H2C C— C : 0
\X 1
TJ /"» p X"^H TJ f
> \x OCHs -^
/
\y
N 0
N
WT
CH3 CH3
J X\
CH3 CH, C
I
II
III
CH
<COOCH3
CH3
I
soon afterwards also adopted by Engeland [1909, 3] after comparing
the properties of the methylation product of proline with those of
stachydrine as given in the literature. Finally Trier [1910] converted
stachydrine by distillation into the isomeric methyl ester of hygric acid 1
(formula II) and obtained stachydrine by hydrolysis of the methiodide
of this ester (formula III) ; the methiodide had already been synthesised
by Willstatter and Ettlinger [1903] starting from trimethylene dibro-
mide and ethyl malonate.
Stachydrine, as obtained from most sources, is optically inactive,
but Yoshimura and Trier [1912] have recently obtained the laevo-
rotatory variety from Galeopsis ochroleuca ; the base prepared by
Engeland by methylating proline (from caseinogen) is optically active.
Stachydrine has an unpleasant sweetish taste and is without marked
physiological action ; taken by the mouth it is excreted unchanged in
the urine. The isomeric methyl ester of hygric acid on the other hand
has a convulsant action (Trier [1910]).
Betonicine and Turicine, C7H13O3N.
Schulze and Trier [1912, I, section on betaine] have found that in
Betonica officinalis, a Labiate closely related to Stachys, stachydrine is
accompanied by a base containing an additional oxygen atom, which
base they have named betonicine. It is the N -dimethyl derivative of
oxypyrrolidine-carboxylic acid (oxyproline), which occurs as a constit-
uent of proteins.
The aurichloride C7H13O8N, HAuCl4 accompanies stachydrine auri-
1 Hygric acid, or N-methyl o-pyrrolidine carboxylic acid, is an oxidation product of
hygrine, an alkaloid accompanying cocaine in Coca leaves.
BETAINES 45
chloride, from which it is separated by its greater solubility in water ;
yield 5*5 grm. per kilo, of air dry Herba Betonicce.
In a later paper Schulze and Trier [1912, 2, section on betaine]
state that another substance of the formula C7H13O3N is also present.
Kiing and Trier [1913] have shown that the latter base is dextro-
rotatory and have named it turicine. It is the enantiomorph of
betonicine, which is laevo-rotatory. Kiing [1913] obtained both these
bases by methylation of oxyproline from gelatin, so that they have the
following constitution : —
H
\C CH2
HO/ | | H
H2C C-C : O
\/ I
N O
/ \
CH3 CH3
Trimethylhistidine, C9H15O2N3.
A base of the above composition was isolated by Kutscher [1910, 4]
from the lysine fraction of a commercial mushroom extract and after-
wards named hercynine. The base gave an intense red coloration
with sodium p-diazobenzene sulphonate, but neither Millon's reaction
nor any reaction for tryptophane. Only the aurichloride was prepared
and of this the melting point was not given. Kutscher considered
that the base was probably a trimethylhistidine and later Engeland
and Kutscher [1912, I] showed its identity with the synthetic betaine
obtained from a-chloro-/3-iminazolyl-propionic acid and trimethyl-
amine. The constitution is therefore
CH-NH>
The same base was obtained more recently by Reuter [1912, Ch. I]
from the arginine fraction of Boletus edulis (8 grm. of the monopicrate
from 2| kilos, of the dried fungus), and Barger and Ewins [1913] have
shown that it is also formed by the oxidation of ergothioneine (see
next section). The direct methylation of histidine with dimethyl-
sulphate leads to the formation of a pentamethyl derivative, since the
imino-group of the glyoxaline ring is also attacked (Engeland and
Kutscher [1912, 2]).
46 THE SIMPLER NATURAL BASES
Ergothioneine, Thiolhistidine-betaine, C9H15O2N3S.
Tanret [1909] isolated from ergot a base of the composition
C9H15O2N3S and named it ergothioneine.
Barger and Ewins [1911] have shown it to be the betaine of
thiolhistidine, as follows : —
I II III
CH— NH
IV V
On heating ergothioneine (I) with concentrated potassium
hydroxide solution, trimethylamine was given off almost quantitatively
and a yellow unsaturated acid (II) resulted, which still contained
sulphur and was almost insoluble m water. On boiling this acid with
dilute nitric acid, the sulphur was removed and iminazolylacrylic acid
(III) was formed and identified by comparison with a synthetic
specimen. This substance was subsequently shown by Hunter
[1912; Ch. II, urocanic acid] to be identical with urocanic acid from
dog's urine (see p. 36). On boiling ergothioneine with ferric chloride
the betaine of histidine itself is formed (IV) (see previous section). On
adding iodine in alcoholic solution two molecules combine to form the
quaternary iodide (V) which is much less soluble than the salts of ergo-
thioneine, to which it bears the same relationship as cystine does to
cystei'ne. By reduction with hydrogen sulphide this iodide is recon-
verted into ergothioneine. The crystals of the dimeric iodide have
the remarkable property of taking up excess of iodine from an aqueous
solution and becoming steel grey or blue, like narceine and other
substances.
The biochemical (interest of ergothioneine is chiefly due to the
sulphur atom contained in the glyoxaline ring. Oddly enough the
BETAINES 47
thiolglyoxalines are intermediate products in the chief method for
synthesising glyoxalines, due to Gabriel. The sulphur of ergothioneine
behaves very differently from that in cystine ; it is not removed by
alkalies and ergothioneine, therefore does not blacken lead hydroxide
solution on boiling. On the other hand the sulphur atom is much
more readily attacked by weak oxidising agents such as ferric chloride.
Bearing this in mind we may perhaps hope to isolate ergothioneine
or similar sulphur compounds from sources other than ergot.
The physiological activity of ergothioneine is slight and it does
not make any significant contribution to the action of ergot.
Hypaphorine, Trimethyltryptophane, C14H18O2N.2.
Hypaphorine is the betaine of tryptophane and has the constitution
CH
CH - r.C-CH,. CH . CO
rr I ll IL
It was discovered by Greshoff [1898] in the seeds of Erythrina
Hypaphorus, Boerl., a tree grown for the sake of its shade in the coffee
plantations of Eastern Java, and known locally as " dadap minjak ".
The constitution of hypaphorine has been investigated by Van Rom-
burgh [1911]. On heating with concentrated aqueous potassium
hydroxide indole and trimethylamine result. The constitution was,
however, determined by the synthesis from tryptophane (Van Rom-
burgh and Barger [1911]). On heating tryptophane in methyl
alcoholic solution with sodium hydroxide and methyl iodide the quater-
nary iodide of methyl-a-trimethylamino-/3-indolepropionate is formed
CH
CH ^ — C— CH2 . CH . COOCH3
CH \/l\/CH i(CH3KI
CH NH
and this, on warming with dilute alkali, yields a substance identical
with the naturally occurring hypaphorine.
Physiological Action of Hypaphorine. — The substance has hardly any
action on rodents and pigeons; thus intravenous doses of 0-5-1 grm.
do not affect rabbits and the unchanged substance is rapidly secreted
in the urine. In frogs, however, doses of 12-1 5 mg. produce increased
reflex irritability and tetanus, lasting for days in non-fatal cases.
Trigonelline, C7H7O2N.
This substance, the betaine of nicotinic acid, is not derived from a
protein fission product ; it contains a pyridine nucleus and is there-
48 THE SIMPLER NATURAL BASES
fore to some extent more akin to the alkaloids. As it is however very
similar to stachydrine, and as it has moreover been found in a number
of species belonging to widely different natural orders, its inclusion
here may be justified. Trigonelline was discovered by Jahns [1885]
in the seeds of Trigonella foenum graecum (the Fenu greek). It has
also been obtained from the seeds and seedlings of Pisum sativum
(Schulze and Winterstein [1910]) ; from the seeds of Phaseolus vulgaris,
Cannabis sativa, Avena sativa (Schulze [1896 ; Ch. IV, choline], Stro-
phanthus hispidus, and 5. Kombe, Thorns [1898, I, 2 ; Ch. IV, choline]
and Coffea arabica, Polstorff [1909, 2; Ch. IV, choline]); from the
tubers of Stachys tuberifera and from potatoes (Schulze [1904 ; Ch. IV,
choline]); from the roots of Scorzonera hispanica and the tubers of
Dahlia (Schulze and Trier [1912, I ; section on betaine]). It is generally
present in very small quantity and will doubtless be found to occur in
many more species.
Nicotinic acid, from which trigonelline is formed by methylation,
occurs in rice polishings (Suzuki, Shimamura and Odake [1912],
Funk [1913] ; both references in Ch. VII, vitamine, oryzanin). When
this acid is given to dogs, trigonelline appears in the urine (Acker-
mann [1912, l]).
The constitution of trigonelline was established by Jahns [1887].
CH CH
CH/^j C . CO CH/^C . COOH
CH" ^'CH — * CH^JCH
N O N
^3
On heating with concentrated hydrochloric acid to 270° nicotinic (/?-
pyridine-carboxylic) acid was formed and trigonelline was shown to be
identical with the " methylbetain " of nicotinic acid, previously synthe-
sised by Hantzsch [1886].
Trigonelline is physiologically inert; given subcutaneously, O'I2
grm. had no effect on frogs, nor O'5 grm. on rabbits (Jahns [1887];
compare also Kohlrausch [1909, 1911; section on betaine]). The
methylation of nicotinic acid to trigonelline in the dog, discovered by
Ackermann [1912, i], is similar to the methylation of pyridine to
methylpyridinium hydroxide (see the next section).
Other Pyridine Bases.
Although they are not betaines, other derivatives of pyridine may be referred to here.
For pyridine derivatives formed in putrefaction see Chapter I, p. 17.
His [1887] showed that when pyridine acetate is given by the mouth to dogs, about
one quarter may be recovered from the urine as the quaternary base, methyl -pyridinium
hydroxide.
BETAINES 49
II I
A
CH3 OH
The isolation was carried out by means of potassium mercuric iodide, and conversion into
the gold and platinum salts. Kutscher and Lohmann [1906, 4 ; section on butyro-betaine]
obtained the same base from normal human urine (at first [1906, 3] they mistook it for
neurine). They [1907] consider that it is derived from the pyridine of tobacco smoke and
of roasted coffee; 10 litres of men's urine yielded 0*17 grm. of the aurichloride, and 100
litres of women's urine 2'6 grm. ; the greater content of women's urine they ascribe to
the " bekannte Vorliebe der Frauen fiir pyridinhaltigen Kaffee ". Roasted coffee beans con-
tain 0*02 per cent, of pyridine [Bertrand and Weisweiller, 1913]. Methyl-pyridinium
chloride has also been obtained from a commercial shrimp extract (Ackermann and Kutscher
[1907, 4 ; under betaine]). The physiological action was investigated by Kohlrausch [1909,
1911 ; under betaine]. The platinichloride (C6H8N)2PtCl6 forms large orange coloured
plates, mp. 205-207°, little soluble in cold water, readily in hot, and the aurichloride
C6H8NAuCl4 yellow needles, mp. 252-253°, very little soluble in cold water.
Achelis and Kutscher [1907] obtained 0-7 grm. of 7-picoline aurichloride mp. 201°
from 10 litres of horse urine. This salt has the same composition as the preceding and is
said to be derived from pyridine derivatives of the fodder.
4. u O CO
7-n-Butyro-betaine, (CH3)3 • NSj >CH2
\CH2— CH/
Among the ptomaines isolated by Brieger [1886, I, p. 27 ; Ch. I]
from horse meat which had putrefied for four months, was a base
C7H17O2N. The chemical and physiological properties, as described
by Brieger, correspond very closely with those of a betaine C7H15O2N
obtained a few years ago by Takeda [1910] from the urine of dogs
poisoned with phosphorus ; Engeland and Kutscher [1910, 3] obtained
Takeda's base by methylating 7-amino-butyric acid, so that there is no
doubt as to its constitution ; the identity with Brieger's base is almost
equally certain, in which case his formula should contain two hydrogen
atoms less.1
7-Butyro-betaine was first synthesised by Willstatter [1902, i ; under
betaine] and was also obtained by Krimberg [1907, 2] by the reduction
of carnitine (see next section). Brieger isolated it from that part of the
precipitate with mercuric chloride, which was the more soluble in water.
After removal of the mercury, the base was precipitated as aurichloride.
The physiological action was studied in some detail by Brieger.
On frogs it has a curare action, in accordance with the fact that it is a
quaternary base and a 7-betaine. In the a-betaines so far described the
1 Brieger's ptomaine and 7-butyro-betaine have a very similar composition, a gold salt of
identical melting point, a soluble picrate and similar reactions to alkaloidal reagents : both
arrest the frog's heart in diastole.
4
50 THE SIMPLER NATURAL BASES
basic properties are more completely neutralised by the carboxyl-group,
which is probably the reason for their physiological inertness (com-
pare also the section on w-amino-acids, p. 33). Brieger found that
10 mg. of his hydrochloride arrested the heart of a frog in diastole.
In rabbits 0-05-0-3 grm. produced mydriasis, salivation, clonic con-
vulsions, often violent lowering of body temperature, dyspnoea,
paralysis and ultimately (after several hours) death with the heart in
diastole (Brieger [1886, I, pp. 29-31 ; Ch. I]).
Brieger obtained two other bases of the composition C7H17O2N. One of these is
gadinine, obtained from putrid cod fish (Bocklisch [1885, Ch. I], Brieger [1885, I, p. 49 ;
Ch. IJ) and isolated as platinichloride. It " appeared " to be physiologically inert and the
solution of the hydrochloride yielded a precipitate with picric acid, but not with gold chloride.
Against these differences we may set the fact that the hydrochloride, like that of y-butyro-
betaine and of betaine itself, was insoluble in absolute alcohol.
The other base C7H17O2N is typhotoxine, obtained from cultures of typhoid bacilli
(Brieger [1886, i, p. 86 ; Ch. I]). The melting point of the aurichloride was identical with
that of the ptomaine from putrid horse meat (176°). Typhotoxine, however, yielded a spar-
ingly soluble picrate, a yellow coloration with diazobenzene sulphonic acid, and amorphous
precipitates with potassium tri-iodide, potassium mercuric iodide and potassium cadmium
iodide. The physiological action of typhotoxine was also somewhat different from that of
the ptomaine from putrid horse meat.
It does not seem wholly impossible, however, that all three bases were identical with
•y-butyro-betaine.
Carnitine (Novaine, a-Hydroxy-7-butyro-betaine),
O --- CO,
CHOH-
Carnitine, C7H15O3N, is a hydroxy-derivative of the base described
in the previous section arid was discovered in extract of muscle by
Gulewitsch and Krimberg [1905]. A few months later Kutscher
[1905] obtained from Liebig's extract of meat a base " novain "
which Krimberg [1908, i] proved to be identical with carnitine ; the
identity has been admitted by Kutscher' s pupils, if not explicitly by
Kutscher himself. According to Kutscher a base C7H16O2N, isolated
by Dombrowski [1902] from normal human urine, was identical with
novaine ; Kutscher thinks that in most cases (except in the dog)
novaine passes into the urine as its reduction product reducto-novaine.
Both carnitine and novaine were found by their discoverers to yield
trimethylamine and crotonic acid (or an isomeride) on heating with
baryta. By boiling with phosphorus and hydriodic acid Krimberg
[1907, 2] reduced carnitine to 7-butyro-betaine.
The only doubt now remaining was with regard to the position of
the hydroxyl group in carnitine. Krimberg at first favoured the
/3-position, but /3-hydroxy-7-butyro-betaine
BETAINES 51
/o — co\
(CH3)3N/ J>CH2
CH2— CHOH
has been synthesised by Rollett [1910] and by Engeland [1910 2]
and was found to differ from carnitine, which is therefore most likely
a-hydroxy-7-butyrobetaine
i N CHOH.
\CH2.CH2/
The a-position of the hydroxyl group seems also to result from the
oxidation of carnitine by calcium permanganate (Engeland [1909, I])
to /?-homobetaine
o— co
(CH3)3 : N/
\CH2CH2
Racemic carnitine has probably been obtained by Fischer and Goddertz
[1910] from 7-phthalimido-a-bromobutyric acid; the melting point
of the platinichloride agrees with that of natural carnitine, but the
aurichloride has a much higher melting point. Carnitine may be pre-
pared from meat extract by Gulewitsch and Krimberg's method, or by
that of Kutscher ; the former method, in which the filtrate from carno-
sine is precipitated with potassium bismuth iodide, gives apparently
the better yield (1-3 per cent, of the Liebig's extract employed).
Smorodinzew [1913; Ch. II, carnosine] obtained O'O2 per cent, of
carnitine from fresh horse meat. Carnitine probably passes unchanged
into the urine, for Kutscher and Lohmann [1906, 2] could isolate
novaine ( = carnitine) from the urine of a dog fed on meat extract but
not from normal dog's urine. In the rabbit carnitine is, perhaps, re-
duced to butyrobetaine, according to Engeland [1908, I]. The physi-
ological action of novaine ( = carnitine) has been studied by Kutscher
and Lohmann [1906, I]. One gram, given hypodermically to a cat,
produced serious disturbance of the digestive tract ; given intravenously
novaine has a slight depressor action. Oblitine, a base obtained by
Kutscher from meat extract, is according to Krimberg merely carnitine
ethyl ester formed from carnitine during Kutscher's process of extrac-
tion (see appendix).
Reductonovaine C7H15ON was isolated as the aurichloride
C7H16ONC1, AuCl3, mp. 155-180°, from women's urine by Kutscher
[1907, 2] who regards it as formed by loss of water from novaine to
which it stands in the same relation as neurine to choline.
4*
52 THE SIMPLER NATURAL BASES
Myokynine (1-Hexamethylornithine ?), CnH28O4N2.
Working with Kutscher's method, Ackermann [1912, 2] has isolated
from the lysine fraction of an extract of dog's muscle a platini-
chloride CnH30O4N2PtCl6, insoluble in ethyl alcohol, mp. 233-234°.
The corresponding base was laevo-rotatory and gave off two molecular
proportions of trimethylamine on heating with baryta. The composi-
tion of the platinichloride agrees with that of a platinum salt of hexa-
methylornithine with 2H2O. Hexamethylornithine was, therefore,
prepared by methylating ornithine, and was found to be dextro-rotatory
and to yield a platinichloride with iH2O melting at 232-233°. It is
not unlikely, therefore, that myokynine is the enantiomorph of the
synthetic base, having the constitution : —
/OH HOX
(CH3)3 : N<^ ^>N : (CH3)3
CH2 . CH2 . CH2 . CH . COOH
Later Ackermann [1913, I] obtained 3 grm. of the same platini-
chloride from 30 kilos, of fresh horse meat. The base contains one
carboxyl group. Unlike the natural base, synthetic hexamethylorni-
thine gives a pyrrole reaction when heated with zinc dust. Ackermann
points out that ornithine to some extent resembles glycine (compare
the formation of ornithuric and hippuric acids) ; trimethyl-glycine
or betaine has already been isolated from the muscles of a number
of animals.
CHAPTER IV.
CHOLINE AND ALLIED SUBSTANCES.
THE previous chapters have dealt with basic substances derived from
the amino-acid units of proteins by various modifications. We must
next consider two bases which enter into the composition of the phos-
phatides ; they are units or " Bausteine " of these compounds, and are
analogous to the amino-acids (described in Plimmer's " Chemical Con-
stitution of the Proteins "). One of these units, choline, is apparently
present (in a combined form) in every living cell ; the other, amino-
ethyl alcohol, is probably the precursor of choline.
Allied to choline there are two bases, neurine and muscarine, which
are derived from choline by dehydration and probably by esterifkation
respectively. These bases do not enter into the composition of phos-
phatides ; their physiological behaviour is different from that of
choline ; they are modified units and are therefore comparable to the
modified amino-acids with which we have been concerned so far.
In this chapter are also included two other bases with pentavalent
nitrogen and without a carboxyl-group ; they are trimethylamine
oxide and neosine ; the latter is perhaps a homologue of choline.
Betaine is generally grouped with choline on account of a more or
less accidental chemical connection, for it can be obtained in the
laboratory by oxidising choline. There is, however, a considerable
physiological difference between the two substances, for choline is a
structural unit of phosphatides, but betaine plays no such part either
in the phosphatide or in the protein molecule. Nor is a genetic
relationship between the two substances apparent in the organism.
It has been suggested that betaine is formed by the oxidation of choline,
but recent work has made the conclusion almost inevitable that
betaine is not formed in this way, but by the methylation of glycine
(glycocoll), like the other betaines described in Chapter III. Choline
and the substances derived from it further differ from the betaines in
being strong bases, having a marked physiological action. To em-
phasise all these points of difference the two groups of substances are
described in separate chapters.
53
54 THE SIMPLER NATURAL BASES
Choline, Trimethyl-/3-hydroxy-ethyl-ammonium Hydroxide,
/PIT N • XT/OH
NCH2.CH8OH.
Strecker [1849] obtained from pig's bile the platinichloride of
a base, of which he later [1862] published the formula and a further
description, and which he then named choline. Meanwhile von Babo
and Hirschbrunn [1852], by hydrolysis of the alkaloid sinapin from
white mustard seeds, had prepared a strong base which was well
characterised by its platinichloride and was named sinkatin (from
Sinapis and alkali). The identity of the base from mustard with
that from bile was established by Claus and Keese [1867], but never-
theless Strecker's (later) name has passed into general use. Con-
fusion was introduced when Liebreich [1865] obtained a base by
the hydrolysis of the brain substance protagon, and termed it
neurin. The analysis of an impure platinichloride led Liebreich
to the erroneous formula C5H12ON, corresponding to vinyl-trimethyl-
ammonium hydroxide, and to this substance the name neurine has
become definitely attached. The identity of Liebreich's protagon
base with choline was established by Dybkowsky [1867] and for
some years neurine was used as a synonym for choline, to which
the name bilineurine was at one time also applied. The true formula
of Liebreich's "neurin" was determined by Baeyer [1866, under
neurine] who also converted it into the vinyl base [1869, under
neurine], and " nevrine " (= choline) was first synthesised by Wurtz
Since choline is a constituent of lecithin, it occurs probably in all
living cells. It has been isolated by Schulze and his collaborators
from every plant extract examined by them for its presence [Schulze
and Trier, 1912, 3], Choline has been found in the following
tissues : —
In the brain: as phosphatide, Liebreich [1865], Gulewitsch [1908, i], Vincent and
Cramer [1904], Cramer [1904], Coriat [1904], Thudichum [1884, 1901 ; under amino-ethyl-
alcohol] ; it is not present in the free state, Kauffmann [1911]. In the cerebro-spinal fluid
in disease (Mott and Halliburton [1899] ; see below for an account of the controversy on
this point). In many viscera (Kinoshita [1910, 2]), in the adrenal gland (Hunt [1899-1900],
Lohmann [1907, 1911]), in the thymus, thyroid and lymphatic glands, and in the spleen
(Schwarz and Lederer [1908]), in blood and in serum (Letsche [1907; Ch. IV, creatine],
Gautrelet and Thomas [1909]), in ox testes (Totani [1910, i]), in semen (Florence [1897]),
in egg-yolk, the most convenient natural source (Diakonow [1868]), in autolysed pancreas
(Kutscher and Lohmann [1903]), in meat extract (Kutscher [1906, i ; Ch. V, creatine]), in
putrid horse meat (Gulewitsch [1884, Ch. I]), in human corpses (Brieger [1885, 2, p. 17;
Ch. I]), in bile (Strecker [1849]), in secretine (von Fiirth and Schwarz [1908]), in cheese
(Winterstein [1904]), in herring brine (Bocklisch [1885, Ch. I]), in salted fish (Morner
CHOLINE AND ALLIED SUBSTANCES 55
[1896, Ch. I]), in carnaubon, a glycerine free monophosphatide from ox kidney (Dunham
and Jacobson [1910]), in sahidin (Frankel and Linnert [1910]), from sinapin by hydrolysis
(von Babo and Hirschbrunn [1852]), in seeds of Vicia sativa and Pisum sativum (Schulze
[1890]), of Strophanthus (Thorns [1898, I, 2]), of Avena sativa (Schulze and Pfenninger
[1911]), in cotton seeds and beechnuts (Boehm [1885, 2]), in seeds of Trigonella feenum
graecum and of Cannabis sativa (Jahns [1885]), in seeds of Artemisia cina (Jahns [1893]),
in etiolated seedlings of lupins and of Cucurbita (Schulze [1887]), in seedlings of Soya
hispida (Schulze [1888]), in malt and wheat germs (Schulze and Frankfurt [1893]), in rice
polishings (Funk [1911]), in potatoes and Dahlia tubers (Schulze [1904]), in tubers of
Stachys tuberifera and in orange leaves (Schulze and Trier [1910, 2; Ch. Ill, stachy-
drine]), in beet molasses (von Lippmann [1887]), in roots of Atropa Belladona, Hyoscyamus
and Ipecacuanha (Kunz [1885, 1887]), in bamboo shoots (Totani [1910, 2]), in the flowers of
Chrysanthemum cineraria folium (Yoshimura and Trier [1912; Ch. Ill, stachydrine]), in
Areca nuts, in pignuts (Arachis hypogcea] and in lentils (Jahns [1890]), in kola nuts (Ilex
Paraguay ensis), Indian tea, and cocoa beans (Polstorff [1909, 2]), in hops and therefore in
beer (Griess and Harrow [1885]), in grape juice and wine (Struve [1902]), in Sesame,
Cocos, and palm seed press cake (Schulze [1896]), in the subterranean parts of Brassica
Napns, Helianthus tubcrosus, Scorzonera hispanica, Cichorium Intybus, Apium graveolens,
Daucus carota and in the aerial parts of Salvia pratensis and Betonica officinalis (Schulze
and Trier [1912, 3]), in ergot (Brieger [1886, 2; Ch. I], Kraft [1906], Rielander [1908,
Ch. I]), in Amanita muscaria (Harnack [1875 '•> under muscarine]), in Boletus luridus,
Amanita pantherina and Helvetia esculenta (Boehm [1885, I » under muscarine]), in Can-
tharellus cibarius, Agaricus campestris, and Boletus edulis (o'oi5-o'oo5 per cent. ; Polstorff,
[1909, i]), in commercial mushroom extract (Kutscher [1910, 4]), in Russula emetica (Robert
[1892]) and in Boletus satanas (Utz [1905]).
The amount of choline obtainable from most sources is very small (in
animal viscera and in seeds often of the order of 0*02 per cent). Schulze
considered that in seeds at least some of the choline is in the free state ;
he showed [1892, I] that in Vicia sativa the choline content increases
during germination from 0*017 per cent, in the seeds to 0*06 per cent,
in the seedlings. The additional choline in the latter is derived from
lecithin, of which the seeds contain 0*74 per cent., but four weeks' old
seedlings only 0-19 percent. We thus see that choline behaves in the
same way as the amino-acids of protein, which are also formed by
hydrolysis during germination. Betaine, which is also present in the
seeds, on the other hand does not change in amount during germina-
tion, for it is not a unit or <( Baustein ".
The choline of the brain does not occur even partially in the free
state. Liebreich [1865] obtained it by the hydrolysis of protagon ;
Gulewitsch [1899] found that at most one-fifteenth of the total
amount is free choline, and KaufTmann [1911] has shown that if
perfectly fresh ox brain is worked up rapidly, no free choline is obtain-
able. According to Coriat [1904] lecithin is not affected by try ps in
or pepsin, but in autolysis choline is slowly split off by a ferment, which
could not be isolated ; during putrefaction choline is liberated more
rapidly.
Mott and Halliburton [1899] found choline in the cerebro-
56 THE SIMPLER NATURAL BASES
spinal fluid in certain degenerative nervous diseases, such as general
paralysis of the insane, and they regard it as a break-down product of
nerve substance. They used platinic chloride for the isolation, but
since the amount of choline to be detected is at most very small, and
since potassium and ammonium salts are also present, a good deal of
controversy has taken place as to the identity of the platinichloride
obtained.
Probably Mott and Halliburton's salt was contaminated with potassium, since even
anhydrous alcohol, as employed by Donath [1905-1906], dissolves ammonium chloride.
Donath has attempted to utilise the double refraction and chromatic polarisation of choline
platinichloride which is not given by the isotropic crystals of the potassium and ammonium
salts. The conclusions of Mott and Halliburton and of Donath have been criticised by
Vincent and Cramer [1904], by Allen and French [1903] and by Mansfeld [1904] ; Rosenheim
[1905-6, 1907] and Allen [1904] have therefore attempted to find a more characteristic test
in Florence's periodide reaction (see below) which may be applied to the platinichloride,
or directly to the crude choline chloride.1 According to Rosenheim and to Allen choline
is indeed present in the cerebro-spinal fluid in certain diseases, but Donath's suggestion
that choline is present in epilepsy and is the cause of the convulsions cannot be upheld
(Allen [1904], Kajura [1908], and especially Handelsman [1908]). At most traces are
present, wholly inadequate to account for the convulsions. Other authors, however, do
not admit that choline has been demonstrated in the cerebro-spinal fluid even in diseases
where there is a break-down of nervous tissue. Webster [1909] considers that no choline
test hitherto employed is satisfactory. Kauffmann [1908, 1910] thinks that if traces of
choline are present they are too small to be recognised with certainty. Kauffmann and
Vorlander [1910] consider that the dimorphism of choline platinichloride (and conversion
of the regular crystals into those of the monoclinic system, see below) affords a most char-
acteristic test, and Kauffmann has concluded that an organic base is present in the cerebro-
spinal fluid, which is not identical with choline. Stanford [1913] has recently arrived at the
same conclusion, that the base present in disease gives alkaloidal reactions, but no tri-
methylamine. Handelsman [1908] has emphasised the fact that on igniting the platini-
chloride the odour of trimethylamine is never observed. It would appear that this con-
troversy can only be ended by a satisfactory analysis of the platinum salt ; the only
published analysis (by Mott and Halliburton) is of little value (Ft found 34-8 per cent. ;
calculated 31*6 per cent.).
According to Mott and Halliburton the choline set free in nervous lesions passes into
the blood, a conclusion shared by Allen [1904], criticised by Vincent and Cramer [1904] and
particularly by Vincent's pupil Webster [1909], and maintained by Halliburton [1905],
Choline has been synthesised by several methods : —
1. By the action of trimethylamine on ethylene oxide in concen-
trated aqueous solution (Wurtz [1867]).
/O\ /OH
(CH3)3 : N + / \ + H20 = (CH3)3 : N/
\CHl.CHJtQH.
2. Trimethylamine combines with dry ethylene dibromide at
1 1 0-112° to yield trimethylamino-bromethylium bromide (Hofmann
[1858, under neurine]).
1 Possibly the very slight solubility of choline nitric acid ester perchlorate might be
utilised with advantage.
CHOLINE AND ALLIED SUBSTANCES 57
/Br
(CH3)3 : N + Br . CH2 . CH3 . Br = (CH3)3 : N(
\CH2.CH2Br.
By acting on the latter substance with silver oxide, Hofmann obtained
the vinyl base instead of choline. Choline is however obtainable from
it in two ways ; —
(a) by boiling for eight days with silver nitrate (Bode [1892])
/Br /Br
(CH3)3 : N( + AgN08 + H,O = (CH3)3 1 N/ + AgBr + HNO3
\CHa . CH2 . Br \CH2 . CH2OH
(fi) by heating with twenty-five parts of water to 160° for a few
hours (Kriiger and Bergell [1903])
/Br /Br
(CH3)3 : N/ + H20 = (CH3)3 j N< + HBr.
\CH2 . CH2Br XCH2 . CH2OH
3. Rather more than one equivalent of trimethylamine gas is passed
into ethylene chlorohydrin cooled to - 12° to - 20° in a tube which
is subsequently warmed to 80-90° ; the yield is almost quantitative
(Renshaw [1910]).
/Cl
(CH3)3 i N + Cl . CH2 . CHoOH = (CH3)3N/
\CH2 . CH2OH.
4. By the methylation of amino-ethyl alcohol (Trier [1912, 2;
under amino-ethyl-alcohol])
/I
3(CH3)I + 2NaOH + NH,CH2 . CHaOH = (CH3)3 1 N/ + 2NaI + 2H2O.
\CH2 . CH2OH
The methods of Kriiger and Bergell and of Renshaw appear to be the
most convenient.
A method for the estimation of choline in animal tissues has been
described by Kinoshita [1910, 2]. For the isolation of choline from
plant extracts, Jahns [1885] has employed potassium bismuth iodide
(Kraut's reagent), Schulze has used phosphotungstic acid and mercuric
chloride and Stanek utilises potassium tri-iodide. The two last named
methods are more or less quantitative. Stanek 's method [1905, 1906,
I, 2] is the most convenient for the quantitative estimation of choline
in the presence of betaine when other bases yielding periodides are ab-
sent (compare Kiesel [1907]). For a description of Stanek's and
Schulze's methods see the appendix (Chapter VIII). The tests for,
and chemical properties and salts of, choline are also described in the
appendix (Chapter VIII).
NH3 + | 0 = I
CH9
58 THE SIMPLER NATURAL BASES
Amino-ethyl Alcohol (Colamine) and the Origin of Choline; the
Possible Presence of other Bases in Phosphatides.
By the hydrolysis of kephalin (a phosphatide from the brain) by
means of baryta, Thudichum [1884, 1901] obtained long ago, in ad-
dition to choline, a base having the composition of "oxethylamin,"
NH2.CH2.CH3OH.
During the last few years Trier has isolated a base of the same com-
position from lecithin of various sources and has definitely identified it
as hydroxy-ethylamine or amino-ethyl alcohol. By hydrolysis of the
phosphatide from beans (Phaseolus vulgaris) Trier [1911] obtained
a fraction, representing one-seventh of the nitrogen content of the
phosphatide, which yielded an aurichloride C2H5ON . HAuCl4 ,
identical with that of a base previously synthesised by Knorr from
ammonia and ethylene oxide : —
CH2\ CH2OH
l>0=|'
CH2/ CH2NH2.
The same base was subsequently obtained from the lecithin of peas
and oats and also from commercial ovolecithin of Merck (Trier [1912, i]).
The amino-ethyl alcohol can be estimated in phosphatides by means
of Van Slyke's method (see Plimmer's " Chemical Constitution of the
Proteins," Part I, p. 69). Trier [1913, 2] concludes from this that the
base is joined to the rest of the phosphatide molecule by means of its
hydroxyl group. In one specimen of ovolecithin the amino-nitrogen
was nearly half the total.
Baumann [1913] and Renall [1913] also used Van Slyke's method
and showed that kephalin from human brain and from that of the
sheep and ox contains as only base amino-ethyl alcohol and that here
too the primary amino-group is free. They could not find choline and
another base, which Thudichum believed to accompany the amino-
ethyl alcohol.
Trier considers that choline is formed from amino-ethyl alcohol
by the biologically common process of methylation, in the same way
that the betaines are derived from amino-acids. Thus there would be
no genetic relationship between choline and betaine.
The question is then : How is amino-ethyl alcohol itself formed ?
Winterstein and Trier [1909, p. 31 1]1 put forward the hypothesis
that formaldehyde is condensed to glycollic aldehyde and that the
latter is converted by ammonia into amino-acetaldehyde. By simul-
1 This and the subsequent references in this section will be found in the bibliography
under choline.
CHOLINE AND ALLIED SUBSTANCES 59
taneous oxidation and reduction (Cannizzaro's reaction) amino-ethyl
alcohol and amino-acetic acid (glycine) are then supposed to be formed
from the aldehyde.
CH3OH + NH, CH2 . NH2
2. CH20 -> | i |
CHO CHO
formaldehyde glycollic aldehyde amino-acetaldehyde
CH2 . NH2 CH3 . NH2 CH2 . NH2
CHO CH2OH COOH
+ H20
CHO
amino-acetaldehyde amino-ethyl alcohol glycine
In his recent book on the simple plant bases Trier [1912, 3, p. 33]
has modified the above hypothesis and imagines that glycollic aldehyde
first undergoes Cannizzaro's reaction and that the two products of this
reaction (glycol and glycollic acid) then condense with ammonia
CH2OH CH2OH CHoOH
2 I + H30 +1
CHO CH2OH COOH
glycollic aldehyde glycol glycollic acid
CH2OH CH,.NH2 + H0O
| + NH* = I
CH2OH CH,,OH
glycol amino-ethyl alcohol
CH2OH CH2 . NH2
I + NH3 = | +. H20
COOH COOH
glycollic acid glycine
Amino-ethyl alcohol and glycine are the simplest units for the forma-
tion of proteins and phosphatides respectively, and hence it becomes
intelligible why, as Stoklasa has pointed out, protein and lecithin
formation are two parallel processes. An argument for the biological
significance of Cannizzaro's reaction is the occurrence of a number of
alcohols as esters of the corresponding acid (e.g. benzyl benzoate and
cinnamyl cinnamate in balsams ; cetyl-palmitate C16H31O2 . C16H33 occurs
in spermaceti and ceryl cerotinate C.27H53O2 . C27H55 in Chinese wax).
A ferment causing Cannizzaro's reaction (" aldehyde mutase") has
been recently found in liver extracts by Parnas and by Batelli and
Stern (see Dakin's " Oxidations and Reductions in the Animal Body,"
pp. 105, 1 06, in this series of monographs).
In addition to Thudichum, Trier, and Baumann, who isolated
amino-ethyl alcohol, other investigators have suggested that phos-
phatides may contain bases similar to choline but containing fewer
alkyl groups. These investigations however require careful scrutiny in
the light of recent knowledge. Koch [1902] applied Herzig and
Meyer's method for the estimation of N-methyl groups to kephalin and
cerebrin and concluded that one N-methyl group is present in kephalin
none in cerebrin, and three in lecithin. Frankel and Neubauer, like
60 THE SIMPLER NATURAL BASES
Koch, failed to isolate Thudichum's non- methylated " ox-ethylamin "
from kephalin, and agreed with Koch that one N-methyl group is
present. Frankel and Linnert [1910] state that sahidin, from human
brain, also contains a base with fewer methyl groups than choline.
On the other hand Cousin [1907] could only obtain choline from
kephalin. Koch, and Frankel and Neubauer did not isolate their
supposed monomethylated base and their results have been criticised
by Baumann [1913]; he and Trier [1913, 5] find that amino-ethyl
alcohol, when heated with hydriodic acid, gives off some ethyl iodide,
thus simulating the presence of an N-methyl group. It should
further be remembered that the accuracy of Herzig and Meyer's
method for determining N-alkyl groups is not sufficiently great for
the certain determination of their number in a molecule of the size of
lecithin, and that its application becomes wholly illusory if more than
one base is present.
Further mention of the presence in phosphatides of bases other
than choline is to be found in papers by Erlandsen [1907] (on
cuorin from ox hearts), by Baskoff [1908] (on the phosphatides of
horse liver), by MacLean [1909], by Njegovan [1911], and in Trier's
book on plant bases [1912, 3, pp. 96-101]. According to Trier,
Njegovan's base "vidine" was merely choline containing a little
ammonia as impurity.
Neurine, Vinyltrimethyl-ammonium Hydroxide,
/OH
(CH3), : N(
\CH:CH2.
Neurine was the name applied by Liebreich to a base obtained in
the hydrolysis of protagon. Baeyer [1866] found that Liebreich's
neurine yielded a mixture of platinichlorides, difficult to separate,
but by means of the aurichlorides he subsequently [1869] showed that
the principal base was identical with Strecker's choline. For the other
base, which Baeyer obtained pure by the elimination of water from
choline by chemical means, he reserved the name neurine, and Brieger
[1885, I, p. 32] sharply differentiated the two bases; for a time
much confusion was introduced by the continued use, by some
authors, of neurine as a synonym for choline, but eventually the term
neurine was restricted to the unsaturated base.
According to Gulewitsch [1899, under choline] protagon does not
yield neurine at all, but only choline. It is very doubtful whether
neurine occurs in the body or body fluids, and apart from the old con-
fusion of nomenclature, statements concerning its presence should be
CHOLINE AND ALLIED SUBSTANCES 61
carefully scrutinised.1 Neurine occurs as a product of putrefaction and
was isolated by Brieger [1885, i, pp. 25-39] from putrid meat (horse, ox,
human corpses). Brieger studied the physiological action of neurine in
some detail and naturally assumed that the base was formed from
choline by bacterial action. This assumption has never been proved
rigidly, but the possibility should be taken into account with reference
to Kutscher's alleged discovery [1905 ; Ch. V, creatine] of neurine in
commercial meat extract. Krimberg [1906, I ; Ch. V, methylguan-
idine] could not find neurine in an extract of perfectly fresh meat
and concludes [1908,2; Ch. Ill, carnitine] that it is not present in
muscle. Lohmann [1909] obtained neurine from the supra- renal gland,
but here again it is not clear to what extent sterility was ensured.
Brieger [1885, I, p. 61] obtained neurine from fresh human brain
by hydrolysis with hydrochloric acid.
Neurine is most readily obtained synthetically and was first pre-
pared by Hofmann [1858] nine years before the synthesis of choline
by Wurtz. Hofmann treated the condensation product of trimethyl-
amine and ethylene dibromide with moist silver oxide, which removes
hydrobromic acid, and forms neurine bromide : —
/Br /Br
(CH3)3 I N/ + AgOH = (CH3)3 i N/ + AgBr + H2O.
\CH2 . CH.2Br \CH : CH,
Baeyer [1869] prepared neurine from choline by heating the
latter with concentrated hydriodic acid and then treating the resulting
iodo-compound with silver oxide as in Hofmann's synthesis. Neurine
is perhaps also formed from choline by boiling with concentrated baryta
and this may have caused it to accompany choline in Liebreich's
hydrolysis of protagon. According to Brieger [1885, i, pp. 33, 34]
neurine appears to be formed from choline by long standing in aqueous
solution.
Physiological Action of Choline and of Neurine.
When given subcutaneously or by the mouth to rabbits in doses of
i grm., choline produces no severe symptoms and is not excreted in
the urine (von Hoesslin [1906]). Riesser [1913; Ch. V, creatine]
found that rabbits often withstood a daily injection of 0*5-1 grm.
choline. Similarly the urine of rabbits, fed on lecithin, does not con-
tain choline, but only a little glycero-phosphoric and formic acids
1 Thus Kutscher and Lohmann's statement [1906, 2, under choline] that neurine occurs
in human urine has passed into the literature (" Biochemisches Handlexicon "), although
these authors subsequently [1906, 4 ; Ch. V, methylguanidine] stated that their supposed
gold salt of neurine was in reality methylpyridyl ammonium aurichloride.
62 THE SIMPLER NATURAL BASES
(Franchini [1908]). Muscarine, neurine and betaine, on the other
hand, are at least partially eliminated in the urine, and in this respect
choline behaves like an amino-acid unit of protein. Whether choline
is oxidised or whether it is synthesised into phosphatides is not known,
but the latter alternative is in agreement with the conception of choline
as a unit (Baustein) of phosphatides. The formation of choline in seed-
lings has been referred to above and its behaviour towards micro-
organisms is mentioned in the appendix.
Riesser [1913 ; Ch. V, creatine] has recently carried out some ex-
periments which suggest that choline, when injected subcutaneously,
may be partially converted into creatine. In some rabbits he increased
the muscular creatine content 10-15 per cent, by this means. Riesser
supposes that choline condenses with urea according to the following
equation : —
CH2OH / 2 CH2OH NH8
+ CO = I S
CH2 . N(CH3)3OH v CH2 . N(CH3) C +2 CH3OH
NH, XH
and that the alcoholic group of the condensation product is then
oxidised to a carboxyl group, yielding creatine. The choline must
therefore lose some of its methyl groups, and in support of this theory
Riesser quotes an experiment in which choline chloride is carefully
heated with sodium tellurite and sodium formate (the latter salt acting
as a reducing agent) ; the garlick-like smell of methyl telluride is pro-
duced ; see also p. 77.
The physiological action of choline has been studied by Gaehtgens,
and by Boehm [1885, 2] who observed salivation, myosis, and diastolic
arrest of the heart ; in frogs Boehm obtained general paralysis with
0*025-0-1 grm. ; in mammals O'Oi-O'O2 grm. injected intravenously
gave a rise of blood pressure. The action is somewhat analogous to
that of pseudo-muscarine (synthetic " muscarine "). Brieger [1885, I,
p. 38] found that the toxic action of choline is inhibited by atropine
(" in pracisester Weise ").
A detailed study of the action was made by Mott and Halliburton
[1899], who found that small doses of choline injected intravenously
cause a fall of blood pressure, but after a preliminary dose of atropine
a rise occurs.
The antagonism between choline and atropine has been confirmed
by all subsequent investigators, but a good deal of confusion and con-
troversy has resulted from a statement by Modrakowski [1908] that
pure choline always produces a rise of blood pressure and that the
CHOLINE AND ALLIED SUBSTANCES 63
depressant action observed by others was the result of an impurity.
Popielski [1910, i], in whose laboratory Modrakowski carried out his
experiments, shares the latter's views, but Mott and Halliburton's
statement that choline has primarily a depressent action has been
confirmed by Busquet and Pachon [1909], Abderhalden and Muller
[1910, 1911], Mendel and Underbill [1910], Pal [1910, 1911], Muller
[1910], Lohmann [1907, 1908], and most recently by Mendel, Under-
bill and Renshaw [1912].
The general conclusion is that Modrakowski's and Popielski's aber-
rant results are not to be explained by impurities in the choline em-
ployed by others, but rather to differences in anaesthesia and dosage.
With small doses up to I mg. per kilo, in dogs and cats under ether
or urethane, a fall of blood pressure always results, which with some-
what large doses may be followed by a slight rise. Larger doses, es-
pecially when repeated, may at once exert a pressor action. With
slight anaesthesia, or with the medulla oblongata cut, small doses may
also produce a rise of blood pressure.
The depressent action is partly due to an effect on the heart and
partly to vaso-dilatation in the limbs and splanchnic area. After
atropine, perfusion of an isolated organ produces only vase-constriction.
According to Muller this vaso-motor reversal depends on a paralysis
by atropine of the dilator elements of the vascular walls, and resembles
the adrenaline vaso-motor reversal by ergotoxine (Dale [1906, Ch. VI]).
Choline has a stimulant effect on the isolated muscle of the in-
testine, uterus and iris, resembling in this respect physostigmine some-
what closely. It further stimulates the secretion of the lachrymal,
salivary, and sweat glands. Salivation is one of the first symptoms of
choline poisoning in an intact animal (Brieger). The physiological
activity of choline is, however, slight, only about TV^j of that of
neurine. The minimal lethal dose for rabbits of I kilo, is 0*5 grm.
according to Brieger, but Mott and Halliburton were unable to kill an
animal by choline injections. Compare also Riesser [1913; Ch. V,
creatine].
The action of choline on isolated nerves and the excised heart of
the frog has been studied by Waller and Sowton [1903].
Hunt and Taveau [1911] have studied the action of a large number of synthetic
choline-like substances and their derivatives. In particular acetyl-choline
(CH3)3 N(OH) . CH3 . CH3 . O . OC . CH,
is remarkable in being 100,000 times as depressent as choline itself. According to Mr. A.
J. Ewins [Bio-Chem. J., 1914, 8, 44] acetyl choline is present in small quantity in some ergot
extracts. The lower homologue formocholine (CH8)8 N(OH) . CHaOH is also more active
than choline. The nitrous acid ester of choline is identical with Schmiedeberg and
Harnack's /s««fo-muscarine (see p. 68).
64 THE SIMPLER NATURAL BASES
Other synthetic substances allied to choline have been described by Schmidt [1891, 1904,
I, 2], Malengreau and Lebailly [1910, under homocholine], Mengefign], and Berlin [1910,
I, 1911, under homocholine] who gives further literature.
The action Q{ neurine shows a general resemblance to that of choline
and muscarine, and like these, it is antagonised by atropine. To
rabbits it is 10-20 times as toxic as choline (Brieger [1885, i, p. 39]) ;
on subcutaneous injection the lethal dose is about 40 mg. per kilo.
Cats are more susceptible and react violently to doses of a few milli-
grams. The effects are profuse salivation, dyspnoea, an initial accel-
eration and then a retardation of the heart beat and death in diastole ;
the intestine is stimulated to violent peristalsis ; there is often myosis
in rabbits and always in cats. Atropine is a powerful antidote. In
frogs there is a curare-like paralysis and diastolic arrest of the heart's
action, after injection of 1-2 mg. into the dorsal lymph sac.
Waller and Sowton [1903] studied the effect of neurine and other
bases on isolated nerves and on the excised heart of the frog ; neurine
was the most toxic, rather more than muscarine, and very much more
so than choline.
Lohmann [1911] finds that neurine in doses of 10 mg. first lowers
the blood pressure of rabbits and then raises it. The general effect of
neurine on the blood pressure is to produce a rise after a preliminary
fall (Mott and Halliburton [1899]; Pal [1911]). Minute doses, of
TTJW mg-> mav k£ either pressor or depressor. The rise of blood
pressure is due to constriction of the peripheral vessels (compare
Samelson [1911] who found, by the Laewen-Trendelenburg method,
that neurine acts on the frog's limb in a dilution of I : 800,000).
The physiological action of synthetic bases allied to neurine has been
described by Schmidt [1891, 1904, i].
Natural and Synthetic Muscarines and their Physiological
Action.1
Muscarine is the name given by Schmiedeberg and Koppe [1869]
to an extremely poisonous base which they obtained from Amanita
muscaria (the Fly Agaric). Very small amounts arrest the frog's heart
in diastole and the action is antagonised by atropine.
Other bases of somewhat similar composition and similar physio-
logical action have been obtained synthetically, and one of these was at
one time considered to be identical with natural muscarine. It seems
certain, however, that this is not so.
1 Compare the important addendum on p. 68.
CHOLINE AND ALLIED SUBSTANCES 65
Schmiedeberg's base was isolated as the gold salt which Harnack
[1875] found to be contaminated with choline ("amanitine ") auri-
chloride ; a separation was effected by crystallisation from hot water,
the muscarine salt being the more soluble. Harnack found muscarine
aurichloride to have the composition C5H14O2N . AuCl4 ; the base
therefore differs from choline in having an additional oxygen atom.
Soon afterwards Schmiedeberg and Harnack [1877] obtained a base
of this composition by heating dried choline chloride with con-
centrated nitric acid on the water bath ; the new base was isolated as
the platinichloride ; the chloride, when left in a desiccator, sets to
a crystalline mass and the base has according to Schmiedeberg and
Harnack the constitution (CH3)3 : NCI . CH2 . CH(OH)2, being
therefore a hydrated aldehyde like chloral hydrate (but compare
addendum, p. 68).
This synthetic, artificial, or flseudo-muscarinQ is chemically very
similar to the natural substance, and the physiological resemblance is
sufficiently close to have induced Schmiedeberg and Harnack to believe
in the identity of the two bases. Boehm [1885, 2] was the first to
point out the differences in the physiological action. He found that
^ mg. of flseudo-muscarine (from choline) was required to stop the
frog's heart in diastole, whereas the corresponding dose of natural
muscarine is only ^VsV mg-> according to Schmiedeberg and Harnack.
Recently this large difference in the activities of the two bases has been
confirmed in Schmiedeberg's laboratory by Honda [1911], who again
prepared natural muscarine and found it active on the frog's heart in
doses of ^VrV mg., according to the season of the year, whereas the
same effect was only produced by -J-i-J mg. of ^seudo-muscarme from
choline. Boehm further found that in larger doses (10 mg.) pseudo-
muscarine produces a curare-effect in mammals, which is not given
even by large doses of the natural base ; moreover there is no com-
plete antagonism between pseudo-muscanne and atropine : cats which
have been poisoned by pseudo-muscanne cannot be kept alive by a
subsequent dose of atropine. The curare-like action of pseudo-
muscarine on frogs is according to Boehm fifty times as great as that
of choline from which it is derived (the minimal paralytic doses being
O'l and 50 mg.), and according to Honda [191 1] flseudo-muscarine
has one-fifth of the activity of pure curarine in this respect. According
to H. Meyer (see below) pseudo-m\\scarmz causes contraction of the
pupil in birds, natural muscarine does not.
Another synthetic substance, much more distantly related to
muscarine than the oxidation product of choline, is trimethylamino-
5
66 THE SIMPLER NATURAL BASES
acetaldehyde, (CH3)3 : N(OH) . CH2 . CHO, which was first prepared
by Berlinerblau [1884] by the action of trimethylamine on mono-
chloracetal and subsequent hydrolysis, and later by Fischer [1893]
by the methylation of acetalamine. The platinichloride has the com-
position [(CH3)3N . CH2 . COH]2PtCl6 . 2H2O ; the water of crystallisa-
tion is given off at 105°. The constitution of this base is quite certain,
for Fischer [1894] oxidised it to betaine and accordingly suggested
for it the name betaine aldehyde. In an abstract of a dissertation
by Nothnagel [1893], E. Schmidt [1904, I, p. 47, under choline]
quotes a report by Hans Meyer, who found that the anhydro-muscarine
of Berlinerblau (= betaine aldehyde of Fischer) does not arrest the
action of the frog's heart in doses of 10 mg., nor does it produce vagus
inhibition in the mammalian heart in doses of several centigrams. It
causes salivation and sweating, however, and kills by respiratory par-
alysis. Betaine aldehyde differs also chemically from muscarine, but on
the other hand natural muscarine and Schmiedeberg and Harnack's
flseudo-muscarine are chemically very similar, according to Schmidt and
Nothnagel. The platinichlorides of both bases have the composition
[(CH3)3N . CH2 . CH(OH)9]2PtCl6 . 2H2O
and do not lose water at 1 00°. The physiological differences observed
by Boehm.were however also found by Hans Meyer ; pseudo-musc^rmo.
in doses of O' 1-0*05 mg- paralyses the intra-muscular nerve-endings of
a frog ; natural muscarine does not. The cardiac effect of the natural
base, even in doses of 6 mg., is counteracted by atropine, but this is
not so with pseudo-muscar'me. Natural muscarine does not affect the
pupil of birds, but maximal myosis is produced by a I per cent, solu-
tion of;to*dfo-muscarine.
Schmidt has suggested that the physiological differences may be
due to stereo-isomerism, but in this case the relationship cannot be
that between an optically active and a racemic modification, for then
the one variety could not be 10-15 times as active as the other.
Further investigation of the chemical properties of natural muscarine
is very desirable, but the base is unfortunately difficult to obtain in
sufficient quantity. Schmiedeberg's process of isolation was a compli-
cated one, and Harmsen [1903] calculates from physiological data
that Schmiedeberg only isolated about 6 per cent, of the muscarine
present in the fungus. According to Harmsen 100 grm. of fresh
fungus (=5 grm. of dried material) contain about 16 mg. of mus-
carine. The amount seems, however, to be very variable, as does also
the amount of choline which accompanies the muscarine. The chief
difficulty in isolating natural muscarine is the separation from choline.
CHOLINE AND ALLIED SUBSTANCES 67
Honda [1911] first separates a good deal of the latter base by means
of its acid tartrate, which is less soluble than the muscarine salt. The
discovery of a muscarine salt which is less soluble than the corres-
ponding choline salt would greatly facilitate the preparation of pure
muscarine.
The fate of pseudo-musczrme. (from choline) in the animal organism
has been investigated by Fiihner [1908, I ; 1909]. The lethal dose for
rabbits of 1-5 kilo, is O'3-O'5 grm. by the mouth and 0^04 -0*05 grm.
subcutaneousty ; the drug is partly secreted in the urine unchanged
(in the toad the whole is so excreted). In this respect ^seudo-muscarine
resembles betaine and differs from choline ; it is not a " Baustein ".
Harmsen has concluded that the muscarine content of Amanita
muscaria is quite insufficient to account for the poisonous effects of
eating this fungus and considers that the effect is mainly due to a
complex toxin insoluble in alcohol and not counteracted by atropine.
From an allied species Amanita phalloides, Abel and Ford [1906]
have prepared a haemolysin which they regard as a nitrogenous
glucoside.
Muscarine occurs in small quantity in Amanita pantherina and in Bol-
etus luridus (Boehm [1885, I, under choline]). Brieger [1885, i, p. 48,
Ch. I] isolated from putrid codfish a platinichloride (C5HUO2N)2 PtCl6 ;
the physiological action of the base was that of muscarine. The physi-
ological action of synthetic bases allied to muscarine has been described
by Schmidt [1891, 1904, I, under choline]; Brabant [1913] has re-
cently synthesised /3-homo-muscarine (CH3)3N(OH)CH2. CH2. CHO.
Trimethylamine Oxide, (CH3)3NO.
This base, the only member of its class known to occur naturally,
was isolated by Suwa [1909, I, 2] from the muscles of Acanthias vul-
garis. One dozen of this fish, yielding 23 kilos, of muscle, gave 20
grm. of the hydrochloride of trimethylamine oxide, together with a
quantity of betaine, but hardly any creatine, or creatinine.
The hydrochloride melts at 205-210°, the pier ate forms thin needles, mp. 197°, sparingly
soluble in ethyl alcohol and cold water ; the platinichloride forms rhombic leaflets, mp.
214° ; the aurichloride C3H9ON . HAuCl4, mp. 250°, is sparingly soluble in hot water.
In concentrated aqueous solutions of the hydrochloride alcoholic solutions of mercury
and cadmium chlorides precipitate C3H10ONC1 . 4HgCl2 . H2O and C;,H10ONC1 . CdCL
respectively.
By putrefaction and also (at least in part) in the organism of the
rabbit, trimethylamine oxide is reduced to trimethylamine from which
it can be produced by oxidation with hydrogen peroxide.
5*
68 THE SIMPLER NATURAL BASES
Neosine, C6H17O2N.
There is still a good deal of doubt concerning the nature of this
base, one of those obtained by Kutscher [1905, Ch. V, creatine] from
extract of meat Krimberg [ 1 906, 1, Ch. V, methylguanidine] could not
find neosine in fresh meat and doubted whether it is present in faultless
meat extract. Ackermann and Kutscher [1907, 4, Ch. Ill, betaine]
afterwards isolated the base from a commercial extract of shrimps
which is the most abundant source. They [1908] found that tri-
methylamine is given off on heating and accordingly surmised that
neosine is a homologue of choline, but various attempts to identify it
with synthetic choline homologues have failed, including the most re-
cent and thorough attempt of Berlin [191 1] who found that Kutscher's
neosine was contaminated with choline.
The uncertainty with regard to this base is shown by the various melting points ascribed
to the aurichloride. Kutscher found 202-205° '•> Kutscher and Ackermann 205° ; Engeland
[1908, i] for the base from meat extract 150-152° ; Berlin, after freeing the crude neosine
from choline, obtained a few grams of a gold salt melting at 244-245° from 6 kilos, of
Liebig's extract of meat.
Berlin has also reinvestigated the synthetic homocholines of previous authors and con-
cludes that Morley, Weiss, Partheil and more recently Malengreau and Lebailly [1910]
obtained £-homocholine (CH3)3N(OH) . CH2 . CHOH . CH3 of which the aurichloride
melts at 163-164°.
By the action of trimethylamine on trimethylene chlorohydrin CH2C1 . CHa . CH2OH and
(less readily) by the methylation of 7-amino-propylalcohol Berlin [1910, 2, 1911] prepared
7-homocholine (CH3)3N(OH) . CH2 . CH2 . CH2OH which yields an aurichloride crystallising
in leaflets and melting at 193°, a mercurichloride C6H16ONC1 . 6HgCl2, mp. 208°, and a picrate
exploding at 255°. The constitution of this base follows from its oxidation to homo-betaine
(CH3)3N(OH) . CH2 . CH2 . COOH, and since it does not contain an asymmetric carbon atom,
neosine, which is optically inactive, was at first regarded as identical with it. But the melting
points of neosine aurichloride (244-245°) and of neosine mercuric chloride C6H16ONC1 . 6HgCl2
(252°) render this hypothesis untenable. The physiological action of 7-homocholine is similar
to that of choline but slightly more intense (Berlin [1910, I, 1911]).
Addendum to Muscarine.
While this book was in the press Dr. H. H. Dale and Mr. A. J. Ewins have, according
to a private communication, established that the />s£7/d0-muscarine of Schmiedeberg and
Harnack and of Schmidt and Nothnagel is not an aldehyde at all, but the nitrous acid ester
of choline. The platinichloride has the formula [(CH3)3N . CH2 . CH2ONO]2PtCl6, instead
of [(CH3)3N . CH2 . CH(OH)2]2PtCl6 . aH2O. This explains why no water of crystallisa-
tion is given off at 100°; the loss of weight at 130° is due to decomposition. The percent-
age composition required by the two formulae is very similar, except as regards nitrogen,
the estimation of which presents difficulties here. This discovery further disposes of the
inherent improbability that two hydroxyls should be attached to the same carbon atom ;
such an arrangement has so far only been observed in compounds in which the carbon atom
is attached to negative groups, as in chloral hydrate, mesoxalic acid and triketohydrindene
hydrate. An analogy for the great modification of the physiological action of choline by
esterification is to be found in the case of acetyl choline, p. 63, and of the nitric acid ester,
p. 153. In its action the latter, according to Dale and Ewins, resembles natural muscarine
even more closely than does the nitrous acid ester, (Comp. PrpQ, Physiol. Soc., March
14, 1914.)
CHAPTER V.
CREATINE AND CREATININE, GLYCOCYAMINE AND GUANIDINES.
A. Creatine and Creatinine.
Creatine was described and named as long ago as 1835, by Chevreul
[1835], in a report to the French Academy of Sciences on commercial
meat extracts. Chevreul did not analyse the substance, but noticed
its resemblance to asparagine. Berzelius later failed to prepare
creatine, but Wohler succeeded, and when Schlossberger [1844] ob-
tained the same substance from the muscles of an alligator, its im-
portance as a general constituent of muscle was recognised.
Our detailed knowledge of creatine dates from Liebig's classical in-
vestigation of the constituents of muscle juice [1847]. Liebig pre-
pared creatine from the flesh of various animals, analysed it and con-
verted it into its anhydride which he named creatinine and found
to be identical with a substance isolated three years previously from
urine by Pettenkofer [1844]. By boiling creatine with baryta, Liebig
further obtained a new substance, sarcosine. Dessaignes [1854, 1855]
showed that creatine is oxidised by mercuric oxide to methyl-
guanidine (" methyl-uramine"). Sarcosine was synthesised by Vol-
hard, who obtained creatine from it [1868].
Our physiological knowledge of creatine and creatinine did not
advance so rapidly as the chemical, largely perhaps owing to the want
of a convenient and accurate method of estimation. Such a method
was, however, supplied by Folin in 1904, and this, together with his
theory of metabolism, has led during recent years to many investiga-
tions on the physiology of creatine and creatinine.
Creatine was synthesised by Volhard [1868] by the action of
cyanamide on sarcosine in alcoholic solution at 100°.
/CH3
CH2.NH.CH3 + CN.NH2 CH-j.N/
<!oOH = ioOH XC<'NH)™*
Horbaczewski [1885] also obtained it by heating sarcosine with
guanidine carbonate to 140-160°. The necessary sarcosine may be
obtained by the hydrolysis of caffeine, but neither of these syntheses is
so convenient as the preparation from natural sources.
69
70 THE SIMPLER NATURAL BASES
Creatine and creatinine are interconvertible. The change from the
former to the latter substance can be brought about quantitatively by
heating with acid or even without a solvent (see appendix).
According to Gottlieb and Stangassinger [1907, 1908, i] creatine
is also converted into creatinine by autolytic ferments. The hydra-
tion of creatinine to creatine is brought about (partially) by alkalies ;
for instance by standing for a long time in solution in lime water
(Liebig), ammonia (Dessaignes), or by boiling with lead hydroxide
(Heintz [1849]).
Although fresh muscle contains at most only traces of creatinine
(Grindley and Woods [1906], Mellanby [1908]), the evaporation
of the extract in the presence of the natural acids of the muscle may
cause a considerable anhydration to creatinine, so that the latter sub-
stance may be abundant in commercial meat extracts. According to
Grindley and Woods [1906] beef contains 0-41 per cent, fish 0-31
per cent., chicken O'24-o*29 per cent, of creatine ; in beef extracts they
found 0-55-479 per cent, of creatine and O'83-5'27 per cent, of creati-
nine ; the total creatine + creatinine in meat extract is however fairly
constant, generally about 6 per cent. Baur and Barschall [1906]
give as maximum 1*25 per cent, of creatine and 3 per cent, of creatinine.
Supposed existence of several creatinines. Johnson [1892] con-
sidered that the creatinine from urine was not identical with that
obtainable from creatine, and Thesen [1898] obtained a yellow " iso-
creatinine " from fish. The supposed differences in these cases are
however due to insufficient purification, as shown by Poulsson [1904],
Toppelius and Pommerehne [1896] and by Korndorfer [1904, i].
Similarly the xantho-, chryso-, and amphicreatinine of Gautier
[1896, Ch. I] were doubtless also impure, as already suggested by
Brieger [1886, i, p. 10, Ch. I], Indeed, no one has apparently thought
it worth while to re-investigate them.
The quantitative estimation of creatinine appears to have been at-
tempted first by Heintz [1849]; Neubauer [1863] then worked
out a method depending on the isolation of the base as zinc chloride
compound. Salkowski showed that Neubauer's method gives results
which are often much too low, and proposed modifications [i 886, 1 890].
Gregor [1900] attempted to utilise the copper reducing power and
Edlefsen [1908] has suggested a method depending on the forma-
tion of creatinine salicylate, but all these methods have been displaced
by Folin's colorimetric method, depending on the use of Jaffa's reaction
(see appendix).
Since creatine can be quantitatively converted into creatinine the
CREATINE AND CREATININE 71
former substance can also be estimated indirectly by Folin's method.
A direct method for estimating creatine, due to Walpole [1911], is
based on the colour reaction with diacetyl (see appendix).
Physiological.
Distribution.
Creatine is a constituent of all vertebrate muscle. It was found
in the muscles of several animals by Liebig [1847], in the alligator
and in man by Schlossberger [1844, 1848], in a whale by Price [1851],
in a snake by Lyman [1908], in the cod and skate by Gregory [1848],
in various fishes and in Amphioxus by Krukenberg [1881], and by
Suzuki and co-workers [1912]. It is not present in invertebrate
muscle [Krukenberg, 1881]. It is absent in the shrimp [Ackermann
and Kutscher, 1907, 1-4; Chapter III, betaine], absent in the cuttle-
fish [Henze, 1910; Chapter III, betaine], [Cabella, 1913], and absent
or present only in traces in Crustacea and Mollusca [Okuda, 1912],
Recently Myers and Fine [1913, I] have shown that for any
particular species the muscle creatine is remarkably constant. They
found 0*522 per cent, in the rabbit, 0*45 per cent, in the cat, 0*39 per
cent, in man, 0-37 per cent, in the dog. Other recent observations are
in close agreement with these determinations ; thus in rabbit's muscle
Riesser [1913] found 0*521 per cent, and Beker [1913] 0*523 per
cent. ; the latter found in dog's muscle 0-364 per cent.
Creatine is most abundant in voluntary muscle (Cabella [1913],
Beker [1913]); there is less in cardiac and least in involuntary muscle.
According to Cabella the pectoral muscle of birds contains more than
that of the thighs ; in voluntary mammalian muscle and in the bul-
lock's heart the creatine nitrogen is 3-4 per cent, of the total ; in birds'
pectoral muscle 4-5 per cent. ; in cardiac muscle of birds and in the
muscle of the bullock's bladder I per cent.
The following table [Beker, 1913] gives the amount of creatinine
in milligrams obtained from 100 grm. of various organs. The figures
must be multiplied by 1*16 to give their content as creatine.
Voluntary muscle, bullock 403
„ „ rabbit 451
pig 338
dog
Cardiac muscle, bullock 215
dog 243
Uterus „ cow 38*18
pig 30-05
Testis, bull 86*8
Liver „ 29-32
„ rabbit 20*05
pig 1671
Pancreas, bullock 14*34
Spleen „ 14-67
Blood „ 2*179
72 THE SIMPLER NATURAL BASES
After two months' gestation, 100 grm. of voluntary muscle of the
foetal calf contained 22 mg., after nine months 250*4 mg. Ac-
cording to Mellanby [1908] creatine is not present in chick's muscle
until the 1 2th day of incubation and the maximum content is only
reached after hatching.
In the rabbit and in the fowl the percentage of muscle creatine
increases during starvation [Mendel and Rose, 191 1, 2], probably owing
to diminution of the non-creatine portion of the muscle. According
to Myers and Fine [1913] it increases in the earlier part of starvation
and afterwards diminishes. In malignant and some chronic diseases,
but not in acute disease, the creatine content of muscle is diminished
[Chisholm, 1912], apparently owing to diminished production.
Letsche [1907] found creatine in the blood serum.
Creatine is generally absent from mammalian urine, but it may be
present in various conditions. It completely replaces creatinine in
birds' urine [Paton, 1910] and occurs also normally in the urine of
infants [Funaro, 1908] and of children [Rose, 1911 ; this paper should
be consulted for further literature], [Folin and Denis, 1912], [Krause,
1913]. In women creatine occurs in the urine immediately after
menstruation, also during and after pregnancy [Krause, 1911 ; Krause
and Cramer, 1910]; its excretion is a concomitant of lactation
[Mellanby, 1913].
In man creatine appears in the urine when no carbohydrates are
taken as food, therefore in starvation [Cathcart, 1 907 ; Benedict and
Diefendorf, 1907; Mendel and Rose, 1911, 2] and also on a diet of
fats and proteins [Cathcart, 1909 ; Mendel and Rose, 1911,1]. Creatine
further appears in the urine in diabetes [Krause and Cramer, 1910;
Krause, 1910; M. R. Taylor, 1910], in phloridzin glycosuria [Cathcart
and Taylor, 1910], in hepatic disease [Mellanby, 1908], in phosphorus
poisoning [Forschbach, 1908], and in toxic fevers, mostly after the
crisis [Myers and Volovic, 1913].
Creatinine is a normal constituent of mammalian urine [Petten-
kofer, 1844; Fiebiger, 1903]. It is absent from muscle or present
only in traces (for precautions to avoid its formation from creatine in
extraction see Mellanby [1908] and Cabella[i9i3]). Small quantities
have been found in cancer tumours [Saiki, 1909] and in egg-yolk
[Salkowski, 1911], but the latter observation is contrary to that of
Mellanby [1908].
Neither creatine nor creatinine occurs in the urine of fish [Denis,
1912] nor in that of cuttle-fish [von Fiirth, 1900]. According to
CREATINE AND CREATININE 73
Sullivan [1911] creatinine (and possibly also creatine) occurs in
wheat, rye, clover and other crops, whence it finds its way into culti-
vated soils, from which it was isolated in the crystalline condition by
Shorey [1912]. According to Skinner [1912] creatine and crea-
tinine have a beneficial effect on plant growth.
Metabolism.
The close chemical relationship between creatine and creatinine
already suggested to Liebig that the former substance is converted
in the animal organism into the latter and is then excreted in the
urine. This view as to a genetic relationship between the two sub-
stances was rejected by Folin, whose colorimetric estimation first
made accurate investigation possible. He [1905, l] was the first to
show that on a creatinine free diet the amount of creatinine excreted
in the urine is remarkably constant for any given individual, and this
important result was soon confirmed by various investigators, e.g. Koch
[1905], van Hoogenhuyze and Verploegh [1905], Closson [1906], af
Klercker [ 1 907], Shaffer [ 1 908], Levene and Kristeller [ 1 909]. Various
authors give slightly different limits for the daily output ; thus Folin
gave 1*3-17 grm. for a man of 70 kilos., i.e. 19-24 mg. per kilo, of
body weight, Closson 15-19 mg. and Shaffer 19-30 mg. of creatinine
per kilo, per diem.
On this constancy of the creatinine output in the individual Folin
[1905, 2] has based a theory of protein metabolism (see Cathcart's
monograph in this series, " Physiology of Protein Metabolism," pp. 94,
95, 98), according to which theory the creatinine excreted is a result
and measure of the "endogenous" catabolism of the tissues and is
independent of the " exogenous " catabolism and of the protein of the
diet. Creatinine given by the mouth is rapidly and almost quantita-
tively excreted in the urine as such and this exogenous creatinine of
the food is thus super-imposed on the constant endogenous amount.
Creatine, on the other hand, as Folin [1906] has shown, when given
by the mouth in moderate quantity, does not appear in the urine,
neither as such, nor as creatinine. This observation has also been
made by many other investigators, e.g. Czernecki [1905] and Plimmer,
Dick and Lieb [1909]; the latter authors found, for instance, that
creatine appeared in the urine after a daily dose of 2-5 grm. but not
after 2-0 grm. In children the power of assimilating creatine is much
smaller and even of doses of O'3 grm. some appears in the urine,
super-imposed on that normally present [Krause, 1913].
In accordance with Folin's theory the amount of endogenous
74 THE SIMPLER NATURAL BASES
creatinine is diminished when the tissue metabolism is decreased.
New-born infants excrete per kilo, of body weight one-third of the
creatinine excreted by adults [Amberg and Morrill, 1907; Funaro,
1908]. Old people excrete less than young adults, and women less
than men [Benedict and Myers, 1907, I], In muscular dystrophy
[Spriggs, 1907], in Basedow's disease [Forschbach, 1908], in hepatic
disease [Mellanby, 1908], in diabetes [Krause, 1910], and in other patho-
logical conditions [Shaffer, 190 8] the creatinine output is diminished.
On the other hand the more rapid metabolism of fevers causes an in-
creased creatinine output [Leathes, 1 907] and this applies also to artificial
hyperthermia [Myers and Volovic, 1913]. The latter authors record
an increase up to 36 per cent. As will be seen, however, the decrease
in creatinine output is also in accordance with the theory which
regards creatine as the precursor of creatinine ; when the output of the
latter substance falls off, the former may take its place in the urine,
as in diabetes and in hepatic disease.
Folin's denial of a genetic relationship between creatine and crea-
tinine has not met with general acceptance. It was endorsed by af
Klercker [1907] and by Lefmann [1908], but, as has been pointed out
by van Hoogenhuyze and Verploegh [1909], Lefmann's results hardly
support his conclusion and rather indicate a partial conversion of
injected creatine to creatinine. Most authors do not agree with Folin's
sharp differentiation between muscular creatine and urinary creatinine ;
there is a good deal of evidence in support of the view that one of these
substances is derived from the other. Mostly creatine has been re-
garded as the precursor of creatinine, but Mellanby [1908] takes the
converse view. According to him creatinine is formed in the liver
from substances brought there by the blood stream, and is subsequently
rendered innocuous by hydration to creatine. In the young chick
creatine is at first absent from the muscles and gradually increases
until the saturation point is reached, and then the excess of creatinine
is excreted as such in the urine. Other investigators agree with
Mellanby in regarding the liver as the seat of transformation, but
consider the change to be in the opposite direction, viz. a dehydration
of muscular creatine to creatinine which is then excreted. When the
activity of the liver is impaired, as in phosphorus poisoning and in
hepatic disease, some creatine escapes dehydration and appears in the
urine as such (see above).
A further argument for the view that creatine is converted into
creatinine and then excreted, has recently been supplied by Myers
and Fine [1913, I], who find that the creatine content of muscle
CREATINE AND CREATININE 75
varies from species to species, but is very constant in the individuals
of the same species ; those species with muscles richest in creatine
show also the greatest output of creatinine in the urine. The con-
stancy of content of muscle and of creatinine output would thus be
the expression of a dynamic equilibrium.
The question whether creatine is formed as the result of muscular
work has been answered in the negative.
Liebig found ten times as much creatine in the muscles of a fox
killed in the chase as in the captive animal, but Voit [1868] found
no increase after work or after tetanising. Van Hoogenhuyze and
Verploegh [1905 ; consult this paper for the earlier literature] only
found an increase when muscular work was done during absolute fast-
ing. Mellanby [1908], Scaffidi [1913] and others have also failed to
change the creatine content of muscle by work ; Brown and Cathcart
[1909] observed a slight increase after stimulation, but only with
isolated frog's muscles.
Although creatine formation is not a function of rapid muscular
contractions, Shaffer [1908] regarded the creatinine output per kilo, as
directly parallel to muscular development or strength (" muscular
efficiency "), and Pekelharing and his pupils have during the last few
years connected creatine formation and creatinine output with muscu-
lar tonus. Weber [1908] had already shown that the surviving
pulsating heart, perfused with Ringer's solution, gave off creatine to
the perfusion fluid, and this observation was confirmed by Howell and
Duke [1908]. Weber also found that in the dog an increased crea-
tinine excretion could be induced by cinchonine convulsions (which
increase the tonus) but not by work ; the creatine in the muscles
decreased. Pekelharing and van Hoogenhuyze [1909, 1911] then
developed a new theory as to the effect of tonus on creatine formation.
They also observed a slight increase of the creatine content of muscle
during rigor ; the additional creatine is excreted in the urine as crea-
tinine. Pekelharing [1911] showed that there is an increase of urinary
creatinine after standing at attention for some hours in a military
position, but not after a long march. During sleep van Hoogenhuyze
and Verploegh [1905] had previously observed a decrease in the crea-
tinine output, which may be connected with the diminished tonus.
Beker [1913] has also supported this theory; he found that in preg-
nancy the creatine content of the uterus increases in the cow from
0-038 per cent, (calculated as creatinine) to 0*084 Per cent- m tne
gravid and 0-060 per cent, in the non-gravid horn. For pregnant and
non-pregnant human uteri the figures were 0*0766 and 0-0446 respec-
76 THE SIMPLER NATURAL BASES
lively. This may be connected with the post partum excretion of
creatine.
Attempts to increase the muscular creatine or urinary creatinine
by giving creatine by the mouth have not been very successful, perhaps
because of bacterial action in the intestine. Thus van Hoogenhuyze
and Verploegh [1908] found only slightly more creatinine in the urine
after taking 2 grm. of creatine. -The destruction of creatine by
bacteria has been studied by von Jaksch [1881], Vandevelde [1884],
and particularly by Twort and Mellanby [1912]. Ackermann [1913]
has shown that in putrefaction creatinine is not broken up like creatine,
but is changed to N-methylhydantoin.
/NH . CO /NH . CO
HN : C/ I + H2O = OC/ + NH3.
\N CH2 \N CH2
CH3 CH:J
When creatine was administered subcutaneously or intravenously,
however, a certain amount of direct evidence of its conversion to
creatinine has been obtained in rabbits [Pekelharing and van Hoogen-
huyze, 1910] and in dogs [Lefmann, 1908]. Recently Myers and Fine
[^S* 3] have shown that of injected creatine 5 per cent, appeared in
the muscles in rabbits; 25-80 per cent, appears in the urine as such,
and 2-10 per cent, as creatinine. Injected creatinine also causes a
slight increase of muscular creatine.
Assuming the conversion of creatine to creatinine, we may next
inquire where this change takes place. Experiments on dogs, in
which the liver was put out of action by an Eck's fistula, have not
proved that the liver has any important function in creatinine metabolism
[London and Boljarski, 1909; Foster and Fisher, 1911 ; Towles and
Voegtlin, 1911]. The last-named authors found that creatine, given
to dogs, increases the creatinine output, but that putting the liver out
of action made very little difference. Paton and Mackie [1912], from
experiments on birds, likewise consider that the liver plays no part
in the conversion of creatine into creatinine. The appearance of crea-
tine in the urine in hepatic disease may suggest incomplete dehydra-
tion to creatinine in the liver, but the formation of creatine might be
increased through the disturbance of the carbohydrate metabolism,
resulting from damage to the liver. When the supply of carbohydrates
in the body is insufficient (in fasting, in diabetes mellitus and in
phloridzin glycosuria) the necessary energy must be obtained from
another source, and this latter process may be accompanied by in-
creased formation of creatine.
CREATINE AND CREATININE 77
As bearing on the function of the liver in creatinine metabolism
the experiments of Gottlieb and Stangassinger [1907; 1908, I, 2]
must be mentioned. They concluded that liver extract dehydrates
creatine to creatinine and then decomposes it further ; these changes
may be brought about by autolysis, and creatinine is also formed by
perfusing the surviving liver with creatine. Mellanby [1908] criticised
the autolytic experiments and considered that in them the destruction
of creatine was due to bacteria.
Rothmann [1908] and van Hoogenhuyze and Verploegh [1908]
supported Gottlieb and Stangassinger, but Beker[i9i3] agrees with
Mellanby that the destruction of creatine was due to bacteria. As
pointed out on page 10 it is very difficult to ensure sterility in auto-
lysis. Gottlieb and Stangassinger's perfusion experiments, on the
other hand, are held by Beker to prove that the liver can dehydrate
creatine to creatinine.
Possible Precursors of Creatine.
The oldest attempts to find a precursor of creatine were directed
to showing that creatine can be formed in the organism from glyco-
cyamine ; Jaffe [1906] and Dorner [1907] adduced evidence in support
of this, but since the transformation is a simple methylation, for which
there are several examples in animal metabolism, and since glyco-
cyamine does not occur in nature, the formation of creatine from this
substance would hardly be a physiological process (see further the
next section on glycocyamine). Suggestions as to the formation of
creatine from muscle protein have been made by Seemann [1907] and
by Urano [1907]. According to Antonoff [1906-7] certain bacteria
(e.g. B. coli} can form from peptone a substance giving Weyl's re-
action (creatinine?). The one known protein constituent containing
a guanidine grouping is arginine, but neither van Hoogenhuyze and
Verploegh [1905] nor Jaffe [1906] could obtain creatine from arginine
in feeding experiments or by subcutaneous injection. The whole of
the administered arginine was excreted in the urine. Dakin [1907]
has shown that creatine is not affected by arginase from the liver.
Lately, however, Inouye [1912] has observed a small formation of
creatine from arginine by liver extract and when arginine is perfused
through the isolated liver. Finally Riesser [1913], in a paper which
contains a useful review of the whole problem, has described experi-
ments in which creatine appears to be formed from choline and from
betaine (see also pp. 62 and 42). By injecting these substances
into rabbits, he increased the creatine content of the muscle, which is
78 THE SIMPLER NATURAL BASES
normally very constant, by 10-15 Per cent- m tne case of choline, and
by 6*3-1 1 '3 per cent, in the case of betaine. Riesser considers that
these two substances are partially demethylated and then condense
with urea, according to the following equations : —
/NH2
/NH2 /OH /
CO/ + (CH3)3 |N/ = C : NH +2 CH3OH
XNH2 XCH2 . CH2OH \N . CH3 . CHaOH
CH
/
NH OH /
NH2
/2 /
CO/ + (CH3)3 : N/ = C : NH + 2 CH'OH
XNH2 XCH2.COOH \N.CH3.COOH
CH,
The two methyl groups would be eliminated as methyl alcohol ; the
condensation product from choline would undergo oxidation to
creatine. Riesser also administered sarcosine and urea by the mouth
and subcutaneously, and in half of the experiments obtained evidence
of creatine formation, which would occur as follows : —
/NH2 /™*
CO/ + NH.CH3.COOH = C : NH + H2O.
NH2 I NxN.CH2.COOH
CH3 j
CH3
B. Glycocyamine and Glycocyamidine.
Although these bases do not occur naturally they may be briefly
referred to on account of their relationship to creatine and creatinine
respectively, from which they differ by having one methyl group less ;
glycocyamine is guanidino-acetic acid and glycocyamidine is the cor-
responding anhydride.
Glycocyamine was first obtained by Strecker, in 1861, by the
addition of cyanamide to glycine. Nencki and Sieber [1878] heated
glycine with guanidine carbonate at 140°, and Korndorfer [1905]
found that heating in the water bath was sufficient and more convenient.
H. Ramsay [1908] has described a convenient synthesis of glycocy-
amine, in which monochloracetic acid is heated with a concentrated
aqueous solution of free guanidine (5 mols.) to 60° for two hours.
The physiological interest of glycocyamine and its anhydride chiefly
depends on their supposed methylation in the organism to form creatine
and creatinine. The question was first studied by Czernecki [1905]
whose results were indecisive or negative; later Jaffe" [1906] found
(by Neubauer's method) that 4-5-14*3 per cent, of the glycocyamine,
GLYCOCYAMINE AND GLYCOCYAMIDINE 79
given to rabbits by the mouth, appears in the urine as creatinine and as
creatine in the muscles. His pupil Dorner [1907] confirmed these
results, using Folin's method. Glycocyamidine given subcutaneously
was also changed in rabbits to creatinine. Mellanby [1908] however
failed to observe any effects of glycocyamine feeding.
NH
C. Guanidine, NH:C<f
\NH2
Guanidine has been isolated from Vicia seedlings by Schulze [1892, 2] (i grm. of the
nitrate from 3 kilos.) but it could not bs obtained from the ungerminated seeds. A small
quantity also occurs in the sap of sugar beets (Von Lippmann [1896]). It is further ob-
tained in the autolysis of pancreas (Kutscher and Otori [1904]) and by oxidation of
guanine and of various proteins with permanganates. Probably the " urea " obtained in
the oxidation of egg white by Be"champ [1857] was in reality guanidine ; its formation in this
manner was first established by Lessen [1880]. Larger quantities were subsequently ob-
tained from various proteins, gelatin, casein, pseudomucin, thymus nucleic acid by Kutscher
and his collaborators [1903, 1904, 1905 ; Otori, 1904, 2] by using calcium permanganate,
and also in the case of pseudo-mucin by hydrolysis with acids (Otori [1904, ij).
The physiological action of guanidine was investigated by Gergens
and Baumann [1876]. The base is a muscle poison affecting the
nerve endings (Camis [1909]). The effect is due to the univalent
guanidinium ion and resembles that of sodium salts (Fiihner [1908, 2]).
TV T-T (~*T-T
D. Methylguanidine, NH : C/
Methylguanidine is of greater physiological importance than
guanidine itself, being a normal constituent of muscle. It is formed
from creatine by boiling with mercuric oxide and dilute sulphuric acid
(Dessaignes [1854, 1855, under creatine], Gulewitsch [1906]) and
from creatinine and potassium permanganate (Neubauer [1861, I]).
Brieger [1886, 1, p. 34] obtained it from putrid horse meat. Kutscher
[1905, under creatine] and Gulewitsch [1906] isolated it from com-
mercial extract of meat (yield of the nitrate 0-38 per cent; Gulewitsch).
According to Krimberg [1906, 1] methylguanidine occurs in fresh beef,
where, however, Brieger [1886, I, p. 41] could not find it. Smoro-
dinzew [1913, Ch. II, carnosine] obtained 0*083 Per cent, of methyl-
guanidine from fresh horse meat. Small quantities of methylguanidine
also occur in normal human urine (Kutscher and Lohmann [1906, 3],
Engeland [1908, 3]), in that of the dog after feeding on meat extract
(Kutscher and Lohmann [1906, 4]) and in that of the horse (Achelis
[1906]). Smorodinzew [1912] recently obtained the base from
liver. In the urine of parathyroidectomised dogs the amount of
8o THE SIMPLER NATURAL BASES
methylguanidirie is greatly increased (up to I -9 gr. of the gold salt
per litre; Koch [1912]) and fairly large quantities are also present in
the urine of animals killed by anaphylactic shock or by burning
(Heyde [1911, 1912]); normal urine only contains traces. The
symptoms of anaphylactic shock cannot, however, be reproduced in
any way by the administration of the base [Loewit, 1913].
Methylguanidine is distinctly poisonous ; 0*2 grm. administered
hypodermically killed a guinea-pig (Brieger [1886, I, p. 38]). The
smallest dose producing a distinct effect in a frog (fibrillar twitchings
of dorsal muscles) is I mg. ; 50 mg. is fatal. The base acts peripherally
on the nerve endings in the muscle ; large doses produce tetanic con-
vulsions (Gergens and Baumann). The action is similar to that of
guanidine, q.v.
Methylguanidine in the organism is probably derived from creatine
and the amount in the urine is increased after feeding with meat ex-
tracts (Achelis [1906]). The mechanism of this change is not clear,
for the simple decarboxylation of creatine would yield dimethylguani-
dine, so that in addition to carbon dioxide a methyl group must be
removed by oxidation. There is however some indirect evidence that
bacteria can bring about the conversion of creatine into methylguani-
dine(Bocklisch[i887]).
E. as-Dimethylguanidine, NH : C/
\NH2
This base appears to accompany the monomethyl derivative in
normal urine; Engeland isolated 0*15 grm. of the aurichloride from 2
litres of dog's urine, and the picrolonate was probably obtained from
human urine by Kutscher and Lohmann [1906, 3, 4].
The formation of as-dimethylguanidine by bacterial decarboxylation
of creatine has not yet been observed (cf. Twort and Mellanby
[1912, under creatine]).
CHAPTER VI.
ADRENALINE (EPINEPHRIN, ADRENINE).
BOTH on account of its powerful physiological activity and its exten-
sive therapeutic application, adrenaline is the most interesting of animal
bases. The physiological importance of the supra-renal glands was
first made clear by Addison [1849] who connected the disease, now
named after him, with a pathological condition of these glands.
Addison's work suggested an experimental investigation to Brown-
Sequard [1856, i, 2, 1857], who showed that extirpation of both
supra-renals soon brings about the death of an animal ; thus, on
the average, rabbits only survived the operation for nine hours. About
the same time Vulpian [1856, I, 2] observed that the medulla of the
supra-renal gland contains a specific substance, which in solution is
coloured green by ferric chloride and rose-red by iodine ; he also ob-
tained the same reactions with blood from the supra-renal vein.
During the next forty years the " chromogen " was investigated by
Virchow [1857] who confirmed Vulpian's results without adding
fresh observations, by Arnold [1866], by Holm [1867], more fully
by Krukenberg [1885], and lastly by Brunner [1892], but none of
these authors were able to prepare the substance in anything like a
pure condition. The physiological action of supra-renal extracts was
the subject of papers by Pellacani [1879] an<^ by Foa and Pellacani
[1884], who, however, failed to observe the rise of blood pressure so
highly characteristic of supra-renal extracts when injected intravenously.
A full account of the earlier investigations on the gland, up to 1895,
was given by Rolleston in his Goulstonian lectures [1895, under
general references]. There is also an extensive bibliography in a
paper by Szymonowicz, published in Pfluger's Archiv [1896], and in
a dissertation by Langlois [1897, under general references].
In 1 894 the subject entered upon a new phase and soon became of
great physiological and biochemical interest. In that year Oliver and
Schafer [1894] observed the remarkable rise of blood pressure caused
by supra-renal extracts on intravenous injection ; they showed that the
effect was due to vaso-constriction and also to a direct action on the
heart. This pressor action was discovered independently and almost
simultaneously by Szymonowicz [1895] wh° found that the low
81 6
82 THE SIMPLER NATURAL BASES
blood pressure caused by extirpation of both supra-renals could be
raised temporarily by an injection of an extract of the gland. Cybulski
[1895], wno continued the investigation, also obtained a pressor
action with blood from the supra-renal vein. The isolation of the active
principle was now attempted by several investigators. Moore [1895-
97] working in Schafer's laboratory, soon found that the physiological
activity of extracts went parallel with the intensity with which they
gave Vulpian's colour reactions and concluded that the chromogen was
identical with the active principle. Attempts to isolate it were made
by Frankel, Muhlmann [1906], Gurber [1897], and especially by
Abel and Crawford [1897], Abel [1898-1901], and by von Fiirth
[1898-1901], but these attempts were all unsuccessful. Von Fiirth,
indeed, obtained a highly active preparation of the substance, which
he termed suprarenin, by precipitating it as iron compound by the
addition of ferric chloride to a purified extract in methyl alcoholic
solution, and Abel separated the active principle as benzoyl derivative,
but he could not recover it in a pure state by subsequent hydrolysis.
Abel's work, however, led to the crystallisation of the active principle
by Takamine [1903, 1-3] who named it "adrenalin," and very soon
afterwards it was obtained independently by Aldrich [1901] who as-
signed to it the correct empirical formula C9H13O3N.
The chemical constitution of adrenaline could now be investigated.
On fusion with potash Takamine had already obtained from it two
substances which he regarded as catechol and protocatechuic acid.
Von Fiirth confirmed the production of the latter substance and also
showed that a methylamino-group and an alcoholic hydroxyl are present.
Abel for a long time defended the erroneous formula C10H13NO3, |H2O
and termed the crystalline active principle " epinephrin hydrate ". The
substance obtained on complete hydrolysis of his benzoyl derivative
he considered to have the composition C10H13NO3, and this he called
" epinephrin," but found later that it was chemically and physiologi-
cally different from the active principle of the gland. Abel's formula
was disproved conclusively in favour of that of Aldrich by Pauly
[1903], who analysed very carefully purified material and also showed
that adrenaline contains an asymmetric carbon atom. Pauly reduced
the number of possible constitutional formulae to two, viz. : —
OH OH
>OH
and II
CHOH CH . NH . CHS
CHg.NH.CHg CH2OH
ADRENALINE (EPINEPHRIN, ADRENINE) 83
Jowett [1904] arrived at results similar to those of Pauly ; on
complete methylation and subsequent oxidation he obtained veratric
acid and trimethylamine ; of the above two formulae he favoured the
first, subsequently shown to be the correct one. Further investigations
were carried out by Abderhalden and Bergell [1904] and by Ber-
trand [1904, I, 2]. In the meantime the problem was being attacked
in a different way by Stolz whose results, although not published until
1904, had already led in August 1903 to a patent application of
the Farbw. vorm. Meister, Lucius und Briining [1904] describing
the synthesis of a substance of the constitution I (above) which
could not at first be obtained crystalline but seemed to be physi-
ologically identical with adrenaline. Similar synthetic experi-
ments were published somewhat later by Dakin [1905, 1-3], but al-
though the identity of the synthetic substance with adrenaline was
rendered extremely probable, this identity could not be proved rigor-
ously, until the former substance had been crystallised and finally
resolved into its optically active components, one of which was found
to be completely identical with natural adrenaline (Flacher [1908]).
Before this, an independent proof of the constitution of adrenaline had
been furnished by Friedmann [1904, 1906] who showed that von
Fiirth's tribenzenesulphonyl adrenaline, which is optically active, lost
its activity on oxidation to the corresponding keto-derivative, which was
crystallised. This proved that adrenaline is a secondary alcohol (for-
mula I) and its constitution was further established by a comparison of
the above-mentioned ketone with a synthetic specimen obtained from
the amino-aceto-catechol of Stolz.
Nomenclature and Synonyms.
It is clear from the above that the active principle of the supra-renal gland has re-
ceived different names from various investigators. The three principal ones are
"epinephrin" (Abel), " suprarenin " (von Fiirth) and "adrenalin" (Takamine), and these
are the only ones in scientific use, together with "adrenine" which has lately been em-
ployed in the " Journal of Physiology ". On grounds of scientific priority the name should be
adopted, which was suggested by the chemist who first isolated the substance in a pure
state ; this was Takamine and we therefore use the name adrenalin(e) in the present mono-
graph ; this name also happens to be the one at present in most general use. The objection
to adrenalin is that it is a proprietary trade-name. For this reason the English Chemical
Society used for some time the name epinephrin, which has also been adopted more recently
by the American Medical Association. Apart from the fact that Abel first applied this
name to an amorphous and probably impure substance there is the additional confusion,
that for a long time he designated by it a supposed artificial alkaloidal anhydride of the
active principle, which latter he called epinephrin hydrate ( = adrenalin) and some of
his papers speak of epinephrin and adrenalin as two distinct substances. Later, when
6*
84 THE SIMPLER NATURAL BASES
the hydrate theory proved to be untenable, epinephrin was made synonymous with
adrenalin.1
Preparation and Purification of Natural Adrenaline.
The various processes depend on the fact that the active principle
is extracted from the glands by water, neutral or acidulated, that it
is not precipitated from its concentrated aqueous solution by alcohol,
nor by neutral lead acetate, and that it separates in a crystalline form
from suitably purified and concentrated aqueous solutions on the addi-
tion of concentrated ammonia. On account of the readiness with which
adrenaline undergoes oxidation various precautions have been sug-
gested, such as preventing the access of air by means of a current of
hydrogen or of carbon dioxide, and carrying out the final precipitation
under a layer of petrol. For the same reason it is very convenient to
extract with water containing sulphur dioxide.
Takamine [1901, 2] extracted the minced gland at 50-80° for five hours with water
acidulated with acetic or hydrochloric acid, shaking at intervals. The extract was then
raised to 90-95° for one hour to coagulate the proteins, using a layer of fat or current of
carbon dioxide to avoid oxidation. The glands were extracted a second time and the mixed
extracts were concentrated in vacuo, and then precipitated with 2-3 volumes of alcohol.
After filtration, the filtrate was again evaporated to a small bulk and was then precipitated
with excess of concentrated ammonia which caused the crude adrenaline to separate in
sphaero-crystals.
Aldrich [1901] proceeded like Takamine, but before precipitating the concentrated
solution with alcohol he added neutral lead acetate, centrifuged and removed the excess of
lead from the solution by means of hydrogen sulphide. Then, after concentration, he
added four to five volumes of 94 per cent, alcohol, evaporated the alcoholic filtrate to a very
small bulk and added ammonia ; after filtration the crude adrenaline is washed with
very dilute ammonia.
Abel [1903, i] recommends a process illustrated as follows : 11*13 kilos, of minced glands
were divided over a number of flasks and to each portion an equal quantity of a solution of
175 grm. trichloracetic acid in 5 litres of absolute alcohol was added, in small quantities at
a time, with vigorous shaking. Next day 5-6 litres of filtrate were collected at the pump
and evaporated to 380 c.c. After filtering off a flocculent precipitate, ammonia (d = 0-94)
was gradually added to the clear filtrate with stirring until the smell of ammonia was per-
manent. The adrenaline, which separated at once, was filtered off and washed with water,
alcohol and ether ; yield 23-79 grm. = 0-2 per cent. The product, although nearly white,
1 Those interested in this question of nomenclature may refer to a letter by T. Maben
in the Pharmaceutical Journal (1907, 78, 388-90 ; "Adrenalin : the Active Principle of the
Suprarenal Gland ") and to a reply by W. Martin in the same journal (1907, 78, 447 and
514 ; " Epinephrin or Adrenalin ? "), and particularly to a correspondence entitled " Pro-
prietary versus Unprotected Names " between the Council on Pharmacy and Chemistry of
the American Medical Association and Messrs. Parke, Davis & Co. (Journ. Amer. Med. Assoc.,
1911, 56, 910-5). It is said that 30-40 different trade names for the active principle of
the supra-renal gland have been in use. Of these adnephrin, adrenalin, adrin, caprenalin,
supra-capsulin and supra-renalin are of American origin ; the following are European : atra-
bilin, chelafrinum, epirenan, haemostasin, hemisine, ischemin, paraganglin, paranephrin,
renoform, supra-nephran, supra-renaden, tonogen, and vaso-constrictin. Suprarenin is used
by the Ho'chst works for their synthetic product.
ADRENALINE (EPINEPHRIN, ADRENINE) 85
contained 10-12 per cent, of ash. A second and a third extract, made from the mass of
glands with 30-40 grm. of trichloracetic acid in 5-6 litres of 60-70 percent, alcohol, yielded
respectively 8*57 and 3 grm. of base ; total = 35*36 grm. or 0*3 per cent, of crude product.
Bertrand [1904, i] extracted 600 grm. of the minced glands (of horses) with 2 litres
of 95 per cent, alcohol, containing 5 grm. of oxalic acid. On evaporation the extract was
shaken with petrol to remove lecithin, etc., and the aqueous layer was exactly precipitated
with neutral lead acetate and centrifuged. After removal of the excess of lead and evapora-
tion to 100 c.c. a slight excess of ammonia was added. 118 kilos, of fresh minced gland
from 3900 horses yielded 125 grm. of adrenaline. This yield is hardly more than one-
third of that obtained by Abel (from bullock's glands).
The purification of the crude adrenaline may be carried out by
dissolving in acid and reprecipitating, but better by Abel's method
depending on the solubility of adrenaline oxalate in alcohol. Pauly
[1903] used it as follows: 12 grm. of crude adrenaline were ground
up with 50 c.c. of 85-90 per cent, alcohol, containing 7 grm. of oxalic
acid ; the inorganic impurities remain behind. After filtration and
dilution with 100 c.c. of water, ammonia precipitated the base in a
crystalline condition ; the base was freed from ammonium oxalate by
thoroughly washing. This process was repeated several times and
finally the base was washed with alcohol and ether. A more compli-
cated process which yielded a substance absolutely free from ash, is
also described by Pauly [1904].
Syntheses of Adrenaline.
Adrenaline has been synthesised by several methods : —
(i) By means of phosphorus oxychloride, catechol is condensed
with monochloracetic acid and the resulting chloracetocatechol (I), thus
first prepared by Dzierzgowski, is suspended in alcohol (50 c.c. for 100
grm. of the ketone).
I II III
CHOH
I
CH2.NH.CH8
A 40 per cent, aqueous methylamine solution (200 c.c.) is then
added and on standing methylamino-acetocatechol separates out ; the
product is washed with water, alcohol and ether. The methylamino-
acetocatechol (II) so obtained is reduced to racemic adrenaline (III) by
means of aluminium amalgam, or electrolytically. The above process is
protected by the German patents Nos. 152814 and I 57300 of the Farb-
werke vorm. Meister, Lucius und Briining [1904] and appears to
86 THE SIMPLER NATURAL BASES
be the only one which is commercially suitable. The resolution of the
racemic adrenaline is effected according to Flacher [1908] by ex-
tracting the bitartrate with methyl alcohol ; d-adrenaline d-tartrate dis-
solves and 1-adrenaline d-tartrate remains behind. The latter yields
commercial synthetic suprarenin.
An attempt to synthesise adrenaline by another method was originated by Barger and
Jowett [1905] and continued by Pauly and Neukam [1908], Barger [1908], Bottcher
[1909] and Mannich [1910], but has not yielded results of practical value (cf. German patents
Nos. 209609, 209610, and 212206). Starting from piperonal (I), Barger and Jowett pre-
pared the bromohydrin (II) which was converted into adrenalin methylene ether (III)
I II III
O— CH2
CHOH CHOH
CH2Br CH2 . NH . CH3.
Adrenaline dimethyl ether was prepared from methyl vanillin by a similar method, but
neither ether is convertible into adrenaline. Mannich showed that on the addition of
methylamine to the bromohydrin, ethers of isoadrenaline^(OH)3C6H3 . CH(NHCH3) . CH2OH
are also formed. The indirect removal of the methylene group by conversion into an un-
stable cyclic carbonate— e.g. OCO2: C6H3 . CH(OH) . CH2C1, has also proved impossible.1
Another synthesis of adrenaline which is theoretically possible and has been referred to
in the patent literature, consists in methylating the primary base 3 : 4-dihydroxy-phenylethanol-
amine (OH)2 . C6H3 . CH(OH) . CH2 . NH2. This base, which is about as active as adren-
aline itself and is known commercially as " arterenol," may be prepared by the reduction of
amino-acetocatechol> (D.R.P. 155632).
(OH)2C6H3 . CO . CH2 . NH2 + 2H = (OH)2C6H3 . CH(OH) . CH2 . NH2
and also by the reduction of the cyanhydrin of protocatechuic aldehyde with sodium
amalgam (D.R.P. 193634).
(OH)2C6H3 . CH(OH) . CN + 4H = (OH)2C6H3 . CH(OH) . CH2 . NH2.
Amino-acetocatechol is obtainable in several ways : —
1. From chloro-aceto-catechol and ammonia (the chief method) : —
(OH)2C6H3 . CO.CH2C1 + 2NH3 = (OH)2C6H3 . CO . CH2 . NH2 + NH4C1.
2. By reduction of w-nitroacetocatechol : —
(OH)2C6H3 . CO.CH2 . NO2 + 6H = (OH)2C6H3 . CO.CH, . NH2 + 2H2O.
The w-nitroacetocatechol is obtained by hydrolysis of the corresponding methylene- or
dimethylether with aluminium chloride in benzene solution. These ethers, co-nitroaceto-
piperone and co-nitroacetoveratrone, may be prepared from piperonal and methylvanillin re-
spectively, by successive treatment with nitromethane, bromine, methylalcoholic potash and
acids (D.R.P. 195814).
3. By hydrolysis with hydrochloric acid of the condensation product obtained from
veratrole and hippurylchloride by means of aluminium chloride (D.R.P. 185598 and 189483)
(CH3O)2C6H4 + C1CO.CH3.NH.CO.C6HB=(CH30)2C6H3.CO.CH2.NH.CO.C6H5 + HC1.
(CH3O)^C6H3 . CO . CH2 . NH . CO . C6H5 + 3HC1 + H2O-» (OH)2C6H3 . CO . CH2 . NHa .
A better yield is obtained by the hydrolysis of the similarly constituted phthalimido-
acetoveratrole (D.R.P. 209962 and 216640).
1 Compare Pauly's repudiation [1909] of Bottcher's claim [1909] to have synthesised
adrenaline by this method and D.R.P. 209609, 209610, 212206.
ADRENALINE (EPINEPHRIN, ADRENINE) 87
In order to utilise the d-adrenaline, obtained as a by-product in
the resolution of the racemic base (according to Flacher [1908]
and D.R.P. 222451), the dextro-variety may be racemised by means
of acids (according to D.R.P. 220355). For example, 1*5 grm.
d-adrenaline is dissolved in 13-5 c.c. normal hydrochloric acid (= 1*65
mol.) and after adding I 5 c.c. of water the solution is heated to 80-90°
for two to three hours, after which the solution is optically inactive
and the crystalline hydrochloride of the racemic base can be isolated
by means of alcoholic hydrogen chloride. When the natural base was
kept for six weeks at 20-30° with the same concentration of hydro-
chloric acid, 75 per cent, had been racemised. By repeated resolution
and racemisation of the d-base, the whole of the synthetic adrenaline
is finally obtained in the 1-form.
For an account of the patents relating to the synthesis of adrena-
line reference may be made to Friedlander's " Fortschritte der Teerfar-
benfabrikation," 1905-7, VIII, 1181-90, and 1907-10, IX, 1024-33;
or to the " Chemisches Zentralblatt ".
Adrenaline Substitutes.
Numerous bases, more or less closely related to adrenaline, have
been synthesised and some of these also resemble adrenaline in
physiological action. Only three of them, however, have been recom-
mended as substitutes for the natural active principle, namely
3 : 4 dihydroxy-phenylethanolamine (OH)2C6H3 . CH(OH) . CH2 . NH2 ("arterenol ")
w-ethylamino-3 : 4-dihydroxy-acetophenone (OH)2 . C6H3 . CO . CH2 . NH . C2H5
(" homorenon ")
3 : 4-dihydroxy-phenylethyl-methylamine (OH2) C6H3 . CHa . CH2 . NH . CH3 (" epinine ")
Of these, arterenol is according to Schultz [1909, I] about as active
on the blood pressure as natural 1-adrenaline (and therefore more
active than the racemic base). Homorenon and epinine are much
less active, the former base having according to Schultz only about
one-eightieth of the pressor action of 1-adrenaline.
Physical and Chemical Properties of Adrenaline. Salts and
Derivatives. Constitution.
Adrenaline, when pure, crystallises in colourless sphaerocrystals consisting of super-
posed lamellae ; crystals suitable for crystallographic measurement have not been obtained.
It melts at 211-212° (uncorr.) with decomposition. According to Bertrand the solubility in
water at 20° is 0-0268 per cent. The base is somewhat more soluble in boiling water, but
less in alcohol ; it is practically insoluble in most organic solvents but dissolves in glacial
acetic acid, in warm ethyl oxalate (Abel) and in benzaldehyde. In the latter solvent
Barger and Ewins [1906] found at 90° the molecular weight 170.
Adrenaline is lasvo-rotatory. The'more trustworthy determinations in solution in dilute
mineral acids are tabulated below : —
88
THE SIMPLER NATURAL BASES
Author.
Source.
Temperature.
Wo
Bertrand [1904, 2] ....
horse ; in N/io H2SO4
- 53'3°
Abderhalden and Guggenheim [1908] .
Flacher (with Korndbrfer) [1908]
bullock
bullock
20°
I9'8°
- 5072°
- 51-40°
Schultz (with Taveau) [1909, i] .
bullock
26-4°
- 53 '4°°
Abel and Macht [1912]
parotid gland of B ufo Agna
20°
- 5i'300
Weidlein [1912] ....
whale
25°
- 52-00°
Flacher [1908]
synthetic 1-adrenahne
- 51*40°
" »»
d-
—
+ 51-88°
d- Adrenaline has the same physical and chemical properties as 1-adrenaline and melts
also at 211-212°, but is much less active physiologically.
Adrenaline is a fairly strong base and can be dissolved in the theoretical quantity of a
mineral acid, or even in somewhat less than one equivalent (Gunn and Harrison [1908]).
Being a phenol, it is also soluble in caustic alkalies, but not in ammonia or sodium carbonate.
The chief chemical characteristic of adrenaline is the readiness with which it undergoes
oxidation, on account of the presence of a catechol nucleus. A large number of mild oxidis-
ing agents colour adrenaline solutions pink, rose red, and brown, and the same change takes
place on exposure to air, slowly in acid, rapidly in alkaline solution. Adrenaline is most
stable in solutions containing a slight excess of acid, for instance one and a half equivalents
of acid to one equivalent of the base. The coloration takes place much more rapidly when
minute traces of iron are present (Gunn and Harrison [1908]). A number of colour reac-
tions, depending on this oxidative change, are described below (pp. 89-91). According to Abel
[1902, 3] extracts of the supra-renal gland are more stable to Fehling's solution than solutions
of the pure active principle. Adrenaline solutions do not give precipitates with the common
alkaloidal reagents, but on heating with dilute acids, or by the action of concentrated hydro-
chloric acid in the cold, adrenaline is transformed into a substance yielding alkaloidal re-
actions (Abel's epinephrine).
The salts of the optically active adrenalines are mostly amorphous and deliquescent ; the
bar ate prepared by evaporating 1-83 gr. of the base and 0*93 gr. of boric acid in 5 c.c. of
water is said to be more stable (D.R.P. 167317). The chief crystalline salt of adrenaline is
the bitartrate, employed in the resolution of the synthetic product, Pauly [1904] prepared
a crystalline urate. The racemic base yields, in addition, a crystalline hydrochloride, mp.
157° (D.R.P. 202169), and a crystalline oxalate, but the corresponding salts of both d-and
1-adrenaline are amorphous (Flacher [1908]).
No crystalline derivatives of adrenaline are known. Abel and Pauly prepared benzoyl
derivatives of somewhat uncertain composition. Von Fiirth obtained a tri-benzenesulphonyl
derivative which contains the alcoholic hydroxyl of the side chain intact, for Friedmann
[1904, 1906] converted it into m-nitrobenzoyl-tribenzenesulphonyl-adrenaline and oxidised
it to tribenzenesulphonyl-adrenalone. Stolz obtained a tri-p-chlorbenzoyl derivative.
The constitution of adrenaline was ascertained from the following
reactions ; —
On fusion with potash catechol and protocatechuic acid are formed ;
on heating with acids or caustic soda methylamine is eliminated. On
methylation and subsequent oxidation with permanganate veratric acid,
vanillin and trimethylamine were obtained. The constitution is further
proved by Friedmann's work (see above, p. 83) and finally of course
by synthesis and resolution.
The alleged production of skatole on potash fusion is probably due
either to the presence of protein impurities, or to that of a benzoyl
ADRENALINE (EPINEPHRIN, ADRENINE) 89
nucleus (in Abel's epinephrine). The constitution of the " alkaloidal "
substance formed by the action of acids on adrenaline has not been
elucidated, nor of the base C3H4ON2 obtained by Abel [1904] on
oxidising adrenaline with nitric acid. Adrenaline is readily attacked
by various oxidases [Neuberg, 1908; Abderhalden and Guggenheim,
1908].
Colour Reactions of Adrenaline.
The principal colour reactions were already observed by Vulpian
and have more recently been used for the estimation of adrenaline.
A general review of the various quantitative colorimetric methods has
lately been furnished by Borberg [1912]. The reactions are as
follows : —
I. Ferric chloride produces in neutral or slightly acid solution a
grass green coloration, changing to violet, reddish violet, and red on
the careful addition of dilute alkali. This is a reaction characteristic
of catechol derivatives. The green coloration is the more fugitive and
the less strongly marked, the more acidic the solution is. The limit of
sensitiveness is about I : 30000, but the addition of sulphanilic acid
increases the sensitiveness tenfold and changes the green colour to
reddish brown or brown yellow (Bayer [1909]). Falta and Ivcovic
[1909] describe another sensitive modification of the ferric chloride
reaction. For the detection of adrenaline in urine Borberg [1912]
gives the limit for the green ferric chloride reaction as I : loopoo.
On standing a red coloration is produced up to I : 300,000.
II. A pink or rose red coloration (" tout a fait remarquable,"
Vulpian) is produced in adrenaline solutions on prolonged exposure
to air and, almost immediately, by various oxidising agents. The
change of colour is less rapid in faintly acid solution than in neutral
solution, and more rapid in alkaline solution. It is also brought
about by oxidases ; from the behaviour of adrenaline to tyrosinase,
Gessard [1904] first deduced a relationship to tyrosine. Neuberg
[1908] found that an enzyme from the ink-bag of Sepia officinalis
produces a black pigment from adrenaline, and Abderhalden and
Guggenheim [1908] observed that adrenaline solutions are coloured
red by a tyrosinase from the fungus Russula delica ; the laevo- , the
dextro- , and the racemic forms are all coloured at the same rate. The
formation of pigments from adrenaline has been considered by some to
be connected with the pigmentation of the skin in Addison's disease.
The oxidising agents employed for the red colour reaction for
adrenaline are : —
90 THE SIMPLER NATURAL BASES
A. Iodine or iodic acid. The excess of iodine may be removed by
shaking with ether and the sensitiveness is then according to Schur
[1909] I : 1,500,000. Abelous, Soulie" and Toujan [1905] removed
the excess of iodine by means of sodium thiosulphate, but according
to Bayer [1909] the reaction, when carried out in this way, is not very
delicate and the red colour is not permanent.
Another modification of the iodine reaction was suggested by L.
Krauss [1909] who used iodic acid. Subsequently Frankel and Allers
[1909], independently of Krauss, employed an equal volume of O'OOi
N-potassium bi-iodate and added a few drops of phosphoric acid ; by
heating the mixture nearly to the boiling point, the reaction is said to
be obtainable at a dilution of I : 300,000. Hale and Seidell [1911]
recommend this test, but do not add phosphoric acid. Frankel and
Allers consider their test to be quite distinct from that of Vulpian ;
they state that at no stage of the reaction is iodine set free, but both
Krauss and Ewins [1910] deny this. Bayer [1909] claims to have
greatly increased the sensitiveness of the Frankel-Allers reaction
by adding sulphanilic acid, which, however, changes the red colora-
tion to an orange or yellow one, which is less specific ; Bayer gives
I : 5,000,000 as the limiting dilution.
B. Another oxidising agent, which colours adrenaline solutions
red, is mercuric chloride, recommended by Comessatti [1909]. Boas
[1909] and Frankel and Allers [1909] could not obtain the reac-
tion at all readily, but Ewins [1910] has pointed out that Comessatti
used solutions of mercuric chloride in tap water, and that the calcium
bicarbonate present in the latter acts as a catalyst ; it may be replaced
by solutions of other salts of weak acids. This observation is of con-
siderable interest in connection with the discovery of Euler and Bolin
that the oxidase from Medicago consists of calcium salts of organic
hydroxy-acids. It was moreover already noticed by Vulpian, that the
spontaneous coloration of the adrenal chromogen by exposure to air
takes place slowly in distilled water, but much more rapidly in tap
water.
Ewins suggests the following conditions for carrying out Comes-
satti's reaction. To I c.c. of adrenaline (i : 100,000) an equal volume
of a I per cent, sodium acetate solution is added and then four to five
drops of a o-i per cent, solution of mercuric chloride in distilled water.
A pale rose tint is produced at room temperature in 4 to 5 minutes.
Here the sodium acetate solution replaces tap water, in order to secure
uniformity.
C. The most sensitive oxidising agent is probably a persul-
ADRENALINE (EPINEPHRIN, ADRENINE) 91
phate. Pancrazio [1909, 1910] has used the sodium salt and Ewins
[1910] the potassium salt. Ewins adds potassium persulphate
solution to the adrenaline solution until the concentration of the per-
sulphate is about o-i per cent, and then immerses the test tube for a
short time in a boiling water bath. Under these conditions a distinct
reaction is still obtained at a dilution of I : 5,000,000. The persul-
phate reaction for adrenaline seems therefore to be more delicate than
any other, with the possible exception of Bayer's modification of the
Frankel-Allers reaction (see above) for which an equal degree of
delicacy is claimed. According to Ewins potassium persulphate has
an additional advantage in the estimation of adrenaline in extracts of
the gland, since it discharges the colour of these extracts to a consider-
able extent, the colour interfering with the Bayer-Frankel-Allers test.
With persulphate a clean and distinct red tint results, which is per-
manent for a considerable time.
D, Other oxidising agents which colour adrenaline solutions red,
are potassium ferri cyanide (Cevidalli [1908]), brown oxides of man-
ganese (Zanfrognini [1909]), sodium nitro-prusside and ammonia,
bleaching powder, chlorine, bromine, ammoniacal silver solutions, and
osmic acid (Mulon [1905]). According to Borberg [1912] all the
" red " colour reactions for adrenaline are similar and depend on the
formation of the same oxidation product. Borberg gives the limit as
I : 300,000, thus perhaps underestimating the sensitiveness of some
of the reactions.
Ewins [1910] examined the effect of iodine and persulphate and
of the Comessatti, Frankel and Allers, and Bayer reagents on a
number of synthetic bases, closely related to adrenaline. He found
that aminoethanol-catechol (arterenol), as well as dihydroxy-phenyl-
ethylamine and its N-alkyl derivatives (including epinine) give the
various reactions with about the same degree of sensitiveness as
adrenaline, but none of these reactions are given by ketone bases, such
as amino-aceto-catechol and its derivatives (including homorenon).
Among these synthetic bases there is therefore no close parallelism
between chemical reactivity and physiological action.
E. Folin, Cannon and Denis [1912] have recently described
a new and very sensitive colour reaction for uric acid, which is
also given by adrenaline with three times as great a sensitiveness
(i : 3,000,000). One hundred grm. of sodium tungstate is dissolved
in 750 c.c. of water, and after adding 80 c.c. of 85 percent, phosphoric
acid, the solution is boiled gently for one and a half to two hours and
then made up to I litre ; -^-^ mg. adrenaline can be detected.
92 THE SIMPLER NATURAL BASES
Colorimetric Estimation of Adrenaline. — The green coloration with
ferric chloride has been employed by Batelli [1902] who found by
this means 0*174 per cent, in fresh bullock's glands. Von Fiirth [1901]
has used the carmin red coloration produced by ferric chloride in the
presence of sodium carbonate and sodium potassium tartrate. The
ferric chloride reaction is, however, not very suitable for quantitative
work (cf. Cameron [1906]) and the same applies, according to the
author's experience, to the iodine-thiosulphate method of Abelous,
Soulie and Toujan [1905]. Comessatti [1909] has employed
the mercuric chloride reaction a good deal for quantitative purposes,
and Cevidalli [1908] and Zanfrognini [1909] have used their re-
actions in the same way ; their methods have been adversely criticised
by Borberg [1912]. Ewins [1910] found a distinct parallelism
between the depth of colour produced by potassium persulphate and
the pressor activity of supra-renal extracts. This physiological control
has not been applied sufficiently to most other colon' metric methods.
A notable exception is found in a recent paper by Folin, Cannon,
and Denis [1913] and the colorimetric method of these authors based
on the reaction described above (under E) appears to be almost or quite
as accurate as the blood pressure method with which its results agree
within a few per cent, of the total adrenaline present. The method
is even sufficiently sensitive to demonstrate the increase of adrenaline
in the supra-renal vein by stimulation of the splanchnic nerve (cf.
p. 95). It is not necessary to have pure adrenaline as a standard, for
uric acid gives an identical coloration with one-third of the intensity.
Amount of Adrenaline in the Supra-renal Gland ; Yield ;
Distribution in other Organs ; Origin.
By the physiological blood pressure method, which is probably the
most accurate, Elliott finds that the adult human gland in health con-
tains about O'l per cent, (unpublished observation, referred to below).
By the same method Elliott [1912] has found that the normal
cafs supra-renal, weighing 0*2 grm., contains on the average 0*22 mg.
of adrenaline, or cm per cent. Folin, Cannon and Denis [1913]
found in the gland of young cats cri 22-0*1 52, of the dog and monkey
0*2-0-25, of the calf 0-25-0-35, of sheep, cattle, rabbits, 0-3 per cent.
Houghton [1902] found Takamine's original adrenaline to be 600 to 800 times as
active as fresh bullock's gland; according to Takamine [1901, 4] the specimen contained
mineral impurities and pure adrenaline is probably 1000 times as active <as the fresh gland,
which would therefore contain OT per cent, of the base.
For the horse we have Bertrand's statement [1904, i] that 118 kilos, of the fresh gland
yielded 125 grm. of adrenaline or 0-106 per cent.
ADRENALINE (EPINEPHRIN, ADRENINE) 93
As an example of actual yields obtained in the manufacture of adrenaline from bullock's
glands, the following figures may be quoted which are percentages of the weight of the fresh
gland after dissecting away the fat : 0*095, 0*086, 0*103. (The weight of a fresh bullock's
gland dissected in this way, is 10-12 grm.)
In manufacture the yield from sheep's is the same as that from bullock's glands, or
slightly less (0*08 per cent. ?).
From 100 bullocks' glands von Fiirth [1903] obtained 0*78-1*74 grm. of adrenaline ;
on the average 1*13 grm.; 100 glands weighed about 1000 grm., therefore the adrenaline
isolated was 0*113 per cent. Weidlein [1912] obtained 0*247 per cent, crude adrenaline
from the whale's supra-renal.
The results of colorimetric determinations, except those of Folin, Cannon and Denis
quoted above, are probably the least reliable. By the persulphate method Pancrazio
[1909] found 0*133 per cent, in the calf's gland and Batelli [1902] by the ferric chloride
method found 0*174 Per cent.
Abel [1903, i j obtained 0*3 per cent, of crude adrenaline from fresh bullock's supra-renals ;
the product contained 10 to 12 per cent, of ash and probably also organic impurities, but never-
theless this appears to be by far the highest yield recorded, and Abel [1903, 2] estimates
that fresh beeves' supra-renals contain at least 0*3 per cent, of the active principle. Hunt
[1906], experimenting with a decoction of dried glands, found by physiological means
(blood pressure) that these glands contained 1*5 per cent, of adrenaline ; according to the
United States Pharmacopeia one part of the dried gland corresponds to six parts of the
fresh gland, so that Hunt's results would indicate a content of 0*25 per cent, in the latter.
For the following observations on the occurrence of adrenaline
in man I have to thank Dr. T. R. Elliott, F.R.S., of University
College Hospital.
At birth adrenaline is almost absent from the supra-renals, but a
large load of it is found in the paraganglion aorticum.1 Thus in a full
term child examined three hours after death : —
paraganglion, O'i'i grm. = -24 mg. adrenalin
left supra-renal, 27 grm. = *oi mg. adrenalin.
The normal weight of each adult supra-renal gland is about 5 grm. ;
in cases of sudden accidental death it contains about 5 mg. of adrena-
line, or about 0*1 per cent.
The adrenaline content rapidly sinks in fevers ; in fatal cases of
pneumonia it may be reduced to I or 2 mg. Similar exhaustion
occurs with the prolonged septicaemia of malignant endocarditis, but
in no fever does it proceed to the minimal values found in Addison's
disease, so that death in fevers cannot be ascribed simply to supra-renal
failure.
In chronic kidney disease, accompanied by high blood pressure,
there is no hypertrophy of the supra-renals, and the glands yield much
1 Compare Elliott [1913]. Fenger [1912, 2] finds on the other hand, by a colorimetric
method, that the gland of the young fcetal calf contains as much adrenalin as the adult
organ. If the discrepancy is not due to the difference in species, it might be that the foetal
gland contains a physiologically inert precursor of adrenaline, giving a similar colour
reaction.
94 THE SIMPLER NATURAL BASES
the same residual load of adrenalin, 2 or 3 mg. , as would be found in
any other individual dying similarly without kidney disease.
The supra-renal gland of mammals is made up by the close asso-
ciation of two tissues, the cortex and the medulla, corresponding
respectively to the inter-renal and adrenal tissues of the lower verte-
brates, in which the two kinds of tissue are less closely associated. In
fishes they occur separately. The medullary substance, also called
chromophil or chromafrin on account of its being stained brown by
chromates, alone contains adrenaline (see for example Gaskell
[1912]). This tissue is also present in the paraganglia, associated
with the sympathetic system of mammals, including the carotid gland,
and the fcetal organs described by Zuckerkandl. An extract of these
paraganglia has been shown to possess the physiological action of
adrenaline. Further details concerning the distribution of chromo-
phil tissue are contained in Vincent's article in the " Ergebnisse der
Physiologic" [1910, under general references to Ch. VI] and Biedl's
" Innere Sekretion " [1913, general references to Ch. VI]. Recently
the remarkable discovery has been made by Abel and Macht [1911,
1912] that adrenaline occurs in the secretion of the so-called "par-
otid gland" (on the skin behind the ear) of a tropical toad, Bufo agua.
The amount of adrenaline in the dried venom is as much as 5 per cent. ;
the substance is chemically and physiologically identical with the
adrenaline from the supra-renal gland of mammals ; in particular the
rotation was found to be [a]D at 20° =--51 -30°, in perfect agreement
with the value given by Flacher (-51 '40°, see above).
Bufo agua is not immune to its own poison and reacts to
adrenaline in the same way as the frog. As might be expected the
tissue of the poison gland gives an intense chromophil reaction with
chromic acid. According to Gunn [1911] cobra venom injected
intravenously has a pressor action like that of adrenaline.
Adrenaline is continuously secreted by the supra-renal gland and
is therefore present in appreciable quantity in the blood of the supra-
renal vein; Cybulski [1895] first demonstrated the pressor action of
the blood from this vein, in which the adrenaline concentration is
of the order of I : 1,000,000. Adrenaline must therefore also be
present in the blood of the general circulation, but the amount is so
small that it cannot be demonstrated with certainty (O'Connor
[1912, I], Stewart [1912]). Adrenaline has been said to occur in
the urine in nephritis, but the evidence is doubtful, and this also
applies to pathological sera.
ADRENALINE (EPINEPHRIN, ADRENINE) 95
It has lately been shown that the secretion of adrenaline is con-
trolled by the splanchnic nerves (Asher [1912], O'Connor [1912, 2],
Elliott [1912], Dale and Laidlaw [1912, 2]). Cutting these nerves
stops the secretion. The supra-renals may be exhausted by fright, by
tetrahydro-/3-naphthylamine and by morphia, but if one of the
splanchnic nerves is cut, the gland on that side is not exhausted
(Elliott). Peripheral electrical stimulation of a cut splanchnic nerve
produces the same effects as an injection of adrenaline. An injection
of nicotine and other alkaloids also stimulates the gland to excrete
adrenaline (Cannon, Aub and Binger [1912], Dale and Laidlaw
[1912,2]).
Asphyxia also increases the adrenaline secretion (Cannon and
Hoskins [1911-2]). The constriction of peripheral blood vessels on
stimulation of the splanchnic nerves (von Anrep [1912]) and the
effect of carbon dioxide on the vascular system (Itami [1912]) are
both due to increased secretion of adrenaline.
Cannon and de la Paz [1911] were the first to show that the
secretion may be stimulated by emotion ; they placed a cat near a
barking dog and found that the blood from the cat's supra-renal vein
contained an increased amount of adrenaline, as shown by its action
on strips of muscle from the rabbit's intestine. It is possible that the
supra-renals obtained from slaughterhouses for this reason contain less
adrenaline than is normally present. Connected with this is emotional
glycosuria (Cannon, Shohl and Wright [1911-2]).
Nothing is known of the nature of the parent substance from
which adrenaline is derived. The base is obviously more closely re-
lated to tyrosine than to any other known constituent of protein, and
Halle [1906] has asserted that the adrenaline content of the supra-
renals is increased when they are incubated with tyrosine, but this
assertion has been disproved by Ewins and Laidlaw [1910, i]. Abel-
ous and his pupils considered at one time that adrenaline is formed by
incubating supra-renals with muscle, but the increased pressor activity
of the mixture was later found to result from the meat alone, which
underwent putrefaction so that p-hydroxyphenyl-ethylamine was
formed (see p. 26). It has been suggested that adrenaline might be
derived from a di-hydroxyphenyl-methyl-serine (by decarboxyl-
ation), but for this there is not the slightest evidence. It should, how-
ever, be noted that Guggenheim [1913] has isolated the amino-acid
3 : 4-dihydroxyphenylalanine, (OH)2C6H3 . CH2 . CH(NH2) . COOH,
from the pods of Vicia Faba.
96 THE SIMPLER NATURAL BASES
Physiological Action of Adrenaline.
A. Action on the Circulatory System.
Oliver and Schafer [1894, 1895, i] and soon afterwards Cybulski
[1895] and Szymonovicz [1895] found that intravenous injection
of supra-renal extracts causes a very marked rise of arterial blood pres-
sure ; this effect is due to the adrenaline contained in such extracts.
Oliver and Schafer showed that the rise of blood pressure is mainly
due to the constriction of the arterioles, but that the action of the
mammalian heart is also accelerated and augmented in a remarkable
manner, the acceleration being most prominent when the vagi have
been cut (cf. Gottlieb [1897]). The vaso-constriction is chiefly of
peripheral origin, due to the action of the drug on the walls of the
arterioles, but some authors have asserted that the vaso-motor centre
also plays a part. Oliver and Schafer [1895, 2] further showed that
the activity is confined to extracts of the supra-renal medulla, those of
the cortex being inactive or nearly so ; the extracts of the gland in two
cases of Addison's disease were also found by them to be inactive.
Cybulski [1895] detected the pressor action of the blood from
the supra-renal vein.
Very minute doses of adrenaline are sufficient to produce a distinct
effect; according to Cameron [1906] 0*0003 mg- per kilo, is enough
in rabbits. The latent period is short and the rise of blood pressure
begins a few seconds after intravenous injection. The rise is very
transitory and the blood pressure soon falls again to the normal level,
at first rapidly, then more slowly. In Oliver and Schafer's experi-
ments, the rise lasted in dogs for at most 4 minutes, and in rabbits
for at most 6 minutes. This rapid cessation of the pressor action,
which is very characteristic of adrenaline, was first attributed to a dis-
appearance of the base from the blood, but Weiss and Harris [1904]
were able to show that after the blood pressure has returned to the
normal, the blood still contains adrenaline, capable of raising the blood
pressure when injected into another animal (cat), and of producing
vaso-constriction, when allowed to flow into a previously ligatured
limb of the animal experimented upon (hind limb of frog).
In man a rise of blood pressure may be produced by subcutaneous
injection of adrenaline, but the effect is much less marked than with
intravenous doses, since the local vaso-constriction, set up at the site of
injection, does not allow a sufficiently rapid absorption of the drug.
This prevents the maintenance of a sufficiently steep gradient of con-
centration between the adrenaline in the blood and that in the arterial
ADRENALINE (EPINEPHRIN, ADRENINE) 97
walls, and it is this gradient which according to Straub's theory is
necessary for the action of certain alkaloids, which only act during and
by virtue of their penetration into the sensitive cells. If the gradient
is maintained by a continuous slow flow of adrenaline into the blood
stream, the pressure may be kept at a high level for hours at a
time, as shown by Kretschmer [1907]. Compare also Straub [1909],
When given by the mouth, adrenaline is without pressor action.
Applied to a mucous surface, it causes marked local vaso-constriction
and blanching ; on this property depends the chief use of adrenaline
as a haemostatic in surgery. The repeated intravenous injection may
cause serious damage to the arterial walls and bring about arterio-
sclerosis.
B. Action on other Organs containing Involuntary Muscle and on
Glands.
Besides affecting the heart and blood vessels, adrenaline acts on
plain muscle in many organs of the body. Thus the muscles in the
wall of the alimentary canal, excepting the sphincters, become relaxed
and their automatic movements cease. The bladder in most animals
is relaxed, but in some it contracts. The uterus is also very sensitive
to adrenaline ; that of the rabbit and of the pregnant cat contract, but
the non-pregnant cat's uterus is relaxed. The amounts of adrenaline
which bring about these effects are as minute as those required for
the pressor action, or even more minute. Kehrer [1908] obtained
tetanic contraction of the pregnant cat's isolated uterus in a bath con-
taining adrenaline in a concentration of I in 350,000,000.
The plain muscle which has perhaps been most commonly em-
ployed as a test object for adrenaline is that of the pupil. The
mydriatic action of adrenaline after intravenous injection was noted
cursorily by Vincent [1897-8] and was first described in detail by
Lewandowsky [1898, 1899]. S. J. and C. Meltzer [1904, i] suggested
the reaction of the frog's eye as a means for determining the strength
of adrenaline solutions, and Ehrmann [1905] subsequently worked
out a method, based on this reaction, which enabled him to detect
quantities of adrenaline as small as 0*000000002 grm.
The above apparently divergent actions of adrenaline on plain
muscular organs may be viewed from a common standpoint if it is
borne in mind that these organs are innervated by branches of the
sympathetic system and that the electrical stimulation of sympathetic
nerves produces effects similar to those caused by adrenaline (Lew-
andowsky, Boruttau, Langley, Elliott). The action of adrenaline (and
7
98 THE SIMPLER NATURAL BASES
of a large number of related amines) resembles that of the sympathetic
nervous system and has accordingly been termed by Dale [Barger and
Dale, 1910, i] " sympathomimetic ". Adrenaline does not, however,
affect the sympathetic nerves themselves, for, as has been shown by Levv-
andowsky[i899, 1900], Langley[ 1901] and Elliott [1905], the reactivity
of plain muscle to adrenaline is not diminished (but rather increased)
by cutting the sympathetic nerve supply and allowing the nerves to de-
generate. Moreover apocodeine, as Dixon has shown, abolishes the
excitability of muscle by sympathetic nervous impulses, and by
adrenaline, but leaves all other irritability unaffected. The blood
vessels of the lungs, which have no sympathetic innervation, are on
the other hand not affected by adrenaline, according to Brodie and
Dixon [I9O4].1 In order to account for the persistence of the
adrenaline action after degeneration of the sympathetic nerve supply,
Elliott [1905] has invoked a hypothetical structure, the " myo-
neural junction," which does not degenerate with the nerve and is the
seat of the action of adrenaline. Langley's conception of a ' ' receptive
substance " for adrenaline is in most essential respects identical with
Elliott's. The nature of the myo-neural junctions determines the re-
sponse to adrenaline, i.e. whether inhibition or augmentation takes
place. Thus these structures would differ in different animals ; in some
species the augmentor elements would predominate, so that adrenaline
causes contraction, in others the reverse condition would prevail. Simi-
larly, during pregnancy, in the cat, the augmentor elements of the
uterine myo-neural junctions would achieve preponderance over the
inhibitor elements, which predominate in the non-pregnant animal.
The existence, side by side, of two kinds of elements, augmentor
and inhibitor, receives considerable support from the discovery by
Dale [1906], that the alkaloid ergotoxine paralyses one set of
elements without greatly affecting the other. Thus the large rise of
blood pressure which adrenaline causes in the normal animal is replaced
by a (smaller) depressor effect, if ergotoxine has been previously ad-
ministered. The ergotoxine paralyses the augmentor elements only
(which normally overcome the inhibitor effect) so that, after ergotoxine,
the inhibition becomes evident and a " vaso-motor reversal " occurs.
1 A different conclusion was reached by Wiggers [1909] who attributes Brodie and
Dixon's results to their use of a perfusion fluid of smaller viscosity than that of the blood.
Older experiments of Plumier and of Langendorff also indicate that adrenaline causes
the pulmonary vessels to contract, but Cow [1911] using O. B. Meyer's method (p. 103)
finds that the intravisceral portion of the pulmonary, the cerebral and the coronary arteries
are not constricted. The action of adrenaline on the pulmonary vessels has also been
studied by Baehr and Pick [1913, 2, Ch. I].
ADRENALINE (EPINEPHRIN, ADRENINE) 99
In this connection it is of some interest that Ogawa [1912] has
recently shown that when the blood vessels of certain isolated organs
(e.g. kidney of dog, cat and rabbit) are perfused with very dilute
adrenaline solutions (i : 50 millions) these vessels are dilated. With
slightly more concentrated solutions a constriction occurs followed by
a secondary dilatation ; larger doses at once produce constriction with-
out subsequent dilatation.
Adrenaline, injected intravenously, causes the bronchioles to dilate
and abolishes the contraction due to muscarine (Januschke and Pollak
[1911]; confirmed by Dixon and Ransom [1912]; see also Jackson
[1912]; Golla and Symes [1913]; Baehr and Pick [1913, I, Ch. I]).
Hence adrenaline is used in the treatment of asthma.
Action on Glands. — Langley [1901] has shown that an injection
of adrenaline excites the secretory activity of salivary and other glands,
and this action, as in the case of plain muscle, apparently persists after
the degeneration of the sympathetic nerve supply.
C. Action on Carbohydrate Metabolism.
As was first shown by Blum [1901], subcutaneous or intravenous
injections of supra-renal extract (in sufficient doses) cause glycosuria ;
this action is due to the adrenaline and does not occur after oral
administration. The latent period is much longer than in the case of
the pressor action and sugar may occur in the urine for several days
after the injection. In other respects there is a close analogy to the
pressor action. Straub [1909] found adrenaline could be injected
continuously at the rate of 0*002 mgm. per minute without causing
glycosuria, but that sugar appeared in the urine when the rate of in-
jection was doubled. This is about the same as found by Kretschmer
[1907] for the pressor action. Although much work has been done
on the subject, the mechanism of adrenaline glycosuria, like that of
other forms of glycosuria, has not yet been cleared up. It appears that
adrenaline causes a greatly increased production of glucose by the liver
and that adrenaline glycosuria is independent of the pancreas. (Com-
pare for instance experiments on birds, after extirpation of the pan-
creas, by Paton [1903, 1904].)
Pollak [1909] concludes from his experiments on hungering rab-
bits that adrenaline causes an accumulation of glycogen in the liver.
Any injection of the drug will also increase the sugar content of the
blood, but glycosuria does not necessarily occur ; it will do so more
probably if diuresis is also set up. In a later paper Pollak [1910]
denies the alleged special protective action of d-adrenaline against the
7*
ioo THE SIMPLER NATURAL BASES
diabetic effect of the natural 1-variety. The minimal dose of the latter
which produces glycosuria in rabbits of 2 kilos, is o*4-O'5 mg.
D. Toxic Action of Adrenaline.
The effects which have so far been described are all brought about
by minute doses of adrenaline. Larger, although still quite small
doses cause death, and adrenaline is therefore a powerful poison. For
guinea-pigs, rabbits, and dogs the fatal intravenous dose is about one-
tenth to one-quarter of a milligram per kilo, of body weight. For cats
the corresponding dose is 0*5 -O'8 mg. per kilo. The subcutaneous
lethal dose is very much higher; for white rats Cushny [1909]
found 10-20 mg. per kilo, arid Schultz [1909, i] for mice 8 mg.
per kilo, of body weight. For guinea-pigs the corresponding dose is
10 mg. according to Crawford [1907]. For the toxicity to dogs and
cats, reference may also be made to Lesage [1904, I, 2].
The Physiological Action of Dextro- and of Racemic
Adrenaline.
Cushny, who discovered the difference in the physiological activity
of optical enantiomorphs in the case of hyoscyamine and hyoscine,
also first drew attention to the quantitative differences in the action
of natural 1-adrenaline and the synthetic racemic sulpstance.1 He
[1908] found racemic adrenaline to be about half as active as the
natural variety and concluded therefore that d-adrenaline is inactive.
Later [1909], having at his disposal a specimen of the dextro-variety,
he was able to estimate its activity directly, instead of by difference,
and he slightly revised his preliminary conclusion. The specimen of
d-adrenaline examined had TVrV of the activity of 1-adrenaline in
raising the blood pressure of dogs and cats. The ratio of the pressor
activities of racemic and natural adrenaline is therefore not I : 2 but
between 13 : 24 and 16 : 30. The ratio of the activities of the two
isomerides in producing glycosuria was very similar, namely I : 12-18,
and the minimal lethal doses for white rats were in about the same
ratio.
The different physiological activity of the two enantiomorphous
adrenalines has also been dealt with in a series of papers by Abder-
halden, in collaboration with Miiller [1908], Thies [1909], Kautzsch
[1909], Slavu [1909], and Kautzsch and Miiller [1909]. Some
of the conclusions arrived at are that 1-adrenaline is fifteen times as
1 Cf. Dixon, Pharm. Journ., 1908, XXVI, 723; Piberfeld, ibid., p. 626; Cushny,,
ibid., p. 668.
ADRENALINE (EPINEPHRIN, ADRENINE) 101
active on the blood pressure as d-adrenaline, that the effects of the
two isomerides on the frog's eye and in producing glycosuria are
different, and that d-adrenaline establishes a tolerance to the toxicity of
1-adrenaline. Of these experiments those on the frog's eye, by Abder-
halden and Thies, and the toxicity experiments, by Abderhalden and
Slavu, have been criticised by Schultz [1909, 2].
Schultz had previously [1909, i] carried out an extensive series
of very careful experiments on the relative activity of racemic and
1-adrenaline; it is a matter for regret that he was not also in possession
of a pure specimen of the dextro-variety. He found the pressor effect
of the natural base to be one and a half times that of the racemic syn-
thetic product. Dale [Barger and Dale, 1910, l] obtained the same
ratio (6*5 : 10) but does not regard the discrepancy from Cushny's
ratio (16 : 30) as having any significance. Biberfeld's original state-
ment [1908] that the racemic base is as active as the laevo-variety is
certainly erroneous. Schultz [1909, 2] states that the ratio of the
activities of dl- and 1-adrenaline on the frog's eye and the toxicity
ratio for white mice is the same as that of the pressor activities,
namely 1:15.
Various authors have suggested that d-adrenaline renders the
organism less sensitive to the action of the natural 1-variety and to
some extent confers an " immunity," so that subsequent doses of
1-adrenaline have a much smaller effect than is normally the case.
This has been claimed for the pressor action by Frohlich [1909], for
the toxicity (to mice) by Abderhalden in collaboration with Slavu
[1909] and with Kautzsch [1909], and for the diabetic action by
Waterman [1909, 1911]. With regard to the last-named effect Pollak
[1909, 1910] has, however, come to a different conclusion and considers
that d-adrenaline is as little able to prevent glycosuria by 1-adrenaline
as a previous dose of 1-adrenaline itself. A phenomenon, similar to
that observed by Frohlich, has recently been described by Ogawa
[1912] who finds that the secondary vaso-dilatation referred to
above (p. 99), when due to d-adrenaline, is not so readily abolished
by 1-adrenaline as the dilatation caused by (smaller doses of the more
active) 1-adrenaline.
Physiological Methods of Estimating Adrenaline.
At a time when little was known of the chemistry of adrenaline,
the methods employed in its estimation were perforce physiological,
and even now the best physiological methods are preferable to the
102 THE SIMPLER NATURAL BASES
colorimetric processes which have been suggested more recently (see
p. 92). The quantitative estimation of adrenaline is of importance
in many physiological and pathological investigations.
The most obvious, accurate and reliable method is based on a
comparison of the pressor effects of intravenous injections ; the
peculiarly evanescent nature of this adrenaline action greatly favours
accurate comparison, and in a suitably prepared animal equal sub-
maximal doses will produce time after time practically identical
effects ; this method is, however, inapplicable to very dilute adrenaline
solutions. The blood pressure of a cat, with brain and spinal cord
destroyed and without anaesthetic, reacts according to Elliott [1912]
" with mechanical accuracy," and by comparison with a standard
solution, Elliott assays the adrenaline content of the cat's supra-renal
gland with an error of croi mg., which is 3-4 per cent, of the total
amount present.
The accurate pharmacological assay of preparations of the supra-
renal gland by means of the blood pressure was first carried out by
Houghton [1901] ; the blood pressure has further been used especially
by Elliott [1912], Hunt [1906], Sollmann and Brown [1906], Cushny
[1908, 1909], Schultz [1909, I], and Dale [Barger and Dale, 1910, I].
Schultz employed dogs (with morphine, ether and curari) and cats
(with ether), Elliott and Dale almost exclusively decerebrate cats. The
doses are TOTT^T mg. fc>r dogs and -^nru mg- f°r cats (which are more
resistant than dogs). Other blood-pressure methods, such as the de-
termination of the dose required to compensate for the vaso-dilator
action of a given quantity of nitroglycerine (Cameron [1906]) and the
determination of the minimal dose necessary to give a perceptible
pressor effect, are much less accurate.
A second method employing the circulatory system but depending
on vaso-constriction instead of on blood pressure is due to Lawen
[1903-4] and has been improved by Trendelenburg [1910]. The rate
is measured at which, under a constant hydrostatic pressure, blood flows
through the vessels of a frog, of which the brain and spinal cord have
been destroyed ; the adrenaline to be estimated is added to the blood.
This method appears to yield moderately accurate results, but is la-
borious when many estimations have to be performed. The significance
of determinations by this method of adrenaline in serum has recently
been questioned by O'Connor [191 1 , 1912, i] who finds that serum itself
causes vaso-constriction, quite apart from the addition of adrenaline
(see also Handovsky and Pick [1913, Ch. I]). Stewart [1912], and
Dale and Laidlaw [1912, 2] agree with O'Connor's objections to the
ADRENALINE (EPINEPHRIN, ADRENINE) 103
use of serum. According to Stewart it is possible to prove the pres-
ence of adrenaline only in the blood from the supra-renal vein.
Besides those on the circulatory system, the other effects of adrena-
line on plain muscle, described in a previous section, are to some
extent available for the quantitative estimation of the drug ; the
methods which have been suggested, based on these effects, are much
less accurate than the blood-pressure method, but, on the other hand,
some of them are more suitable for the very rough estimation of
extremely minute quantities of adrenaline, such as may occur in the
blood or in tissue extracts. In such cases it is, however, necessary to
avoid confusion with other ill-defined substances (such as vaso-dilatin,
p. 30) which may produce similar effects in plain muscle (cf. Hoskins
[1911] and O'Connor [1912, i]).
O. B. Meyer [1906] has employed isolated rings of the sub-
clavian or carotid artery of the ox, which contract in solutions of
adrenaline up to I : 1,000,000,000 (0-000015 mg. in 15 c.c. Ringer's
solution). Cow [1911] has investigated other arteries by this
method and finds that the only arteries not constricted by adrenaline
are the intravisceral portion of the pulmonary, the coronary and the
cerebral arteries. Argyll Campbell [1911] also finds by this method
that adrenaline causes marked constriction of the vessels of all
organs, except those of the heart and lungs. A slight constriction
occurs occasionally in the heart and more frequently in the lung
vessels.
A. Frankel [1909] used the isolated uterus of the rabbit, which
still reacts to adrenaline at a dilution of I : 20,000,000, but Hoskins
[1911] states that this reaction is not specific and that contractions
are caused by a large number of glandular and tissue extracts ; the
use of the rabbit's uterus for testing serum has also been criticised by
Stewart [1912].
Cannon and de la Paz [1911] employed longitudinal strips of muscle
from the rabbit's intestine and Hoskins [191 1] a short length of small
intestine from the same animal. These two methods depend on the
inhibition, by adrenaline, of the spontaneous contractions. In Hoskins's
experiments this inhibition occurred regularly at I : 100,000,000 and
sometimes even at I : 500,000,000. Hoskins considers his method
and that of O. B. Meyer (above) to be the most sensitive methods
known. According to O'Connor [1912, I] substances are formed
during the coagulation of blood with actions simulating this and other
effects of adrenaline, but by using the plasma, instead of the serum,
and rabbit's intestine as test object, he finds that the blood from the
104 THE SIMPLER NATURAL BASES
supra-renal vein contains one part of adrenaline in I to 5 millions ; he
could not demonstrate adrenaline with certainty in the peripheral
blood. Stewart, who employed this method and that depending on the
contraction of the rabbit's uterus, also concludes that adrenaline is not
detectable in the general circulation, or indeed in blood from the supra-
renal vein, except during massage of the gland or stimulation of the
splanchnics, when there was respectively I : 500,000 and I : 1,000,000.
Dale and Laidlaw [1912, 2] have used as a test object another
organ which is inhibited by adrenaline, viz. the non -pregnant uterus
of the cat. In a cat under chloroform and ether they find that the
blood from the supra-renal vein contains one part of adrenaline in from I
to 2 millions. • After injection of pilocarpine this amount was increased
tenfold.
The method which has been most widely used for the detection of
small quantities of adrenaline is based on mydriatic action, particu-
larly as applied to the excised eye of the frog. This test object was
first employed by S. J. and C. Meltzer [1904, i, 2] ; later Ehrmann
[1905] brought it into prominence by his experiments on body fluids
and by his claim that the excised eye, being much more sensitive than
the intact eye, can reveal adrenaline in a concentration of I : 10,000,000.
According to Borberg [1912] the sensitiveness is only one-tenth of
this. Schultz [1909, i] has elaborated the technique of this method
by measuring the pupil under the microscope. Hoskins [191 1] dis-
sected the eye, removed the lens and applied the fluid under examina-
tion directly to the iris ; in this way results were obtainable at a dilu-
tion of i : 5,000,000 and sometimes a positive result was noted at
I : 100,000,000, but a mydriatic effect is also shown by pituitary ex-
tract, iodothyrin, etc., which renders the method very uncertain when
applied to the detection of adrenaline in the blood. Schultz [1909, 2]
considers that Ehrmann overstated the sensitiveness of the method.
He writes : " At its very best the excised frog's eye as a pharmaco-
logical assay for adrenaline is inferior to the blood-pressure method.
As a qualitative test it is perhaps one of the most sensitive test-objects
known, but it is not a characteristic test (Comessatti, Meltzer) and
observations convince me that too much weight ought not to be at-
tached to results with it in clinical diagnosis ". This adverse opinion
is shared by Cameron [1906] and by Borberg [1912], but the
method at least has the advantage that it is applicable to very dilute
solutions and that it can be used by the chemist who cannot undertake
more elaborate animal experiments. According to Schultz the dilata-
tion time is a better index than the degree of mydriasis and one should
ADRENALINE (EPINEPHRIN, ADRENINE) 105
aim at making this time equal for both of a pair of eyes. In a recent
article on the estimation of adrenaline in the blood, Gottlieb and
O'Connor [1912] place the blood-pressure method first in point of
accuracy, provided the adrenaline solution is sufficiently concentrated.
Next comes the perfusion of the frog's blood vessels, which may be
used quantitatively and is more sensitive (up to I : 30,000,000). For
the qualitative recognition of the minutest quantities the inhibition of
the cat's small intestine is very specific and, in particular, it is not pro-
duced by serum (limit I : 400,000,000).
CHAPTER VII.
BASES OF UNKNOWN CONSTITUTION.
THE constitution of nearly all the bases dealt with in the preceding
chapters is known with certainty. In addition a large number of bases
of unknown constitution have been described at various times. In
many cases even their composition has not been fully established.
Nevertheless some of the latter class will be included here on account
of their great physiological interest. It is of course impossible to say
whether they have a " simple constitution," but in any case the methods
by which their isolation may be attempted are similar to those used
for the other bases of this monograph.
Spermine.
The phosphate of this base crystallises out when semen dries, and
constitutes over 5 per cent, of the solids. It has been most fully in-
vestigated by Schreiner [1878] who prepared it in a pure condition
by boiling fresh human semen with alcohol, filtering off and drying
the precipitate so formed, extracting the latter with very dilute warm
aqueous ammonia and then concentrating. The phosphate is hardly
soluble in cold, and only a little in hot water, but soluble in dilute acids
and alkalies. The salt contains two atoms of nitrogen to one of phos-
phorus, and at 100° 3H2O are given off; it melts at 170°.
Schreiner found that the crystals on the surface of old anatomical
preparations (Bottcher's crystals) are identical with spermine phosphate ;
he obtained them by scraping them off the surface of calves' livers and
hearts and bulls' testes, kept in alcohol for three months.
It has further been suggested that the crystals discovered by Charcot
in the spleen, liver, and blood in cases of leucocythaemia, and also found
in the sputum in cases of bronchial asthma, are identical with spermine
phosphate, but this does not appear to be the case.
Schreiner assigned to spermine the formula C2H5N ; Ladenburg
and Abel [1888] considered it to be most probably identical with
piperazine C4H10N2, which has the constitution : —
x
NH(
\CH2.CH/
106
BASES OF UNKNOWN CONSTITUTION 107
By direct comparison with a specimen of Schreiner's preparation
they found a great similarity to piperazine but also some differences.
Schreiner's specimen was found to be slightly impure and to contain
calcium. Ladenburg and Abel considered that Schreiner's phosphate
might conceivably be (C4H10N2)2CaP2O8 which agrees better with his
analyses. Poehl [1891] arrived at the formula C10H.,6N4 for spermine
after analysing the platinichloride and the aurichloride, but the formula
C5H12N2 would also fit his results.
Bases from Muscle.
In addition to creatine, methylguanidine, carnosine, carnitine,
neosine, betaine, myokynine, and trimethylamine-oxide, all described
previously, the following may be mentioned : —
Vitiatine, C5H14N6, has been obtained by Kutscher [1907] from
meat extract and is regarded by him as a guanidine derivative of the
possible constitution : —
/
: C
HN : C C : NH
\N(CH3) . CH2 . CH2 . NH/
Crangitine, C13H2oO4N2, and crangonine, C13H26O3N2, have been ob-
tained by Ackermann and Kutscher [1907, 4, Ch. Ill, betaine] from
shrimps.
Creatosine has been obtained from commercial meat extract by
Krimberg and Izra'ilsky [1913] and yields an aurichloride
CuH2804N3Au2Cl8.
Bases from Urine.
The following bases, already described, have been isolated as
normal or occasional constituents of human or animal urine : trime-
thylamine, isoamylamine, putrescine, cadaverine, iminazolylacetic acid,
urocanic acid, kynurenic acid, methylpyridinium hydroxide, ^-picoline,
butyrobetaine, carnitine (= novaine), reductonovaine, creatine, creati-
nine, methylguanidine, dimethylguanidine, vitiatine. In addition the
following may be mentioned : —
Mingine, C13H18O2N2. Kutscher [1907, Ch. Ill, butyrobetaine]
obtained 0-45 grm. of the di-aurichloride from 100 litres of women's
urine.
Gynesine, C19H23O3N3. Kutscher and Lohmann [1906, 4, Ch. Ill,
butyrobetaine] obtained 1*5 grm. of the aurichloride C19H2SO3N3,
2HAuCl4, from 100 litres of women's urine.
Kynosine, Cl3H.,6O4N4, was isolated from normal dog's urine as the
aurichloride C13H26O4N4, 2HAuCl4 by Kutscher [1906].
xoB THE SIMPLER NATURAL BASES
Putrefaction Bases.
In addition to the amines of Chapter I and some other bases
mentioned in the previous chapters a large number of less well char-
acterised putrefaction bases have been described. A few of these
may be mentioned here : —
Viridine^ C8H12OaN2, was obtained by Ackermann [1908, 2] from
putrid pancreas. The hydrochloride has an intense green colour ; on
heating the odour of quinone is perceptible. The aurichloride is
blackish green to yellow and melts at 176° ; the platinichloride is intense
yellow and melts at 212-216°.
Marcitine, C8H19N3, also obtained by Ackermann [1907, 2] from
putrid pancreas, gives an aurichloride C8H19N3, 2HAuCl4 melting at
175-178°. It is perhaps a guanidine derivative.
Putrine, CnH26O3N2, likewise isolated by Ackermann [1907, 2] from
putrid pancreas, gives a dark orange aurichloride melting at 109-1 10°.
The formula of this base contains one carbon atom and two oxygen
atoms less than the so-called diamino-trihydroxy-dodecanic acid
C12H26O5N2 of Fischer and Abderhalden from which it is perhaps de-
rived by decarboxylation.
Skatosine, C10H16O2N2, has been described by Baum [1903] and
Swain [1903] as a product of pancreatic autolysis. It is stated to
give a benzoyl derivative melting at 169° and a hydrochloride forming
leaflets melting at 345°. To the latter the improbable formula
C10H16O2N2, 3HC1 was given. Mr. A. J. Ewins (private communica-
tion) has lately failed to obtain this base by Baum's process.
The Active Principle of the Pituitary Body.
Soon after their discovery of the pressor action of supra-renal ex-
tracts Oliver and Schafer [1895, 3] found that an extract of the pituitary
body or hypophysis cerebri (a small appendage at the base of the brain)
has the power of raising the blood pressure, when injected intravenously.
The active principle is only contained in the infundibular or posterior
lobe of this organ. At first stress was laid in the literature on the
similarity of the action to that of adrenaline, and some authors even
imagined that the two active principles must have a similar chemical
constitution. During the' last few years pituitary extracts have come
more and more into therapeutic use on account of their great power of
producing contractions of the uterus, and the isolation of the active
principle has been attempted. Although these attempts have perhaps
not been wholly successful as yet, they seem to prove that the active
substance is a base ; little else is definitely known about its chemical
BASES OF UNKNOWN CONSTITUTION 109
constitution. Its physiological action has, however, been studied in
some detail and such correspondence as exists between the action of
the pituitary body and of adrenaline has been found to be " superficial
and illusory".
The chemical investigation of the pituitary active principle is greatly
hampered by its instability and by the difficulty of procuring enough
material. The infundibular portions, dissected clean from fresh glands,
are ground up with sand and boiled with water acidulated with acetic
acid. After filtration a clear colourless extract is obtained, which
contains a little protein and some phosphates. By the addition of
uranyl acetate the phosphates may be precipitated and most of the
protein is carried down with the precipitate, but the solution remains
physiologically active. Almost the only precipitant for the active
principle itself is phosphotungstic acid, as has for instance been found
by Engeland and Kutscher [1911] and by Meister, Lucius and
Briining (see Fiihner [1913]). The chemists of the Hoechst firm,
on decomposing the phosphotungstate with baryta, and removing the
excess of baryta with sulphuric acid, obtained on concentration in vacua
a pale yellow crystalline sulphate, which was physiologically active
and apparently homogeneous, but was afterwards separated by fractional
crystallisation into four different substances, all crystalline, and all
having some physiological activity. Two of these were more active
than the others ; the more abundant of the two is a colourless sulphate,
readily soluble in water, but only slightly so in alcohol, acetone, or
ethyl acetate. It gives Pauly's histidine reaction with p-diazobenzene-
sulphonic acid and also the biuret reaction. Its picrate is readily soluble
in water. In contact with alkali a volatile amine is at once given off.
According to Fiihner, who has examined physiologically the various
substances from the phosphotungstate, they all contribute to the
activity of the gland ; thus there would be four active principles.
The facts at present available do not, however, absolutely exclude
the possibility that these four substances all owe their activity to con-
tamination, in various degrees, with one and the same highly active
substance which has so far escaped isolation. The further chemical
examination of the most active of the four substances should prove of
great interest. That this substance gives the biuret reaction may be
considered in conjunction with an observation by Dale [1909] that
the activity of pituitary extracts is rapidly destroyed by trypsin and
much less rapidly by pepsin. This would point to a polypeptide struc-
ture. The activity is also fairly rapidly lost when an aqueous solution
is evaporated to dryness ; perhaps this is owing to hydrolysis.
i io THE SIMPLER NATURAL BASES
The fact that the bases from a pituitary extract give the Pauly re-
action suggests a connection with histidine, and moreover /3-imina-
zolylethylamine, which is obtained from histidine by decarboxylation,
also causes powerful contractions of the uterus. Possibly, therefore,
the pituitary active principle is a polypeptide-like derivative of
histidine.
Guggenheim [1913] has lately synthesised a number of bases
by combining amines with chloracetylchloride and treating the pro-
duct with ammonia. In this way, for example, glycyl-/3-iminazolyl-
ethylamine
NH . CH^
^C . CH2 . CH2 . NH . CO . CH2 . NH2
CH = NX
was prepared. The bases of this type, for which the name pep famine
is suggested, are therefore decarboxylated polypeptides ; their physio-
logical action is of the same kind as the amine from which they are
derived, but much weaker.
The physiological action of pituitary extracts has been in-
vestigated chiefly by Schafer, in conjunction with Oliver [1895, 3],
Magnus [1901], Herring* [1906] and Mackenzie [1911], and further
by Dale [1909], von Frankl-Hochwart and Frohlich [1910], Pankow
[1912] and others. Pituitary extract produces a direct stimula-
tion of involuntary muscle, without any relation to innervation.
Here there is, therefore, an important difference from adrenaline which
stimulates sympathetic nerve endings (see p. 98). The action of
pituitary is most nearly allied to that of the digitalis series, but the
effect on the heart is slight, that on plain muscle intense. The rise
of blood pressure caused by pituitary is thus due to the stimulation of
the plain muscle of the arterioles. The rise is much smaller than in
the case of adrenaline and lasts much longer. A further difference is,
that when the blood pressure has returned to the normal, the rise
caused by adrenaline can at once be reproduced by a second dose, but
in the case of pituitary the effect of a second dose is much smaller, un-
less it is administered after a considerable interval of time. In the
birds pituitary extract causes a fall of blood pressure, which is anta-
gonised by adrenaline and by barium (Paton and Watson [1912]).
The powerful stimulation of uterine plain muscle was first pointed out
by Dale [1909] and also studied by von Frankl-Hochwart and
Frohlich [1910] and was first applied clinically by Bell [1909] in
England and soon afterwards by Foges and Hofstatter in Germany.
The supposed pure substances have been used clinically by Herzberg
[I9I3J
BASES OF UNKNOWN CONSTITUTION in
Pituitary extracts bring about contraction of the uterus in the cat,
dog, guinea-pig, rat, and rabbit, in all functional conditions. Adrena-
line, on the other hand, in some of these species has a motor effect
on the pregnant uterus only and inhibits the non-pregnant organ.
The effect of pituitary extracts on the uterus can be shown both by
intravenous injection into the anaesthetised animal and by means of the
surviving uterus in a bath of oxygenated Ringer's solution. The
latter method, applied to the uterus of the young virgin guinea-pig, has
been worked out by Dale and Laidlaw [1912, i] to a process for
standardising pituitary extracts and has also been used more recently
by Fiihner [1913]. It has the great advantage over blood pressure
experiments that tolerance is practically absent. Dale and Laidlaw
find that o-J^- c.c. of an extract obtained by boiling infundibula with five
parts of water will produce almost maximal tonus of the uterus in a
bath of 250 c.c. Ringer solution. Since such an extract only contains
about O'6 per cent, of solids, this represents a concentration of little
more than cri mg. of solid matter per litre, most of it being inert
material. The pituitary active principle is therefore a very powerful
uterine stimulant, the activity being probably at least of the same
order as that of y@-iminazolyl-ethylamine.
In addition to the above effects on plain muscle, pituitary extracts
bring about a profuse flow of urine and also greatly increased secretion
of milk. The diuretic action was discovered by Schafer in conjunc-
tion with Magnus and with Herring and was at first attributed to a
different substance from that causing the rise of blood pressure ; later
observers, however, consider that the active principle is the same in
both these cases. According to Houghton and Merrill [1908]
diuresis is merely a secondary effect of the rise in blood pressure and
is also brought about by injecting adrenaline. The galactagogue
action was first observed by Ott and Scott [1911] and has subse-
quently been described by Schafer and Mackenzie [1911], and Ham-
mond [1913]. For the effect on the mammary gland in the human
subject see Schafer [1913].
Vitamine, Oryzanine, Toruline.
A polyneuritis, resembling the tropical disease beri-beri, can, as
Eykman discovered, be induced artifically in fowls by feeding them on
an exclusive diet of polished rice. The condition is due to the lack
of a substance present in the outer coating of the rice and removed in
the process of polishing. During the last year or two several attempts
have been made to isolate this curative substance from various sources.
ii2 THE SIMPLER NATURAL BASES
Funk [1911] in England, and Suzuki with Shimamura and
Odake [1912] in Japan, showed independently and about the same
time that the substance is a base, is present in very small amount, and
has great curative action. To Funk belongs the further credit of
having been the first to analyse the substance and to isolate the same
or a similar body from yeast. Chemical work in this direction has
also been done by Schaumann [1912, I], Moore and his collaborators
[1912], Cooper [1913] and others.
In spite of the discrepancies which exist between the statements of
various authors, it seems fairly well established that the curative sub-
stance in rice polishings, for which Funk has suggested the name
vitamine and which Suzuki and his collaborators call oryzanine, is a
base which can be extracted by water and by alcohol, but not by
acetone or ether. It is precipitated by phosphotungstic acid, by
tannin, by mercuric chloride in alcoholic solution and by silver nitrate
and baryta. The latter property indicates the presence of an imino-
group. The mercurichloride is soluble in boiling water.
Suzuki, Shimamura and Odake describe a crystalline picrate of
their substance, which they did not however analyse. Funk, by
utilising the properties indicated above, obtained from rice polishings
a minute yield of a crystalline substance, to which he assigned the
formula C17H20O7N2, but more recently [1913], by fractional crystal-
lisation, he separated it into two substances ; one of these was found
to give the following average analytical results: C = 58*85 per cent,
H = 3-9 per cent, N = 10*6 per cent ; it melted at 233°. The other
gave on the average C = 58*4 per cent, H = 4*0 per cent, N = 11*05
per cent, and melted at 234°. The latter was identified as nicotinic
acid, C6H5O2N, which, in the pure state, is inactive and had already
been obtained from rice by Suzuki. To the former substance Funk
gave the formula C26H20O9N4 and he stated that it is a tetrabasic acid.
It is considered by Funk to be the chief curative substance in rice
polishings. Funk separated the " vitamine " fraction of yeast, which
he at first considered to be identical with that of rice, into nicotinic
acid and an active principle melting at 229° (corr.) which when dried in
vacua at room temperature has the formula C26H21O9N5, but dried at
1 00° changes to C24H19O9N5, implying the somewhat unusual loss of
two carbon and two hydrogen atoms.
It will be seen that the substance C26H20O9N4 obtained from rice
has a very close resemblance to nicotinic acid, both as regards melting
point and chemical composition, and at present the possibility does not
seem completely excluded, that this body is merely nicotinic acid con-
BASES OF UNKNOWN CONSTITUTION 113
taminated with a small quantity of a highly active substance richer in
carbon. Further work will therefore be of the greatest interest.
Funk and also Schaumann consider that there are a number of
substances capable of preventing and curing polyneuritis. The former
[1912, 2] has found that certain purine and pyrimidine derivatives have
a weak activity in this direction. The crystalline and apparently
homogeneous vitamine fraction from rice and from yeast is active in
doses of a few centigrams, and when injected subcutaneously such
doses will restore a severely paralysed pigeon within a few hours. A
substance curing polyneuritis is also present in ox brain, in milk (Funk
[1912, i]), and in muscle (Eykman [1897], Cooper [1913]). Edie,
Evans, Moore, Simpson, and Webster [1912] have given the name
toruline to an antineuritic base from yeast having the formula
C7H17O5N2. A concomitant effect of a diet of polished rice is a loss
of body weight which has been taken into account more particularly in
the experiments of Suzuki and his colleagues. In this connection
attention may be drawn to the work of Hopkins [1912] which shows
that growth is greatly influenced by some as yet undetermined con-
stituents of food.
Sepsine.
The name sepsine was given more than forty years ago by
Schmiedeberg to a poisonous putrefaction product which was more
recently isolated by Faust [1903-4] as a crystalline sulphate. Faust
used putrid yeast and obtained under the most favourable conditions
only 0-03 grm. of sepsine sulphate from 5 kilos, of yeast. The pro-
cess of isolation is a complicated one, one of its chief features being
that the sepsine is precipitated by mercuric chloride from an aqueous
solution rendered strongly alkaline by means of sodium carbonate.
Later the sulphate separates out in a crystalline condition by fractional
precipitation of the alcoholic solution of the base by means of
sulphuric acid dissolved in alcohol. The sulphate can be recrystal-
lised and then forms well-developed crystals having according to
Faust the composition C5H14O2N2, H2SO4 ; his analyses, however, fit
equally well or slightly better the formula C5H12O2N2, H2SO4. The
free base is a syrup readily soluble in water.
Sepsine is very unstable ; on repeated evaporation of the aqueous
solution of the sulphate on the water bath this salt is transformed
according to Faust into cadaverine sulphate, and the substance loses
its physiological activity. This transformation, which involves the
loss of two oxygen atoms, is without any analogy and very difficult to
8
ii4 THE 'SIMPLER NATURAL BASES
understand. Perhaps the identification of the inactive substance as
cadaverine is erroneous, as it is apparently only based on the platinum
content of a platinichloride. Perhaps the analyses of sepsine sulphate
have been wrongly interpreted. However this may be, it seems clear
that a crystalline substance of remarkable physiological properties was
obtained, corresponding to those originally possessed by the putrid
yeast and described by Schmiedeberg.
Twenty mg. of sepsine sulphate injected into a dog of 7-8 kilos,
weight very soon cause vomiting and defecation ; finally almost pure
blood is passed and the poisoning ends fatally ; sepsine is a capillary
poison.
Fornet and Heubner [1908] have isolated organisms which
they imagined produce sepsine and the chief of these they named
Bacterium sepsinogenes, but in a later paper [1911] they greatly
modified their original conclusions. The organism referred to was
found not to produce sepsine but a colloidal poison having a similar
action and being in some respects comparable to the toxin formed in
anaphylaxis.
A further chemical investigation of Faust's sepsine appears to be
very desirable, particularly if it could reveal the constitution of this
interesting substance.
Secretine.
This substance, which causes secretion of pancreatic juice when
injected intravenously, appears to be a base, judging from a method of
purification described by Dale and Laidlaw [1912, 3]. This is founded
on the solubility of the mercury compound in moderately dilute acid
and its insolubility in neutral or weakly acid solution. Dale and
Laidlaw's method may be given as an additional example of the tech-
nique of using mercuric chloride for the separation of bases (cf. p. 1 19).
The mucous membrane of the intestine of dogs is scraped off
weighed and ground up with one-fifth of its weight of solid mercuric
chloride to a smooth paste ; then two parts of water are added for every
part of the mucous membrane taken. This mixture can be accumu-
lated and kept indefinitely ; the mercuric chloride coagulates the pro-
tein and acts as an antiseptic. To work up the mixture it is boiled,
filtered through paper or muslin, and pressed dry. The press cake is
suspended in an aqueous I per cent, mercuric chloride solution
containing acetic acid ; 4 c.c. of this are used for every gram of moist
mucous membrane taken. The mixture is boiled and filtered, and the
filtrate should be nearly clear. Ten per cent sodium hydroxide is added
BASES OF UNKNOWN CONSTITUTION 115
until the filtrate is nearly neutral, i.e. until the yellow mercuric oxide
just fails to be permanent. The white flocculent precipitate formed
is collected at the pump, suspended in hot water, and decomposed by
hydrogen sulphide ; after neutralising and boiling off the hydrogen
sulphide the solution is filtered and then furnishes a strongly active
secretine solution. The active substance can further be precipitated
from this solution by excess of picric acid, but attempts to obtain it
chemically pure have so far been unsuccessful.
8 *
CHAPTER VIII. (APPENDIX.)
PRACTICAL CHEMICAL METHODS AND DETAILS.
A. GENERAL METHODS FOR THE SEPARATION AND ISOLATION OF
BASES.
WITH few exceptions the simple natural bases are readily soluble in
water, but not in ether or chloroform. As a rule they cannot therefore be
extracted from alkaline solution by shaking with organic solvents, and
the methods of Stas and DragendorfT, employed for the isolation of
vegetable alkaloids and based on the use of solvents immiscible with
water, are therefore not applicable. The earliest work on putrefaction
bases, therefore, suffered from too close adherence to the methods used
for alkaloids ; amylamine and phenyl-ethylamine which are readily
soluble in ether and in chloroform, and p-hydroxy-phenyl-ethylamine
which dissolves in amylalcohol, are among the few simpler bases which
can be isolated in this manner.
In general, therefore, the isolation of these bases is effected by
means of an insoluble salt or other derivative, a method which in the
case of putrefaction bases was first extensively used by Brieger, with
conspicuous success.
The simplest (aliphatic) monamines are volatile with steam and can
therefore easily be separated by steam distillation , first from acid solution
in order to remove non-basic volatile products and subsequently from
alkaline solution. Some non-volatile bases, particularly betaines, are
decomposed by strong alkalies with evolution of trimethylamine ; if
such bases are present the solution should only be made alkaline with
magnesium oxide and the distillation should be carried out at a low
temperature under reduced pressure. This precaution is for instance
important in the estimation of trimethylamine in urine.
When bases have to be isolated from a complex mixture such as a
tissue extract, it is necessary to remove first proteins and peptones as
far as possible. The oldest method employed for this purpose is to
evaporate the aqueous extract to a small bulk and add alcohol which
precipitates the proteins, but leaves the salts of organic bases in solution.
The separation is, however, not very complete ; in some cases it may be
improved by using acetone instead of alcohol. The aqueous solution
116
GENERAL METHODS FOR ISOLATING BASES 117
containing proteins and bases is evaporated to a thin syrup, and this
is mixed with sand and then ground up under acetone. Dry acetone
does not dissolve the salts of most organic bases, but enough water
remains behind in the aqueous extract to prevent precipitation of the
salts by acetone.
The preliminary purification of a tissue extract after removal of
coagulable protein is, however, best effected by means of lead acetate
or by tannin. In the former case the solution is first treated with
normal lead acetate and then with the basic salt ; the joint precipitate
of these reagents is then filtered off and the excess of lead is removed
from the filtrate as sulphide, sulphate, or phosphate. The tannin
method has been largely employed by Kutscher and his pupils ; it
completely removes peptones and proteoses, but bases are also carried
down by the bulky precipitate ; according to Krimberg the yield of
bases from meat extracts is much smaller after purification with tannin
than with lead acetate. Many bases form tannates insoluble in neutral
solution, so that the reaction before precipitation should be made
distinctly acid by adding phosphoric acid, if necessary. A 20 per
cent, aqueous tannic acid solution is then added until no further pre-
cipitation occurs ; at this stage the precipitate ceases to be milky and
flocculates ; a considerable excess of tannic acid must be avoided since
it redissolves the precipitate (it is a case of the mutual precipitation of
two colloids). On standing overnight the bulky precipitate shrinks to
the consistency of pitch and the clear supernatant solution can easily be
poured off. In order to remove the excess of tannin, a warm saturated
baryta solution is added until, after stirring, the surface of the liquid
shows a reddish or purple colour. The barium tannate is filtered off
at the pump, the filtrate is acidified with sulphuric acid, and without
removing the barium sulphate formed, freshly prepared lead hydroxide,
suspended in distilled water, is stirred in. This removes the last
traces of tannin and the excess of sulphuric acid, and now, after
filtration, the solution should contain at most only traces of lead and
should be alkaline to litmus.
The last operations illustrate the general principle that as far as
possible no ions should be introduced into the solution which cannot
afterwards be removed, for the separation of bases from inorganic salts
is often difficult.
Kossel and Weiss [1910] use a solution containing 70 grm. of
tannic acid, 100 grm. of sodium chloride and 50 c.c. of glacial acetic
acid per litre for the precipitation of peptones.
The solution of bases which has been purified by one or other of
u8 THE SIMPLER NATURAL BASES
the above methods is now evaporated to a small volume, when on
standing some bases, such as creatine, may crystallise out. Generally,
however, they are too soluble in water and must be separated by some
general precipitant. The most important reagent for this purpose is
phosphotungstic acid, introduced into physiological chemistry by
Drechsel. The acid is readily soluble in ether, in acetone and in
water. It precipitates all nitrogen bases from their aqueous solution
if the latter contains 5 per cent, by weight of sulphuric acid. Ammonia
is also precipitated and should therefore be expelled, if present in
quantity. It is important to employ a good preparation of phospho-
tungstic acid, such as that of Kahlbaum, which dissolves in water with
hardly any opalescence. A method for preparing the acid has been
given by Winterstein (" Chemiker Zeitung," 1 898, p. 539). In order to
obtain the bases from an aqueous solution, sulphuric acid is added to
the latter to make 5 per cent, and a concentrated aqueous solution of
phosphotungstic acid, which should also contain 5 per cent, of sulphuric
acid, is added until no further immediate precipitation occurs. After
standing for a day the precipitate is filtered off at the pump and
thoroughly washed with 5 per cent, sulphuric acid. Often the pre-
cipitate is partially or wholly soluble in acetone, and more readily in
a mixture of acetone and water. (Compare Wechsler, below.) By
pouring the solution of the precipitate into a large bulk of 5 per
cent, sulphuric acid, the phosphotungstates of the bases are reprecipi-
tated and in this way they can be purified more readily than by
washing at the pump. In synthetic work, and when only one or two
bases are present, a phosphotungstate may occasionally be crystallised
from a large volume of boiling water (for instance in the case of
iminazolyl-propionic acid).
The bases are again liberated from their phosphotungstates by
means of baryta, finely powdered or dissolved in water. For this
purpose the phosphotungstate precipitate must be carefully suspended
in water in as fine a state of division as possible ; where possible it is
much quicker to dissolve the precipitate in dilute acetone and then
add an aqueous baryta solution. Wechsler [1911] recommends a
mixture of three volumes of acetone with four volumes of water ; this
dissolves arginine phosphotungstate to the extent of 120-130 per cent,
and of the histidine salt even 160 per cent, of its own weight, but
albumose phosphotungstates only to the extent of 2-7 per cent. The
precipitate of barium phosphotungstate and sulphate settles down
rapidly. Several drops of the clear supernatant fluid are sucked up
into a capillary pipette and tested on a glass plate. When they no
GENERAL METHODS FOR ISOLATING BASES 119
longer give a precipitate with baryta, but precipitate both with sul-
phuric acid and with sodium carbonate solutions, enough baryta has
been added to liberate the bases. The barium phosphotungstate is
then filtered off on the pump and washed out thoroughly with hot
water until the washings no longer give a precipitate with a phos-
photungstic-sulphuric acid solution. The excess of barium is at once
removed from the filtrate and washings by passing carbon dioxide
through them ; on filtration and evaporation the organic bases are
obtained either in the free state or as carbonates.
Should it be necessary to remove the excess of phosphotungstic
acid from the filtrate, after precipitation of bases as phosphotungstates,
this can be done either by precipitation with excess of baryta, or, ac-
cording to Jacobs [1912], by extracting the acid solution with amyl-
alcohol, which may be conveniently mixed with up to four parts of
ether. This method may also be used for decomposing the phos-
photungstates of bases if they are soluble in hot water.
Mercuric chloride is next in importance to phosphotungstic acid
as a precipitant of bases. It is not so universal a precipitant and
is most frequently used after phosphotungstic acid to separate the re-
covered bases into several fractions. With suitable precautions merT
curie chloride may, however, often replace phosphotungstic acid
altogether. It was first used extensively by Brieger for isolating
putrefaction bases, before phosphotungstic acid had come into general
use.
Mercuric chloride is generally used in saturated alcoholic solution
which is added to an alcoholic or sometimes to an aqueous solution
of the bases to be precipitated. Some bases are precipitated from
neutral solution, but others only after the solution has been made
slightly alkaline. In aqueous solution sodium carbonate is used, in
alcoholic solution fused sodium acetate, dissolved in alcohol, is added,
or the solution is saturated with powdered sodium acetate. If such a
solution is afterwards also saturated with powdered mercuric chloride,
very few bases escape precipitation. Generally the mercuric chlorides
are much more soluble in hot water than in alcohol ; Brieger extracted
the precipitate formed in alcoholic solution with boiling water, when
the mercuric chloride compounds of peptones remained undissolved.
On filtration and cooling choline mercurichloride crystallised out.
Another example of the use of mercuric chloride is the preparation of
histidine from blood, by Frankel's method. After the blood (or haemo-
globin) has been hydrolysed by boiling with concentrated hydrochloric
acid, most of the acid is distilled off and the residue, after being nearly
120 THE SIMPLER NATURAL BASES
neutralised with sodium hydroxide, is filtered. The filtrate is then
made alkaline with sodium carbonate and the histidine is precipitated
by adding alcoholic mercuric chloride solution. Engeland has worked
out a method for separating the bases of meat extract in which all the
bases are first precipitated by the alternate addition of cold saturated
solutions of mercuric chloride and of sodium acetate. The precipitate
dissolves for the most part in hot water acidulated with hydrochloric
acid and is freed from mercury by means of hydrogen sulphide. After
evaporation of the aqueous filtrate the residue is dissolved in alcohol
and alcoholic mercuric chloride is added ; finally the solution is satur-
ated with the powdered salt. This precipitates neosine, carnitine and
vitiatine as mercurichlorides which are removed by filtration. Alcoholic
sodium acetate solution is now added and precipitates the mercury
salts of histidine, methyl guanidine and /3-alanine. Cf. also p. 114.
Silver nitrate is principally used to precipitate bases containing an
imino-group and is of great value for their separation. As in the case
of mercuric chloride, the degree of acidity or alkalinity of the solution
is the determining factor. In the presence of (nitric) acid only purine
bases are precipitated as insoluble silver compounds ; in a slightly
alkaline solution, i.e. after the addition of a limited quantity of baryta,
the silver compounds of histidine and allied bases are thrown down ;
excess of baryta then precipitates the silver compound of arginine.
The separation of arginine and histidine in this manner may be
rendered quantitative and if silver sulphate is used instead of the
nitrate, the process affords a means of estimation by determination of
the nitrogen in the various fractions (see Plimmer's " Chemical Constitu-
tion of the Proteins," Part I, pp. 35-8). The practical details in the
application of silver nitrate may be illustrated by a description of
Kutscher's method for the isolation of bases from meat-extract. After
purification by means of tannin, as described above, and concentration
to a small volume, creatine and some creatinine crystallise out. Then,
after filtration, the solution is acidified with sulphuric acid and the
resulting precipitate of lead sulphate is filtered off. Now a 20 per cent,
silver nitrate solution is added to the filtrate and this causes the pre-
cipitation of the purine bases (as compounds with silver nitrate), together
with a little silver chloride. After standing for some time this pre-
cipitate is filtered off and enough silver nitrate is added to the solution
to enable the whole of the bases capable of forming silver compounds
to be precipitated as such by subsequent addition of baryta. Enough
silver nitrate has been added for this purpose when a drop of the
solution, mixed on a watch glass with cold saturated baryta water,
GENERAL METHODS FOR ISOLATING BASES 121
shows no longer a white precipitate (silver compound of bases) but at
once a brown precipitate (of silver oxide). The addition of barium
hydroxide in excess would now precipitate both the histidine and the
arginine fraction, but a separation of these may be effected by utilising
the fact that histidine silver is precipitated by an ammoniacal silver
solution but arginine silver is not. Hence, after adding enough silver
nitrate, baryta is added in small quantities until a drop of the clear
supernatant or filtered solution no longer gives a white precipitate
with a reagent which is prepared by adding ammonia to 10 per cent,
silver nitrate until the silver oxide has just dissolved.
The histidine fraction, which is thus precipitated by baryta, is
filtered off, and the precipitate, after washing, is suspended in water
in as fine a state of division as possible. If a suitable centrifuge is
available this means of separation is greatly to be preferred. The
silver is then removed with hydrogen sulphide, or with hydrochloric
acid, a little sulphuric acid being first added to precipitate adherent
baryta. The barium sulphate formed can be readily filtered off with
the silver sulphide or chloride.
Baryta in excess is now added to the filtrate of the " histidine "
fraction, and precipitates the silver compounds of the " arginine " frac-
tion, which are treated in the same way.
The former fraction may contain histidine, /3-iminazolyl-ethylamine,
carnosine and creatinine, the latter arginine, agmatine and methyl-
guanidine. The separation is not always quite sharp, however. Thus
Reuter found adenine (a purine base) in the histidine fraction of the
bases from Boletus edulis and trimethyl-histidine in the arginine fraction
from this same fungus. In Kutscher's examination of mushroom
extract trimethyl-histidine altogether escaped precipitation by silver
and appeared in the lysine fraction.
After the silver precipitate of the arginine fraction has been filtered
off, the solution may still contain various bases constituting the so-
called " lysine " fraction. The excess of baryta is removed by sul-
phuric acid and that of silver by hydrochloric acid ; then the bases
remaining in solution are precipitated by phosphotungstic acid, and
after recovery from the phosphotungstic precipitate, they are separated
by mercuric chloride or by other means.
Potassium bismuth iodide and potassium tri-iodide are more or less
general precipitants for bases and have been chiefly used in investi-
gations on plant alkaloids, but only to a slight extent for the separation
of animal bases. Potassium bismuth iodide (Dragendorff's reagent,
modified by Kraut) gives brick red and generally amorphous precipitates
122 THE SIMPLER NATURAL BASES
with organic bases. The reagent is prepared by dissolving 80 grm. of
bismuth subnitrate in 200 c.c. of pure nitric acid of density n8, and
pouring this solution slowly, with stirring, into a concentrated aqueous
solution of 227 grm. of potassium iodide. A precipitate forms and
dissolves on stirring to a deep orange solution. This is cooled strongly
to allow potassium nitrate to crystallise out as far as possible. The
clear solution is poured off and made up to I litre; the more concen-
trated solution may also be employed. The reagent should be kept
in the dark. Kossel and Weiss [1910] recommend a solution of 50
grm. sodium iodide and 100 grm. bismuth iodide in 100 c.c. of 0*5
per cent, aqueous hydriodic acid.
To regenerate the bases, the precipitate caused by addition of
Dragendorff's reagent is ground up with freshly precipitated lead
hydroxide, which is transformed to lead oxyiodide. After filtration
the last traces of lead are removed by hydrogen sulphide ; the solution
is then concentrated to a syrup, which is extracted with alcohol.
To precipitate bases as periodides a concentrated solution of iodine
in potassium iodide is employed (compare the estimation of choline
and betaine by Stanek's method). The periodides may be decomposed
by sodium bisulphite or thiosulphate, but this introduces into the solu-
tion a good deal of inorganic matter. It is better to grind up the per-
iodide in warm water with finely divided copper, so-called " molecular
copper," prepared by Gattermann's method, as follows : Zinc dust is
added through a sieve to a cold saturated solution of copper sulphate
in a porcelain dish, until the solution is only faintly blue. The pre-
cipitated copper settles down and is repeatedly washed by decantation.
To remove traces of metallic zinc, the copper is placed under several
times its volume of distilled water and quite dilute hydrochloric acid
is added until no more hydrogen is evolved and the copper is no longer
carried up to the surface of the solution but remains quietly at the
bottom. The copper is then collected on a filter at the pump, washed
until neutral and kept in a well-stoppered bottle in the moist state.
It is very easily oxidised.
For the isolation of individual bases from the fractions obtained
by any of the above methods, it is necessary to prepare a crystalline
derivative. Bensoylation is occasionally resorted to (in the case of
diamines from urine, p-hydroxyphenyl-ethylamine, etc.) but generally
a salt of the base is crystallised. The hydrochlorides of putrescine and
of betaine are almost insoluble in alcohol, in contradistinction to the
corresponding cadaverine and choline salts. The nitrates of some
bases (guanidine, methylguanidine, arginine, hypaphorine, certain
GENERAL METHODS FOR ISOLATING BASES 123
purine bases) can be readily crystallised from water and are particu-
larly little soluble in dilute nitric acid.
Much more frequently picrates are prepared. The picric acid is
added in aqueous and also in alcoholic solution ; the precipitated
picrate is recrystallised from water, from dilute or from strong alcohol.
Often, on cooling a hot solution, it separates first in oily drops which
only become definitely crystalline on standing. Ammonium salts,
when present, may sometimes lead to confusion owing to the forma-
tion of ammonium picrate, which is not very soluble in water and
forms long thin pale yellow needles ; these have no proper melting
point, but decompose suddenly on heating. When a base is insol-
uble in ether (as is the case with most of the simpler natural bases) it
can be readily recovered from its picrate by dissolving the latter in
hot dilute hydrochloric acid and, after cooling, extracting the picric
acid with ether or with benzene. On the large scale most of the
picric acid generally separates and can be filtered off. The estimation
of picric acid in picrates can be carried out very conveniently and
with enough accuracy by means of the " nitron " reagent of Busch
[1905]. This process has the further advantage over a combustion
that the base is recovered unchanged.
Picrolonates are much less soluble than picrates and generally
crystallise well, but to some extent this advantage is neutralised by
the slight solubility in water of picrolonic acid itself. An alcoholic
solution of the acid is generally added to an aqueous solution of the
base. The precipitate is at first often amorphous, but readily crystal-
lises from hot water in some cases. The high molecular weight of
picrolonic acid renders the melting points and analyses of picrolonates
of less significance than those of picrates.
Platinic chloride is used in concentrated aqueous or (more frequently)
alcoholic solution. The platinichlorides of the simplest bases are
often readily soluble in water, but not in alcohol, and may be crystal-
lised from dilute alcohol.
Gold chloride is generally used in a 30 per cent, aqueous solution.
Aurichlorides sometimes partially decompose on recrystallisation, gold
being set free. In order to avoid this and obtain a gold salt of normal
composition, the salt should be recrystallised from -j- - I per cent,
hydrochloric acid to which a little gold chloride has been added.
In special cases zinc chloride or cadmium chloride are used for
forming double salts in alcoholic solution, or the base is isolated
as chrornate, perchlorate or metaphosphate.
124 THE SIMPLER NATURAL BASES
B. SPECIAL METHODS. PROPERTIES OF INDIVIDUAL BASES AND
OF THEIR SALTS.
4
Bases Volatile with Steam.
Methyl-, dimethyl-, and trimethylamine, isobutyl- and the amyl-
amines can all be readily distilled by passing steam into their alkaline
solutions. The last two can be separated from the others by extract-
ing an alkaline solution with chloroform or ether and distilling ;
isobutylamine boils at 68°, isoamylamine at 95°.
The separation of the first three bases from one another can be
accomplished in various ways. Delepine [1896, Ch. I] dissolves the
mixture of their salts in cold concentrated formaldehyde solution.
An equal volume of potassium hydroxide is added and the solution
is distilled. Trimethylamine passes over as such, dimethylamine
forms CH2[N(CH3)J2 and CH2(OH)N(CH3)2, b.p. 80-85°, and
monomethylamine yields (CH2 : NCH3)2, b.p. 166°.
For the quantitative determination of trimethylamine and ammonia,
Budai (Bauer) [1913] has worked out a titration method with for-
maldehyde. The neutral aqueous solution of the mixed hydrochlorides
is treated with an excess of formalin (10 c.c.), previously neutralised
to phenolphthalein. The solution is then titrated with standard
potassium hydroxide until pink with phenolphthalein ; this gives the
amount of ammonia present. The solution, together with the hexa-
methylene tetramine formed from the ammonia, is strongly acidified
with concentrated hydrochloric acid and boiled down to one-third of
its original volume. It is then distilled with excess of potassium
hydroxide. This gives ammonia + trimethylamine ; the latter is
estimated by difference.
The quantitative separation of ammonia, mono-, di-, and
trimethylamine is carried out by processes due to Bresler [1900],
Bertheaume [1910, i, 2], and Francois [1907, I, 2] and is chiefly based
on the fact that trimethyl- and dimethylamine hydrochloride alone are
soluble in boiling chloroform. 1-2 grm. of the mixed hydrochlorides
are dried at 1 10°, weighed out, dissolved in a little very dilute hydro-
chloric acid, mixed with at least 20 grm. of pure silver sand, dried in
vacuo over sulphuric acid, and extracted with hot chloroform in a
small funnel tube over glass wool.
The chloroform is evaporated, the residue is weighed and dis-
solved in 2000 parts of water ; 200-300 c.c. of the solution are
measured, cooled to o° and for every 100 c.c. of solution taken, at least
30 c.c. of an ice cold solution of 127 grm. of iodine and 15 grm. of
potassium iodide in 100 c.c. of water are added. After one hour the
APPENDIX TO CHAPTER I— AMINES 125
crystals of the periodide of trimethylamine are sucked off on to glass
wool, washed with 3-4 c.c. of a mixture of one part of the above potas-
sium tri-iodide solution with three parts of water. The crystals are then
dissolved in sodium thiosulphate solution, and after adding excess of
sodium hydroxide, the trimethylamine is distilled ; the distillate is
titrated with acid. The mother liquor of the crystals of trimethyl-
amine periodide yields by a similar treatment the dimethylamine on
distillation.
The separation of ammonia and monomethylamine, which are
left behind as hydrochlorides mixed with the sand, is effected by
Frangois's process, of which the following is an example : 70 grm. of
methylamine + 7 grm. of ammonia (both in the free state) in 2000 c.c.
of water are shaken for one hour with 200 grm. of yellow mercuric
oxide. The solution is decanted and the precipitate is washed. The
filtrate and washings contain all the methylamine, but almost the
whole of the ammonia is in the mercury precipitate. To remove the
remainder, 40 c.c. of caustic soda and 40 c.c. of saturated potassium
carbonate solution are added, together with 100 grm. of mercuric
oxide. The solution now only contains monomethylamine.
Methylamine can be distinguished from ammonia by means of
Nessler's reagent ; the amine gives a cream-coloured precipitate,
ammonia a brown one.
The estimation of small quantities of amines in the presence of
much ammonia has been described by Bertheaume [1910, 2].
Fleck [1896] recommends the separation of trimethylamine from
ammonia by means of the sulphates, rather than the chlorides.
Ammonium sulphate is insoluble in absolute alcohol, in which am-
monium chloride is distinctly soluble ; trimethylamine salts dissolve
readily in alcohol.
de Filippi [1906] has estimated trimethylamine in urine by destroy-
ing ammonia, primary and secondary amines by means of sodium hypo-
bromite ; this reagent leaves tertiary amines intact. Doree and Golla
[1910] by a slightly modified method found 0*014 Per cent, trimethy-
lamine in urine. They state that this amine cannot bedistinguished from
choline by the alloxan test, nor by the bismuth iodide or periodide test.
Melting points and solubility of trimethylamine salts :—
Hydrochloride 271-275° soluble in boiling chloroform.
Picrate . . 216° soluble in 77 parts of cold water.
Picrolonate . 250-252° in 1121 parts of cold and 166 parts of boiling water,
794 of cold and 233 of boiling alcohol.
Aurichloride . 228° yellow monoclinic crystals, readily soluble in hot alcohol,
slightly in water.
Platinichloride 240-245° regular orange crystals, little soluble in boiling alcohol.
126 THE SIMPLER NATURAL BASES
Isobutylamine hydrochloride does not melt at 160°, as stated in
Beilstein, but at 177-178° (Thorns and Thiimen [1911]).
The platinichloride forms golden yellow crystals, very soluble in
alcohol and in water, decomposing at 224-225° and melting at 230-
232°.
Isolation of isoamylamine from putrid horse meat. — The material
had undergone putrefaction anaerobically for eight to ten days at 37°.
The proteins were coagulated, the filtrate was evaporated to a syrup,
mixed with sand and extracted with acetone. After distilling off the
acetone, hydrochloric acid was added to the residue, which was washed
with chloroform to remove fatty acids, etc., and then rendered alkaline
and again extracted with chloroform. After evaporation of the solvent
the base was distilled and converted into the crystalline oxalate.
Isoamylamine hydrochloride forms deliquescent crystals ; the hydro-
bromide is non-deliquescent. The acid oxalate C5H13N, H2C2O4 is ob-
tained by mixing ethereal solutions of oxalic acid and of the base ;
m.p. 169°; it slowly loses amylamine at 100° and should be dried in
vacuo.
^^platinichloride forms golden yellow leaflets, readily soluble in
hot water.
Isolation and Separation of Putrescine and Cadaverine.
Both bases are very common in putrefaction. They are not
readily volatile with steam, nor can they readily be extracted from
aqueous solution by ether or by chloroform. They can be precipi-
tated by phosphotungstic acid, and after treatment with silver nitrate
and baryta they are found in the lysine fraction (see above). From
this they can be precipitated by mercuric chloride in alcoholic solution,
or they may be precipitated directly by this reagent, as was done by
Brieger, without previous use of phosphoturigstic acid. He precipi-
tated both bases from an alcoholic extract of a putrefaction mixture
by means of alcoholic mercuric chloride and afterwards fractionally
crystallised the platini- and aurichlorides (putrescine aurichloride
is the less soluble in water). It is, however, more convenient to
separate the hydrochlorides, that of putrescine being but little soluble
in 96 per cent, alcohol, whereas the corresponding cadaverine salt
dissolves readily.
From urine Udranszky and Baumann [1888, I, 1889] separated
both bases as dibenzoyl compounds by shaking with benzoyl chloride in
sodium hydroxide solution ; this process is quantitative even in a I :
10,000 solution of the base. The benzoyl derivatives are washed with
APPENDIX TO CHAPTER I— AMINES 127
water and dissolved in a little boiling alcohol. After concentration
the alcoholic solution is poured into thirty volumes of water when the
benzoyl compounds crystallise. The concentrated alcoholic solution
of the crystals is then poured into twenty volumes of ether when
dibenzoyl putrescine separates and the cadaverine compound remains
dissolved. Another method is due to Loewy and Neuberg [1904].
After filtering off the cystine the bases in the urine are precipitated
with phosphotungstic acid and after regeneration are treated in
alkaline solution with phenylisocyanate. The precipitated com-
pounds of the diamines are very little soluble in most organic solvents,
and are boiled out with alcohol, dried and dissolved in warm pyridine.
On adding dry acetone the putrescine compound crystallises at once,
the cadaverine compound only on standing.
Properties and Compounds of Putrescine.
The base is obtained synthetically by reduction of ethylene di-
cyanide (succino-nitrile), but more conveniently by reduction of
succindialdoxime (Willstatter and Heubner [1907]).
Putrescine is a liquid of semen-like odour; m.p. 27-28°; b.p. 158-
160°; slightly volatile with steam; very soluble in water, miscible
with alcohol, very little soluble in ether.
The dihydrochloride, C4H12N2 . 2HC1, crystallises in leaflets and
needles and is insoluble in absolute alcohol. On destructive distilla-
tion it yields pyrrolidine (rigid proof of the constitution) (Ackermann
[1907, I])-
The platinichloride, C4H12N2 . H2PtCl6, needles or six-sided plates,
is sparingly soluble in water (Brieger [1885, 2, p. 26]).
The aurichloride, C4H12N2 . 2HAnCl4 . 2H2O, is less soluble than
the cadaverine salt (Brieger [1886, I, p. 51]).
The mercur i chloride is readily soluble in water, but not in alcohol.
The dipicrate, C4H12N2 . 2C6H8O7N3, silky needles, hardly soluble
in cold water, decomposes at 250°.
The dipicrolonate, C4H12N2. 2C10H8O5N4, dissolves in 13,157 parts
of cold and 65 3 parts of boiling water, and in 17,857 parts of cold and
954 parts of boiling alcohol ; decomposes at 263° (Otori [1904, 3]).
The dibenzoyl derivative, C4H8(NHCOC6H5)2, crystallises in long
needles ; m.p. 178° ; almost insoluble in ether ; sparingly in cold, readily
in hot alcohol.
The phenylisocyanate, C4H8(NH . CO. NH . C6H6)2, forms sheaves
of needles from pyridine acetone ; m.p. 240° (corr.). Insoluble in water
and most organic solvents ; hardly soluble in boiling alcohol.
128 THE SIMPLER NATURAL BASES
Properties and Compounds of Cadaverine.
Cadaverine or pentamethylene diamine was obtained by Ladenburg
[1886] by the reduction of trimethylene dicyanide, but is now
most easily obtained from potassium phthalimide and pentamethy-
lene dichloride ; the latter compound is readily formed from
benzoyl piperidine and phosphorus pentachloride, by von Braun's
method [1904]. Cadaverine is also formed in small quantity by
the destructive distillation of lysine (Neuberg [1905]). Cadaverine
is a liquid with the odour of semen and of piperidine ; b.p. 178-179° ;
somewhat volatile with steam, readily soluble in water and in alcohol,
hardly in ether ; is precipitated by alkaloidal reagents.
The dihydro chloride, C5H14N2 . 2HC1, needles, non-deliquescent
according to Gulewitsch [1894], is readily soluble in 96 per cent,
alcohol, sparingly in absolute alcohol. On destructive distillation it
yields piperidine.
^\\Q. platinichloride, C5H14N2 . H2PtCl6, forms orange coloured rhom-
bic prisms, somewhat resembling ammonium platinichloride (for details
see Brieger [1885, 2, p. 37]) ; they blacken at 195° and decompose at
215°; soluble in 70*8 parts of water at 21° (Gulewitsch [1894]), in
113 to 114 parts of water at 12° (Udranszky and Baumann).
The aurichloride, C5H14N2 . 2HAuCl4, forms long needles and also
flat prisms; m.p. 186-188°; fairly readily soluble in water and contain-
ing water of crystallisation.
The mercurichloride, C5H12N . 2HC1 . 4HgCl2, prepared with excess
of mercuric chloride, crystallises from hot water and melts at 214*5°
(Gulewitsch [1894]). It already loses mercuric chloride at 95°.
Soluble in 32-5 parts of water at 21°; not appreciably soluble in
alcohol.
The dipicrate, C5H14N2 . 2C6H3O7N3, forms long needles ; m.p. 221° ;
sparingly soluble in hot water, hardly at all in boiling alcohol.
The dipicrolonate, C5H14N2 . 2C10H8O5N4^ darkens at 220° and melts
at 250°; soluble in 7575 parts of cold water and 357 parts of boiling
water, 5952 parts of cold and 475 parts of boiling alcohol (about twice
as soluble as the putrescine salt) (Otori [1904, 3]).
The dibenzoyl derivative, C5H10(NHCOC6H5)2, long needles, hardly
soluble in ether, melts at 135°.
The phenylisocyanate, C5H10(NHCONHC6H5)2, is somewhat more
soluble in pyridine acetone than the putrescine compound and melts
at 207-209° (corr.).
APPENDIX TO CHAPTER I— AMINES 129
Tetramethyl-putrescine, C8H20N2.
This base occurs along with hyoscyamine, in Hyoscyamus muticus.
It is a strongly alkaline liquid, boiling at 169° and miscible with water,
alcohol and ether in all proportions. Pharmacologically it is inert
(0-05 grm. given as salt hypodermically to frogs and 0-5 grm. intra-
venously to rabbits was without effect).
The dihydro chloride, m.p. 273°, is neutral and deliquesces in moist
air ; the dipicrate is fairly readily soluble in water; m.p. 198°.
^hzplatinichloride, C8H2()N2 . H2PtCl6 . 2H2O, is readily soluble in hot,
but much less in cold water; m.p. 234°. The aurichloride, of similar
solubility in water, dissolves very readily in acetone and forms golden
yellow anhydrous prisms decomposing at 206-207°. The constitution
(CH3)2 : N . CH2 . CH2 . CH2 . CH2 . N : (CH3)2 was established by syn-
thesis (Willstatter and Heubner [1907]).
Agmatine.
On treatment with silver nitrate and baryta, in the way described
in section A of this chapter, this base is precipitated in the arginine
fraction.
Agmatine salts. — The sulphate, C5HUN4. H2SO4, forms long needles,
m.p. 229° ; the dipicrate, C5HUN4 . 2C6H3O7N3, forms crystals melting at
238° and decomposing at 244°; the aurichloride, C5H N4. 2HAuCl4,
crystallises in yellow needles. The carbonate separates from aqueous
solution on concentration as a chalky mass.
Phenyl-ethylamine.
From a putrefaction mixture this base is best isolated in the manner
described above for isoamylamine, from which it is separated by its
much higher boiling point.
Phenyl-ethylamine and its salts. — The base is easily obtained
synthetically, by the reduction of benzylcyanide ; the highest recorded
yield by this reaction is 53 per cent, of the theory (Wohl and Berthold
[1910]). It is also obtainable from phenyl acetic acid, via the
amide, by Hofmann's reaction and via the hydrazide and urethane, by
Curtius's method ; it is further one of the products of the destructive
distillation of phenylalanine.
The synthetic base is a liquid of slight amine-like odour and
readily absorbs carbon dioxide from the air, forming the crystalline
carbonate. The boiling point of the base is 196° at 747 mm., 197-
198° at 754 mm. ; it is somewhat lighter than water, and dissolves
in 24 parts of water at 20° ; it is miscible with alcohol and with ether.
9
130 THE SIMPLER NATURAL BASES
The hydrochloride, C8HUN . HC1, is soluble in alcohol and melts at
217° ; with mercuric chloride a sparingly soluble crystalline compound
is formed. Other salts are the acid oxalate, C8HUN . C2H2O4, m.p. 1 8 1 ° ;
the normal oxalate, (C8HnN)2C2H2O4, m.p. 2 1 8° ; and ttizpicrate, C8HnN.
C6H3O7N3, tetragonal prisms, m.p. 171-174°, readily soluble in warm
water.
The benzoyl derivative, C6H5 . CH2 . CH2 . NH . CO . C6H5, melts
at 114°.
p-Hydroxy-phenyl-ethylamine.
Small quantities of this amine are most readily prepared by heat-
ing tyrosine under reduced pressure in test tubes dipping into a bath
of fusible metal at 260-270° ; the amine sublimes ; the yield is
50 per cent. (cf. F. Ehrlich and Pistschimuka [1912]). For the
isolation from complex mixtures such as are obtained in putrefaction,
the base can be precipitated with phosphotungstic acid, but the
phosphotungstate is rather soluble. On fractionation with silver and
baryta, the base is obtained as platinichloride from the lysine fraction.
A better way is to utilise its phenolic properties by washing its
solution in *5N sodium hydroxide with amyl alcohol, neutralising, add-
ing sodium carbonate and extracting the amine with amyl alcohol.
After distilling off the solvent with steam, the dibenzoyl derivative
is obtained by the Schotten-Baumann method.
In sufficient quantity p-hydroxy-phenyl-ethylamine is best purified
by distillation ; it boils at 161-163° at 2 mm- an(* 175-181° at 8 mm. It
is also readily purified by crystallisation from boiling xylene in which
it is very sparingly soluble. It forms colourless hexagonal leaflets
melting at 161°, soluble in 95 parts of water at 15° and in about 10
parts of boiling ethyl alcohol. The base is fairly soluble in amyl
alcohol, but hardly at all in ether or chloroform. It gives Millon's
and Morner's reaction for tyrosine, but no coloration with triketo-
hydrindene hydrate.
The hydrochloride, C8HnON . HC1, is very soluble in water and may
be crystallised from concentrated hydrochloric acid; m.p. 268°.
The phosphate, C8HnON . H3PO4 . i|H2O, forms white prisms,
readily soluble in water; m.p. 209-210°.
The picrate, C8HnON . C6H3O7N3, forms short prisms; m.p. 200°.
The platinichloride, (C8HnON)2HJ?tCl6, forms six-sided leaflets.
The N-monobenzoyl derivative crystallises from alcohol in hexagonal
plates; m.p. 162°.
The dibenzoyl derivative, C6H5CO.O.C6H4.CH2CH2.NH.CO.C6H5,
APPENDIX TO CHAPTER I— AMINES 131
is the most useful and characteristic derivative of the base. Formed
by the Schotten-Baumann reaction, it crystallises readily from alcohol
and melts at 170°; this derivative gives Morner's reaction, but not
Millon's.
Yeast transforms p-hydroxy-phenyl-ethylamine to the correspond-
ing alcohol, tyrosol, OH . C6H4 . CH2 . CH2OH (Ehrlich and
Pistschimuka [1912]). p-Hydroxy-phenyl-ethylamine is attacked by
various oxidases and converted to pigments, but does not always
behave in the same way as its parent substance tyrosine. Thus
Neuberg [1908, Ch. VI] found that a ferment from a melanoma at-
tacked the amine, but not the amino-acid, whereas an extract of the
ink-bag of Sepia acts on tyrosine more readily than on the amine.
Compare also J. Chem. Soc., Abstr., 1908, 94, i., 236.
Hordenine.
Gaebel's process of isolation was as follows : The extract of 3 kilos,
of malt germs with 95 per cent, alcohol was evaporated to a syrup
and extracted with I litre of water. After filtration the aqueous
extract was made alkaline with sodium carbonate, shaken once with
a little ether to remove a colouring matter, and then ten times with
large quantities of ether. The concentrated ethereal extract was
dried with potassium carbonate and evaporated, when the residual
syrup soon crystallised. On recrystallisation from dry ether, with
charcoal, the pure base is obtained; the yield is O'2 per cent, of the
air dry germs.
Properties: — Hordenine forms colourless crystals melting at II7'8°
(corr.) and boiling at 173-174° and 1 1 mm. Distillation under reduced
pressure is the most convenient method of purification. The base
dissolves readily in alcohol and in chloroform, and fairly readily in
ether and in water ; it is hardly soluble in benzene. Hordenine gives
Millon's and Piria's reactions for tyrosine ,and reddens phenolphtha-
lein ; it is not coloured by concentrated sulphuric acid, but reduces
potassium permanganate in the cold and ammoniacal silver nitrate on
warming.
Ite sulphate, (C10H15NO)2. H2SO4 . H2O, the hydrochloride and the
hydrobromide are sparingly soluble in alcohol. The quaternary iodide,
hordenine methiodide, obtained by the action of methyl iodide in
methyl alcoholic solution on hordenine (or on p-hydroxy-phenyl-ethyl-
amine), forms large glassy prisms, sparingly soluble in cold water ;
m.p. 230-231°.
9*
I32 THE SIMPLER NATURAL BASES
Indolethylamine.
The free base, on recrystallisation from a mixture of alcohol and
benzene, forms long colourless needles, melting at 145-146°. It is
readily soluble in alcohol and in acetone, but is almost insoluble in
water, ether, benzene and chloroform. It gives very intensely Hopkins
and Cole's reaction with glyoxylic and sulphuric acids, characteristic of
tryptophane ; the bluish-violet coloration is still obtainable with the
base in a dilution of I : 300,000. Unlike tryptophane, aminoethyl-
indole is not coloured by bromine water, nor does it react with
triketohydrindenehydrate.
The hydrochloride > C10H12N2 . HC1, forms thin prisms melting at
246° and is soluble in about 12 parts of water at 18°.
The picrate is the most characteristic salt of the base. It has the
composition C10H12N2 . C6H3O7N3 and is obtained by adding a cold
saturated solution of picric acid to a solution of the hydrochloride in
water ; the mixture at once becomes turbid and orange-red in colour,
and dark red crystals, consisting of fern-like aggregates of needles or
prisms (resembling in shape those of ammonium chloride) rapidly
separate. This picrate is almost insoluble in water and very sparingly
so in alcohol and most organic solvents, but dissolves readily in
acetone ; it melts and decomposes at 242-243°.
The picrolonate crystallises readily from hot water in deep chrome-
yellow prisms melting at 231°.
The monobenzoyl derivative of 3-/3-amino-ethylindole is difficult to
crystallise, and therefore not suitable for characterising the base ; it
forms stout prisms melting at 137-138°.
/3-Iminazolyl-ethylamine.
Bacterial Preparation.
Ackermann [1910, l] dissolved 49 grm. of histidine hydrochloride
in 4 litres of water, added 10 grm. of Witte peptone, 20 grm. of
glucose, a few drops of magnesium sulphate and sodium phosphate
solutions, and excess of calcium carbonate to keep the reaction
alkaline. After inoculation with putrid pancreas the solution was
kept fifty- two days at 35°. It yielded 6 1 '6 grm. of iminazolyl-
ethylamine dipicrate which is 42 per cent, of the theoretical ; a very
small quantity of iminazolyl-propionic acid was also formed.
When working with small quantities of histidine and pure cultures
of certain bacteria one can occasionally obtain solutions of which the
physiological activity indicates an almost complete conversion. How-
APPENDIX TO CHAPTER I— AMINES 133
ever, it seems generally impossible to isolate more of the amine than
Ackermann obtained and the yield is often very much less. The
mode of action of one and the same organism seems to depend on
conditions which areas yet imperfectly understood, so that this method
is rather uncertain.
Mellanby and Twort [1912] have isolated a bacillus of the
typhoid-coli group from the intestine of various mammals (from the
duodenum downwards) which is capable of decarboxylating histidine.
The best yields of the amine were obtained by inoculating histidine
solutions with adequate quantities of a vigorous twenty-four hours'
culture of the organism on glycerine-agar and incubating for one week
at 37°. The solutions contained histidine I per cent., ammonium
tartrate I per cent., dipotassium phosphate O'l per cent, magnesium
sulphate O'O2 per cent., calcium chloride 0*01 per cent., but no peptone.
Solutions containing cri per cent, histidine give a better yield. See
also patents by Hoffmann, La Roche & Co. [1912] and papers by
Berthelot and Bertrand [1912, i, 2; 1913, I, 2] and by Bertrand and
Berthelot [1913] describing the isolation of Bacillus aminophilus intes-
tinalis, a Gram-negative capsulated organism, resembling B. lactis
aerogenes and the bacillus of Friedlander, but differing from these in
its great power of decarboxylating amino-acids. For isolation of the
organism they used o '2 grm. K2SO4, 0'2 grm. MgSO4, 0*5 grm. K2HPO4,
0*25 grm. KNO3, 0*02 grm. CaCl2 and I -5 grm. histidine hydrochloride
per litre.
To isolate /3-iminazolyl-ethylamine when pure histidine has been
submitted to putrefaction, it is hardly necessary to precipitate with
phosphotungstic acid. Instead one can precipitate at once with picric
acid, having removed ammonia, and recrystallise the picrate. For the
isolation of the base from complex mixtures such as ergot, it is neces-
sary to fractionate with silver nitrate and baryta. The base is then
found in the histidine fraction. Its hydrochloride is conveniently
separated from inorganic salts by extraction with methyl alcohol.
Salts of $-iminazolyl-ethylamine. — The dihydrochloride, C5H9N3.
2HC1, is extremely soluble in water and sparingly soluble in ethyl
alcohol; it crystallises in prisms; m.p. 240°. The dihydrobromide has
similar solubilities and forms stout prisms sintering at 265° and
melting at 284° (corr.). The acid phosphate, C5H9N3 . 2H3PO4, is some-
what less soluble in water and crystallises very well ; it decomposes
indefinitely at 120-140°.
rI\\Qplatmichloridet C5H9N3. H2PtCl6, orange coloured prisms readily
soluble in hot water and hardly at all in alcohol, blackens and de-
134 THE SIMPLER NATURAL BASES
composes between 200° and 240° without melting. The aurichloride,
C5H9N3 .(HAuCl4)2, melts with decomposition at 200-210°.
The dipicrate, C5H9N3 . (C6H3O7N3)2, is the most convenient salt for
purposes of isolation. It forms deep yellow rhombic leaflets, melting
at 238-242° (corr.) according to the rate of heating. It is very spar-
ingly soluble in cold water and can be recrystallised from hot water.
The monopicrate, C5H9N3 . C6H3O7N3, m.p. 233-234°, forms bunched,
slightly curved, pointed needles.
The dipicrolonate, C5H9N3(C10H8O5N4)2, dissolves in about 450 parts
of boiling water, from which it crystallises in sheaves of needles, melt-
ing at about 264°.
Reactions of $-iminazolyl-ethylamine. — In common with histidine,
this amine gives Pauly's reaction with p-diazobenzene sulphonate ; a
very distinct rose pink coloration is still obtainable at a dilution of
I : 10,000. It also gives Knoop's histidine reaction, a claret colora-
tion, on boiling with bromine water. It is precipitated by ammoniacal
silver oxide, by mercuric chloride in the presence of potassium
hydroxide, and by phosphotungstic acid. On the other hand, it is
distinguished from histidine in not giving the biuret reaction, nor
Ruhemann's reaction with triketohydrindenehydrate, and it further
behaves differently on benzoylation. When shaken with benzoyl-
chloride in potassium hydroxide solution, the glyoxaline ring is rup-
tured and tribenzoyl-butentriamine is formed, of the following con-
stitution : —
CH . NH . CO . C6H6
C . NH . CO . C6H6
CH2 . CH2 . NH . CO . C6H5.
Histidine, on the other hand, yields a monobenzoyl derivative.
BASES OF CHAPTER II.
/9-Alanine.
The substance may be obtained synthetically in several ways, the
best being from succinimide (Holm [1904]); I mol. of succinimide
in 10 per cent, potash solution containing 6 mol. of KOH and I mol.
of KOBr is warmed for two hours to 50-60°. The resulting /3-alanine
is purified by esterification.
Abderhalden and Fodor [1913] isolated /3-alanine according
to Fischer's ester method. The free ester boils at 54° and 10 mm.
The hydrochloride melts at 64°. On distilling the ester at ordinary
pressure it gives the pungent smell of ethyl acrylate which is a good
mode of recognition of /3-alanine.
Synthetic yS-alanine forms prisms, melting at 206-207° and decom-
posing into acrylic acid and ammonia.
The hydrochloride melts at 1 22 -5°.
The sulphate, (C3H7O2N)2H2SO4, decomposes at I 50°.
The platinichloride, (C3H7O2N)2H2PtCl6, crystallises from water or
hydrochloric acid in deep yellow needles, m.p. 180°; it is soluble in
alcohol (Engeland [1908, i]).
The copper salt, (C3H6O2N)2Cu + 6H2O, forms azure crystals
(Holm [1904]).
7-Aminobutyric Acid.
According to Engeland and Kutscher [1910, 3, Ch. Ill, buty-
robetaine] ry-aminobutyric acid is precipitated in dilute solution by
phosphotungstic acid and also by mercuric chloride in the presence of
sodium acetate, but not by mercuric chloride alone. These properties
it shares with histidine and methyl-guanidine, from which it may be
separated by silver nitrate and baryta, when it appears in the lysine
fraction. It can also be separated by distillation of its ester, prepared
by Fischer's method.
^-Amino-butyric acid was first obtained by Schotten [1884] by
oxidising piperidylurethane with fuming nitric acid and subsequently
135
136 THE SIMPLER NATURAL BASES
hydrolysing the oxidation product (for details see Abderhalden and
Kautzsch [1912]). The free acid forms leaflets melting at 183-184°
XCH2 . CH2
with conversion into the anhydride pyrrolidone, NHV |
^CO . CH2.
The kydrochloride crystallises in stout prisms ; m.p. 135°-
The platinichloride forms orange prisms; m.p. 220°.
The aurichloride crystallises in glistening plates; m.p. 138°. The
ethyl ester boils at 75-77°/12 mm.
£-Amino-valeric Acid.
E. and H. Salkowski obtained this substance from putrid blood
fibrin by evaporating the mixture repeatedly with water, adding barium
chloride to remove some fatty acids as soaps, acidifying the nitrate,
washing with ether, evaporating to dryness, and extracting the residue
with alcohol. On standing for a long time in a desiccator the residue
from the alcoholic solution gave the crystalline hydrochloride of S-
amino-valeric acid, from which the platinichloride and finally the
aurichloride was isolated.
Formation from proline. — Ackermann [1911, 2] obtained 3-6 grm.
of S-amino-valeric acid aurichloride from 34 grm. of proline after
putrefaction for nine days with glucose, peptone and salts. Neuberg
isolated the acid by means of a-naphthylisocyanate and obtained at
the same time n-valeric acid, which would result from the deamina-
tion of S-amino-valeric acid. Neuberg [1911, I] used a I percent,
proline solution, made and kept alkaline by repeated addition of
sodium bicarbonate, and containing a few drops of saturated magnes-
ium sulphate, potassium chloride and sodium phosphate solutions, but
no glucose or peptone; from 23 grm. of proline I2T grm. was re-
covered unchanged, together with 27 grm. S-amino-valeric acid hydro-
chloride, and 2 -3 grm. of silver n-valerate.
S-Amino-valeric acid crystallises in pearly leaflets, extremely soluble
in water, and melting at 1 57-1 58° when they undergo transformation to
piperidone. The aqueous solution is faintly acid and has an astringent
taste. The substance is precipitated in dilute solution by phospho-
tungstic acid, but not by cupric acetate or ammoniacal silver solution.
The hydrochloride, C5HnO2N . HC1, forms rhombic leaflets which on
heating distil for the most part without change.
The platinichloride, (C5HnO2N)2H2PtCl6, forms long rhombic leaf-
lets, readily soluble in hot water but only slightly in cold water and
in alcohol.
APPENDIX TO CHAPTER II— w-AMINO-ACIDS 137
The normal aurichloride, C5HnO2N . HAuCl4. H2O, crystallises in
monoclinic orange coloured crystals ; m.p. 86-87° ; an abnormal auri-
chloride, C5HUO2N . AuCl3, is also known; it forms pale yellow crystals
decomposing at 130° and is transformed to the more deeply coloured
normal salt by recrystallisation from dilute hydrochloric acid.
Benzoyl-§-amino-valeric acid is formed by the oxidation of benzoyl
piperidine with potassium permanganate and by the benzoylation of
S-amino-valeric acid. It melts at 94° and at 105°.
S-Amino-valeric acid does not yield a blue copper salt on boiling
with cupric oxide or on adding cupric acetate.
/3-Iminazolyl-propionic Acid.
This substance was isolated by Ackermann from the filtrate of
the /9-iminazolyl-ethylamine picrate [1910, i] obtained in the putrefac-
tion of histidine. The picric acid was removed from this filtrate, the
solution was evaporated, the residue was extracted with alcohol and
to the alcoholic solution platinic chloride was added. A slight pre-
cipitate was filtered off and the alcoholic solution was evaporated to
dryness. The residue, dissolved in a minimum quantity of boiling
water, deposited the crystals of the platinichloride of /3-iminazolyl-
propionic acid.
/3-Iminazolyl-propionic acid is readily soluble in water, less so in
alcohol and crystallises from dilute acetone ; m.p. 208-209°.
The nitrate, C6H8C>2N2 . HNO3, readily soluble in methyl alcohol,
forms elongated six-sided leaflets; m.p. 143-148°.
The platinichloride, (C6H8O2N2)2. H2PtCl6, melts at 209°.
The phosphotungstate crystallises from hot water in characteristic
rectangular leaflets, decomposing above 300°.
The copper salt forms blue needles.
Carnosine (Ignotine).
Carnosine is obtained from the regenerated phosphotungstic acid
precipitate (after neutralisation with nitric acid) by means of silver
nitrate and excess of baryta. After decomposing the silver precipitate
with hydrogen sulphide and removing the baryta by carbon dioxide,
the solution is neutralised with nitric acid and concentrated ; carnosine
nitrate crystallises out after the addition of alcohol.
Krimberg, by Gulewitsch's method, obtained 15-3 grm. of the free
base from I Ib. of Liebig's extract, or 3-4 per cent.; by Kutscher's
process he only obtained 3 grm. of carnosine from the same quantity
138 THE SIMPLER NATURAL BASES
of meat extract, and Kutscher himself obtained 3 grm. of ignotine
from I Ib. of Liebig's extract.
The free base, C9HUO3N4, crystallises in needles, soluble in 3-2 parts
of water at 25° and appreciably so in alcohol ; m.p. 248-5 - 250° ; [a]D2°c
= 21°, independent of the dilution. A 2*5 per cent, aqueous solution
gives no precipitate with platinic chloride, but it causes turbidity with
picric acid and a precipitate with gold chloride and potassium bismuth
iodide.
The nitrate, C9HUO3N4 . HNO3, melts at 219° and dissolves in 1*04
parts of water at 25°; [aD20°] in 1-48 per cent, solution = +24-2°, in 8
per cent, solution = +22 '8°; excess of nitric acid lowers the rotation
[Gulewitsch, 1913].
The copper salt, C9HUO3N4 . CuO, forms deep blue six-sided plates,
resembling cystine crystals in shape. It is sparingly soluble in hot
water and results when carnosine is boiled with copper carbonate.
Carnosine yields a sparingly soluble dipicrolonate, of which
Mauthner [1913] has attempted to use the mono-sodium salt as a
means of estimating carnosine in the histidine fraction of muscle
extracts.
Carnosine resembles arginine and differs from histidine in requir-
ing a fixed alkali for its precipitation as silver compound from a solu-
tion of carnosine nitrate containing an equimolecular amount of silver
nitrate. With silver nitrate in excess the silver compound is also
precipitated by careful addition of ammonia, but is soluble in excess.
Demjanowski [1912, Ch. V, methyl-guanidine] gives the following
limits of precipitation in aqueous solution : mercuric chloride,
I : 2000 ; mercuric sulphate, I : 100,000 ; mercuric nitrate, I : 100,000 ;
25 per cent, phosphotungstic acid, I : 20,000.
Urocanic Acid.
Preparation. — Jaff£ obtained the substance by a very simple
method. The urine was evaporated to a syrup and the latter was
extracted with hot alcohol ; after evaporation of the alcohol the
residue was acidified with sulphuric acid ; after washing with ether to
remove impurities the urocanic acid crystallised from the aqueous
layer. Hunter used phosphotungstic acid for the isolation of urocanic
acid and this is probably also the most certain method of obtaining
it from urine. The amount when present in urine is not inconsider-
able ; Jaff6 obtained 2-3 grm. per day and Siegfried found the urine
to contain O'i8 per cent, of the substance.
Jaffe gave the formula C12H12O4N4, 4H2O to the free acid, but this
APPENDIX TO CHAPTER II— w-AMlNO-ACIDS 139
must be halved. The free acid is slightly soluble in cold water (0-15
per cent, at 18° according to Siegfried) and readily soluble in hot
water. The melting point depends greatly on the rate of heating ;
after crystallisation from dilute acetone Barger and Ewins found
235-2360 (uncorr.). Hunter gives 231-232° (corn), Jaffe 212-213°,
Siegfried 229°. Hunter obtained the acid in slender, beautifully iri-
descent needles or tetragonal prisms. With sodium p-diazobenzene
sulphate it gives the red coloration of histidine. The acid is pre-
cipitated from solution by silver nitrate ; the precipitate dissolves in
excess of ammonia and in nitric acid.
The barium salt, (C6H5O2N2)2Ba . 8H2O, crystallises in needles and
loses 6H2O at 100° and the rest at 150°.
The nitrate, C6H6O2N2- HNO8, is the most characteristic salt. It
is sparingly soluble in dilute nitric acid and crystallises in small
sickle-shaped plates frequently united to cross- or rosette-shaped aggre-
gates (figured by Hunter, p. 541); m.p. 198° with explosive decom-
position (Barger and Ewins).
The picrate, C6H6O2N2. C6H3O7N3, forms golden yellow prisms;
m.p. 213-214°, 224-225° (corr.).
The picrolonate, C6H6O2N2 . C10H8O5N4, crystallises from dilute
alcohol ; m.p. 268° (corr.).
The phosphotungstate forms small rectangular plates from dilute
acetone or from hot water.
Kynurenic Acid.
To obtain kynurenic acid, Kretschy [1881] fed a dog of 34
kilos, weight daily with I kilo, of horse meat, 70 grm. of bread and
I litre of water. At first the daily production of the acid was cri
grm. but after I month O'8 grm. The best method, however, is to
give tryptophane by the mouth. The urine is acidified and the pre-
cipitate formed in twenty-four hours is filtered off and purified by
dissolving in ammonia, acidifying slightly with acetic acid and leaving
for twenty-four hours to allow a brown impurity to precipitate. After
filtration the solution is acidified with 4 per cent, hydrochloric acid.
Adherent uric acid may be removed by Hopkins's method and the
kynurenic acid may be finally recrystallised from 800 parts of boiling
alcohol (Homer [1913]). The pure acid forms long glistening
needles, of the formula C10H7O3N, H2O. The water of crystallisation
is given off at 140-145°. The highest melting point obtained by
Miss Homer was 288-289° (uncorr.). The acid is practically insoluble
in cold water and 100 parts of boiling water only dissolve 0*09 parts ;
I4o THE SIMPLER NATURAL BASES
100 c.c. of boiling alcohol dissolve cri grm. The following salts
are crystalline: C10H6O3NK + 2H2O, (C10H6O3N)2 Ba + 4iH2O,
(C10H6O3N)2 Ca + 2H2O and (C10H6O3N)2 Cu + 2H2O. The barium
salt is fairly soluble in hot water, but the copper salt is almost in-
soluble in it. The crystalline hydrochloride C10H7O3N, HC1 easily
loses hydrochloric acid (Brieger [1879]); the basic properties of the
substance are further evident from its precipitation by phosphotungstic
acid (Hofmeister [1880, Ch. V, creatine]).
Kynurine> formed in a 90 per cent, yield by heating kynurenic
acid to 253-258°, is little soluble in cold water, more so in alcohol
The hydrated substance C9H7ON, 3H2O melts at about 52°, the anhy-
drous substance at 202°. It is a feeble base yielding a platinichloride
(C9H8ON)2PtCl6 + 2H2O and a crystalline hydrochloride ; with bro-
mine the substance C9H4Br3ON is formed (Brieger [1879]).
Jafffs reaction for kynurenic acid '[1883]. — A solution of the acid
is evaporated on the water bath with hydrochloric acid and potassium
chlorate ; the red residue becomes brownish green with ammonia, soon
changing to an intense emerald green ; the chief product is tetrachloro-
oxykynurine, C9H3O2NC14.
A convenient method of estimation has been described by Capaldi
[1897,2].
BASES OF CHAPTER III— BETAINES.
Betaine (Acetobetaine).
The isolation by Schulze's method is described along with that of
choline (p. 150) as is also StaneVs method of estimation (p. 151).
For the estimation in crude sugar and in molasses Stanek [1904]
dissolves 20-30 grm. of the former or 3-5 grm. of the latter in 50 c.c.
of I o per cent, sulphuric acid previously saturated with sodium chloride.
This yields in either case a 1-3 per cent, solution of betaine which is
completely precipitated by the potassium tri-iodide reagent (if the
precipitate is oily, it may be rendered filterable by adding finely
powdered iodine); the nitrogen is determined in the precipitate as
described in the section on choline (p. 151).
For the estimation of betaine in plants Stanek and Domin [1910]
may also be consulted.
In order to prepare betaine from molasses Stanek [1901-2] utilises
the great stability of the base by mixing the molasses with an equal
volume of concentrated sulphuric acid and heating for three hours to
130°. After neutralisation with lime, evaporation to dryness and ex-
traction of the residue with alcohol, the alcoholic extract is treated
with charcoal, concentrated to a syrup and saturated with gaseous
hydrogen chloride, when betaine chloride crystallises out.
A method of isolating betaine from the desaccharified strontium
liquors as the phosphate is given by Andrlik [1903-4] and as
the chloride by Stoltzenberg, German patent No. 243332 and [1912],
The last-named method is similar to that given by Urban [1913],
but the best method of all is apparently that due to Ehrlich [1912 and
D.R.P. 157173 of 1904], From the desaccharified residue (" Melasse
Schlempe ") the betaine is extracted as base by means of 96 per cent,
alcohol, and after evaporation of the alcohol, the free base is converted
into the chloride which is crystallised. The commercial product acidol
is prepared according to this method.
Chemical Properties and Derivates of Betaine.
Betaine crystallises from alcohol in deliquescent crystals containing
one molecule of water which is lost at 100°. The hydrated substance
141
142 THE SIMPLER NATURAL BASES
probably has the constitution (CH3)3N(OH) . CH2. COOH, of which
the other substance is a cyclic anhydride.
Betaine and its isomeride, the methyl ester of dimethyl-ammo-
acetic acid, are interconvertible at temperatures between 135° (the
boiling point of the ester) and 293° ; over this range betaine is the
more stable and it is formed in good yield by heating the ester in a
sealed tube to 200°. On the other hand a 50 per cent, yield of the
ester is obtainable by heating betaine to 300°, when the ester distils
out. At or above 293° betaine begins to be decomposed into tri-
methylamine and other substances (Willstatter [1902, I]).
Betaine is a very feeble base, forming a series of stable salts. The
salts with mineral acids have a strongly acidic reaction, and for this
reason the chloride is sold as a solid substitute for hydrochloric acid
under the name " acidol ".
The chloride^ C5H12O2NC1, forms leaflets, melting and decomposing
at 227-228° (243°); it is very soluble in water and differs from the
hydrochlorides of most organic bases in being almost insoluble in
absolute alcohol (i grm. dissolves in 365 c.c. of absolute alcohol at
room temperature; Schulze [1909, Ch. IV, choline]).
The iodide, C5H12O2NI, non-deliquescent crystals; m.p. 188-190°;
very soluble in hot alcohol, but little in cold (Willstatter [1902, i]).
The periodide, C5H12O2NI . I5, loses iodine on exposure to the air
[Stanek, 1912]. Compounds with potassium iodide of the formulae
C5HU02N . KI . 2H2O and (C5HnO2N)2 . KI . 2H2O have also been de-
scribed (see Willstatter [1902, i]).
The/^<?j^<2/£,C5HnO2N . H3PO4, melts at 199-200° and decomposes
at 234°(Andrlik [1903-4]).
Iteperchlorate, C^^N . HC1O4, is much less soluble than the
corresponding choline salt; at 19° 1773 parts dissolve in 100 parts
of water (Hofmann, Roth, Hobold and Metzler [1910, Ch. IV,
choline]; Hofmann and Hobold [i9ii,Ch. IV, choline]).
The/zVrate, C5HnO2N . C6H3O7N3, forms yellow needles ; m.p. 180-
181°; it is suitable for the separation of the base from mixtures
(Schulze and Trier [1910, i]).
Th&picrolonate, C5HnO2N . C10H8O5N4, forms yellow needles readily
soluble in alcohol and in water, and decomposes at 200° (Otori [1904, 3,
Ch. I]).
The platinichloride, (C6HnO2N)2H2PtCl6 . 4H2O., crystallises from
concentrated aqueous solution in the cold in large rhomb-shaped
tables with truncated angles, and effloresces in air; m.p. 242°; insol-
yble in alcohol, very soluble in hot water from which it crystallises
APPENDIX TO CHAPTER III— BETAINES 143
in pale orange-yellow prisms with varying water content. In con-
tact with the aqueous mother liquor, the anhydrous needles which
separate from a hot solution are transformed into the four-sided tables
with 4H2O. This constitutes a test for betaine [Trier, 1913, 5]. It
is possibly dimorphous (Willstatter [1902, 2]).
The aurichloride, C5HnO2N . HAuCl4, is the most characteristic salt
and is dimorphous (Willstatter [1902, 2]).
(a) Regular system ; from a 5 per cent, solution in hot water on slow
cooling, best in the presence of a slight excess of gold chloride ; it
generally separates in dull yellow, star-shaped aggregates ; m.p. 2OO-
209° (uncorr.) according to the rate of heating.
(£) Rhombic system ; bright yellow leaflets, prisms and plates with
one truncated angle ; m.p. 248-250° (uncorr.). This form always
separates in the presence of hydrochloric acid.
By recrystallisation from pure water a pale yellow salt of an inferior
gold content is obtained, possibly due to admixture with a hydrated
salt. For purposes of identification it is therefore best to recrystal-
lise from O'5-i per cent, hydrochloric acid, in order to obtain the
rhombic variety of high melting point (Willstatter [1902, 2], Fischer
[1902]).
The mercurichloride, (C5HUON . HC1)2. HgCl2, is fairly readily
soluble in water, sparingly in alcohol.
Stachydrine.
The preparation of stachydrine from Stachys tubers and from
orange leaves was carried out bySchulzeand Trier [1909, i] by purify-
ing an aqueous extract with basic lead acetate, precipitating the bases in
the filtrate with phosphotungstic acid, removing the " histidine " and
" arginine " fractions of the recovered bases by means of silver, again
precipitating the bases from the filtrate of these fractions as phospho-
tungstates, extracting the recovered hydrochlorides with absolute
alcohol and then precipitating with mercuric chloride ; the stachy-
drine is separated from choline by Stanek's method (see p. 151).
The yield from fresh tubers of Stachys was 0-036 per cent, of
stachydrine; from dried orange leaves 0*19 per cent. The tubers
also contain a minute quantity of trigonelline. Jahns [1896] isolated
stachydrine by means of potassium bismuth iodide (Kraut's reagent).
For the properties of the base and its salts consult Schulze and
Trier's paper [1910, 2]. Like other betaines, the base loses a mole-
cule of water of crystallisation at 100° ; the anhydrous base has the
composition C7H13O2N and melts at 235°. Stachydrine is readily
i44 THE SIMPLER NATURAL BASES
soluble in water and in alcohol, but not in cold chloroform or in
ether ; its aqueous solution is neutral.
The hydrochloride, C7H13O2N . HC1, crystallises in large prisms and
dissolves in 127 parts of cold absolute alcohol at 17-18°; it is therefore
much more soluble than betaine hydrochloride.
The acid oxalate, C7H13O2N . C2H2O4, forms needles, insoluble in
cold absolute alcohol; m.p. 105-107°.
The picrate, C7H13O2N . C6H3O7N3, m.p. 195-196°, is only precipi-
tated from a concentrated solution.
The aurichloride, CyH^O^N . HAuCl4, precipitated in aqueous solu-
tion, soon crystallises and forms characteristic four-sided leaflets of
rhombic habit ; m.p. 225° on rapid heating.
The platinichloride, (C7H13O2N)2H2PtG6, with o, 2 and 4H2O,
readily soluble in water, insoluble in alcohol; m.p. indefinite at 210-
220°.
Mercuric chloride causes a precipitate in solutions of the hydro-
chloride (best in alcoholic solution), but not in those of the free base.
Stachydrine methyl and ethyl esters are only soluble in acid solu-
tion.
Betonicine and Turicine.
The hydrochloride of betonicine is less soluble in absolute alcohol
than the hydrochloride of turicine, but the free bases have a reverse
order of solubility.
Betonicine, C7H13O3N + H2O has [«]D= - 36*60° and decomposes
at 243-244°. Turicine [a] has D = +36*26° and decomposes at 249°.
Betonicine hydrochloride gave [a]D = - 2479° and turicine hydro-
chloride [a]D = +24*65°.
Betonicine aurichloride decomposes at 242°, that of turicine at 232°.
Betonicine platinichloride crystallises with 2H2O and decomposes at
226°, turicine platinichloride contains only iH2O and decomposes at
223°. Both bases heated with zinc dust give a pyrrole reaction with
pine wood.
Trimethyl-histidine.
Reuter found this base in the arginine fraction, Kutscher curiously
enough in the lysine fraction. It is best isolated as aurichloride.
The base from Boletus has [a]o= +41 *i° (in the presence of 8 mol.
HC1).
The nitrate forms large transparent plates and octahedra. The
monopicrate, C9H15O2N3 . C6H3O7N3. H2O, thin felted needles, m.p. 201°,
is readily soluble in water; the dipicrate, C9H16O2N8. 2C6H3O7N3 . 2H2O,
APPENDIX TO CHAPTER III— BETAINES 145
is much less soluble in water (in 25 parts at 100°); it melts at 123°;
when anhydrous the melting point is 213-214°.
The normal aurichloride, C9H15O2N3. 2HAud4, forms large orange
yellow crystals, m.p. 184°, by crystallisation in the presence of dilute
hydrochloric acid and excess of gold chloride. Reuter mentions two
other gold salts of abnormal composition.
Ergothioneine.
For the preparation of the base according to Tanret [1909]
ergot is extracted with 90 per cent, alcohol ; after evaporation of the
alcohol, the aqueous residue is freed from fat and resin by filtration ;
20 per cent, sulphuric acid is then added to precipitate colouring
matters, and after removal of the acid by baryta, the filtrate is precipi-
tated with basic lead acetate. After filtering again, the excess of lead
is removed with sulphuric acid and the solution is made alkaline and
extracted with chloroform to remove the complex ergot alkaloids. It
is then acidified with acetic acid and precipitated completely with a
warm 8 per cent, solution of mercuric chloride. The mercury precipi-
tate is filtered off, washed, suspended in a large bulk of water and de-
composed by hydrogen sulphide. After removal of the mercuric
sulphide, the filtrate is evaporated under reduced pressure to a syrup
from which ergothioneine hydrochloride soon crystallises. After
washing with alcohol the substance is recrystallised from water. The
yield is cri per cent, of the ergot employed. From the hydrochloride
the base can be obtained in various ways, for instance by boiling with
excess of calcium carbonate, filtering, concentrating and adding alcohol.
The free base is recrystallised from boiling 60 per cent, alcohol.
Ergothioneine crystallises in leaflets and needles containing two
molecules o£ water of crystallisation. It is soluble in 8.6 parts of
water at 20°, but requires more than a thousand parts of boiling 95
per cent, alcohol, and is insoluble in ether, chloroform and benzene.
The base is dextro-rotatory, [a]D = + 1 10°. The melting point on the
Maquenne block is 290°.
Ergothioneine does not act on litmus ; the salts are precipitated
even in dilute solution by potassium mercuric iodide, by iodine in
potassium iodide and by mercuric chloride. With sodium p-diazo-
benzene sulphonate a cherry-red coloration is produced (Pauly's
histidine reaction). The most characteristic reaction is with excess
of alcoholic iodine solution which forms crystals of a less soluble iodide
(p. 46). On evaporation of the alcohol these crystals take up iodine
and become steel grey or blue.
10
146 THE SIMPLER NATURAL BASES
Hypaphorine.
The isolation from the seeds of Erythrina Hypaphorus is carried
out, according to GreshofT, by adding dilute nitric acid to an aqueous
or alcoholic extract of the powdered cotyledons ; this causes the very
sparingly soluble nitrate to crystallise out. The yield is 3 per cent, of
the dried seeds.
The free base is obtained from the nitrate by adding concentrated
sodium carbonate solution ; the base then separates as an oily upper
layer which soon crystallises. Hypaphorine is also obtainable from
an aqueous extract, after purification with lead acetate and concen-
tration. The mother liquor of the crystals of the free base is treated
with nitric acid and yields a further quantity as nitrate.
Hypaphorine crystallises from water in large monoclinic trans-
parent crystals of the composition CUH18O2N2 . 2H2O which effloresce
in a desiccator.
The anhydrous substance melts at about 255° with decomposition.
It is dextro-rotatory; in 1-3 per cent, solution in water [a]o = +93°-
Hypaporine dissolves very readily in water and also readily in alcohol,
but not in other organic solvents. The aqueous solution is neutral to
litmus and, if not very dilute, yields precipitates with most alkaloidal
reagents. Gold chloride is reduced and coloured red even by dilute
solutions ; potassium permanganate is decolourised and a solution
containing ferric chloride and potassium ferricyanide yields Prussian
blue. -The solution of the base in concentrated sulphuric acid yields
with various oxidising agents (potassium dichromate, ferricyanide,
etc.) an intense violet coloration which soon disappears. The close
relationship between hypaphorine and tryptophane is shown by the
fact that the former substance also gives Hopkins and Cole's reaction
with glyoxylic and sulphuric acids, but hypaphorine does not react with
triketohydrindenehydrate. In spite of the similarity of its structure to
that of tryptophane, hypaphorine yields on oxidation with ferric chloride
only traces of yS-indole aldehyde.
The most characteristic salt is the nitrate, C14H18O2N2 . HNO3, which
melts with decomposition at 215-220° and dissolves at room tempera-
ture in about 170 parts of water; other crystalline salts and the free
base are much more soluble.
The quaternary iodide, C16H21OaN2I, obtained by methylation from
both tryptophane and hypaphorine, forms glistening plates from boiling
water and dissolves in 200 parts of water at 18°.
APPENDIX TO CHAPTER III— BETAINES 147
TrigoneUine.
Jahns extracted Trigonella seeds with 70 per cent alcohol, purified
with basic lead acetate, concentrated to a syrup and precipitated with
potassium bismuth iodide. The precipitate was decomposed with
soda, and after filtration the solution was exactly neutralised ; mer-
curic chloride was then added until mercuric iodide appeared. This
precipitates only choline, but on acidification the crystalline double
salt of trigonelline separates.
Schulze [1909; Ch. IV, choline] used phosphotungstic acid and
alcoholic mercuric chloride for approximately quantitative estimation
of trigonelline.
Trigonelline, C7H7O2N. H2O, becomes anhydrous at 100°, when the
crystals become opaque without losing their shape. The hydrated
base melts at about 130°, the anhydrous at 218°.
The platinichloride, hardly soluble in alcohol, crystallises from
water. There are two characteristic aurichlorides ; one, of normal
composition, C7H7O2N . HAuCl4, leaflets, m.p. 198°, changes on re-
crystallisation from water to the basic salt (C7H7O2N)4 . 3HAuCl4,
needles, m.p. 186°, which recrystallised in the presence of gold chloride
and hydrochloric acid, may be reconverted to the normal salt.
Butyrobetaine.
Brieger precipitated the alcoholic mother liquors of putrescine
hydrochloride with alcoholic mercuric chloride and extracted the pre-
cipitate with boiling water. On cooling cadaverine mercurichloride
crystallised out, while the butyrobetaine salt remained in solution.
After removal of the mercury with hydrogen sulphide and concen-
tration to a syrup, the butyrobetaine was precipitated as sparingly
soluble aurichloride.
According to Willstatter the free &w<? crystallises from dilute alco-
hol in leaflets, probably with three molecules of water. Dried over
sulphuric acid, the composition is C7H15O2N ; the crystals begin to
soften at 130° and froth up at 222°, decomposing into trimethylamine
and 7-butyrolactone.
The hydrochloride^ C7H15O2N. HC1, forms needles, almost or quite
insoluble in absolute alcohol ; m.p. 200° (Takeda), 203° (Engeland and
Kutscher).
The aurichloride, C7H15O2N . H AuCl4, is precipitated on adding gold
chloride to an aqueous solution of the hydrochloride ; it crystallises
in needles and leaflets and melts at 176° (Brieger; his formula con-
tains two more hydrogen atoms).
10*
148 THE SIMPLER NATURAL BASES
^ (C7H15O2N)2 . H2PtCl6, is readily soluble in warm
water, but hardly in hot alcohol, and forms light red plates, melting at
224-225°.
The ethyl ester yields a characteristic platinichloride (C9H19O2N)2 .
H2PtCl6, melting at 220° (Takeda, Engeland and Kutscher).
Apart from the synthesis, the constitution is established by the
formation of an ester, by the optical inactivity (Takeda) and by the
liberation of trimethylamine on distillation with baryta.
Solutions of the hydrochloride are not precipitated by picric acid,
but by phosphomolybdic and phosphotungstic acids, by potassium
mercuric iodide, potassium cadmium iodide, and potassium tri-iodide ;
in all cases the precipitate, which is at first amorphous, soon crystallises
in needles (Brieger). Takeda also observed the gradual crystallisation
of the precipitate with potassium bismuth iodide.
Carnitine.
This substance is best prepared from meat extract by Gulewitsch and
Krimberg' s method. After removal of carnosine and other bases by
means of silver nitrate and baryta, the solution is freed from silver and
barium, and the carnitine is precipitated with potassium bismuth
iodide (see p. 1 21).
The free base, the hydrochloride C7H15O3N . HC1 and the nitrate
C7H]5O3N . HNO3 are all readily soluble in water ; a 10 per cent, solution
of the hydrochloride in excess of free acid has [a]D = - 20-9°.
The platinichloride, (C7H15O3N)2 . H2PtCl6, crystallises from 80 per
cent, alcohol in short prisms; m.p. 214-218°.
The aurichloride, C7H15O3N . HAuCl4, forms citron yellow needles ;
m.p. I53-I540.
There are two double salts with mercuric chloride : C7H15O3N .
2HgCl2, from the free base and mercuric chloride, both in alcoholic
solution ; sparingly soluble in water and crystallising fairly readily ; m.p.
204-205°. C7H15O3N . HC1 . 6HgCl2 is formed in the presence of a
slight excess of hydrochloric acid ; it is an oil, crystallising with diffi-
culty ; m.p. 211-215°.
Carnitine ethyl ester, C0H19O3N, was according to Krimberg [1908, 2]
mistaken by Kutscher for a new base from meat extract under the
name oblitine ; Kutscher gave it the formula C18H38O5N2. Krimberg
[1907, 2] showed that oblitine is formed by evaporating an alcoholic
solution of carnitine with hydrochloric acid, which is one of the steps
in Kutscher's process of separation. At first Krimberg considered
oblitine to be the ethyl ester of an anhydride, formed from two
APPENDIX TO CHAPTER III— BETAINES 149
molecules of carnitine by loss of one molecule of water, but the com-
position of the base is not C18H38O5N2 but C9H19O3N and the substance
is merely carnitine ethyl ester. It is therefore not surprising that
"novaine" ( = carnitine) is formed from oblitine by bacterial action,
and is the only product which can be isolated (Kutscher [1906, 2]), nor
that oblitine is partially transformed in the intestine to "novaine"
(Kutscher and Lohmann [i
BASES OF CHAPTER IV— CHOLINE AND ALLIED SUBSTANCES.
Preparation of Choline from Natural Sources.
The best source is egg-yolk. Crude lecithin, obtained by extract-
ing the yolk with alcohol and ether, is hydrolysed by boiling with
saturated baryta solution for one hour; after removal of the barium,
the solution is evaporated and the residue extracted with alcohol.
After acidification of the alcoholic solution with hydrochloric acid,
the choline is precipitated by alcoholic platinic chloride solution.
According to the German patent No. 193449 of J. D. Riedel
[1908] lecithin is heated with twice its weight of 40 per cent, sul-
phuric acid, and after removal of the acid with baryta, choline is
precipitated with mercuric chloride (cf. also Moruzzi [1908] and
MacLean [1908]). To convert the platinichloride into the hydro-
chloride, the aqueous solution of the former salt is evaporated after
adding the calculated quantity of potassium chloride, and then the
choline chloride can be extracted by absolute alcohol.
Schulze's Method of Separating Choline and other Plant Bases.
This method [1909] for the more or less quantitative isolation
of choline, betaine and trigonelline, is more trustworthy than that of
Stanek (described below) when other bases are present, and is corres-
pondingly more complicated.
An aqueous extract of the material (which is preferable to an
alcoholic one since it excludes phosphatides more completely) is puri-
fied with lead acetate, strongly acidified with sulphuric acid and
precipitated with phosphotungstic acid. After regeneration of the
precipitate with baryta, the purine bases and the histidine and arginine
fractions are removed by means of silver nitrate in the usual manner,
and the last filtrate is again precipitated with phosphotungstic acid ;
after regeneration the mixture of chlorides is dissolved in 95 per cent,
alcohol and precipitated with alcoholic mercuric chloride. Choline
mercurichloride is very little soluble in boiling water, the betaine
compound more so. The separation is completed by converting the
more and the less soluble mercurichlorides into the dry hydrochlorides
150
APPENDIX TO CHAPTER IV— CHOLINE 151
and extracting with anhydrous alcohol, which leaves betaine hydro-
chloride undissolved. The method may be shortened by omitting the
second precipitation with phosphotungstic acid, and in place of it
precipitating the filtrate from arginine at once with mercuric chloride
(after removal of the silver). It is also possible to combine Stanek's
process with mercuric chloride precipitation.
The properties of trigonelline are similar to those of betaine and
the separation from choline is effected in the same way. According
to Schulze 3-4 per cent, of these bases escape precipitation with phos-
photungstic acid. In alcoholic solution 5 per cent, of the trigonelline
and choline escaped precipitation by mercuric chloride, but in the case
of betaine the loss was more than double this amount, so that it is
advisable to concentrate the filtrate.
Stanek's Method for the Estimation of Choline and Betaine.
The method [1905, 1906, i, 2] is based on the fact that betaine,
being a very weak base, is set free from its salts by sodium bicarbonate,
while choline is not. It is carried out as follows : To 25-40 c.c. of the
aqueous solution, containing at most 5 per cent, of the mixed hydro-
chlorides of choline and betaine, sodium or potassium bicarbonate is
added to make 5 per cent, and then a solution of 153 grm. of iodine
and 100 grm. of potassium iodide in 200 grm. of water is added until
precipitation is complete ; the precipitate consists of brown choline
ennea-iodide and soon becomes crystalline. It is collected on a paper
disk in a Gooch crucible, washed with water and transferred to a
Kjeldahl flask for nitrogen determination. If desired, the choline may
instead be recovered from the periodide by adding finely divided
(" molecular ") copper (see p. 122), boiling with cupric chloride and
copper and, after filtration, treating the filtrate with hydrogen sulphide.
The solution then contains the choline as hydrochloride.
The betaine is estimated by concentrating the filtrate which passed
through the Gooch crucible to 25 c.c. and adding enough sulphuric
acid to make 10 per cent. ; the solution is then saturated with sodium
chloride, and the betaine is now precipitated with the potassium
tri-iodide solution (already used for choline). After standing for three
hours the precipitated betaine per-iodide is collected, washed five
times with 5 c.c. of saturated sodium chloride and transferred to a
Kjeldahl flask in which its nitrogen content is determined.
For the estimation of choline (and betaine) in plants Stanek ex-
tracts the air dry material with 96 per cent, alcohol which is distilled
off; the aqueous residue is boiled with baryta and the barium is re-
152 THE SIMPLER NATURAL BASES
moved by carbon dioxide; the filtrate is then treated with tannin, of
which the excess is removed by baryta. The choline and betaine are
then precipitated together as periodides from acid solution, and after
successive treatment of the precipitate with copper powder and with
cupric chloride, the mixture of chlorides is separated as described
above. If much betaine is present it is preferable to effect a prelimin-
ary separation of the dry chlorides by means of absolute alcohol, in
which betaine chloride is insoluble.
Tests, Chemical Properties and Salts of Choline.
An admirable account of choline is given by Gulewitsch [1908, i].
The free base is very soluble in water, from which it cannot be ex-
tracted by organic solvents. (Only amyl alcohol extracts more than
traces from an alkaline solution.) Choline is a strong base, liberating
ammonia from its salts and preventing the coagulation of proteins.
The most delicate precipitant is potassium tri-iodide (limit accord-
ing to Gulewitsch i : 20,000; according to Kinoshita [1910,2] the
limit (with Stanek's concentrated potassium tri-iodide, see above) is at
i : 2,000,000. The choline per-iodide on standing forms rhomboidal,
almost quadratic, leaflets.
Phosphotungstic acid precipitates at I : 20,000 (Gulewitsch). Less
sensitive precipitants in aqueous solution are potassium bismuth iodide,
mercuric chloride, saturated cadmium chloride and gold chloride.
Tannin precipitates only in strictly neutral solution. In absolute
alcoholic solution mercuric chloride and platinic chloride are the most
delicate reagents (i : 2,000,000).
The/*ftft&& test was used by Florence [1897] as a reaction for
semen; Bocarius [1901] showed that it is due to choline. The test
may be applied in a characteristic way to crystals of choline platinic
chloride. After evaporating the solution of this platinum salt in
1 5 per cent, alcohol on a microscope slide at 40°, potassium tri-iodide
solution (20 grm. iodine and 60 grm. potassium iodide per litre) is
added ; the yellow crystals of the platinichloride disappear and are
replaced by dark brown doubly refractive and dichroitic prisms and
plates of choline periodide. When the excess of reagent evaporates,
the periodide dissociates and the brown crystals liquefy and disappear,
but they can be reformed by again adding the reagent (cf. Rosen-
heim [1905-6]). Joesten [1913] considers that Florence's crystals
are perhaps merely iodine, without any choline. His paper should be
consulted for an account of the literature of the reaction.
Alloxan reaction. — When a drop of choline hydrochloride is eva-
APPENDIX TO CHAPTER IV— CHOLINE 153
porated with a drop of a saturated alloxan solution, a reddish violet
colour results, which becomes more blue on the addition of caustic
soda. The reaction is not characteristic and similar colorations are
produced by ammonium salts, proteins and amino-acids (cf. Hurtley
and Wootton, Journ. Chem. Soc., 1911, 99, 288).
Choline, as free base, deliquesces in the air and absorbs carbon
dioxide. According to Gulewitsch aqueous solutions may be con-
centrated by boiling to 4 per cent, concentration, when trimethylamine
is given off. The base is not changed rapidly by boiling with alkalies
in dilute solution ; on keeping for a long time in aqueous solution
neurine is formed. Concentrated nitric acid converts it to its nitrous
acid ester, pseudo-muscarine (cf. addendum, p. 68). It may be
oxidised to betaine (" Oxyneurin," Liebreich [1869, i]).
The organisms from a hay infusion probably to some extent con-
vert choline into neurine (Schmidt [1891]). Brieger had already
surmised that this change takes place in putrefaction and found that all
the choline disappeared within the first week, but Gulewitsch [1864,
Ch. I] isolated choline from putrid horse meat after four months' putre-
faction at 15°. According to Ackermann and Schutze [1910, 1911,
Ch. I] Bacterium prodigiosum forms trimethylamine and a little mono-
methylamine from choline, but Bacillus vulgatus does not decompose it.
Prolonged anaerobic putrefaction yields CO2, CH4, N2, NH3 and
CH3NH2 (Hasebroek [1887, Ch. I]).
All known choline salts are readily soluble in water, except the
periodide, the phosphotungstate and the double salts with gold and
with mercury. Some, as for instance the chloride C5H14ONC1, are
deliquescent. The chloride also dissolves readily in absolute alcohol
(distinction from betaine).
The sulphate (C5HUON)2SO4, the acetate C5HUON . C2H3O2, and the
monophosphate C5HUON . H2PO4 are all readily soluble in water, and
crystallise in needles. The first two are readily soluble in alcohol, but
the phosphate is not. The acetate is deliquescent (Renshaw [1910]).
The perchlorate C5H14ON . C1O4, m.p. 273°, dissolves in 2-9 parts of
water at 1 5° and is not birefringent. The perchlorate of the nitric acid
ester of choline is, however, only very slightly soluble (0*62 parts in 100
parts of water at 1 5°). It is obtained by evaporating O'l grm. of choline
perchlorate in 50 c.c. of water with 2 c.c. of 65 per cent, nitric acid,
dissolving the residue in a little water and adding a few drops of a
concentrated aqueous solution of perchloric acid. This latter salt is
characteristic; it is strongly birefringent, melts at 185-186° and is
suitable for the isolation of choline (Hofmann and Hobold [1911]).
154 THE SIMPLER NATURAL BASES
The acid chromate, C5H14ON . HCrO4, is on the other hand much
more soluble than the neurine salt (Cramer [1904]).
Th&picrate, C5H14ON . C6H2O7N3, is fairly soluble in water and more
so in alcohol (Brieger [1885, 2, p. 56 ; Ch. I]).
The ptcrolonate, C5HUON . C10H7O5N4. H2O, loses water of crystal-
lisation at 130°, melts at 158° and decomposes at 241-245° (Otori
[1904, 3]).
^.}\^ platinichloride y (C5H14ON)2PtCl6, is dimorphous. It crystallises
from a mixture of equal volumes of absolute alcohol and water in the
regular system (octahedra, cubes) and from water in rhomb-shaped six-
sided or pyramidal crystals of the monoclinic system ; on slow eva-
poration the latter kind may attain considerable size (Kauffmann and
Vorlander [1910] ; Gulewitsch [1891, i] gives crystallographic details).
Both forms of the salt are anhydrous arid orange red in colour ;
they are stable in the dry state, but readily interconvertible by recry-
stallisation from the proper solvent. Since one form is isotropic and
the other anisotropic, the dimorphism of choline platinichloride is
readily detected in polarised light and affords according to Kauffmann
the surest qualitative means of identification. The platinichlorides of
potassium, ammonium, trimethylamine and neurine all crystallise from
dilute alcohol in the regular system only ; if, after adding water and
evaporating, crystals become anisotropic, choline is probably present.
At 21° one part of choline platinichloride dissolves in 5-82 parts
of water (Gulewitsch). The melting point is not characteristic ; both
forms melt at 209-211° on slow heating and at 240-241° when heated
rapidly.
The aurichloride, C5H14ON AuCl4, crystallises in deep yellow needles
and also (from very dilute alcohol) in octahedra and cubes ; it dis-
solves in 7 5 -2 parts of water at 21° and in hot alcohol (Gulewitsch).
The melting point has been variously given as 238-239°, 249°, 244-
264°, etc.
The mercurichloride, C5H14ONC1 . 6HgCl2 . H2O, forms crossed
hexagonal prisms, loses water above 1 00° and melts at 249-251°; it is
soluble in 56^6 parts of water at 24-5° (Gulewitsch). According to
M6rner[i896; Ch. I] the melting point is 242-243° and it dissolves
in 67 parts of water at I9'5°. Schulze [1909] found that one part
of the mercury salt dissolves in about fifty parts of water at room
temperature ; the solubility determinations were not concordant, pro-
bably owing to hydrolytic dissociation.
The slight solubility of choline mercurichloride in cold water was
used by Brieger for its isolation ; after complete - precipitation by
APPENDIX TO CHAPTER IV— CHOLINE 155
mercuric chloride in alcoholic solution, the precipitate was extracted
with boiling water, in which the mercury compounds of peptones and
proteins were completely insoluble. The choline mercurichloride
crystallised out almost completely from the filtrate on cooling, and the
mercury salts of other putrefaction bases remained in solution.
Double salts of choline chloride with cadmium and with zinc
chloride are precipitated in alcoholic solution.
Stanek [1905] has described two periodides. With excess of
iodine in potassium iodide an ennea-iodide C5H14ONI . I8 is formed as
a brown precipitate, changing to shiny green crystals; in a O'l-i per
cent, choline solution only 2-3 per cent, of the total escapes precipita-
tion. When choline is in excess a hexa-iodide C5HUONI . I5 results.
Amino-ethyl Alcohol.
The free base distils at 160-165° a"d 718 mm. Ihzhydrochloride
C2H5ON . HC1 is hygroscopic. The aurichloride C2H5ON . HAuCl4
crystallises slowly from concentrated hydrochloric acid containing
excess of gold chloride in large crystals, melting at 186-187°. The
platinichloride is anhydrous.
Amino-ethyl alcohol differs from choline in not being precipitated by
potassium bismuth iodide, and not by phosphotungstic acid except in
concentrated solutions. Heated with hydriodic acid, as in Herzig and
Meyer's method for the determination of N-methyl groups, it gives
off a little ethyl iodide [Trier, 1913, 5].
Neurine.
The separation of neurine from choline may be carried out by
fractional crystallisation of the platinum salts ; the large crystals of
choline platinichloride are readily obtained pure, but the small, less
soluble crystals of the neurine salt are only purified with difficulty
(Gulewitsch [1899 ; under choline]).
The chemical properties of neurine and some of its compounds
have been described in detail by Gulewitsch [1898, 2]. It is a strong
base and, like choline, it liberates ammonia from its salts and prevents
the coagulation of protein. It may be boiled in dilute solution with-
out decomposition, and is not changed by boiling with concentrated
baryta. Its behaviour with alkaloidal reagents is very similar to that
of choline; generally the reactions are more delicate; thus with
phosphotungstic acid and with potassium tri-iodide a micro-crystalline
precipitate is produced which is even indicated at a dilution of I :
200,000.
156 THE SIMPLER NATURAL BASES
The chloride, C5H12NC1, forms deliquescent needles, the iodide is
non-deliquescent ; m. p. 196°.
The perchlorate, C5H12NC1O4, forms characteristic aggregates of
short prisms, which are scarcely birefringent ; 100 grm. of water at
20° dissolve 5 764 grm., at 145° 4*89 grm. Hence this salt is much less
soluble than the corresponding choline salt, but six times as soluble as
the perchlorate of choline nitric acid ester, q.v. [Hofmann and Hobold,
191 1 ; under choline].
The acid chromate, C5H12N . HCrO4 . H2O, forms orange needles
from water; m.p. 278° on rapid heating; heated slowly it decomposes
explosively at 140-150°. In contradistinction to choline chromate it
is little soluble in cold water (Cramer [1904, under choline]).
The picrate, C5H12N . C6H2O7N3, forms long feathery golden yellow
needles ; m.p. 263-264° ; soluble in 91 '6 parts of water at 23°, more so
in hot water, readily in hot alcohol (Gulewitsch [1898, 2]).
The platinichloride, (C5H12N)2PtCl6, forms cubes and octahedra of
the regular system; m.p. 196-198° (but according to Nothnagel the
melting point is 15-20° higher); the salt is anhydrous and at 20*5° dis-
solves in 37*6 parts of water (Gulewitsch [1898, 2]). The solubility is
considerably less than that of the corresponding choline salt.
The aurichloride, C5H12N . AuCl4, forms large golden yellow
acicular crystals; m.p. 232-238°; soluble in 336*5 parts of water at
2 1 -5°; not very soluble in hot water.
There are two mercurichlorides formed by precipitation with
alcoholic HgCl2 and not readily separated, (a) C5H12NC1 . 6HgCl2,
plates and prisms; m.p. 230-234°; is but little soluble in hot water.
(fr) C5H12NC1 . HgCl2, triclinic plates, more readily soluble in water
(Gulewitsch [1898, 2]).
BASES OF CHAPTER V.
Creatine and Creatinine.
Preparation of creatine for muscle. — Liebig mixed minced meat
repeatedly with an equal volume of cold water and pressed out. In
the extract the protein was coagulated and, after straining, the solu-
tion was treated with baryta until no more precipitate occurred.
After filtration and concentration creatine crystallised out in the
course of a few days.
It is, however, better to start with commercial meat extract and
after dissolving in twenty parts of water, to precipitate peptones, etc.,
either with basic lead acetate (Mulder and Mouthaan [1869]) or
with tannin (Kutscher [1905]). After removal of the excess of lead
or of tannin (see p. 117) the filtrate is concentrated to a thin syrup ;
on standing creatine crystallises and is then washed with absolute
alcohol to remove creatinine and is recrystallised with charcoal ; the
creatinine crystallises from the alcoholic washings on the addition of
ether. Creatinine, abundantly present in most commercial meat
extracts, is also obtained by Kutscher's method as a silver com-
pound in the histidine fraction. Here it is accompanied by carnosine,
from which it is separated by solution in alcohol, which leaves the
carnosine behind.
Preparation of creatinine. — Creatinine is most conveniently obtained
from urine by precipitation with picric acid (Folin and Blanck [1910]).
To each litre of urine 18 grm. of picric acid, dissolved in 45 c.c. of
boiling alcohol, is added.
The resulting precipitate, mostly of creatinine potassium picrate,
is decomposed by grinding with potassium bicarbonate and, after
filtration, the solution is slightly acidified, mixed with two volumes
of alcohol, decolourised with a little charcoal and treated with concen-
trated alcoholic zinc chloride. The crude creatinine zinc chloride,
which separates on standing, may be boiled with lead hydroxide,
when about equal quantities of creatine and creatinine are obtained ;
or it may be dissolved in warm 10 per cent, sulphuric acid, when the
addition of acetone causes the separation of pure creatinine zinc
sulphate, (C4H7ON3)2H2SO, . ZnSO4 . 8H2O.
157
158 THE SIMPLER NATURAL BASES
The use of zinc chloride alone was introduced by Pettenkofer
[1844], the discoverer of creatinine ; Neubauer [1863] and Salkowski
[1886, 1890] attempted to make this method a quantitative one, but
as such it has been entirely superseded by Folin's colorimetric estima-
tion. The use of picric acid for the precipitation of creatinine from
urine was introduced by Jaffe [1886]; other precipitants are mercuric
chloride (Maly [1871]) and phosphotungstic acid (Hofmeister [1880]).
Quantitative conversion of creatine to creatinine. — Benedict and
Myers [1907, 2] heated a dilute creatine solution containing 6-7 per
cent, hydrochloric acid (i.e. \ volume of the concentrated acid) in an
autoclave to 117° for forty-five minutes. Dorner [1907] warmed a
OT per cent, creatine solution for 3-4 hours on the water bath with
twice its volume of normal hydrochloric acid (hence concentration of
acid = 2*44 per cent). Thompson, Wallace and Clotworthy [1913]
recommend adding an equal volume of normal hydrochloric acid and
heating on the water bath for 3 hours or in the autoclave to 117-1 20
for 25 mins.
According to the last named authors pure dextrose, up to 10 per
cent., does not affect the estimation of creatine, although 3 per cent,
phosphoric acid has been recommended instead of hydrochloric acid,
in order to avoid the formation of coloured products. Creatine figures
for diabetic urine may come 5 per cent, too low, probably owing to the
presence of aceto-acetic acid. The darkening of the urinary pigment
by treatment with acid may increase the creatine readings in human
urine by \-2\ per cent., in dog's urine by 10 per cent.
According to Folin and Blanck [1910] creatine crystals may be
converted quantitatively into creatinine by heating without a solvent
in an autoclave for three hours at 4-5 atmospheres ; the water of
crystallisation appears to be the active agent.
Physical and chemical properties of creatine. — This substance forms
lustrous transparent monoclihic prisms of the composition C4H7O2N3,
H2O. The 12*08 per cent, of water of crystallisation is given off
quantitatively at 100-110°, and the crystals become opaque (a deter-
mination of the loss of weight may be used for identification).
Creatine dissolves in 74 parts of water at 18°; it is much more
soluble in hot water, but hardly at all in absolute alcohol (i : 9400).
The aqueous solution is neutral. The basic properties of creatine
are very feeble (dissociation constant 1*81 x io~u at 40*2°, Wood
[1903]) and its salts with mineral acids are hydro lysed by water.
Creatine is precipitated from aqueous solution by mercuric nitrate,
but not by phosphotungstic acid, nor by basic lead acetate • crystal-
APPENDIX, TO CHAPTER V 159
lisable compounds with zinc chloride and cadmium chloride are known
and are dissociated by water.
Creatine reduces Fehling's solution without separation of cuprous
oxide and is oxidised by boiling with mercuric oxide to methyl
guanidine oxalate (Dessaignes [1854, 1855]) and also by Fenton's re-
agent (hydrogen peroxide and ferrous sulphate, Dakin [1906]); in the
latter case glyoxylic acid is the chief other product. When it is
heated with dilute mineral acids, with water, or by itself, creatinine
is formed. On boiling with barium hydroxide it forms urea and
sarcosine (Liebig [1847]) and also methyl hydantoin (Neubauer
[1866, l]). Heating with soda lime causes it to give off methyl-
amine.
Physical and chemical properties of creatinine. — Creatinine generally
forms anhydrous monoclinic prisms ; on slow evaporation of a cold
saturated solution it also crystallises with 2H2O in large tables and
prisms, which easily effloresce (Worner [1899]). It is considerably
more soluble in water than creatine, the solubility being i : io§6 at 14°
and I : 1078 at 1 7° (Toppelius and Pommerehne [1896] ; according to
Liebig one part dissolves in 1 1 '5 parts of water at 1 5°). Creatinine
is also more soluble than creatine in cold absolute alcohol, namely
I : 625 (Toppelius and Pommerehne [1896]). In hot alcohol much
more dissolves, but hardly any in ether.
Creatinine solutions have an acrid taste and are hardly alkaline to
litmus. The substance is, however, a stronger base than creatine
(dissociation constant 3-57 x io~n at 40*2°; Wood [1903]) and is
precipitated by phosphomolybdic acid, phosphotungstic acid (limits
I : 12,000 on prolonged standing, according to Hofmeister [1880], and
I : 25,000 according to Demjanowski [1912, under methylguanidine]),
mercuric nitrate, mercuric chloride (l : 3000) and by silver nitrate after
careful addition of ammonia (hence it occurs in the histidine fraction of
bases ; Kutscher [ 1 905 ]). It is not precipitated by potassium tri-iodide.
The reducing properties of creatinine are similar to those of creatine.
On boiling with Fehling's solution the cuprous oxide formed at first
remains dissolved as a compound with unattacked creatinine (Maschke
[1878], Korndorfer [1904, 2]), but after prolonged boiling with
excess of the reagent cuprous oxide separates — creatinine is the chief
cause of the slight action of normal urine on Fehling's solution. Un-
like glucose, creatinine does not reduce alkaline bismuth solutions.
Mercuric oxide, potassium permanganate, lead peroxide and sulphuric
acid oxidise creatinine to methylguanidine and oxalic acid ; Fenton's
reagent produces methylguanidine, formaldehyde, formic, carbonic,
160 THE SIMPLER NATURAL BASES
and glyoxylic acids. On boiling with baryta methylhydantoin re-
sults. Dry distillation of creatinine chloride yields hydrocyanic acid,
pyrrole, and dimethylamine (Engeland [1908, 4]). On standing or
boiling with very dilute alkalies, creatine is formed.
Compounds of creatine. — The nitrate, C4H9O2N3 . HNO3, is less
soluble than the hydrochloride or the sulphate. The compounds
C4H9O2N3 . ZnCl2 and C4H9O2N3 . CdCl2 . 2H2O are crystalline (Neu-
bauer [1862, 2]). All these salts are hydrolysed by water.
Compounds of creatinine. — The hydrochloride, C4H7ON3.HC1,
separates in anhydrous prisms and tables when a solution of creatinine
in hydrochloric acid is evaporated on the water bath ; from cold
solution it crystallises with iH2O. It is not precipitated by zinc
chloride except in the presence of excess of sodium acetate.
Creatinine zinc chloride, (C4H7ON3)2ZnCl2, is the most characteristic
derivative and separates immediately as a micro-crystalline precipitate
on adding a concentrated neutral zinc chloride solution to an alcoholic
or not too dilute aqueous solution of creatinine ; on standing, a dilute
solution deposits needles and prisms. It is soluble in 53*8 parts of
water at 15° and in 2774 at 100°; it is insoluble in absolute alcohol,
readily soluble in hydrochloric acid, from which sodium acetate causes
a double salt of creatinine hydrochloride and zinc chloride C4H7ON3 .
HC1 . ZnCl2 (Neubauer [1861, 2]) to crystallise in long needles, readily
soluble in water. Creatinine zinc chloride dissolves in warm 10 per
cent, sulphuric acid and then the addition of acetone causes the separa-
tion of a double sulphate of zinc and creatinine (C4H7ON3)2H2SO4 .
ZnSO4 . 8H2O (Folin and Blanck [1910]).
Creatinine may be regenerated from its double compounds with
zinc by boiling with freshly precipitated lead hydroxide.
The mercury salt (C4H7ON3 . HC1 . HgO)4sHgCl2 is formed on the
addition of mercuric chloride and sodium acetate to a creatinine solution.
The picrate, C4H7ON3. C6H3O7N3, forms long yellow needles spar-
ingly soluble in cold water ; m.p. 213-214° (Toppelius and Pommerehne
[1896]), 215-217° (Korndorfer [1904, 2]).
Creatinine potassium picrate, C4H7ON3 . C6H3O7N3 . C6H2O7N2K
(formed by saturating urine with picric acid) crystallises in citron
yellow needles or thin prisms, and explodes on rapid heating : 100 c.c. of
water dissolve 0*1806 grm. at 19-20° ; it is also very slightly soluble in
hot alcohol (JarTe [ 1 886]).
An acidpicrate C4H7ON3 . (C6H3O7N3)2, m.p. 161-166°, has been de-
scribed by Mayerhofer [1909].
Creatinine aurichloride, QH7ON8 . HAuCl4, separates in yellow
APPENDIX TO CHAPTER V 161
leaflets on adding a slight excess of gold chloride to a concentrated
solution of creatinine hydrochloride at 40-50° ; the gold salt is readily
soluble in water and in alcohol and, after drying at 100°, melts at
170-174° (Worner [1899]), 182-185° (Korndorfer [1904, 2]).
Creatinine platinichloride, (C4H7ON3)2H2PtCl6, crystallises in orange
red prisms and needles ; from water with 2H2O, from alcohol anhydrous
(Worner [1899]). It is soluble in about 36 parts of water (Top-
pelius and Pommerehne [1896]); hardly soluble in cold alcohol;
m.p. 220-225° on rapid heating.
Creatinine oxime, C4H6O2N4, m.p. 250°, is according to Schmidt
[1912] identical with " nitroso-creatinine" of Kramm.
Colour reactions and estimation of creatine and creatinine. — The only
colour reaction for creatine is the pink coloration produced by diacetyl,
CH3 . CO . CO . CH3 (Harden and Norris [191 1]). This reaction is
also given by arginine and some other guanidine derivatives, but not
by creatinine. Walpole [1911] has used it for the direct estima-
tion of creatine in pathological urines. The usual method, however, is
an indirect one ; the creatine is converted into creatinine by heating
with acids (see above) and then estimated by Folin's method, described
below.
The following are colour reactions for creatinine: —
(a) Weyl's reaction [1878]; a freshly prepared very dilute
solution of sodium nitroprusside is added and then a few drops of
dilute caustic soda. In the presence of creatinine a ruby red colour
is produced ; acetone gives a similar coloration, and if present should
first be boiled off. The red colour due to creatinine is fugitive and
soon changes to yellow ; if then glacial acetic acid is added and the
solution is boiled, it becomes green and on standing a deposit of
Prussian blue is formed (Salkowski [1879]). This reaction is given
by hydantoins but not by creatine, and is still obtainable with pure
creatinine solutions containing 0*03 per cent, and urine containing
O'o66 per cent, of creatinine.
(fr) Jaffe's reaction [1886]. The addition of aqueous picric
acid and a few drops of caustic soda produces in creatinine solutions
an immediate red coloration (orange to blood red). The colour in-
creases during the first few minutes and afterwards fades very slowly.
Limit i : 5000. Acetone gives a somewhat similar but much feebler
reddish yellow coloration, and if present should first be boiled off.
Aceto-acetic ester, hydrogen sulphide and particularly aceto-acetic
acid are the only other pathological substances which may interfere.
According to Chapman [1909] the coloration in Jaffe's reaction
II
1 62 THE SIMPLER NATURAL BASES
is due to the reduction of the picric acid and is also caused by acetone,
acetaldehyde, hydroxylamine and titanium chloride in the cold, and
by dextrose, maltose, laevulose, and urea on warming.
(c) Maschke's reaction [1878]. The creatinine solution is satur-
ated with sodium carbonate ; on warming with Fehling's solution
the blue colour is discharged and a white precipitate of creatinine
cuprous oxide appears, which is readily soluble in water, but only
slightly so in sodium carbonate solution.
Folirts method [1904]. Since the coloration produced by picric
acid and sodium hydroxide gradually fades, a half normal solution of
potassium bichromate (24*54 grm- Per litre) is employed as a permanent
standard of colour; this accurately matches the creatinine coloration.
Since the intensity of coloration is further influenced by dilution, it is
necessary to work within certain limits and the solution to be ex-
amined should contain 7-15 mg. of creatinine in 500 c.c.
Folin adds to 10 c.c. of urine in a 500 c.c. measuring flask 15 c.c.
of saturated (1*2 per cent.) aqueous picric acid solution and 5 c.c. of
10 per cent, sodium hydroxide; after shaking, the solution is allowed
to stand for five minutes to let the colour develop fully, and is then
made up to 500 c.c. The solution thus diluted is now matched with
a column of the 0*5 N bichromate solution 8 mm. high. If the column
of creatinine solution required to do this has a height of x mm. there
are present in the 10 c.c. of urine employed — x 10 mg. of creatinine.
If more than 15 mg. of creatinine is present, only 5 c.c. of urine are
taken, if less than 7 mg. 20 c.c. are employed. For substances which
interfere with the test, see above, under Jaffe's reaction.
According to Thompson, Wallace and Clotworthy [1913] the
maximum colour develops in 5 minutes at 17-20°; at 15-17° seven
minutes are required, at 10-15° eight minutes.
The necessity of a constant temperature has been emphasised by
Mellanby [1908], Chapman [1909] and others. Mellanby has
plotted a curve showing the variation of colour with dilution and
Cook [1909] has suggested a correction for dilution, namely the
addition of 0*19 mg. to the value found for every 10 c.c. of dilution
above the original 10 c.c. ; thus for a 100 c.c. solution 9 xo'19 mg.
should be added. For factors influencing the estimation in urine
consult Taylor [1910] who considers that the variation in the light
and in the pigmentation of the urine constitute the chief sources of
error, and also Thompson, Wallace and Clotworthy [1913]. Under
ideal conditions 10 mg. of creatinine may be estimated to within O'l
APPENDIX TO CHAPTER V 163
mg. ; under bad conditions to within I mg. Weber [1908] puts the
error at 4 per cent. Rona [1910] purifies solutions by means of
colloidal ferric hydroxide, which does not adsorb any creatinine.
The estimation of creatine and creatinine in meat and meat extracts
by Folin's method has been carried out by Baur and Barschall
[1906], Grindley and Woods [1906], Emmett and Grindley [1907],
Chapman [1909] and Cook [1909]. It affords a means of distinc-
tion from the very similar commercial yeast extracts which contain
no creatine or creatinine (at most cro8 per cent).
Chapman [1909], for the estimation of creatine + creatinine,
mixes 10 c.c. of a 10 per cent, meat extract solution with 10 c.c. of
normal hydrochloric acid, and heats to 120° in an autoclave for half
an hour. After cooling to 20°, 30 c.c. of saturated picric acid and
15 c.c. of i o percent, sodium hydroxide are added ; after five minutes
the solution is made up to 500 c.c. and estimated colorimetrically.
For the actual isolation of creatinine from small quantities of ex-
tracts, see Micko [1910].
Glycocyamine and Glycocyamidine,
Glycocyamine, C3H7O2N3, forms anhydrous crystals which gradually
decompose above 220° without melting. At 14*5° I part dissolves
in 218 parts of water (Ramsay). The substance is a stronger base
than creatine (dissociation constant 2*32 x io~n at 40*2°; Wood [1903,
under creatine]) and yields a hydrochloride, C8H7O2N3 . HC1, m.p. 191° ; a
picrate, C3H7O2N3 . C6H3O7N3, m.p. 199-200°, very little soluble in water ;
a readily soluble platinichloride, (C3H7O2N3)2. H2PtCl6. 2H2O, m.p.
198-200°, and an aurichhride, m.p. 173°. Glycocyamine solutions give
with copper acetate a pale blue precipitate (C3H6O2N3)2Cu . H2O,
and with HgCl2 in the presence of sodium acetate a white precipitate ;
no compound with zinc chloride is known.
Glycocyamidine, C3H5ON3> is formed by heating glycocyamine
hydrochloride to 160-170° ; small quantities are more readily prepared
by heating I grm. of this hydrochloride with 5 c.c. of concentrated
hydrochloric acid to 140° in a sealed tube. The free base is obtained
by boiling the resulting hydrochloride with freshly precipitated lead
hydroxide. An alcoholic solution (but not an aqueous solution) of
glycocyamidine hydrochloride gives with alcoholic zinc chloride a
crystalline salt (C3H5ON3)2ZnCl2. The picrate, C3H5ON3 . C6H3O7N3,
forms yellow needles ; m.p. 206-210°. The normal aurichloride is very
soluble and easily changes to the less soluble gold salt C3H6ON3 . AuCl3 ;
m.p. I53-I540 (Korndorfer [1905]).
n *
1 64 THE SIMPLER NATURAL BASES
Glycocyamidine, like creatinine, gives Weyl's and Jaffa's reactions ;
there is, however, this point of difference, that whereas the red or yellow
coloration produced by creatinine, sodium nitroprusside, and caustic
soda is discharged by acetic acid or changed to green on boiling
(formation of Prussian blue), glycocyamidine yields with acetic acid a
stable burgundy red coloration.
Guanidine.
Guanidine is a strong base, absorbing atmospheric carbon dioxide
to form the well crystallised carbonate, (CH5N3)2. H2CO3, soluble in
water but not in alcohol. Of the salts with mineral acids the nitrate
CH5N3 . HNO3 is among the least soluble ; it forms large plates, melt-
ing at 214°.
The picrate> CH5N3 . C6H3O7N3, when pure forms characteristic ir-
regular aggregations of leaflets ; m.p. 315°, on rapid heating up to 320°.
The solubility in cold water is I : 2630 at 9° and the salt may be used
for the estimation of guanidine (Emich [1891]). From complex mix-
tures, particularly when arginine is present, guanidine is not so readily
precipitated by picric acid ; the arginine should first be precipitated
by alcoholic picrolonic acid solution, and then, after removal of the
excess of picrolonic acid from the filtrate, the guanidine may be pre-
cipitated by aqueous picric acid (Kutscher and Otori [1904]).
The picrolonate, CH5N3 . C10H7O5N4, dissolves in excess of alcoholic
picrolonic acid solution (separation from arginine, above). With
aqueous picrolonic acid an amorphous precipitate is formed, which
crystallises from hot water in clusters of thin needles; m.p. 272-274°
(Schenck [1905, 2]).
The aurichloride, CH5N3 . HAuCl4, forms deep yellow needles, little
soluble in water.
With alcoholic cadmiumchloride a double salt CH5N3 . HC1 . 2CdCl2
results; m.p. 390-395° (Schenck [1904]).
Guanidine is precipitated in the " arginine" fraction by silver
nitrate and baryta as a silver compound CH5N3 . Ag2O which may be
crystallised (Kutscher and Otori [1904]). Guanidine salts in con-
centrations down to O'Oi per cent, give a white or pale yellow precipi-
tate with Nessler's reagent ; arginine gives a similar precipitate.
Methylguanidine.
Methylguanidine may be synthesised by heating cyanamide and
methylamine hydrochloride in alcoholic solution to 60-70°. It forms
deliquescent crystals. The nitrate^ C2H7N3 . HNO3, forms rhombic
APPENDIX TO CHAPTER V 165
leaflets, melting at 150° (i$5°), not very soluble in cold alcohol, and
less in water and particularly in dilute nitric acid. The picrate
C2H7N3. C6H3O7N3, m.p. 201-5°, crystallises in two modifications ac-
cording to Gulewitsch [1906] and is more soluble than guani-
dine picrate.
The picrolonate, C2H7N3 . C10H7O5N4, dissolves in 4000 parts of cold
water; m.p. 291° (Wheeler and Jamieson [1907]). The aurichloride
C2H7N3 . HAuCl4, m.p. 198°, is soluble in ether. The platinichloride
(C2H7N3)2. H2PtCl6 forms monoclinic prisms and dissolves in 1 4-3 parts
of water at 18-19°.
Benzene-sulphonyl-methyl-guanidine, C2H6N8 . SO2 . C6H5, m.p. 184°,
soluble in 2500 parts of cold water, is suitable for the isolation (Acker-
mann [1906]). Aqueous mercuric chloride does not precipitate the
nitrate of methylguanidine even in 5 per cent solution ; mercuric
sulphate precipitates a I per cent, solution, phosphotungstic acid a
solution of i : 9000 (Demjanowski [1912]).
Dimethylguanidine-
The aurichloride, C3H9N3 . HAuCl4, melts at 144°, decomposes at
150° and forms thin leaflets or plates.
IL\\Q ptcrolonate, C3H9N3 . C10H7O5N4, m.p. 275-278°, was probably
obtained from human urine by Kutscher and Lohmann [1906, 3, 4]
and forms four-sided prisms. The picrate, C3H9N3 . C6H3O7N3, forms
small pointed needles or branch-like growths ; m.p. 224° (Wheeler and
Jamieson [1907]).
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ABDERHALDEN, E., und A. FODOR (1913). Versuche uber die bei der Fdulnis von l-Aspara-
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ABDERHALDEN, E., und A. SCHITTENHELM (1907). Studien uber den Abbau raccmischer
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Zeitschr. physiol. Chem., 51, 323-33.
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ACKERMANN, D. (1910, 3). Uebcr ein neues, auf baktericllem Wegegewinnbares Aporrhegma.
Zeitschr. physiol. Chem., 69, 273-81.
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Zeitschr. Biol., 57, 104-11.
ACKERMANN, D. (1911, l). Ueber das &-Alanin als bakterielles Aporrhegma.
Zeitschr. Biol., 56, 87-90.
ENGELAND, R. (1908, i). Ueber Liebig's Fleischextract.
Zeitschr. Unters. Nahr. Genussm., 16, 658-64.
FISCHER, E., und G. ZEMPLEN (1909). Neue Synthese von Amino-oxysduren und von
Piperidinderivaten.
Ber. deutsch. chem. Gesellsch., 42, 4878-92.
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Beitr. chem. Physiol. Pathol., 7, 144-47.
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Ber. deutsch. chem. Gesellsch., 42, 3S4-76-
MICRO, K. (1905). Hydrolyse des Fleischextraktes.
Zeitschr. Unters. Nahr. Genussm., 10, 393-415.
NEUBERG, C. (1911, i). Biochemische Umwandlung von a-Pyrrolidincarbonsdure in
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NEUBERG, C. (1911, 2). Wird d. Ornithin bei der Fdulnis racemisiert ?
Biochem. Zeitschr., 37, 507-9.
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Ber. deutsch. chem. Gesellsch., 16, 1191-95.
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Ber. deutsch. chem. Gesellsch., 31, 776'83-
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Ber, deutsch, chem. Gesellsch., 17, 2544-47.
176
BIBLIOGRAPHY OF CHAPTER II 177
CARNOSINE.
GULEWITSCH, WL., und S. AMIRADZ!BI (1900, i). Zur Kenntniss der Extraktivstoffe der
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GULEWITSCH, WL., und S. AMIRADZIBI (1900, 2). Ueber das Carnosin, eine neue organische
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GULEWITSCH, WL. (1913). Zur Kenntniss der Extraktivstoffe der Muskeln. XIV. Ueber
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KRIMBERG, R. (1906, i). Zur Kenntniss der Extraktivstoffe der Muskeln. IV. Uber das
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KUTSCHER, F. (1905). Ueber Liebig's Fleischextract.
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UROCANIC ACID.
BARGER, G., and A. J. EWINS (1911). The constitution of Ergothioneine, a betaine related
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HUNTER, A. (1912). On urocanic acid.
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KYNURENIC ACID.
ABDERHALDEN, E., E. S. LONDON, und L. PINCUSSOHN (1909). Ueber den Ort der
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BRIEGER, L. (1879). Zur Kenntniss der Kynurensdure.
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CAMPS, R. (1901, 2). Von der Amido-phenylpropionsdure zur Kynurensdure und deren
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12
1 78 THE SIMPLER NATURAL BASES
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Monatsh., 2, 57-85.
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LIEBIG, J. (1853). Ueber Kynurensaure.
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Monatsh., 15, 453-68.
REFERENCES TO CHAPTER III.
BETAINE (TRIMETHYLGLYCINE).
ACKERMANN, D., und F. KUTSCHER (1907, 1-4). Ueber Krabbenextrakt I.-IV.
Zeitschr. Unters. Nahr. Genussm., 13, 180-84, 610-13, 613-14; 14, 687-91.
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ANDRLIK, K., A. VELICH, und VL. STANEK (1902-3). Ueber Betain in physiologisch-
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BEBESCHIN, K. (1911). Zur Kenntniss der Extraktivstoffe der Ochsennieren.
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DELEANO, N. T., und G. TRIER (1912). Ueber das Vorkommen von Betain in griinen
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Zeitschr. physiol. Chem., 79, 243-46.
EHRLICH, F. (1912). Uber die Gewinnung von Betainhydrochlorid aus Melasse. Schlempe.
Ber. deutsch. chem. Gesellsch., 45, 2409-13.
EHRLICH, F., und F. LANGE (1913). Uber die biochemische Umwandlung vcn Betain in
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Ber. deutsch. chem. Gesellsch., 46, 2746-52.
EWINS, A. J. (1912). The constitution and synthesis of damascenine, the alkaloid of
Nigella damascena.
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FISCHER, E. (1902). Ueber Betainchloraurat,
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GRIESS, P. (1875). Ueber eine neue Synthese des Betains (Oxyneurin).
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HENZE, M. (1910). Ueber das Vorkommen des Betains bei Cephalopoden.
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HUSEMANN, A. (1875). Identitdt der PJlanzenbasen Lycin und Betain.
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HUSEMANN, A., und W. MARM£ (1863). Vorlaufege Mittheilung uber Lycin, ein neues
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Liebig's Annalen, II Supplementsb., 383-87.
HUSEMANN, A., und W. MARM£ (1864). Ueber Lycin.
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KOHLRAUSCH, A. (1909). Ueber das Verhalten von Betain, Methylpyridyl-am-
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KOHLRAUSCH, A. (1911). Untersuchungen uber das Verhalten von Betain, Trigonellin
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KUTSCHER, FR. (1909). Notiz zu der Arbeit der Herren U. Suzuki und K. Joshimura:
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KUTSCHER, FR. (1910, 3). Ueber einige Extraktivstoffe.
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179
12 *
i8o THE SIMPLER NATURAL BASES
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Ber. deutsch. chem. Gesellsch., 3, 161-63.
RITTHAUSEN, H., und F. WEGER (1884). Ueber Betain aus Pressriickstanden der
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J. prakt. Chem., [ii.j, 30, 32-37-
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SCHULZE, E., und N. CASTORO (1904). Beitrage zur Kenntnis der in ungekeimten
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BIBLIOGRAPHY OF CHAPTER III 181
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STACHYDRINE.
ENGELAND, R. (1909, 2). Ueber Hydrolyse von Casein und den Nachweis der dabei
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BETONICINE AND TURICINE.
KUNG, A. (1913). Die Synthese des Betonicins und Turicins.
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1 82 THE SIMPLER NATURAL BASES
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HYPAPHORINE.
GRESHOFF, M. (1898). Mededeelingen uit 's Lands Plantentuin.
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TRIGONELLINE.
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Ber. deutsch. chem. Gesellsch., 19, 31-40.
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OTHER PYRIDINE BASES.
ACHELIS, W., und FR. KUTSCHER (1907). Der Nachweis organischer Basen im Pferde-
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7-BUTYROBETAINE AND CARN1TINE.
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BIBLIOGRAPHY OF CHAPTER III 183
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MYOKYNINE.
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CHOLINE.
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184
BIBLIOGRAPHY OF CHAPTER IV 185
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1 86 THE SIMPLER NATURAL BASES
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BIBLIOGRAPHY OF CHAPTER IV 187
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1 88 THE SIMPLER NATURAL BASES
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Pfliiger's Archiv, 128, 142-44.
MUSCARINE.
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190 THE SIMPLER NATURAL BASES
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TRIMETHYLAMINE-OXIDE.
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HOMOCHOLINE AND NEOSINE.
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A. CREATINE AND CREATININE.
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DAKIN, H. D. (1906). The formation of glyoxylic acid.
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191
192 THE SIMPLER NATURAL BASES
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13
194 THE SIMPLER NATURAL BASES
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B. GLYOCYAMINE AND GLYCOCYAMIDINE.
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ADRENALINE.
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J. Pharm. Exp. Therap., 4, 59-74.
JANUSCHKE, H., und LEO POLLAK (1911). Zur Pharmakologie der Bronchialmusku-
latur.
Arch. exp. Path. Pharm., 66, 205-20.
JOWETT, H. A. D. (1904). The constitution of epinephrine.
]. Chem. Soc., 85, 192-97.
KEHRER, E. (1907). Physiologische und pharmakologische Untersuchungen an den
uberlebenden und den lebenden inneren Genitalien.
Arch. f. Gynakologie, 8l, 160-210.
KEHRER, E. (1908). Der uberlebende Uterus als Testobjekt fur die Wertigkeit der
Mutterkornprdparate.
Arch. exp. Path. Pharm., 58, 366-85.
KRAUSS, L. (1909). Die Jodsaurereaktion des Adrenalins.
Biochem. Zeitschr., 22, 131.
KRETSCHMER, W. (1907). Dauernde Blutdmcksieigerung durch Adrenalin und uber den
Wirkungsmechanismus des Adrenalins.
Arch. exp. Path. Pharm., 57, 423-40.
KRUKENBERG, C. FR. W. (1885). Die farbigen Derivate der Nebennierenchromogene.
Virchow's Archiv f. path. Anat. u. Physiol., 101, 542-61.
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Arch. exp. Path. Pharm., 51, 415-40.
LANGLEY, J. N. (1901). Observations on the physiological action of extracts of the
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J. Physiol., 27, 237-56.
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Compt. rend. Soc. de Biol., 56, 632-34 ; . . . pour le chat, ibid., 56, 665-66.
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Zentralbl. f. Physiol., 14, 433-35.
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BIBLIOGRAPHY OF CHAPTER VI 205
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206 THE SIMPLER NATURAL BASES
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Proc. Physiol. Soc., 14 Dec., xxix.-xxx. ; J. Physiol., 27.
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Therapeutic Gazette, 25, 221-24.
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Compt. rend. Soc. de Biol., 3, 223.
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Pfluger's Archiv, 103, 510-14.
BIBLIOGRAPHY OF CHAPTER VI 207
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REFERENCES TO CHAPTER VII.
SPERMINE.
LADENBURG, A., und J. ABEL (1888). Ueber das Aethylenimin (Spermin ?).
Ber. d. deutsch. chem. Gesellsch., 21, 758-66.
MAJERT, W., und A. SCHMIDT (1890). Ueber das Piperazin (Hofmann's Didthylendiamin*
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Ber. d. deutsch. chem. Gesellsch., 23, 3718-23.
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Ber. d. deutsch. chem. Gesellsch., 24, 359-60.
SCHREINER, P. (1878). Ueber eine neue organische Basis in thierischen Organismen.
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MUSCLE-, URINE-, AND PUTREFACTION BASES.
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Zeitschr. physiol. Chem., 57, 28-29.
ACKERMANN, D. (1907, 2). Bin Beitrag zur Chemie der Fdulnis.
Zeitschr. physiol. Chem., 54, 1-31.
BAUM, FR. (1903). Ueber ein neues Produkt der Pankreasselbstverdauung.
Beitr. chem. Phys. Path., 3, 439-41.
KRIMBERG, R., und L. IZRAILSKY (1913). Zur Kenntniss der Extraktivstoffe der Muskeln*
Uber das Kreatosin, eine neue Base des Fleischextraktes.
Zeitschr. physiol. Chem., 88, 324-30.
KUTSCHER, FR. (1906). Bemerkungen zu unserer ersten Mitteilung : Der Nachweis toxischer
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KUTSCHER, FR. (1907). Zur Kenntniss von Liebig's Fleischextract II.
Zentralbl. f. Physiol., 21, 33-35.
SWAIN, R. E. (1903). Weiteres uber Skatosin.
Beitr. chem. Physiol. Path., 3, 442-45.
THE PITUITARY ACTIVE PRINCIPLE.
BELL, W. BLAIR (1909). The pituitary body and the therapeutic value of the infundi-
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Brit. Med. J., ii., 1609-13.
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Biochem. J., 4, 427-47.
DALE, H. H., and P. P. LAIDLAW (1912, i). A method of standardising pituitary (infundi-
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J. Pharm. Exp.Therap., 4, 75-95.
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Zeit. f. Biol., 57, 526-33.
FRANKL-HOCHWART, L. VON, und A. FROHLiCH (1910). Z ' ur Kenntniss der Wirkung des
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Nervensystem.
Arch. exp. Path. Pharm., 63, 347-56.
FROHLICH, A., UND E. P. PICK (1913). Zur Kenntniss der Wirkung der Hypophysen-
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Arch. exp. Path. Pharm., 74, 92-106, 107-13, 114-18.
Fi?HNER, H. (1912). Das Pituitrin und seine wirksame Bestandteile.
Munch, med. Wochenschr., 59, 852-53.
208
BIBLIOGRAPHY OF CHAPTER VII 209
FtJHNER, H. (1913). Pharmakologische Untersuchungen uber die wirksamen Bestandteile
der Hypophyse.
Zeitschr. f. d. ges. exp. Medizin., i, 397-443.
GUGGENHEIM, M. (1913). Proteinogene Amine. Peptamine : Glycyl-p-oxyphenylathylamin,
Alanyl-p-oxypheny lathy lamin. Glycyl-&-imidazoly lathy lamin.
Biochem. Zeitschr., 51, 369-87.
HAMMOND, J. (1913). The effect of pituitary extract on the secretion of milk.
Quart. Journ. exp. Physiol., 6, 311-38.
HERZBERG, S. (1913). Klinische Versuche mit den isolierten wirksamen Substanzen der
Hypophyse.
Deutsch. med. Wochenschr., 39, 207-10.
HOUGHTON, E. M., and C. H. MERRILL (1908). The diuretic action of adrenalin and the
active principle of the pituitary gland.
J. Amer. Med. Assoc., 51, 1849-54.
MAGNUS, R., and E. A. SCHAFER (igoi). The action of pituitary extracts upon the kidney.
Proc. Physiol. Soc., 20 July; J. Physiol., 27, ix.
OLIVER, G., and E. A. SCHAFER (1895, 3)* On the physiological action of extracts of the
pituitary body and certain other glandular organs.
J. Physiol., 18, 277-79.
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mammary gland,
Therap. Gazette, 35, 689-91.
PANKOW, O. (1912). Ueber Wirkungen des Pituitrins (Parke, Dames &> Co.), auf KreislauJ
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Pfluger's Archiv, 147, 89-99.
PATON, D. N., and A. WATSON (1912). The actions of pituitrin, adrenalin, and barium on
the circulation of the bird.
J. Physiol., 44, 413-24.
SCHAFER, E. A. (1913). On the effect of pituitary and corpus luteum extracts on the
mammary gland in the human subject.
Quart. J. exp. Physiol., 6, 17-19.
SCHAFER, E. A., and P. T. HERRING (1906). The action of pituitary extracts upon the kidney.
Phil. Trans. Roy. Soc., 199, B, 1-29.
SCHAFER, E. A., and K. MACKENZIE (1911). The action of animal extracts on milk secretion.
Proc. Roy. Soc., 84, 16-22.
VITAMINE, ORYZANIN, TORULIN.
COOPER, E. A. (1913). Tnc preparation from animal tissues of a substance which cures
polyneuritis in birds induced by diets of polished rice.
Biochem. J., 7, 268-74.
EYKMAN, C. (1897). Eine Beri Beri-ahnliche Krankheit der Huhner.
Virchow's Archiv, 148, 523-32.
EDIE, E. S., W. H. EVANS, B. MOORE, G. C. E. SIMPSON, and A. WEBSTER (1912). The
antineuritic bases of vegetable origin in relationship to beri-beri, with a method of
isolating torulin, the antineuritic base of yeast.
Biochem. J., 6, 234-42.
FUNK, C. (1911). On the chemical nature of the substance which cures polyneuritis in
birds induced by a diet of polished rice.
J. Physiol., 43, 395-400.
FUNK, C. (1912, i). The preparation from yeast and certain foodstuffs of the substance the
deficiency of which in diet occasions polyneuritis in birds.
J. Physiol., 45, 75-81.
FUNK, C. (1912, 2). Further experimental stiidies on beri-beri. The action of certain
purine and pyrimidine derivatives.
J. Physiol., 45, 489-92.
FUNK, C. (1913). Studies on beri-beri. VII. Chemistry of the vitamine fraction from
yeast and rice polishings.
J. Physiol., 46, 173-79-
HOPKINS, F. G. (1912). Feeding experiments illustrating the importance of accessory
factors in normal dietaries.
J. Physiol., 44, 425-60.
14
210 THE SIMPLER NATURAL BASES
SCHAUMANN, H. (1912, i). Ueber die Darstellung und Wirkungsweise einer der in der
Reiskleie enthaltenen, gegen experimentelle Polyneuritis wirksamen Substanzen.
Arch. f. Schiffs- und Tropenhygiene, 16, 349-61.
SCHAUMANN, H. (1912, 2). Zu dem Problem der Beri-beri Atiologie.
Arch. f. Schiffs- und Tropenhygiene, 16, 825-37.
SUZUKI, U., T. SHIMAMURA, und S. ODAKE (1912). Ueber Oryzanin, ein Bestandteil der
Reiskleie und seine physiologische Bedeutung.
Biochem. Zeitschr., 43, 89-153.
SEPSINE.
FAUST, E. S. (1903, 1904). Ueber das Faulnisgift Sepsin.
Arch. exp. Path. Pharm., 51, 248-69.
FORNET, W., und W. HEUBNER (1908, 1911). Versuche iiber die Entstehung des Sepsins.
Arch. exp. Path. Pharm., Schmiedeberg Festschrift, 176-80, and 65, 428-53.
SECRETINE.
DALE, H. H., and P. P. LAIDLAW (1912, 3). A method of preparing secretin.
Proc. Physiol. Soc., 18 May; J. Physiol., 44, xi.-xii.
REFERENCES TO CHAPTER VIII.
BUSCH, M. (1905). Gravimetrische Bestimmung der Salpetersaure.
Ber. deutsch. chem. Gesellsch., 38, 861-66.
JACOBS, W. A. (1912). A note on the removal of phosphotungstic acid from aqueous solutions.
J. Biol. Chem., 12, 429-30.
KOSSEL, A., und F. WEISS (1910). Vber die Einwirkung von Alkalien auf Proteinstoffe.
Zeitsch. physiol. Chem., 68, 165-69.
E. WECHSLER (1911). Zur Technik der Phosphorwolframsdurefdllungen.
Zeitschr. physiol. Chem., 73, 138-43.
211 14*
INDEX.
ACETYL choline in ergot, 63.
Acids produced in putrefaction, 8.
Addison's disease, 81, 89.
Adrenal gland, see Suprarenal Gland.
Adrenaline, 81-105.
Agmatine, 16, 29, 129.
/3-Alanine, 34, 36, 135.
Alkaloid, definition of, 6.
Amanitine, 65.
Amphicreatinine, 70.
Amino-acids, behaviour of, in putrefaction,
7-io, 33.
7-Amino-butyric acid, 34, 135.
e-Amino-caproic acid, 35.
Amino-ethyl alcohol, 58, 59, 155.
disulphide, 13.
glyoxaline, see Iminazolyl-ethyl-
amine.
indole, see Indolethylamine.
8 -Ami no- valeric acid, 35, 136.
Amylamines, 13, 126.
Anaphylactic shock, 30, 31, 80.
Aporrhegmata, 3.
Arginine fraction of bases, 121.
Arterenol, 86, 87, 91.
Arteriosclerosis, 27, 97.
Aurichlorides of abnormal composition, 123,
137, 143, 145, 147.
Autolysis, difficulty of securing sterility in,
io, 15, 77.
BACILLUS aminophilusintestinalis,?, 25, 133.
— botulinus, 5.
— liquefaciens, 12.
— putrificus, 8.
— vulgatus, 12.
Bacterium prodigiosum, 11, 12.
— sepsinogenes, 114.
Base, definition of, 5.
Betaine, 12, 40-43, 77, 78, 141-143, 150-152.
Betaines, general properties of, 39, 40.
Betonicine, 44, 144.
Bilineurine, 54.
Blood pressure, action of adrenaline on, 96,
97, 98, 102.
of amines on, 26-32.
— — — of choline and neurine on, 62-64.
of pituitary extracts on, no.
— — persistent high, 25, 27.
Botelus edulis, bases in, io, 13, 15, 19, 45.
Bronchioles, affected by adrenaline, 99.
— affected by j8-iminazolyl-ethylamine, 29
32.
Bufo agua, adrenaline in, 94.
Butylamine, 12, 126.
7-Butyrobetaine, 39, 49, 147, 148.
CADAVERINE, 14-16, 126-128.
Carnitine, 50, 51, 148, 149.
Carnosine, 36, 137, 138.
'.phalopoda, p-hydroxy-phenyl-ethylamine
in salivary gland of, 20, 28.
— creatine absent from, 71.
— creatinine absent from, 72.
Cerebro-spinal fluid, alleged choline content
of, in disease, 56.
Cheese, bases in, 16, 19.
Choline, n, 12, 54-64, 78, 150-155.
— physiological action, 61-63.
— of acetic acid ester, 63, 68.
— of nitrous acid ester, 68.
— nitric acid ester, 68, 153, 156.
Chromaffin or chromophil tissue, 94.
Chromogen of suprarenal gland, 81.
Chrysocreatinine, 70.
Cod liver oil, bases in, 12, 13, 18.
Colamine, 58.
Collidine, 17.
Crangitine, 107.
Crangonine, 107.
Creatine and creatinine, 69-78, 157-163.
Creatosine, 107.
Curare action, 49, 65.
Cyclic vomiting, 25.
Cystine, amine from, 13.
Cystinuria, 15.
DEAMINIZATION, 8, 33, 35.
Decarboxylation, conditions favouring, 7, 9,
12, 14, 16, 25.
— by bacteria, 7, 8, io.
— by ferments, io.
Dimethylamine, n, 124, 125.
Dimethylguanidine, 80, 165.
Dissociation constant, of amino-acids, 33.
of creatine, 158.
of creatinine, 159.
Dragendorff's reagent, 121.
EPINEPHRIN, see Adrenaline, 81.
Epinephrin hydrate, 82, 83.
Epinine, 87, 91.
Ergamine, see #-iminazolyl-ethylamine.
Ergot, io, 13, 15, 16, 19, 28, 46, 63.
Ergotoxine, cause of vaso-motor reversal, 98.
Estimation of adrenaline, colorimetri , 92.
physiological, 101-105.
— of amino- ethyl alcohol, 58.
— of betaine in the presence of choline, 150,
151-
in crude sugar and molasses, 141.
— in plants, 141.
— of carnosine, 138.
213
2I4
THE SIMPLER NATURAL BASES
Estimation of choline, 150, 151.
— of creatine directly, 161.
indirectly, as creatinine, 163.
— of creatinine, 70, 161-163.
— of guanidine, 164.
— of kynurenic acid, 140.
— of the methylamines, 124, 125.
— of the pituitary active principles, HI.
— of trigonelline, 147, 151.
Ethylamine, 11.
GADININE, 50.
Germination, formation of betaine, 43, 55.
of choline, 55 ; of guanidine, 79.
of hordenine, 21.
— absence of primary amines, 15, 18.
Glycocyamine, 78, 79, 163, 164.
Glycocyamidine, 78, 79, 163, 164.
Guanidine, 79, 164.
Gynesine, 107.
HERCYNINE, 45.
Herring spawn, 16.
Histamine, see Iminazolyl-ethylamine.
Histidine, decarboxylation of, by bacteria,
132, 133-
— formation of, from carnosine, 36.
— fraction of bases, 121.
— in human urine, 37.
— lower homologue of, in human urine, 37.
— polypeptide of, in human urine, 37.
— preparation of, from blood, 119.
Homobetaine, 51, 68.
Homocholine, 68.
Homomuscarine, 67.
Homorenon, 87, 91.
Hordenine, 20, 21, 131.
— physiological action, 28.
p-Hydroxy-phenyl-ethylamine, 18-20, 130-
131-
— physiological, action, 26-28.
p-Hydroxy-phenylacetic acid, 27.
Hypaphorine, 47, 146.
Hypophysis cerebri, 108.
IGNOTINE, see Carnosine.
Imidazolyl-ethylamine, see Iminazolyl-ethyl-
amine.
Iminazolyl-acrylic acid, 36, 46, 138, 139.
Iminazolyl-ethylamine, 22-24, 132-134.
— physiological action, 29-32.
Iminazolyl-methylamine, 24.
Iminazolyl-propionic acid, 35, 137.
Indolaceturic acid, 29.
Indolethylamine, 21, 22, 132.
— physiological action, 28, 29.
Iso-amylamine, 13, 126.
— physiological action, 26.
Isobutylamine, 12, 126.
Isocreatinine, 70.
KRAUT'S reagent, 121.
Kynosine, 107.
Kynurenic acid, 37, 139, 140.
Kynurine, 37.
LEUCOMAINES, 6.
Lycine, 40.
Lysine, destructive distillation of, 128.
— fraction of bases, 121.
— in cystinuric urine, 15.
— in putrefaction, 14, 35.
MARCITINE, 108.
Meat, bases in, 107.
Mercuric chloride, use in the isolation of
bases, 49, 113, 114, 119, 145, 150, 158.
Metchnikoffs sour milk treatment, 25.
Methylamine, n, 124, 125.
— formation from choline by putrefaction,
153.
Methylation by the animal organism, 48, 49,
77, 78, 79-
Methylguanidine, 69, 79, 159, 164, 165.
Methylhydantoin, 159, 160.
Methylpyridinium hydroxide, 48, 49, 61.
Methylpyrroline, 13.
Mingine, 107.
Muscarine, 64-67, 68.
Muscle, bases in, 107.
Mydine, 19.
Myokynine, 52.
NEOSINE, 68.
Neurine, 54, 60, 61, 155, 156.
— physiological action, 64.
Nicotinic acid, 48, 112.
Nitric acid ester of choline, 153, 156.
Nitrosocholine, nitrous acid ester of choline,
63, 68.
Novaine, see Carnitine, 50, 149.
DBLITINE, 51, 148, 149.
Ornithine, behaviour in putrefaction, 14, 35.
— methylation, 52.
Oryzanine, 112.
Ox-ethylamine, 58, 60.
Oxyneurine, 40.
Oxyproline, 44, 45.
PARAGANGLION aorticum, 93.
Pentamethylene diamine, see Cadaverine.
Peptamines, no.
Periodides, 122, 125. 142, 145, 151, 152 155.
Phenyl-ethylamme, 16-18, 129.
— physiological action, 26.
Phosphotungstic acid, 6, 118, 119, 150.
•y-Picoline, 49.
Picric acid, 123.
Picrolonic acid, 123.
Pituitary active principle, 108-111.
Placental extracts, supposed activity of, 19.
Potassium bismuth iodide, 121, 122.
— tri-iodide, 121, 122.
Preparation of bases, general methods, 116-
123.
special methods, 84, 106, 109, 113,
114, 124-165.
Proline, 13, 33, 35, 44.
Proteus vulgaris, 12, 25.
Proto-alkaloids, 13.
Pseudo-muscarine, 63, 65-67, 68.
Ptomaines, 2, 5, 6, see also Putrefaction Bases.
Putrefaction, 7-9, and Ch. I ; 33-35, 61, 67.
— bases, Ch. I; 33-35, 49, 50, 54, 61, 67, 79,
108, 113.
INDEX
215
Putrescine, 14-16, 126, 127.
Putrine, 108.
Pyridine bases, 17, 48, 49.
Pyrrolidine, 13.
REDUCTION by putrefaction, 8, n, 33, 67,
153-
Reductonovaine, 51.
SALIVARY gland, action of adrenaline, 99.
— of -y-butyrobetaine, 50.
of choline, 63.
of p-hydroxyphenyl-ethylamine,
27.
of j8-iminazolyl-ethylamine, 32.
of muscarine, 66.
secretes p-hydroxy-phenyl-ethylamine
in Cephalopoda, 20, 28.
Sarcosine, 69, 78, 159.
Secretine, 114.
Sepsine, 113.
Silver nitrate method of separating bases,
120, 121.
Sinkaline, 54.
Skatosine, 108.
Spermine, 106.
Stachydrine, 43, 44, 143, 144.
Streptococcus, production of amines by, n,
J7-
Suprarenal gland, 81, 82.
— adrenaline content of, 92-95.
Suprarenin, 82-84 ; see Adrenaline.
Sympathomimetic action, 26, 98.
Synthetic amines, physiological action of,
26, 28, 87.
TANNIN method for purifying extracts con-
taining bases, 117.
Tetramethylene diamine, see Putrescine.
Tetramethyl putrescine, 16, 129.
Toruline, 112.
Toxins of bacteria, 4-6.
Trigonelline, 47, 48, 147, 150, 151.
Trimethylamine, n, 12, 41, 124, 125.
— oxide, 67.
Trimethylhistidine, 45, 46, 144, 145.
Trimethyltryptophane, 47, 146.
Tryptophane, bases from, 21, 22, 47.
Turicine, 45, 144.
Typhotoxine, 50.
Tyramine, see p-Hydroxy-phenyl-ethylamine.
Tyrosamines, 18.
Tyrosol, 131.
URINE, adrenaline in, 89.
— list of bases from, 107.
Urocanic acid, 36, 138, 139.
Urohypertensine, 27.
Urohypotensine, 30.
Uterus, action of adrenaline, 97, 103, 104.
of agmatine, 29.
of p-hydroxy-phenyl-ethylamine, 27.
of j8-iminazolyl-ethylamine, 29.
of pituitary, in.
VASO-DILATIN, 30.
Vaso-motor reversal, 63, 98.
Viridine, 108.
Vitamine, 111-113.
Vitiatine, 107.
XANTHOCREATININE, 70.
YEAST, action on amines, 25, 131,
betaine, 43.
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