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DEFENSIVE FERMENTS OF THE
ANIMAL ORGANISM

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DEFENSIVE FERMENTS OF
THE ANIMAL ORGANISM

against substances out of harmony with the body, the
blood-plasma and the cells ; their demonstration, and
their diagnostic significance for testing the functions

of different organs

BY

EMIL ABDERHALDEN

Director of the Physiological Institute of the University at Halle a\S.

With ii Text Figures and one Plate

THIRD ENLARGED EDITION

ENGLISH TRANSLATION BY

J. O. GAYRONSKY, L.R.C.P., M.R.C.S., M.D., Halle a/S.

AND

\Y. F. LANCHESTER, M.A.

LONDON :

JOHN BALE, SONS & DANIELSSON, LTD.

OXFORD HOUSE

83-91, GREAT TITCHFIELD STREET, OXFORD STREET, W.

1914
All Rights Reserved

TO
MY FAITHFUL COLLABORATORS.

Preface to the English Edition.

IT is more than three years since Abderhalden first
published his results in regard to the sero-diagnosis
of pregnancy. In the course of his general studies
on the nature and properties of the ferments in the
blood, and on their relations to metabolism, he came
across an instance of their specific action, which
suggested the possibility of diagnosing the condition
of pregnancy by their means. And it was on the
basis of the same methods as had been employed
years before by himself and his followers in their
preliminary theoretical investigations, that he was
led to the great discovery of the demonstration of
specific ferments in the blood-serum ; that is to say,
by the use of the optical method and of the dialysation
process.

In view of the possibility of the practical appli-
cation, in medicine, of these new methods of research,
for the purpose of making differential diagnoses and
of testing the functions of organs in various diseases,
they have been taken up by many members of the
medical profession. They were first employed by the
gynaecologist ; but there is now hardly any branch
of medicine left in which the application of these new
methods has not been attempted, and in the course

Viii PREFACE TO THE ENGLISH EDITION

of time interest in these new weapons of research
has rapidly increased. Much has been done, for the
popularization of these methods, by the kindness
which has been shown by Abderhalden and his
assistants to all those who were willing to acquaint
themselves with the rather complicated technique
involved in them. He found room in his institute
for all who wanted to come; every written inquiry
was promptly answered ; and reagents, such as
placenta-albumen and peptone, in the preparation of
which some difficulty is met with, were freely sup-
plied from his laboratory.

The conception of " harmony and disharmony '
has been employed by us, in order to represent the
meaning we attach to Abderhalden 's terms " fremd '
and ' eigen.' These phrases, though they have
been translated literally by some, do not seem to us
to be amenable to direct translation.

In presenting this translation of the latest edition
of Abderhalden 's work on defensive ferments, I
have been inspired by the hope of being able to excite
or further, in regard to this important line of modern
research, the interest of many to whom the German
text may be inaccessible.

J. O. GAVRONSKY.

7, Cambridge Terrace,

Regent's Park.
February 23, 1914.

Preface to the Third Edition.

IT took less than three months for the Second
Edition to be exhausted, a pleasing sign that this new
field of research has excited much interest. The
number of works which have been completed on the
basis of the principles there laid down, and of the
methods there disclosed, exceeds one hundred and
twenty ! Every week brings forth new works. I am
not sure whether that ought to give me entirely un-
broken satisfaction. The fundamental works, which
have arrived at a definite conclusion in regard to the
elaboration of the dialysation process and of the
optical method, have been produced during the last
twelve years or so. The " theoretical : part, which
pointed to the possibility of a sero-diagnosis of the
functions of organs, was practically established six
years ago. Experiments on animals were started
on a large scale, so as to allow for all possibilities.
Over and over again doubts cropped up which had
to be settled. The astonishing result was found that,
in disturbances of certain organs, only their albu-
minous constituents suffer decomposition. These
discoveries were not made public, and only those
results, which were established in investigations on

X PREFACE TO THE THIRD EDITION

pregnancy, were published. Pregnancy is a con-
dition which allows of no misinterpretation. In
almost every case the clinical diagnosis can be
compared, with absolute certainty, with the result of
the serological diagnosis. The actual diagnosis
either corresponds with the former, or it does not.
These clear conditions, however, are not presented by
the other morbid processes. A certain disease may
be accompanied by all kinds of other disturbances of
the functions of the organ. Very seldom are we faced
with the presence of " pure " disease. Therefore, we
are bound to conclude that only the worker in a
hospital is in a position to judge, to what extent sero-
logical investigations can be applied for testing the
functions of an organ. In this case two aims have to
be distinguished. The serological diagnosis can, in
many cases, widen our understanding of the disturb-
ances occurring in a given disease. We gain an in-
sight into long suspected functional troubles of certain
organs, or discover that others, which had never
been thought of, regularly produce disturbances in
a certain disease. It is an entirely different question
to ask whether the serological diagnosis of an organ
can be applied to differential diagnosis, i.e., whether
we are entitled to accord a preference to this, as
against any other, method.

Many years may be required before the question
of the practical value of the methods worked out can

PREFACE TO THE THIRD EDITION XI

be decided for each separate case. Every research,
which has not been carried out with absolutely
unobjectionable technique, delays our arrival at a
clear appreciation of the suitability of the methods.
There are practically no methods which, on first
acquaintance, will lead anyone to good results.
Often weeks have to be spent in preliminary
studies, before facts are acquired which entitle us to
apply the required methods to certain questions. Xo
conscientious student would publish these preliminary
studies, but would treat them as exercises. Owing
to a very extensive experience of my own, I cannot
deny that many preliminary studies of this kind have
been published. It is only work that is deliberate,
and that is based on a complete command of methods,
that can lead to satisfactory results. It is, besides,
the duty of the clinical worker to thoroughly study
each case, and to follow it out to the end.

As yet, it is too early to criticize the works that
have appeared, and I have contented myself merely
with summarizing such as have come to my notice.
Then the results of recent experimental researches
have been referred to. The question of the specificity
of the substrates is discussed, and, finally, in the
description of the technique, some recent experiences
have been considered.

EMIL ABDERHALDEN.

November, 1913.

Preface to the Second Edition.

THOUGH scarcely a year has elapsed since the
publication of the First Edition, yet it has been
possible to widen the scope of the Second in many
points. Since then, numerous investigations in
different fields have been either begun or completed.
The most important results of these investigations
are given at the end of this little volume.

The term " protective ferment ' has been dropped,
because it may easily convey the idea that these
ferments, which are called into action by the presence
of substances out of harmony with the plasma, are
unconditionally protective. The term " defensive
ferment will more readily suggest the notion, that
the animal organism attempts to defend itself. By
means of decomposition it often deprives dishar-
monious substances of their specific character, but in
many cases the defensive ferments form decom-
position stages which are more dangerous than the
substrates they attack.

May this new edition find the same friendly
welcome as the first.

EMIL ABDERHALDEN.

Halle a/S.,

June 15, 1913.

Preface to the First Edition.

lx my text-book on Physiological Chemistry, pub-
lished in 1906, I made an attempt to harmonize
the defensive measures, adopted by the animal
organism against products generated by cells out of
harmony with the body, with the metabolic processes
of the individual cells of the body. I was of the
opinion that, when an invasion takes place of cells
which are out of harmony with the bodv. the blood

j j '

or plasma, and the cells, the cells of the body respond
with counter-measures which are not entirely new to
the cells of the particular organ or of the blood ; on
the contrarv, I tried to bring the whole question of
the so-called reactions of immunity into close line
with processes that are normal, and consequentlv
familiar, to the cells. From the point of view stated
in the above-mentioned text-book, I attacked
experimentally the problem of the method of
defence, used by the animal organism, against the
invasion of substances out of harmony with the body,
the blood plasma, and the cells. In the first place
I studied the question whether normal blood plasma
contains definite ferments; and, in the second place,
whether the introduction of disharmonious substances
is followed by the appearance of ferments which were
not there before. I found, in fact, that, after the

xiv PREFACE TO THE FIRST EDITION

introduction of substances out of harmony with the
blood plasma, ferments appeared which are capable of
transforming these products, and of depriving them
thus of their specific character. These facts estab-
lished beyond doubt one means of defence possessed
by the animal organism against the invasion of
disharmonious substances.

My thoughts then turned at once to the relation
of these facts to immunity, and especially also to
anaphylaxy, and I undertook experiments to decide
the question as to whether the animal organism
develops any ferments of a specific nature against
substances produced by micro-organisms. And I
w^as particularly interested in the question whether
the stages that arise, in any given case, during the
decomposition of a particular substrate vary with the
species of the invading cell, and whether this may
not give us the explanation of many phenomena that
appear in the course of certain infections. Finally,
I was able to demonstrate that, during pregnancy
also, the organism defends itself, by means of
ferments, against certain constituents which are
passed into the blood, most probably from the cells
of the chorionic villi, and which, though in harmonv

o *

with the species, are out of harmony with the plasma.
This observation renders possible a diagnosis of
pregnancy.

The above statements have a bearing on a great

PREFACE TO THE FIRST EDITION XV

number of particular problems connected with
immunity, which still await solution ; nor is there any
doubt that many well-ascertained facts are closely
connected with the results of our researches. Even
now it would be tempting to select suitable instances
from the mass of my particular observations, with a
view to giving a more general signification to the
views I have formulated on the means of defence
possessed by the organism against the invasion of
substances or cells that are out of harmony with the
body. For the time being I have refrained from
doing so, as the mere enumeration of closely related
observations, quite apart from a discussion of all the
hypotheses put forward, would enormously increase
the scope of this little volume, and incidentally would
interfere with a clear insight into our subject.

Again, it is very difficult, for those not actually
engaged in research work on immunity, to keep in
touch with all the communications made at different
times concerning ideas and theories that are con-
stantly changing, and above all to find a sure footing
amongst the somewhat pleonastic terminology and
nomenclature employed. Theory and actual fact
form, in this field of research, a closely interwoven
net of conceptions ; so much so, that only those, who
have already acquired, by actual corroboration, a
thorough knowledge of all the problems connected
with the subject, are able to trace sharp limits

PREFACE TO THE FIRST EDITION

between hypotheses and facts. For these reasons I
have limited myself to making mention of those
works which either are closely connected with my
own researches, or else will be of special service to
the reader, in that the full lists of references they con-
tain will be a guide to further study in this field of
research. This limitation alone has enabled me to
present a picture which, I hope, is quite clear of
the development of my own investigations, and to
show ho\v I arrived at the doctrine of the active part
played by ferments in connection with disharmonious
substances.

The comprehensive survey, which I now present,
has resulted from the fact that many problems have
been so far advanced, recently, by means of experi-
mental work, that it seemed advisable to take stock of
the observations that lie stored in numerous pub-
lications. And, on the other hand, I find that the
further study of particular problems can be carried
on only in institutions supplying means and
apparatus which I cannot command. One man
by himself is able in certain problems to reach
only a certain point. He takes over, as it were,
an edifice which has been built up to a certain
height from all possible sides. He tests the
scaffolding the existing working theories to see
whether it will last any longer, or whether it must be
replaced ; and, more important than that, decides

PREFACE TO THE FIRST EDITION XV11

whether the structure itself is perfectly sound. He
then builds further, but in most cases makes only a
tiny addition. It is very easy for a single observer
to lose a clear view of the whole, through using too
complicated a scaffolding. Others follow ; they test
the solidity of the structure, they move the misplaced
bricks into their correct position, and give a finishing
touch to the parts that are insufficiently trimmed.
Each new workman brings new tools, new ideas, and
his own extensive experience with him, and tackles
the whole structure from different points of view.
Then the scaffolding is removed, and a mighty build-
ing appears, which scarcely gives any idea how
diverse were the plans on which it was founded. So,
too, this contribution to our knowledge of the
functions of the cells may be considered only as an
attempt to adjust a new stone in the already existing
structure, and to construct a scaffold from which
further progress may be made.

In conclusion, may I be permitted to express my
heartiest thanks to my collaborators, whose untiring
energy has made it possible to accomplish so many
single experiments in such a relatively short time,
and to work out different problems from various
standpoints at the same time.

EMIL ABDERHALDEN.

Halle a/S.,

April 15, 1912.

Contents.

PAGE

Means of defence of unicellular organisms against substances

out of harmony with the cell ... ... ... ... 2

Progressive decomposition of the units of nutrition ... 8, 32

The co-operation of different unicellular organisms ... ... 10

Division of labour in multicellular organisms ... ... 11

The value of milk to the suckling 13-15

The transformation of nutritive material into products

in harmony with the body, plasma, and cells ... 16, 23

The specific structure of each cellular species ... 17, 24

The signification of digestion in cell-metabolism ... ... 16

Observations, in the fields of physiology and pathology, sug-
gesting a specific structure in different cellular species 16
Hermaphroditismus verus ... ... ... ... ... 17

Results of experiments on transplantation ... ... ... 18

Cell-specific therapy ... ... ... ... ... ... 19

Distinction between substances that are in, or out of, harmony

with (a) the body ; (b] the blood ; and (c) the cells ... 22
Demonstration of the probability of the existence of cell
constituents, that are specific to the species and the

()l,j(tllo * *T

Cell constituents specific to the species and to "function" ... 27
Transformation of the units of a given cellular species into

constituents of other cells ... ... ... ... ... 29

The regulation of the harmony of the metabolic processes in

the organism ... ... ... ... ... ... 31*42

Inter-relations of different cellular species ... ... ... 35

The invasion of cells out of harmony with the body. Means

of defence of the organism against such cellular species 42

CONTENTS XIX

PAGE

Means of defence of the animal organism against substances

out of harmony with the blood plasma ... ... ... 45

Preliminary -study -on the ferments normally Contained an -;r

the blood .'.'? tci..i ;v. ,. ... 49

Methods for-the study of ferments ..-. ... ... . J. 49

The formation of defensive ferments ... ... ... ... 55

1. After mtrodUction of albuminous substances' out of
harmony with the body and the 1 plasma, and their

' initial staged : of <deccVnipositJonv : with 'special refer--

ence to anaphylaxy ... ... ... ... ... 58

2. After introduction of carbohydrates out of harmony

with the body and the plasma ... 72

3. After introduction of fats ... ... ... ... 80

4. After introduction of nucleo-proteids and nucleins ... 83
A survey of the significance of defensive ferments ... ... 85

Origin of defensive ferments ... ... ... ... ... 87

Demonstration of substances that are in harmony with the

body but not with the plasma ... ... ... ... 90

Biological diagnosis of pregnancy ... ... ... ... 91

Question of specificity of defensive ferments ... ... ... 96

Sero-diagnosis of organic functions ... ... ... ... 106

Doubt concerning the uniformity of the so-called proteolytic

ferments ... ... ... ... ... ... ... 108

Study of the correlations of particular organs ... ... 112

Defensive ferments which appear in the constituents of the

blood corpuscles ... ... ... ... ... ... 114

The application of the methods in pathology ... ... 116, 133

Their application in infectious diseases ... ... ... 116

Methods ... ... ... ... ... ... ... ... 147

1. The dialysation process ... ... ... ... 147

Testing of the dialysing tubes ... ... ... ... 156

Preparation of the substrates (organs) ... ... 162

Means of obtaining blood serum ... ... ... 173

Performance of the experiment ... ... ... 176

Sources of error in the dialysation process ... ... 189

2. The optical method ... ... ... ... ... 202

The application of the optical method ... ... 203

Preparation of peptones ... ... ... ... 204

Standardization of the peptone ... ... ... 210

XX CONTENTS

PAGE

1.1 1 C! I * 1 1 U 1 C* . . . ** rf 1 ^

Personal researches . 215-224,240

Complete list of works of other authors 224

Literature in connection with problems related to our

suDject ... * 4

Researches, published during the year 1912, and up to
November 15, 1913, in which the dialysation process
and the optical method have been made use of 227-240

Personal researches during the years 1912-13 ... 240-242

Defensive Ferments of the Animal

Organism.

THE question has frequently been raised, whether
unicellular organisms exhibit simpler processes, in
their general organization and metabolism, than do
organisms composed of numerous cells. A priori,
it is conceivable that organisms of a morphologically
simpler construction are composed of simpler com-
binations, and that their metabolic processes follow
a simpler path, than is the case in those forms of
life in which the body is built up by the co-operation
of different cells. But all our experience, hitherto, has
proved, that even those cells which are constructed
on a simple plan morphologically do, when studied
from a purely chemical point of view, show exceed-
ingly complicated relations. Indeed, the study of
the processes of metabolism in unicellular forms of
life is a study all the more difficult, in comparison
with that of more complicated organisms, in that, in
the former, it is so difficult to separate the actually
i

2 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

absorbed materials from the metabolic by-products,
and these again from the secretory or excretory pro-
ducts. Absorption and secretion merge in each
other. The higher we climb in the scale of organi-
zation, particularly in the animal kingdom, the more
do we meet with cells which are entrusted with
special functions. For instance, we find cells which
receive matter from the exterior. Others transform
particular compounds into products of a special
nature. Others, again, have the function of carrying
the final products of metabolism to definite points for
excretion.

A unicellular organism stands constantly in rela-
tion with numerous substances of the outer world,
which differ from place to place and from time to
time.. Some of these it makes use of as nutriment.
Others, on the contrary, are entirely useless to the
cell in question, while many would cause consider-
able harm, if allowed to penetrate its wall cells. To
these substances the single cell does not yield itself
helplessly, but has at its disposal various arrange-
ments for its own defence. It has, in the first place,
a cell wall which is impermeable by many substances.
Further, the cell, by means of different processes, is
capable of altering substances, which may in any way
be injurious to it, in such a way that the active group
is rendered harmless. Often a simple hydrolytic
decomposition is sufficient to deprive the compli-

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 3

cated substance of its specific properties. The dis-
harmonious product is decomposed into indifferent
by-products which are harmless to the cell. More
energetic means are often employed, and the material
is oxidized or reduced according to the special needs
of the cell. Even in these simple forms of life it is
probable that many substances are rendered harm-
less by combining to form fresh compounds, just as,
in the metabolism of a more complicated organism,
rearrangements of different kinds are undergone
which alter such materials as are undesirable, so
that they may be excreted in this form out of the
body. Very often a given substance is incapable
of combination, in which case it must first be
so transformed by special processes as to be sus-
ceptible to combination. We thus see how the
cells of the body oxidize, reduce, or decompose,
until a product is reached that is capable of com-
bination. There is no reason to doubt that uni-
cellular organisms have similar means of defence at
their disposal, but they are not so easily traced, owing
to the fact that it is more difficult to add certain
substances to a single cell, without damaging it, than
it is in the case of a more complicated organism.
The latter are able to modify profoundly the action
of substances introduced by the mouth, owing to the
fact that they are gradually absorbed. Further, these
substances are considerably diluted in the lymph

4 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

and the blood. Finally, they may be easily with-
drawn from the body before they have had any
opportunity of penetrating into the interior of the
cells.

As a principal defence a single cell always has the .
cell wall, with its characteristic construction and its
specific physical properties. Besides this, there is no
doubt that ferments play a considerable role. They
allow the cell to make a choice from amongst the
substances which are continually acting upon it.
These ferments, as Emil Fischer (Lit. 6) 1 has proved
from his exact researches on the subject, are directed
in a specific manner against definite substrates. Only
those substances, which are capable of being decom-
posed by the cell into simpler groups, are in general
found to be of use to it. Throughout, our positive
knowledge has led us to the conclusion that cells
supply their vital needs only from the simplest
units of the nutritive material, and that thev

j

probably never break down such complicated sub-
stances as fats, polysaccharides, and proteins, directly
into their final metabolic products. Even the
simplest units are not at once completely broken
up. The cell works in stages. First of all it splits
up large molecules into smaller particles, and
so sets free from the rest one fraction of the entire

1 The numbers refer to the Bibliography given at the end
of the book.

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 5

energic contents of the original material, until finally
-at least, with carbohydrates and fats the whole
amount of contained energy is released. The cell
regulates its own metabolism down to the minutest
details. In the proper preparation of the material
to be decomposed, and in the gradual liberation
of the amount of energy required, lies the real impor-
tance of those substances formed by the cell, which
we at present comprise under the name of ferments.

The ferments have yet another value for the cell,
in that they help it to regulate its own structure.
Not every product which is taken up by the cell
passes into its structure. Sometimes the decomposi-
tion must be carried on further; in other cases the
particles must be synthesized suitably for the pro-
duction of the necessary structural unit; after
which it sets about the recombination of all the
numerous constructive units, so as to form the
complicated characteristic structure of the cell.
Though w y e do not yet know the precise nature of
the ferments, yet their specific activities, and their
great importance in regard to cell metabolism and
cell structure, are well known.

Without energy no cell can do work or produce
heat, and it is in energic metabolism that we find
a true picture of the functions of the cell. How the
cell procures the required energy, in what manner
it makes use of it, and so on, we can learn only by

6 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

an accurate and possibly exhaustive study of the
finer metabolic processes that go on in the cell. In
these processes the so-called ferments play the most
important role. With their aid we have succeeded
in following 1 up, outside the cell, processes which
seemed to be exclusive properties of the cell. The
more these experiments are extended, the more we
meet with observations which show that we have been
in the habit of picturing far too schematically the
processes within the cell body. Thus, for instance,
the simply formulated process of the fermentation of
grape sugar into alcohol and carbonic acid gas-
C 6 H 12 O 6 = = 2C 2 H 5 OH + 2CO 2 has been found to
be a most complicated process. A whole chain of re-
actions takes place, before the grape sugar is finally
converted into alcohol and carbonic acid gas. There
are many more intermediate stages present than
were ever suspected. It will be an important duty
of future research to ascertain what importance
alcoholic fermentation, in all its stages, has for the
yeast cell. We are indebted to recent researches,
in which Knoop, Neubauer, Friedmann, Embden,
Dakin, Schittenhelm, Jones and others have taken
a prominent part, for a knowledge of numerous
intermediate stages in the decomposition of the
ammo-acids, of grape sugar, of purin bases, &c.
Every time we demonstrate fresh intermediate links
in the decomposition of certain compounds we get a

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 7

deeper insight into the working of the metabolic
processes of the cells, and obtain important clues as
to the means by which the cells of the animal organ-
ism produce, from compounds of a definite kind,
substances that belong to a different group. We
may mention here, for instance, the conversion of
amino-acids into grape sugar, and of carbohydrates
into fats.

Some of the unicellular forms of life, and some
organisms consisting of a few groups of cells, are, at
least in part, equipped with agents (ferments) which
are not so precisely directed against certain substrates
"as the ferments of the higher species, such as plants
and animals. While the ferments of the latter, so
far as we know, principally decompose substrates
consisting of units which are found in the cell-con-
stituents that constantly recur in Nature, cases h'ave
been observed amongst the lower organisms (i.e.,
morphologically lower) where the latter split up
compounds, which have been prepared in the labora-
tory from units which are not known to exist in a
free state in Nature. Owing to this wider indepen-
dence these organisms are assured of better conditions
of existence. These cells can live where others, being
unable to secure the energic contents of the material
supplied to them, and being also unable to form from
this substrate the elements required for their bodies,
are bound to perish through lack of nourishment. In

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

this way a cell dies, though it be plentifully sur-
rounded by a material rich in energy, which, how-
ever, cannot be used because it lacks the proper form
-both structural and configurative. It does not suit
the organization of the cell. There is an abundance
of oxygen at its disposal, but the latter cannot find
any point of attack ; and so the necessary preparation
is wanting.

Some of the substances cannot be absorbed bv the

j

cell, simply because they are physically too coarse to
penetrate through the cell wall. Such is the case
with many colloidal substances, which must be first
decomposed into simpler groups before they can
pass into the interior of the cell. In these cases the
presence of ferments is essential, and they have TO
be of such a kind as to be capable of decomposing
the complicated molecules into a form which may
easily penetrate through the cell wall. Often, how-
ever, such conditions may suffice as enable the
coarse complex substance to be simply broken up into
finer particles, which may be ingested in this form by
the cell, without any molecular decomposition being
necessary. Further decomposition takes place during
absorption or, even later, at a suitable point within
the cell.

Even a unicellular organism does not enter into
intimate relations with substances which have
not previously been remodelled. This remodelling

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 9

generally takes place in such a manner that the sub-
strate is decomposed into simpler indifferent con-
stituents, after which the cell builds from the base
upwards. 1 '* In many cases this rebuilding is unneces-
sary. Such is the case when the cell only requires the
energy contained in the absorbed substance. As
soon, however, as substances are required as vital
units of the cell, then they have to be adapted to
the whole structural plan in all its minutest details.
This is also the case when secretory substances,
having a characteristic structure and a specific action,
are to be formed.

\Ye know of unicellular organisms which produce
their body substance from very simple elements
indeed. Thus, we know of organisms which
produce their cell plasma from carbonates, nitrates,
water, and salts. Others can draw their nitrogenous
supply from any substance which will supply them
with ammonia. Others, again, make use of the free
nitrogen of the air. There are, however, even
amongst unicellular organisms, some that are very
fastidious and will only thrive in the presence of
certain peptones. Others even require certain forms
of protein from which to obtain their derivatives.

An exhaustive study of the sources of nitrogen

1 a See in connection with this, Emil Abderhalden, " Syn-
these der Zellbausteine in Pflanze und Tier," Julius Springer.
Berlin, 1912.

IO DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

necessary for each separate organism, paying due
attention to other nutritious materials and conditions,
\vill no doubt lead to exact methods for the culture of
separate cells in the laboratory. Following this
line of inquiry we could surround certain micro-
organisms with peptones whose composition we are
familiar with, and so acquire a deeper insight into
the processes of their metabolism. 2 Even the mode
of decomposition of the substrate, and of the inter-
mediate stages, may furnish some important hints as
to the specific functions of the cell and, in many
cases, allow us to recognize particular organisms. 2 ' 2
We shall, by this means, understand why certain
germs thrive upon certain media, while on any other
substrate they either cease to grow or perish entirely.

It will also be possible to determine exactly, which
of the decomposites and by-products formed from the
culture medium produce harmful effects.

There is no doubt that, in the organic world, cer-
tain species prepare the soil for others, and in this way
one organism acts as a pioneer to the others. It is a
most interesting task to follow up this co-operation
of different living beings in all its details. To a
certain degree we have, in the co-operation of single

If the nitrogenous basis from which different micro-
organisms obtain their nourishment is not known, it might
be possible to obtain a culture medium by decomposition of
the micro-organisms themselves.

2 a A firm at Hochst a/M. supplies peptones of definite
composition for this purpose.

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM II

cells, a forecast of the division of labour found
amongst the organs of the higher forms of life. In
the former case we have the cells as yet free, while in
the latter they are combined into tissues. From this
point of view we may look upon the symbiosis of
heterogeneous species of cells as a first experiment in
the building up of a cell state. The single cells are
still independent and their duties multifarious.
There is no strong bond uniting the organisms into
any one "organ,' and yet they depend upon each
other for mutual support. Unicellular beings begin
to organize themselves into combinations. Another
step further and we arrive at cell complexes having
definite functions, which we call organs. But even
the most developed organisms, both of the animal and
plant worlds, have relations with cells which stand
outside the common organization. By means of
micro-organisms the plant gains access to otherwise
inaccessible sources of nitrogen, while by means of
bacteria the animals make use of the important carbo-
hydrate, cellulose. The bacteria convert the latter,
within the intestines, into products which can be
further decomposed by the ferments secreted by the
glands.

In those organisms in which division of labour has
been instituted amongst the cells, and particularly
in those in which definite cells have closed in to form
an alimentary canal, these are the only cells which

12 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

are in communication with the outer world. They alone
know, so to speak, what food is ingested. Even these
have no direct relation with the ingested material,
seeing that the latter, before being taken up by the
cells of the gut, has been subjected to the action of
the ferments poured into the alimentary canal, and
been disintegrated into simpler and indifferent par-
ticles. All nutriment of a composite nature is dissolved
in stages, until finally products of decomposition
result which no longer exhibit any special characters.
Generally speaking, food supplies the material for
the building up of the cell, and we must remember
that w r e are dealing with the complicated tissues of
animals and plants. Each cell has a specific fabric
of its own, which is dependent on the nature of its
separate units, and on the manner in which they
combine together. We must not look upon this
from a purely chemical point of view alone, but
should pay attention to its physical aspects as
well. The sum of the properties resulting from
the special structure of the cell conditions its special
functions. When such cells, with their specific
structure and functions, are taken in by an individual
organism, the latter can at first do nothing with
the material supplied. The special character of the
different products that make up the particular cells
must first be destroyed. Unit must be separated
from unit, so as to leave only a mixture of simple

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 13

compounds, from the elements of which the body
cells may construct their own material, or else renew
their supply of energy. In the latter case, too, as
has been mentioned before, a preparatory decom-
position (a kind of adaptation to the cell) is necessary.

An analogy may be used to make clear this kind
of reconstruction. Suppose an architect is called
upon to convert a certain building, which has been
specially designed for a particular purpose, into one
suitable for an entirely different object. He would
only be able to carry out this work on the condition
that he might pull down the original structure. He
would naturally be able to work some of the bricks
of the old building into the plans of his new one.
Some of the bricks, or even combinations of bricks,
may be used as they are, others will have to be recut,
while others, again, are of no value whatever. In
just the same w^ay does the animal organism act
towards the specifically constructed parts of the cells
which are used as food. First of all comes the dis-
integration into simple compounds, and then a recon-
struction, according to entirely new plans, on the
other side of the intestines.

The simplest conditions, in this respect, are to be
seen amongst mammals during the suckling period,
when, under normal conditions, the animal imbibes
the milk peculiar to its species. This, as G. von
Bunge first demonstrated very precisely, is in every

14 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

respect adapted to the growing organism (Lit. 2, 3).
The great point is, that the suckling is constantly sup-
plied with the same mixture of salts and the same
organic nourishment, namely, albumen, carbo-
hydrates, and fats. Later, when mixed nourishment
is taken, the conditions become much more compli-
cated, according as greater quantities of this or that
unit are introduced during digestion. The cells of
the intestines are continually confronted with new
duties, and have to adapt themselves gradually to the
new conditions.

The cells of the milk glands are charged with the
proper choice of food. They prepare the food for
the developing organism, and simplify in particular
the task of the intestines which, with the help of the
liver, prepare the ingested food for the other cells
of the body. Even the components of the milk,
before they can be of any use to the organism, must
be first considerably altered in the intestine, just as
later, in the case of mixed food, a complete decom-
position by means of the ferments precedes absorp-
tion. The difference in respect to the latter mode of
nourishment only lies in the fact that, with the milk
food, the same stages of decomposition, giving rise to
the same by-products, always recur. Day after
day, to a certain extent, the cells of the intestines and
of the organism have to perform the same task.

From this point of view we may discern three

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 15

important stages in the nutrition of the young of the
mammal. Right up to its birth, which is the first
stage, the foetus has received from the mother only
food which is in harmony with her body, and it makes
this harmonize with its own blood and its own cells.
Its organism has never come into contact with entirely

o >

disharmonious substances, and thus its metabolic
processes run on definitely balanced lines. But birth
supervenes, and with it the first change in the mode
of nourishment. The individual has become inde-
pendent. Respiration begins, and the cells of the
lungs immediately enter upon their duty of exchang-
ing gases. With equal rapidity the cells and glands
of the intestinal walls undertake their new functions,
which are, with the help of ferments, to prepare new
nourishment for the cells of the body. The mother
facilitates this task by giving off a supply of milk
that is adapted to the requirements of the infant. In
the first place, the intestinal cells have their task
simplified. They never come into contact with a con-
tinually changing mixture of ions, nor are they over-
whelmed with all kinds of disintegrated organic
by-products. In this way the as yet inexperienced
being is gradually accustomed to its new functions,

C? <5

and finds itself at last well prepared when it has to
deal with a new kind of food which requires it to exer-
cise its functions in a more variable, and, consequently
more difficult manner. From the moment of parting

l6 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

with the milk as sole nourishment, from the moment
of passing on to the mixed nourishment that is
peculiar to its species, the second important change
in the feeding of the growing individual is accom-
plished. The third stage of its evolution has begun. 3

The cells must function quickly to prevent dis-
harmonious substances from entering the circulation.
To ensure the proper discharge of a duty so important
to the organism, the liver is placed between the
intestines and other organs. Within this important
organ the blood, still laden with the absorbed and
partly metamorphosed food-stuffs, comes into contact
with the liver cells. This material is once more
thoroughly sifted, and the blood is finally discharged
into the general circulation, freed from all substances
that would be out of harmony with the body and the
blood.

The knowledge that digestion is the means by
which unsuitable products are prevented from passing
into the blood and the cells of the body is of the
greatest importance for our comprehension of the
whole metabolism of the animal organism. Thus, to
a certain extent, we may look upon the animal
organism as a whole in itself. All the cells of the

From this point of view it is easy to see why lack of
its proper milk sets up disturbances in the suckling, and
particularly how dangerous are continual changes in the
composition of the food, seeing that the young animal is
not yet prepared for the reception of mixed nutriment.

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM IJ

body have a common architecture, which is bequeathed
from generation to generation by means of the sexual
cells. The cells which combine into an organ have,
besides that, a structure specific for the organ. We
are bound to accept this view, otherwise it would be
incomprehensible why, for instance, the cells of the
liver should produce only bile, and the cells of the
medulla of the suprarenal bodies adrenalin, c. All
the cells of the body have certain functions to perform
which are of use to the whole organism. It is quite
certain that the different organs supply substances to
the blood, which set up definite processes in other
particular parts of the organism. If these substances
are to act effectively, they must have a definite specific
structure. The cells, too, on which they are destined
to act, must also be characterized by a special struc-
ture, otherwise it would be difficult to understand
why a special secretion acts only upon certain cells,
and leaves a number of other cells quite unaffected.
A particularly fine example of the specific action
of gland secretions upon cells of specific structure is
supplied by such cases of hermaphroditismus verus
as that, for instance, in which the bullfinch is found
to have a testicle on one side and an ovary on the
other. These peculiar animals have on the one side
' male, and on the other female, plumage, each being
delimited accurately, and without any transition, along
the middle line of the body. It is absolutely
2

1 8 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

impossible to imagine that the gland secretions of the
two different glands, which bring about the full
development of secondary sexual characters which are
obviously present from the first, should remain only
on one side of the body. They must, in fact, be
carried by the blood to all the cells of the body.
Nevertheless, the secretions of the male gland pass
only to those cells which have "male' properties,
and vice versa, the secretions of the ovary affect only
the cells of the " female ' half of the body.

Strong support for this view of a specific cell
structure is supplied by the numerous experiments
on transplantation. The surgeon nowadays tries, as
much as possible, to retain the full strength of the
functions of every organ, and, if some of the tissues
are missing, he seeks aid in substitutes. It is found
that only those tissues graft which are taken from the
same species, while still better results are obtained
by the use of parts of the same individual. Hetero-
plasty, i.e., the attempt to graft foreign tissues, has
never succeeded. A body requires cells in harmony
with itself. If they are in close relation, as is the
case in tissues of the same species even the indi-
vidual has its own type then it is very probable that
with time the newly grafted tissue will, by means of
reconstruction, assimilate itself with the other cells
of the same organ, and so eventually with the entire

organism.

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 19

Finally, pathology provides us with a large number
of cases supporting our view of the specific structure
of the different cells belonging to a given organism.
We know that certain poisons have an injurious effect
only upon very definite kinds of cells. \Ye might
here refer to the well-known system diseases of the
central nervous system. The so-called metasyphilitic
phenomena, for instance, manifest themselves, only in
very special regions of the spinal cord and the brain.

The idea that each kind of cell has its own struc-
ture, and to some extent its own metabolism, opens
up a wide vista for therapy also. So far as the
organism always forms products which act upon
certain cells and only upon these, it must be possible
to find substances which will act only upon those cells
whose metabolism we may wish to alter in some way
or other, or whose complete destruction is desirable.
The latter is the aim of the battle waged against
germs of infectious diseases and tumour cells,
especially cancer. There is a great future for cell-
specific therapy, which will pay special attention to
the structure and the configuration of the means
employed, or else attempt generally so to modify the
chemical and physical conditions in certain cells that
life will be impossible for them.

The admission of a certain specific structure, for
each cell species with special functions, implies that
each separate cell possesses special means enabling it

2O DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

to regulate its own structure. The components of
the blood plasma, which serve as the deriving material,
are the same for all cells. The formation of a speci-
fically acting secretion also requires that every kind
of cell should have means and arrangements at its
disposal for the specific transformation, under certain
circumstances, of the same product. From this
point of view we should expect to find that
each kind of cell controls particular ferments,
of which, however, some will be common to all
the cells of the body. These ferments have
the task of decomposing the nourishment, brought
by the blood plasma to the cell, into simpler
products. Investigations on the peculiarities of
cell ferments the tools of the cells are already in
progress, and we shall deal with this question later
on. It may be that the result of these investigations
will supply the most unequivocal and sure support
for the theory of the dependence of cellular function
on cellular structure.

For the maintenance of a regular and undisturbed
flow in the varied processes of the cell, we must
assume that within certain limits constant conditions
prevail. When we carry out certain experiments in
a laboratory and try to study, for instance, the inter-
action of two substances upon each other, we choose
the most favourable conditions possible, and take
particular precautions against the presence of any

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 21

other substances than those essential to the reaction.
It is a well-known fact that the slightest contamina-
tion may influence the reaction to a very great extent.
It may either fail altogether, or be retarded, or may
even be diverted into quite a different direction.
We meet with great difficulties if we have to follow
up several reactions in one and the same medium.
Intermediate products mav act, one upon the other, to
such an extent that we arrive at a series of final pro-
ducts whose origin it would be extremely difficult to
account for. Now, if in an animal organism the
separate processes were not regulated in a very strict
manner, and if, for instance, the blood did not receive
substances which are in harmony with it, that is,
always transformed in a definite and regular manner,
it would be difficult for us to understand how
the separate secretions always attain their aims
in a very certain way, and how they are able
locally to attack particular metabolisms, and either
retard, or hasten, or initiate them.

There is not the slightest doubt that the course of
this metabolism, as well as the inter-relations of the
cells of a particular organ, is only imaginable under
the supposition that the metabolism of the whole
organism is regulated in the most precise way, not
only quantitatively, but also qualitatively. We are
bound to imagine that, in the work of the cells, the
same stages of decomposition recur regularly, and

22 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

that it is at a quite definite stage that the by-products
of metabolism are passed by the cells into the lymph
channels, and so into the blood system. The indivi-
dual cell is in this sense responsible for the constant
composition of the contents of the blood, in the same
way as the cells of the bowels with their respective
ferments.

Here, again, the animal organism controls im-
portant weapons of defence which may correct any
possible errors. Between the blood and the cells of
the body lies the lymph. The latter is the first to
receive the substances supplied by the individual
cells, and controls them by means of its accessory
apparatus, namely, the lymph cells and the glands.
Some of the substances are further disintegrated or
transformed in some other way, and, perhaps, even
utilized for various syntheses. From this point of
view we may look upon the lymph as a powerful
means of defence, whose aid is particularly valuable
in preventing the infusion into the blood of com-
pounds that are both quantitatively and qualitatively
unsuitable. From all sides care is taken that only
normally suitable substances shall appear in the
blood.

From this point of view we may distinguish
substances that are " out of harmony with the body,'*
i.e., such compounds as, in their structure and
configuration, show no correspondence with the con-

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 23

stituent parts of the organism. To these belong all
such substances as are received from the outside as
nutriment, with the exception of those products
which may be ranged amongst the most simple
units, as, for instance, grape sugar. As sub-
stances " in harmony with the body,' we would then
term those which, when entirely recast, correspond
in their structure to the essential composition of the
particular species or individual. In addition to this
general conception, which only means that a sub-
stance is not absolutely disharmonious to the body
in general, we have undoubtedly to make a still finer
distinction according to the special features of the
compound in question. As early as the year 1906*
we had suggested the advisability of distinguishing
between substances which, though they are adapted
to the blood, are nevertheless out of harmony with the
varied cells of the body, and those which show any
features characteristic of the structure of the cells of
a particular organ. If our ideas concerning the
structure of the particular cells of an organ, and the
dependence of its functions on this peculiarity, prove
correct, then it follows that, as we have alreadv
emphasized, each kind of cell must have at its dis-
posal units of its own kind. We may then speak
of substances that are " in harmony with " an organ,

4 " Lehrbuch der physiologischen Chemie," i Auflage,.
S. 292, Urban and Schwarzenberg. Berlin-Wien, igo6.

24 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

or even more precisely, ' with the cells " ; or ' with
the blood.' Substances that are specifically elabo-
rated for the blood would then be " out of harmony
with ' the cells, and conversely the substances ' in
harmony with ' the cells are " out of harmony
with ' the blood, or better, with the plasma, because
the components of the form elements of the blood
are out of harmony with the plasma, and inversely.
Products in harmony with the cells will only be
in harmony with one another in so far as they belong
to cells with similar functions, so that from this
point of view, for instance, the specific elements
of the thyroid gland must be regarded as out of
harmony with those of the suprarenal bodies, and
inversely. The idea of an entirely specific structure
for each cell of an organ both from the chemical
and physical points of view is based not only on
the supposition that, without such a notion, the
special duties and functions of the separate cells of the
body would appear incomprehensible, but, above
all, on the above-mentioned fact that definite
secretions given off by particular organs act con-
stantly and only upon cells of a definite system.
This implies that the cells in question must have a
structure which distinguishes them sharply from all
other kinds of cells.

The view that each animal species is capable of
building up complicated compounds of peculiar

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 2$

structure, and further, that every cell with special
functions is formed of specially constructed com-
ponents, is very often met with doubt. How is it
possible for the animal and plant worlds to produce
such an enormous number of different compounds ?
There would have to be formed millions and millions

of different substances. Only think of the enormous

j

amount of animal and plant species, and just put
against this the fact that in general always the same
and similar components reappear ! In each cell
we meet with carbohydrates, fatty substances, and
albuminous particles. If these compounds are de-
composed into their units, we find the same com-
pounds resulting. All the albumens give, for in-
stance, with very few exceptions, the same, that is,
some twenty amino-acids. This obvious contra-
diction on one side cell constituents based on
similar elements, and on the other the idea of speci-
fically constructed cells disappears immediately we
begin to make a calculation. Suppose we synthesize
three elements A, B, and C; we at once obtain, by
merely altering the sequence of the particular com-
binations, the following six different products :-

A B C B A C C A B.

A C B B C A C B A

If we start with four different elements we get

o

twenty-four different compounds, while five elements

26 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

correspond to one hundred and twenty isomeric com-
binations. We give below the number of possible
compounds which result from simply altering the
sequence, the form of combination remaining the
same.

The number of Number of resulting compounds, the

different units sequence only being changed

8 40,320

10 3,628,800

12 479,001,600

15 ... 1,307,674,368,000

18 ... 6,402,373,705,728,000

20 2,432,902,008,176,640,000

This enormous number of different compounds
is solely produced by the manner in which the twenty

elements follow one another. If hydrolysed. all these

j j

compounds would give the same elements to the
same amount. These reflections may serve as :i
warning to those investigators who are inclined to
infer the identity of particular compounds from the
presence of the same elements.

Nor is it only the sequence of the individual units
that needs to differ ; for the mode of combination of
the different compounds may also vary. The number
of possible combinations is infinite. Again, the
units are present in unequal quantities. Finally,
one very important factor must be allowed for.
No cell is composed of only one albumen particle,
one carbohydrate, and one fatty substance ; on the

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 2/

contrary, we always rind mixtures of these. So

J > m>

that, given quite similar compounds, e.g., several
albumens, the cell has the power of making up mix-
tures of various kinds which give it a special stamp.
By these means we see then that the possibilities for
the production of specifically constructed kinds of
cells are infinite. Xo one would be able to calculate
the number that would account for all these possi-
bilities.

We take it as probable, on the strength of numerous
observations, that all through the animal kingdom

T5 O

similar organs show, besides their specific, and
possibly individual characters, certain features which
are common to all species of animals. All that is
required is the recurrence of a particular albumen in
the cell. We conjecture this from the fact that experi-
ments have shown that certain ferments, when they
act on albumen of a special kind, show specificity
for the organ, but yet are not specific for any par-
ticular animal species. It is probable that we are
here on the track of an important biological law.

Yet, in spite of these similar or kindred features,
each species and individual retains, by means of the
mixing of its cell components, the cell organization
peculiar to its kind. If a single group be repeated
but once only, the ferment that acts on it finds a
point of attack. We lay stress on these points,
because a casual consideration of the fact that in the

28 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

dialysation process, as well as in the optical method,
human organs may sometimes be replaced by those
of animals, might easily lead one to argue against
the existence of specifically constructed cell units,
as well as of the ferments that act on them.

A special place is occupied, at least qualitatively,
by all those substances which, like the units of the
different organic nutritive and tissue materials as
well as the inorganic constituents, the salts, water,
&c. exhibit no specific structure, and which are
common to the most different kinds of cells, as well as
to the blood and lymph, as intermediate and final
products. In this case disturbances can only be
caused by quantities. Rapid secretion, or synthetic or
analytic processes, may in such cases act in a regu-
lating manner and again restore normal conditions.
All substances, however, which have a specific struc-
ture, are peculiar either to the blood or else to
specific cells. From this point of view we must con-
sider substances, which leave the cell and pass into
the blood in a state of insufficient decomposition, as
being out of harmony with the blood, or rather with
the plasma; and, inversely, disturbances would cer-
tainly occur in the metabolism of certain cells, if, for
instance, the insufficiently decomposed constituents
of muscle cells were to penetrate the cells of the
kidneys. The units of the muscle cells are out of
harmony with the cells of the kidneys, and only a

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 2Q

radical reconstruction could make them harmonious
therewith.

That, in an animal organism, the formation of
material for definite cells can be effected by the com-
ponents of absolutely different cells, we can learn
from experiments on the starvation of animals, and
particularly from the well-known observations made
by the Basle physiologist, Friedrich Miescher, on
salmon. This observer was able to prove that the
sexual glands of this fish become extraordinarily
developed in fresh water at the expense of the
muscles. It can be demonstrated microscopically
that the components of the muscle tissues are
gradually decomposed until they pass into the blood
circulation; and Miescher speaks quite plainly of
a liquidation of the units of the muscle cells.
At the same time it may be observed that the sexual
glands gradually begin to grow, without the animal
taking anv nourishment. But in the cells of the sexual

f> J

glands we do not meet with the specific muscular
constituents in an unmodified state; on the contrary,
we meet with quite new substances, chiefly albumens
in a state in which they are never met with in the
muscle cells. We notice in this case that histones
appear in place of the muscle albumens. These are
albuminous bodies of a basic nature, containing the
so-called di-amino-acids in large quantities. Soon
we find the histones, the more the sexual organs, and

3O DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

especially the lestes, approach maturity, replaced by
protamines, which consist nearly exclusively of di-
amino-acicls. \Ve see, in this example, how cells of a
characteristic structure transfer their material to the
blood circulation in a profoundly modified form.
First of all substances are produced that are in
harmony with the plasma, and these are transferred
to the cells of the sexual glands by means of the cir-
culation. These glands take up the indifferent
substances, and from them build up products specific
to themselves. There is no doubt that similar pro-
cesses play a part in normal metabolism. Sometimes
one group of cells will help another in this way, par-
ticularly in cases where the supply of nourishment is
delayed for some time.

The reconstruction of substances of every kind
from products that are harmonious to the plasma and
the lymph is demonstrated by every growing hair and
every growing nail. Every new blood corpuscle tells
us of far-reaching transformations ; and every secre-
tion whether produced directly, as in the case of
saliva or milk, or manifested when a fistula is pro-
duced by surgical means, or whether it forms a
so-called internal secretion, choosing the blood or the
lymph for its path of action every one of these gives
evidence of powerful disintegrations, integrations, or
transformations. When thousands and thousands of
leucocytes hurry forth against an invasion of

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 3!

micro-organisms, for the purpose of limiting their
sphere of action or of subduing them, no more con-
vincing picture could be presented to us of the
synthesizing capacities of the animal organism.
Even the full-grown organism is able at any moment
to completely equip a vast army of cells and endow
them with special functions.

If the ingested food materials, with .their peculiarly
disharmonious structure, were passed directly into the
circulation and handed over to the cells in this state,
then the organism would be subjected to continual
surprises. The control of its metabolism would be
utterly impossible under such conditions. Some-
times one substance, sometimes another, would pre-
dominate in the circulation, and the blood would be
correspondingly affected sometimes in one way,
sometimes in another. The cells would have to dis-
integrate all these disharmonious materials. In such
_>

a case they would have to be provided with all sorts
of arrangements for the continual modification of
these materials. Each separate cell of an organism
would be in exactly the same state as a unicellular
organism. Just as these have to make a selection
from amongst the disharmonious substances by which
they are continually bathed, so, too, would the cells
of the body have to pick out the substances they need,
according to the conditions presented. Not only
would the work of the single cells be enormously

32 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

increased, but also, without doubt, the mutual
influence of different kinds of cells, by means of
certain secretions, would be much hindered. And
not infrequently we should find, that some substance,
that was quite specific in its structure, would be
caught up by disharmonious substances circulating
in the blood, and would be either altered or com-
pletely annihilated. In a short time the extra-
ordinarily delicate regulation of the general meta-
bolism would be thrown out of gear, and all kinds
of injuries would inevitably result. The intermediate
products in particular, which may vary in any given
case, would give rise to disturbances.

The cell, as has already been mentioned, always
works by degrees, for it is quite incapable of suddenly
decomposing a complicated molecule, and of directly
transforming it by means of combustion into its final
products. The cell builds step by step, and so pre-
serves the equilibrium of its energic metabolism.
The rapid combustion of albumen, fats, and poly-
saccharides would, in certain places, suddenly pro-
duce a great deal of energy, which would appear in the
form of heat, and under certain circumstances would
destroy the life of the cell itself. In consequence, the
gradual acquisition of the energic contents of the
food is of the greatest value for the maintenance of
all the finely graded processes of metabolism, as well
as for the functions of the individual cell ; while, on

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 33

the other hand, the decomposition of some dis-
harmonious, unsuitable, material may give rise to
some intermediate stages which are the cause of
serious disturbances. Here and there a cell would
be seriously injured. Complete disintegration could
never be effected, either because the cell would refuse
to act, or because it would lack the particular agent
with which to dissociate the compounds presented to
it. All this would lead to numerous possibilities,
which would exclude all regularity in the metabolism
of the cells, as well as in the general metabolism of
the body.

The animal organism prevents all these possi-
bilities by allowing only material which has been put
in harmony with the body, and particularly the
plasma, to reach the circulation. The nutritive
material of the tissue cells, which from this point of
view can be considered homogeneous, gives decom-
position stages with which the cells have been long
familiar. Nothing that is disharmonious appears on
the scene. Just as in a workshop, in the production
of an article, one machine prepares the material for
another, and one workman transfers to another
material which is finished up to a certain degree, so
do the tissue cells mutually support each other in their
task. The cells of the gut and the liver continually act
as important sorters for the whole organism. One may
imagine the chaos and disturbance which would be

3

34 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

produced in a workshop if machines were suddenly
supplied with unsuitable material. All of them would
soon refuse to work and come to a standstill. The
single workman, who, with his knowledge and his
tools, is trained only for a single phase in the pro-
duction of a complicated whole, would be helpless if
he were suddenly ordered to undertake a new task.
He would require new tools, and be forced to acquire
new experience. If his duties changed without any
regularity at all, i.e., were he restricted in his
activities to any casual work that might be given
to him, then any successful results would be entirely
out of the question. We find exactly the same
relation in the collective mass of cells which compose
our organism. The single cells represent the
machines and the workmen who, in an enormous
workshop, pursue common aims in separate groups.
The cells of the gut and its accessory glands,
especially those of the liver, superintend in a certain
degree the supply of raw material, which is
first prepared in a proper manner, and then recast
so as to be ' palatable ' to all the cells ; after which
it passes from hand to hand from one cell to
another.

Tn these considerations it is not only the purely
chemical processes that have to be taken into account ;
the physical processes also play an important role.
Every cell possesses substances which have an

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 35

influence upon osmotic pressure, together with
others which are without this influence. In this
respect, too, the cell is always laid down on the most
delicate lines. Sometimes it decomposes colloidal
substances and transforms them into others, which
increase the osmotic pressure of the cell ; at other
times it synthesizes materials in solution into larger,
more complicated molecules, until a body appears
which is more and more extracted from the solution,
and by this means loses its influence upon the
osmotic pressure of the cell. This variety of function
is of great importance to the cell in quite a different
direction. We know that single ions exhibit very
specific activities. Here also the cell must be
equipped with arrangements to accelerate in one case
the action of a separate ion and to check those of
another, or else to entirely exclude them. The cell
is able to effect this in diverse ways. Sometimes an
ion is combined with a protein, for instance, or with
other substances, and so is robbed of its own
characteristics ; at other times an ion is set free
through decomposition or simple dissociation. Or else
the cell induces antagonistically acting ions to react
mutually on each other in finely graduated stages.
Numerous experiments have shown, as has already
been mentioned, that definite cells depend upon
definite secretions having their origin in other organs.
If we remove certain organs, for instance the thyroid

36 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

gland, the accessory thyroids, the sexual glands,
the suprarenal bodies, and so on, we get definite
degenerative phenomena appearing. In many in-
stances, indeed, the absence of these organs is
incompatible with life itself. The same phenomenon
manifests itself when the organ is left in its proper
place, but through some cause or other gradually
discontinues its proper functions. In such cases
there is no need for the organ to be destroyed ; it is
sufficient if the production of a specific secretion
entirely ceases, a condition which is equivalent, to a
certain extent, to the complete absence of the organ.
These observations, which are supplied to us by
pathology, together with facts which may be produced
at any time as when we extirpate certain organs
and, after the results of such extirpation have mani-
fested themselves, make a fresh transplantation-
give an extremely varied picture of the reciprocal
relations of the different organs towards each other.
Each group of cells each organ has certain func-
tions to fufil in regard to the rest of the cell organiza-
tion, and in this respect it possesses a certain inde-
pendence of its own. There are also, of course,
reciprocal relations within the cells themselves of an
organ. Many observations point to the possibility
that apparent morphological unity of an organ does
not always mean unity of function. The independence
of a given organ is only a relative one. As we have

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 37

repeatedly indicated before, all the cells stand in
actively reciprocal relations with each other. We
have plenty of proofs for the acceptance of this view ;
while, on the other hand, we have no clear insight,
at present, into the signification of this reciprocal
dependence. Probably unicellular organisms alone
are wholly dependent upon themselves. They per-
form all the processes necessary for life independently
of other cells, except when, as sometimes happens,
a conjunction of these simple organisms rises to the
level of a symbiosis. The latter, as we have already
pointed out, must have a value corresponding exactly
}l o the reciprocal interactions of the cells of the more
highly organized forms of the vegetable and animal
kingdoms. For there is no doubt that in plants, too,
the cells have actively reciprocal relations.

Doubtless there are, in an organism composed of
cell groups, numerous kinds of cells which can
live without having reciprocal relations with other *
cells, exactly in the same way as a single individual
can isolate itself from its stock and still continue life
for a certain time. But in the same manner as the
well-being of a people or a State finally depends
upon the regular collaboration of the many, so each
kind of cell expresses its full value only by associating
its work with that of the other cells in the organism.
Only then is a cell capable of developing all
its capacities. In many particular functions, indeed,

3S DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

so much division of labour is found, and to such
an extent, that a large number of cells are entirely
dependent upon the functions of others. Were such
cells to cease to work this would result, as has already
been mentioned, in the sickness and finally in the
death of many other cells. In this direction there
still lies an extensive field of research before us. The
" whys ' and the " wherefores ' in this case extend
indefinitely.

The possibility of breeding single cells and
pieces of tissues in the blood plasma outside the
organism, and keep them alive for a certain time,
opens out a prospect of answering many problems
by experimental means. We shall see in due course
why some of the cells lose their normal functions
when the secretion of certain organs is lacking. The
number of possibilities is almost unlimited. For
example, some substances, such as grape sugar, can
only be dissociated by the cells into final products-
carbon dioxide and water after they have been pre-
pared in a certain manner. A gradual dissociation
takes place. The cell is equipped with appliances
for the alteration of a given substance, but they are
not at first in a condition suitable for use. A second
agent must first of all make them capable of their
respective functions just as a hammer without a
handle, or a screw without a screwdriver, are only
useful when the missing parts are at our disposal.

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 39

These agents are probably supplied by the cells of
other organs.

It is quite probable that, at present, being too
much concerned with the phenomena of structural
chemistry, we observe the processes in the cell from
a too one-sided point of view, and think too little of
the physical state of the cell. We know that many
reactions depend entirely upon the conditions present,
if the action is to take place. For instance, a change
in the reaction of the medium is sufficient to annihilate
the activity of n ferment. The addition of the least
trace of an electrolyte will, under certain circum-
stances, accelerate certain reactions; and alterations
in the conditions may even upset a reaction entirely,
and lead to totally different end products. The pro-
cesses in the interior of the cells are surely subjected
to a much greater extent to the influences of the
physical state of the cell. Colloidal substances and
electrolytes the ions and perhaps the rest of the
substances in solution, certainly play a considerable
role in their reciprocal relations. Here we meet with
regulations of a kind which we are at present unable
to discern. Might it not be in this direction that the
collaborations of different body cells would appear of
the greatest significance? Many a process, which
manifests itself and attracts our attention most
strongly on account of the ease with which it can be
demonstrated, may perhaps be of quite a secondary

4O DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

nature. The cause the primurv process escapes
our notice, partly because at the time we do not know
how best to state the problem, partly because we have
no methods at our disposal for an experimental
investigation of the case.

In all biological problems it is remarkable how
entirely dependent we are upon the philosophy and
the methods employed in the exact natural sciences.
\Ye transfer all that is there obtainable to the
problems of biology. For some years certain ideas
prevail, only to recede as soon as a new impulse or
a new success in the domain of physics and chemistry
directs a host of workers into new paths. -We drill
and work until a new gallery is driven into the rock
of puzzles which is found in every cell. Very often
the gallery ends blindly, but on its way has given
rise to numerous interesting discoveries. Sometimes,
however, the pioneer work is crowned with success.
An important stage is left behind, and a new outlook
gained. The final aim, however a complete in-
sight into the metabolism of the cell still lies far
ahead. Yet the knowledge we have acquired serves
as a compass to keep us on the right road. The
careful traveller will never leave anything unnoticed,
for observations which often seem but trifles mav

m>

point the way to entirely new problems.

In studying the functions of the cell we must never
forget that there is not a single substance which is of

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 4!

no value to the cell. It would be quite erroneous if
we were to consider any substance for instance,
albumen as the paramount life substance. A single
ion can in certain cases decide the life or death of a
cell. An aggregation of molecules may combine to
form a powerful complex a colloid and by means
of its properties dominate the whole function of a
cell. The structure and configuration of the separate
compounds, and of the separate units of the
cell are of the greatest importance for its indivi-
duality. To this we must add, and as partly con-
ditioned by the above, their structure and configur-
ation in the physical sense. A. separation of the
chemical and physical properties of the cellular
units is impossible, since they constitute mutually
the conditions of life for the cell. They stamp it with
its own character.

Substances, which may be indifferent products for
one kind of cell, may be injurious to another kind.
Each cell produces secretions of its own, in the forma-
tion of which many intermediate stages are passed
through. If the whole transformation into substances
that will be in harmony with the plasma be performed
inside the cell, then any by-products that may appear,
even though they be not indifferent in regard to
other cells, will display no injurious activity in the
organism as a whole. If, however, such insufficiently
transformed substances penetrate into the general

42 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

circulation, then we must expect troubles of all kinds.
Such a case may arise, for instance, when certain cells
cannot complete a decomposition that they have
initiated, owing to the absence of the necessary
agent, i.e., the ferment; so that the incomplete
action of a particular organ may be the cause
of numerous disturbances of every kind. If
continuity of function be broken but once, then
one disturbance, like an avalanche, is followed by
another. It is true that the organism defends itself
in such a case. It produces compensatory activities
and tries to adapt itself to the new conditions,
often succeeding in a most amazing fashion,
and repairing the damage for a long period
of time. Pathology supplies us every day with
examples of this kind. The study of cellular func-
tions under variable conditions is one of the most
attractive that we know. Experimental pathology
is a field which will be of undoubted importance for
the whole of physiology, and to an extent as yet
unrealized.

Thus all observations on the structure and meta-
bolism of the individual cells of the body lead us, in
the most unequivocal manner, to the conclusion that
within a given organism large aggregates of
cells work together harmoniouslv for the benefit of

<r?

the whole. Complete harmony of relations is guar-
anteed let us emphasize the fact once more by

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 43

having on the one hand the cells of the gut and the
liver ready to prevent anything, that is not completely
deprived of its own characteristics, from passing
into the circulation, and, on the other hand, by having
the cells of the body passing on to the blood only
such substances as have been so far disintegrated
as to have lost those features which harmonize them
with the cells. Blood which is in circulation thus
always shows the same metabolic products and the
same substances ; and from this point of view we may
consider the contents of the blood as being always
constant. Xo doubt the duty of the lymph, which is
placed between the cells of the body and the blood,
is to guard the blood against an excess of individual
products of metabolism. Probably, also, some of
the products, which have been insufficiently disinte-
grated, are finally decomposed by the lymphatic
glands, or by the lymph itself.

We are bound in this sense to look upon the lymph
system, as indeed we have already pointed out, as an
important control station. By means of its own
cells, and especially by means of the glands, the
lymph watches that no material shall reach the blood
which is out of harmony with it.

From the above point of view we gather an insight
into the significance of the invasion of organisms

r> o

of all kinds into an animal organism. The isolation

O

of the whole organism is immediatelv disturbed when

44 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

disharmonious cells settle on any spot within the
hitherto harmonious cell complex. From this
moment the harmoniously organized cells of the
tissues are subjected to the influence of a kind of
cell which has an utterly strange organization of its
own. These new cells have a characteristic metabo-
lism corresponding with their whole structure and
configuration, and this they bring with them definitely
into the new organism. They pass into the blood
numerous end-products of their metabolism. Further,
some cells decay here and there, and partial products
reach the blood which are out of harmony with the
species, and, of course, entirely so with the plasma
and the cell. The whole regulation of tho normal
metabolism is seriously injured. The cells of the
gut-wall will still be on the watch to prevent any
disharmonious material from entering the organism ;
and the single cells of the body will still struggle to
supply the blood only with properly altered sub-
stances. But the whole organization has been
damaged, in regard to the collaboration of its various
cells, by the fact that disharmonious substances are
continually given off by the invaders. The very
same thing occurs if, through any cause whatsoever,
the cells of the body change their structure and
acquire a metabolism which is entirely foreign to
the rest of the cells of the body. If cancer
cells or sarcoma cells, for instance, appear, then we

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 45

have cells before us which are neither subordinate
to, nor co-ordinate with, the rest of the complex
of cells. These cells have obviously reached
a definite state of independence, nor do thev
maintain any direct relations with the different
cells of the body. They are, so to say,
outside the association of the cells of a particular
organ, nor is there any doubt that they produce
secretions, the products of their metabolism, which
are out of harmony with the blood plasma. And we
can well believe that here, too, cells decay, and
products pass into the blood which are quite out of
harmony with the plasma.

These ideas afford the possibility of studying,
within the body, the action of disharmonious organ-
isms of every description, especially of micro-
organisms, and their relations towards the rest of the
body cells, from a purely physiological point of view.
It seems to us well worth while to follow up these
conceptions, and to attempt, by means of direct
experiments and observations, to bind together in
closer relations the two fields of research that are
covered by physiology and the study of immunity.

In the first place, we set ourselves the question :
To what measures does an animal organism resort if
substances penetrate into its body, and particularly
into its blood, which are out of harmony with the
species as a whole, or else only with the blood or

40 DEFENSIVE FEKMKNTS OF THE ANIMAL ORGANISM

plasma ? Is it deprived of the possibility of defend-
ing itself against such substances, or have the
cells of the body also, excluding those of the
intestines, retained the capacity of attacking com-
plicated substances which are out of harmony with the.
organism, and of reducing them by profound decom-
position to indifferent particles, which the cells may
use for the construction of new material, or else as a
source of energy ?

To solve this problem, in a satisfactory manner,
preliminary experiments on a very large scale were
required. First of all, it was necessary to ascertain
in what manner the individual cells of the body use
up the nourishment which is normally brought to
them by the blood. Does the individual cell decom-
pose the complicated nutritive material directly into
its encl-products, or does it always disintegrate them
first into simpler fragments, which are then reduced
by successive stages, until finally the whole of the
stored energy which the organism is capable of
setting free is at the disposal of the cell, and the
final products of the decomposition appear? All
experiments that have hitherto been carried out in
this direction lead us, as we pointed out at the
beginning, to the idea that each separate cell of the
body in general, with very few exceptions, disposes
of the same, or of similar, ferments as those
secreted by the digestive glands into the intestinal

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 47

canal. These ferments may not be identical in all
details. It is quite possible that the ferments passing
from the glands of the intestinal canal differ more or
less in nature, because, in the case of food, a much
more heterogeneous mixture of separate products is
introduced from the outside than is found in the
already transformed nutritive material of the cells of
the body, which circulates in the blood and lymph
channels. It is also possible that differences prevail
in the mode of disintegration, and consequently in
the resulting decomposites. It is quite certain that
the cells of the body are capable of hydrolytically
splitting fats into alcohol and fatty acids. Further,
they are able to decompose carbohydrates of a com-
plicated structure, especially glycogen, through dex-
trines to maltoses. The maltose formed is reduced,
by the ferment known as maltase, into two molecules
of grape sugar. We know also that very dissimilar
cells of the body contain ferments which decompose
albumen into peptones. The latter are further
reduced to still simpler products, and eventually
amino-acids are left, which again may be subjected to
further reductions.

It could, further, be easily shown that the cells of
the body are able to decompose into their structural
units the so-called polypeptides, that is, amino-acids
linked in the manner of acid amides. These ferments
have acquired the name of peptolytic ferments. Their

48 DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM

presence has been demonstrated in animals and
plants inside the most varied kinds of cells. In plants
they are not always found in an active state. In
seeds, for instance, they appear only when these are
beo-jnnino' to o-erminate. In the same way they are

c5 O J J

absent, as Iwanow has shown at my Institute, when
plants are resting during the winter. In the foetus
their presence can be demonstrated fairly early. They
can be detected, for instance, in a chicken on the
seventh day of development, while in embryos of
swine active peptolytic ferments appear on about the
fortieth day.

The demonstration of the peptolytic ferments may
be performed in various ways. One way is to treat
them in the manner adopted by Edward Buchner,
namely, to entirely destroy the cells of certain tissues
or even single cells by trituration with quartz sand,
so as to squeeze out the internal fluid of the cells.
This fluid is afterwards mixed with kieselguhr,
which readily absorbs moisture from the cell frag-
ments, and produces a compressible plastic mass.
The absorbed juice is then extracted out of the latter
under pressure- -up to 300 atmospheres and filtered
through a filter candle. We get a clear juice, which
contains many components of the cells ; the original
structure of these having, of course, disappeared. In
a juice obtained in this manner the presence of various
ferment activities can be demonstrated, and it may be

DEFENSIVE FERMENTS OF THE ANIMAL ORGANISM 49

shown that many processes go on exactly in the same
way, qualitatively, as if the cell were intact. But the
principal life process, the oxidation to carbon dioxide
and water, is not found. Even slight injuries to the
cells are sufficient to annul this important process.
In such a juice it may be said that only the
preparative functions remain all of them processes

which we usually ascribe to ferments. If to the juice

j j

obtained in this manner a peptone containing very
sparingly soluble amino-acids is added as, for
instance, tyrosin or cystin or else a kind of peptone
in the building up of which an amino-acid takes
part and this may be easily detected at the moment
of decomposition by means of a colour reaction 5
then it is very easy to ascertain whether the juice con-
tains any ferment that is capable of splitting the
peptone in question. The precipitation of the respec-
tive amino-acids, or the appearance of the colour
reaction, announces the presence of the decomposing
agent.

Still more conclusive results are obtained if comr
binations of a know^n structure for instance, poly-
peptides, in the building up of which the above-
mentioned amino-acids take active part- -be chosen
for the experiment. Or one may follow the decom-
position in a polariscope tube. A certain quantity of

5 This is the case, for instance, with tryptophane.
4

5O METHODS FOR THE STUDY OF FERMENTS

the expressed juice is mixed with a measured solution
of an optically active polypeptide of known compo-
sition. The mixture is poured into a polariscope tube
and the rotation for the solution is ascertained as
quickly as possible. If one then determines the
rotation from time to time, an insight into the nature
of the decomposition is acquired. Instead of optically
active polypeptides we can employ racemic bodies.
The latter are optically inactive, because they consist
of two halves equally strong as regards their respec-
tive rotations in opposite directions. The peptolytic
ferments generally decompose only such polypeptides
as are built up out of the optically active amino-
acids as thev are found in nature. If we have to

j

deal with a racemic polypeptide, of which one-half
complies with this condition, then this part is
reduced to its component parts, and we are left with
the other half of the racemic body, which consists
of amino-acids not found in nature. We recognize
this asymmetric splitting through the fact that the
original optically inactive mixture becomes optically
active.

An example may convey a clear idea of these
conditions. In nature we meet the amino-acids
l-leucin and d-alanin, while d-lcucin and l-alanin
have never yet been found amongst the products of
reduction of the proteins. If we allow peptolytic
ferments to act on the racemic bodies d-alanvl

METHODS FOR THE STUDY OF FERMENTS 5!

l-leucin + l-alanyl d-leucin, then we obtain the
amino-acids l-leucin and d-alanin, and are left with
the compound l-alanyl d-leucin. This is optically
active.

Most interesting results are obtained when optically
active polypeptides are chosen for examination in the
building up of which several amino-acids take part.
As in these bodies the rotation of every possible
reduction stage is well known, it is easy to find out,
in the most exact and unequivocal manner, at what
particular stage the peptolytic ferment of a particular
tissue attacks the substrate employed. We have
thus a means at hand of comparing ferments of
different origins, together with the possibility of
recognizing, in the most exact way, all the specifically
active peptolytic ferments. Further development
of this field of research, by the use of the greatest
variety of substrates from all kinds of substances,
is required, in order to give an answer to the question
of the peculiarities of certain kinds of cells in
many directions. It will be possible in future to
recognize certain cells by the manner in which they
reduce substrates, the synthesis of which, as a matter
of course, must be previously fully known to us.

An example will make clear this method of study-
ing cell ferments. 6 The subjoined scheme supplies

6 'Here we have an enormous field, promising very fruitful
results with respect to the most varied problems connected

52 METHODS FOR THE STUDY OF FERMENTS

information on the power of rotation of three poly-
peptides composed of three amino-acids. At the same
time the optical relations of the individual decom-
posites are given.

(l) +20 (2) - 64

1-Leucyl-glycyl-d-alanin Glycyl-d-alanyl-glycin

(3)

d-Alanyl-glycyl-glycin

50

The explanation of our example (3) illustrates the
others as well. The tripeptide d-alanyl-glycyl-glycin
rotates + 30. If glycin (==glycocoll) were first split
off by a ferment, then the dipeptide d-alanyl-
glycin (see p. 53 (i)) would appear. The rotation
of the solution would rise towards the right,
because d-alanyl-glycin turns further to the right
than the original material. If, on the contrary,
d-alanin were set free first, then the rotation would
soon decrease to o, as the resulting dipeptide glycyl-
glycin is optically inactive (see p. 53 (2)).

with the chemistry of albumen, with studies in immunity, with
bacteriology, and so on, but which fails simply through lack of
means for the upkeep of a small army of capable young
chemists.

METHODS FOR THE STUDY OF FERMENTS 53

0)

d-alanyl-glycyl-glycin

+ 2 /

/ N

d-alanyl-glycin glycocoll

/~+**\

d-alanin glycocoll
, ^ v

+ 2

(2) + 3 I

d-alanyl-glycyl-glycin

d-alanin glycyl-glycin

/

f ^

glycocoll glycocoll

<? * ?

Finally, we may, for the purpose of tracing pepto-
lytic ferments in tissues, inject into the tissues
peptones and polypeptides, which contain sparingly
soluble amino-acids, and observe directly whether
any amino-acids are set free.

In all these experiments the co-operation of micro-
organisms was most carefully excluded, and there
can be no doubt that these ferments belong to the
tissues themselves. The same holds for ferments

54 METHODS FOR THE STUDY OF FERMENTS

that act on fats, carbohydrates, nucleo-proteids,
nucleic-acids, phosphatides, and so on. Everything
points to the fact that the cell has agents at its dis-
posal which render it capable of splitting up, into
their simplest units, all the complicated substances
which are brought to it, or which it itself builds up.
In favour of such a view, we may more particularly
cite, besides the direct proof of the existence of
ferments, the observation that in the metabolism of
the cell all the units, out of which the complicated
nutritive substances and the components of the cells
are built up, are found to occur.

At the present time there is no doubt that an
important part of the metabolic processes of the cells
is furthered by ferments. In general, we may say
that complicated substances are hydrolytically reduced
in stages until the simplest structural units are
formed. Once the latter appear, then the further
reduction continues in stages, through various inter-
mediate products, right down to the end-products of
metabolism, or else the resulting products of decom-
position form the starting-point for new .syntheses.
From these products very varied links are forged
between very different groups of substances.

It is thus proved that, in a certain sense, each
separate cell of the body is capable of digestion. This
holds particularly for the white and red blood cor-
puscles ; even the platelets are able to produce hydro-

METHODS FOR THE STUDY OF FERMENTS 55

lytic decompositions. The blood plasma is unable,
either in the majority of animals or in man, to produce
decomposition of albumens, peptones, and poly-
peptides, at least not in any degree which can be
demonstrated by available methods. The capacity
for decomposing fats is also apparently absent in
most cases. On the other hand, we often meet with
assertions that the blood always has a diastatic action,
i.e., the capacity for splitting complicated carbo-
hydrates. Under normal conditions the blood plasma
does not generally seem to be constructed for the
reduction of complicated substances. Only in the
l case of guinea-pigs do we find conditions that are
undoubtedly exceptional ; here the blood plasma
shows other properties, and even under normal con-
ditions can partly break down polypeptides which are
not in the least acted upon by the blood plasma of
other animals. The cause of this peculiar behaviour
of the plasma in guinea-pigs cannot yet be explained.
That the blood plasma in general is lacking in diges-
tive powers must obviously be construed in the sense
that, under normal conditions, substances which are
out of harmony with the plasma, and require a quick
chemical reduction, never have access to the blood.
As soon as these observations had been made it
become possible to study the question, whether the
blood plasma exhibits new properties in cases where
substances that are out of harmony with the plasma,

56 FORMATION OF DEFENSIVE FERMENTS

and particularly with the body, find their way into
the plasma of an organism by any other way than
through the intestinal canal. The order of these
experiments was of the following character :-

In the first place we determined the composition of
the blood plasma, or of the serum, in an animal, in
regard to the proteolytic and peptolytic ferments it
contained under normal conditions, that is to say,
when the nourishment is normal. The manner in
which this is done is as follows : 10 c.c. of blood is
taken from the animal under experiment, for instance,
from a dog, from the vena jugularis externa or from
a vein of the leg. Either this is left to clot of its own
accord, so as to separate out the serum ; or else o'i gr.
of ammonium oxalate is added to the test-tube con-
taining the blood, so as to prevent it from clotting.
The form-elements are centrifu^ed out, and the

o

clear plasma can then be withdrawn easily by
means of a pipette. In both cases serum and
plasma we must test for the absence of haemo-
globin, for, if it is present, then the red blood cor-
puscles have been broken up, in which case we may be
quite certain that the ferments belonging to the red
corpuscles have passed into the fluid derived from
the blood. Only serum and plasma which are
absolutely free from hemoglobin must be used for
these experiments. To a measured quantity of serum
or plasma a certain quantity, in cubic centimetres, of

FORMATION OF DEFENSIVE FERMENTS 57

an albumen, peptone, or polypeptide solution, having
a known composition in regard to substrates, is added ;
a polariscope tube is filled with the mixture, and the
rotation is quickly ascertained by means of a good
polariscope. The tube is then placed in an incu-
bator, and from time to time the angle of rotation
is again noted. To avoid mistakes another tube
is filled with the same quantity of plasma or serum,
and normal salt solution is added in the same quantity
as the substrate solution employed ; this mixture is
then observed in the polariscope under the same con-
ditions as the former. Finally, another test, with the
substrate solution alone, is arranged in the same way.
It is further essential to add to the mixture a measured
quantity of a phosphate mixture for the purpose of
preventing the action of the ferment from being in
anv wav influenced bv changes in the reaction

- o

of the mixture. To prevent cooling of the
polariscope tube its jacket is filled with water at
37 C., or an incubator is used, which can be fitted
to the polariscope (see below, the technique of the
optical method). Decomposition of proteins or pep-
tones could never be observed in these experiments,
so long as the blood was taken from healthy,
normally fed animals.

We now take the animal under observation, i.e.,
the animal whose plasma or serum we are investi-
gating, and introduce selected substances directly

58 FORMATION OF DEFENSIVE FERMENTS

into the organism, so as artificially to avoid the dis-
integrating action of the ferments of the intestines.
These substances are injected either subcutaneously,
or into the abdominal cavity, or else intravenously.
After a certain lapse of time blood is extracted, and
its serum, or plasma, is treated exactly in the same
way as we have described above.

The first experiments were made with dogs and
rabbits. White of egg, or horse blood-serum, was
introduced parenterally into these animals, that is to
say, avoiding the intestinal canal ; tests were then
made to see whether the plasma of the animals
under experiment either decomposed certain poly-
peptides, or whether it decomposed them quicker
than the plasma of the same animal did before the
injection of the disharmonious substance. The very
first experiments gave a positive result. It was found
that the contents of the blood increased in peptolytic
ferments. In a further experiment the substance used
for the injections was silk-peptone. It was found
that the serum of normal rabbits did not reduce
this peptone at all, the angle of rotation of the mixture
of plasma + peptone remaining constant. But if
silk-peptone be injected into an animal, and
the serum of the latter be then brought into contact
with this peptone, then, if we take a rapid reading
of the rotation in a polariscope, we find that the
initial rotation alters in the course of time.

FORMATION OF DEFENSIVE FERMENTS 59

Experiments were then made with gliadin and with
peptones obtained from gelatine, from edestin, and
from casein. Edestin and casein were also injected
by themselves. In all cases the result was the
same. When substances, that are out of harmony
with the plasma, are introduced into the plasma or
serum of a given animal, we always find developed a
special power of decomposing bodies belonging to
the protein series, especially protein itself and its
peptones. A specific activity of the injected sub-
strates could be traced only so far as that, after the
injection of proteins and peptones, ferments appeared
in the plasma which were able to chemically reduce
the derivatives of this group, but not, for instance,
fats or carbohydrates. Conversely, no splitting
of protein could be traced after injections of
fats, of carbohydrates, or of amino-acids. On the
other hand, after injection of a particular protein, or
of a particular peptone mixture derived from a definite
protein, not only were the injected substances
decomposed by the plasma, but the decomposition
extended to the whole group of proteins and their
nearest derivatives.

That the process actually depends upon the pres-
ence of ferments can be proved in two ways. In the
first place the splitting of a particular peptone
solution, by the plasma of suitably prepared animals,
was compared with the action of extracted yeast

60 FORMATION OF DEFENSIVE FERMENTS

juice on the same peptone. It was possible to
show that the decomposition in both cases was
very similar, i.e., that the initial rotation varied in
the same direction and to the same extent, whether
the plasma of specially treated animals was used, or
the active extract of yeast.

The following experiment proved, with excep-
tional clearness, that the plasma of an animal, when
specially treated, actually reduces proteins. The
plasma was mixed with gelatine or with white of egg,
and the mixture was placed in a dialysation tube.
Very shortly the presence of peptones could be
demonstrated in the outer fluid distilled water being
chosen for this purpose by means of the biuret re-
action. But when plasma of normal animals was
mixed with albuminous bodies and placed in a
dialysation tube, no substances giving the biuret
reactions could be traced in the outer fluid, even after
standing for many days. Finally, it has been proved
quite recently that, by mixing the plasma or serum
of specially treated animals with albumen, the nitro-
genous contents of the outer fluid increase to a con-
siderably greater extent than when the plasma of
normal animals and albumen are mixed together. In

o

the last case the increase of nitrogen in the outer fluid
is no greater than when the same quantity of plasma
is brought into the dialysation tube by itself, i.e.,
without addition of albumen. It must be understood

FORMATION OF DEFENSIVE FERMENTS 6l

that, in this experiment, the albumen has to be pre-
viously freed from all nitrogenous and crystalloid
admixtures by dialysis or by boiling.

When the plasma of specially treated animals,
which experiment showed to be active, that is, to split
up proteins and peptones, was raised to the tempera-
ture of 60 C. it became inactive, i.e., its decomposing
action could no longer be demonstrated.

The facts we have enumerated have been repeatedly
verified by numerous experiments. In all these
experiments on specially prepared plasma or serum
control experiments have, of course, been carried out,
-on the one hand with peptone solutions alone, on the
other hand with the plasma alone. Further, the
serum, or plasma, was made inactive each time,
for the purpose of avoiding any error, by raising
the temperature to 60 C. Finally it was shown,
by the use of the dialysation method, that the
observations made by means of the so-called optical
method were absolutely correct. It mav also be men-

j *

tioned that iodized albuminous substances were also
injected, and that no splitting action of the blood
plasma could be produced in this case. We know,
from other experiments, that iodized albuminous sub-
stances are decomposed with difficulty or not at all.
It seems likely that they are so intensely dis-
harmonious with the body that the organism, even
though armed with the proper ferments, can find no

62

FORMATION OF DEFENSIVE FERMENTS

point of attack from which to initiate their decom-
position.

Some examples which, in the form of curves, repre-
sent the results obtained from the disintegration of
mixtures of plasma, or serum, with some substrate
(albumen or peptone), may serve to illustrate the
above statements.

(i) A dog, whose serum did not decompose pep-
tones, was given 0*5 gr. of casein by means of a
subcutaneous injection on November 25 and 29 and
December 4. The blood used in the experiment
below was withdrawn on December 6. The polari-
scope tube was filled with a mixture of 0*5 c.c. of
serum, 0*5 c.c. of silk-peptone solution (10 per cent.),
and 7 c.c. of normal salt solution (see fig. i).

o

0,10

0,7J

Q2O"

Holt; s-

6 7

FIG. i.

(2) A dog was given repeated subcutaneous in-
jections of crystallized albumen obtained from seeds
of the gourd. The last injection was made on
December 8, 8 gr. of albumen being injected. The
serum was examined on the following day. For this
experiment i c.c. of serum was mixed with 0*5 c.c.

FORMATION OF DEFENSIVE FERMENTS

of a 10 per cent, gelatine-peptone solution, and 2-5 c.c,
of normal salt solution (see fig. 2).

0,30

Hou

>-.i

(3) The dog in this experiment was given on
October 18, 3 c.c. of a 10 per cent, silk-peptone
solution by subcutaneous injection. On October 21
blood was taken. The serum split up both silk-
peptone (curve a in fig. 3), and gelatine (curve c in
fig. 3). At a temperature of 60 C. the serum became
inactive (curve b in fig. 3).

\Ve may point out here that we thought it possible,
at first, that the phenomena observed by us might
have some connection with what is called anaphy-
laxy, or supersensibility. 7 By this we understand the

7 Hermann Pfeiffer, of Graz, at about the same time as, and
independently of, ourselves, has demonstrated the existence
of proteolytic ferments in the blood plasma of sensitized
animals, after we had already established the fact of the
appearance of peptolytic ferments subsequent to the intro-
duction into the blood of disharmonious derivatives of
albumen, and had in this way systematically treated the
whole problem. The first experiments were made with
albumen. They have since been abandoned, because the
results of an alteration in rotation seemed particularly
ambiguous in cases where the serum of animals, treated pre-
viously with albumen, was brought into contact with albumen
or peptone. For this reason polypeptides are preferable for
reactions on ferments, as being compounds whose exact struc-
ture is known to us.

6 4

FORMATION OF DEFENSIVE FERMENTS

extraordinary property possessed by the animal or-
ganism of responding with certain typical symptoms
to a second injection of the same material as was used
in the first injection. A certain time elapses in the
case of a guinea-pig, about fifteen to twenty days
before this state is overcome.

0,60

0,70

-0.60

-o,w

-0,30

Hours

3 V J

FIG. 3.

6 8 1O 15 2O

{a} i c.c. serum.

o'5 c.c. of a 10 per cent, silk-peptone solution.
5 c.c. normal salt solution.

(b) i c.c. serum at a temperature of 60.

o's c.c. of a 10 per cent, silk-peptone solution.
5 c.c. normal salt solution.

(c) i c.c. serum.

i c.c. of a i per cent, gelatine solution.
4'5 c.c. normal salt solution.

Cramp can be observed within different groups of
muscles, as well as a sudden fall of temperature,
&c. Peptones also can be demonstrated in the blood
after reinjection of the original protein. Various
authors have supposed that anaphylaxy is directly
connected with the production of derivatives of pro-
teins, particularly peptones, without, however, having

FORMATION OF DEFENSIVE FERMENTS 65

succeeded in supplying definite proofs for such a
view. It is onfly recently that experiments have
been made, by means of injections of peptones and
derivatives of amino-acids, especially of amines,
with a view to producing phenomena resembling
those of an anaphy lactic shock. It is difficult to
decide with any certainty what part is played, by the
ferments we have observed, in the setting up of
anaphylaxy. Several facts run counter to the suppo-
sition of a direct relation between the presence of
active ferments and the particular substrate against
which thev are directed. It has been proved, beyond
doubt, that these ferments exist in the blood at a time
when the anaphylactic shock cannot yet be produced
by a second injection of the same material as was used
in the first case. Further, it has already been pointed
out that these ferments are specific only in respect of
the group of substances which are used for th'e in-
jection, but not for the particular body that has been
introduced. To produce the shock, on the contrary,
the substrate, with which the animal under experiment
was rendered sensitive, must be present. A certain
importance, in regard to the setting up of the state
of shock, may be attached to the power possessed by
the plasma of decomposing albumen ; as is shown by
an observation which was made by Hermann Pfeiffer
and confirmed by ourselves, according to which the
proteolysis in the plasma disappears during the

66 FORMATION OF DEFENSIVE FERMENTS

moments following the anaphylactic shock, i.e.,
during so-called antianaphylaxy a state in which
the animal becomes absolutely insensitive towards
further injection.

If we summarize all the results obtained up to date,
we arrive at the conclusion that our observations with
regard to the appearance of ferments in the blood
plasma, after injection of disharmonious proteins and
peptones, undoubtedly stand in some kind of relation
to anaphylaxv. The special significance of these
ferments, however, remains uncertain. It would
appear possible that these ferments acquire some
special properties in the course of time, and then, by
decomposition of the second dose of albumen, give
rise to derivatives of a highly specialized nature and
activities. 8

There are many other possibilities to be considered.
The decomposition may not necessarily take place
only in the blood. Our method has at present only
demonstrated the appearance of ferments in the
plasma or serum, and that could only be done
because the ferments, which we find after parenteral
introduction of proteins and peptones, cannot nor-
mally be traced in the blood plasma of certain
animals. It is not unlikely that, after the introduction

Other substrates, which are also decomposed, may not
give the same derivatives, in which case a specific activity of
the material first injected would be assured.

FORMATION OF DEFENSIVE FERMENTS 6/

of substances out of harmony with the species, new
properties appear also in the cells of the body, and
that the latter undertake likewise the decomposition of
these disharmonious substances. In a certain sense
each individual cell would act in the presence of the
disharmonious material exactly in the same way as an
unicellular organism, and fight them to the extent
with which it is provided with the necessary weapons
-the ferments that enable it to make a successful
attack on the substrate. Like primitive .organisms,
too, it is able to protect itself against the penetration
of these substrates by means of the constitution and
quality of its walls, and so to wait until the modi-
fication of the substances has been effected elsewhere
to such a degree, that all their disharmonious pro-
perties have disappeared, and only an indifferent
product remains.

Finally, it may be that the whole problem of
anaphylaxy will not be resolved by purely chemical
considerations only. Why should not disturbances
originating from dislocations of osmotic equilibrium,
or activities of special ions, be taken into account,
and associated with the other observed phenomena.
(Cf. on this point Lit. 13.)

The more widely the limits of these problems are
extended, the more probable does it become that the
experimental testing of all possibilities will put us on
the proper road for an explanation of the phenomena

68 FORMATION OF DEFENSIVE FERMENTS

observed. Surely it would be absurd to limit the
study of anaphylaxy only to a study of the behaviour
of the blood ; for it is more than likely that it is
the cells of the body which ultimately play the chief
part in the appearance of anaphylaxy. The behaviour
of the blood plasma is possibly only a reflection of the
defensive measures adopted by the cells of the body ;
while, in any given case, it may be only a special type
of cell that has to be considered.

Special interest attaches to the proof of how 7 the
organism reacts when blood of its ow 7 n kind, or from
another animal species, is introduced into its cir-
culation. In the latter case ferments appeared in the
plasma, which decomposed albumens and peptones.
If harmonious blood were chosen from an animal
of the same race, no reaction whatever w r as noticed
when it was transmitted directly, i.e., without leaving
the blood-vessels. When, on the contrary, blood
which belonged to an entirely different race was
introduced into a dog, then a decomposition could
be demonstrated within the circulation.

Ap-ainst the results thus obtained one micrht raise

c? o

the objection that the appearance in the circulation
of active reducing ferments would give rise to enor-
mous disturbances, because even those albuminous
bodies that are in harmony with the plasma are
liable to be attacked by them. But this is evidentlv
not the case, since the plasma, though containing an

FORMATION OF DEFENSIVE FERMENTS 69

active ferment, retains its initial angle of rotation ;
and it is only in very exceptional cases that dialysis
shows the presence, in the outer fluid, of substances
that give the biuret reaction. It is only after proteins
or peptones are added to the plasma, that the activity
of the ferments first manifests itself.

How can we explain a behaviour that is, a priori, so
peculiar ? There are already, before the addition of
the proteins or peptones, large quantities of albumen
in the plasma in the presence of an active ferment.
We must always remember, in this connection, that
the ferments are directed, in a more or less explicitly
specific manner, against certain substrates. A slight
alteration in structure and configuration suffices to
remove a substrate from the influence of a cnven

o

ferment. Just as the ferments themselves are first
transformed into their active form by means of a
special agent, so, without doubt, the substances in
the blood and the cells which are presented to the
ferments need special agents to bring them into a
condition suitable for attack.

The substrates, too, are rendered active in a certain
sense. The body defends its cells, and the substances
contained in them, against disintegration by ferments
by giving them a structure and configuration it may
be that their physical condition also plays a part-
which are out of harmony with the ferments; and
from this point of view we can understand whv

70 FORMATION OF DEFENSIVE FERMENTS

the harmonious proteins of the plasma are not
attacked by the ferments which circulate in the
blood.

Finally, the question may be raised, why the
decomposition of parenterally introduced proteins
and peptones cannot be followed up directly, by
observations on the rotating power of the plasma,
without the addition of proteins or peptones. If the
appearance of proteo- and peptolytic ferments in the
plasma has the object of undertaking the decom-
position of the substrates introduced into it, then we
ought to be able to follow up the digestion the
decomposition in. the plasma itself. As a matter
of fact it has been found possible to demonstrate, by
means of intravenous introduction of large quantities
of proteins and peptones, after the animals have
been prepared by previous injections, that, when the
blood is withdrawn immediately, not only has an
alteration taken place in the original rotation of
the plasma, to which nothing has been added, but
also that peptones may be found in the outer
fluid in the dialysation test. That this demon-
stration does not generally succeed i.e., that
the decomposition of the substances that are out of
harmony with the body cannot be followed up by
means of observations on the plasma alone, without
the addition of substrates depends primarily upon
the fact, that the injected substances suddenly become

FORMATION OF DEFENSIVE FERMENTS /I

very much diluted, and then probably pass straight
into the lymph, and possibly also into the cells of
the body. The optical method is not so exact as to
permit us to establish very minute changes in
rotation, and, even if it were possible to observe such
rotations, it would be impossible to know for certain
whether the fluctuations were not within the limits
of errors of observation. Moreover, the decom-
position undoubtedly proceeds quickly, so much so
that we are really indebted to a lucky chance \vhen
we are able to follow up the decomposition of the
injected matter in the plasma itself. These are
the reasons why we have to prove the presence of the
ferments by means of substrates, against which the
respective ferments are directed. The substrate is
the reactive for its corresponding ferment, and the
decomposition of the former betrays the presence of
the latter.

It may be remarked that the clear establishment
of the presence of proteo- and peptolytic ferments in
the blood plasma, after injection into the circulation
of albuminous substances that are out of harmony
with the body, has supplied a real explanation of the
behaviour of parenterally injected proteins during
metabolism. There is no longer any doubt that
they are made use of, that is, that they are utilized
in the metabolism of the cells of the body, so far
as experience has shown decomposition to be

FORMATION OF DEFENSIVE FERMENTS

possible. Different observers (Lit. 4, 8, 10, u,
12, 16, 17, 18, 19), who have instituted experiments
on metabolism subsequent to parenteral introduction
of proteins, have suggested that decomposition by
means of ferments takes place outside the intestines.
This is most clearly stated by Heilner. This
suggestion, however, was only proved by the
direct demonstration of the ferments by means of the
experiments and methods we have described.

The positive knowledge that it is possible to induce
a splitting activity in the blood plasma of animals,
the plasma of which is otherwise unable to decompose
albuminous substances, by means of parenteral injec-
tions of these substances, led of itself to the problem
whether analogous phenomena appear when other
substances, which are out of harmony with the body
and the plasma, but do not belong to the albumens,
are used in such injections. We began with the
parenteral introduction of disharmonious forms of
sugar. In the first place it was ascertained that the
plasma or serum of dogs is unable to split up cane
sugar. If blood serum, or blood plasma, of a dog
be brought into a solution of cane sugar, it can
easily be demonstrated, by means of analytical
methods, that the cane sugfar does not undergo

j r*>

any alteration. Certainly no decomposition takes
place. The contents of the blood plasma are not
increased in respect of reduced substances. If,

FORMATION OF DEFENSIVE FERMENTS /3

however, in this experiment we use the blood plasma,
or serum, of a clog to which cane sugar has been
administered as an injection, either subcutaneously
or directly into the circulation, then, on bringing this
plasma and cane sugar together, we observe that
the reducing potentialities of the mixture are con-
siderably increased. Simultaneously, it is possible
to show that the quantity of the admixed cane sugar
diminishes.

These experiments give very positive results when
the splitting action of the plasma is investigated with
the aid of the optical method. In this case plasma is
taken from a normal dog in a certain quantity, and a
known amount of cane sugar solution is added ; a
polarization tube is filled with the mixture, and the
rotation of the latter is ascertained. The readings of
the polariscope are taken from time to time, and the
tube is kept during the intervals in an incubator at
37 C. It is found that the initial rotation keeps
constant.

Xow, if an injection of cane sugar is made into the
circulation of the same dog from which the plasma
was taken, it may be demonstrated after a very short
time that its plasma is now capable of breaking up
cane sugar. The strong rotation to the right, which
we observe at first, decreases continuously. It
approaches zero, and finally, passing zero, it travels
to the left. We obtain eventuallv a left-handed

74 FORMATION OF DEFENSIVE FERMENTS

rotation, the cane sugar being converted into invert
sugar. The latter consists of one molecule of grape
sugar and one molecule of fruit sugar; that is, of the
units of the disaccharide, cane sugar. Since the
fruit sugar turns more to the left than the grape sugar
does to the ri^ht a final rotation results to the left.

o

Many observations point to the fact that, at the same
time, part of the products of the decomposition suffer
further disintegration.

Parenteral introduction of cane sugar does not
always succeed in effecting the appearance of invertin
in the blood plasma. Obviously, the time during
which the disharmonious substance remains in the
blood plays an important part in the formation of the
defensive ferments. The cane sugar is very quickly
excreted through the kidneys. 9

The following examples will give an idea of the
results of these experiments :-

(i) A dog was given subcutaneous injections of
cane sugar (5 gr. at a time) on October 22 and 23.
The blood taken on October 24 was used for testing

9 It has been pointed out in original communications
that, in the parenteral introduction of carbohydrates, no such
regular results can be obtained as is the case with proteins :
for the latter remain longer in the circulation, and are
not usually excreted by the kidneys. The organism is, in this
case, directly dependent on the composition of the products for
its freedom from disharmonious substances. In the case of
cane sugar, the kidneys are able of themselves to deal with
the disharmonious compound.

FORMATION OF DEFENSIVE FERMENTS

75

the behaviour of the serum towards cane sugar.
To i c.c. of serum was added i c.c. of a 10 per cent,
solution of cane sugar and 5 c.c. of normal salt
solution. The initial rotation of the mixture was
+ 0*45. At the end of the experiment the rotation
had sunk to - 0*50 (see fig. 4).

(2) Blood was taken from a dog before the paren-
teral introduction of cane sugar, and the behaviour of

V,JV

1

QW
O,30"
O?0
QtO

o a

0,10
0,20

Q30

qw

/).V)

>-.*.

~^

x

\

1

v

\

"v

N

s.

\

V

X.

V

^x

>

*x.

,

Hours

5 JO 12 IV Iff

20 ZZ 21 26 2d 3O 35 W tS SO

FIG. 4.

the serum towards this disaccharide was ascertained.
Decomposition did not take place (curve i in fig. 5).
Then 10 c.c. of a 5 per cent, solution of cane sugar
were given to the animal by intravenous injection.
The sample of blood, taken fifteen minutes after the
injection, already showed hydrolysis of the cane sugar
that had been added (curve 2 in fig. 5). For the pur-
pose of control the rotation of the serum without the

FORMATION OF DEFENSIVE FERMENTS

addition of cane sugar was noted (curves .4 and B in
fig. 5). The arrangement of the experiment is shown
in the following summary :-

(i) o'5 c.c. serum (blood taken before the injection

of cane sugar).

o'5 c.c. of a 5 per cent, solution of cane sugar.
7 c.c. normal salt solution.

v,v& -

0,00
0,01
0,02
0,03
0,04
0,05

0,07
0,05
0,09
0,10
0,15
0,20
0,25

\

\

,

\

s

s

s^

s

^^^

~-~.

* ta

=5a

=^;

*

h-.

v.

^

*-

-^

(4

JJ

1

*3 & <s> <? -0 <>. *SJ J5 ^ <<5 T <s <"> N "> ^ ^ ^ C5 JJ

FIG. 5.

(2) o'5 c.c. serum (blood taken fifteen minutes after

intravenous injection of a solution of cane

sugar).

0*5 c.c. of a 5 per cent, solution of cane sugar.
7 c.c. normal salt solution.
A and B. 0*5 c.c. serum.

7'5 c.c. normal salt solution.

(3) Further experiments were undertaken for the

FORMATION OF DEFENSIVE FERMENTS

77

purpose of studying the question, how long after the
actual parenteral introduction of cane sugar the
presence of invertin in the blood serum may be
demonstrated. After a single subcutaneous injection
of cane sugar the power of decomposing this di-
saccharide was still traceable at the end of fourteen
davs (curve I in fig. 6). In a dog, which received a

4-0,35
4-0,30
4-0,25
4-0,20

4-0,15

+ 0,10
4-0,05
0,00
0,05

/) 7 ft Q

">^

^vJ=U

1 1

T

I

~>>_

_Svj

i

14pays~after-1 s -flnj

SOi

\

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tion/

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s,

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- U,1U
15

I

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w,a j

ft on o

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0,25
0,30
0,35
-fl 4/00

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FIG. 6.

subcutaneous injection of cane sugar on two occa-
sions, it was still possible to bring about an energetic
splitting of this disaccharide with blood serum after
nineteen days (curve II in fig. 6). The property
once acquired does not therefore disappear at once.
The individual experiments were conducted with the

78 FORMATION OF DEFENSIVE FERMENTS

following quantities of serum and cane sugar
solution :-

(1) 0*5 c.c. serum (blood taken fourteen days after

injection of cane sugar).

o'5 c.c. of a 10 per cent, solution of cane sugar.
7 c.c. normal salt solution.

(2) 0*5 c.c. serum (blood taken nineteen days after

the second injection of cane sugar).
0*5 c.c. of a 10 per cent, solution of cane sugar.
7 c.c. normal salt solution.

Control Test.

A and B. 0*5 c.c. serum.

7'5 c.c. normal salt solution.

These results, without our knowing it, confirmed
experiments which Weinland had made before us.
He had already been able to show that the blood
plasma of a dog is able to split up cane sugar;
that is to sav, it contains invertin as soon as cane

mf '

sugar is parenterally introduced. These experi-
ments were then later extended to other kinds of
sugar, and especially to milk sugar. It was pos-
sible to show that the latter also undergoes alteration,
but it seems that here, alongside of a hydrolysis, a
decomposition takes place in another direction.

Very extraordinary is the observation that, after the
introduction of soluble starch, and also of milk su^ar,

<T^

the blood plasma or serum is able to decompose

FORMATION OF DEFENSIVE FERMENTS 7Q

cane sugar. It seems as if here also, after the intro-
duction of a disharmonious kind of sugar, fer-
ments appear which are not exclusively directed
against the carbohydrate injected. Further, the
ability of the organism to supply ferments seems to
have limits, because, after an injection with raffinose,
no definite reaction could be traced. Very likely this
material is too markedly disharmonious with the cells
of the body.

It is interesting to note that ferments contained in
the plasma persist for a long time after the intro-
duction of disharmonious substances, whether they
be products of the albumen order or of the carbo-
hydrates. The splitting activity of the plasma could
still be clearly traced in individual cases up to three
weeks after the injection. Important, too, is the fact
that, after an intravenous injection of cane sugar,
invertin could be demonstrated in the blood plasma
within a quarter of an hour. When albuminous sub-
stances were introduced subcutaneously, then three
to four days elapsed before the formation of the
ferments attained its full value. After intravenous
injections they manifest themselves within twenty-
four hours. The fact that there are individual differ-
ences is also important. Moreover, the appearance of
the defensive ferments is very much delayed after the
introduction in large quantities of substances out of
harmony with the blood.

8O FORMATION OF DEFENSIVE FERMENTS

Finally, the behaviour of fatty products was tested.
In this case difficulties of method were encountered
at first. The experiment made to ascertain the decom-
position of fat in the blood, by simple titration of
the acids produced, failed entirely. The question,
whether, after the introduction of fats that are out
of harmony with the body and the plasma, an increase
follows of the amount of lipase in the plasma, could
only be attacked after Michaelis and Rona had
selected the alteration of surface-tension during dis-
sociation as the basis of a method for the study of
the decomposition of fats. The fats belong to a
group of substances that are strongly surface-active,
while the products of disintegration produced by their
decomposition, such as alcohol and fatty acids,
possess no marked influence over surface-tension. . If
plasma of a normal animal be mixed with any kind
of fat, such as tributyrin, and the mixture be allowed
to flow from a capillary tube, a certain number of
drops escape in a given time. But if any fat gets
into the animal's circulation by whatever means, then
the number of drops escaping through the capillary
tube decreases.

As far as we can judge from the experience gained
up to the present, it seems that the conditions met
with in fats are much more complicated than in the
case of proteins and polysaccharicles. This experi-
ence tells us that while, under normal conditions,

FORMATION OF DEFENSIVE FERMENTS 8 1

proteins of a definite kind, and in obviously definite
quantities, are always circulating in the blood, and
the amount of carbohydrates also varies only within
narrow limits, the fats behave quite differently. The
amount of fats in the plasma varies within a wide
range. After a meal rich in fat we find so much fat in

C5

the blood plasma that it may be seen with the naked
eye ; and, if we let the plasma stand, the fat
separates out directly and appears as a layer on
the surface of the plasma. A short time after the
meal the fat disappears again from the blood. It is
transmitted to the different cells of the body, and is
, there used up, transformed, or even directly stored
as reserve material. It seems that the blood, with
every increase in the amount of fat, responds with an
increase of lipase. From the point of view we have
laid down the excess of fat has to be considered as
being out of harmony with the plasma. Only in an
animal whose stomach is completely empty do we find
no, or very little, capacity for splitting fats. After
a meal rich in fat active lipase can be demonstrated
in the blood. It can also be shown that during a
more or less prolonged hunger period the splitting
power of the blood increases. This corresponds with
the experience that, during fasting, an active trans-
portation of material is going on. In many cases of
fasting large quantities of fat could be shown to be
present in the blood. If a fat be introduced that is
6

82 FORMATION OF DEFENSIVE FERMENTS

out of harmony with the species, we rind in the plasma
an exceedingly high capacity for splitting fats.

In the case of fats we meet with some difficulties
when we try to introduce, into the circulation, fats
that have not been modified so as to be in harmony
with the plasma. If they are injected subcutaneously
they remain at the point of injection for a long w r hile,
and are probably only transported further after the
actual splitting has begun. In cases of intravenous
injection one runs the risk of killing the animal,
owing: to fatty embolisms. The introduction, into

f^ J

the blood, of a fat that is out of harmony with
the species could only be effected for the first time
.after an old experiment of J. Munk had been made
use of, namely, to gorge an animal with an excessive
quantity of fat, so that it may easily be demonstrated
in the tissues and, naturally, also in the blood. We
fed in this w r ay on large quantities of rapeseed oil
and mutton suet, and. then found in the plasma a
very strongly marked capacity for splitting fats.
We may mention here, also, that the same effects
may be produced, with proteins and peptones, and
also with carbohydrates, as in the case of paren-
teral injection, if an excess of these substances is
forced through the intestines bv flooding the intes-

J O

tinal canal with the particular nutriment. We would
also emphasize the fact that a state of anaphylaxy
may be successfully set up by this means. If we

FORMATION OF DEFENSIVE FERMENTS 83

introduce into an animal a large amount of white of
egg, there is no doubt that unaltered protein passes
into the circulation. It is also possible that peptones
are absorbed, which still have the specific structure
of the white of egg. This transfer may be ascer-
tained by the so-called biological reactions, precipitin
reaction, &c., but chiefly and most exactly by demon-
stration of the existence of peptolytic ferments in the
circulation. If, after a certain interval, white of egg
is once more introduced by either parenteral or
enteral means in the latter case the supply must be
very copious the state of shock is obtained. 10

Seeing that harmonious fats, as we have already
mentioned, also produce in the circulation an in-
creased power of splitting fats, it is rather difficult to
determine whether disharmonious fatty products give
rise to a definite specific activity. Further experi-
ments will be needed to determine this point.

Finally, we have also made injections of nucleo-
proteids, nucleins, and nucleic acids, introducing
them into the organism in such a way as to prevent
them passing through the intestine. We found that,
after introduction of these bodies, ferments appear in
the blood plasma in increased quantity, and quickly
reduce these substances (see also Lit. 21). Moreover,
it could be shown that it was possible, by means of

10 Enteral sensitization and subsequent enteral shock have
been successfully accomplished by us on two occasions only.

84 FORMATION OF DEFENSIVE FERMENTS

certain nucleo-proteids as well as nucleins, to excite
anaphylactic phenomena of a very specific character.
Experiments on guinea-pigs, which were made in
collaboration with Kashiwado, showed that a second
injection of the same substance as was used in the
first instance produced specific cramps of the muscles
of the neck and of the jaw. Moreover, an increased
peristalsis was regularly present, the animals ex-
creting faeces continuously. Symptoms of lameness
soon set in, and a marked fall of temperature always
took place. We injected, for instance, nucleo-
proteids, nuclein substances which had been obtained
from the thymus, and finally nucleo-proteids from the
blood corpuscles of a goose ; the reaction was in all
cases a strictly specific one. In the case of nucleic
acids we were unable to get definite results, and it
would appear that these cannot produce anaphylactic
phenomena. It may be that, in the nucleo-proteids
and in the nucleins, their albuminous components are
the deciding factor. A systematic study of the nuclear
structure of the various cells of the same individual
may enable us to determine the question, whether
albumens that are in harmony with the nucleus take
part in its construction, or whether the nucleus plays
a part, in the cellular metabolism, which is repeated
in an identical manner in the various cells of the same
individual.

So long as purely chemical research is unable to

FORMATION OF DEFENSIVE FERMENTS 85

answer questions concerning the finer structure of the
cell elements, we are bound to resort to indirect
methods. The latter have, in a relatively short time,
opened up a vast field, and discovered views of the
widest interest in regard to every kind of cellular pro-
cess. It is the duty of the future to follow up with
exact methods all the observations that have been
made, and to replace with known quantities the many
unknowns with which our present-day methods have
still to reckon.

If we sum up all the phenomena observed, we get
the following picture : By introducing substances
which are out of harmony with the species, and more
particularly with the plasma, we bring into the
organism bodies which are, in their whole structure,
absolutely disharmonious with the cells of the body.
No alteration has as yet taken place. In order that
these substances may be utilized by the cells of the
body, the products that are suitable for the organism
must be so far decomposed as to entirely lose their
specific character. This decomposition is effected bv
ferments, and is certainly initiated very quickly, for
the disharmony with the blood or plasma and the
body may extend also to the cells, and have an in-
jurious effect on them. During the decomposition
of these substances products arise at first which are,
in part at least, definitely disharmonious with the

86 SIGNIFICANCE AND ORIGIN OF (DEFENSIVE) FERMENTS

organism, and these may be equally injurious under
certain conditions. If these products of a gradual
decomposition constantly appear only in small quan-
tities and further decomposition is very rapid, then
the injury will be but trifling and transitory. When,
on the contrary, a large quantity of these products
of decomposition suddenly appear, they can produce
serious disturbances by their combined action. In
these processes it is not only their chemical nature,
their structure and confio-uration, that is of im-

O

portance ; we have to remember that, during the
decomposition of colloid substances, products arise
which exert an influence upon the osmotic pressure,
and may, in consequence, disturb the existing equili-
brium. What we observe in the plasma also takes
place, as we have already emphasized, in the interior
of the cells, and probably in a similar way. It may
be pointed out here that, when bodies of a simple
constitution, such as crystalloids, are introduced, the
organism is able to defend itself, not only by decom-
posing such disharmonious substances, but also by
excreting a part of them, at any rate, through the
kidneys. The same method of defence may be
resorted to when, during the decomposition of com-
plicated substances, simpler particles are produced.
In this case the excretion accelerates the ejection of
the disharmonious substance from the body. It is
true that, in doing this, the organism loses valuable

SIGNIFICANCE AND ORIGIN OF (DEFENSIVE) FERMENTS S/

fuel on the one hand, and certain constructive
units on the other.

Many observations point to the fact that parenter-
ally introduced substances, in so far as they can be
modified, are utilized by the organism ; that is, they
serve as nourishment. The digestion, which would
otherwise take place in the intestine and prevent the
passage of disharmonious material into the body, is
performed by the blood.

It is an open question from what source these fer-
ments, which we are going to call defensive ferments,,
take their origin. Many facts accord with the sugges-
tion that the leucocytes play a part in this connection
(see also Lit. 23). They probably give off these
ferments to the circulation. If so, we should then
have in the blood plasma phenomena more or
less analogous to those observed, for instance, by
B. Friedrich Miiller, during the dissolution of the
fibrin that is excreted into the alveoli in cases of pneu-
monia. We see here numerous leucocytes penetrating
into the solid exudate and dissolving it, after which an
absorption of the products of decomposition begins,
a kind of digestion taking place in the interior of
the alveoli. Here also, as can be shown by special
experiments, ferments can be demonstrated in the
contents of the alveoli (in the expectorated sputum) *
and these ferments take their origin from the leuco-
cytes. The old view, whereby the leucocytes take up

88 SIGNIFICANCE AND ORIGIN OF (DEFENSIVE) FERMENTS

substances from the outside and digest them, must
now be completed by the observation, that ferments
can be given off to the exterior, and that therefore
digestion may be accomplished outside the cell. We
would like for the present to leave the question open,
whether any importance can be ascribed, in this con-
nection, to the white blood corpuscles generally, or to
any special forms of these. We presume that the red
blood corpuscles as well, and very likely also the blood
platelets, play an important part in these processes.
The presence of ferments in these cells must not, it
is true, be unconditionallv connected with the forma-

./

tion of defensive ferments, because it is clear that
these cell elements must have means of reducing
their nutriment to simpler molecules, and construct-
ing their own bodies. In any case, it is extra-
ordinary that, in these kinds of cells, there are
such active ferments present, and in such large quan-
tities. According to our experiments, the splitting
processes in these cells take place much quicker than
in the other cells of the body. It is certain that the
red blood corpuscles have, besides the function of
transporting the oxygen, other duties to fulfil in the
'economy of the organism. We further consider it
quite possible that the same cells, which give off
insufficiently harmonized products to the blood, also
supply the ferments which are able to complete the
decomposition in the circulation.

SIGNIFICANCE AND ORIGIN OF (DEFENSIVE) FERMENTS 89

According to our observations, there is not the
slightest doubt that the animal organism is not left
without means of defence against disharmonious sub-
stances. If such products make their way into the
body, the latter sends out defensive ferments that are
directed against special kinds of substrates. Not only
do they effect the destruction of the specific character
of the parenterally introduced substance by means
of an extensive decomposition, but they render
possible the utilization of the products of the decom-
position in the general metabolism. The reaction
we have demonstrated enables us at any time to decide
whether a certain substance is in harmony with the
body or not. We have already emphasized the fact
that we must distinguish not only substances that are
in, or out of, harmony with the body, but also those
which are in, or out of, harmony with the blood or its
plasma, or again with the cells. We have already
described how the intestine, with its ferments and
those of its accessory glands, decomposes all dis-
harmonious substances until an indifferent mixture of
only the simplest units is left; and how then the cells
of the gut- walls, and of the liver, carefully test the
absorbed products for the absence, or transforma-
tion, of all substances that are out of harmony with
the body and blood. Moreover, all the cells of the
body take care that nothing shall pass from them
into the circulation which has not attained a certain

9O BIOLOGICAL DIAGNOSIS OF PREGNANCY

grade of decomposition. For further protection, the
lymph, with all its complicated arrangements, is inter-
posed between the circulation and the cells of the
body. Here everything is tested afresh ; and nothing
is let loose into the circulation that has not been
rendered harmonious with the blood and its plasma.
For ourselves, we have scarcely any doubt that the
lymph system plays an important part in metabolism,
along the lines we have indicated. Sometimes sub-
stances are reduced and converted into harmonious
material ; sometimes products of a definite type are
built up. The lymph is, as we have pointed out
before, to be considered as a sort of buffer between
the cells of the blood and those of the body ; as a
neutral zone, in which evervthinor is assimilated as

J O

far as possible.

If these views are correct, it should be possible
to trace such substances as are in harmony with the
body, but not with the blood and its plasma, by look-
ing for definite ferments. It is quite conceivable
that, in certain diseases, the cells only partially effect
the decomposition of the nutritive material and of
the constituents of the body, and that, to a certain
extent, materials that are harmonious only with the
cells are handed on to the lymph. The lymph, as
already pointed out, would in many cases do its best
to correct this failure bv means of its cells, the leuco-

j *

BIOLOGICAL DIAGNOSIS OF PREGNANCY

cytes, and its special organs, the lymphatic glands,
and would attempt to decompose some of the dis-
harmonious products before they reach the blood. In
many cases, however, disharmonious material will get
into the blood, and produce all kinds of disturbances.
We know of at least two conditions in which dis-
harmonious substances undoubtedly circulate in the
blood, namely, Bence Jones's albuminuria, and preg-
nancy. In the latter case it is certain that we are
deal in cj 1 with substances that are in harmony with the

O

species, while in the first case there is a possibility
that disharmonious substances are present. Bence
Jones's albuminuria is nearly always found in con-

J *

junction with sarcoma of the bones. Whether the
sarcoma is to be regarded as a harmonious tissue or
not, we are not yet in a position to decide.

As to pregnancy, we know, thanks to the important
observations of Schmorl, that the cells of the chorionic
villi may be torn off, and be carried into the circula-
tion. Yeit lias indeed proved that these cases are rela-
tively frequent. 11 Weichardt, and later also Richard
Freund, tried to connect the appearance of chorionic
villi in the circulation with eclampsia. Weichardt was
thinking of a dissolution of the cells cytolysis in
which case toxic products are supposed to appear.

11 Cf., on this point, Hans Hinselmann, " Die angebliche,
physiologische Schwangerschaftsthrombose von Gefassen der
uterinen Plazentarstelle/' Ferdinand Enke, Stuttgart, 1913.

92 BIOLOGICAL DIAGNOSIS OF PREGNANCY

To us these observations and views had this much
value : that they directed our attention to the fact that,
during pregnancy, the appearance of substances,
that were in harmony with the species but not with
the plasma, might be possible. If our view is correct,
that the animal organism sets free ferments of a
special kind, as soon as material that is in harmony
with the species, but not with the plasma, passes
into the blood, then it ought to be possible to demon-
strate the existence of such ferments during preg-
nancy. As, however, the ferments, as we know from
experience, disappear fourteen to twenty-one days
after the actual introduction of disharmonious sub-
stances, it was scarcely possible to expect that defen-
sive ferments should be met with during the whole
period of pregnancy. One had to experiment with a
great many cases, before striking that one in which
dissolution of the cells of the villi had just taken place.

Experience, however, soon showed that the serum
of pregnant individuals always contains defensive
ferments which are directed against placenta
albumen. We cannot, therefore, maintain that the
tearing off of the epithelium of the chorionic villi is
the only cause of the appearance of the defensive
ferments. It must be remembered that even the
mare exhibits, during pregnancy, defensive ferments
which are directed against placenta albumen ; yet the
placenta of the mare is related to the circulation in

BIOLOGICAL DIAGNOSIS OF PREGNANCY 93

such a way as to exclude the possibility of the cells
of the chorionic villi getting into the blood.

How can we then explain the existence of the
defensive ferments during pregnancy ? It can only
be in very exceptional cases that they are produced by
the invasion of morphological elements. In most
cases it must be the result of the transfer of particular
bodies the constituents of particular cells, or the
products of their decomposition. It may be, of
course, that the extraordinarily active metabolic pro-
cesses, which arise at the junction of the maternal and
foetal organisms, result in an insufficient reduction
of many of the products of the cells of the placenta;
the metabolism, in short, overreaching itself through
its own rapidity. It is, however, also conceivable
that the cells themselves break up easily.

The following view is probably the correct one.
The organism of the mother has at its disposal, up to
the appearance of pregnancy, a certain amount of cells
of a certain kind, which all harmonize, in their meta-
bolism, with each other. Now, with conception,
comes the appearance of an entirely new kind of
tissue, which has to perform particular duties.
Although the impregnated egg and the developing
placenta, with all its various cells, are in harmony
with the species, nevertheless the metabolism of all
these cells appears as something quite new and
strange to the complex of cells composing the

94 BIOLOGICAL DIAGNOSIS OF PREGNANCY'

organism of the mother. The blood probably receives
substances perhaps also secretions which are out of
harmony with the plasma, and remain so ; and the time
is too short for the blood to accustom itself entirely to
these new kinds of substances. The placenta, accord-
ing to this point of view, together with the foetus,
never settles clown completely within the organism of
the mother. With the expulsion of the placenta, in
which process, again, ferments probably play a pre-
paratory part, the ferments which are directed against
its albumen disappear fairly quickly. It is, of course,
quite possible that these placentally active ferments
are conditioned by many different forces.

The view we have just discussed gives us an oppor-
tunity of demonstrating the initiation of the function

J O

of a particular organ. Were an organ to suddenly
take up a particular function at a particular moment
-say the delivery of a particular secretion then it
is quite conceivable that its activities would, at first,
be disharmonious with the plasma. We might pos-
sibly expect something of this kind from the sexual
glands at the onset of puberty ; but experiments
carried on in this direction upon animals in heat have
not as yet given any certain result. On the other
hand, the cessation of the functions of a particular
organ might lead to the appearance of substances out
of harmony with the blood; for these functions clo not
cease suddenly, and it may be that their gradual dis-

BIOLOGICAL DIAGNOSIS OF PREGNANCY 95

continuance leads to the appearance of insufficiently
decomposed products. Involution, too, may be the
cause of the formation of products that are out of
harmony with the blood ; in which connection we have
in mind more especially the degeneration of the
thvmus, and the climacterium.

J

Many of our own researches, and those of various
j

observers, have shown that, during the whole period
of pregnancy, defensive ferments circulate in the
blood, which are able to reduce placenta albumen.
These ferments may be demonstrated within about
eight days after impregnation. Their presence is,
without any doubt, dependent upon the circulation
of disharmonious substances originating in the
placenta ; since the defensive ferments disappear
within fourteen to twenty-one days, when the
relations of the placenta with the maternal organism
have ceased.

Attempts have also been made to decompose
placental tissues by means of the blood serum of the
foetus. No digestion could be induced; nor can the
serum of pregnant individuals be made to attack
the tissues of the foetus. In any case these obser-
vations must be carefully followed up. It might
be imagined, a priori, that there are develop-
mental stages, in which the tissues of the foetus are
as yet so little differentiated, that they are still of a
generalized character. Xor did umbilical blood

96 SPECIFICITY OF DEFENSIVE FERMENTS

serum show any decomposition, when mixed with the
serum of impregnated individuals. Had that been
the case, then a very simple method for the diagnosis
of pregnancy would have been found.

From the experience we have already gained, we
may venture to say that the behaviour of blood
serum towards coagulated placental tissue, or to
placenta peptone, enables us to diagnose a state of
pregnancy in the clearest possible way ; or, expressed
more correctly, to decide whether a placenta is in
existence which is still in communication with the
organism of the mother. A limitation is only neces-
sary, because the defensive ferments can still be traced
for a certain time after the expulsion of the placenta.
For the practical sero-diagnosis of pregnancy this
circumstance is of little importance, because the
case under observation can be examined clinically.
Normal non-pregnant individuals do not show any
disintegration of the placental tissue.

We now had to decide the extremely important
question, whether defensive ferments of a more
general nature appear, when the organism contains
other substances that are out of harmony with the
plasma. This problem may be precisely expressed in
the following manner : Does the serum of individuals,
who suffer from infectious diseases, or from carci-
noma, or who exhibit a disease of any other kind,
decompose placental tissue ? A priori, one was driven

SPECIFICITY OF DEFENSIVE FERMENTS 97

to the conclusion that they would do so, because it
had been noticed that, after the introduction of dis-
harmonious substances into the circulation, not only
do ferments appear which reduce the particular com-
pound inoculated, but the defensive ferments pro-
duced attack many other substances of the same order.
There was no production of strictly specific ferments,
but only of those which are limited in their action
to a particular class of compounds. And when sub-
stances that were in harmony with the body, but not
with the plasma, were chosen for these experiments,
no ferments appeared that were strictly specific.

We tested the sera of tuberculous individuals, of
sufferers from carcinoma, of persons with salpingitis,
and others, for their behaviour towards placental
tissue, but in not a single case did decomposition
take place. To our great surprise it appeared, that
the animal organism onlv sets free strictly specific fer-
ments, when particular cells are themselves giving off
substances which are not in harmony with the plasma.

How can we explain this different mode of
behaviour, according to whether we introduce
such disharmonious substances, or whether the
organism itself supplies them? There are various
possibilities to be considered. In the first place
the cell may give off the substances in question
only in minute quantities, a condition which we are
unable to imitate. Our methods of interference are
7

SPECIFICITY OF DEFENSIVE FERMENTS

always brutal, and at once produce pathological con-
ditions. We cannot imitate this method of supplying
a substance in minute quantities, simply because we
have no means of controlling the essential mechanism
of regulation, owing to our ignorance of its nature.
By our injections of disharmonious material
we suddenly alter the composition of the blood,
and injuriously affect the whole organism. It is,
in this respect, most interesting that defensive fer-
ments which only decompose cane sugar, can be ob-
tained when, for instance, cane sugar is injected in
very small quantities. As the quantity of cane sugar
is increased then, very often, the blood serum is able
to dissociate milk sugar as well. But, if too much of
the above kind of sugar be introduced into the blood,
then as a rule defensive ferments do not appear at all.
Further, it is possible that the substances, which we
artificially introduce into the bodv r , are not sufficiently
fine in structure to produce specific ferments, specially
directed against them. The cell gives off its
particular disharmonious substances with their
specific features stamped on them, while we, on the
contrary, bring into the circulation material that has
already been altered. This difference may be illus-
trated in the following manner. Suppose, on the one
hand, that two persons start to fight each other with
two ' specifically ' chosen weapons. Such a fight
is premeditated ; the weapons are precise, and the

SPECIFICITY OF DEFENSIVE FERMENTS 99

defensive measures adopted depend on their nature.
In another case two individuals throw themselves
brutally one upon the other without any previous
choice of weapons ; then any means is good, if it only
succeeds in overcoming the opponent somehow.

One fact must always be kept in mind. When
we introduce proteins or peptones and the like into
the blood-vessels, these are never pure compounds.
Together with the peptones we certainly introduce
into the circulation a number of different stages
of the decomposition of proteins. Suppose we take
the white of an egg, then without doubt innumerable
albuminous substances are present, which greatly
differ one from the other. We must not forget the
fact, that traces of particular substances are quite
sufficient to cause the formation of ferments. The
cells, on the contrary, set free substances that are
probably of a very precise character.

If we artificially introduce material that is out of
harmony with the blood, the animal organism reacts
against it with a whole complex of defensive ferments,
because our material is always a mixture of sub-
stances. It is to a certain degree prepared for all
eventualities. It has no actual knowledge of the
characteristics of the introduced product. If, at anv
point, particular cells exhibit a deficiency of function
in respect of their metabolism, then there always
appears in the blood plasma, from one moment to

IOO SPECIFICITY OF .DEFENSIVE FERMENTS

another, just a trace of some specifically organized
substance, and this is at once deprived of its essential
properties by means of the opposing defensive
ferment. It might be thought that the same cells
which give off the disharmonious material would
themselves supply the ferments, and transfer their
further reduction into the plasma ; but at present there
are no proofs for such a suggestion.

This view may be opposed on the ground, that it
is difficult to understand how specifically directed
ferments can be distinguished in boiled tissues.
Many of the iiner features in the structure of the
substrate must surely be obliterated in the pro-
cess of boiling. This probably holds only for the
physical properties and hardly at all for the chemical.
We may boil a substance having a composition
A B C D, and another having a structure B C D A,
for a long time ; both would still retain their original
composition or structure, although their physical
properties might undergo alteration. Thus, for
instance, their rotation mi^ht alter, and to this extent

o

their biological behaviour might be affected. Thus
there is nothing against the idea, that the substrate
against which the ferment is directed may, in spite
of its newly acquired properties, be still liable to
attack by the ferment. With a key corresponding to
a particular lock we can still unlock the latter, even
though it has been badly damaged, provided only

SPECIFICITY OF DEFENSIVE FERMENTS IOI

that the key can fit into the guide, and push back the
bult. The whole of the rest of the lock may, in this
case, have suffered considerable alterations.

The ferments attack a particular substrate at a
particular point, and very probably always combine
with the groups against which they are directed. It
is only secondarily that disturbance occurs of the
equilibrium of the compound. So long as this point
of attack remains unchanged the ferments are able to
act, but the conditions become very different when
the much altered product, with all its groupings, is
brought into the circulation. If the decomposition is
to be a complete one, then a number of ferments has
to act. The new conditions, caused by the denatur-
ation, produce their full effect after the introduction
into the circulation. When we search for ferments by
means of boiled tissues, we expose a variety of proteins
to the action of ferments. Only that grouping of
atoms, towards which the ferment is directed, has to
be considered here. All other groups may be neglected,
because it would be scarcely possible to presume that
the boiling has produced structural conditions which
are accessible to ferments, although the natural sub-

o

strate was not so accessible. Rather must we reckon
with the possibility that a too extensive denaturation
will so strongly modify an original grouping, that is
accessible to ferments, that the ferment then becomes
inactive. The grouping has now become foreign to it.

IO2 SPECIFICITY OF DEFENSIVE FERMENTS

Much weightier still, at first sight, is the following
objection. In the dialysation process we make use
of coagulated albumens, when looking for defensive
ferments. In the optical method peptones are used
which are produced from these. Is there not a con-
tradiction between our methods of research, and the
ideas developed above with respect to the cause of
the origin of the defensive ferments ? If we surmise
that the defensive ferments serve the purpose of de-
composing disharmonious substances, built up from
numerous units, into their single units, then we
must further agree that ferments are present which
can carry on the decomposition at least so far, that
the specific characteristics of the cells are entirely
destroyed. Therefore we ought to expect that, where
proteins are decomposed by defensive ferments, pep-
tones will also be destroyed provided we take, as
substrates, those which correspond with the normal
fermentative reduction stages of the original material.
If, then, we agree that a substance that is out of
harmony with the plasma always has an albuminous
character, i.e., that the defensive ferments commence
their decomposition at this stage, then there is
no difficulty in imaefinino- that the dialvsation pro-

j O O -

cess and the optical method lead to similar results.
In the first case we allow the defensive ferment to
commence the decomposition at the albuminous stage,
and establish the appearance of the crystalloidal, dif-

SPECIFICITY OF DEFENSIVE FERMENTS IO3

fusible stages (peptones). In the second case, we pre-
pare the way for the ferments by producing peptones,
which we then allow the ferments to decompose. So
we allow serum of pregnancy to act upon a boiled
placenta albumen, while in the optical method we
use peptone produced from placenta albumen. In
the first case, the appearance of diffusible stages of
decomposition in the dialysate shows us that the
decomposition has begun. In the second case, the
change that takes place in the rotation enables us to
infer a change in the composition of the added sub-
strate, namely, of the peptone mixture.

Now very often, and probably in the great majority
of cases, albumen itself does not pass into the circu-
lation, but rather the products of its decomposition.
We may easily understand that the optical method
Drives us trustworthv results, because it is quite

r?

possible that the peptone mixture used may also
contain those stages of decomposition which have
acted disharmoniously with the plasma. How, then,
can we establish a decomposition of albumen ? In
those cases, for instance, where the cells of the body
give off to the blood plasma peptones which still bear
the characters of the cell, we start, in the dialvsing
process, at a higher stage of decomposition than was
the case in the blood itself.

Unfortunately, we have no knowledge whatever

. o

concerning the nature of the ferments. We realize

IO4 SPECIFICITY OF DEFENSIVE FERMENTS

them exclusively by the manner in \\iiich they act, and
that is whv, in must questions relating to ferments
and their activity, we are only able to answer with con-
jectures. We may imagine that a ferment is directed
against a simpler product of decomposition, and yet
that it attacks at the same time a more complicated
molecule, in so far as the group through which it
attacks the substrate is actually present therein and
within reach. It only depends on whether the
ferment is able to find a group, corresponding to its
own structure and configuration, in the particular
molecule. We must, also, not forget that, in a
body of high molecular structure, the same group-
ing may recur many times. In any case we consider
it very possible that cases are met with in which
the optical method shows a decomposition, while the
dialysation process gives a negative result ; although
it must be admitted that, up to the present, not a
single case of this kind has been satisfactorily proved.
All these discussions would be superfluous, if we
only knew, on the one hand the nature of the fer-
ments, and on the other the components that are
out of harmony with the plasma. As it is, we are
simply faced with the fact that ferments, directed
against particular substrates, are to be found in the
blood serum under very definite conditions. What
is entirely new is the clear demonstration of the fact
that the animal organism, within certain limits,

SPECIFICITY OF DEFENSIVE FERMENTS IC>5

generally defends itself, by means of ferments, against
compounds that are capable of decomposition and
that consist of many elements. New, too, is the idea
that by means of these ferments we can judge as to
the functions of particular organs. Finally, the idea
that the animal organism sets free specifically directed
ferments, and that, in so doing, it registers the fact
that the components of its various kinds of cells have
an exclusive structure corresponding to each kind of
cell, is also new.

Objection has been taken to the idea of strictly and
specifically directed ferments on the ground, that it
is impossible to accept the idea that specific reactions
take place, because the so-called " antitrvptic power '
of Henkel-Rosenthal, the " cobra-poison haemolysis '
of Heynemann, and finally the " catalysator
influence ' of Weichardt, are not specific. It is for-
gotten that the fermentative decomposition represents
the primary activity, and that those substances, which
are in question in the above-mentioned methods,
are produced secondarily by the defensive ferments.
That, during the process of decomposition, the
original characteristic structure of a compound is soon
destroyed, we have repeatedly affirmed. All possible
stages of decomposition, of the most different origin,
can in many respects act identically. Thus, for
instance, it is possible to prove conclusively that the
hydrolysis of the dipeptide, d-alanyl-glycin, can be

IO6 SERODIAGXOSIS OF ORGANIC FUNCTIONS

retarded by the addition of optically active a-amino-
acids. It is a matter of indifference what kind of
a-ami no-acid is used, provided only it belongs to the
units of the protein. We demonstrate the decom-
position of a specifically constructed substrate, while
the methods in question are concerned only with the
influence of the products that are split off.

Of course, the mere fact, that it has been found
possible to found a sero-diagnosis of pregnancy on
the principles described above, would not give us the
right to speak of a sero-diagnosis of the functions
of organs. Further research, based on these prin-
ciples as well as on the methods described, has,
however, given us results which entitle us fairly to
presume, that a new road has already been found for
the development of our knowledge of the structure
and metabolism of cells, under normal and patho-
logical conditions.

As, meanwhile, our knowledge of the physical and
chemical properties of the complicated constituents
of the cell, and the products of its metabolism, is still
very scanty, while in addition the disharmonious
components of the plasma only appear in minute
quantities, we are not in a position to seek them out
by direct means. We have, therefore, to hit upon
indirect means, and to find out whether a particular
blood serum has ferments at its disposal, which are

SERODIAGXOSIS OF ORGANIC FUNCTIONS

able to decompose the substrate peculiar to a par-
ticular organ. In a certain sense we give the serum a
-definite question to answer, when we add all kinds
of organs to it, and observe which, or how many, of
them are decomposed by it. If we find a decom-
position, then we infer a somewhat abnormal activity
of the cells of the corresponding organ. We presume
that substances have been passed out, primarily from
the cells of the organ in question, which have not
vet been made sufficiently harmonious with the
plasma, and that they still exhibit characteristic
features peculiar to the cells in question.

In the future we shall undoubtedly avoid using

<7

whole organs and tissues for our researches, but
shall select particular types of cells ; and we shall
have to be particularly careful in deciding, whether
the tissue used is normal or modified. It is quite con-
ceivable that, in certain diseases, only those tissues
are decomposed which have been modified in a
particular manner. In such a case the diseased tissue
would be altered in such a way, that the substances
that are out of harmony with the plasma would be
more or less in disharmony with the cells of the
normal organ ; by which we mean, that compounds
and decomposites would appear, which have com-
pletely disharmonious activities. And indeed, it
would be possible to imagine that products would be
formed that are out of harmony with the entire body,

108 NATURE OF PROTEOLYTIC FERMENTS

because the whole organ has become similarly dis-
harmonious.

The fact that the animal organism replies to the
invasion of disharmonious substances which, either
taking their origin from the metabolism of certain
cells of its organs, or being normal constituents of
the cells, pass directly into the blood plasma by
means of specifically directed ferments, is of the
greatest importance to physiology as well as to
pathology.

Up to the present we have only been able to dis-
tinguish three different proteolytic ferments, namely,,
pepsin, trypsin, and erepsin. In addition to these,
we may perhaps reckon as proteolytic the ferments of
rennet, and of fibrin. Strictly speaking, erepsin must
be excluded, because it is principally directed against
the products of decomposition of albumen. Our
experience of the defensive ferments induces the
supposition that trypsin, for instance, is not uniform
in its nature. Of course, it may be possible that
there are ferments which, just as a master-key can
open various kinds of locks, are able to decompose
very different substrates, when these belong to the
same type of compound. But it is more likely that,
in trypsin, ferments of different kinds are combined,
and that in the blood the different components each
act separately.

The defensive ferments are, as we have already

NATURE OF PROTEOLYTIC FERMENTS KX)

pointed out, reagents acting on the characteristic,
typical structure of the components of definite kinds
of cells. We may illustrate this idea by an example.
A great sensation was caused at one time bv the

O

observation, that there were unicellular organisms
which apparently showed signs of intelligence. It
could be seen under the microscope how the uni-
cellular organism, called Vumpyrella spirogyra*,
hurried from one alea thread to another, until it

O

stopped at a particular kind of alga in order to use it
as food. However many kinds of alga were offered
to it, it would always pick out the same kind. This
.phenomenon, which seems so amazing at first sight,
may doubtless be explained in the following way :
Every living being has ferments at its disposal which,
as Emil Fischer pointed out, may be compared with
keys, and the substrate, against which they are
directed, with locks. Just as a particular key gener-
ally unlocks and locks only a particular lock, so can
particular ferments only decompose or reconstruct
substrates of a particular constitution.

The Vampyrella spirogyrce, then, hurries from alga
to alga, bearing with it ferments, by means of which
it intends to convert nutriment into a suitable form.
It is always trying to effect an entry by means of its
'keys,' and it only succeeds in certain cases,
namely, when the key fits the lock, which is to
say, when the cell-wall of the particular alga is of

110 .NATURE OF PROTEOLYTIC FERMENTS

such a structure, that the decomposition can be
effected by means of the ferments it possesses. A
breach is made into the cell-wall ; its contents are laid
bare, and can then be utilized as nutriment.

This unicellular organism shows us, then, that
the various alga? have a very different cell structure.
The defensive ferments prove the same thing, and
provide us with knowledge which we should be
unable at present to arrive at by any other means.

Exhaustive researches are now in progress, which
seek to ascertain whether the separate kinds of cells
of an organism have the command of specifically
directed ferments. We know that every cell requires
ferments for the purpose of breaking up the food that
is brought to it, or of constructing new compounds
out of it. Further, we know that the cell is able to
disintegrate parts of its own contents, and to replace
them by new material. Is it not probable that
specific activities are indicated in this case also ? In
the course of our experiments in this direction pep-
tones were produced from certain cells, and the
decomposition of these peptones by the correspond-
ing cell ferments was then attempted. As a matter
of fact, juices obtained from certain organs by
extraction or maceration decomposed only peptones
or albumen from the corresponding tissues ; that is
to say, extracted thyroid juice broke down peptones
obtained from that organ, but not liver peptones

NATURE OF PROTEOLYTIC FERMENTS III

(E. Abderhalclen, A. Fodor, and E. Schiff). The
kidneys alone were exceptional, their ferments attack-
ing peptones originating from the most different
organs. In all probability this result points to a new
function of the kidney, namely, the duty of intercept-
ing all disharmonious substances of a complicated
nature, which have been brought to it by the
blood, but have escaped the action of the defensive
ferments of the blood. The kidney decomposes
these, and by so doing renders them useful to the
organism. The observations we have quoted suggest
the possibility, that the kidneys may be instrumental
in supplying defensive ferments to the blood. It
would be very interesting to determine the contents
of diseased kidneys in respect of ferments, and to find
out whether they are still able to perform their duties.
By such studies new points of view might be supplied,
which would give us a better understanding of the
particular diseases affecting this organ. Moreover,
exhaustive studies on the specificity of the ferments of
cells as such should enable us to prove that each kind
of cell has its own structure. We shall also be able,
by means of the dialysation process and of the optical
method, to make a much better study of cell ferments
than has hitherto been the case.

The number of problems, that arise from the facts
we have brought forward, is so immense that we shall
content ourselves with drawing attention to only a

112 CORRELATIONS OF PARTICULAR ORGANS

few of them. In the first place it would be desirable
to find out where the defensive ferments arise, and
whether they can be met with inside the individual
cells themselves. It is, for instance, conceivable that
the walls of the intestine, and perhaps also the cells
of the liver, are always provided with definite fer-
ments for the purpose of further reducing- substances
which, though insufficiently decomposed, pass
through the gut epithelium ; and it is very probable
that the leucocytes play an important part in this con-
nection. They circulate rapidly through the w^hole
organism. They are to be looked on as protective
organs, which, to use a metaphor, overlook every-
thing with a view to finding out whether order
prevails. Some products are eliminated by being
absorbed into the body of the leucocytes (phago-
cytosis) ; others are attacked by their ferments, and
so broken up, and deprived of their characteristic
structure. Finally, as mentioned before, the separate
organs have to be considered, particularly the
kidneys.

But the most important advantage of the methods
we have described is, that they will enable us to
study the reciprocal dependence of individual organs.
Suppose, for instance, we remove the thyroid gland ;
we then anticipate that another organ, some of whose
functions depend on this gland, Avill have its meta-
bolism interfered with, and will in consequence give

CORRELATIONS OF PARTICULAR ORGANS 113

off disharmonious material. The failure of this
organ is followed by that of a second, which was
accustomed to obtain secretions from the first, and
thus we are led to the discovery of wheels within
wheels.

Or it may be that investigation of a large material
shows that certain dys-f unctions were attributed, on
the basis of our earlier experience, to disease of a
particular organ, when all the time it was functioning
quite .normally. For instance, the following case is
quite conceivable. Let us suppose that a very definite
function of organ B depends upon organ A. The
latter may work quite normally, although B is so
modified that it passes into the blood constituents of
its own cells. Let us suppose that it is these pro-
ducts against which the secretion, originating from
organ A, is directed ; then it finds the substances,
which it ought to affect within organ B, already
in the blood. It combines with these substances,
and in consequence never reaches organ B.
We then observe the same phenomena as would
result if organ A were diseased. The dialysation
process and the optical method would, in this case,
give the apparently astounding result, that defensive
ferments are present in the blood serum which are
directed against the components of organ B, whilst
those which correspond to the components of organ A
would, against all expectation, be entirely absent.
8

114 DEFENSIVE FERMENTS IN BLOOD CORPUSCLES

Organ A only appears to refuse to work because, as
the result of a primary dys-function of organ B, the
secretions are unable to achieve their aim in the
proper place. They are caught up too soon.

We must not forget to mention, that it is, perhaps,
more often than we imagine, that substances which
are out of harmony with the plasma circulate in the
blood. We are thinking particularly of disinte-
gration of the form-elements of the blood. That

o

ferments, directed against the components of the red
blood corpuscles, can be found in animals that are
apparently quite normal, is shown by the fact that,
amongst horses and cattle, about 40 per cent, of cases
investigated gave a decomposition of albumen which
originated from the form-elements (E. Abderhalden
and A. Weil).

The following experiments supply a striking
indication of the probable cause of this phenomenon.
Blood was taken from a rabbit. The serum neither
decomposed any organ that was free from blood, nor
any organ that contained blood traces of blood
being quite sufficient. Without any further treat-
ment, blood was again taken from the animal after
two days, and again the serum gave no decomposition
with any organ free from blood. On the other hand,
the reaction gave positive results with all organs that
contained blood. It was certainly not the albumen
of the organs that was decomposed, but the blood

DEFENSIVE FERMENTS IN BLOOD CORPUSCLES 11$

contained in the tissue. The appearance of defensive
ferments, after the extraction of blood, is without
doubt to be ascribed to the destruction of red blood
corpuscles that results therefrom. Is it not possible
that the absorption of plugs of fibrin that occlude the
walls of the vessels may be connected with defensive
ferments, and may not the latter also have a share
in the organization of thrombi?

Defensive ferments, which decompose the albumen
of blood corpuscles, may be produced by means of
injections of haemolytic blood, and herein lies a
source of error which should never be under-
estimated. Only organs that are absolutely free
from blood give conclusive results. Defensive fer-
ments, which decompose the albumen of blood
corpuscles, are frequently met with in carcinoma.
Any extravasation of blood into the tissues, however
slight, will give rise to this kind of defensive ferments.

Within the domain of pathology there is no field
which would not lend itself to researches based on the
methods we have described. We will call attention
to some. In the first place we can try, by means of
defensive ferments directed against certain kinds of
cells, to discover those organs which are giving off
substances that are out of harmony with the blood
or the plasma. This would be the case, when a
particular organ fails to complete its otherwise
normal metabolism. But it is also possible that

Il6 APPLICATION OF METHOD IN INFECTIOUS DISEASES

decomposites, or secretions, are formed, which are
in themselves disharmonious. The future must
teach us whether quantitative conditions are decisive
or not, but it is at least possible that a secretion of
quite normal composition may act disharmoniously
with the plasma, when it passes into the blood in too
large quantities.

In pathological cases, too, we shall be able, by
following up a particular disease, to determine the
nature of the reciprocal relations in which different
organs stand towards each other. It may be noticed,
perhaps, that at the beginning only one organ shows
signs of dys-function, that another then follows suit,
and so on. We shall also be able to make thera-
peutical studies. If a therapeutical measure should
result in the disappearance of the defensive ferments,
the therapy would have to be estimated otherwise
than if this were not the case.

A large field of study is presented by all cases of
degeneration, such as muscular and nervous degen-
erations, as well as by processes which result in the
formation of decaying products of every kind, such
as putrefaction of tissues, or absorption of exudates,
of extravasations of blood, or of thrombi, &c.

The infectious diseases obviously supply us with
an extensive field of study. On the one hand we
shall have to decide whether defensive ferments exist
that are directed against specific micro-organisms,

APPLICATION OF METHOD IN INFECTIOUS DISEASES I IJ

and, further, whether the tissue attacked is decom-
posed by blood serum. Either the micro-organisms
will be able to decompose the tissue their nutritive
medium in a manner harmonious with themselves
and disharmonious with the body, and so produce
decomposites out of harmony with the plasma, or else
the injured tissue will be altered in such a way as
to be no longer able to continue the normal processes
of its metabolism. A mass of observations are wait-
ing to be made in this direction.

We may point out that it has been ascertained that,
in cases of miliary tuberculosis, defensive ferments
exist which are directed against tubercle bacilli. It
seems that the serum of cattle suffering from tuber-
culosis is able to decompose the bovine type only.
Caseated lung tissues were not decomposed by the
serum of animals which suffered from miliary tuber-
culosis, but only of those which exhibited caseous
pneumonia. These experiments, which were per-
formed with the assistance of Andryewsky, on cattle
and cows, are an inducement to further studies.

We may take this opportunity of pointing out, that
the dialysation process for the demonstration of defen-
sive ferments offers the great advantage of toxi-
logically testing the products of decomposition. We
may use the dialysate, which must, of course, contain
the products of decomposition, either directly, or after
complete concentration at a low temperature and

Il8 APPLICATION OF METHOD IN INFECTIOUS DISEASES

decreased pressure, for all kinds of experiments on
animals. It is a pity that hardly any investigations
have been made in this direction.

Once we have demonstrated the existence of fer-
ments directed against particular micro-organisms,
then the question naturally arises, what influence is
attributable to the defensive ferments in a special case.
They may act defensively. But it is also possible
that it is they which first produce the poisonous
substances, when decomposing disharmonious
material. The ferment is unable to ' know what
will be the result, when it breaks down a particular
substrate. It may be that the attacked substrate is
quite harmless to the organism, and that injurious
substances first appear during decomposition.

If further researches show that the organism
defends itself successfully by means of definite
ferments, then a road is marked out for therapy to
follow. By the direct addition of the necessary
micro-organisms, or of certain parts of them, we shall
produce defensive ferments which are directed against
them, and try to transmit these with the serum. We
can determine, exactly, the moment when the defen-
sive ferments appear. A particularly fine basis for
experiments seems to be supplied by thromboses,
which one mi Hit be able to attack effectively by

<T5 - */

means of properly directed ferments, and so bring
about their absorption.

APPLICATION OF METHOD IN INFECTIOUS DISEASES I

The following observation is important. If
albumen is injected into the circulation of an animal
for the first time, defensive ferments are found to
appear, in the case of intravenous injection, at the
end of about one day. If the injection is repeated,
say, a month after the defensive ferments have dis-
appeared, then the ferments reappear very much
earlier. (Abderhalden and Schiff.) May it not be
that immunity partly rests upon the fact that an
organism is able to set free its defensive ferments
quicker than usual ?

Syphilis, also, may certainly be studied from the
standpoint we have laid down. We have to con-
sider the affected tissue on the one hand, and the
spirocha?te on the other, as substrates.

It is also clear that' we may suspect the presence,
in the serum, of defensive ferments which are able to
reduce fats, carbohydrates, nucleo-proteids, c. The
demonstration of proteolytic defensive ferments repre-
sents only one special case. We have selected this
case because, up to the present, no methods exist for
satisfactorily demonstrating lipolytic or amylolytic
ferments in short, those ferments which are directed
against the particular constituents we have mentioned
-unless one has rather large quantities of serum at
one's disposal. We are, meanwhile, engaged in
extending our researches to other ferments.

With regard to the infectious diseases, we should

I2O APPLICATION OF METHOD IN INFECTIOUS DISEASES

like to discuss more closely what is, in our concep-
tion, the relation of the micro-organisms to the cell
complex of the host.

Let us return for a moment to the conception we
developed at the commencement of this work, accord-
ing to which the organism, under normal condi-
tions, represents a whole closed in itself. We have
already pointed out that the harmony of all the

processes going on within the whole complex is per-
turbed, as soon as disharmonious kinds of cells settle
inside it ; that is, species of cells which have their
own metabolism, and their own specific structure.
On the one hand these cells have to be fed, and on
the other they give off the products of their meta-
bolism, and perhaps also secretions of various kinds,
to the exterior. In order to be able to make use of
the nutritive material supplied by the host, which is
at first disharmonious with their cells, they also must
possess ferments which will make the food accessible.
It is conceivable that the substances belonging to the
host are first absorbed by the cells, and then trans-
formed in their interior, but it is more likely that the
invaders give off ferments externally, which decompose
the nutritive media around them, and prepare it in that
way for absorption. The resulting decomposites are
then taken up by the cell. A reconstruction must be
made in any case, and especially when the sub-
stances are intended, for the building of new cells.

APPLICATION OF METHOD IX INFECTIOUS DISEASES 121

Experiments which have been carried on with
different so-called toxins have shown, without any
doubt, that splitting agents are present in them.
These experiments, however, do not make it
absolutely certain that the micro-organisms secrete
ferments, because it is difficult to decide whether the
so-called toxins of the trade represent uniform pro-
ducts, and particularly whether they always contain
secretions only. As a preliminary condition, then,
for the possibility of the existence of micro-organisms
amidst a particular complex of cells foreign to them,
we require the presence of ferments which enable
them to build up the food they require from sub-
stances that are in harmony with the cells and the
blood of their host. In this case the relations between
the configuration of the ferments and that of the sub-

O

strates are no doubt expressed in their most distinctive
form. How often may not a micro-organism pene-
trate into an organism and die out, only because it is
unable to feed on the nutritive medium supplied,
while in other cases it is able to settle down because
the substrates presented to it can be rendered acces-
sible bv its ferments. If all the substances are used

m,'

up, and none of the same kind are supplied anew by
the host at the proper place, then the conditions
of its existence are withdrawn from the micro-
organism, and it must either perish or find a new
'pasture.' It may also happen in many cases that

122 APPLICATION OF METHOD IN INFECTIOUS DISEASES

the cells of the host catch the secreted ferments of
the micro-organism, or render them inactive in some
other way, and in this manner either increase the
difficulties of existence for the invaders, or else com-
pletely destroy them.

How sensitive individual organisms are, in regard
to these nutritive substrates, is shown, by numerous
laboratory observations on the cultivation of the
most varied micro-organisms. We know that

o

many of them only thrive, when very definite sub-
strates are offered to them. The fact, that an altera-
tion of the nutritive medium deprives certain organ-
isms of their means of existence, is shown in the
clearest manner by the observation that an infection
with trichophyton fungi cures itself at the time of
puberty. Evidently the cells of the skin become so
modified, at the onset of sexual maturity, that the
substrate of the host the components of the skin-
can no longer act as a means of subsistence for the
fungus. From this point of view we may well imagine
that medicines and other therapeutical means effect
a curative action, without directly attacking particular
kinds of cells which may be living as parasites in the
animal organism, for they need only to destroy the
conditions of existence required by the organism in
question by means of a modification of its nutritive
substrate. It is imaginable that certain means do
modify certain cells to such an extent, that their com-

APPLICATION OF METHOD IX INFECTIOUS DISEASES 123

ponents can no longer be considered as supplying
nutritive material for the organism that lives in them.
The fact that the cells which are out of harmony
with the body are dependent upon nutritive material
of the most varied nature for the opportunity of ex-
tending their existence, and more particularly for
maintaining their species, gives us an insight into
the kind of influence exercised by these parasites on
the host. In the first place they may act injuriously
by the simple removal of nutritive substrates, while,
in addition, the preparatory decomposition of the
nutritive material may give rise to by-products which
are harmful to the organism. We can well imagine
that particular cells have ferments at their disposal,
which decompose particular substrates in a thoroughly
characteristic fashion, and so, for instance, produce
stages of decomposition which are quite out of
harmony with the cells of the host. The same sub-
strate may be decomposed in the most various
ways, right clown to its simplest structural units.
The idea of an atypic decomposition of substances in
harmony with the body, cells, and plasma, by fer-
ments of disharmonious cells, suggests the possibility
that micro-organisms, though not actually themselves
passing into the circulation substances that are
directly poisonous, may act injuriously, simply from
the fact that, by means of fermentative decomposi-
tions, they form products out of the material

124 APPLICATION OF METHOD IN INFECTIOUS DISEASES

of the host, which seriously affect the metabolism
of the latter. It is certainly not necessary in
every case that the poisonous substance, the so-
called toxin, should originate in the cell of the
micro-organism itself; it is just as likely that it is
formed, outside the cell, by ferments given off by the
latter. When we introduce substances out of har-
mony with the species or the plasma, we have
similarly to reckon with the presence of stages of
decomposition, which may be disharmonious with the
organism, and capable of producing injurious effects.
In this case the disharmonious substrate is the
cause of the appearance of a substance, that is dis-
harmonious both in structure and configuration. In
cases of invasion by bacteria, we have, on the
contrary, a destruction of generally harmonious sub-
stances, yet the decomposition is brought about by
ferments which are probably of a different kind. The
cause of the appearance of disharmonious decompo-
sites is thus to be explained, not by the substrate, but
by the nature of the ferments. It is possible that, in
time, we may succeed in tracking down in the organ-
ism these ferment-like agents that are given off by
parasites. For the time being we must be satisfied
with being able to point to the possibility of a decom-
position of this kind being a cause of injury.

Disharmonious cells may also act injuriously upon
the organism, owing to their decomposition inside the

APPLICATION OF METHOD IN INFECTIOUS DISEASES 125

body. The death of a cell of this kind results in the
appearance, within the circulation, of substances which
are disharmonious. We can make a comparison be-
tween a case of this kind and the parenteral injection
of substances out of harmony with the body and the
plasma ; for, in this case, the organism will surely
defend itself against the very disharmonious sub-
strate, by depriving the substrate of its specific struc-
ture by means of an extensive decomposition. We
should then be presented with conditions entirely
analogous with the parenteral injection of various
substances, or with the invasion, into the circulation,
.of chorionic cells which are out of harmony with the
blood plasma, and the resulting reaction would be
entirely similar. But here, too, during decompo-
sition, it may happen that the organism will produce
decomposites which are naturally injurious, so that,
in any given case, it would mainly depend on whether
the intermediate products appeared only in small
quantities and were promptly reduced, or whether,
conversely, the power of decomposition possessed by
the organism is inhibited either because the decom-
posites cannot be further reduced or be rejected, or
because the ferments necessary for further reduction
are not present in sufficient quantity. We can well
imagine that the decomposition of the bodies of dead
micro-organisms, without the direct participation of
the microbes themselves, will give rise to various

126 APPLICATION OF METHOD IX INFECTIOUS DISEASES

disturbances ; and we might certainly expect secondary
disturbances in the harmonic processes of the whole
metabolism of the host, without the micro-organisms
as such exerting any direct action.

Finally, we have to consider yet another possibility,
that certain micro-organisms produce poisonous
substances within themselves, and give them off
externally. It is, at present, very problematical, what
view we are to take of these substances. Are they
substances which play a part in the metabolism of
the micro-organisms themselves, or are we dealing
with agents which, when passed to the exterior, affect
the nutritive medium in some way or another, that
is, by either decomposing or reconstituting it? It is
quite conceivable that certain micro-organisms have
agencies at their disposal, which are able to modify
particular nutritive cultures in a particular way.
Many pathological observations have proved, that
certain micro-organisms require a so-called mixed
infection, that is, that certain bacteria alter the cell
substance of the host in such a way, that some other
kind of bacterium finds conditions favourable for its
existence. It seems that this is also the case with
some kinds of tumours, such as sarcoma and carci-
noma, where a preparation of the medium by certain
substances is of great importance. In the future we
shall be compelled to appreciate all these possibilities.
If we could succeed in limiting more precisely the

APPLICATION OF METHOD IX INFECTIOUS DISEASES I2~

conditions under which certain bacteria can exist,

mainly on the basis of more exact studies on the

j

composition of the medium, then we should undoubt-
edly be in a position to employ more objective
therapeutic methods. Further, it would be possible
to formulate a conception of the injurious activities
of certain kinds of bacteria more clearly than we
can at present. Unfortunately, it would scarcely be
possible to make use of direct methods in this case,
unless we were to succeed in cultivating individual
micro-organisms on substrates the composition of
which we were thoroughly acquainted with. Our
^ progress in the field of the chemistry of the different
kinds of cell units, and of their nutritive bases, has
brought us nearer to this objective, but a large
part of the road lies before us yet, before we shall
obtain such an exact knowledge of the composition
of certain albuminous substances, such as phospha-
tides and nucleoproteids, &c., as to be able to properly
appreciate differences of configuration, as well as
differences of structure. Once we have advanced so
far, we shall be able to replace our present conception
of " disposition ' by definite facts.

The train of thought we have been pursuing is
only intended to show that, in considering the ques-
tion of the injuries which bacteria may inflict on their
host, we must not only consider the bacteria as such,
but must realize that their entire metabolism is of

128 APPLICATION OF METHOD IN INFECTIOUS DISEASES

pre-eminent importance. It is not the bacteria alone,
and the so-called toxins, that have to be considered in
the whole question of immunity reactions, but most
probably the intermediate products of their meta-
bolism, as well as certain decomposites which are,
at any rate partly, formed quite outside the cells in
question. And, above all, \ve have to consider the
structure of the organism. The host directs its
struggle, not only against the living micro-organism,
but also against the particles which appear with the
decay of the dead organism, and more particularly
against the intermediate products which originate
during the preparation of the nutritive medium. The
organism attacks every point with its ferments, and
tries to decompose or reconstruct anything that is
disharmonious with its structure or configuration, or
even its physical properties. The more it succeeds
in this respect, the more does it deprive the micro-
organisms of the conditions required for their
existence, and protect its own cells against the
injurious action of these substances.

We come, then, to the conclusion, that at least one
part of the means of defence, possessed by an organ-
ism against infections of any kind, depends on its
power of liberating ferments, which attack the dis-
harmonious substances be they by-products or
end-products of metabolism, or products of cellular
disintegration and deprive them as quickly as

APPLICATION 01- METHOD IX INFECTIOUS DISEASES 129

possible of those specific properties which are out
of harmony with the host. Of course, other
processes come into play in this connection. The
decomposites are oxidized, reduced, methylated,
acetylated, benzolated, &c., and are also, without any
doubt, coupled in very different ways with various
compounds. The defensive ferments prepare the
disharmonious material in a proper manner, so that
the individual cells of the body may attack it
in their own specific way. The ferments remain
unaltered during all these processes. They enter
temporarily into combination with the substrate to
'be altered. Once the decomposition is completed,
the ferment is again ready to initiate new reactions-
mostly decompositions. An over-production of fer-
ments, in response to an invasion of disharmonious
bodies, is therefore unnecessary. The importance
we ascribe to these means of defence of the organism
against the invasion of disharmonious substances
may, however, be objected to on the ground that
little is gained by demonstrating the existence of
ferments in the blood plasma, and by agreeing that
they play an important part in connection with in-
fectious diseases, so long as the ferments themselves
are not known by us. We know nothing of their
structure, their nature, or their special modes of
action ; and only become aware of their existence
through their activities. The fact, that they are
9

130 APPLICATION OF METHOD IN INFECTIOUS DISEASES

specifically directed against particular substrates,
makes it possible for us to demonstrate their exis-
tence, and the knowledge, that ferments play an im-
portant part amongst the means of defence of the
animal organism against disharmonious substances,
indicates this much progress, that we are enabled
thereby to follow up, experimentally, phenomena
which we meet with under normal conditions in the
individual cells of the body. With the help of the
ferments the cell is continuously remodelling in a suit-
able manner the food, which the blood brings to it
still in harmony with the plasma, either by way of
completing a further decomposition, or of setting up a
synthesis. The ferments are the tools of the cells, bv

^ ^

means of which fuel is brought into a suitable form,
the structure of the cell is completed, and various
substances are prepared, which, as secretions,
have a more or less definite part to play through-
out the organism. If the organism liberates
defensive ferments, then its cells effect nothing
actually new. A normal process is simply allocated
to a specific case. The ferments are adapted to the
new kind of substrate, and, if necessary, passed out
into the circulation. So that this form of defence
against disharmonious substances, which the cell

utilizes, mav be ranked directlv with the normal

j j

processes of cellular metabolism an idea which
has always dominated the researches of Paul

APPLICATION OF METHOD IX INFECTIOUS DISEASES

Ehrlich. At the same time, a careful analysis of the
processes effected by the ferments gives us the chance
of determining, with more certainty than has hitherto
been the case, what is the nature of the injuries set
up by the presence of cells that are out of harmony
with the body. Sometimes the parasite takes an
active part, sometimes only a passive one, while at
other times its influence is extremely varied.

j

The proof, that ferments play an important part in
the means of defence of animal cells against dishar-
monious substances, opens up new paths for ex-
perimental research. It may be long before the true
nature of ferments is elucidated ; nevertheless, we
shall always be getting nearer to the possibility of
excluding the second unknown the substrate. The
more we extend our knowledge of the composition and
structure of the food material, and of the components
of the cells, the more do we find ourselves in a
position to make use of substrates of a known
structure, which enable us to investigate the ferments
in a much surer manner, and to determine exactly how
they decompose a particular product. We shall be
able to get hold of individual decomposites, and study
their properties, so as gradually to penetrate into the
mysteries of the effects of infectious diseases, as well
as into the principles of immunity.

There is scarcely, in the whole domain of biology,
a more stimulating task than that of finding

13- APPLICATION OF METHOD IN IXrECTlOUS DISEASES

oat liu\v the organism defends itself when dishar-
monious 'substances introduce a disturbing element
into its metabolism, so delicately poised and har-
monized even in its finest details. In these problems
the most varied hypotheses regarding cellular meta-
bolism are to be met with. The more the biologist
enlarges the limits of his researches, and the more he
follows up all general phenomena, the more may he
venture to hope that he will gain new means and
new lines for the study of special processes. The
appearance of the defensive ferments in the animal
organism, when it is invaded by substances that are
out of harmony either with its body, with its blood,
or even with some of its cells, gives us an insight into
many problems of pathology, and particularly into
those of immunity. Every approximation of fields
of thought that are at first sight dissimilar, arising
cut of observations which allow us to presume
common reactions and common processes, must be
met with approval. It will then be possible that,
by an exchange of results based on very different
methods and hypotheses, we shall acquire a wide
outlook over the fundamental properties of cells of
different origin.

Prolonged observations on a particular case of
definite disease will be of the greatest importance.
It would be absurd to investigate, sav, a hundred

APPLICATION OF METHOD IX INFECTIOUS DISEASES 133

cases of tuberculosis, of paralysis, or of dementia
pra?cox, <xc., without carefully considering the clinical
aspect of each of them. Above all, one ought to
make a continuous study of certain types of disease
in their different stages. Thus, for instance,
epilepsy ought to be observed before, dtiring and
after its onset, at the period of remission, and so on.
The normal individual also offers opportunities for
such studies, on such occasions as the advent of
pubertv, the climacterium, <xe.

All the various forms of nephritis supply another
important field of research. Does the kidnev plav an
^active part in individual cases, or does it only excrete
albumen that is out of harmony with the blood plasma
-that is, does it, in the main, play only a passive
part? The following observation illustrates a case f
this kind. Serum, from a female patient suffering fr i
nephritis gravidarum, decomposed placenta-albumen
and placenta-peptone with great difficulty. The rota-
tion of the serum was unusually hi^h. When this

O

serum was mixed with that of a normal pregnr.
patient, a change was observed in the rotation of
the mixture; and the fact that this was clue to a
decomposition was proved by the dialysation method.
Neither serum, dialysed by itself, showed any de-
composites of albumen. When both sera the one
from the case of nephritis gravidarum, and the other
from tile normal pregnant person were subjected

134 APPLICATION OF METHOD IN INFECTIOUS DISEASES

to dialysis together, peptone appeared in the dia-
lysate. This case evidently has to be explained as
follows :

As in every case of pregnancy, the blood was filled
with substances out of harmony with the plasma in
this case, obviously, proteins. Normally, these com-
pounds are removed by decomposition, with the help
of the ferments. In the case of the female patient
suffering from nephritis gravidarum the decompo-
sition was evidently very incomplete, in consequence
of which the disharmonious proteins accumulated
rapidly, and had eventually to be removed by the
kidneys.

This observation agrees very closely with some
results obtained by Aschner, who observed that the
albumen found in the urine during eclampsia is
decomposed by the serum of pregnant individuals,
although this is not the case when albumen is taken
from a case of ordinary nephritis. It is obvious that
traces of a specifically constructed albumen are quite
sufficient to produce the effects of a decomposition by
specifically directed ferments. Of course, we have
no right to conclude, on the strength of this fact,
that, because the serum of pregnancy reacts quite
specifically towards particular urine-albumens, there-
fore all the excreted proteins belong to a particular
type. The eclampsia may be coupled with an ordinary
case of nephritis, or follow it.

APPLICATION OF METHOD IN INFECTIOUS DISEASES 135

Each individual case of albuminuria presents
analogous problems. Can the serum decom-
pose urine-albumen, or does it disintegrate the
tissues of the kidneys? Is the normal tissue of
kidneys decomposed, or only pathologically altered?
Let us point out, at this stage, that the determination
of the power of rotation of the serum may itself be
sufficient to direct us towards various important
observations. May there not be a hyper- and
a hypoproteinaemia ? Is there any albuminuria
which is based exclusively on heteroproteinasmia ?
Bence Jones's albuminuria and the typical albumin-
uria of pregnancy very likely represent such cases.
It would obviously be incorrect to designate such
kinds of albuminuria as being conditioned by
nephritis.

In this connection we would lay stress on the fact
that eclampsia, and the toxaemias of pregnancy,
present us with a fruitful field for researches on
their special conditions. Up to the present it would
appear that the prognosis of eclampsia is the more
unfavourable, according as the decomposition of the
proteins disharmonious with the plasma is the less
complete. It goes without saying that we must not
jump to broad conclusions on the strength of these
observations. We have never ventured to assert that
the deciding factor in eclampsia is the insufficient
decomposition of the disharmonious substances ; and,

136 APPLICATION OF METHOD IX INFECTIOUS DISEASES

indeed, it is quite likely that this is a secondary
phenomenon. The fact, that in cases of eclampsia a
disturbance may occur in the functions of the liver,
deserves much attention. In two cases the only two
which have been investigated along these lines a
disturbance of the functions of the thyroid gland was
also found.

A very useful field of research is also supplied by
the tumours. Carcinoma, in particular, should
give rise to substances that are out of harmony with
the blood plasma, and so to defensive ferments.
Our own experience has shown, that the serum of
carcinomatous patients decomposes boiled carcinoma
tissues, but not placenta. On the other hand, decom-
position of carcinoma by the serum of pregnancy was
never observed. According to our experience it was
possible to obtain an early diagnosis of carcinoma.
Moreover, the method may perhaps be of use for
controlling the therapeutic methods used in cases of

j .1

carcinoma, whether they be of an operative or of
any other character. From a fortnight to three weeks
after the disappearance of the carcinoma cells, the
ferments that are directed against cancer should also
disappear, if we are to judge from present experience.
Finally, we shall have to consider the diseases of
metabolism, and all such phenomena as are etiologic-
ally wrapped in obscurity, as, for instance, various
dermatoses, sympathetic ophthalmia, cS:c. Here we

APPLICATION OF METHOD IX INFECTIOUS DISEASES 137

shall have to test one organ after another, until we
meet with one against which defensive ferments are to
be found. The exact study of the so-called nutritive
disturbances of the suckling will, from this point of
view, be of much interest.

Moreover, we shall certainly be in a position to
determine experimentally, as well as by means of
clinical observations, the best method of attacking
certain poisons, such as lead, nicotine, alcohol
(methyl, ethyl, &c.), ether, chloroform, morphia, &c.
We shall be able to delimit primary and secondary
injuries, and to shed new light upon many problems
of toxicology and pharmacology.

In conclusion, we should like to summarize the
researches which have been published since the appear-
ance of the first edition of this book. We ourselves
have at our disposal more than 500 cases of differen-
tial diagnosis between pregnancy and non-pregnancy.
In these cases complications of all kinds were
present, salpingitis, carcinoma, myoma, parametritis,
tuberculosis, &c. With the exception of one un-
certain case of probable abortion, no mistakes have
been made in our diagnosis. In addition, forty-two
cases of carcinoma have been investigated. The

o

serum of carcinomatous patients decomposed similar
carcinomatous tissues after boiling, but never tissues
of placenta.

We ought to point out here, that we must never

138 RKVIKW OF RECENT RESEARCHES

rely, in these investigations, upon one particular
boiled organ. The great advantage of the whole
method of investigation is the fact, that it is possible
to make each case absolutely sure by means of control
experiments. Placenta, for instance, can always be
tested by means of serum from definitely non-
pregnant individuals, as well as from males. Further,
all kinds of organs should be subjected in turn to
the action of a particular serum.

The sero-diagnosis of pregnancy has been recentlv
employed with success by numerous scientists. 12
While many observers agree with our point of view,
that the defensive ferments which appear during
pregnancy are directed against the albumen of
placenta, other authors are of the opinion that no
strictly specific action exists. But surely positive
facts have more value than negative ones.

We are so strongly persuaded, from our own ex-
periences, of the certainty of our methods, that we do
not hesitate to demand that no one should deal with
pathological cases by means of the dialysation
method and the optical method, who has not given
evidence of having been able to produce 100 per cent,
of correct diagnoses from pregnant, and particularly
from non-pregnant, individuals, using placenta as
his substrate. Should the technique of the student be

12 See list of literature.

REVIEW OF RECENT RESEARCHES 139

found wanting in this branch of practical application,
then it is quite certain that he has not mastered the
method, and no special investigations should be under-
taken without such a certificate of capacity. No con-
scientious student should make use of a particular
method without having made sure, by some means
or another, that he is master of it; and the diagnosis
of pregnancy supplies these means.

A further important rule is, that we must never
allow current theories to mislead us. The assump-
tion of a specific action of the defensive ferments has
been established on the strength of the facts derived
from experiments, but it has still to be supported by
means of exhaustive investigations. Nor should any
final conclusion ever be expressed in regard to a
particular case, until it has been definitely determined
by clinical means. It is in this respect that preg-
nancy offers such a sure basis. \Ve do not know of
any other condition which allows of such a clear and
indubitable estimate of method. It either exists or
it does not, and is never open to a discussion of
11 probabilities.' 1

Valuable service is rendered to research as a whole,
if every case, which gives an unexpected result in
respect of its reaction, is thoroughly investigated. In
the first place the dialvsation tubes should be
changed, or else another organ should be used. If
the result alwavs recurs, we still have to exclude the

I4O RKVIEW OF KKCKNT RESEARCHES

possibility that, in the organ used, substances are
inherent which are also contained in other organs. In
saying this we are thinking of such a case as the
following. It may be that an individual has
some diseased connective tissue in some part of his
body. The supporting tissues of the organism also
have their metabolism, and are quite able to give off
disharmonious products into the blood, especially if
they are profoundly altered or broken down. Every
organ contains connective tissues, though whether
they possess an organically specific structure is
highly doubtful. It would certainly be of the
greatest importance, if so-called failures were tested
with connective tissue pure and simple, and then with
the corresponding organ without its connective
tissue. It would also be advisable, in the case of
unexpected results, to test with the albumens of
blood serum, and those of blood corpuscles. Studies
of this kind will lead much more quickly to clear
results than the hurried collection of data which have
probably been all subject to the same source of error.
Amongst other observations which have been

o

communicated up to the present time we shall refer
to the following. Epstein found that out of thirty-
seven cases of cancerous patients all but one,
originating from a cachectic patient, aged So,
decomposed the coagulated albumen of carcinoma,
while in no one case was the albumen of placenta

REVIEW OF RECENT RESEARCHES

attacked. Further, the serum of forty-seven indivi-
duals who undoubtedly had no carcinoma, but who,
on the other hand, had at least partly suffered from
serious illnesses resulting in general loss of strength,
was tested for their action upon the tissues of carci-
noma. In forty-six cases no decomposition took
place. Ludke and Gambaroff also announce favour-
able results in regard to the diagnosis of carcinoma.

o o

Some interesting observations of Paltauf may be
quoted, as specially advocating the acceptance of
ferments which are specifically directed against
particular substrates. The tissue of a tumour from
a woman, aged 61, was not attacked by the
serum of a carcinomatous patient which decomposed
carcinomatous tissues, although at the same time the
coagulated tissue was decomposed by the serum of
pregnancy. The pathological diagnosis was as
follows : Malignant chorionic epithelioma.

Bauer draws attention to the demonstration of
defensive ferments in the blood serum, in cases of
endemic goitre, which are able to decompose the
tissues of the thyroid gland. These ferments have
also been observed, even when no goitre was present,
while the clinical phenomena pointed to a disturbance
of the functions of the thyroid gland. We, too, have
found a decomposition of the tissues of the thyroid
gland in one case of myxcedema. There is no doubt
that in these diseases we are dealin, not with an

142 REVIEW OF RECENT RESEARCHES

athyreosis, as was once supposed, but with a
dysthyreosis.

The symptoms in Basedow's disease are of par-
ticular interest. Here we find decomposition of the
thymus gland, of the thyroid gland, and very often
of the ovary, as has been established by Lampe,
Papazolu, and Fuchs, on the basis of a large amount
of material. No other organ was decomposed. It
is interesting to note, that normal thyroid gland was
decomposed only in very exceptional cases, while
Basedow thyroids were always attacked.

This observation points to -two possibilities in
regard to the production of substances out of harmony
with the plasma. Sometimes the normally con-
structed cell is incapable of completing the otherwise
normal decomposition of particular substances, so
that materials appear in the blood which still show
the characteristic features of the cell, from which they
originated. The decomposition is discontinued at a
particular stage. A certain analogy with this kind
of disturbance of cellular metabolism is presented by
those anomalous cases, in which simpler products
are not fully reduced. We may refer to cystinuria,
alkaptonuria, pentosuria, &c. In the first case cystin,
in alkaptonuria, homogentisic acid, and in the last
case a pentose, are excreted in the urine. Some
forms of glycosuria also belong here. The cells are
unable to attack the grape sugar, because no active

REVIEW OF RECENT RESEARCHES 143

ferment is present which can act on this carbo-
hydrate.

Further, the cause of disharmony with the blood
plasma may depend upon the fact that cells of a
particular kind have degenerated, in consequence of
which they have a pathological structure, and give
off a disharmonious kind of material, to which the
blood plasma is unused.

We may also mention the experiments of Bauer
and Reines, made for the purpose of clearing up the
etiology of sclerodermy. The actual experiments
point to a disturbance of the functions of the thyroid
gland; but it is probable that other organs are
affected sympathetically. In these experiments, also,
it appears that certain organs are decomposed, while
others are not.

Fauser, Wegener, Job. Fischer, Kafka, and others
report numerous observations on cases of dementia
pra?cox, paralysis, and melancholia. In the first-
named disease defensive ferments appear against the
sexual glands and the cortex of the brain. Accord-
ing to our ideas this means that these organs exhibit
a disturbance of function, though which organ
primarily discontinues its functions cannot at present
be ascertained. In cases of melancholia no defensive
ferments have as yet been found. In paralysis the
cortex of the brain is nearly always decomposed.

These researches naturally do no more than imply

144 REVIEW OF RECENT RESEARCHES

a rapid advance into unknown territory. There are
still many stages to go over again, before we can
arrive at a definite picture. A serological examina-
tion must be first employed, and the clinical
diagnosis, which is generally more uncertain, will
be compared with it. Often later observations alone
will show, whether the serological diagnosis is
really correct. It will be particularly necessary to
make our clinical description of individual cases as
thorough as possible. The serological diagnosis of
diseases of the nervous svstem will attain a oreat

J O

measure of success, when it enables us to isolate
diseases, hitherto considered identical, according to
the presence of particular defensive ferments, and to
find that the clinical course of the disease corre-
sponds with this. In any event, each separate case

must be carefullv and continuouslv studied.

j

Moreover, the above investigations are of special
importance for this reason, that they definitely indi-
cate the specific action of the defensive ferments.
Fauser and Wegener report that female patients
suffering from dementia prascox never decomposed
testes, but only ovaries. Conversely, males only
attack testicular tissue, but never the ovary. Both
authors have also experimented with placenta, and,
on the one hand, confirmed the sere-diagnosis of

O

pregnancy, and on the other showed that the male
serum never decomposes tissues of placenta. Of

REVIEW OF RECENT RESEARCHES 145

great importance is Wegener's communication, in
which he states that, in cases of neuritis, muscle
tissue is decomposed.

Finally, we may refer to the fact that experiments
have been made with a view to throwing light upon
the etiology of diseases of the eye, the causes of
which are not yet known with certainty. Sympathetic
ophthalmia provides features of particular interest.
The researches carried on in this connection, by
v. Hippel and Hegener, show distinctly that the
defensive ferments exhibit specific activities. The
number of observations is as yet too small to enable
us to draw definite conclusions therefrom. In con-
nection with these latter observations we may point
out, how important it is to follow up every case,
clinically, over a long period, and in no case to pay
attention onlv to the disease which o'ave rise to the

<_?>

investigation. In particular, the further course of
the disease should be followed up in all its phases.
We have, in the dialysation process and in the optical
method, means which allow us to test the functions
of organs over long periods of time.

The observations, which we have here brieflv

/

sketched out, will certainly undergo rapid extension
in various directions. Perhaps it will be found later
on, that there are other explanations than those which
have been developed here ; and it is highly probable
that some of the earlier results, which were obtained
10

146 REVIEW OF RECENT RESEARCHES

by means of other methods, will be brought into line
with those acquired by means of the new methods.
Thus the discovery, by Hermann Pfeiffer and his
pupils, of the existence of toxic compounds in the
urine in certain diseases and in certain conditions,
should suggest to us that the products, which are
formed by the defensive ferments, are also finally
excreted. The comparative investigation of the
dialysates, however, will have to decide whether there
are direct relations of any kind between such products
of decomposition and the poisonous components of
the urine, and whether we are right in speaking of
toxaemias that result from the decomposition of
albumens.

THE DIALYSATION PROCESS 147

Methods in Use.

I.- -The Dialysation Process.

The principle of the method: Albumen being a
colloid does not diffuse through animal membranes,
while on the other hand peptones the first products
of its decomposition are diffusible. If we put
albumen in a dialysing tube and place the latter in
water, no albumen appears in the surrounding fluid
even after a considerable time. If, however, sub-
stances such as pepsin and hydrochloric acid are
added to the albumen in the tube, we can soon trace,
in the water surrounding the tube, substances which
are produced from the decomposition of the albumen.
These substances are the so-called peptones and
some other simpler products of decomposition. If
we desire to test any liquid to ascertain whether it
contains any proteolvtic i.e., albumen-decomposing
-ferments, we place it in a dialysing tube together
with albumen, and note whether peptones appear in
the liquid surrounding the dialysing tube. If none
are present, we may be sure that the tested liquid

148 THE DIALYSATION PROCESS

contains none of the active ferments capable of de-
composing albumen. Should we detect the presence of
peptones, we may be certain that some decomposition
of the albumen has taken place. In our special
case the fluid to be tested is blood serum. It is
obvious that the method is exactly the same, when
we test, for their capacity of decomposing albumen,
such substances as cerebro -spinal fluid, lymph, or
extracts from various organs e.g., juices obtained
by means of high pressure.

Dialysing Tubes.- -The result of tests for albumen-
decomposing ferments by the dialysation process
depends in the first place upon the quality of the mem-
brane used. The latter must above all answer two
requirements. First of all it must be absolutely im-
permeable to albumen, and further, evenly permeable
to decomposites of albumen. If the tube allows
albumen to pass through it, the latter may be mis-
taken for peptones, unless we apply special tests for
albumen. Should dialysing tubes be used which
allow peptones to diffuse through at a variable rate,
then we should be at a loss in our judgment upon
the results of a test, because, as will presently be
shown, a control test of the fluid to be tested must
always be made, without the presence of albumen, and
the results of this test be compared with those of the

tests in which albumen has been mixed in the dialv-

j

sing tube with the fluid under research. Should one

THE DIALYSATION PROCESS 149

tube be very dense and allow little or no passage at
all of the peptones, we should naturally have in this
a considerable source of error.

Numerous dialysing membranes are known, of
which very few have any real value for our purpose.
The dialysation process requires dialysing tubes which
can be used over and over again. The best are
those supplied by Schleicher and Schiill, of Diiren in
Rhineland. The tubes of this firm should in no case
be used without a thorough preliminary examination,
because tubes are nearly always met with which allow
albumen to pass through, while others are found
'through which peptones diffuse with difficulty, so
that careful testing of the tubes is indispensable. 13
Further, the tubes must be short ones. No. 57QA
is a tube specially prepared for our purpose. If
tubes be used, which project too much over the
surface of the surrounding fluid towards which
the dialysing process acts, this gives rise to a very
uneven evaporation of the dialysate. The latter
soaks into the tube, is carried upwards, and
evaporates. Indeed, as we shall see later, everything
depends upon the fact that, in comparative experi-
ments, the concentration of the dialvsates shall not

Tested tubes are supplied by Schops, of Halle a/S.,
but still it is advisable to test them, previous to use, as a
matter of security.

150 THE DIALYSAT1ON PROCESS

be affected by unequal evaporation ; and every pre-
caution must be taken to avoid this source of error.

The first duty to be undertaken in making use of
the dialysation process, is the testing of the tubes,
the so-called standardization of the dialysing tubes.
This standardization, as we have already emphasized,
implies the impermeability of the tubes towards
albumen, and a perfectly equal permeability for the
products of its decomposition.

(a) Test for Impermeability by Albumen. A solu-
tion of albumen is prepared. The simplest way is
to take the white of a new-laid egg. 5 c.c. of per-
fectly fresh white of egg are diluted with distilled
water in a graduated tube to 100 c.c., and thoroughly
mixed by shaking. Of the white of egg, which must
be absolutely fresh, only the more fluid portion is
used, while all flaky matter or bits of skin in short,
all solid parts are rejected, as otherwise it is im-
possible to get a good mixture. Instead of the white
of an egg, blood serum may be employed.

Now the tubes to be tested are prepared. They
are soaked in cold water for about half an hour. The
tubes are then placed in small Erlenmeyer flasks
(fig. 7) and 2*5 c.c. of the thoroughly mixed solu-
tion of white of egg in water are poured into them.
The solution is measured by means of a pipette.
While filling the tubes the pipette is placed far down
in them, and the greatest precautions must be taken

THE DIALYSATION PROCESS I5 1

not to spill any of the egg solution upon the exterior
of the dialysing tube. Should this occur, the dialy-
sate would incorrectly show a positive reaction for
peptones, when, for instance, the biuret test is
applied, since both albumens and peptones give this
reaction. To avoid any chance of such an error, the
dialysing tube, after having been filled, is closed at the
top between the thumb and forefinger and well rinsed
in running water. Then the tube is closed in the
same manner half way down, and water is allowed

FIG. 7.

to enter the upper part of the tube, so as to wash that
part of the dialysing tube which, during the dialysing
process, projects out of the dialysate and above the
layer of toluol. By moving the thumb and fore-
finger towards the upper end of the tube we expel
the water remaining after washing. All these manipu-
lations have the following object :-

When filling the tube with albumen its interior,
near the free edge, rnav easilv come into contact

fj / */

with the pipette. Some of the albumen may adhere
to the edge of the tube and dry up in time. At the

152 THE DIALYSATION PROCESS

conclusion of the test some parts of this albumen
may fall into the dialysate and pollute it. During
the operation of cleansing the inside of the tubes,
care must be taken to prevent water from entering
the tubes. Before touching the tubes the hands
should be thoroughly cleansed. The use of forceps
is much recommended, and these must have wide,
parallel, smooth arms.

The rinsed tubes are again put into Erlenmeyer
flasks which contain 20 c.c. of sterile distilled water.
The filling of the dialysation tubes must never be
done in the same flasks in which it is intended to
carry out the dialysation ; something out of the
pipette may too easily get into the flask. In order
to prevent contamination the surrounding fluid, as
well as the contents of the tubes, is covered with a
layer of toluol about \ cm. thick (fig. 7, p. 151). It
is best to cover the flasks with watch glasses, unless
one is prepared to use stoppered vessels. The
dialysation is carried on at the temperature of the
room, or, better still, in a closed space at a constant
temperature i.e., in an incubator.

After about sixteen hours time is of no import-
ance in this test, since the tubes are in this case
merely tested for their permeability towards colloids
-the dialysation is interrupted. The Erlenmeyer
flasks, which should bear corresponding numbers,
are placed in a row. By means of a pipette, which is

THE DIALYSATIOX PROCESS 153

closed at its upper extremity by the finger and rapidly
passed through the layer of toluol, 10 c.c. of the
dialysate are taken out, and placed in a test-tube
bearing the same number as the corresponding
Erlenmever flask. This is the best way to avoid

/

mistakes. Of course, for each dialysate a separate
and absolutely clean pipette must be used. We do
not recommend transferring the dialysates to the
test-tubes by means of the same pipette, rapidly
cleansed each time after use, because by this means
some impurity or other may easily be introduced into
the dialysate. Some saliva may very easily enter
that part of the pipette which, during the so-called
cleansing, remains untouched by the water, alcohol
and ether. On the contrary, new saliva is drawn in
at each operation if the suction is made by the mouth.
Xo\v, when the dialysate is taken up, it is almost
certain to be drawn above the level marked upon the
pipette, and may then become mixed with the saliva.
If test-tubes, graduated to 10 c.c., are to hand, then
these tubes may be employed in the following man-
ner : After removing the dialvsing tubes, the toluol
is drawn off and the dialysate is poured directly into
the test-tube. It is of no great importance, in the
biuret reaction, to consider quantities to the minutest
exactness, nor does a little toluol do any harm.

Now, to each test-tube is added about 2*5 c.c. of a
33 per cent, caustic soda solution. The whole is shaken

154 THE DIALYSATION PROCESS

sideways to and fro. The mouth of the tube should
not be closed with the linger, as in this manner some
impurities may easily enter the mixture. Very often
the dialysates become turbid upon the addition of the
caustic soda solution, but this does not interfere
with the reaction. In order to test for diffused albu-
men we have different methods at our disposal, of
which the biuret reaction has been found to be the
best. One could also make use of the precipitin
formation that appears when prepared serum is
employed, but such serum is not always at hand.
Further, we may use ninhydrin, but it is not so
sensitive to albumen.

Ninhydrin reacts, amongst others, with com-
pounds which carry an amino group in a position to
the carboxyl group ; when it produces a bluish-violet
colour, if the concentration of the reacting compounds
is sufficiently strong. The albumen molecule contains
a few free amino and carboxyl groups, and as soon
as it is decomposed, these groups are set free. The
ninhydrin reaction becomes stronger the more the
albumen is decomposed, provided the various stages
of decomposition are not withdrawn. At each stage
an amino and carboxyl group are set free. The
biuret reaction manifests itself quite differently. The
greater the fractional decomposition of the albumen,
the weaker is the biuret reaction. As soon as we pass
a certain limit of decomposition the reaction ceases.

THE DIALYSATION PROCESS 155

The biuret reaction is unfortunately rather difficult
to detect when it is a case of demonstrating slight
traces of the reddish-violet coloration. This is due
to the fact that the eye is but slightly sensitive to these
tints. Again there are great individual differences.
If the observer is unable to detect a light biuret
reaction then he has to rely on standardized tubes ; or

j

else he must make use of the ninhydrin reaction and
try, by means of lengthy dialysation, to. increase the
quantity of albumen in the dialysate, so far as
the tubes are permeable to albumen. Seeing that
white of egg, as well as serum, always contains
substances which diffuse and react with ninhy-
drin, we are bound to find out, by means of a
standardized tube, what quantity of a given albumen
solution we may use without running the risk 'of the
dialysate showing a ninhydrin reaction. How to
perform the ninhydrin test we shall describe later,
when we give the test for equal permeability to decom-
posites of albumen.

The biuret reaction is performed as follows : To
the mixture of the dialysate with caustic soda about
i c.c. of a very much diluted copper sulphate solution
-e.g., i in 500 c.c. is added. This solution is run
down by means of a pipette along the inside of the
test-tube, so as to obtain a surface layer. Then we
observe by transmitted light the dividing line
between the blue layer, which often, however, appears

156 TESTING OF THE DIALYSING TUBES

turbid owing to the deposition of copper hydroxide,
and the quite colourless liquid below. The slightest
trace of a pinkish-violet colour is a proof that the
tube from which the dialysate was procured is un-
suitable. Often the presence of albumen is shown by
the fact that the precipitated copper oxide dissolves
after a time in about half an hour and a clear violet
layer appears which gradually diffuses into the other
liquid. With this test it is better to be over-cautious,
and the tubes should be rejected each time the biuret
reaction gfives doubtful results.

o

(b) Testing of the Dial y sing Tubes for equal Per-
meability to Decomposites of Albumen.- -Tubes,
which do not allow the passage of albumen,
must first of all be thoroughly cleansed. Their con-

O J

tents are poured out, and they are then placed on a
sieve and rinsed for about half an hour in clean
running water.

For the sake of security they are put in boiling
water for not more than half a minute. We may also
point out that experience has shown that boiling
the tubes, is not very good for them, for they easily
become too dense. After this, 2*5 c.c. of a i per cent,
solution of silk-peptone are poured into them ; the
tubes are again carefully rinsed in cold water, one
by one, and are then placed in Erlenmeyer flasks filled
with 20 c.c. of sterilized distilled water (compare
pp. 150-152). The latter is covered with toluol. In

TESTING OF THE DIALYSIXG TUBES 157

this case also the dialysis is carried on in an incubator,
in order to expose all the tubes to approximately equal
conditions,

After some sixteen hours the ninhydrin re-
action is applied. As this reaction depends so
much upon the degree of concentration, it is
advisable to carefully guard against the following
sources of error. First of all, the dialysate must
not be allowed to evaporate unevenly. To avoid this,
an excess of toluol is added, and the Erlenmeyer
tube is preferably covered with a watch glass. It is
clear that, should the different dialysates evaporate
unevenly, the ninhydrin reactions would be of vary-
ing intensity. The second source of error lies in the
boiling of the separate test-tubes, which is applied
in order to produce the formation of the colouring
substances. We shall return to this presently.

In the application of the ninhydrin reaction we must
never forget the fact, that ninhydrin is a most delicate
reacting agent for albuminous substances, peptones,
polypeptides, and amino-acids. Perspiration reacts
very readily with ninhydrin, as do also the epidermic
scales, &c. It is most important to avoid any contact
of the dialysing tube with the hand ; only sterilized
forceps should be employed for holding them, and
all the apparatus in use should be absolutely clean
and dry. One must never rely upon any rapid
drying methods. In the first place, it will not do 10

158 TESTING OF THE DIALVSIXG TUBES

transfer the dialysates into the test-tubes by means
of one pipette. It is essential to have at one's dis-
posal for the actual tests as many different pipettes,
graduated to 10 c.c., as there are dialysates to be
handled. The test-tubes must also be absolutely
clean and dry, and they must be of exactly the same
width. Pouring the dialysates into the test-tubes is
not admissible, because the toluol may easily spoil the
reaction, chiefly by preventing satisfactory boiling.

In detail one proceeds as follows : As before, the
pipette, closed at the top with the finger, is passed
through the toluol layer, and 10 c.c. of the dialysate
are withdrawn. The pipette is kept closed when
passing through the toluol layer, in order to prevent
any toluol from entering it. After transferring
10 c.c. of all the dialysates into the test-tubes, using
separate pipettes for each, we add to each test exactly
o'2 c.c. of an accurately prepared i per cent, solution
of ninhydrin.

For accurate measurements a capillary pipette of
i c.c. is used. The ninhydrin solution is prepared
as follows : ninhydrin is usually sold in o'i-gr.
packets, and this quantity is shaken out of the tube
into a measuring flask marked to 10 c.c. The tube
is best emptied by tapping it against the inside of
the mouth of the measure, though it is not possible
by this means to transfer the whole of the o'i gr. of
ninhvdrin into the measure. The rest of the nin-

TESTING OF THE DIALY5IXG TUBE- 159

hvdnn must be dissolved with distilled and sterilized
water; this solution is poured into the measure, and
the tube is again rinsed several times, after which

_

the measure is filled up nearly to the mark. Xin-
hydrin dissolve - - aringly in water, and in order I
dissolve it quickly it must be heated a little. F r
this purpose it is best to stand the measure in the
incubator. As soon as the solution is effected, the
contents are . and filled up to the mark on th^

flask.

The ninhvdrin solution is not absolutely stable.
I: is liable to infection, and is a".- nsitive to the
action of liglv . I: mav be kept in a brown flask,
but thi- is not ne- ess - > long as one prepar -
<~>nlv 10 c.c. at a time, a quantitv which is quickly-
used up.

After all the test-1 es containing 10 c.c. of the
dialvsate have been filled with o'2 c.c. of the ninhv-
drin solution, a boiling-stick is placed in each. The
latter is absolutelv essential, because onlv a verv
even ebullition will produce a properly comparable
colour reaction. The boiling-sticks used in the trade
are divided into segmen'- about 10 cm. long; these
must be boiled in distilled water, dried at 6>
~ - ' *.. and kept in a tightlv closed glass vessel. Boil-
ing-sticks must not be stored in a damp condition :
for on the one hand their use in this state mav give

o

rise ' error, owing 1 to the uneven amount - :'

I6O TESTING UF THE D1ALYSING TUBES

water present, while on the other hand mould may
easily appear. Again, the boiling-sticks should never
be dried at too high a temperature, otherwise they may
turn brown, and in that case they give off a brown
colouring matter during boiling, and thus render
an exact reading impossible. They must never be
touched with the hands, but should always be placed
in the test-tubes by means of forceps.

The process of boiling is now started, and the
manner in which this is carried out is of the greatest
importance. Boiling must be intensive; at the same
time every precaution must be taken to avoid the
slightest spilling, as also to prevent uneven evapora-
tion. When all the liquids to be tested have been
boiled, we must assure ourselves that they are at the
same level in all the test-tubes. It is best to use
large test-tubes upon which the volume of 10 c.c.
is conspicuously marked. It is then easy to ascer-
tain whether the very important point of even boiling
has been accurately carried out.

The test-tube is first held by means of a holder in
the centre of a Bunsen burner, the flame of which
must be a full one. One then watches carefully for

/

the moment when the first bubbles of gas appear on
the sides of the test-tube, which only takes a few
seconds, and calculating from this moment one boils
for exactly one minute. After ten to fifteen seconds a
vivid ebullition is observed, and as soon as this point

TESTING OF THE DIALYSING TUBES

161

is reached the test-tube is brought to the edge of the
flame, and the boiling is continued at the middle
height of the flame (see fig. 8). In this way it is
possible to carry out the boiling continuously and
energetically, so that the liquid travels over more than
half of the test-tube, without any danger of over-
boiling. Not for a single moment should the atten-
tion be allowed to wander from this process, for

FIG. 8.

(a) Test-tube holder; (b) boiling-stick.

everything depends upon the accuracy of the
operation. If the ebullition is too weak, then under
certain circumstances the reaction may fail altogether,
while, if the rate of ebullition differs in the different
tests, we get a difference in the intensities of the
colorations. The results, in short, are inaccurate.
After the lapse of half an hour a comparison of the

I I

l62 PREPARATION OF THE SUBSTRATES

intensity of the blue coloration is made in each
case. It is soon found that a particular intensity
prevails. All the tests which show a greater or
smaller intensity than this are carefully noted, and
the tubes from which the dialysates in question were
obtained are rejected. In this case, too, it is neces-
sary to be very accurate, otherwise the actual experi-
ments may easily lead to deceptive results. Thus it
may happen that serum alone, and serum added to
a given organ, contain diffusible compounds which
react with ninhydrin with precisely equal difficulty ;
yet on testing the dialysate in the experiment serum +
organ, we may get a positive reaction, because the
tube was more permeable to decomposites of albumen
than the tube used as a control.

The tubes that are equally permeable in this respect
are now carefully rinsed, then plunged into boiling
water for thirty seconds, and finally brought into a
sterilized flask. Sterilized water is added, together
with an equal quantity of toluol, the fluid being
calculated to fill the flask exactly. The tubes are
now ready for use. They are taken out of the flask
with sterilized forceps and must, if possible, not come
into any contact with the fingers during all the
manipulations.

Preparation of the Substrates (Organs'). As material
for these tests we use either an albuminous body or
else a mixture of these bodies e.g., an organ. The

PREPARATION OF THE SUBSTRATES 163

manner in which a substrate is prepared is of the
greatest importance for the whole success of the
dialysation process, and, unless one adheres to the
directions in every particular, one is bound to meet
with unsuccessful results. These, however, can be
successfully avoided if the preparation of the sub-
strate be carried out with proper attention. The
principle of the matter is, to obtain substrates which
contain coagulated albumen and are absolutely free
from diffusible substances which react with ninhydrin.
We shall demonstrate the method of obtaining the
substrates by means of the preparation of coagulated
placenta. Other organs are treated in exactly the
same way ; only, those which are rich in fats and
lipoids have to be previously extracted with carbon
tetrachloride in a Soxhlet apparatus. The same
applies also to tubercle bacilli. Placenta can always
be procured in a fresh state, whereas in other cases
we have to deal with organs from dead bodies. In
the latter case the dissection should be made at the
earliest opportunity. The best corpses are those of
accidents. If prolonged agony has been undergone
previous to death, the organs are almost useless.
It is very important to test the organs for patho-
logical changes; and it is absolutely essential
to state in what condition the organ used was
found, for different results could easily be obtained
if one observed used normal organs while another

164 PREPARATION OF THE SUBSTRATES

made use of abnormal ones. The question
whether the organs of animals may be used will be
discussed later. 14

The organ must be absolutely .freed from blood,
a condition that can be attained in the case of
different organs with varying facility. Placenta and
lungs, for instance, can be easily washed so as to free
them from blood, or the blood may be rinsed out
through the large blood-vessels ; whilst the liver,
kidneys, and particularly the uvea, are freed from
blood with great difficulty. With the latter there is
scarcely any other means of proving its suitability,
than by experimenting comparatively with serum
from individuals with healthy and diseased uvea
respectively. The pigment prevents us from dis-
covering the last traces of blood.

The fresh and still warm placenta is first freed
from blood clots by mechanical means, the mem-
branes and the umbilical cord being removed at the
same time. Then the placenta is cut into small pieces,
about one inch square or less, and these are crushed
in a current of water, for which purpose they are
best placed on a sieve. Water is allowed to run
continuously upon the pieces of placenta, each piece
being pressed between the fingers. From time to
time the pieces are placed in a cloth and squeezed
in it. The washing of the placenta must never be

14 See also p. 27.

PREPARATION OF THE SUBSTRATES 165

interrupted. Pieces to which coagulated blood
adheres, which cannot easily be removed, are rejected.
Finally, they are placed in a mortar and broken up
with a pestle, by which process the last traces of
blood are eliminated ; and then the connective tissue
can be removed. We now have a snow-white tissue,
which is immediately boiled. The whole process
takes from one to at most three hours, according to
the kind of tissue employed.

The extraction of blood can also be effected by
thoroughly washing out the organ through the blood-
vessels ; but in this case, the organ must be washed
out again after being broken up. If the extraction
presents any difficulties, one can often attain one's
object by covering the tissue in the fresh state with
a very thick layer of common salt. The mixture
is allowed to stand for two to six hours in
an ice-chest; the salt is then dissolved, and
the washing carried on in the usual manner.
One must never preserve an organ from which
the blood has been incompletely abstracted, in
any particular manner, with the intention of com-
pleting the process later on. All preservation media
produce coagulation and alteration of the blood. The
smallest blood-vessels always contain, in that case,
small quantities of blood constituents. We must
also give particular warning against the use of bleach-
ing agents, as, for instance, hydrogen peroxide. The

l66 PREPARATION OF THE SUBSTRATES

red colour of the blood indicates to us that it is still
present. If we use hydrogen peroxide, then we lose
any control over the blood contained in the tissue.
If one is not quite certain of the fact that the organ
is free from blood, one should squeeze out a few
pieces of it in a little water, and examine the fluid
with the spectroscope.

About a hundred times more distilled water than
there is of the tissue is placed in an enamelled vessel
and then brought to the boil. The tissue, having
been absolutely freed from blood, is placed in the
boiling water, for every litre of which it is advisable
to add about five drops of glacial acetic acid. This
is boiled for ten minutes, and the boiling water is
passed through a sieve; the tissue is thoroughly
rinsed for about five minutes with distilled water,
and the same process of boiling is repeated, using
fresh water without the addition of acetic acid. The
boiling, the pouring off of the boiled water, the
rinsing of the tissues, and the renewed boiling are
repeated about six times without interruption. If it
is necessary to cease boiling, then one must never
forget to pour a fairly large quantity of toluol on the
top of the boiled water containing the tissue. If
this be omitted the tissue is liable to become infected,
and then some hours of boiling may be necessary in
order to free the organ again from extractive sub-
stances which react with ninhydrin.

PREPARATION OF THE SUBSTRATES 167

If a centrifuge be at one's disposal, the boiling
water is centrifuged at a suitable speed. This is
still more necessary when one is working with finely
minced organs or bacteriological cultures and the
like, otherwise too much of the material would be
lost when pouring off the water.

After the sixth boiling only five times the amount
of water at most is used. The smaller the amount of
water employed, the more exact is the result of the test
for the extractive substances that react with ninhydrin.
In every case as much water must be present as will
be needed to continue active boiling for five minutes
without the risk of burning, the smallest possible
vessels being used. Then a certain quantity of
boiled water is filtered through a hardened filter paper.
To 5 c.c. of the filtrate is added at least i c.c. of a
i per cent, aqueous solution of ninhydrin, and the
mixture is boiled (as described on p. 160 seq.) for one
minute. If, after half an hour, not the slightest trace
of a violet coloration manifests itself, the organ may
be considered as suitable, provfded it still remains
snow-white. Only the tissues of the liver, the spleen,
and the kidneys do not appear quite white. Should
the tissue turn grey, or even brown, during boiling,
this is a proof that it was not absolutely freed from
blood, or that the boiling was not conducted properly.
Should the particular test prove positive, the boiling
must be continued i.e., the water must be poured

1 68 PREPARATION OF THE SUBSTRATES

off, the organ be thoroughly rinsed in distilled water,
and boiled over again for five minutes with not more
than five times its own quantity of water. It is
filtered again through a hardened filter; to 5 c.c. of
the filtrate is added at least i c.c. of ninhydrin
solution, and the mixture is boiled for one minute.

Before the organ is put by for keeping, it is spread
upon a white glass plate or a sheet of white paper,
and every separate piece is thoroughly examined.
Should brown spots or other doubtful points, which
cause one to suspect the presence of coagulated blood,
be noticed, the pieces affected must be thrown away.
Only by conscientiously and carefully adhering to
these rules can results be expected which are free
from all objection. An organ, which has given a
whole series of correct results, may lead us astray if
even one single piece containing blood happens to be
used.

As soon as the organ has been tested in the above
manner for the absence of any piece that may contain
blood, and as being free from extractives which
react with ninhydrin, it is immediately placed
in a bottle, with a well-ground stopper ; the bottle
having been previously sterilized. Then a little
sterilized distilled water and a good deal of chloroform
and toluol are added, the bottle beino- filled in such

7 O

a way that the stopper comes into contact with the
liquid. A thoroughly well-prepared organ should

PREPARATION OF THE SUBSTRATES 169

preserve indefinitely, and it only becomes useless
again by being contaminated. There are various
contingencies that may spoil a perfect organ. In the
first place, it must be taken out of the bottle only by
means of sterilized forceps. None of the sample taken
should be put back into the bottle if it has been
exposed to the risk of infection, or been left lying
about, and so on. The bottle must be kept filled with
toluol, otherwise part of the tissue may adhere to the
neck of the bottle. If such a piece protrudes from
the level of the toluol it decays, and finally drops
down on to the rest of the tissue. The bottle con-
taining the organ should be kept in an ice cupboard.

Bacteria and other living organisms may be pre-
pared exactly in the same way as tissues. Boiling is
also resorted to in these cases ; and the same rules hold
good. It is obvious that organs can be separated
into their tissues. The more special the problems
to be dealt with, the more does one limit oneself to a
very definite tissue.

All organs which are very dense in structure, and
which become hard when boiled, require special
treatment. Carcinomas, myomas, &c., may appear
snow-white and still contain blood, so that in these
cases the pieces have to be cut into very minute
particles in order to prevent mistakes.

Every organ must be standardized. Placenta is
only useful so long as it is not decomposed by the

I/O PREPARATION OF THE SUBSTRATES

serum of carcinomatous subjects, or of individuals
with salpingitis, tuberculosis, and the like. Carci-
noma is correctly prepared if it is not attacked by the
serum of pregnant individuals.

Above all, the organ should be tested by means of
cases which contain ferments acting against the com-
ponents of the red blood corpuscles. Cases of blood
effusion are excellent testing agents for the absence
of blood in the prepared organ. Or disharmonious
blood in this case human blood is injected into
an animal, and its serum is tested against
coagulated red blood corpuscles and the organ to be
employed.

In conducting these experiments we must be able,
with absolute certainty, to prevent the decomposition
of all proteins other than those belonging to the
actual organ itself. It is clear that serum, which
contains a defensive ferment against the components
of the form-elements of the blood, will decompose every
organ containing blood that is, it will split up, not
the proteins of the organ, but the components of the
blood within the organ. The importance of a clear
recognition of this circumstance may be gathered
from the fact that serum of normal horses and cattle
decomposed red blood corpuscles in about 40 per cent,
of cases. Further, it was found that serum taken
from animals that exhibited hagmatoma produced
decomposition with every kind of organ containing

PREPARATION OF THE SUBSTRATES

blood, whilst organs freed from blood and subjected
to parallel tests were left unattacked. This funda-
mental rule, of completely freeing the organ in ques-
tion from its blood, is often transgressed. If the
serum does not contain any defensive ferments against
the form-elements of the blood, then, of course, even
an organ containing blood may give correct results.
As, however, mistaken results are liable to occur,
such an organ should, as a rule, never be used.

It is advisable never to use one particular organ
exclusively for testing a definite problem ; and one
should always work with controls. For instance,
placenta is always tested with serum from obviously
non-pregnant persons. Male serum should also be
employed. Should cases of diabetes, for instance, be
tested exclusively with faulty preparations of pan-
creatic gland, then in most cases a ' decomposition J
would be found. Such mistakes are avoided bv

/

using thoroughly prepared organs on the one hand,
and by means of control experiments on the other.

It is of fundamental importance to establish the
morphological state of the organ used, and its origin.
It is possible that, in a given disease, a normal organ
is not decomposed, although the same organ is readily
attacked, if it has already undergone particular
pathological changes. Thus it is quite possible
that, for instance, a normal thvroid Hand would not

J O

be decomposed by Basedow serum, while a gland

IJ2 PREPARATION OF THE SUBSTRATES

originating from a morbus Basedowi would be subject
to decomposition. Just as every case examined has to
be thoroughly tested by clinical means, and its further
course closely followed up, so also must the substrate
to be employed be characterized exactly. A bare
statistical compilation of cases, with percentage
accounts of faulty diagnoses is unworthy of scientific
publication. Each separate case must be clinically
investigated. This is the reason why the fruits of
these researches are bound to fall into the hands of
clinical observers. The physiologist can only note
one case after another without being able to charac-
terize them individually, or even to observe them
continuously, and, in consequence, we can expect
little help from his side.

A very important question is whether, instead of
human organs, the corresponding organs of animals
may be used in experiments with human serum. 15 It
would naturally be a great advantage in all these
researches if this were the case. Our earliest re-
searches enabled us to state the fact that human
placenta can be replaced by that of animals, and vice
versa. 16 We have made further experiments with the
brain and other organs, and have obtained good
results. It seems that organs which have the same

15 Compare here also p. 27.

16 Compare also the works of Schlimpert and Issel (Lit.
74), of v. Hippel (Lit. IIQ), Fuchs (Lit. in).

PREPARATION OF THE SUBSTRATES 1/3

function to fulfil, in the animal kingdom have common
properties in their structure. In spite of our favour-
able experiences we have not ventured generally to
recommend the use of animal organs. It is still very
difficult to find a proper balance amongst the contrarv
results of many observers, and were we to change
the type of substrate without sufficient experience,
we should arrive at still more divergent results.
This is the reason why it is particularly necessary to
use organs of the same species as that to which the
serum under investigation belongs, as well as those
of a different type. Only when it is established that
harmonious results are obtained ought we to be
satisfied with non-specific organs, and always under
the condition that no substrate is used which shows
definite pathological alterations.

Means of Obtaining Blood Serum.- -Three condi-
tions have to be complied with. The serum must be as
poor as possible in diffusible substances which react
with ninhydrin, and this is attained by taking the
blood in a fasting condition. In all cases in which
the albuminous metabolism is very rapid, in cases of
disease which are accompanied by decay of the
tissues, as in the case of carcinoma, in cases of
absorption of exudates and transudates, in all purulent
processes, and lastly, in effusions of blood, the blood
always contains a larger quantity of such compounds.
The blood serum must further be absolutely free from

174 OBTAINING THE BLOOD-SERUM

haemoglobin, and in doubtful cases the spectroscope
should be used.

The serum must be completely freed from its form-
elements, a point which is often neglected. A serum
may appear absolutely clear, and yet contain millions
of red blood corpuscles. The serum must be treated
with a good electric centrifuge until the tube shows
no trace of blood corpuscles, either on its sides or
at the bottom. The serum, after each treatment with
the centrifuge, is drawn off with a pipette and trans-
ferred to another tube, and during this operation, in
order to avoid any contact of the pipette with the red
blood corpuscles, the tube is placed upon a mirror.
One can then see exactly where the end of the pipette
is at any moment. The blood is best taken with an
absolutely dry needle and placed directly into a
sterilized centrifuge tube, or, better still, into a
small Erlenmeyer flask. The blood is allowed to
clot spontaneously, and is watched until the serum
separates out. Any mode of procedure which
accelerates the separation of the serum increases the
risk of haemolysis. The blood should not be placed
either in an ice-chest or in an incubator, but should be
left simply at room temperature. In the first case, the
risk of haemolysis is very great ; in the second, auto-
lysis of the form-elements generally results. Serum
is generally obtained in a considerable quantity
after five or six hours, but if enough has not

OBTAINING THE BLOOD-SERUM 175

separated out one makes use of the centrifuge. In the
first case the serum is poured into a centrifuge tube,
and centrifuged for about five to ten minutes. It is
then easy to ascertain that the serum, which was ap-
parently free from solid elements, has now given off
a whole layer of red blood corpuscles during the
process of centrifuging a second time. Should this
remain in the serum, then during the dialysis
haemolysis would take place in the dialysing tube,
and the experiment would give faulty results.

It happens, usually, that the experiment is so
arranged that, say, 1*5 c.c. of serum are taken from
.the centrifuge tube and employed as a control.
Only after this do we remove more for the test,
organ + serum. If at this point the directions
are not followed exactly, it may easily happen that
red corpuscles are found in the test, organ + serum.
Haemolysis appears during dialysis, and then we
have exactly the same conditions as arise when
organs are used which contain blood; only in
this case the contents of the corpuscles are
found, not in the tissue, but in the serum. It is
from non-observance of the rules given that we get
the observation that a serum, which is absolutely free
from haemoglobin, appears quite red at the end of
the experiment. It is the diffusion of water from the
outer fluid into the tube that has led to the haemolysis
of the red corpuscles which, though present, have
been overlooked.

1/6 PERFORMANCE OF THE EXPERIMENT

It is sufficient to use 15 to 20 c.c. of blood. For
sending away, only serum should be used which has
been centrifuged completely. The latter must in any
case be centrifuged again. The serum should not
be more than twelve hours old, even though it
has been collected and preserved in a really sterile
way. The taking of the blood, its collection, and its
manipulation must be done aseptically.

PERFORMANCE OF THE EXPERIMENT.

In carrying out a dialysation test the following
fundamental rules must be obeyed, of which not one
is unimportant :

(1) Extreme cleanliness is the first condition for
ensuring success in the experiment. This applies
to the surroundings, and to all the utensils employed.
Pipettes, test-tubes, Erlenmeyer flasks, &c., must
be thoroughly cleansed and absolutely dry.

(2) The water used must be thoroughly sterilized
distilled water. So-called distilled water often proves
to contain numerous germs of every description. If
one uses water like this as the outer fluid in dialysis,
then the way is laid open for all kinds of mistakes.

(3) The work is performed, as far as possible,
aseptically and antiseptically.

(4) In the room in which the experiments are in
progress neither bacteriological nor chemical work
should be allowed. Above all, an incubator must be

PERFORMANCE OF THE EXPERIMENT I//

specially reserved for these experiments, nor is it
possible to allow the incubator to be used at the same
time for bacteriological purposes.

(5) Before starting it must be ascertained that all
utensils are to hand and in perfect condition.

(6) Experiments can only be carried on with good
light. It is impossible to carry on more than five or
six experiments at the same time with the necessary
care.

(7) Before successful tests can be expected, one
must not only be certain of a perfect knowledge of all
the details of the method, but a thorough knowledge
of their fundamental principles is most essential.
It is not sufficient to know the method thoroughly,
one must have a perfect command of it, and, as it were,
live in it. No one is able to stain tissues perfectly
for the first time, even though he be guided by the
strictest directions. Even simple chemical methods
require practice, and the most elementary analyses
sometimes fail. Even the Kjeldahl method, which
is so easily handled, requires to be thoroughly learnt.
Should a failure result, no one would think of
communicating it while blaming the method; he
would never rest until the cause of the error was
found. The statement, "We have been working
in the strictest manner according to the given
directions,' I treat with scepticism on the basis
of a rich experience. Such great offences are often

12

PERFORMANCE OF THE EXPERIMENT

committed against the fundamental rules of the
whole method, that errors are bound to occur.
Therefore, we must not ignore a method because it
requires careful working. It is quite possible that
with time we shall arrive at more simplicity in our
manipulations, and technique may place sonic
further means at our disposal. But it is as yet
too early to try to introduce modifications in the
manner of working of the two methods, after a whole
number of observers have obtained good results by
their means in their present form. The principal
requirement of any method is, that we should not rest
until the cause of error is found in each case that
occurs. This is the only way of avoiding them.

First of all blood is taken. If there be any doubt
regarding the suitability of the substrate it is ad-
visable first to test the organ, so as to avoid with-
drawing the blood unnecessarily. This test should
be repeated immediately before performing the
experiment. The blood is allowed to coagulate
spontaneously at the temperature of the room.

Immediately before beginning each experiment the
organ is tested, and this important rule must never
be neglected. It may so happen that all the parts of
an organ have been freed from all extractive sub-
stances reacting with ninhydrin, except a piece here
or there. It should be the duty of the observer to
note down each time in his record : " Organ tested."

PERFORMANCE OF THE EXPERIMENT 1/9

So much of the tissues as are necessary for the
experiments to be performed are taken, and to them
is added at. most five times their quantity of water.
If any difficulty arises in the boiling, which may be
traced to the insufficient quantity of tissue used, then
more tissue is added, the excess of the organ being
immediately put back into the bottle that contains
the rest, in case it may be wanted later on. If the
organ is left Ivino- about for anv time it becomes

o o

infected. An organ should never be boiled without
being previously tested. It should not show any
places that contain blood.

Further, the tissue must be shredded into small
particles before it is boiled. It would be a great
mistake to boil the tissues in large pieces and
to use them later in the form of little pieces, for
it might often happen that inside the big pieces pro-
ducts were enclosed which diffuse and react with
ninhydrin, and they would not be noticed because
they have not reached the outside. If, for instance,
a lentil is boiled as a whole, the water does not readily
show any ninhydrin reaction, but as soon as the lentil
is broken up and boiled an intense reaction is
observed. In the process of boiling the outer part
coagulates, and thus tightly encloses the inner con-
tents. Exactly the same thing may happen with
other tissues. Therefore, before the experiment, the
organ must be boiled in the same way as it is intended
to be used, i.e., in a shredded form.

180 PERFORMANCE OF THE EXPERIMENT

The best way is to carry out the boiling in a test-
tube for live minutes. It must be boiled energetic-
ally, and then filtered through a small hardened
filter; after which, at least i c.c. of the i per cent,
ninhydrin solution is added to 5 c.c. of the filtrate.
Should one have less than 5 c.c. of the filtrate there
is no harm in boiling with i c.c. of ninhydrin,
because the stricter the conditions of these tests the
better.

Boiling is performed (as described on p. 161) for one
minute with the aid of a boiling rod. Only in cases,
where the solution gives no traces whatsoever of a
violet coloration, can the organ be used, and one must
wait half an hour before one can establish its presence
or absence. Should the organ not be required for
immediate use, it must at once be covered with a
layer of toluol. Should this test still give a colora-
tion, then the substrate must be boiled over again
with five times as much distilled water, until the test
shows negative results.

Now, as many standardized dialysing tubes as are
required are placed into empty, dry Erlenmeyer
flasks, and about J gnri- of the organ is poured
into the tubes. This quantity is previously placed
upon a piece of blotting paper, and dried by squeezing
it strongly. Were the organ placed in a wet state
directlv into the tubes, a reaction which would eive

o

a weakly positive result might turn out negative,

PERFORMANCE OF THE EXPERIMENT iSl

i

owing to the dilution of serum so caused. The tissue
should never be handled with the fingers.

To the tubes containing the tissue i to 1*5 c.c. of
serum are now added. A rule should always be made

j

of arranging this experiment first. Afterwards from
i to i '5 c.c. of serum are placed in an empty tube
(control test). Then the tubes are thoroughly rinsed
with distilled water (as described on p. 151), and
placed in Erlenmeyer flasks which have previously
been filled with about 20 c.c. of sterilized water. Then
a large amount of toluol is poured into the tubes and
over the liquid outside, care being taken that the
part of the tube which projects from the liquid should
be soaked with toluol. At this stage of the experi-
ment the following sources of error may arise. First
of all, water may get into the tubes while they are
being rinsed. If the work is not carried on in a
scrupulously accurate manner considerable dilutions
may occur. The tube must be completely closed
during this operation. I have lately been in a
position to observe a second source of error which
may arise. Contrary to instructions the flask was
filled with 20 c.c. of water and a large quantity of
toluol, and only then was the tube and its contents
immersed. In this case the liquid in the flask was
raised to such a level that it passed from the outside
to the inside of the tubes. Besides, the tube came
into contact with the neck of the Erlenmever flask

l82 PERFORMANCE OF THE EXPERIMENT

in many places, and here part of the liquid became
enclosed by capillary action, thus forming a kind of
communication between the contents of the tube- and
the liquid outside. From these observations it
follows, that the toluol should never be introduced
before the dialysing tube has previously been im-
mersed in the 20 c.c. of water, in which case the
quantity of toluol added can be accurately controlled,
and care can be taken that both the inner and outer
surfaces of the tubes shall project at least 0*5 c.c.
over the toluol layer. Moreover, only wide-mouthed
Erlenmeyer flasks should be used.

Then the flasks are placed in an incubator at a
temperature of 37 C. At a higher temperature the
ferments would be destroyed, and at a lower tem-
perature the decomposition would be too slow.

After about sixteen hours the experiment is stopped.
A thick layer of toluol must still be found upon the
contents of the tubes, as well as on the surrounding
liquid, at the end of the experiment. The Erlenmeyer
flasks, carefully numbered, are best arranged in no
special order. Then the tubes are taken out of the
flasks, and placed, right up to the end of the experi-
ment, into empty Erlenmeyer flasks. In withdraw-
ing the tubes one at the same time effects a uniform
mixing o f the dialysate. Particular care must be taken
to avoid a source of error that often arises at this
point, which is, that if the flask has been supplied

PERFORMANCE OF THE EXPERIMENT 183

with too much toluol, or if the tube at the beginning
of the test has not been immersed sufficiently deeply,
then it may easily happen that, during the intro-
duction of the pipette, some of the liquid passes from
the outside into the dialysing tube. While, if one
sucks strongly with the pipette at that moment, then
the reverse may occur, and the contents of the tube
may enter the pipette.

Ten cubic centimetres of the dialysate are taken, by
means of a closed pipette passed through the toluol
layer, and poured into a dry, wide, and absolutely
clean test-tube. For each dialysate, as a matter of
course, a separate, absolutely clean and dry pipette is
used. One's work should never be arranged in such
a manner that the pipette has to be hurriedly cleansed
with alcohol, water, or ether after use, for the clean-
ing in this case may very easily be insufficient.

The danger of soiling the pipette with saliva is
particularly great. (Compare p. 153.)

Then o'2 c.c. of the i per cent, aqueous solution of
ninhydrin are added to each test, together with a dry
boiling rod (see p. 159), and one test after another is
boiled absolutely evenly for a whole minute (see
p. 1 60). After half an hour we ascertain which tests
show a coloration and which do not, and only then do
we compare our results with the original dialysates.
If there be any tests which have evaporated more than
the others, they are rejected, if they show a positive

184 PERFORMANCE OF THE EXPERIMENT

reaction. It may sometimes happen that, for
instance, the dialysate of the serum gives a negative
reaction, while serum + organ shows a slight
violet coloration. If both samples have been boiled
equally according to the directions, then both
will have evaporated equally, and in this case the
slightest coloration may be considered as uncon-
ditionally positive. 17 If, on the contrary, the sample,
organ + serum, has evaporated more, then we are con-
fronted with the possibility that the stronger concen-
tration is the cause of the coloration. In spite of the
presence of absolutely equal quantities of substances,
capable of reacting with ninhydrin, in the dialysate of
the serum and that of the sample serum + organ, a
higher concentration has been obtained owing to
stronger evaporation. If it is impossible to effect even
boiling by any other means, then it is necessary to
resort to a water bath. The samples to be compared are
placed in a stand, and immersed in a water bath. The
boiling must be continued longer than when heating
in an open flame, but two to three minutes are

17 If the reaction is very weak, one may try to make it
stronger in the following manner : To each of the cooled solu-
tions dialysate from the experiment serum alone, and serum
+ substrate one again adds o'2 c.c. of the ninhydrin solution,
and boils for one minute. The reaction then often becomes
stronger. Obviously we must in this case, too, make a com-
parison with the dialysate of the serum only. Our present
experience is still too small to enable us to recommend this
process for general use.

PERFORMANCE OF THE EXPERIMENT 185

sufficient. As this method has not yet been applied
to large quantities, the most suitable time has yet to
be actually found.

Exact comparisons are only possible, when the
test-tubes are of the same dimensions and have
exactly the same thickness. For this purpose we
must always arrange to have a sufficient stock of test-

o

tubes answering perfectly to this requirement. In
order to realize the importance of this proceeding, we
only have to pour a slightly bluish solution into a
wide and a narrow test-tube respectively, when we
see that the former will show a much deeper blue
colour than the latter. A faulty diagnosis would
thus result.

The following cases are possible. The usual
result of the reaction is either, dialysate of serum
and of serum + organ negative, in which case
no decomposition has taken place ; and, if placenta
had been used, we should assert that there was no
placenta in the living state that had any connection
with the particular organism ; or else serum alone
gives negative, and organ - serum positive, results.
The diagnosis would indicate pregnancy, or, better
still, the existence of a placenta that still stands in
effective relations with the organism of the mother.

o

It may happen that the serum alone gives off sub-
stances to the dialysate, in sufficient quantity for the
positive reaction to appear under the conditions

1 86 I'KRFOiniAXCE OF THE EXPERIMENT

selected. If, in such a case, the sample of organ +
serum shows a markedly stronger blue coloration,
then the case has to be looked upon as positive in
respect to the reaction. Should, however, the differ-
ence in the intensity of the coloration be very small,
the experiment has to be performed again, using a
less quantity, say i c.c., of serum. It would then be
possible to ascertain whether decomposition had
taken place or not, as the serum sample would be
negative.

The appearance of the reaction should on no
account ever be determined by artificial light. Again,
it is not advisable to compare the test-tubes in their
stands, but each one should be taken out separately,
and examined against white paper by both trans-
mitted and reflected light.

Breaches of this rule are very often committed.
Many reactions are declared positive which, when
thoroughly investigated, show not the slightest
coloration. If a sample is marked as being just
perceptibly positive, then a number of other samples
should be changed about in the hand, and, only if
the same sample can be unhesitatingly picked out as
showing a coloration, should one's judgment con-
cerning the reaction be relied on.

Difficulties are only experienced with reddish and
yellowish-brown tones, but these have no relation
whatever with the ninhydrin reaction. They can

PERFORMANCE OF THE EXPERIMENT

easily be recognized by diluting a truly violet solution
with water until the intensity of the colour corre-
sponds with that of the sample, when one can see at
once that, though the solution has been very much
weakened, the colour still appears violet. A reddish,
or, rather, yellowish-brown tint means that either the
work has not been properly carried out, or else that
the blood contained acids or alkalies in excess. The
experiment must be repeated, otherwise it may
happen that the existing conditions conceal a positive
reaction. We shall deal with this point in fuller
detail, when we return to the question of the sources
of faulty observations.

Under certain conditions a special control test may
be needed. Such would be the case in dealing with
micro-organisms cultivated on a medium which
could not be readily separated by centrifuging.
In this case the germ-free medium must be boiled by
itself, until the filtered boiled water gives no traces
of coloration with ninhydrin. Then the cultures are
prepared in exactly the same way, and the following
tests are performed: (i) Serum alone; (2) serum +
medium ; and (3) serum + culture. Should the experi-
ment (2) produce decomposition, then a positive
reaction in experiment (3) would certainly not prove
that the micro-organisms had been decomposed.

A very important control test, for proving the suit-
ability of the organ or the substrate used, is the

l88 PERFORMANCE OF THE EXPERIMENT

following : About five to ten times more of the sub-
strate than has been used for the test is taken together
with 5 c.c. of water, and the whole is dialysed in an
incubator for sixteen hours, against 20 c.c. of distilled
water. Then the dialysate is evaporated on the water
bath to 5 c.c., and the latter is boiled in the usual way
with i c.c. of ninhydrin solution. The solution must
remain absolutely colourless. According to my own
experience this test always results negatively, when
the substrates have been prepared in accordance with
the directions. It is only necessary for the first test-
ing of the organ, and is carried out if doubts arise
as to the suitability of the latter. As the same organ
is always used over and over again for experiments
in which no decomposition is expected, we have a
concurrent control over the suitability of the organ.
Should errors occur in these experiments, then the
dialysing tubes are immediately tested, as well as the
organ, in the manner laid down. The statement that
for the control experiment organ alone was used-
'5 g r - f the organ and that 10 c.c. of the dialysate
have given a negative result, always proves that the
principles of the whole method have been misunder-
stood. An organ must have been very unsatis-
factorily prepared, if the 20 c.c. of the dialysate con-
tain such a quantity of substances reacting with
ninhydrin that the reaction, after being conducted in
the usual way, gives positive results.

PERFORMANCE OF THE EXPERIMENT 189

\Ye have described the performance of the experi-
ment as it is carried out at the present time.
Previously it was usual to make use of the biuret
reaction for proofs of decomposition of albumen. To
10 c.c. of the dialysate 2*5 c.c. of a 33 per cent,
solution of caustic soda were added, and this was then
covered with a layer of very dilute copper sulphate
solution. (See here p. 155.) If a violet to red ring
appeared, the reaction was recorded as positive.

The biuret test has been entirely given up for the
ninhydrin test, because the majority of observers have
a difficulty in distinguishing with certainty a feeble
biuret reaction. Those, however, who are capable
of distinguishing a biuret reaction, however slight,
should adhere to this test as well under all conditions.

SOURCES OF ERROR ix THE DIALYSATIOX PROCESS.

There are many possibilities leading to erroneous
results. It is best to consider them from the point
of view of utensils employed and manipulations
adopted, and to refer again to the sources of error
mentioned in the description of the method.

(i) Tubes.--\Ye take it for granted that all tubes
are thoroughly and accurately tested before anything
else. On the average about 20 to 30 per cent, of the
dialysing tubes supplied by the firm Schleicher and
Schiill will be useless, because there are nearlv

19O SOURCES OF ERROR (IN THE PROCESS)

always some which allow the passage of albumen. 1
Or they may become useless subsequently, generally
becoming permeable to albumen. This, however,
only occurs when they are handled improperly.
They must not be cleaned with a rough brush, nor
must they be boiled for too long a time. Tubes
may become impermeable to peptones through over-
boiling, so that, though they should be thoroughly
washed, they should be boiled but slightly. They
must be kept in sterilized water with a thick layer of
toluol (see p. 162), and must never be left for a long
time unemptied of their contents.

A great source of error which is, however,
impossible with proper manipulation, is due to
tubes being insufficiently cleaned. The result of this
is, that the wall of the tubes will contain traces of
substances, which react with ninhydrin if sufficiently
concentrated. They may be present in such minute
quantities as to be unable of themselves to produce
a coloration ; yet they will, when added to the
analogous substances that are present in the serum,
convert a negative reaction into a positive one.
Therefore the utmost possible care must be exercised
in the manipulation of the tubes.

18 We have recently observed up to So per cent, of useless
tubes. It would be very desirable if a dialysing tube could
be produced which was, at the least, indubitably impermeable
to albumen.

SOURCES OF ERROR (IX THE PROCESS)

Tubes must be tested a^ain about every four weeks.

O -

Should any error in diagnosis have occurred before
this time, and other possible errors have been ex-
cluded, then the tubes must be immediately tested for
permeability to albumen and for even permeability
to peptones.

(2) Serum. Here we have to deal only with its
age, the possibility of an infection, haemolysis, and
the contents of the serum in respect of red blood
corpuscles and of other form-elements. (See

pp. 173-175-)

(3) The Organ. --This is nearly always the cause
-.of errors in diagnosis. It is nearly always forgotten

o r?

that, in the arrangement and execution of the experi-
ment, we are dealing with quantitative conditions.
Two cases have to be distinguished :-

(a) The Biuret Reaction.- -The serum alone dors
not give off substances which diffuse and produce a
biuret reaction ; so that, as regards compounds which
give a biuret reaction, it must be reckoned as com-
pletely indifferent. It is comparatively easy to boil
the organ in such a way, that the water in which it was
boiled will not sfive any biuret reaction. If the ninhv-

O . ^

drin reaction turns out negative, one can never obtain a
biuret reaction. If such an organ be mixed with serum,
and the dialysate now gives a positive biuret reaction,
then we may be sure that decomposition has taken
place. The conditions here are very simple.

19- SOURCES OF ERROR (iX THE PROCESS)

(b) The Xinhydrin Reaction. In order to under-
stand the propositions that follow, we must be clear
concerning the fact, that blood serum always contains,
in varying quantities, substances which are to be
found within the peptone group, and therefore react
with ninhydrin. After a meal at which albumen
has been taken, the quantity of such substances
appearing in the serum immediately increases, in
consequence of which the blood must be taken during
a state of hunger.

A great many experiments have been necessary
to determine what quantity of serum, in general, will
give off to the dialysate only so much of the
substances referred to, as is required for a negative
reaction with ninhydrin. An insufficient quantity of
serum must not be used, if the decomposition of the
organ's albumen is to be as complete as possible. It
has been found that, in general, 1*5 c.c. of the serum
may be used. It is obvious that, under certain
circumstances, an even greater quantity of serum mav
give off so few substances reacting with ninhydrin
that the reaction of the dialysate still remains negative.
Conversely it may happen that 1*5 c.c. of serum
alone will give a positively reacting dialysate, which
is the reason why a control test with serum alone is
absolutely essential. The latter test indicates whether
the serum in use answers the condition of not giving
off, of itself, a sufficiencv of substances to react with

SOURCES OF ERROR (iN THE PROCESS) 193

ninhydrin. It is obvious, for the reasons mentioned,
that exactly the same quantity of serum must be
added to the organ, as has been used for the control
test with serum alone. We must never, on the
strength of the fact that the test with the serum alone
gives a positive reaction, jump to the conclusion that,
during the test, proteins have been decomposed in
the serum. The substances producing this reaction
were present from the beginning. If the reaction
with serum alone turns out negative, then it simply
means that the dialysate contains those compounds,
which react with ninhydrin, in a state of concentration
insufficient to produce a coloration; and this is the
only conclusion we are entitled to draw from the
result. It certainly does not indicate that there are
no such compounds present. If one concentrates a
dialysate of this kind, it eventually gives a positive
reaction.

We therefore arrive at the fact, that we can only
determine whether there are sufficient .compounds
present to give the coloration, but not what the
quantities actually are. If, however, the following
conditions are complied with, then this circumstance
offers no difficulties. The organ must be absolutely
free from substances, reacting with ninhydrin, which
can be boiled out and so passed over to the filtrate.
When the tubes are rinsed, no water should be allowed
to enter them. The organ must be perfectly dried,

13

194

SOURCES OF ERROR (IN THE PROCESS)

before the tube is tilled with it. During storage
in the incubator no evaporation must take place.
Further, when boiling the actual samples, uneven
ebullition must not be allowed. An example may
help us to make these conditions clear. We will
assume that twelve experiments have been made with
serum obtained from non-pregnant individuals, and
that the serum has in every case given negative
results. We conclude that none of the dialysates
have attained the necessary concentration in com-
pounds that produce coloration with ninhydrin. Only
from a certain concentration onwards is the coloration
possible ; and this limit we designate by the number T .
Then, to give an example, the cases quoted in the
annexed table are possible :

Contents in the

Case

Test with
serum alone.
Nit/hydrin
test.

serum of compounds
which, with
ninhydrin at a suffi-
cient concentration,

Test with
organ + serum
organ = o.
Ninhydrin

Test with
organ + serum
organ = o. 10
Ninhydrin

Test with

organ -f- serum
organ = 0*50.
Ninhydrin

react so as to pro-

test

test

test

duce a coloration

I

-

O'I2

-

2

0-45

3

0-84

+

4

0-65

--

+

5

0-89

+

6

0-98

+

+

7

0-87

+

8

099

+

+

9

O'42

10

0-86

+

ii

078

-f

12

o'75

_

+

SOURCES OF ERROR (IX THE PROCESS)

Three series of experiments were conducted with
the same sera, and with equal quantities of these.
In the first experiment the organ was -- ; o, i.e., it was
absolutely free from substances which could be boiled
out and filtered, and which, under the strictest con-
ditions, would produce a coloration with ninhydrin.
In every case we had to add to the quantity of
substances emanating from serum alone, and passed
into the dialysate, o gr. of these compounds. Then,
in the experiment serum organ, the ninhydrin
reaction obviously remains negative.

For the second experiment an organ was taken,
which passed over to the boiled water just a trace
of reacting substances. \Ve will assume that it
contained o'lo gr. 19 of these compounds. This quan-
tity is added to that which the serum gives off, and
we have the positive reaction of Cases 6 and 8. The
limital value, i, has been exceeded. Thus, by means
of a simple addition, a positive reaction has been
obtained and, in consequence, two errors in dia-
gnosis. The third column shows us how the ninhydrin
reaction results, when we use an organ prepared in a
still more imperfect manner.

Exactly the same position is reached, if the dialysate

We take this here merely by way of an example.
Obviously, in actual tests, the same quantity, i.e., o"io gr. ,
would never be transferred to the dialysate, if the organ can
only give off that amount; some lesser quantity would pass
over.

196 SOURCES OF ERROR (IN THE PROCESS)

evaporates unevenly in the incubator. Take, for
instance, Cases 6 and 8. In both cases the serum
alone nearly reaches the limit, i. Then, should
the dialysate, in the experiment organ + serum,
evaporate more strongly, or should the correspond-
ing dialysate become more strongly concentrated,
during boiling, than that of the relative control
experiment, then we shall get a positive reaction
owi-ng, entirely, to the concentration ; in which case
we shall get a wrong result. These examples may
be a warning to those who make use of a particular
technique in an imperfect manner.

It is easy to understand that errors in diagnosis
have often occurred, and that, on the other hand,
brilliant results have been reported.

As a matter of fact our limit value, i, is seldom
attained. Unfortunately, this occurs just when
carcinoma, myoma, salpingitis, exudates, suppur-
ations, or haemorrhages are present, that is, just
when the method should diagnostically give the most
valuable differential results. It is obvious that the
investigation of such cases requires double care.

The performance, under absolutely equal conditions,
of a particular experiment, and its control, is of deci-
sive importance in regard to the results obtained. In
the first place, absolutely pure distilled water must be
used. Water, which gives an acid or alkaline reac-
tion, leads inevitably to erroneous results. Ninhydrin

SOURCES OF ERROR (iN THE PROCESS) 197

reacts not only with albumen and albuminous
decomposites, but under certain conditions with other
compounds as well, for instance, sugar. 20 No trouble
can be caused by these, if distilled water be used.
The organ cannot give off any non-albuminous sub-
stances, which will affect the reaction of the fluid, if it
is boiled in the manner prescribed. There could not
possibly be any carbohydrates left, and we have the
control test with serum to fall back on, in any case.
Were this to contain much sugar, and, in conse-
quence, to interfere with the reaction of the outer
fluid, then it is conceivable that a coloration might
take place, which could not be referred to albuminous
decomposites. This result, however, would appear in
the test with serum alone, and also in the one with
serum substrate. Even the blood serum from
cases of diabetes does not show any positive reaction
ascribable to the presence of sugar. Non-compliance
with the directions respecting water generally mani-
fests itself in the fact, that a really positive reaction
turns out negative ; the reaction being, in fact, very
sensitive towards acids and alkalies, i.e., towards H
and OH ions.

For the reasons laid down we must always boil

j

the organs in distilled water, and preserve them, as

20 Vgl. \V. Halle, E. Loewenstein und E. Pribram :
" Bemerkungen iiber Farbreaktionen des Triketohydrinden-
hydrats (Ninhydrin), 3 ' Biochem. Zeitschr., lv, 357. 1913.

198 SOURCES OF ERROR (IX THE PROCESS)

\\cll as the tubes, in this medium. The rinsing ot
the dial \\sino' lubes must also oe done with distilled
water.

Finally, we must bear in mind another source of
error, which we have not yet specially referred to. It
may sometimes happen, that the substrate added to
the serum absorbs some Constituents of the latter, and
retains them. Such a case would manifest itself in
the fact, that the serum alone would react positively,
while the dialysate of the experiment, organ + sub-
strate, would give a negative reaction. Further, a
reaction might give a negative result, although
decomposition had actually taken place. The optical
method would easily detect such sources of error.

There is no single point in the rules which lacks a
definite foundation. Researches have generally
been wrecked owing to trifling details. A glance
at the literature, however, shows that at present the
method is properly used in many places, and leads to
surprisingly beautiful results.

Further sources of error are : The use of vessels
that are not dry, and of boiling-sticks that have been
touched by the hands, soiling the pipettes with
saliva, inaccurate measurement of the ninhydrin
solution, the use of infected water, the cultivation of
bacteria in the same incubator as is used for experi-
ments on the action of ferments, covering the con-
tents of the tubes, and the outside fluid, with an

SOURCES OF ERROR (IX THE PROCESS)

insufficient layer of toluol, changes of temperature in
the incubator, and working in places where acid or
alkaline vapours are developed.

These sources of error ought, properly, to occur
very seldom.

On the other hand, the following point is often
overlooked. After the tube has been filled with the
organ, and the serum and the toluol have been added,
it is absolutely necessary to make sure that the whole
of the tissue is covered with the serum and toluol.
Should the slightest portion of the tissue project
above the toluol, it is then liable to decay in the course
of sixteen hours, and so become a source of serious
error.

In conclusion, we will add the following supple-
mentary' details, which are not at present in general
use, because they are not considered as being abso-
lutely necessary. We may, instead of the control
with serum, use a control with organ + inactivated
serum. The serum is heated for thirty minutes
at a temperature of 60 C. This kind of control is
capable of indicating an insufficiently prepared organ.

Starting with the idea that a certain Hmital value

O

must be present, in order to give a colour reaction
with ninhydrin, one might conclude that it would not
suffice to test the filtered water, in which the organ
was boiled, with i c.c. of ninhydrin solution. We
have therefore produced a solution of silk-peptone,

2OO SOURCES OF ERROR (IN THE PROCESS)

which has been so strongly diluted, that 5 c.c. of the
solution just fails to show any coloration with i c.c.
of ninhydrin solution. 2'5 c.c. of this solution were
then added to 2*5 c.c. of the filtrate obtained from the
water in which the organ was boiled, and 2 c.c. of the
ninhydrin solution were added to this. The mixture
was boiled in the usual way for one minute, and the
reaction remained negative. It would always have
been possible for the limital value to be attained by
means of additions. Further, a volume of 10 c.c.
was reduced to 5 c.c. After the addition of i c.c., and
later of 2 c.c. of the ninhydrin solution, no coloration
appeared.

Finally, we may once more insist on the fact that
an organ containing blood frequently fails to act,
even when it fully complies with the conditions with
reference to the water, in which the organ has been
boiled (see pp. 164-168).

A desire has often been expressed, that we might
have a special colour-scale for estimating the results
of the ninhydrin reaction, with a view to recording the
strengths of the reaction in a generally equivalent
manner ; but this cannot well be effected, because the
ninhydrin reaction does not allow of sharp delimita-
tion. With experience, each observer will soon be
able to judge whether the reaction is strong, medium,
slight, or very slight. Besides, we must not lay too
much stress upon the intensity of the reaction. It is

SOURCES OF ERROR (iX THE PROCESS) 2OI

quite possible, for instance, that in any given case a
quantity of highly molecular peptones is present in
the dialysate. The biuret reaction is surprisingly
strong, while the ninhydrin reaction, on the con-
trary, is very weak. Conversely, we can imagine the
extreme case, in which the decomposition lies below
the peptone limit. We obtain a deep blue ninhydrin
reaction, which means that many compounds, having
the structure of amino-acids, are present ; whilst the
biuret reaction gives a negative result. These facts
show clearly, that the ninhydrin reaction enables us
to recognize many more compounds of the group
of albumen decomposites than the biuret reaction.
Certainly many points in the whole method of the
dialysation process might be modified. In the first
place, the apparatus used could be improved. One
might consider, for instance, the possibility of con-
structing an apparatus, which would enable us to boil
the solutions of the ninhydrin reaction simultaneously
and equally, and at the same time to prevent anv
evaporation. We have purposely made no propo-
sitions in this direction, because it seemed to us, that
the great advantage of the present method is just
that it is simple, clear and concise. We have also
made experiments for simplifying the preparation of
the organs, and more particularly for shortening that
process. Studies on organs, that had been dried
and pulverized at 37 C. with special precautions,

2O2 THE OPTICAL METHOD;

gave good results, but the risk of infection is great.
In any case, organs prepared in this way have
also to be tested each time before use. The boiling
process has this advantage over the other, that the
tissues are loosened, and in this way are more easilv
acted upon by the ferment.

II. The Optical Method.

The principle of the Method. --The optical method
enables us to demonstrate alterations in optically
active substrates by a determination, with the aid of
a polariscope, of changes in their angle of rotation.

The aim of the optical method is, in principle,
exactly the same as that of the dialysation
process. In the latter we determine the transform-
ation of a colloid into a diffusible crystalloid. This
transformation is the result of a hydrolytic decom-
position. In the optical method we start, for purely
technical reasons, not with albumen, but with pep-
tone produced from the latter. We cannot use
albumen, because it would prevent us determining the
angle of deviation of the substrate-serum mixture.
It would either give rise to precipitates, or else render
the mixture so heterogeneous, that slight changes of
rotation would be very difficult to follow. When
using the optical method, we allow the decomposition,
produced by the ferments present in the serum, to set

AND ITS APPLICATION

in later, than in the dialysation process. We remove
part of the decomposition from the influence of the
ferment, when we convert albumen into peptone in the
test-tube. It must be our aim to maintain the peptone
mixture in as high a molecular state as is possible,
as experience has shown, that decomposites of too low
molecularity are not attacked by some kinds of serum,
which decompose more highly molecular peptones.
It is very clearly shown, in this connection, that the
conception of the unity of the proteolytic ferments
does not correspond at all with the reality. There is
not the slightest doubt, that different ferments exist
for different stages of decomposition. The principal
problem, in the application of the optical method to
biological questions, was the elaboration of a method
of dealing with highly molecular peptones, which are
very closely related to the albumens.

The Application of the Optical Method.- -This
is very simple, i c.c. of serum, absolutely free from
haemoglobin, is placed in a test-tube. It must not
contain any form-elements, and must be sterile.
To this is added i c.c. of a 5 to 10 per cent, solution
of peptone, prepared from the organ in question.
Of course, peptones may also be prepared from bacilli,
or else from certain proteins. The serum is mixed
with the peptone solution and poured into a polar-
ization tube, of a capacity of 2 c.c., and the
angle of rotation of the mixture, at a temperature of

2O4 PREPARATION OF PEPTONES

37 C., is determined. The deviations are noted at
certain intervals. If there is no change in the
deviation, then we conclude that no decomposition
has taken place. Should there be an alteration in the
rotation after some time, then we must infer a
fermentative decomposition, such as has been demon-
strated by special experiments with ferment solutions.
We shall now give a description of the preparation
of the peptone.

PREPARATION OF PEPTONES FOR USE IN THE OPTICAL

METHOD.

Organs are first deprived of their blood, in exactlv
the same way as has been described on p. 164.
They can then be subjected directly to hydrolysis,
after the pieces of tissue have been dried, as much as
possible, between filter papers. If it is desired to
collect larger quantities of the same tissue, then the
tissue, freed from blood, is boiled for ten minutes in
water, and is subsequently preserved in sterilized
water with chloroform and toluol. It is, of course,
not necessary, in this case, to boil the organ to
such an extent, as to deprive it of all substances
reacting with ninhydrin. Boiling is merely resorted
to here, in order to destroy any cell ferments that may
be present; otherwise autolysis may manifest itself.
As soon as enough of the organ has been collected,

PREPARATION OF PEPTONES 2O5

then it is similarly freed from water, as far as possible,
before being placed in sulphuric acid, which is kept
cool by means of ice. Nervous tissue, after it has
been deprived of all blood and boiled, must first be
extracted with carbon tetachioride, as otherwise its
lipoidal sheath makes decomposition very difficult.
Tubercle bacilli must also be freed from lipoids.

For hydrolysis, we use 70 per cent, (by weight) of
sulphuric acid, which must be cold. We take three
times as much of this, as of the tissue to be decom-
posed. The vessel is energetically shaken, and then
carefully stoppered. From time to time it is shaken
again. The tissue is soon dissolved, the solution
becoming more or less brown. After standing for
exactly three days, at the temperature of the room
(20 C. at most), the vessel containing the hydroly-
sate is placed into iced water, and diluted with ten
times its quantity of distilled water. The addition
must be made very gradually. The temperature of
the solution is controlled bv means of a thermometer,

J

and must never be allowed to rise above 20 C. If
the vessel is too small, then the solution is trans-
ferred into a larger one, and the water with which
we are diluting is used to rinse the first vessel.

We now begin the neutralization of the sul-
phuric acid with barium hydroxide. Pure crystalline
hydroxide is employed for this purpose, and this
is gradually added, until the solution gives no

JO6 PREPARATION OF PEPTONES

precipitate, either with barium hydroxide solution or
with sulphuric acid. In the test with barium hydroxide
it may happen, that a precipitate appears, even though
no more sulphuric acid is present. These are barium
salts of peptones, which separate out. They can be
dissolved in nitric acid, while barium sulphate is
insoluble in this.

Neutralization is carried out in such a way, as to
calculate the quantity of barium hydroxide necessary,
by the amount of sulphuric acid used. The barium
hydroxide is best added in the solid form, and is well
stirred until the action is complete. The neutral-
ization of the sulphuric acid is first tested by means
of litmus paper. Finally, small samples are filtered
through a small funnel, 21 and then one sample is
tested with barium hydroxide, 22 and another with
sulphuric acid. If, in the first case, the solution
becomes turbid, or precipitates are formed, then nitric
acid is added, and the solution is slightly warmed.

21 If there be a centrifuge at one's disposal, then we recom-
mend centrifuging samples of the mixture. In this way a
clear solution is obtained immediately without any loss of
material.

22 For testing purposes an aqueous solution of barium

chloride gives better results than barium hydroxide, because
the baryta water becomes turbid, owing to its affinity for
carbonic acid, with consequent formation of barium carbonate.
When using the above solution, the sample employed must
never be returned to the original solution, but must be thrown
away.

PREPARATION OF PEPTONES 2O/

If the sediment remains, it is a sign that more barium
hydroxide is to be added to the original solution. It
is advisable, always to work with very dilute solutions
of sulphuric acid and barium hydroxide, otherwise
one may easily overshoot the mark.

When the solution is free from sulphuric acid and
baryta, we proceed to filter it through a doubled
sheet of folded filter paper, or, by means of a filter
pump, through a hardened filter impregnated with
animal charcoal. This process can be hastened by
the use of a centrifuge. The precipitate of barium
sulphate is stirred up with distilled water, well
kneaded in a mortar with water, and then filtered
again. It is advantageous, in order to ensure a good
output of peptone, to repeat this washing out with
cold water many times. The ninhydrin test can be
applied at this stage, as a test of the satisfactory
washing out of the precipitate. To a portion of the
filtrate about i c.c. of ninhydrin is added, and the
mixture is boiled for one minute. If the coloration
is faint, or even negative, then the process of washing-
out is discontinued.

In the meantime, the process of concentration has
been begun. As solutions of peptones produce a
great deal of scum, the apparatus represented in
fig. 9 is used. The latter allows the peptone solution
to evaporate to dryness, at about 40 C., under
highly reduced pressure. The drop funnel serves the

208

PREPARATION OF PEPTONES

purpose of conducting the peptone solution, in drops,
into the flask. These drops evaporate immediately,
and no scum is formed.

FIG. 9.

The peptone solution must never be strongly
evaporated, until we. have repeatedly satisfied our-
selves, that it is actually free from sulphuric acid and

PREPARATION OF PEPTONES 2(X)

barium. With very dilute solutions traces of these
compounds may escape detection. During the con-
centration of the solution, that of the sulphuric acid
and barium hydroxide naturally increases, so that we
may eventually get an hydrolysis of the peptone
mixture.

Finally, we are left with a light yellow syrupy
residuum. The latter is mixed with about 100 times
its amount of methyl-alcohol, and the mixture
is boiled. The boiling hot solution is filtered
through a filter paper into about five times its
amount of cold ethyl alcohol. It is well to put
the latter into iced water. Precipitation is aided by
the addition of ether. The whole is filtered, directly
the precipitate begins to be formed. During the
filtration, the filter should not be allowed to become
empty. It is best to use a filter pump. Only at the
end is the liquid allowed to pass entirely through the
filter, after which the latter is immediately placed in
a vacuum exsiccator. After a day or two the peptone
is absolutely dry, and may then be weighed. First,
a 10 per cent, solution, in 0*9 per cent, solution of
common salt, is prepared, and the deviation of rota-
tion of the solution is determined. If this is more
than i, the solution is diluted, until it shows a rota-
tion of about 075. The higher degree of rotation
would not be injurious. Dilution is only effected in
order to make the best use of the costly material.

2IO STANDARDIZATION OF PEPTONES

Standardisation of the Peptone. Let us assume
that we have to deal with placenta peptone. This is
mixed with the serum of individuals, who are
certainly not pregnant, and then there should be no
alteration of the original rotation. Should this
not be the case, then the peptone is certainly not free
from sulphuric acid or barium. With the serum of
pregnant individuals a decomposition is bound to
take place. At first, readings are taken every hour,
and tests are made with many sera. A normal curve
for the peptone is constructed, from the separate read-
ings, by marking the angle of rotation on the abscissa
and the time on the ordinate (cf. the curves given
on pp. 62, 64, and 75-77). Once the normal altera-
tions of rotation of the serum peptone mixture are
known, then the readings for the diagnosis of normal
cases need only be taken every four to six hours. If
one has a special object in view, then the readings are
taken more often.

The optical method supplements the dialysation
process in many directions. In the first place, it is
possible to determine quantitative differences in the
speed of the decomposition. Further, qualitative
differences may be observed. In the dialysation
process, on the other hand, the dialysate may
be used for experiments on animals and, for
instance, be injected, in a state of concentration,
for the purpose of 'deciding, whether : certain

STANDARDIZATION OF PEPTONES

211

products of decomposition, contained in it, have a
toxic effect.

To determine the range of rotation, a perfect
instrument is necessary. The polarizing apparatus of
Schmid and Hansch, of Berlin, is one that answers

(a]

FIG. 10.

(a) Ocular for taking readings; (b) polarization tube;
(c) sodium flame; (d) for illumination; (e) battery.

all the requirements (fig. 10). It allows of read-
ings to the hundredth part of a degree. Since
everyone makes individual errors in taking readings,
i.e., the range of rotation of the same solution is

212 STANDARDIZATION OF PEPTONES

differently observed, it has to be determined how
great the limits of error are, on the average. It has
been found, that most observers are capable of read-
ing with accuracy to o'O2 of a degree. In order to
attain greater certainty, we shall consider even a
difference of 0*04 of a degree as the limit of error.
Only with a change of rotation of 0*05 of a degree can
decomposition be assumed to have taken place. The
limit can thus be fixed without any danger, because,
when an hydrolysis of the peptone does take place,
the alteration of rotation is certainly more than 0*04
of a degree.

This method, as such, presents hardly any sources
of error. At most, errors may be occasionally pro-
duced through turbidity, precipitates, and the like,
Fortunately, however, in such cases, which actually
very seldom happen with proper working, the reading
of the rotation is impossible, and so this source of
error disappears of itself. Of course, we should get
no result if we were to try to polarize a cloudy solution.

A very important source of error would arise, if the
range of rotation of the cold solution were taken for
the initial value. The readings must be taken from
the moment the contents of the tubes reach a tem-
perature of 37 C. It is best to take the reading at
the end of one hour, and take another at the end
of the second hour. Values obtained in such a
manner should, in general, not be too distant one

STANDARDIZATION OF PEPTONES

from the other, as decomposition begins, and
manifests itself for a certainty, only after about six
hours. Readings should not be followed up for more
than thirty-six to thirty-eight hours.

Great progress would be made, if the reading of
angles of rotation could be taken by means of some
kind of automatic device. Objective values could be
obtained, and we should be in a position to follow

FIG. ii.

up details which, during the long intervals between
readings, at present escape observation. Experi-
ments in this direction, with the collaboration of Dr.
Wildermuth, are in progress.

To obviate the cooling of the polariscopic tubes,
during polarization, tubes have been constructed with
water jackets (fig. n). Lately, an electric heating
apparatus 2; has been added to the polariscope itself.

23 Emil Abderhalden : " Ueber eine mit Polarisations-
apparat kombinierte elektrisch heizbare Vorrichtung zur
Ablesung und Beobachtung des Drehungsvermogens bei
konstanter Temperatur. Zeitschr. f. physiol. Chem., 84, 300
(1913).

214 STANDARDIZATION OF PEPTONES

The former holds six polarization tubes, which can
be brought into the field of observation without
opening the heated incubator. In this way, all
variations of temperature are avoided during the
test. (See Plate.)

The most important source of error lies in the
observer himself. The eye soon becomes tired, and
it is impossible to take many readings at a time. One
must become so experienced, as to be able to record
a reading in at least thirty seconds. So soon as the
eyes become weary, the readings become dubious. It
is not advisable to resort to the optical method, until
one has attained to sufficient certainty in taking
readings.

LITERATURE 215

Literature. 1

Complete List of Works on Cell-metabolism, and on the Peculiar
Structure of the Cells of Certain Species, of Individuals,
and especially of Single Organs.

EMIL ABDERHALDEN. Die Bedeutung der Verdauung fur den
Zellstoffwechsel im Lichte neuerer Forschungen auf dem
Gebiete der physiologischen Chemie. Zeitschr. des Oster-
reichischen Ingenieur- u. Architekten-Vereins, 1911,
Nr. ii u. 12, und im Verlag Urban u. Schwarzenberg,
Berlin-Wien, 1911.

EMIL ABDERHALDEN. Neuere Anschauungen iiber den Bau
und den Stoffwechsel der Zelle. Julius Springer, Berlin,
1911.

EMIL ABDERHALDEN. Les conceptions nouvelles sur la struc-
ture et le metabolisme de la cellule. Revue generale des
sciences pures et appliquees, 23 Jahrg., Nr. 3, S. 95.
February, 1912.

EMIL ABDERHALDEN. Synthese der Zellbausteine in Pflanze
und Tier. February, 1912. Julius Springer, Berlin.

EMIL ABDERHALDEN. Lehrbuch der physiologischen Chemie.
i und 2 Aufl. Urban u. Schwarzenberg, Berlin-Wien.
1906 u. 1909. The concluding chapters give expression to
views bearing specially on the intimate relations subsisting
between the metabolic processes of the body-cells on the
one hand, and of the parasitic cells (micro-organisms) on
the other.

1 Compare the most recent works, pp. 227-240.

2l6 LITKKATt'KK

Comparative Researches on the Composition of Milk, and

of the Suckling.

EMIL ABDERHALDEN. Die Beziehungen der Zusammensetzung
der Asche des Sauglings zu derjenigen der Asche der
Milch. Zeitschr. f. physiol. Chem., Bd. xxvi, 1899, S. 498.

EMIL ABDERHALDEN. Die Beziehungen der Wachstumsge-
schwindigkeit des Sauglings zur Zusammensetzung der
Milch beim Kaninchen, bei der Katze und beim Hunde.
Zeitschr. f. physiol. Chem., Bd. xxvi, 1899, S. 487.

EMIL ABDERHALDEN. Die Beziehungen der Zusammensetzung
der Asche des Sauglings zu derjenigen der Asche der
Milch beim Meerschweinchen. Zeitschr. f. physiol.
Chem., Bd. xxvii, 1899, S. 356.

EMIL ABDERHALDEN. Die Beziehungen der Wachstumsge-
schwindigkeit des Sauglings zur Zusammensetzung der
Milch beim Hunde, beim Schwein, beim Schaf, bei der
Ziege und beim Meerschweinchen. Zeitschr. f. physiol.
Chem., Bd. xxvii, 1899, S. 408 und 594.

The Use of various Sources of Nitrogen by Lower

Organisms.

EMIL ABDERHALDEN and PETER RONA. Die Zusammensetzung
des " Eiweisses " von Aspergillus niger bei verschiedener
Stickstoffquelle. Zeitschr. f. physiol. Chem., Bd. xlvi,
1905, S. 179.

EMIL ABDERHALDEN and YUTAKA TERUUCHI. Kulturversuche
mit Aspergillus niger auf einigen Aminosauren und Poly-
peptiden. Zeitschr. f. physiol. Chem., Bd. xlvii, 1906,
S. 394-

Examination of Animal and Plant Tissues for the Presence of
Proteo- and Peptolytic Ferments.

1. On the Technique of the Demonstration of Proteo- and

Peptolytic Ferments.

EMIL ABDERHALDEN and ALFRED SCHITTENHELM. Uber den
Nachweis peptolytischer Fermente. Zeitschr. f. physiol.
Chem., Bd. Ix, 1909, S. 421.

LITERATURE 2I/

EMIL ABDERHALDEN. Notiz zum Nachweis peptolytischer Fer-
mente in Tier- und Pflanzengeweben. Zeitschr. f. physiot.
Chem., Bd. Ixvi, IQIO, S. 137.

EMIL ABDERHALDEN and HANS PRINGSHEIM. Beitrag zur
Technik des Nachweises intracellularer Fermente. Zeit-
schr. f. physiol. Chem., Bd. Ixv, igio, S. 180.

EMIL ABDERHALDEN. Die optische Methode und ihre Verwen-
dung bei biologischen Fragestellungen. Handbuch der
biochem. Arbeitsmethoden, Bd. v, IQII, S. 575.

2. Experiments on the Action of the Peptolytic Ferments.

EMIL FISCHER and EMIL ABDERHALDEN. Uber das Verhalten
verschiedener Polypeptide gegen Pankreasferment. Sit-
zungsberichte der kgl. preussischen Akademie der Wissen-
schaften X. 1005.

EMIL FISCHER and EMIL ABDERHALDEN. Uber das Verhalten
verschiedener Polypeptide gegen Pankreassaft und Magen-
saft. Zeitschr. f. physiol. Chem., Bd. xlvi, 1905, S. 52.

EMIL FISCHER and EMIL ABDERHALDEN. Uber das Verhalten
einiger Polypeptide geben Pankreassaft. Zeitschr. f.
physiol. Chem., Bd. li, 1907, S. 264.

EMIL ABDERHALDEX and A. H. KOELKER. Die Verwendung
optisch-aktiver Polypeptide zur Priifung der Wirksamkeit
proteolytischer Fermente. Zeitschr. f. physiol. Chem.,
Bd. li, 1907, S. 2Q4.

EMIL ABDERHALDEN and LEONOR MICHAELIS. Der Verlauf der
fermentativen Polypeptidspaltung. Zeitschr. f. physiol.
Chem., Bd. Hi, 1007, S. 326.

EMIL ABDERHALDEN and ALFRED GIGON. Wieterer Beitrag zur
Kenntnis des Verlaufs der fermentativen Polypeptidspal-
tung. Zeitschr. f. physiol. Chem., Bd. liii, 1907, S. 251.

EMIL ABDERHALDEN and A. H. KOELKER. Weitere Beitrage
zur Kenntnis der fermentativen Polypeptidspaltung. IV
und V Mitteilung. Zeitschr. f. physiol. Chem., liv, igo8,
S. 363 und Bd. Iv, igo8, S. 416.

EMIL ABDERHALDEN and CARL BRAHM. Zur Kenntnis des Ver-
laufs der fermentativen Polypeptidspaltung. VI Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ivii, 1908,
S. 342-

2l8 LITERATURE

EMIL ABDERHALDEN, G. CAEMMERER and L. PINCUSSOHN. Zur
Kenntnis des Verlaufs der fermentativen Polypeptidspal-
tung. VII Mitteilung. Zeitschr. f. physiol. Chem.,
Bd. lix, 1909, S. 293.

3. Researches on the Presence of the Peptolytic Ferments.

(a) IN ANIMAL AND PLANT TISSUES.

EMIL ABDERHALDEN and PETER RONA. Das Verhalten des
Glycyl-1-tryosins im Organismus des Hundes bei subkutaner
Einfiihrung. Zeitschr. f. physiol. Chem., Bd. xlvi, 1905,
S. 176.

EMIL ABDERHALDEN and YUTAKA TERUUCHI. Das Verhalten
einiger Polypeptide gegen Organextrakte. Zeitschr. f.
physiol. Chem., Bd. xlvii, 1906, S. 466.

EMIL ABDERHALDEN and ALFRED SCHITTENHELM. Die Wirkung
der proteolytischen Fermente keimender Samen des
Weizens und der Lupinen auf Polypeptide. Zeitschr. f.
physiol. Chem., Bd. xlix, 1906, S. 26.

EMIL ABDERHALDEN and PETER RONA. Das Verhalten von
Leucyl-phenylalanin, Leucyl-glycyl-glycin und von Alanyl-
glycyl-glycin gegen Presssaft der Leber vom Rinde.
Zeitschr. f. physiol. Chem., Bd. xlix, 1906, S. 31.

EMIL ABDERHALDEN and ANDREW HUNTER. Weitere Beitrage
zur kenntnis der proteolytischen fermente der tierischer
Organe. Zeitschr. f. physiol. Chem., Bd. xlviii, 1906,

S. 537-
EMIL ABDERHALDEN and YUTAKA TERUUCHI. Studien iiber die

proteolytische Wirkung der Presssafte einiger tierischer

Organe sowie des Darmsaftes. Zeitschr. f. physiol.

Chem., Bd. xlix, 1906, S. i.
EMIL ABDERHALDEN and YUTAKA TERUUCHI. Vergleichende

Untersuchungen iiber einige proteolytische Fermente

pflanzlicher Herkunft. Zeitschr. f. physiol. Chem.,

Bd. xlix, 1906, S. 21.
EMIL ABDERHALDEN and FILIPPO LUSSANA. Weitere Versuche

iiber den Abbau von Polypeptiden durch die Presssafte

von Zellen und Organen. Zeitschr. f. physiol. Chem.,

Bd. Iv, 1908, S. 390.

LITERATURE 219

EMIL ABDERHALDEN and AUGUSTE RILLIET. uber die Spaltung
einiger Polypeptide durch den Presssaft von Psalliota
campestris (Champignon). Zeitschr. f. physiol. Chem.,
Bd. lv, 1908, S. 395.

EMIL ABDERHALDEN and DAMMHAHN. Uber den Gehalt unge-
keimter und gekeimter Samen verschiedenerPflanzenarten
an peptolytischen Fermenten. Zeitschr. f. physiol.
Chem., Bd. Ivii, 1908, S. 332.

EMIL ABDERHALDEN and HANS PRINGSHEIM. Studien Uber die
Spezifizitat der peptolytischen Fermente bei verschiedenen
Pilzen. Zeitschr. f. physiol. Chem., Bd. lix, 1909, S. 249.

EMIL ABDERHALDEN and ROBERT HEISE. Uber das Vorkommen
peptolytischen Fermente bei den Wirbellosen. Zeitschr.
f. physiol. Chem., Bd. Ixii, 1909, S. 136.

EMIL ABDERHALDEN and EUGEN STEINBECK. Weitere Unter-
suchungen liber die Verwendbarkeit des Seidenpeptons
zum Nachweis peptolytischer Fermente. Zeitschr. f.
physiol. Chem., Bd. Ixviii, 1910, S. 312.

EMIL ABDERHALDEN. Uber den Gehalt von Eingeweidewiir-
mern an peptolytischen Fermenten. Zeitschr. f. physiol.
Chem., Bd. Ixxiv, S. 409.

EMIL ABDERHALDEN and HEINRICH GEDDERT. Darstellung
optisch-aktiver Polypeptide aus Racemkorpern. Zeitschr.
f. physiol. Chem., Bd. Ixxiv, 1911, S. 394.

(b) IN THE BLOOD.

EMIL ABDERHALDEN and H. DEETJEN. Uber den Abbau
einiger Polypeptide durch die Blutkorperchen des Pferdes.
Zeitschr. f. physiol. Chem., li, 1907, S. 334.

EMIL ABDERHALDEN and BERTHOLD OPPLER. Uber das Ver-
halten einiger Polypeptide gegen Blutplasma- und serum
vom Pferde. Zeitschr. f. physiol.' Chem., Bd. liii, 1907,
S. 294.

EMIL ABDERHALDEN and H. DEETJEN. Wekere Studien liber
den Abbau einiger Polypeptide durch die roten Blut-
korperchen und die Blutplattchen des Pferdeblutes. Zeit-
schr. f. physiol. Chem., Bd. liii, 1907, S. 280.

22O LITERATURE

EMIL ABDERHALDEN and PETER RONA. Das Verhalten von
Blutserum und Harn gegen Glycyl-1-tryosin unter ver-
schiedenen Bedingungen. Zeitschr. f. physiol. Chem.,
Bd. liii, 1907, S. 308.

EMIL ABDERHALDEN and WILFRED MANWARING. Uber den
Abbau einiger Polypeptide durch die roten Blutkb'rperchen
und die Blutplattchen des Rinderblutes. Zeitschr. f..
physiol. Chem., Bd. Iv, igo8, S. 377.

EMIL ABDERHALDEN and JAMES MCLESTER. Uber das Ver-
halten einiger Polypeptide gegen das Plasma des Rinder-
blutes. Zeitschr. f. physiol. Chem., Bd. Iv, 1908, S. 371.

(c) IN SPUTUM DURING THE RESOLUTION IN PNEUMONIA.

EMIL ABDERHALDEN. Zur Kenntnis des Vorkommens der
peptolytischen Fermente. Zeitschr. f. physiol. Chem.,
Bd. Ixxviii, 1912, S. 344.

4. Test of the Mode of Action of the Proteo- and Peptolytic Ferments

of Tumour Cells and Bacteria.

EMIL ABDERHALDEN. Neue Forschungsrichtungen auf dem
Gebiete der Storungen des Zellstoffwechsels. Arch. f.
wissenschaftl. und praktische Tierheilkunde, Bd. xxxvi,
1910, S. i.

EMIL ABDERHALDEN. Studium liber den Stoffwechsel von
Geschwulstzellen. Zeitschr. f. Krebsforschung, Bd. ix,
1910, 2 Heft.

EMIL ABDERHALDEN and PETER RONA. Zur Kenntnis der pepto-
lytischen Fermente verschiedenartiger Krebse. Zeitschr.
f. physiol. Chem., Bd. Lx, 1909, S. 411.

EMIL ABDERHALDEN, A. H. KOELKER and FLORENTIN MEDI-
GRECEANU. Zur Kenntnis der peptolytischen Fermente
verschiedenartiger Krebse und anderer Tumorarten.
II Mitteilung. Zeitschr. f. physiol. Chem., Bd. Ixii, 1909,
S. 145-

EMIL ABDERHALDEN and FLORENTIN MEDIGRECEANU. Zur
Kenntnis der peptolytischen Fermente verschiedenartiger
Krebse und anderer Tumorarten. Zeitschr. f. physiol.
Chem., Bd. Ixvi, 1910, S. 265.

LITERATURE 221

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Zur Kenntnis
der peptolytischen Fermente verschiedenartiger Krebse
und anderer Tumorarten. Zeitschr. f. physiol. Chem.,
Bd. Ixvi, 1910, S. 277.

EMIL ABDERHALDEN, LUDWIG PINCUSSOHN and ADOLF WALTHER.
Untersuchungen liber die Fermente verschiedener Bak-
terienarten. Zeitschr. f. physiol. Chem., Bd. Ixviii, 1910,
S. 471.

The Application of the Optical Method in Biological Problems.

Technique of the Method.

EMIL ABDERHALDEN. Die Anwendung der u optischen
Methode J: auf dem Gebiete der Immunitatsforschung.
Med. Klinik. Jahrg. 1909, Nr. 41.

EMIL ABDERHALDEN. Die Anwendung der optischen Methode
auf dem Gebiete der Physiologic und Pathologic. Zen-
tralbl. f. Physiol. XXIII, Nr. 25.

EMIL ABDERHALDEN. Die optische Methode und ihre Verwen-
dung bei biologischen Fragestellungen. Handbuch der
biochemischen Arbeitsmethoden, Bd. v, 1911, S. 575.

Defensive Ferments after the Introduction of Albuminous
Substances and Peptones out of Harmony with the Body.

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. XJber den
Gehalt des Kaninchen- und Hundeplasmas an peptoly-
tischen Fermenten unter verschiedenen Bedingungen.
I Mitt. Zeitschr. f. physiol. Chem., Bd. Ixi, 1909, S. 200.

EMIL ABDERHALDEN and WOLFGANG WEICHARDT. Uber den
Gehalt des Kaninchenserums an peptolytischen Fermenten
unter verschiedenen Bedingungen. II Mitteilung. Zeit-
schr. f. physiol. Chem., Bd. Ixii, 1909, S. 120.

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Uber den
Gehalt des Hundeblutserums an peptolytischen Fermenten
unter verschiedenen Bedingungen. Ill Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixii, 1909, S. 243.

222 LITERATURE

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Serologische
Studien mit Hilfe der " optischen Methode." IV Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ixiv, IQIO.,
S. 100.

EMIL ABDERHALDEN and K. B. IMMISCH. Serologische Studien
mit Hilfe der " optischen Methode." V Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixiv, 1910, S. 423.

EMIL ABDERHALDEN and A. ISRAEL. Serologische Studien mit
Hilfe der " optischen Methode." VI Mitteilung. Zeit-
schr. f. physiol. Chem., Bd. Ixiv, 1910, S. 426.

EMIL ABDERHALDEN and ]. G. SLEESWYK. Serologische Studien
mit Hilfe der " optischen Methode." VII Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixiv, 1910, S. 427.

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Serologische
Studien mit Hilfe der " optischen Methode." IX Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ixiv, 1910,
S. 433-

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Serologische
Studien mit Hilfe der " optischen Methode." X Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ixvi, 1910,
S. 88.

EMIL ABDERHALDEN and LUDWIG PINCUSSOHN. Serologische
Studien mit Hilfe der "optischen Methode." XIII Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ixxi, IQII,
S. no.

EMIL ABDERHALDEN and E. RATHSMANN. Serologische Studien
mit Hilfe der " optischen Methode." XIV Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixxi, IQII, S. 367.

EMIL ABDERHALDEN and BENOMAR SCHILLING. Serologische
Studien mit Hilfe der " optischen Methode." XV Mit-
teilung. Zeitschr. f. physiol. Chem., Bd. Ixxi, IQII,
S. 385-

EMIL ABDERHALDEN and ERNST KAMPF. Serologische Studien
mit Hilfe der " optischen Methode." XVI Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixxi, IQII, S. 421.

LITERATURE 223

Defensive Ferments after the Introduction of Carbohydrates
out of harmony with the Body and the Blood.

EMIL ABDERHALDEN and CARL BRAHM. Serologische Studien
mit Hilfe der " optischen Methode." VIII Mitteilung.
Zeitschr. f. physiol. Chem., Bd. Ixiv, 1910, S. 429.

EMIL ABDERHALDEN and GEORG KAPFBERGER. Serologische
Studien mit Hilfe der " optischen Methode. " XI Mit-
teilung. Parenterale Zufuhr von Kohlehydraten. Zeit-
schr. f. physiol. Chem., Bd. Ixix, 1910, S. 23.

Appendix.

EMIL ABDERHALDEN and JULIUS SCHMID. Bestimmung der
Blutmenge mit Hilfe der " optischen Methode." Zeitschr.
f. physiol. Chem., Bd. Ixvi, 1910, S. 120.

EMIL ABDERHALDEN and ARTHUR WEIL. Beobachtungen iiber
das Drehungsvermogen des Blutplasmas und -serums ver-
schiedener Tierarten verschiedenen Alters und Gesch-
lechts. Zeitschr. f. physiol. Chem., Bd. Ixxxi, 1912,
S. 233.

EMIL ABDERHALDEN and T. KASHIWADO. Studien iiber die
Kerne der Thymusdriise und Anaphylaxieversuche mit
Kernsubstanzen. Zeitschr. f. physiol. Chem., Bd. Ixxxi,
1912, S. 285.

EMIL ABDERHALDEN. Weitere Studien iiber Anaphylaxie.
Zeitschr. f. physiol. Chem., Bd. Ixxxii, 1912, S. 109.

Defensive Ferments after the addition of Fats.

EMIL ABDERHALDEN and PETER RONA. Studien iiber das
Fettspaltungsvermogen des Blutes und Serums des Hundes
unter verschiedenen Bedingungen. Zeitschr. f. physiol.
Chern., Bd. Ixxv, S. 30.

EMIL ABDERHALDEN and ARNO ED. LAMPE. Weitere Versuche
iiber das Fettspaltungsvermogen des Blutes und des
Plasmas unter verschiedenartigen Bedingungen. Zeitschr.
f. physiol. Chem., Bd. Ixxviii, 1912, S. 396.

224 LITERATURE

Defensive Ferments after the Introduction of Substances in
harmony with the Body but not with the Blood.

Demonstration of the Existence of Proteolytic Ferments in the

Blood during Pregnancy.

EMIL ABUERHALDEN, R. FREUND and LUDWIG PINCUSSOHN.
Serologische Untersuchungen mit Hilfe der " optischen
Methode " wahrend der Schwangerschaft und speziell bei
Eklampsie. Praktische Ergebnisse der Geburtshilfe und
Gynakologie. II Jahrg., II Abt., 1910, S. 367.

EMIL ABDERHALDEN and MIKI KIUTSI. Biologische Unter-
suchungen iiber Schwangerschaft. Die Diagnose der
Schwangerschaft mittels der " optischen Methode " und
dem Dialysierverfahren. Zeitschr. f. physiol. Chem.,
Bd. Ixxvii, 1912, S. 249.

Review of the Problems involved in Researches on immunity,
with special reference to Anaphylaxy.

E. FRIEDBERGER and collaborators. Zahlreiche Arbeiten iiber

Anaphylaxie in der Zeitschr. f. Immunitatsforschung und

experimentelle Medizin.
E. FRIEDBERGER. Die Anaphylaxie mit besonderer Beriick-

sichtigung ihrer Bedeutung fiir Infektion und Immunitat.

Deutsche med. Wochenschr., 191 1, Nr. u.
E. FRIEDBERGER. Die Anaphylaxie. Fortschritte der Deutsch.

Klinik, Bd. ii, 191 1, S. 619.
E. FRIEDBERGER. Uber das Wesen und die Bedeutung der

Anaphylaxie. Miinchener med. Wochenschr., 1910, Nr. 50

und 51.
ERNST MoRO. Experimentelle und klinische Uberempfind-

lichkeit (Anaphylaxie). J. F. Bergmann, Wiesbaden,

1910.
HERMANN PFEIFFER. Das Problem der Eiweissanaphylaxie.

Gustav Fischer, Jena, 1910.
CLEMENS VON PIRQUET. Allergic. Julius Springer, Berlin,

1910.
ROBERT ROSSLE. Fortschritte der Cytotoxinforschung. J. F.

Bergmann, Wiesbaden, 1910.

LITERATURE 22$

WOLFGANG WEICHARDT. Jahresbericht iiber die Ergebnisse
der Immunitatsforschung. In course of publication since
1906. Ferdinand Enke, Stuttgart. In addition to a
general synopsis contains individual references to all
works bearing on Immunity.

ALFRED SCHITTENHELM. Uber Anaphylaxie vom Standpunkt
der pathologischen Physiologic und der Klinik. Jahres-
bericht iiber die Ergebnisse der Immunitatsforschung,
1910. Ferdinand Enke, Stuttgart.

EDGAR ZUNZ. A propos de TAnaphylaxie. Bruxelles, 1911.

1. BRUNO BLOCK and RUDOLF MASSINI. Studien iiber

Immunitat und Uberempfindlichkeit bei Hyphomyze-
tenerkrankungen. Zeitschr. f. Hygiene, Bd. Ixiii, S. 68.

2. GUSTAV VON BUNGE. Der Kali-, Natron- und Chlorge-

halt der Milch, verglichen mit dem anderer Nahrungs-
mittel und des Gesamtorganismus der Saugetiere.
Zeitschr. f. Biol., Bd. x, 1874, S. 295 und 323.

3. GUSTAV VON BUNGE. Lehrbuch der Physiologic des

Menschen, Bd. ii, 1901, S. 103.

4. W. CRAMER. On the Assimilation of Protein introduced

Parenterally. Journ. of Physiol., vol. xxxvii, 1908,
p. 146.

5. P. ESCH. Uber Harn- und Serumtoxizitat bei Eklampsie.

Miinchener med. Wochenschr., Bd. lix, 1912, S. 461.

6. EMIL FISCHER. Bedeutung der Stereochemie fiir die

Physiologic. Zeitschr. f. physiol. Chem., Bd. xxvi,
S. 60.

7. RUPERT FRANZ. Uber das Verhalten der Harntoxizitat in

der Schwangerschaft, Geburt und im Wochenbett.
Arch. f. Gynakol., Bd. xcvi, 1911, Heft 2.

8. U. FRIEDEMANN and S. ISAAC, tiber Eiweissimmunitat

und Eiweissstoffweichsel. Zeitschr. f. exper. Path. u.
Therap., Bd. i, 1905, S. 513; Bd. iii, 1906, S. 209; und
Bd. iv, 1907, S. 830.

9. G. B. GRUBER. Peptolytische Stoffe und Immunstoffe im

Blut. Zeitschr. f. Immunitatsforschung und exper.
Therap., Bd. vii, 1910, S. 762.

15

226 LITERATURE

10. ERNST HEILNER. Uber die Wirkung grosser Mengen

artfremden Blutserums im Tierkorper nach Zufuhr per
os und subkutan. Zeitschr. f. Biol., Bd. 1, 1907, S. 26.

11. ERNST HEILNER. Versuch eines indirekten Fermentnach-

weises ((lurch Alkoholzufuhr) ; zugleich ein Beitrag
zur Frage der Uberempfindlichkeit. Miinchener med.
Wochenschr., 1908, Nr. 49.

12. ERNST HEILNER. Uber das Schicksal des subkutan einge-

fiihrten Rohrzuckers im Tierkorper und seine Wirkung
auf Eiweiss- und Fettstoffwechsel. Zeitschr. f. Biol.,
Bd. Ixi, 1911, S. 75.

I3A. ERNST HEILNER. Uber die Wirkung kiinstlich erzeugter
physikalischer (osmotischer) Vorgange im Tierkorper
auf den Gesamtstoffumsatz mit Beriicksichtigung der
Frage von der " Uberempfindlichkeit." Zeitschr. f.
Biol., Bd. 1, 1908, S. 476.

13. HERTLE and HERMANN PFEIFFER. Uber Anaphylaxie

gegen artgleiches blutfremdes Eiweiss. Zeitschr. f.
Immunitatsforschung und exper. Therap., Bd. x, 1911,
S. 541.

14. TH. HEYNEMANN. Eine " Reaktion ' im Serum Schwan-

gerer, Kreissender und Wochnerinnen. Arch. f. Gynak.,
Bd. xc, 1910, Heft 2.

15. G. KAPSENBERG. Studien iiber Immunitat und Zellzerfall.

Zeitschr. f. Immunitatsforschung, Bd. xii, 1912, S. 477.

16. KORNEL VON KOROSY. Uber parenterale Eiweisszufuhr.

Zeitschr. f. physiol. Chem., Bd. Ixii, 1909, S. 76; Bd.
Ixix, 1909, S. 313,

17. L. LOMMEL. Uber die Zersetzung parenteral einge-

fiihrten Eiweisses im Tierkorper. Verhandl. des Kon-
gresses fiir innere Medizin, Bd. xxiv, 1907, S. 290, und
Arch. f. exper. Path. u. Pharm., Bd. Iviii, 1908, S. 50.

1 8. LEONOR MICHAELIS and PETER RONA. Untersuchungen

iiber den parenteralen Eiweissstoffwcchsel. Pfliigers
Arch, fiir die gesamte Physiologic, Bd. Ixxi, 1908,
S. 163; Bd. Ixxiii, 1908, S. 406; Bd. Ixxiv, 1908, S. 578.

19. CARL OPPENHEIMER. Uber das Schicksal der mit Um-

gehung des Darmkanals eingefiihrten Eiweissstoffe im
Tierkorper. Hofmeisters Beitrage, Bd. iv, 1903, S. 263.

LITERATURE 22J

20. H. PFEIFFER and S. MITA. Experimentelle Beitrage zur

Kenntnis der Eiweiss-Antieiweissreaktion. Zeitschr. f.
Immunitatsforschung und exper. Therap., Bd. vi, 1910,
S. 18.

21. HERMANN PFEIFFER and A. JARISCH. Zur Kenntnis der

Eiweisszerfallstoxikosen. Zeitschr. f. Immunitatsfor-
schung und exper. Therap., Bd. xvi, 1912, S. 38.

22. H. PFEIFFER. Neue Gesichtspunkte zum Nachweis von

Eiweisszerfallstoxikosen. Mitteil. des Vereins der Arzte
in Steiermark, Xr. 8, 1912.

23. GlACOMO PIGHIXI. Uber die Bestimmung der enzyma-

tischen Wirkung der Nuclease mittels " optischer
Methode." Zeitschr. f. physiol. Chem., Bd. Lxx,
1910-11, S. 85.

24. GOTTLIEB SALUS. Versuche iiber Serumgiftigkeit und

Anaphylaxie. Med. Klinik., Jahrg., 1909, Xr. 14.

25. HEINRICH SCHLECHT. Uber experimentelle Eosinophylie

nach parenteraler Zufuhr artfremden Eiweisses und
liber die Beziehungen der Eosinophylie zur Anaphy-
laxie. Habilitationsschrift, F. C. AY. A'ogel, Leipzig,
1912.

26. AA'OLFGAXG AA^EICHARDT. Uber Syncytiolysine. Hygien.

Rundschau., 1903, Nr. 10. See also Miinchener med.

AA r ochenschr., 1901, Xr. 52, und Deutsche med.
Wochenschr., 1902, X'r. 35.

27. AA'OLFGANG AA T EICHARDT. Studien iiber das AA T achstum

und den Stoffwechsel von Typhus- und Colibacillus und
liber die Tatigkeit ihrer Fermente. Zentralbl. f. die
gesamte Physiol. und Path, des Stoffwechsels. X. F.
Jahrg., 5, 1910, S. 131.

28. E. AA'EIXLAXD. Uber das Auftreten von Invertin im

Blut. Zeitschr. f. Biol., Bd. xlvii, 1907, S. 279.

Researches published during the year 1912 and up to November,
1913, in which the Dialysation Process and the Optical
Method have been made use of.

i. ERICH FRANK and FRITZ HEIMANN. Die biologische
Schwangerschaftsdiagnose nach Abderhalden und ihre
klinische Bedeutung. Berliner klin. AA'ochenschr.,
1912, Xr. 36.

228 LITERATURE

2. R. FRANZ and A. JARISCH. Beitrage zur Kenntnis der

serologischen Schwangerschaftsdiagnostik. Wiener klin.
Wochenschr., Bd. xxv, 1912, Nr. 39.

3. J. VEIT. Bewertung und Verwertung der Serodiagnostik

der Schwangerschaft. Zeitschr. f. Geburtshilfe u.
Gynakologie, Bd. Ixxii, 1912, S. 463.

4. A. FAUSER. Einige Untersuchungsergebnisse und klin-

ische Ausblicke auf Grund der Abderhaldenschen
Anschauungen und Methodik. Deutsche med. Wochen-
schr., 1912, Nr. 52.

5. M. HENKEL. Zur biologischen Diagnose der Schwan-

gerschaft. Archiv. f. Gynak., Bd. xcix, 1912, S. i.

6. P. LlNDlG. Uber Serumfermentwirkungen bei Schwan-

geren u. Tumorkranken. Miinchener med. Wochen-
schr., 1913, Nr. 6. Vgl. dazu E. ABDERHALDEN.
Ebenda, 1913, Nr. 8.

7. A. FAUSER. Weitere Untersuchungen (3. Liste) auf

Grund des Abderhaldenschen Dialysierverfahrens.
Deutsche med. Wochenschr., 1913, Nr. 7.

8. BRUNO STANCE. Zur Eklampsiefrage. Zentralbl. f.

Gynak., Bd. cxxxvii, 1913.

9. HANS FALK. Das Dialysierverfahren nach Abderhalden,

eine Methode zur Diagnose des Friihmilchendseins der
Kiihe. Berliner tierarztl. Wochenschr., 1913, Nr. 8.

10. ERNST ENGELHORN. Zur biologischen Diagnose der

Schwangerschaft. Miinchener med. Wochenschr., 1913,
Nr. u.

11. FRITZ HEIMANN. Die Serodiagnostik der Schwanger-

schaft. Die Naturwissenschaften, Bd. i, 1913, S. 283.

12. HENRY SCHWARZ. Abderhalden's Serodiagnosis of Preg-

nancy and its Practical Application. Interstate Med.
Journ., vol. xx, 1913, p. 195.

13. CARLO FERRARI. Ricerche sulla diagnosi della gravi-

danza col metodo polariscopico e col metodo della
dialisi. Liguria medica, Bd. vii, 1913, Nr. 5-6.

LITERATURE 22Q

14. HANS SCHLIMPERT and JAMES HENDRY. Erfahrungen

mit der Abderhaldenschen Schwangerschaftsreaktion
(Dialysierverfahren und Ninhydrinreaktion). Mlin-
chener med. Wochenschr., 1913, Xr. 13.

15. A. FAUSER. Zur Frage des Vorhandenseins spezifischer

Schutzfermente im Serum von Geisteskranken. Mlin-
chener med. Wochenschr., 1913, Nr. n.

16. P. GAIFAMI. Sulla serodiagnosi della gravidanza col

metodo della dialisi secondo Abderhalden. Bolletina
della R. Acad. med. di Roma, Bd. xxxix, 1913, Nr. 3-4.

17. CESARE DECIO. Prime ricerche sulP applicazione della

reazione di Abderhalden nel campo ostetrico. Annali
di Ostetricia e Ginecologia, 1913.

1 8. HlRSCHFELD. Die Schwangerschaftsdiagnose nach Ab-

derhalden und ihre \vissenschaftliche Grundlage.
Schweizerische Rundschau f. Med., 1913, Nr. 13.

19. JULIUS BAUER, t'ber organabbauende Fermente im

Serum bei endemischem Kropf. Wiener klin. Wochen-
schr., Bd. xxvi, 1913, Nr. 16.

20. EMIL EPSTEIN. Die Abderhaldensche Serumprobe auf

Karzinom. Wiener klin. Wochenschr., Bd. xxvi, 1913,
Nr. 17.

21. RUDOLF EKLER. Erfahrungen mit der biologischen

Diagnose der Schwangerschaft nach Abderhalden.
Wiener klin. Wochenschr., Bd. xxvi, 1913, Nr. 18.

22. REINES. Bericht liber Versuche bei Sklerodermie. Vgl.

Vgl. Wiener klin. Wochenschr., Bd. xxvi, 1913, Nr. 18,
S. 729.

23. PALTAUF. Untersuchung eines Falles von Chorion-

epitheliom. Vgl. Wiener klin. Wochenschr., Bd. xxvi,
1913, Nr. 18, S. 729.

24. OTTO W. LEDERER. Bericht liber Serodiagnose der

Schwangerschaft. Vgl. Wiener klin. Wochenschr.,
Bd. xxvi, 1913, Nr. 18, S. 728.

25. ERNST FREUND. Uber die Serodiagnose des Karzinoms.

Vgl. Wiener klin. Wochenschr., Bd. xxvi, 1913, Nr. 18,
S. 730-

230 LITERATURE

26. FRITZ HEIMANN. Zur Bewertung der Abderhaldenschen

Schwangerschaftsreaktion. Miinchener med. Wochen-
schr., 1913, Nr. 17.

27. N. MARKUS. Untersuchungen iiber die Verwertbarkeit

der Abderhaldenschen Fermentreaktion bei Schwanger-
schaft und Karzinom. Berliner klin. Wochenschr.,
1913, Nr. 17.

28. JOHANNES FISCHER. Gibt es spezifische, mit dem Abder-

haldenschen Dialysierverfahren nachweisbare Schutz-
fermente im Blutserum Geisteskranker ? Sitzungs-
berichte u. Abhandlungen der Naturforschenden Gesell-
schaft von Rostock, Bd. v, 3 Mai, 1913.

29. CESARE DECIO. Untersuchungen iiber die Anwendung

der Abderhaldenschen Reaktion auf dem Gebiete der
Geburtshilfe. Gynak. Rundschau, 1913.

30. CAREY PRATT and McCoRD. The Employment of Pro-

tective Enzymes of the Blood as a means of Extra-
corporeal Diagnosis. Serodiagnosis of Pregnancy.
Surg. Gynec. and Obstetr., vol. xvi, 1913, No. 4, p. 418.

31. WILLIAMS and PEARCE. Abderhalden's Biological Test

for Pregnancy. Surg. Gynec. and Obstetr., vol. xvi,
1913, No. 4, p. 411.

32. HENRY SCHWARZ. The Practical Application of Abder-

halden's Biological Test of Pregnancy. The Inter-
State Med. Journ., vol. xx, 1913.

33. RICHARD FREUND and CARL BRAHM. Die Schwanger-

schaftsdiagnose mittels der optischen Methode und des
Dialysierverfahrens. Miinchener med. Wochenschr.,
1913, Nr. 13, S. 685.

34. BRUNO STANCE. Zur biologischen Diagnose der Schwan-

gerschaft. Miinchener med. Wochenschr., 1913, Nr.
20, S. 1084.

35. ERICH FRANK and FRITZ HEIMANN. Uber Erfahrungen

mit der Abderhaldenschen Fermentreaktion beim Kar-
zinom. Berliner klin. Wochenschr., 1913, Nr. 14.

LITERATURE 23!

36. BEHNE. Ergibt das Dialysierverfahren von Abderhalden

eine spezifische Schwangerschaftsreaktion ? Zentralbl.
fiir Gynakologie, 1913, Nr. 17.

37. TH. PETRI. Uber das Auftreten von Fermenten im Tier-

und Menschenkorper nach parenteraler Zufuhr von art-
und individuumeigenem Serum. Miinchener med.
Wochenschr., 1913, S. 1137.

38. C. A. HEGXER. Zur Anwendung des Dialysierverfahrens

nach Abderhalden in der Augenheilkunde. Miinchener
med. Wochenschr., 1913, S. 1138.

39. W. RUBSAMEN. Zur biologischen Diagnose der Schwan-

gerschaft mittels der optischen Methode und des Dia-
lysierverfahrens. Miinchener med. Wochenschr., 1913,
S. 1139.

40. WEGENER. Serodiagnostik nach Abderhalden in der

Psychiatric. Miinchener med. Wochenschr., 1913,
S. 1197.

41. ERWIN SCHIFF. 1st das Dialysierverfahren Abderhaldens

differentialdiagnostisch verwertbar ? Miinchener med.
Wochenschr., 1913, S. 1197.

42. VICTOR L. KING. Uber trockenes Plazentapulver und

seine Anwendung bei dem Abderhaldenschen Dialysier-
verfahren beziiglich der Diagnose der Schwangerschaft.
Miinchener med. Wochenschr., 1913, S. 1198.

43- ^ JONAS. Beitrage zur klinischen Verwertbarkeit der
Abderhaldenschen Schwangerschaftsreaktion. (Dialy-
sierverfahren.) Deutsche med. Wochenschr., 1913,
S. 1099.

44. FRANZESCO MACCABRUNI. Uber die Verwendbarkeit der

Abderhaldenschen Reaktion bei der Serumdiagnose der
Schwangerschaft. Miinchener med. Wochenschr., 1913,
S. 1259.

45. FAUSER. Pathologisch-serologische Befunde bei Geistes-

kranken auf Grund der Abderhaldenschen Anschau-
ungen und Methodik. Psychiatrisch-neurol. Wochen-
schr., 31 Mai, 1913.

232 LITERATURE

46. ARNO ED. LAMPE and PAPAZOLU. Serologische Unter-

suchungen rnit Hilfe des Abderhaldenschen Dialysier-
verfahrens bei Gesunden. Miinchener med. Wochen-
schr., 1913.

47. BERNARD ASCHNER. Untersuchungen iiber die Serum-

fermentreaktion nach Abderhalden. Berliner klin.
Wochenschr., 1913.

48. ARNO ED. LAMPE and PAPAZOLU. Serologische Unter-

suchungen mit Hilfe des Abderhaldenschen Dialysier-
verfahrens bei Gesunden und Kranken. Stiidien iiber die
Spezifizitat der Abwehrfermente. 2 Mitt. Untersuch-
ungen bei Morbus Basedowii, Nephritis und Diabetes
melitus. Miinchener med. Wochenschr., 1913.

49. GEBB. Die Untersuchungsmethoden nach Abderhalden

in der Augenheilkunde. Bericht iiber die 39. Versamm-
lung der ophthalmol. Gesellschaft zu Heidelberg,

50. VON HIPPEL. Uber sympathisers Ophthalmic und

juvenilen Katarakt. (Das Abderhaldensche Dialysierver-
fahren bei diesen beiden Erkrankungen, sowie bei
Keratitis parenchymatosa.) Bericht iiber die 39. Ver-
sammlung der ophthalmol. Gesellsch. zu Heidelberg.

51. LUDWIG PlNCUSSOHN. Untersuchungen iiber die fer-

mentativen Eigenschaften des Blutes. Biochemische
Zeitschrift, Bd. li, 1913, S. 107.

52. K. JAWORSKI and Z. SZYMANOWSKI. Beitrag zur Sero-

diagnostik der Schwangerschaft. Wiener klin. Wochen-
schr., Nr. 23, 1913.

53. LICHTENSTEIN. Zur Serumreaktion nach Abderhalden.

Miinchener med. Wochenschr., 1913.

54. SlGMUND GOTTSCHALK. Zur Abderhaldenschen Schwan-

gerschaftsreaktion. Berliner klin. Wochenschr., 23
Juni, 1913, S. 1151.

55. HERMANN LUDKE. Diagnostic precoce du carcinome au

moyen du procede de dialysation d'apres E. Abder-
halden. Gazette des Hopitaux, 86 Annee. Nr. 65,
10 juin, 1913, S. 1064.

LITERATURE 233

56. EVLER. Beitrage zu Abderhaldens Serodiagnostik.

Medizin. Klinik. 29 Juni, 1913. Nr. 26 u. 27, S.
1043. Compare with this EMIL ABDERHALDEN, in the
same journal, 1913, Xr. 29, S. 1171.

57. ARTHUR LEROY. Essai sur le mecanisme probable de la

crise dans 1'epilepsie et dans Fasthme. Paris Medical.
23 Mai, 1913, S. 70.

58. H. MlESSNER. Die Amvendung des Dialysierverfahrens

nach Abderhalden zur Diagnose der Trachtigkeit und
von Infektionskrankheiten. Deutsche tierarztl. Wochen-
schr., 1913, Nr. 26.

59. Diskussion zu HEGNER. Cber das Dialysierverfahren

in der Augenheilkunde :: von Binswanger u. Ahrens.
Miinchener med. "Wochenschr., 1913, Nr. 27, S. 1518.

60. O. PARSAMOOR. Die biologische Diagnostik der Schwan-

gerschaft nach Abderhalden. Zentralblatt f. Gyna-
kologie, 1913, Xr. 25.

61. ARNO ED. LAMPE. Basedo\vsche Krankheit und Genitale.

Untersuchungen mit Hilfe des Abderhaldenschen Dia-
lysierverfahrens. Monatsschr. f. Geburtshilfe u. Gyna-
kologie, Bd. xxxviii, 1913, S. 45.

62. J. VEIT. Die Serodiagnostik der Graviditat. Berliner

klin. Wochenschr., 1913, Nr. 27.

63. G. A. PARI. Sulla sierodiagnosi della gravidanza secondo

1'Abderhalden. Ace. Med. di Padova, 28 Febr., 1913.

64. G. A. PART. Sulla sierodiagnosi della gravidanza

secondo 1'Abderhalden. Gazzetta degli Ospedali e delle
Cliniche, 1913, X T r. 69, S. 727.

65. ERNST HEILNER and TH. PETRI. Uber kunsthch herbei-

gefiihrte und natiirlich vorkommende Bedingungen zur
Erzeugung der Abderhaldenschen Reaktion und ihre
Deutung. Miinchener med. AVochenschr., 1913, Xr. 28,
S. 1530.

234 LIT KR. \TURE

66. ZDZISLAW STEISING. Uber die Natur des bei der Abder-

haldenschen Reaktion wirksamen Ferments. Miinch-
ener med. Wochenschr., 1913, Nr. 28, S. 1535.

67. ERICH FRANK, FELIX ROSENTHAL, and HANS BIBERSTEIN.

Experimentelle Untersuchungen liber die Spezifiziatat
der proteolytischen Abwehr-(Schutz-)Fermente (Abder-
halden). Miinchener med. Wochenschr., 1913, Nr. 29,
S. 1594.

68. LAMPE. Gesellschaft fiir Geburtshilfe u. Gynakologie.

Leipzig, 610. Sitzung, 1913. Zentralbl. f. Gynak., 1913,

Nr. 30.

69. KARL KOLB. Gelingt es mittels der Abderhaldenschen

Fermentreaktion, den Nachweis eines persistierenden
oder hypoplastischen Thymus zu fiihren ? Miinchener
med. Wochenschr., 29 Juli, 1913, Nr. 30, S. 1642.

70. G. vox GAMBAROFF. Die Diagnose der bosartigen Neu-

bildungen und der Schwangerschaft mittels der Abder-
haldenschen Methode. Miinchener med. Wochenschr.,
29 Juli, 1913, Nr. 30, S. 1644.

71. ERNST FRAENKEL and FRIEDRICH GUMPERTZ. Anwendung

des Dialysierverfahrens (nach Abderhalden) bei der
Tuberkulose. Deutsche med. Wochenschr., 14 Aug.,
1913, S. 1585.

72. FRANZ BRUCK. Uber den diagnostischen Wert der Abder-

haldenschen Serumreaktion (Fermentreaktion). Miin-
chener med. Wochenschr., 12 Aug., 1913, S. 1775.

73. M. E. GOUDSMIT. Zur Technik des Abderhaldenschen

Dialysiervenfahrens. Miinchener med. Wochenschr.,
1913, S. 1775.

74. HANS SCHLIMPERT and ERNST ISSEL. Die Abderhalden-

sche Reaktion mit Tierplazenta und mit Tierserum.
Miinchener med. Wochenschr., 1913, S. 1759-

75. JULIUS BAUER. Uber den Nachweis organabbauender

Fermente im Serum mittels des Abderhaldenschen Dia-
lysierverfahrens. Wiener klin. Wochenschr., 1913,

Nr. 27.

LITERATURE 235

76. ALEX. PAPAZOLU. Sur la production des substances biure-

tiques dans les centres nerveux malades (epilepsie,
demence precoce, paralysie generale) et dans le corps
thyroide (goitre), le thymus et 1'ovaire des basedowiens,
par le serum des individus atteints de ces memes mala-
dies. C. r. de la Soc. de Biol., Bd. Ixxiv, 3 janv., 1913,
S. 302.

77. G. MARIXESCO and MME. ALEX. PAPATOLU. Sur la speci-

ficite des ferments presents dans le sang des Parkin-
soniens. C. r. de la Soc. de Biol., Bd. Ixxiv, 29 Mai,
1913, S. 1419.

78. HEINRICH XEUE. Uber die Anwendung des Abderhalden-

schen Dialysierverfahrens in der Psychiatric. Monats-
schr. f. Psychiatric u. Neurologic, Bd. xxxiv, 1913,

S. 95.

79. AHRENS. Uber Abderhaldenreaktion bei Nervenerkran-

kungen. Miinchener med. Wochenschr., 1913, S. 1857.

80. MICHELE BOLAFFIO. Contributo alia diagnosi di gravi-

danza col metodo ottico di Abderhalden. Patologica,
Bd. v, 1913, Nr. in, S. 352.

81. CHARLES C. W. INDD. The Serum Diagnosis of Preg-

nancy. Bull, of the Amer. Med. Assoc., vol. Ix, 1913,
No. 25, p. 1947.

82. FRITZ HEIMANX. Die Abderhaldensche Schwangerschafts-

reaktion. Berliner klin. Wochenschr., 1913, S. i.

83. R. G. LURRIE. Abderhaldensche Reaktion. Russkji

AVratsch., Bd. xii, 1913, S. 697.

84. DAUXAY and ECALLE. De Pexamen du serum de la femme

enceinte et du serum de la femme non enceinte, par la
methode de dialyse d'E. Abderhalden. C. r. hebd. des
seances de la Soc. de Biol., Bd. Ixxiv, 1913, S. 1190.

85. POLAXO. Zur biologischen Schwangerschaftsdiagnose.

Monatsschr. f. Geburtsh. u. Gynak., Bd. xxxvii, 1913,
S. 857-

86. PETRI. Uber die Spezifizitat der gegen Plazenta gerich-

teten Schutzfermente des Schwangerenserums. Monats-
schr. f. Geburtsh. u. Gynak., Bd. xxxvii, 1913, S. 859.

236 LITERATURE

87. JOHANNA LEVY. Zum Nachweis der Schwangerschaft

durch das Dialysierverfahren nach Abderhalden. Der
Frauenarzt., Bd. xxviii, 15 Juli, 1913, Heft 7.

88. A. MAYER. Uber die klinische Bedeutung des Abder-

haldenschen Dialysierverfahrens. Zbl. f. Gynak., 1913,

Nr. 32.

89. M. URSTEIN. Die Bedeutung des Abderhaldenschen

Dialysierverfahrens fiir die Psychiatrie und das korre-
lative Verhaltnis von Geschlechtsdriisen zu anderen
Organen mit innerer Sekretion. Wiener klin. Wochen-
schr., Bd. xxvi, 1913, Nr. 53, S. 1325.

90. A. MAYER. Uber das Abderhaldensche Dialysierven-

fahren und seine klinische Bedeutung. Miinchener med.
Wochenschr., 1913, S. 1972.

91. G. PLOTKIN. Zur Frage von der Organspezifitat der

Schwangerschaftsfermente gegeniiber Plazenta. Miin-
chener med. Wochenschr., 1913, S. 1942.

92. C. F. JELLINGHAUS and J. R. LOSEE. The Sero-diagnosis

of Pregnancy by the Dialyzation Method. Based on the
examination of serum from 563 different individuals.
Bull, of the Lying-in Hospital of the City of New York,
vol. ix, 1913, p. 68.

93. M. J. BREITMANN. Uber die Diagnose der Leberkrank-

heiten mit Hilfe der Methode von Prof. Abderhalden,
mit spezieller Beriicksichtigung der Selbstandigkeit der
beiden Leberlappen. Zbl. f. innere Medizin, Bd. xxxiv,
1913, Nr. 34.

94. B. TH. KABANOW. Beziehungen der Magen-Darmaffek-

tionen zu der perniziosen Anamie nach dem Dialysier-
verfahren von Prof. E. Abderhalden. Zbl. f. innere
Medizin, Bd. xxxiv, 1913, Nr. 34.

95. N. KAFKA. Uber den Nachweis von Abwehrfermenten

im Blutserum vornehmlich Geisteskranker durch das
Dialysierverfahren nach Abderhalden. I Mitt. Zeit-
schr. f. d. gesamte Neurologie und Psychiatrie, Bd. xviii,
1913, S. 341-

LITERATURE 237

96. F. DEUTSCH and R. KOHLER. Serologische Untersuchun-

gen mittels des Dialysierverfahrens nach Abderhalden.
Wiener klin. Wochenschr., 1913, Nr. 34.

97. A. FAUSER. Die Serologie in der Psychiatric. Miin-

chener med. Wochenschr., 1913, Nr. 36, S. 1984.

98. JACOB GUTMANN and SAMUEL J. DRUSKIN. Experiences

with the Abderhalden Test in the Diagnosis of Preg-
nancy. Medical Record, vol. Ixxxiv, p. QQ, July 19,

99. A. FAUSER. Pathologisch-serologische Befunde bei Geis-
teskranken auf Grund der Abderhaldenschen Anschau-
ungen und Methodik. Allgem. Zeitschr. f. Psychiatric
Medizin, Bd. Ixx, 1913, S. 719.

100. WlLHELM MAYER. Die Bedeutung der Abderhaldenschen

Serodiagnostik fur die Psychiatric. Miinchener med.
Wochenschr., 1913, Nr. 37, S. 2044.

101. P. JODICKE. Zum Nachweis von organabbauenden Fer-

menten im Blute von Mongolen. Wiener klin. Rund-
schau, 1913, Nr. 38.

102. CARLO FERRAI. Sulla specificita dei peptoni placentari

nella diagnosi della gravidanza col metodo polari-
metrico. Patol., Bd. v, 1913, S. 449.

103. ARNO ED. LAMPE. Untersuchungen mit Hilfe des Abder-

haldenschen Dialysierverfahrens bei Lungentuberkulose.
Deutsche med. Wochenschr., 1913, Nr. 373.

104. J. B. PORCHOWNICK. Die Serodiagnostik der Schwanger-

schaft. Zbl. f. Gynak., Bd. xxxvii, 1913, S. 1226.

105. ERNST FRANKEL. Uber Spezifitat und Wesen der Abder-

haldenschen Abwehrfermente. Wiener klin. Rund-
schau, 1913, Nr. 38.

106. C. F. BALL. A new Sero-diagnostic Test for Pregnancy

(Abderhalden's). Vermont Medical Monthly, August,
1913-

107. FRITZ HEIMAXX. Die Abderhaldensche Schwangerschafts-

reaktion. Berliner Klinik, Heft 301, Jahrg., 25 Juli,
1913.

238 LITERATURE

108. ARNO ED. LAMPE and ROBERT FUCHS. Serologische Un-
tersuchungen mit Hilfe des Abderhaldenschen Dialysier-
verfahrens bei Gesunden und Kranken. Studien liber
die Spezifitat der Abwehrfermente. 3 Mitt. Weitere
Untersuchungen bei Schilddriisenerkrankungen : Mor-
bus Basedowii, Basedowoid, Myxodem, endemische
Struma. Miinchener med. Wochenschr., 1913, Nr. 38
und 39, S. 2 1 12 und 2177.

109. B. TH. KABANOW. Uber die Diagnose der Magendarm-

affektionen mit Hilfe des Abderhaldenschen Dialysier-

verfahrens. Miinchener med. Wochenschr., 1913, S.

2164.
no. H. DEUTSCH. Erfahrungen mit dem Abderhaldenschen

Dialysierverfahren. Wiener klin. \Vochenschr., 1913,

Nr. 38.
in. ADOLF FUCHS. Tierexperimentelle Untersuchungen iiber

die Organspezifitat der proteolytischen Abwehrfermente

(Abderhalden). Miinchener med. Wochenschr., 1913,

Nr. 40, S. 2230.

112. P. SCHAFER. Der Abderhaldensche Fermentnachweis im

Serum von Schwangeren. Berliner klin. Wochenschr.,
1913, Nr. 35.

113. TSCHUDNOWSKY. Zur Frage iiber den Nachweis der

Abwehrfermente mittels der optischen Methode und des
Dialysierverfahrens nach Abderhalden im Blutserum
bei Schwangerschaft und gynakologischen Erkran-
kungen. Miinchener med. Wochenschr., 1913, Nr. 41,
S. 2282.

114. M. RUBINSTEIN and A. JULIEN. Exameri des serums des

chevaux atteints d'ascaridiose par la methode d'Abder-
halden. C. r. des seances de la Soc. de Biol., Bd. Ixxv,
26 Juli, 1913, S. 180.

115. SCHATTKE. Die Anwendung des Abderhaldenschen Dialy-

sierverfahrens zur Diagnose der Trachtigkeit bei Tieren.
Zeitschrift f. Veterinarkunde mit besonderer Beriick-
sichtigung der Hygiene, Bd. xxv, 1913, S. 425.

116. M. ZALLA. I metodi sierodiagnostici di Abderhalden.

Rivista di Patol. nervosa e mentale, Bd. xviii, 1913,
Fasc. 9 (Literaturiibersicht).

LITERATURE 239

117. F. EBELER and R. LOXXBERG. Zur serologischen Sch \van-

gerschaftsreaktion nach Abderhalden. Berliner klin.
AYochenschr., 1913, Xr. 41.

118. OTTO BIXSWAXGER. Die Abderhaldensche Seroreaktion

bei Epileptikern. Miinchener med. \Vochenschr., Bd.
Ix, 1913, Nr. 42.

IIQ. E. v. HIPPEL. Zur Atiologie des Keratokonus (Unter-
suchungen mit dem Abderhaldenschen Dialysierver-
fahren). Klinische Monatsblatter fiir Augenheilkunde,
Bd. li, 1913, S. 273.

120. ANTOX SUXDE. Die Abderhaldensche serologische Reak-

tion der Schwangerschaft. Xorsk Magaz. for La?ge-
Avidenchaben, Bd. Ixxiv, 1913, S. 1234.

121. PAOLOS AR. PETRIDIS. Ferments protecteurs de Forga-

nisme animal. Diagnostic biochimique de la grossesse
par la reaction d'Abderhalden. Precede du dialyseur.
Progres med., Bd. xliv, 1913, S. 451.

122. JACOB GUTMAX and SAMUEL O. DRUSKIX. Experiences

with the Abderhalden Test in the Diagnosis of Preg-
nancy. Medical Record, vol. Ixxxiv, 1913, p. 99.

123. HUSSELS. Uber die Anwendung des Abderhaldenschen

Dialysierverfahrens in der Psychiatric. P?ychiatrisch-
neurologische Wochenschrift, Bd. xv, 1913, Nr. 27,
S. 329-

124. JAMISON CHAILLE and J. C. COLE. The Sero-diagnosis of

Pregnancy. Xew Orleans Med. and Surg. Journ.,
vol. Ixvi, 1913, p. iSS.

125. F. JESSEX. Uber Untersuchungen mit dem Abderhalden-

schen Dialysierverfahren bei Tuberkulosen. Medizin.
Klinik, Bd. ix, 1913, S. 1760.

126. X'AUMAXX. Experimentelle Beitrage zum Schwanger-

schaftsnachweis mittels des Dialysierverfahrens nach
Abderhalden. Deutsche medizin. Wochenschr., Bd.
xxxix, 1913, S. 2086.

127. KASIMIR JAWORSKI. Klinische Bemerkungen betreffend

die Abderhaldensche Reaktion. Gynakol. Rundschau.,
Bd. vii, 1913, S. 582.

24O LITERATURE

128. RUDOLF BUNDSCHUH and HANS ROMER. tiber das Ab-

derhaldensche Dialysierverfahren in der Psychiatric.
Deutsche med. Wochenschrift, 1913, Nr. 42.

129. EDMUND WALDSTEIN and RUDOLF EKLER. Der Nachweis

resorbierten Spermas im weiblichen Organismus.
Wiener klin. Wochenschr., Bd. xxvi, 1913, Nr. 42.

EMIL ABDERHALDEN. Diagnose der Schwangerschaft mit
Hilfe der optischen Methode und des Dialysierverfahrens.
Miinchener med. Wochenschr., 1912, Nr. 24.

EMIL ABDERHALDEN. Wekerer Beitrag zur Diagnose der
Schwangerschaft mittels der optischen Methode und des
Dialysierverfahrens. Miinchener med. Wochenschr.,
1912, Nr. 36.

EMIL ABDERHALDEN. Weiterer Beitrag zur biolog. Feststel-
lung der Schwangerschaft. Zeitschr. f. physiol. Chem.,
Bd. Ixxxi, 1912, S. 90.

EMIL ABDERHALDEN. Die Diagnose der Schwangerschaft
mittels der optischen Methode und des Dialysierver-
fahrens. Berliner tierarztl. Wochenschr., 1912, Nr. 25.

EMIL ABDERHALDEN. Nachtrag zu : Weiterer Beitrag zur bio-
logischen Feststellung der Schwangerschaft. Miinchener
med. Wochenschr., xl, 1912, und Berliner tierarztl.
Wochenschr., 1912, Nr. 42.

EMIL ABDERHALDEN and ARTHUR WEIL. Tiber die Diagnose
der Schwangerschaft bei Tieren mittels der optischen
Methode und des Dialysierverfahrens. Berliner tierarztl.
Wochenschr., 1912, Nr. 36.

EMIL ABDERHALDEN. Die optische Methode und das Dialysier-
verfahren als Methoden zum Studium von Abwehrmass-
regeln des tierischen Organismus. Die Diagnose der
Schwangerschaft bei Mensch und Tier mittels der genann-
ten Methoden. Handb. der biochemischen Arbeits-
methoden, Bd. vi, 1912, S. 223.

EMIL ABDERHALDEN. Die Serodiagnostik der Schwangerschaft.
Deutsche med. Wochenschr., 1912, Nr. 46.

LITERATURE 24!

EMIL ABDERHALDEN. Ausblicke liber die Verwertbarkeit der
Ergebnisse neuerer Forschungen auf dem Gebiete des
Zellstoffwechsels zur Losung von Fragestellungen
auf dem Gebiete der Pathologic des Nervensystems.
Deutsche med. Wochenschr., 1912, Xr. 88.

EMIL ABDERHALDEN. Der Nachweis blutfremder Stoffe mittels
des Dialysierverfahrens und der optischen Methode und
die Verwendung dieser Methoden mit den ihnen zugrunde
liegenden Anschauungen auf dem Gebiete der Pathologic.
Beitrage zur Klinik der Infektionskrankheiten und zur
Immunitatsforschung, Bd. i, 1913, Heft 2, S. 243.

EMIL ABDERHALDEN. Zur Frage der Spezifizitat der Schutz-
fermente. Miinchener med. Wochenschr., 1913, Nr. 9.

EMIL ABDERHALDEN. Uber eine mit dem Polarisationsapparat
kombinierte elektrischheizbare Vorrichtung zur Ablesung
und Beobachtung des Drehungsvermogens bei konstanter
Temperatur. Zeitschr. f. physiol. Chem., Bd. Ixxxiv,
1913, S. 300.

EMIL ABDERHALDEN and ARNO ED. LAMPE. Uber den Einfluss
der Ermiidung auf den Gehalt des Blutserums an dialy-
sierbaren, mit Triketohydrindenhydrat reagierenden Ver-
bindungen. Zeitschr. f. physiol. Chem., Bd. Ixxxv, 1913,
S. 136.

EMIL ABDERHALDEN and HUBERT SCHMIDT. Einige Beobach-
tungen und Versuche mit Triketohydrindenhydrat (Ruhe-
mann). Zeitschr. f. physiol. Chem., Bd. Ixxxv, 1913,
S. 143-

EMIL ABDERHALDEX. Die Diagnose der Schwangerschaft
mittels des Dialysierverfahrens und der optischen
Methode. Monatsschrift f. Geburtshilfe u. Gynakologie,
Bd. xxxviii, 1913, S. 24.

EMIL ABDERHALDEN and PETER ANDRYEWSKY. Uber die Ver-
wendbarkeit der optischen Methode und des Dialysierver-
fahrens bei Infektionskrankheiten. Untersuchungen iiber
Tuberkulose bei Rindern. Miinchener med. Wochen-
schr., 29 Juli, 1913, Nr. 30, S. 1641.

16

242 LITERATURE

EMIL ABDERHALDEN and ARTHUR WEIL. Beitrag zur Kenntnis
der Fehlerquellen des Dialysierverfahrens bei sero-
logischen Untersuchungen. Uber den Einfluss des Blutge-
haltes der Organe. Miinchener med. Wochenschr., 1913,
S. 1703.

EMIL ABDERHALDEN and ANDOR FODOR. tiber Abwehrfer-
mente im Blutserum Schwangerer und von Wochnerinnen,
die auf Milchzucker eingestellt sind. Miinchener med.
Wochenschr., 1913, Nr. 34, S. 1880.

EMIL ABDERHALDEN and ERWIN SCHIFF. Weiterer Beitrag zur
Kenntnis der Spezifitat der Abwehrfermente. Das Ver-
halten des Blutserums schwangerer Kaninchen gegeniiber
verschiedenen Organen. Miinchener med. Wochenschr.,
IQIS, S. 1923.

EMIL ABDERHALDEN and ANDOR FODOR. Studien iiber die
Spezifitat der Zellfermente mittels der optischen Me<thode.
Zeitschrift f. physiol. Chemie., Bd. Ixxxvii, 1913, S. 220.

EMIL ABDERHALDEN and ERWIN SCHIFF. Studien iiber die
Spezifitat der Zellfermente mittels der optischen Methode.
Zeitschrift f. physiol. Chemie., Bd. Ixxxvii. 1913, S. 231.

EMIL ABDERHALDEN and ERWIN SCHIFF. Versuche iiber die
Geschwindigkeit des Auftretens von Abwehrfermenten
nach wiederholter Einfiihrung des plasmafremden Sub-
strates. Zeitschr. f. physiol. Chemie., Bd. Ixxxvii, 1913,
S. 225.

EMIL ABDERHALDEN. Gedanken iiber den spezifischen Bau der
Zellen der einzelnen Organe und ein neues bio-
logisches Gesetz. Miinchener med. Wochenschr., Bd. Ix,
1913, S. 2386.

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