The Biochemical journal, 1909

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_ BENJAMIN MOORE, M.A., D.Sc.
“EDWARD WHITLEY, M. A..

VOLUME IV
1909 Pte
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Bie os UNIVERSITY OF LIVERPOOL

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CONTENTS OF VOLUME IV

Observations on Certain Marine Organisms of (a) Variations in Reaction to
Light, and (}) a Diurnal Periodicity of Phosphorescence. By Benjamin Moore, M.A.,
D.Sc., Johnston Professor of Bio-Chemistry, University of Liverpool :

The Relative Importance of Inorganic Kations, especially those of Sodium and

“Calcium in the Causation of Gout and Production of wid ne Bak By William

Gordon Little, M.A. (Aber.), M.D. (Edin.)

On the Nitrogen-containing Radicle of Lecithin and cetild Phosphates By
Hugh MacLean, M.D., Carnegie Research Fellow .

Some Observations on the Haemolysis of Blood by Syebslote on iver:
osmotic Solutions of Sodium Chloride. By U. N. Brahmachari, M.A., M.D., Lecturer
on Medicine at the Campbell Medical School, Calcutta

Further Observations on the Action of Muscarin and Plocarpin on a ey
By Hugh MacLean, M.D., Carnegie Research Fellow

The Occurrence and Distribution of Cholesterol and allied Bodies i in he Nite
Kingdom. By Charles a M.A., B.Sc., Lindley Student of the b geivieyd of
London

Allyl sihininase Sina Aspect o of its Physiological celal By E. Wace

Carlier, M.D., F.R.S.E.

Choline in Animal Tissues and Fluids. “By W. Webster, M. D., ©. M. Haleais
strator of Physiology in the University of Manitoba, Canada

The Biuret Reaction and the Cold Nitric Acid Test in the Recognition of
Protein. By Karl A. van Norman, M.B. (Toronto)

The Properties and Classification of the Oxidising Sasi and heieaies
between Enzymic Activity and the Effects of Immune Bodies and Complements.
By Benjamin Moore, M.A., D.Sc., Johnston Professor of Biante University
of Liverpool, and Edward Whitley, M.A. (Oxon.) ;

On the Occurrence of a Mon-amino-diphosphatide Lecithin-like Body i in he
Yolk of Egg. By Hugh i 5 M.LD., a Saree Research sb uae
of Aberdeen . :

Iodo-Eosin as a Test fe Pi ree Alkali j in 0 dried-up Plant aaa. By A. C. Hof,
Héchst a, Main

On the Growth of the Bacillus Tuberculosis he sale Micte-Onseata in
different Percentages of yg 4 om By Benjamin Moore, M.A., D.Sc., Johnston
Professor of Bi emistry, University of Liverpool, and R. Stenhouse Williams,
M.B., D.P.H., Lecturer on Public Health Bacteriology, University of Liverpool

The Electrical Forces of Mitosis and the Origin of Cancer. By A. E. and A. C.
Jessup; E. C. C. Baly, F.R.S., Fellow of University College, London; F. W.
y, M.D., M.R.C.P. ; and E. Prideaux, M.R.C.S., L.R.C.P. ;

The Estimation of Phosphorous in Urine. By G. C. Pata, M.B., B. s.

(Melb.), Sharpey Scholar
On the Nitrogen-containing Radicle of Lecithin ed Pee Phosphatides. By
Hugh Maclean, M.D., Carnegie Research Fellow, University of Aberdeen

A Polarimetric Study of the Sucroclastic Enzymes in Beta Vulgaris. By R. A.
Robertson, M.A. ; James Oe ean Iving, D.Se., Ph.D.; and Mildred E. Dobson,
M.A., B.Se., Carnegie Sch

The Output of ic Phosphorus in Urine. ay 3 “Mathison, M. B.,
B.S. (Melb nae Or : sg

On the Relative Haemoglobin Value of the éditent Ery ehisotees tiles he
Haemolysis of Blood with Hyposmotic Sodium Chloride Solution, and on the
Permeability of the Erythrocytes to Water as a Factor in the production of Haemolysis.
By U.N. Brahmachari, M.A., M.D., Ph.D., Lecturer in begeeag —* Medical
School, Calcutta

PAGE

CONTENTS

The Isolation of Conium Alkaloids from Animal Tissues, and the Action of

- Living Cells and Decomposing Organs on these Alkaloids.
M.B., Ch.B. (Aber.), Carnegie Scholar in Pharmacology

By Walter J. Dilling,

Some Observations upon the Error in the Opsonic Technique. By Ernest E.
Glynn, M.A., M.D. (Cantab.), M.R.C.P., Lecturer in Morbid Anatomy and Clinical
Pathology, University of Liverpool, Pathologist, Royal Infirmary, Liverpool, and
G. Lissant Cox, M.A, M.B., B.C. anne ), Holt Fellow in. Pethelogy, sia
of Liverpool .

The Relationship of ee of a Dow to the i size of re Animal Treated, aida
in regard to the Cause of the Failures to Cure Trypanosomiasis and other Protozoan
Diseases in Man and in large Animals. By Benjamin Moore, M.A., D.Sc., gion
Professor of Bio-Chemistry, University of Liverpool :

Proposals for the Nomenclature of the Lipoids. By ras poesaliien

A Comparison of the Methods for the Estimation of Total setae in Urine,
By Stanley Ritson, A.K.C. :

The Use of Barium Peroxide in the heiadon of Total Sulphur in Urine:
Stanley Ritson, A.K.C.

A Contribution to the Bio- Chaaety a ean is Chaiiges.{ in ie
Solubilities of the Lipoids in presence of one another, and of certain unsaturated

organic substances; (+) The Balancing Action of Certain Pairs of Haemolysers i in

By

PAGE

Preventing Haemolysis ; ; (c) The Protective Action of Serum Proteins against |
Haemolysers ; (d) The Effects of Oxidising and Reducing Enzymes upon Haemolysis,
By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bio~-Chemistry, University

of Liverpool ;

Frederick P. ae M.D. So . ; and Lancelot purest: as
M.D. (Liverpool) :

Observations on the Hsemidbyiic mee of Ceftsin Bile ‘Deriestives By Hugh
MacLean, M.D., Carnegie Research mm os fame pr of ap sas and Lancenrt
Hutchinson, M. D. (Liverpool)

The Pharmacology of ge bare Citiebaaehe By J.C. w. Graham, MA,
M.D., B.C. (Cantab.)

The Physiological Effects of Bclesitid Cieiiscuidil with Relation to ine po .

on Glycogen and Sugar Derivatives in the Tissues. ed Charles O. dares M.D.

(Liverpool)

The Effect of Work on rank ecl. Colic of Muscle.
and E. P. Cathcart

The Action of Extracts of the Pivcitary Body. By H. H. Dale, M.A., M D.

By T. Gish Bind .

A Method for the Estimation of the Urea, Allantoin, and Amino Acids in the
Urine. By Dorothy E. Lindsay, B.Sc., Carnegie Research Scholar _ ,

On the Nature of the so-called Fat of Tissues and Organs. By Hugh MacLean, e
M.D., Carnegie Fellow, University of Aberdeen, and Owen T..Williams, M.D., |
B.Sc. "(Lond .), M.R.C.P., Hon.. Assistant he lacie: Hoepita} for Consumption, 4

Lecturer in Pharmacology, University of Liverpool
The Osmotic Pressure of Liquid Foods. By Judah L. ie B. Sc. (Adel) “J
The Relationship of Diastatic Efficiency to Average Glycogen Content in the.+~< 52 a =

Different Tissues and Organs.
Fellow, University of Aberdeen _

tt $554

By — Beieet M.D.,, ee Revearthiy

The Osmotic Pressure of the Egg of he Gascon Fowl pe its Changs during

Incubation.

By W. R. G. Atkins, M.A. (Tri righty College, Dublin) .

»

462

‘dhe ‘ss .

SERVATIONS ON CERTAIN MARINE ORGANISMS OF
= _ (@) VARIATIONS IN REACTION TO LIGHT, AND
(6) A DIURNAL PERIODICITY OF PHOSPHORESCENCE

ei sex JAMIN “MOORE, -M.A., D.Sc., Johnston Professor of Bio-
me aeeeeese U ey of Liverpool.

tag rom the Marine Biological Station, Port Erin, Isle of Man

oe fie (Received November 6th, 1908)

“he observations recorded in this paper were chiefly conducted upon
) yanisms taken by means of a fine silk tow-net in Port Erin Bay during
the spring and summer of 1908.1 In addition certain observations are
added upon the reaction to light of young larvae of the plaice
_ (Pleuronectes platessa) taken from the Hatchery of the Station.

2 te: ~The experiments on the action of light were made in April, and the
~ attempt in September to investigate the action of light upon the phos-
ent organisms then present in the Bay, based on the supposition
that organisms which themselves emitted light might possibly show
- interesting variations in reaction to incident light from without, led to
- the accidental discovery of the diurnal periodicity in the phosphorescence
-s of these organisms, which furnishes the subject of the second section of
paper.

_ The two sets of experiments on the variations in a to light,
aay upon the diurnal periodicity in phosphorescence, are really distinct,
and will be described in two separate sections.

ma

A. Vartarioys wy rue Reactions or OrGanisms (Cuierty Nave oF
. Batanvs) vo Daytieutr ann Arviricta, Licutr

Since the very existence of all living organisms, either directly or
directly i is dependent upon the energy of light, and the transformation
E this into other types of energy, it is not surprising that reactions to
it are amongst the most fundamental and most widely spread
‘throughout the whole world of organized living creatures. Such
reactions must have been developed in the very beginning of the dawn
when the first living cells commenced to synthesize organic products
from the inorganic materials of their environment by the use of the store
oe energy from the sunlight. Later on organisms arose which were only

Soek., All the sdvesibebhieig eat tn Section B were takén in Port Erin Bay and were surface
tow. Prot. Herdman. some of those used in Section A were kindly taken for me outside the Bay by

2 BIO-CHEMICAL JOURNAL

dependent upon the light at second-hand, since they were able to
consume the synthesized organic products formed by other organisms
converting the light energy directly, and so were only indirectly
dependent upon the light for their existence. Even for this type of
organism, utilizing the light energy indirectly, reactions to light
remained essential in the search for food and for other physiological
functions, and also there would be an inheritance of relationships to
light derived from the earlier ancestry with direct dependence upon light.

At a later stage structures or organs arose specially adapted for
light reactions, and in those living creatures possessing such organs there
probably came a deterioration of the sensitiveness to light of the
remaining cells of the body. But in spite of all such decline in direct
sensitiveness to light, there must have remained some trace of their old
primeval relationships to light. .

Experimental evidence of this persistence of relationship to light of
all cells exists of two kinds; there is first the deleterious effects of
complete withdrawal of light for prolonged periods, and the necessity of
sunlight for healthy existence; and, secondly, there is the direct evidence
of the effects of application of strong light to animal cells seen in the
Finsen effects, and in other forms of radiant energy allied to light.

It is, however, in the more lowly organized types of both animal and
vegetable organisms that the strongest and most direct reactions to light
are observable—apart from the particular case of the reaction of chemical
synthesis in the chlorophyll-containing cells of the green parts of the
higher plants.

Examples of this reactivity are seen in “Te effects of sunlight | upon
nearly all types of bacteria; in the sudden outburst of vegetable life in
the form of diatoms in the spring of each year as the length of the day
increases and the more vertical light reaches and penetrates the water
before there is much increase in the temperature of the sea—an outburst
upon which the whole life of the sea is as thoroughly dependent as that
of the terrestrial world is upon the similar outburst of activity in land
plants; and in the most marked movements which occur towards or from
the light according to varying circumstances of the minute organisms,
either larval or adult, which chiefly constitute the plankton or floating
life of the ocean.

It is hence clear that the observation of the reactions of living cells
to light is of importance both to the student of biology, and to the student
of medicine who makes practical applications of the discoveries of biology,
using the term in its widest sense.

VARIATIONS IN: REACTION TO LIGHT 3

Recent discoveries have proven the value of light treatment as a
practical adjunct of medicine, and the study of light effects upon the
_ simpler organisms must sooner or later yield a key, both for the rational
understanding of such effects, and their extension to further utility. In
addition to these utilitarian advantages, the study is one of the most
"<<a from its own intrinsic interest in the whole wide field of
mn ge One of the most obvious lines of attack in-inv pagakeas the reactions

ae . change in mS scien. or orientation, in fixed or sessile
organisms.
Tt must, however, be clearly borne in mind that this movement is an
__ index of other things, that the underlying problem is ultimately and
__ essentially a chemical one, or, better expressed, one of chemical
transformation of light energy.! The organisms move because of an action
of light upon chemical constituents in the cells, that is to say, there is a
_ change in the metabolism of the cell stimulated, giving rise to the
movement of the organism. Also, according to the nature and condition
_ of both cell and light-stimulis, which form the two inter-acting factors,
the character and sense of the movement of the organism will vary.
Thus, we shall see that with the same condition and previous treatment
of the organism, the reaction varies and becomes positive or negative
with varying intensities of the light-stimulus, and, secondly, with the
same constant intensity of light-stimulus, the reaction varies when the
_-_——s previous history and condition of the reacting organism have been
P ms artificially varied. That is to say, the light induces chemical alterations
in the cell, and the nature and amount of the chemical changes vary with
____ the two factors, the condition of the cell at the time, and the intensity of
the incident light.
‘Tt has been clearly pointed out by Loeb that the orientations or
_ tropisms of sessile organisms, and the movements of free organisms
__ towards or away from light, are essentially the same in character, the free
organism being first orientated and then, by the action of its locomotor
organs, carried in either direction according to the sense of the previous
orientation.
This is a fundamental observation which to a certain extent unifies
the problem, but there still remain the questions of why the light induces

1. This view has been also put forward by Loeb, Dynamics of Living Matter, 1906, pp. 112
seq.

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j BIO-CHEMICAL JOURNAL

orientation, the conditions under which orientation varies with the
condition of the organism and the strength of stimulation, and also the
remarkable fact that in higher organisms at any rate there is developed
what might be described as resistance to orientation, so that the organisms
accumulate either at the proximal or distal point to the light and yet lie
in all possible planes of orientation, and, further, that they move about
within a certain zone in all possible directions.

It is in fact self-evident, and may be taken as axiomatic, that there
must have been a certain degree of orientation, or steering, or the
organisms would never have been able to move either to or from the light.
But this, it is to be observed, is quite different from the organism being
turned round when the movement first begins, being definitely held there
by the influence of the light in a fixed plane, and then as a result moving
towards or away from the light. .

The experiments to be recorded later show clearly that there is no
such fixed or rigid orientation keeping the organisms in a constant plane,
but rather a continually directed control bringing the organism back
more or less towards the same direction as it darts about under other
varying influences and stimuli, and this on the whole gives steering to the
course, so that the animal as a net result moves towards or away from
the light.

Taking this movement then as a sign of chemical change in the cells
of the organism, or certain of those cells, the effects were observed—of
exposure of organisms to light of varying intensities, of change in
reaction as a result of keeping in light of about constant intensity, of
velocity of movement in light of varying intensity, of the effect of light
of different colours, and of velocity of movement in such lights, of the
effects of converging and diverging light, of the effects of light and shade
on organisms in the same vessel, on the association of upward or down-
ward movements in level with positive or negative phototaxis, and on |
movement in presence of more than one source of light.

A very considerable literature exists dealing with heliotropism and
phototaxis, but no attempt need be made to quote from this, further than
relates to the organisms used for the research, or in incidental relationship
to the variations in reaction to light described in the present experiments.!

The experiments were made with a free-swimming larval stage of the
Barnacle (Nauplii of Balanus), obtained in by far the largest quantity in

1. For a general survey and for literature, reference may be made to Verworn, General
Physiology, translation by Lee, 1899; Holt & Lee, American Journal of Physiology, Vol. TV,
p. 460, 1901 ; and Loeb, Dynamics of Living Matter, 1906.

VARIATIONS IN REACTION TO LIGHT 5

the tow-nettings, mixed with a much smaller number of copepods, and
larval spirochaetes
The manner in which the organisms congregate at the points of the
dish nearest to and farthest from the light was used to pipette them off
and separate them from other organisms indifferent to the light, and the
positive and negative groups of organisms so obtained were examined
separately. Many of the Nauplii were found in both the positive and
_ negative groups, but no difference in average size or degree of development
_ could be found in the two types to differentiate them, and later experiment
_ showed that the same separated group might be artificially varied back-
ward and forward between positive and negative according to their
previous treatment by light.
_ The phototaxis of the Nauplius of Balanus has been examined by
, “Loeb, and Loeb and Groom, and Loeb! states that they are positively
heliotropic upon leaving the egg, but soon become negatively heliotropic.
This I consider is entirely due to over-stimulation by the light, for on
keeping for some time in darkness the negative organisms become strongly
positive to the same intensity of light in which they were previously
negative, and in which part of them left during the same interval have
continued negative. The statement of Loeb and Groom that they remain
positive in artificial light (gas flame) is confirmed by the results of my
experiments, but holds up to a certain intensity of illumination only,
for if the light of a small lamp was converged by means of a cylindrical
museum jar in which the organisms were contained, the organisms in the
strongly illuminated area gradually became negative and passed into the
shaded parts or to the distal pole.
In later experiments made at Berkeley, U.S.A., Loeb found that
tae there behaved differently from those examined in his earlier
experiments made at Naples, and showed more complicated reactions.
Working with the larvae of Polygordius, and with those of Limulus,
Loeb noticed a phenomenon which was also conspicuous throughout the
present series of experiments, namely, that the positively phototactic
organisms gathered in a group towards the top of the vessel, while the
negative organisms at the same time as they gathered away from the light
congregated at the lower part of the vessel near the bottom.

This I have also invariably observed when a tow-netting is brought
into the Station and placed in the diffuse light of a window in a glass jar.
___ The positive organisms are in a compact group nearest to the window, and
almost ai the surface of the water; while the negative ones are at the most
. 1. Loe. cit,

:.
=
-
e

DONE Ce Ee a ee

6 BIO-CHEMICAL JOURNAL

distal point of the jar from the window and down near, or on, the bottom.
The same arrangement holds even in a shallow dish, well illuminated
throughout its depth, the positive organisms are up close to the surface,
und the negative ones on the bottom of the dish. The arrangement
continues when the organisms are lit in the dark room by a candle on the
same level as the water—still the positive ones are near the surface and
the negative ones near the bottom.

I consider that the most probable explanation of this is the constant
association, in the natural habitat of the organisms (the sea), of swimming
upwards towards the light when positively phototactic, and downwards
towards the darker regions of water when negatively phototactic.

In addition to the interest of this association on its own account, it
seems to me to be valuable as a sign that the light not only affects the |
sensitive area on which it acts, but also indirectly affects the whole
organism, the chemical changes set up at the sensitive area communicating
changes to the whole organism, which stimulate it and cause it to rise or
sink in the medium.

Loeb, working with Gammarus, found that traces of acid made the
organisms more strongly positive, and traces of alkali tended to produce a
negative heliotropic effect. I have not been able to obtain similar results
with hydrochloric acid, or caustic soda, in Nauplius, although both
reagents were pushed to the limits compatible with life, viz., 545 normal.
The organisms in the dishes to which either acid or alkali was added
seemed to behave exactly like the untreated control. I do not, however,
consider this any contradiction of Loeb’s results, since the organisms used
were different. Moreover, Loeb’s results are stch as would be expected
from the knowledge that alkalies, within the compatible range, excite the
activity of living matter, while acids depress it. For, if we regard the
light effect as producing an increased chemical activity, then the optimum
value of reaction, at which the change would occur from positive to
negative as the intensity of the illumination was increased, might be
expected to be reached sooner in the case of an organism already made
hyperactive by alkali than in the case of an organism where the activity
was depressed by added acid.

Throughout the whole series of my experiments I have consistently
found that the phototaxis is positive with very feeble illumination, and
becomes negative as the strength of the light is increased. Further,
continued illumination, either by diffuse daylight or by a very bright

1. The limit of acidity or alkalinity compatible with life seems to have nearly the above
value for all unprotected minute organisms of either vegetable or animal origin.

VARIATIONS IN REACTION TO LIGHT 7

artificial illumination, causes an increasing number of organisms to
become negative, and keeping in darkness or in a feeble illumination
causes this negativity to pass back to a positive phototaxis.

This again is compatible with the view that the effect of light upon
the sensitive substances of the organism is always the same whether the
effect is shown by a positive or negative phototaxis. The degree of the
stimulus determines the reaction of the organism towards it, as shown by
the direction of the orientation and consequent movement, but the
chemical nature of the stimulus is the same. Below a certain optimum
the organism reacts so that the sensitive surface is turned towards the
light, that is to say so as to increase the amount of light energy reaching
it, and so increase the reaction towards its optimum value for the organism
in its condition at the given moment. Above the optimum value of
stimulation, the organism conversely reacts so as to turn the sensitive
surface into a region of diminished light intensity, and so also to decrease
the velocity of the reaction towards its optimum for the organism.

This supports the view expressed by Holt and Lee,' that direction of
light is only effective in a secondary manner in so far as it alters intensity
of light falling upon different parts of the organism, and the orientation
is hence primarily a question of intensity of light.

The very ingenious experiment of Loeb, showing that an organism
which is positively phototactic to direct sunlight will pass from this
onward into diffuse sunlight, that is, into a region of lower intensity of
illumination, and will not reverse its direction when it finds itself in this
region of lower illumination, is quite susceptible of explanation on this
view, as well as the result of the experiments given below in this text,
upon the movement of negatively phototactic organisms away from the
source of illumination in converging light, and still onward past the focus
of the light in now diverging light with decreasing intensity.

Loeb’s experiment consisted in placing an organism (young cater-
pillars of Porthesia chrysorrhaea) in a test-tube the axis of which was
horizontal and at right angles to the plane of a window near by, through
the upper part of which direct sunlight fell on the more distal portion of
the test-tube, while the portion of tube near the window was lit only by
diffused daylight. Under such conditions these animals, which react
positively, did not halt at the junction of diffuse light and direct sunlight

and turn again backwards to the stronger light, but proceeded on in the
feebler light toward the incident point right up to the end of the tube,
From this experiment, Loeb argues strongly against the anthropo-

1. Loe. cil,

8 BIO-CHEMICAL JOURNAL

morphic point of view which would assign any choice to the animal as to
whether it sought, or turned from, the light because the light was pleasant
to it or the reverse, and urges that the whole process is mechanical or
automatic, the animal’s head being turned by the stimulus irresistably
towards the light, and the whole movement following inevitably upon
this turning.

Without assuming any extravagantly anthropomorphic point of view,
it may be maintained that the ingenious experiment scarcely supports the
interpretation placed upon it, and that the whole matter depends upon the
force of the stimulus outweighing the degree of development, of what
represents the intelligence of the animal, or, if the expression is more
suitable, the development of the nervous system, or, in more general terms
still, the co-ordination of the organism.

When the animal's body or the sensitive area of it passes from the
area of direct sunlight into the less illuminated area of diffuse daylight,
in order to turn back into the brighter area of sunlight, the sensitive
surface would require for a time to be turned away from even the diffuse
light into a region of shadow from its own body, that is to say, it would
require for the time to behave as a negatively phototactic animal, and
reduce the intensity of illumination of the sensitive area. This supposes
a degree of intelligence and of memory for the ‘ pleasanter ’ (or more near
the optimum) stimulus which the organism does not possess, and hence it
does not turn; but a more highly organized animal would turn, and once
more seek the stimulus which suited the organism best.

It is such excess of stimulus over organization which makes the moth
burn itself in the flame or the bird dash itself to pieces against the
lighthouse lantern, and in my opinion this differs in degree of complexity
only, but not in kind, from the strength of the irresistible impulse which
forces the victim of any drug habit to keep on drugging himself, or leads
the unfortunate human being with an incoordinated or improperly
balanced nervous system into committing crimes against himself or others.
The germs of resistance to stimuli, or rather of reacting so as to alter
strength of stimuli, must be present in all living creatures, or life and
continuance of the species would speedily become impossible; and it
appears to me that denial of this would be nearly as great an error as the
view which appears to be held by some opponents of the advance of
physiological science, that all organisms and animals are about enna HT
sentient to stimulation and to pain.

The experiments conducted with organisms under different aaal
glasses, described below, in which the relationship of the two halves of the

VARIATIONS IN REACTION TO LIGHT 9

dish to the direction of the incident light was identical, also show, from
the selection of one-half of the dish by the organisms in preference to the
other, that the organisms seek that region where the light activity
possesses an optimum for them although there is nothing in the incident
- direction of light to lead them to swim under one particular glass as a
result of orientation.

‘The same is seen in the experiments of Oltmann! and of Holt and Lee,
in which a range of varying intensity of light was arranged by means of a
prism placed along the long side of a long glass trough containing
organisms. The incident light came in varying intensity perpendicu-
larly through the prism, and the organisms were then found to place
themselves in certain intermediate positions where the intensity of light
suited their optimum, although they had to move to this position
practically at right angles to the direction of incidence of the light.
_. Experiments were made in the present series of observations upon the
velocity with which the organisms moved in light of varying intensity,
and also under glasses of varying colour, and it was found that within
the limits of the experiment, the velocity of movement was practically
constant, thus showing that the chemical reactions set up by the light did
not affect the locomotor organs.

Description or EXPERIMENTS

Experiment I.—A tow-netting was taken in Port Erin Bay, April
2ist, 12-1 p.m. After stirring up in sea-water, it was divided into
five portions of 300 ¢.c. each, which were placed in white soup plates and
treated as follows :—

No. 1.--Control, untreated.

N N
“ff No. 2,—Added 3 ¢.c, of io HCl, making 7000 solution.

ie N . N
_ No. 3-Added 6 c.c. of 0 HCl, making 500 Solution.

N N
No, 4.--Added 3 ¢.c. of 7g NaOH, making [99 solution.

y

N wie. iN
No, 5.-—Added 6 c.c, of i NaOH, making es solution,

_ The dishes were left in the diffuse daylight of a north window, and
examined after two hours (3 p.m.), the arrangement of the organisms is

1, Quoted by Holt & Lee, loc cit,

10 BLO-CHEMICAL JOURNAL

found to be the same in all five plates, showing no change due to acid or
alkali, and this persisted throughout the experiment.

In each of the dishes there are two prominent groups of organisms,
a larger group at the part nearest the window and close to the surface of
the water, a smaller, but well-marked group at the diametrical pole
farthest from the window and at the bottom of the plate.

On shading, for a few minutes, half of one dish with a cardboard,
the line of shade of edge of cardboard being at right angles to the plane of
the window, in the illuminated half of the plate there is a thick group at
the nearest point to the window; in the darkened semicircle, immediately
on lifting the card, a smaller group is seen at the point distal to the light,
and also there is a diffusely scattered but increased number over all this
previously dark half, much greater than in corresponding areas of the
illuminated half.

Examined again at night (8—9 p.m.) by lamp-light when nearly all
the organisms in the plates come to the point nearest the light. Shading
as before with shadow parallel to direction of incidence, gives a compact
group in the illuminated half near the light, but a great many are in the
darkened half which possesses a diffuse group at farthest point from light.

Examined again April 23rd, noon (about forty-eight hours from
commencement of experiment). ‘Took the control plate of organisms into a
south room having direct strong sunlight from an open window. The
organisms after a time collect very slightly to sun side, but in the quite
open unshaded plate are fairly indifferent, being distributed all over.
Now one-half of the dish was shaded by cardboard, the line of shade, as on
previous occasions, being arranged parallel to mcidence of the light; at
once all the organisms came out into the sunlit half, somewhat more at the
point nearest to the sun. On reducing the sunlit part to a very small
space, it became crowded with organisms accumulating more densely at
the point nearest tosun. This compact group of organisms was pipetted
off from the white plate into a black vulcanite half-plate photographic
developing dish, containing sea-water, when the organisms, at once almost,
accumulate at the part of the dish farthest from the sun. The half of the
black dish farthest from the sun, after stirring up, was covered over
(that is, with the line of shade.at right angles to plane of incidence), and
the organisms all collect, at remotest end of shaded part, away from sun.

This peculiar reversal in the black dish is difficult to explain, unless
it was due to the absence of reflection. The result could not be repeated
in other experiments because the organisms were never again found

1, This is a very exceptional behaviour in sunlight.

VARIATIONS IN REACTION TO LIGHT 11

indifferent in sunlight, but always strongly negative, even in the white
pistes. :

The organisms in this experiment were observed for four days longer ;
at the end of the third day they had become very strongly negative in the
diffuse daylight of the north window, a reversion, it will be observed, from
their original mixed condition with a large preponderance of positive

organisms. While in this strongly negative condition they were taken

into the dark room and tested with lamplight from a small oil lamp. At
once there was a change; three of the five, viz., Nos. 3, 4, and 5, were now
altered to positive, while Nos. 1 and 2 were mixed, partially positive and
partially negative.

The plates were left in the dark room over-night, the only trace of
illumination being a very faint ruby light, coming from a small borrowed

light through the double thickness of a ruby window and ruby photo-

graphic screen.
The following morning, as soon as a light was struck in the dark-

room, it was seen that all the organisms in all the plates were collected
at the points nearest to the faint ruby light.

The small oil lamp was lit and the plates arranged round it; all five
showed the organisms strongly positive. Taken out immediately from the
lamplight to the diffuse daylight of the north window again, the organisms
in all five are found to be strongly negative. No interval save the time
of shifting the plates out from dark room to window bench elapsed
between these two observations with reversed results.

Experiment I1.-Tow-netting taken by Professor Herdman, on April
25rd outside the Bay. On standing in diffuse daylight of north window,

_ two large groups separate in the glass jar; as usual, one towards light

and at top, the other away from light and at bottom of jar. These two
groups were separated off by pipetting into two soup plates, one containing
the positive group, the other the negative group, and both were found to
consist chiefly of Nauplii of Balanus.

The negative group was taken first for examination in the dark room.
On lighting one candle the organisms swim to the opposite pole; on
placing two candles at opposite diameters of the plate, the organisms lie
in the middle, half-way between the two lights; with four candles placed
around equi-distant, the organisms are clustered compactly at the centre
of the plate. The positive organisms similarly examined show a grouping
dround the periphery of the plate accentuated opposite each candle.

The organisms were left in the dark room overnight and examined
in it next morning. On first striking a light, both sets of organisms were

ee
OO ee

: = ; re :

Pais R :

12 BLO-CHEMICAL JOURNAL

seen clustered at nearest point of each plate to the exceedingly faint ruby
light. One candle was lit and placed close to the plate containing the
previously negative organisms, these are now nearly all positive to this
intensity of light. Next morning (11 a.m.) both sets of organisms, which
had remained in the dark room overnight, when tested by candle light
were strongly positive. They were at once taken out of the dark-room
and placed on the window bench in the north room in fairly strong diffuse
daylight (it had been snowing, and the hill across the Bay from the —
Station was covered with snow). All the organisms in the originally
negative plate were now negative again; those in the originally positive
plate were mixed about three-fourths negative and the remainder positive.

The two plates were once more carried back to the dark room and
tested to candle light. The originally negative plate, which a few
minutes before had been conrpletely positive in the dark room to candle
light, had now, on account of its short sojourn in the diftuse daylight,
turned to partially negative and partially positive in about equal groups.
The originally positive group was still all positive to the candle light,
although a few minutes previously in the diffuse light of the window
about three-fourths of the organisms had been positive.

Three points are shown clearly in this experiment.

First, that the reaction varies in the same organism at the same time
with the intensity of the light, and that feeble illumination gives a
positive reaction and strong illumination a negative one.

Secondly, with the same intensity of illumination the reaction varies
with the previous history and exposure to light of the organism.
Exposure to darkness or feeble illumination turns the organism so that it
reacts positively, and previous bright illumination changes it so that it
reacts negatively to a strength of stimulus to which it before acted
positively. 3 | }

.. ‘Thirdly, throughout these series of changes the original bias of the
particular set of organisms persists, the other effects being superposed in
a roughly algebraic summation. Thus the original trends towards positive
and negative in the two sets of organisms dawn out again at the end of
the experiment. :

Experiment L11.—On velocity of movement in light of varying intensity
and colour.

This experiment on the velocity of movement in light of different
intensity and of different colour, was made by observing the time required
for the organisms to swim from one end to the other of a flat, black
vulcanite dish of rectangular shape. The length of the dish was 17 cm.,

VARIATIONS IN REACTION TO LIGHT 13

and the organisms were first brought to a compact mass at one side by
placing the light to be used at one end and then the time noted for them
to swim across and form a similar compact mass at the other end when the
source of light was shifted to that end.

Then the time was again noted which they require to swim back to
their original position; these times are denoted by ‘ Out” and ‘ Back’
the following table. In taking the time ‘ out’ one does not wait for sell
organism, but waits till the great majority are in a compact group, this,

after a little practice can be done accurately within a quarter of a minute

- For different intensities of light, one ordinary paraffin wax candle

was used in one case, and four similar candles in the other case. For

white light, the dish was simply uncovered, and for coloured lights it was

covered completely over with slips of coloured glass, through which the

coloured light passed to reach tle organisms. The coloured glasses

e which it was possible to obtain were red, green and blue. Regarding the

fi total intensity of light passing through the three slips, it appeared to the

eye as if the red strip was most obscure, and the green most transparent,

the blue being intermediate, but no exact photometric instrument was

available. The organisms used were a strongly positive group obtained by
pipetting off in diffuse daylight.

1. Illumination intensity = one candle.
Redlight ... Time ‘Out’ ... 3 min. 0 sees.
Time * Back’ ... 3 min. 0 sees.
Blue light ... Time ‘Out’ ... 3 min. 30 sees.
Time ‘ Back’ ... 3 min. 20 sees.
Green light... Time ‘Out’ ... 3 min. 40 sees.
Time ‘ Back’ ... 3 min. 0 sees.
2. Illumination intensity = 4 candles.
White light... Time ‘Out’ ... 3 min. 0 sees.
Time * Back’ ... 2 min. 50 sees.
Red light ... Time ‘Out’ ... 3 min. 0 secs,
a Time * Back’ ... 3 min. 0 sees,

Experiment IV Selection of position under different coloured glasses
with the same direction of incidence.

In this experiment, diffuse daylight was used on some occasions and
candlelight on others, the long side of the dish being placed parallel to the
surface of the window, or next to the candles. Then the two glasses of the
two different colours to be compared were placed edge to edge, each covering

4 BIO-CHEMICAL JOURNAL

one-half of the dish, the edge where the two slips of glass touched being at
right angles to plane of window, so that each half was situated exactly the
same as to direction of incidence and intensity of light from the window ;
and a similar arrangement was used with the candlelight, the candles
being so placed opposite the middle of one of the long sides of the dish that
they shed equal light on the two different coloured halves. Before placing

the two slips over the dish, the contents were stirred so as to uniformly
distribute the organisms, but care was taken that the contents were not

rotating when the slips were put over. Also, after the organisms had
distributed themselves selectively, and the result had been noted, the two
slips were reversed in position, each to each, and the change in distribution
observed; the organisms at the time were strongly positive in the candle-
light, and strongly negative in the diffuse daylight.

First, using four candles in the dark room, and with the red glass on
the left-hand half and the blue glass on the right-hand half, in 2 min.
30 secs. from the commencement all the organisms are under the blue
glass and next the candles, none under the red glass. The red and blue
glasses are now reversed without disturbing candles or organisms, and in a
very short time all the organisms have shifted and are once. more under
the blue glass in its new situation.

Second, similar results obtained with diffuse daylight, except that
organisms now swim from the light; with blue and red most of the
organisms under blue, a few only under red; with blue and green, two
groups form at the two corners distal to the light, the larger of the two
groups being under the green. Thus the organisms move with equal
velocity under all coloured glasses, but when two colours are offered for
selection they accumulate chiefly under one. Further, the direction of
movement to pass from one colour to another is across the direction of
incidence, and not to or from the light, and the relation to the light of the
two halves being the same, it would appear that a preference for a
particular colour or wavelength (or the greater or lesser stimulus of
different wavelengths), caused the different distribution. If the organisms
are carefully watched when they are becoming distributed, it is seen that
they do not move directly across from one half to the other, but are moving
about apparently freely, an organism every now and then leaving a group
and darting off; but there is a certain amount of steering and controlling
during these apparently free movements, which ultimately settles them
down in their final distribution. .

In this type of experiment, observation of the grouped animals shows,
as in all the other experiments where the animals are grouped either

VARIATIONS IN REACTION TO LIGHT 15

- positively or negatively under the influence of the light, that there is no

such thing as fixed and continuous orientation of the minute animals. In

every group a great many are moving about in and out amongst one

another, and a good many are entering and leaving the group like bees
from a hive, but each individual, after a short trip about soon returns to
the group. The source of light is, in fact, a strong directive influence, but
there is no rigidly fixed orientation, any more than there is in a cluster of
midges, or a brood of chickens around their mother.

Experiment V.—Movement in converging and in diverging light.

In order to obtain converging and diverging light, cylindrical
museum jars, about 10-5 centimetres in diameter and 18 centimetres high,

were used, which happened to be in stock at the Station.

Two such jars were used; the first, filled with clear fresh water, was
used only as a cylindrical water lens, and contained none of the
organisms; the second jar contained the organisms in sea-water. The
first cylinder was placed a variable short distance, up to about one foot,
from a small oil lamp with a circular wick, and the second cylinder was
placed close up against it, on the other side from the lamp. The lamp and
two cylinders were so arranged that the diverging light from the lamp
became slightly convergent in passing through the first cylinder, and being
still further converged by the second cylinder, it formed a caustic about
two-thirds to three-fourths of the way through the second cylinder, and
from that onward to the concave surface of the second cylinder the light
was diverging.

By this arrangement any organism moving along the path of the rays,

either towards or away from the light, is forced in one part of its path to

travel in converging light, and the remaining part it travels in diverging
light. Experiments were carried out both with white light and with
coloured lights. The first set of organisms examined were negative; these
swam away from the light into light of increasing intensity towards the
caustic, and then through this onward in light of decreasing intensity till
they reached the glass surface most remote from the light. Positive
organisms were next tried, and swam in the exactly reverse direction, first
from the most distal part towards the caustic in converging light, and
therefore of increasing intensity, and then onward in diverging light,
therefore of decreasing intensity, up to the glass surface nearest to the
light.

At first sight it looks proven from this that intensity of light is of no
effect, and the direction of incidence the whole matter, because the

16 BIO-CHEMICAL JOURNAL

organisms appear to swim in one direction indifferently, whether the
illumination is increasing or decreasing. In reality, however, such a
conclusion would be fallacious, for in order that, say, a positive organism
should turn when it began to swim in light of gradually decreasing
intensity, it would be necessary for it to turn its sentient surface away

from the light, and that would plunge it into darkness. 1
The true conclusion is shown by what might be termed secondary —
effects seen on carefully watching the above experiment with negative he

organisms. These organisms at first accumulate in the narrow band of
light at the distal glass surface from the light, where they dart about in

sh

small curves, keeping close to the glass; but in a few minutes it is found &

that a great many of them have accumulated in the two shady margins rs
just outside this strongly illuminated band, and on either side of it. The ;
probable explanation of this is that for these negative organisms the 2
feebler light outside the band is nearer the optimal stimulus, and when | -
they escape from the direct light beam in the course of their “
peregrinations, they find a suitable stimulus in the feebler light. But b
when any accident, such as a chance movement stimulated by some other = “4
cause, sends them again into the beam, they are stimulated to turn away ; i
from the light, and must again return via the distal glass surface to the | di
refuge of the shade again. My
This effect is seen still more strikingly when the red glass strip is a
interposed on the path of the incident light; then scarcely a single bes
organism is seen on the illuminated strip, but two packed masses are seen . ie
on each side of it in the shade, and gradually tailing off as the distance

«
<=

from the illuminated strip increases. Similar results are seen with
negative organisms if a narrow opaque white strip, such.as a strip of
cardboard, be lowered into the jar and held in a vertical position at the
caustic. When the light is now placed in position, any organisms in the
course of the beam, or swimming into it from the two dark zones at either
side of it, turn at once away from the light, and swim along the path of
the rays towards the caustic and the card; but they do not accumulate to i
any appreciable extent at the card, they swim round its edges and alll
accumulate in the narrow feebly-lit space behind it. 4

»
oo ge

Experiment VI.-With young larvae of the plaice (Pleuronectes ae
platessa). or :

A number of young plaice larvae, which were five to seven days” ‘lf 3
were taken from the Fish Hatchery attached to the Station, and placed i in ?
sea-water in a flat, oblong pie-dish. It was found that they were faintly

VARIATIONS IN REACTION TO LIGHT 17

“negatively phototactic in diffuse daylight. Contrary to the case of the
Nauplii, this appeared to be increased in lamplight as well as in direct
sunlight. When the dish is brought into lamplight in the dark room, it
is found that most of the larvae after some time are accumulated in the
half of the dish farthest from the lamp, decreasing to a clear space directly
e. under the lamp. There is, however, no such tight packing up as in the
ease of the Nauplii.
‘ - ‘The interesting point, however, is that there is no evidence whatever
_ of orientation in regard to the light; the larvae lie at rest with their
nz long axes at all possible angles with the line from the lamplight, some
= directly facing it, some straight away from it, others nearly at right
angles, and many indiscriminately at all angles. The arrangement is not
a chance one, as it looks at first sight, for no matter how often the larvae
are disturbed and stirred up, they finally settle with the great majority in
oe the distal half, and lying there at rest at all angles to the direction of
incidence. On shading one-half of the dish with cardboard, the line of
_ shade being parallel to the plane of incidence, the great majority of the
larvae are found in the shaded half, more in the distal quadrant, and in
all lines of orientation. If cards are arranged so that one quadrant of the
dish only is illuminated, that quadrant becomes almost free.

ee ee

_ Eaperiment VII.—Indifference of phosphorescent organisms to
movement in light from without.
ry It was thought that organisms which themselves emitted light might
" show interesting results in their reactions to light from without, and this
2 led to the work of Section B about to be described; but it was found that
_____ the phosphorescent organisms present, probably certain copepoda, were
___ entirely indifferent to incident light, at any rate as far as movement was
concerned.
Since the organisms could not be made to phosphoresce in the dark
room during the day, the procedure was adopted of taking a tow-netting
4 during the day, when the Bay was known, by observations made during the
od _ previous night, to contain abundance of phosphorescent organisms. This
___ tow-netting was placed in diffuse daylight, and nearly all the positive
organisms were pipetted off into one dish containing sea-water, nearly all
_ the negative organisms were similarly pipetted into a separate dish, and
finally, a good number of indifferent organisms were pipetted off into a
third dish, from the middle of the bottom of the stock jar.
The three sets of organisms were then examined for phosphorescence
_ after dark, when phosphorescence where organisms were present had

18 BIO-CHEMICAL JOURNAL

spontaneously set in and could be further intensified by stirring. It was
then found that the positive and negative portions each contained only one
or two phosphorescent organisms taken up unavoidably with the others;
but the indifferent set contained a large number of phosphorescent
organisms. The indifferent set containing the majority of the
phosphorescent organisms were also practically indifferent to candle-light.
In regard to numbers of organisms in each set, the positive set were by far
the most numerous, and the numbers in the indifferent and negative sets
were about equal.

The experiment was varied in a fresh tow-netting by placing several
flat pie-dishes containing the organisms (not separated off on this oceasion
as to phototaxis) around the lamp in the photographic room, just after
nightfall, until the usual phototactic groups had separated, then
extinguishing the lamp, and watching the spontaneous appearance of
phosphorescence without disturbing the dishes. There is no spontaneous
phosphorescence for a period of about two minutes under such
circumstances, then it commences, and it is seen that the phosphorescent
organisms are scattered about indiscriminately in each dish, and not
arranged in any relationship to where the light had previously been.
Sometimes the phosphorescing organisms are moving about rapidly while
illuminated, but in the majority of cases they are almost or quite at rest,
and it is probable that if there had been any previous movement of a
phototactic character while the lamp was lit, the arrangement would not
have quite disappeared in the short interval after the light was
extinguished before the spontaneous phosphorescence reappeared.

The only conclusion from the experiments appears to me to be that
these particular phosphorescent organisms are almost or quite indifierestt
to incident light.

B.—Drvrnat Pertopicrry tv PHosPHORESCENCE

The suggestion of the work described in this section arose incidentally, -
as above-mentioned, and at the time the experiments were made it was
unknown to me that a diurnal periodicity in phosphorescence had
previously been observed and described. :

A search through the earlier literature, however, revealed a
description of its occurrence in Pyrophora by Aubert and R. Dubois,!
and in Noctiluca by Massart.2 Henneguy? states that Noctiluca does not

21. Compt. rend, acad., T. XLIX, p. 477, 1884; Compt. rend. soc. d. biol., P 661, 1884.
See also papers i in both these Journals by R, Dubois, 1884-6.

Bulletin scientifique de la france et de la belgique, T. XXV, p. 72, 1898.

Compt. rend. soc. d. biol., XL, p. 707, 1884.

re

‘DIURNAL PERIODICITY OF PHOSPHORESCENCE 19

li , h up until it has been kept in the dark for half an hour, and that the
ntensity is not at the maximum for another additional half-hour.

ae The following passage from Massart describes the variations as
in Noctiluca :—
_ ~~ +‘ The experiments show that the irritability is dependent on the
alternations of day and night, the Noctiluca is hardly excitable on shaking
ring the day and shines only during the night. Fact still more curious,
the organisms are submitted to the alternations of day and night,
a » whether they are maintained in constant illumination or constant
_ obscurity, they still remain much more excitable during the night than
during the day. It is a veritable phenomenon of memory, everything
looks as if the Noctilucae preserved the recollection of the regular
succession of the days and nights.’
______ Massart compares this to the change in position of the leaves of plants
. during day and night in the Oxalis and certain Papilionaceae, but adds
_ that while the phenomenon lasts only some days in plants, in the
‘ | Bettitncee it lasts until the death of the animal.

His experiments at the outside limit, however, lasted for one week
“aay, when the organisms died; in the present set of observations the
- diurnal alternation of activity was followed with organisms kept in
continuous darkness for twelve days, and although the number of living
Organisms was decreasing all the period, a few were still left alive and
__ phosphorescent at night at the end of the period.

Since the fact of this diurnal periodicity is one of the most striking of
those alternating habits or functions of the lower invertebrates which bear
_ such a curious resemblance to memory in higher vertebrates, and, indeed,
have been regarded as a rudimentary memory,! it may be regarded as
sufficiently interesting to merit a detailed description. It appears to stand
some danger of being forgotten, since it is not mentioned even in the
. of the modern text-books, and to the best of my knowledge it has
ae " ot been shown to exist in the phosphorescent copepoda, nor demonstrated
as persisting for such a long period as in the present experiments. Also
its onset at the close of the day and gradual extinction at dawn have not
previously been followed with any exactitude.

1. . See F, Darwin, Presidential Address, Brit. Association, Dublin, 1908.

20 BIO-CHEMICAL JOURNAL

Diary or ExperIMEN’S

Monday, September 21st, 1908 (8-30 p.m.)—Calm night, and sea very
phosphorescent. Collected plant (Polysiphonia nigrescens) from the rope
of an old mooring buoy. The plant is covered over with phosphorescent
organisms which flash most brilliantly. The specimen is preserved in
sea-water and examined ashore. It shows most brilliant phosphorescence
when rubbed. When a piece is put in fresh tap water in the dark it
lights up most brilliantly all over for about three minutes, then gradually
the light fades out, and cannot now be evoked by any process of shaking or
rubbing. |

Tuesday, September 22nd.—The plant was taken into the dark room
at 11 a.m. and examined; no phosphorescence could now be evoked by any
process, either shaking in air, stirring up in the sea-water, rubbing, or
applying fresh water.

A tow-netting had just been taken in the Bay (12 noon). This was
taken into the dark room at once, but no trace of phosphorescence could
be obtained from it, even with most vigorous stirring.

In the evening, from 9 to 9-30 p.m., a tow-netting was taken in the
Bay, the sea being very phosphorescent wherever touched by the oars. The
haul, when taken into the boat, scintillated most brilliantly while being
washed into sea-water in a jar. The contents of the jar, taken into the
dark room at the Station, are showing spontaneous phosphorescence, and
give a vivid show when stirred. Left in the dark room oyer-night.

Wednesday, September 23rd—Vixamined the previous night’s tow-
netting at 11 a.m.; there is not a trace of phosphorescence to be elicited,
even on stirring briskly. Examined at intervals all day in the dark
room. There is not a trace of phosphorescence seen till about 6-30 p-m.,
when sparking first starts on stirring, just as it is growing dusk outside,
and at 7 p.m. there is spontaneous phosphorescence.

Took also during the day three tow-nettings from a row-boat, each of
15 minutes’ duration, at 12-45 to 1 p.m., 3-45 to 4 p.m., and 5-15 to
5-30 p.m. As each tow-netting was finished, it was taken to the Station,
at once emptied into a flat pie-dish, and taken to the dark room to be
examined for phosphorescence. On each such occasion the tow-nettin

previously there were also examined, as also at other intervals during the _

day. In none of the three was any phosphorescence seen till about
5-40 p.m., when a single organism was seen to spark in the second tow-
netting (taken 3-45 to 4 p.m.), but nothing in the first or third.

Examined at 6-45, when it is dusk outside, all three are

DIURNAL PERIODICITY OF PHOSPHORESCENCE 21

a phosphorescing spontaneously, bright sparks showing up, sometimes three
or four at once in each dish. On stirring there is a bright display lighting
up each dish. “All three left over-night in dark room.
~ Thursday, September 24th—Examined at 10 a.m., none of the three
tow-nettings show any phosphorescence in the dark room. Nos. 1 and 3
were kept in the dark room all day, while No. 2 was kept in the daylight,
but taken at intervals to the dark room for examination. No
___ phosphorescence seen in any of the three at any time during the day; but
sat night (7 p.m.) all three are sparking spontaneously, showing bright
___ sparks at intervals. The phosphorescence is increased on stirring, so that
six to ten phosphorescent spots are visible at once, but the display is not
so brilliant as on the previous night, probably owing to deaths.
All three left in dark room till Friday morning; the faint ruby light
_ from the dark room window is completely shut off by banking it up with
eardboard on the outside.
This same day, being a bright day with good sunlight, three
additional tow-nettings were taken, at 11 to 11-15 a.m., 12-45 to 1 p.m.,
and 4-45 to 5 p.m., and examined in future along with the other three,

4 being kept in dark room also. Examined as follows in dark room :—
. No. 1 observed at 11-30 a.m. efi ... No phosphorescence.
a _ Nos. 1 and 2 observed at 1-15 p.m. ... ... No phosphorescence.
" Nos. 1, 2 and 8 observed at 5-10 p.m. ... No.1, Nil; No. 2, single
- 4 | spark on vigorous
a stirring; No. 8, Nil.
Nos. 1, 2 and 3 observed at 5-35 p.m. ... Nil; single-spark ; Nil.
(Good light outside).
Nos. 1, 2 and 3 observed at 6-35 p.m. ... Spontaneous sparking
(Almost dark outside). in all three, No. 8
most brilliant. Dish
lit up in each case
) on stirring.
_ Nos. 1 2 and 3 observed at 6-50 p.m. ... All spontaneously phos-
(Quite dark outside). phorescing most
brilliantly.

Also at 1 p.m. to-day, a further supply of Polysiphonia nigrescens
was collected from the old mooring rope, and examined in the dark room.
It showed no phosphorescence during the day. On placing in distilled
water it gives a feeble sparkling, but incomparably less brilliant than on

22 BIO-CHEMICAL JOURNAL

similar treatment at night. After dark, from 6-30 p.m. onwards, the
same sample sparkles when stirred, and a piece put in distilled water lights
up brilliantly all over for from three to five minutes; then the light dies
away, and cannot further be evoked in that piece by any of the Se
mentioned,

All six of the tow-nettings of yesterday and to-day examined again at
7-15 p.m.; all spontaneously phosphorescing, and showing up me
on stirring. Same result when again examined at 8-40 p.m.

Friday, September 25th.—Arrived at Biological Station at 4-50 a.m;
there is just a trace of dawn in the dull, grey sky. Organisms examined
at once in the dark room, where they have all still been kept over-night;
all six dishes are flashing spontaneously.

On standing quietly by and watching the phosphorescence, the minute
organisms are not moving about rapidly in most cases, and one can observe
that each active organism is emitting a series of flashes at about the rate
of one per minute, and between the flashes there is a dimmer light showing
which regularly becomes increased by a flash. The effect on the eye is
very similar to that of a revolving light seen at sea at some distance off.
There is an almost constant dim light lit up by repeated and fairly regular
flashes.

Many of the more active organisms are so still that one is able to
observe clusters of four or five in nearly constant positions for some
minutes, so as to give an impression of constancy of shape to the group for
the time resembling a stellar constellation.

The effect in the complete darkness of the dark room is very beautiful
as the undisturbed organisms spontaneously flash out in the darkness.

The organisms were now observed at frequent intervals of about ten
minutes, in order to accurately note the decline and disappearance of the
phosphorescence. It was observed that the number of organisms flashing
out was decreasing all the time. The rate of decrease became very rapid
about 5-30 a.m., when the daylight was just beginning to grow rapidly |
brighter outside. At 6 a.m. there was only an occasional odd flash in each
dish, showing that only a few organisms in each were still active. At
6-15 a.m. only one dish (the third of those collected on Thursday)
was still showing an occasional gleam; all the other five dishes had
stopped spontaneous phosphorescence. At 6-30 a.m. all spontaneous
phosphorescence had disappeared, but a faint display could still be
elicited in all six dishes by vigorous stirring. At 7 a.m. no sparking
obtainable in any dish, even on most vigorous stirring; same result
repeated at 7-30 a.m.

DIURNAL PERIODICITY OF PHOSPHORESCENCE 23

‘The organisms on the Polysiphonia nigrescens behave similarly to the
: free organisms in the dishes throughout. It was feared that the organisms
___ would perish if the sea-water were not changed, so Nos. 1 and 2 of the

Wednesday tow-nettings were filtered in the dark room through the silk
of the tow-netting, and then the net being turned (so that no fresh
organisms could be introduced), the organisms were washed into a fresh
; quantity of sea-water poured on to the net. Hence there were in future
_ five dishes to observe instead of six, but no alteration in rate of survival
- on ‘account of the changing was observed, and, as the other dishes of
_ organisms appeared to be doing well, the process of washing into a fresh
supply of sea-water, which was exceedingly difficult and awkward in the
quite dark room, was abandoned.

The organisms were next examined at 1 p.m., when vigorous stirring
failed to call forth a single spark in any of the tow-nettings or on the weed.
Bi _ The next examination was at 9 p.m., when every one of the dishes
ag showed spontaneous phosphorescence. ‘The display in the three Thursday
2g nettings is not so vivid as on the previous night, there being fewer
sso ganisms phosphorescing. It is also noticeable that the phosphorescence

ig not so vigorous in each individual organism. The flare out is perhaps
as great, but the light completely dies out in all cases after each flare, and
the period between the flares seems to be lengthened, so that one cannot
pick out a particular organism by its flashes and keep track of it. The
two dishes from the Wednesday tow-nettings, which are to-night showing
for the third time, are not much decreased in vigour from the second
night, either in frequency of spontaneous flashing or in vividness on
stirring them. Nearly as many phosphorescing organisms appear to be
present, and the flashes are about as bright as on the preceding night.!
_-—C hese Wednesday organisms have now lit up for the third time,
_ having been quite quiescent in the intermediate periods of daylight in the
_ outer world. One of the two dishes has been in complete darkness
_ throughout the period. From this onward all the sets of organisms are
ome _ kept in complete darkness the whole time.
__—‘Saturday, September 26th.—The organisms were examined at 11 a.m.,
_ and again at 1 p.m., when no sparking was occurring, nor could any be
____ evoked by vigorous stirring. The next observation was commenced at
6-07 p.m., when the daylight was commencing to fade outside. The dishes
were not stirred, but quietly watched in the complete darkness. When the
first spontaneous flash occurred, the dark room was quitted and the time

1. This was observed in nearly all the ts, t drop during the first twent
four hours, and then a very slow re Paes in the residue. a great drop ig the first twenty-

24 BIO-CHEMICAL JOURNAL

noted; it was 6-13 p.m. Between 6-15 and 6-30, six flashes were counted ;
between 6-30 and 6-45, twenty-two flashes; between 6-55 and 7-15 p.m.,
there were twenty flashes. The display is much less marked than on the
previous evenings. On stirring the dishes, three or four organisms can
be made to phosphoresce at once in each case.

The organisms on the Polysiphonia nigrescens are also phospherssai
on stirring.

Sunday, September 27th—Kxamined at 10-30 a.m.; no _phos-
phorescence, either spontaneous or on stirring, from any of the dishes.
Re-examined at 7 p.m., four of the dishes show spontaneous
phosphorescence, the rate of sparking being extremely slow. The
remaining dish (the third of the Thursday tow-nettings) has undergone
putrefaction, and shows no phosphorescence, even on stirring. It is
taken from the dark room, and all the organisms in it are seen to be dead.

Stirring elicits two to four phosphorescent organisms at the same
time in the remaining four dishes.

Monday, September 28th.—Examined the four dishes at 2-30 pam. ;
no phosphorescence obtainable from any of them. Examined again at
7-30 p.m. ‘Two spontaneous sparks seen in the Wednesday dishes in an
interval of about five minutes; no spontaneous phosphorescence seen in -
the Thursday dishes. On stirring, about six phosphorescent organisms
seen in one of the Wednesday dishes, and three or four in the other; one
seen in the first of the Thursday dishes, and three or four in the second.

Tuesday, September 29th.—Examined at 3-30 p.m.; no phos-
phorescence visible or obtainable. Examined again at 9 p.m., there is
spontaneous phosphorescence in both of the Wednesday dishes, and in one
there is an organism which remains steadily phosphorescent with a dull
glow all the time. On stirring, about six phosphorescent organisms are
visible in each of the Wednesday dishes, and the sparking is brilliant.
In the Thursday dishes, on stirring, there is less display, only two or three
organisms showing up at-once in either. The few organisms are,
however, quite active, and a single organism in each case lights up so as
to illuminate the contents and sides of the whole dish.

Wednesday, September 30th—Examined at 11 a.m.; no _phos-
phorescence, spontaneous or otherwise. Examined again at 7-30 p.m., no
spontaneous phosphorescence during a period of about 5 minutes, but on
stirring there is a good display in all four dishes. This is the eighth
night of appearance of phosphorescence in the Wednesday lots, and
seventh night for the Thursday organisms.

Thursday, October 1st—Examined the four dishes at 11 a.m.; no

DIURNAL PERIODICITY OF PHOSPHORESCENCE 26

phorescence, spontaneous or on stirring. Examined again at 7 p.m.,
a fare is spontaneous phosphorescence at a slow rate in three (two

* Wednesday and one Thursday), and in all four on stirring.

{ Friday, October 2nd.—Examined at 3 p.m.; no phosphorescence,
either spontaneous or on stirring. Examined again at 9 p.m.; in one of
the Wednesday dishes there is an organism which remains permanently
lit up the whole time of observation, about seven minutes. Spontaneous
phosphorescence seen in the other Wednesday dish, and in one of the
gems dishes. All four give phosphorescence on stirring.

This is the second time a continuously phosphorescent organism has

: ee observed. It may be a pathological condition of the organism..

: Saturday, October 3rd.—Examined at 4 p.m., no phosphorescence of
2 my kind; did not examine after nightfall this day.
i ae - Sunday, October 4th——Examined at 12 noon, no phosphorescence in
ny dish, either spontaneously or after vigorous stirring. Examined
again at 6-20 p.m., and watched at intervals till 8-30 p.m., but there is no
g ‘spontaneous flashing. On stirring, however, there is phosphorescence
obtainable in each of the four dishes, one or two organisms only flashing in
each case.
‘The experiments were brought to an end at this date. When the
dishes are taken to the light it is found that only a small number of
a organisms are visible and alive in each, and there is much débris of dead
| organisms.
_ The diurnal periodicity of the phosphorescence had been observed for
_ twelve days and nights in the case of the organisms collected on
Wednesday, September 23rd, and for eleven periods in the case of those
collected on Thursday, September 24th, without any exception. During
___ this interval, with the exception of one of the Wednesday dishes which had
been exposed to light on the first Thursday of the period, all the dishes
were kept in continuous darkness, yet at the close of the day the
ga always lit up, and lights were extinguished about daylight in
. > morning.
____ ‘The four dishes of organisms were now filtered one after the other into
RS the small end of the same tow-net, washed out into a little sea-water, and
_ fixed with five per cent. formol.
a The fixation was carried out in the dark room in order to observe if
there was any phosphorescence. About six bright points shone out, two
of which persisted brilliantly for about three minutes, and then faded out.
a The weed (Polysiphonia nigrescens) was kept in the dark from the
Thursday (September 24th) till Wednesday (September 30th), showing

26 BIO-CHEMICAL JOURNAL

phosphorescence at night and none during the day. Fearing that it would
decompose, it was then placed in ordinary diffuse daylight in a vessel
with running sea-water. This treatment increased the amount of
phosphorescence enormously, and in a day or two it Was quite as
phosphorescent as at first. Taken from the diffuse daylight to the dark
room for examination, it was never phosphorescent, but at night it always
phosphoresced most brilliantly. It, also, at the end, was fixed in 5 per
cent. formol, and in this process lit up about twenty seconds after the
application of the formol, and shone vividly for about three minutes before
dying out.

The examination of the united tow-nettings was difficult on account of
the majority of the organisms being dead and in a broken-up condition
through the long duration of the experiment, but the following account
was kindly given me by Mr. A. Scott, to whom my best thanks are
due : —

Diatoms.—-Biddulphia mobiliensis, 1,000; Chaetoceros densum, 50;

Coseinodiseus radiatus, 50; Trochisea sp., 250,

Corrrops.—Calanus helgolandicus, 20; Pseudocalanus elongatus, 680;
Temora longicornis, 100; Centropages hamatus, 10;
‘Paracalanus parvus, 100; Isias clavipes, 100; Copepod
nauplii, 100; Copepod Juv., 200. .

Moxtvsca (larval).__Gasteropods, 150; Lamellibranchs, 500.

No Noctilucae were present.

It is not probable that ‘the diatoms or molluscan larvae were
phosphorescent, so that there is little doubt that the phosphorescence was
due to the copepods present, or certain species of these.

The following is a statement of the contents of the routine tow-
nettings always taken of the plankton of the Bay, for the statistical work -
of the Biological Station, on the date (Thursday, September 24th) when
the second set of tow-nettings were collected for the observations :—

Diatoms.—Biddulphia mobiliensis, 800;° Chaetoceros decipiens, 600;
Ch. densum, 440; Coscinodiscus radiatus, 50; ite
thamensis, 150; Trochisea sp., 50.

DInoFLaGELLatTa, &e.—Ceratium furca, 50; C. fusus, 100; C. tripos,
100; Tintinnopsis sp., 600.

DIURNAL PERIODICITY OF PHOSPHORESCENCE 27

_ Corgropa.—Calanus helgolandicus, 50; Pseudocalanus elongatus, 3,180;
Temora longicornis, 280; Centropages hamatus, 65;
_ Aeartia clausi, 2,200; Oithona similis, 1,750; Paracalanus
parvus, 830; Isias clavipes, 160; Copepod nauplii, 3,960;
d Copepod juv., 2,180.
- Morzusca, &c.—Lamellibranch larvae, 280; Oikopleura, 3,800.

Whether this diurnal periodicity has the same physical basis in a
rudimentary fashion as memory in higher animals, is still an open
question, for it is open to believe that the alternating play of light and
darkness upon those cells which produce the phosphorescence may have

induced in them a periodicity of activity and rest which still persists after
the alternating stimulus is withdrawn. The process may, for example, be
__ due to a secretion by certain cells which phosphoresces as each drop is

produced, and this process of secretion may have a period of rest during
_ the day and activity during the night. The rhythm of this activity may
be timed daily under ordinary conditions, and regulated by alternation of
___ light and darkness. During the day there would be storage in the cell,
and at night discharge. On the removal of the stimulus of light during
the day this state of alternation of rest and action might persist for a long

‘li

CoNncLUSIONS

1. The characters of the response of an organism by movement to
light are not constant for a given organism, but vary for the same
organism at the same time according to the intensity of the light and the
____ previous history of the organism in regard to light. As a general rule,
____ the organism is positive to feeble light and negative to stronger light, and
_ for a constant intensity of light at a given moment previous darkness or
_ weak stimulation tends to turn organisms positive, and previous exposure
to bright light turns them negative.
2. Both the positive and negative behaviour to light may be
explained on the basis of one chemical action of light upon the cell
_ (akatabolic one). The positive state indicates that the speed of reactions
in the cell lies below a certain value, which may be called the optimal
value, and the negative state corresponds to a speed of reactions in the
cell above the optimal value. In the former case the sentient surfaces
are turned into the light to increase velocity of reaction up towards the
optimal value; in the latter case the sentient surfaces are turned away

28 BLO-CHEMICAL JOURNAL

from the light so as to decrease the velocity of the reactions down towards
the optimal value.

3. Asa result of the orientation so caused, there arises movement of
the organism towards or away from the source of light, but such
orientation is not a fixed orientation, but rather a steering action; the
animals as a result do not remain in one fixed plane or direction of
movement, but the net result of the movement is that the organisms move
to or from the light. When the movement is finished the organisms
(plaice) may lie in all possible planes of orientation to the light.

4. Movement towards or away from the light has in some organisms
(Nauplii of Balanus) an associated movement upwards or downwards.
These two movements would coincide together in natural movements of
the organisms under the influence of light alone in the sea.

5. In the case of Nauplii of Balanus, addition of small amounts of
acid or alkali was not found to alter the reactions to light.

6. The rate of movement of the organism (Nauplii) is almost the
same with different intensities of light and different coloured lights,
showing that the locomotor apparatus is not affected by the light, but
continues to work at the same rate.

7. The particular organism used (Nauplius of Balanus) moves from
red light to blue light, and from blue to green, under such circumstances
that the incident light is the same in direction for both coloured regions.
This would indicate that with the particular total intensities being used
for the experiments, green is a more suitable or optimal stimulus than
blue, and blue in turn more optimal than red.

8. Movement in converging and diverging light is described and
shown to be explicable on the basis of intensity of light alone, and that
direction produces its effects in a secondary manner on account of the
light and shade effects of the animal’s own body.

9. The phosphorescent organisms experimented with (certain
copepods) were shown to be indifferent, in regard to movement, to light
from without.

~ 10. That light from without has another type of influence upon these
phosphorescent organisms is shown, however, by the fact that their
periods of activity and rest in regard to phosphorescence follow
respectively the hours of daylight and darkness.

11. It is shown that this alternating diurnal periodicity can persist
for a long period (twelve days) in absence of the accustomed recurring
stimulus of the light and darkness of day and night.

12. The phosphorescence of these copepods in captivity is

“4 DIURNAL PERIODICITY OF PHOSPHORESCENCE 29

neous, and although increased by mechanical stimulation, it goes on
sie even. ee the organisms are undisturbed and quite still.
; the organisms are freshly taken, the character of the
»sphorescence is such that a faint light persists, which is increased at
als by bright flares or flashes. Ata later period the light disappears
ely between the flashes, which have a longer interval between them.
ide "probably pathological conditions, after the organisms have been
pt confined for a considerable period, there may be lighting up of the
gan nisms with a continuous glow.

_ The appearance of the spontaneous phosphorescence at nightfall,
gpa at dawn, are characterised by the same changes in a
reversed order in the two cases. Before the appearance of spontaneous

osph 0. nce at night, and after its disappearance in the morning,
| eta: of minimal excitability of about half an hour during
tiring still calls out phosphorescence. here this the organisms
om mnpet refractory. :
; _ Addition of fresh water, or formol, produces, during the period
| which the organism is dying, a most vivid phosphorescence, which lasts
1 two to three minutes, and then fades and disappears.
ris display is very feeble during a daylight period, compared to what
er » after dark when spontaneous phosphorescence is present.

ae
: ee ———

30

THE RELATIVE IMPORTANCE OF INORGANIC KATIONS,
ESPECIALLY THOSE OF SODIUM AND CALCIUM, IN
THE CAUSATION OF GOUT AND PRODUCTION OF
GOUTY DEPOSITS'

By WILLIAM GORDON LITTLE, M.A. (Aber.), M.D. (Edin.)
From the Bio-Chemical Department, University of Liverpool

(Received November 14th, 1908)

As long ago as 1844, Ure? suggested that calcium salts and sodium
salts were deposited in gouty conditions; the sodium as biurate in the
synovial membranes and tendons, and the calcium as phosphate in the
arterial walls.

From the predominance of sodium salts in the fluids of the body it is
to be expected that the bulk of any salt should contain that kation either
in solution or as a deposit; but the more insoluble any salt-forming anion
is, such as that of uric acid, the more important does the presence not of
one but of several kations become; for upon such multiplicity of kations
does the carrying power of the solvent for the feebly soluble anion depend.

The great importance of the relative effects upon one another of these
kations, in common solution with uric acid, in dissolving or precipitating
the uric acid anion, has not been sufficiently realised, and the solubilities
under such conditions have not been sufficiently investigated.

The object of the present paper is to supply some further information
on this question of solubility and precipitation of uric acid anion as acid
salt, from the common solution containing more than one kation.

The various salts of calcium and uric acid have been described by
Delepine? at length, as well as their occurrence in urine and tissues. He
describes (1) an acid salt (biurate) which is comparatively soluble in water,
and (2) a basic or neutral salt (normal urate) comparatively insoluble.
He then quite justly remarks that this is a reversal of what obtains in the
case of alkaline urates, presumably those of sodium and potassium, and
calls attention to the evident importance of this in relation to the reaction
of the solvent medium.

Following Heintz, Delepine and other authors, quote calcium
biurate as a highly soluble biurate, being even more soluble than the
potassium biurate.

1. Part of the expense of this research has been defrayed by a grant from the British
Medical Association.

2. Medical Times, Vol. XI, p. 145, 1844.

3. Journ. of Physiol., Vol. VITI, 1887.

INORGANIC KATIONS IN GOUT 31

Thus Neubauer and Vogel give the following figures for solubility of
_ different biurates in water in the cold and at boiling point :—

————

Lithium biurate, 1 part dissolves in 370 parts cold, and 39 parts boiling water.

Calcium » 1 9s . 603 - 276
Potassium ,, Em 3 oa 790 ~ 75
Sodium a 1 ee mn 1,150 = 112
Magnesium ,, =|, bs 3,750 n 160

The fact that calcium biurate occurs in all tophi, as shown by Ebstein
and Sprague,! and usually to the extent of 12 to 15 per cent, alongside of
about 57 per cent. of sodium biurate, is sufficient to imdicate that the
statements and figures given above as to the solubility of calcium biurate,
do not represent the true state of affairs accompanying deposition in the
body, which must occur at body temperature, and from media which,
me rich in sodium chloride, may behave quite differently as a solvent
- from distilled water.

It is peculiar that no experiments have been recorded as to the
~ solubility at body temperature, and from salt solutions. Such experi-
ments I have undertaken, and have found, first, that caleium biurate
must be placed even lower than magnesium biurate at the bottom of the
_ seale of solubility of biurates, when the solubility is measured in
distilled water kept at body temperature in a thermostat; secondly, that
mere traces of a calcium salt added to solutions of sodium biurate causes
precipitation ; and thirdly, that the presence of so little as 0-5 per cent.
of sodium chloride enormously lowers the solubility of sodium biurate.

These facts taken together appear to me to explain why sodium
biurate and calcium biurate appear together in tophi, and give a
significance to calcium biurate in gout, similar to that shown by O. T.
Williams? for the insoluble calcium soaps secreted by the intestinal mucosa
in mucous colitis and appendicitis, and by Klotz and others for the
_ealeium saponification in arterio-sclerosis, where the insoluble soap
aie ‘ sooner or later passes into the form of the likewise insoluble calcium
7 carbonate.

The biurates used for our experiments were prepared from Merck's
‘extra pure’ uric acid. In the case of the calcium and magnesium
biurates, the composition was controlled by incineration and weighing
the calcium oxide and magnesium oxide respectively. The calcium salt
yielded 12 per cent. of CaO, theory requiring in the biurate 13°6 per cent.,

1. Ergebnisse der Physiologie, Bd. Il, 1903.
2. This Journal, Vol. IT’ p. 395.1907; Vol. TIL p. 391, 1908.

82 BIO-CHEMICAL JOURNAL

and in the normal urate 27 per cent. Similarly, the magnesium salt

yielded 11-7 per cent. of MgO, the theoretical yield being 11°2 per cent..

for biurate and 21 per cent. for normal urate. It is, therefore, obvious
that in each casé We were dealing with the biurate.

The results obtained for solubility in distilled water of the four

biurates were as follows : —
One part of potassium biurate dissolves in 64 parts boiling water and in 550 parts at body

temperature
» sodium bd = 117 Pa i 1,030 a ”
Bt magnesium ,, “4 148 < = 2,440 *» wn
23 calcium * > 666 = x 4,760 * »

The last figures are the average of two independent titrations with
potassium permanganate, which were carried out most carefully, and the
result corroborates the findings of those observers who give calcium
biurate a permanent place amongst the constituents of tophi. If it were
so soluble as Delepine, and the other authors whom he quotes, would place
it, then it could not be present in such gouty deposits. w

In the body, however, the various biurates are not dissolved in
distilled water, but in a saline solution containing as the chief constituent
sodium chloride, along with other inorganic salts in lesser concentrations.
Accordingly an attempt was instituted to approach more closely to natural
conditions by determining the solubilities of the above biurates in a halt
per cent. solution of sodium chloride.

Here the most interesting result was obtained that while the solu-
bilities of calcium and magnesium biurates were actually somewhat
increased in the saline solutions, that of the sodium biurate was reduced
almost to zero.

This result gives probably the key to two things, first, that in the
body the preponderating salt in gouty concretions is sodium biurate, and
secondly, on account of the increased solubility of the biurates of the
alkiline earths (Ca and Mg) ‘in salines, we see, perhaps, a rational basis
for understanding the improving effects of certain saline mineral waters
empirically used in gout.

In any case, the variation in the figures according to whether the ~

solvent medium for the biurates is water or a dilute sodium chloride
solution, gives an indication of the great value which would attach to the
study of the solvent action of solutions containing a number of inorganic
salts in varying proportion. rai

Attention may be specially called to the small amount of soll
chloride which produces such a considerable change in the solubilities,
illustrating that small variations in relative distribution of the salts of the

; i. sl = i teen ile
5 -* - ie a
2

INORGANIC KATIONS IN GOUT 33

_ plasma, such as might naturally occur from individual to individual, may
' have profound effects on the solubilities of the biurates in the body.

4 __'Phe enormous effects of the sodium chloride on the solubility of the
sodium biurate is, in the language of physical chemistry, probably to be
referred to the mass action of the common sodium ion of the sodium
chloride and sodium biurate, tending to throw out of solution the less
soluble of the two salts, viz., the sodium biurate. At least, such an action

a has certainly been shown to occur in countless cases in pure solution of
4 salts possessing a common ion in their constitution. Hopkins and Hope!
y quote Nernst’s. generalization to the effect that any two salts susceptible

of dissociation, which contain an electric ion in common, naturally
a diminish each other’s solubility. But the converse of this proposition has
; been found to hold true in certain cases, and salts possessing no electric
ion in common may mutually increase each other’s solubility in a fluid.
_ The possibility exists, therefore, that the ingestion of a mixed dietary may
_ produce such a temporary increase in the proportion of salts other than
those of sodium (especially potassium salts) as to increase the solubility of
any retained sodium biurate, and so accelerate its excretion. They
- eonsider this an important principle in lithiasis.

For ease of comparison the results of titration of the saturated
solutions of the four different biurates, (a) in distilled water, and (6) in
half per cent. sodium chloride solution, in each case at body temperature,

n

' are given in the following table. The figures show c.c. of 20 KMn0O,,

required to oxidize the urie acid, and to get quantities of uric acid
dissolved these must be multiplied by the factor 000375. In the
_-_—_—_— second part of the table this has been done and the results re-stated in
terms of the amount of fluid in each case required to dissolve one part of
uric acid in the form of the respective biurates at body temperature.
Amount dissolved by 100c.c. | Amount dissolved by 100 c.c.

Biurate taken of distilled water at body of 0-5 per cent sodium
temperature chloride solution at body
as uric acid temperature * ~
Calcium biurate se 5&7 78
Sodium i ais 25°9 sas 0-6
Magnesium ,, pal 110 ies 13-6
Potassium ,, es 48-4 a 48-0
. Parts of distilled water Parts of 0-5 per cent. sodium
Biurate taken required to dissolve one part chloride required to dissolve
one part
Calcium biurate op 4,760 jes 3,420
Sodium “ a 1,030 i. 44,400
Magnesium ,, Ey 2,440 an 1,960
Potassium —_,, es 550 és 555

1. Journal of Physiology, 1898-99, p. 284.

34 BIO-CHEMICAL JOURNAL

Precrerrarine Errecrs or Cancrum Sats on Soprum Brurate Sonvrions

Another important relationship is the precipitating effeet of calcium
salts on biurate solutions of sodium. It was found that the addition of
even small percentages of calcium salts to distilled water had the effect
of very much lessening its solvent power on sodium biurate. Kp

Experiment I,—Calcium chloride— ;

At 87° ©. 100 c.c. distilled water alone in 12 hours took up an equivalent of 25-9 c.c. 5= * KMnO,

a 100 c.c. +01% CaCl . ” 3:5.0.0. 2»
2H 100 c.c. * +03 % CaCl, = cs 108 ¢.c. ,,
4 100 ¢.c. 7 + 05 % CaCl, s sf 1-9 ¢.c, Pa

So that calcium chloride exerts a decidedly deterrent influence on the
solubility of sodium biurate in aqueous solution.

y
4

Experiment II.—Calcium sulphate :-—

Control in distilled water ... ahi ie is 25-9 .0.35 KMnO,
is . + 0-1 % CaSO, ... «190 ee. .
re 7 0-2 % CaSO, _.... oaks 15-0 c.c. *.

Calcium phosphate also had a decided effect in lowering the solubility,
and this has a special interest in being so frequently found in tophi.

Experiment I1I1.—Calcium Phosphate.—The following titrations give detailed quantitative
results to show the markedly deterrent effect of small percentages of calcium phos on the
biurates in aqueous solution.

Three solutions containing distilled water (a) with potassium, and (b) sodium bieliabes
in excess, and (c) a mixture of the two in excess were placed in the incubator at 37°C. To three
other samples of these same solutions } per cent. of calcium phosphate was added, and these were
similarly treated. Three titrations were made with the following results per 100 c.c, :— .

s
(a) Distilled water with KHU in excess after 2 hours at 37° C.
had dissolved an equivalent of 63 c.c. SD KMnO,

(b) ” ” NaHU ” ” ” 34 ©.c. ”
(ec) co a both biurates ,, oe =f 75 c.c. me

: Il.
(a) Distilled water with both biurates in excess after 72 hours at 37° C.

had dissolved an equivalent of 60 c.c. i KMn0,

(b) ob eo » pe pe oe 38 c.c. ae
(e) *” 9 ” % PA >” 75-5 c.c. ou
Til.
(a) + 4% CaHPO, in excess after 72 hours at 37° C. had dissolved .
an equivalent of ... bas vas .. 4650.0. 6 KMn0,
(b) ., - 9 os = 27-5 c.c. >

(e) ” ” ” ” ” 54-0 cc, ”

INORGANIC KATIONS IN GOUT 35

_ These results derive added importance from the fact that CaHPO, is
_ constantly found in tophi, and emphasises the value of recent research in
_ demonstrating the excretory function of the intestine for calcium and for
_ phosphates. They also go towards giving a rational explanation for the
e, empirical treatment by mercurials, salines, etc., which prevailed in the
past, and is still recommended, for constipation is a classical symptom in
ee eehan).
_ Complementary to the general question of solubility comes that of
. the formation of deposits and tophi, and the influence of calcium salts
in this direction. There is a presumption that its influence is decided,
for we have already pointed out that calcium is usually present as a
biurate or a phosphate.
. Without wishing to exaggerate the value of experiments in vitro,
: as applied to body processes, one is tempted to suggest some analogy
4 een an experiment like the following and the conditions prior to an
acute attack of gout, for, as Osler states, ‘the formation of tophi must
rest upon some physico-chemical basis of precipitation and crystallization.’
B This experiment is intended to demonstrate the powerful precipitating
effect of a calcium salt in minute percentages on a solution of sodium
- biurate containing amounts varying from 1 in 819 to 1 in 3,275, which
Roberts believed to approximate to the amounts possibly occurring in a
supersaturated condition of the blood and sera preceding an acute attack.
A solution was made of uric acid (Merck’s extra pure) in a ‘2 per cent.
_ sodium of bicarbonate of sodium. This was found by direct titration

. with 3 KMn0, to require 36 c.c. per 100 c.c. of solution, corresponding

to a strength of 1 in 655 of uric acid. Into eight stoppered bottles, the

_ following quantities of this solution were poured, 80, 70, 60, 50, 40, 30,
_ 20, respectively, and distilled water added to bring each volume up to
100 cc. These represent strengths of biurate of sodium varying from
1 in 819 to 1 in 3,275. To each was added a weighed amount of
_ erystallized sulphate of sodium, equivalent to ‘2 per cent. of the
anhydrous salt. The bottles were then placed in the incubator at
_ 87°C., and left for twenty-four hours. At the end of that time they were
still clear. Another 1 per cent. of anhydrous sulphate was added, and as at
the end of three hours they were still found to be clear, ‘1 per cent. acid
phosphate of soda was added.!. Three hours afterwards they were still
clear, and they remained so during the next twenty-four hours. At the end

1. These salts were employed so as to minimize the error in the final titration due to
oxidizing of the chloride.

36 BIO-CHEMICAL JOURNAL

of that time ‘01 per cent. CaCl, was dropped from a stronger solution, and
after sixteen hours they were found to be still clear. At the end of that
time another ‘01 per cent. Ca@l, was added. This produced no
immediate effect, but after one hour slight floceuli were visible in certain:
of the bottles. Ten c.c. of each solution (previously filtered) gave the
following results on titration, which show clearly the precipitating effect
of CaCl, even in ‘02 per cent.

(1) 80 ¢.c. solution + 20 ¢.c. water’+ 0-28{Na,8O. Wa N Milligrammes °% loss of
10 H,O) + 0-2 % eH found theoretical Uric acid
+ 0-02 CaCl, 2-1 2-9

(2) 70c.c. _,, + 30 c.c. af a4 20 2-4 ia

(3) Bec. ,, + 40 c.c. m os % 1-9 2-2 13-6

(4) 50 c.c. a + 50 c.c. > me $s 1-7 18 55

(5) 400.c. __,, + 60 cc, oe *” is 1:3 1-4 yf

(6) 30ec. ,, + 70c.c. Ps = - 1-0 1-08 70

(7) Mec. ,, + 80 c.c. ts > ~ 0-7 (0°72) 2-8

Regarding the precipitating effect of sodium chloride in gout, and
the use of common salt in the diet, the statements in the literature are
somewhat contradictory. Sir W. Roberts! advised abstinence from the
use of much culinary salt in the gouty. He believed it could be stored
up, especially in the serous fluids, to a concentration sufficient to impede
solubility, and presumably by inference cause precipitation. Hofmeister’s
work (quoted in Schaefer’s Physiology) tends to corroborate this suggestion
by proving the power for adsorption of sodium chloride which colloids of
the chondrin and mucin type possess. Roberts himself quotes figures in
his published Croonian lectures to show that a maximum sodium content
is found in cartilaginous tissues.

Mendelssohn? mentions experiments with some of the well-known
uric acid solvents. He dissolved piperazin and lysidin in blood serum
and ‘ found such solutions to have as great a solvent power for uric acid
as their aqueous solutions. If sodium chloride be added precipitation
occurs, and in the form of sodium biurate.’ He continues: ‘ The sudden
nature of attacks of gout seems to point to sudden formation of some such
urate precipitant, and the fact that they generally occur after mistakes
in diet, over-feeding, etc., where much salt is consumed seems to indicate
sodium chloride to be the cause.’

Dapper and von Noorden, in a monograph recently published on the
effect of sodium chloride on metabolism, discuss the effect of sodium
chloride waters upon the excretion of uric acid. They find in therapeutic
literature the greatest confusion on the point as to whether it increases
or diminishes the output of uric acid in gout. They cite quantitative ©

1. Croonian Lectures, 1882.
2. Deutsch. med. Wochenach., 1895, p. 283.

INORGANIC KATIONS IN GOUT 37

ts from several cases to prove that such saline waters do increase the
output of uric acid, arid state that these facts point to the use of saline
mineral waters in our treatment of gout as worthy of consideration.

Two cases in my own practice have afforded suggestive evidence that
sodium chloride has something to do with gouty-joint phenomena. In
_ the first of these cases a child of five years developed acute arthritic
_ symptoms in the feet and thighs, simulating acute rheumatism, after an
overdose of sodium chloride administered as a home cure for intestinal
: oo Previously, too, a fairly strong salt solution had been injected
per rectum daily for the same purpose. That the sodium chloride had
some pathogenic significance was proved by the fact that as soon as its
- administration ceased, with no other medication except small doses of
_— citrate, the child became rapidly well and has been well ever

ae toe joint and then in the other.

In this case two samples of 24-hours’ urine (150 c.c.’s in volume)
_ were analysed for the quantitative estimation of the bases, (1) one when
_ the attack was at its worst, (2) the second when recovery was well
_ advanced, at an interval of several days. The exact amount of chlorides
was not tabulated. Sodium chloride, however, was found in excess, but
_ in regard to the bases, the figures are—

a Na,O — K,0O CaO MgO
(1) * 2-041 grs. 0-091 grs. =: 0-067 grs. 0-085 grs. | in 150 c.c,
wh (2) 1-375 grs. 0-183 grs. 0-122 grs. 0-093 grs. | of urine

This, so far as it goes, would suggest that sodium chloride in the
acute stage had something to do with arthritic symptoms, and that an
we increase of urinary calcium, magnesium, and potassium are at least com-
; patible with recovery from an attack of gout, whilst the reverse is true
= Whether sodium chloride can exist in such concentration as to cause
_ precipitation or not, it is much more likely that a metal of higher valency
than sodium, especially one such as calcium, which can produce a higher
insoluble biurate, will cause precipitation.
It may be noticed also that a gouty condition is often associated with
lead poisoning, as Garrod pointed out in his early researches.

In conclusion I have to express my thanks to Dr. Charles E. Harris
for valuable assistance in carrying out the solubility experiments.

38

ON THE NITROGEN-CONTAINING RADICLE OF LECITHIN
AND OTHER PHOSPHATIDES

By HUGH MacLEAN, M.D., Carnegie Research Fellow.

Irom the Department of Physiological Chemistry, Institute of
Physiology, Berlin

(Received November 18th, 1908)

Part |

Since the investigations of Diaconow and Strecker! it has generally
been assumed that lecithin is a compound of fatty acids with glycero-
phosphoric acid and a base choline. This assumption is based on the
results of elementary analysis combined with the fact that hydrolytic
decomposition of the lecithin yields the above-mentioned constituents.
From this it is obvious that the total amount of nitrogen present is
represented by the nitrogen of the choline radicle, and in this way a
knowledge of the total amount of nitrogen yielded by any pure lecithin
makes it easy to deduce the amount of choline (C,H,,;NO,) actually
present, from a theoretical standpoint? Many experiments have been
made in order to obtain the choline content of different lecithins, but
in every case the results actually obtained fell far below the theoretical
values. Thus Erlandsen? obtained from pure heart lecithin, which had
been split up by boiling with barium hydrate, only about 42 per cent. of
the theoretical amount, and Heffter,? using lecithin extracted from
liver, obtained under similar conditions only 25 per cent.

In order, if possible, to elicit the cause of these losses, the following
investigation was undertaken; here a comparison of the amounts of
choline actually obtained from different lecithins, saponified and
manipulated under exactly ‘similar conditions, suggests much as to the
real nature and cause of this loss in certain lecithins.

Marteriat Usep

For the first set of experiments a lecithin sold by the firm of J. D.
Riedel, Berlin, under the trade name of ‘lecithol’ was employed.
Afterwards, I extracted and purified lecithin from the heart muscle of the
ox, as described below.

Annal. d, Chem. u. Pharm., 148.

L.
2. Zeits. j. Physiol. Chem., Ba. LY, 8. 113.
3. Arch. f. exp., Pathol. 4. Pharm., Ba. XXVIIL, 8. 100.

NITROGEN-CONTAINING RADIULE OF PHOSPHATIDES 89
* Leerrnot ’

t first the lecithin salt of cadmium chloride was made use of, the
lecithin being dissolved in alcohol, and the solution carefully filtered,
and then precipitated by cadmium chloride. This lecithin salt, however,
while much more convenient to weigh and handle than lecithin itself,
ave rise to much difficulty on attempting to split it up with solution of
m hydrate, either in water or alcohol; here it constantly adhered to
: : 2s of the flask above the liquid, so that, even by constant shaking,
was difficult to ensure complete saponification. For this reason it

Si me discarded, and the lecithin itself used. The lecithin was found,
however, to contain traces of ammonium compounds, and in order to get
vi of these impurities it was treated as follows. Small quantities were
radually added to some water in a mortar and ground up until a
pletely homogeneous emulsion was formed. This emulsion was
) inte by means of acetone, the lecithin carefully filtered, kneaded
a plastic mass with some more acetone, and again emulsified as
re. The filtrate gave abundant evidence of the presence of ammonia
her n treated with caustic alkali and heated. After the process had been
pe ited three times it was found that acetone failed to give a precipitate.
is difficulty was overcome by the addition of a few drops of sodium _
chloride solution, when the lecithin separated out quite readily. This
ay process of emulsification and precipitation by acetone was carried out
five: times, the last two filtrates giving no indication of the presence of
ammonia. The lecithin was then thoroughly washed with acetone, and
dried in vacuo over sulphuric acid. After drying, it was dissolved in
____ absolute alcohol, and the solution preserved in a well-stoppered dark
of bottle. For each experiment 5 c.cm. of this solution was used, the same
1 » each time thoroughly cleaned and dried, being used for
This lecithin solution gave the following results on

Nitrogen (Kjeldahl’s Method)
Tite 6 ca. solution = 10-2 10-0 109 and 10-2 o.°. To

Average 10-1 oom. = 14:14 mgrs. N

Phosphorus (Neumann's Method)
In five experiments 5 c.c. solution = 52-0, 52-5, 51-9, 52:1, 52-3, 0.0, = NaOH

Average 52-2 c.om, = 28-89 mgr. P
Ratio of P: N = 1: 1-08
_+ 5 -e.c. aleoholic sol. = 14:14 mgrs. N & 0-31073 grm. cholin platinum chloride,

In another solution used—
5 cc. = 17-4062 mgr. N = 0-38250 grm. cholin platinum chloride.

40 BIO-CHEMICAL JOURNAL
Experiments with * Lecrrso.’

Five c.c. of the above alcoholic solution was taken and added to 100 c.c.
of methyl alcohol, saturated with barium hydrate, and boiled in a flask
on the water bath for varying periods; the flask, which was fitted with a
reflux condenser, was shaken from time to time. After heating for
periods of one to ten hours,the mixture was allowed to stand for some time,
when a well-marked precipitate separated out and fell to the bottom of
the flask, the fluid above remaining fairly clear. This fluid was filtered
off and the precipitate returned to the flask; to the flask was added
100 c.c. of ethyl alcohol, and after being well shaken up with the residue
was boiled for five minutes. ‘The mixture was then allowed to stand
and the clear alcohol filtered off as before. This process was repeated
usually about four times, and sometimes oftener, in order to ensure
thorough extraction of any choline remaining in the insoluble residue.
All these alcoholic extracts were then mixed together and carefully
evaporated on the steam bath to about the bulk of the original amount of
alcohol (100 c.c.) or sometimes rather less. The barium was then separated
by treatment with hydrochloric acid; the barium chloride was filtered off
and the fluid evaporated to dryness. In order to avoid losses from
bumping, it was found better to perform all these evaporations in flasks.

When carried out in this way it was easy to ensure that nothing was
lost. The dried residue was then thoroughly extracted with absolute
alcohol, evaporated to small bulk and treated with a saturated solution
of sublimate in absolute alcohol. This was left to stand till next day
when the precipitate was separated by filtration; the filtrate was then
evaporated to dryness, and the residue, after being waslied with cold
alcohol, added to the first precipitate; the combined precipitates were
then dissolved in hot water. This solution was treated with sulphuretted
hydrogen and filtered; filtrate was evaporated to dryness and dissolved in
a little absolute alcohol. The choline, which was present in the form of —
choline chloride, was now precipitated by a solution of platinum chloride
in absolute alcohol; after eighteen to twenty-four hours the precipitate
was filtered off, washed with cold absolute alcohol, dried and weighed.

Sublimate was introduced on the assumption that possibly the
presence of impurities might interfere with the precipitation of the
choline by platinum chloride, and that those impurities might to a greater
extent be got rid of by sublimate. Subsequent results did not bear out
this view, and in all my later experiments sublimate was omitted.

PROGEN-CONTAINING RADICLE OF PHOSPHATIDES 41

_ An extended series of observations with the above method has been
ly published! by the writer, but the following short extract serves
) show the general results obtained. In fifteen experiments the average
yercentage of the lecithin nitrogen obtained as choiin nitrogen was only
73 per cent :—

CHOLLN E-PLATIN UM-CHLORIDE

No. of Percentage actually
hours Actual amount found, Theoretical amount, found, in terms of
hydrolysed in grms. calculated from N theoretical amount
of lecithin, in grms.
1 0-2700 0-38250 70-59
lk _ 02450 0-31073 78°85
“S 0-2941 0-38250 76-89
ant 4 0-2991 0°38250 78-19
2 7 0-2421 0-31073 77-91
‘ ” 10 0-2390 0-31073 76-91
mde):

ae / That the substance obtained was pure choline platinum chloride is
evident from the following figures :—

1. 02439 grm. left on ignition 0-0771-grm. Platinum = 31-61 °%

2. 0-1740 grm. * 0-0550 grm. » = 31-61%

Calculated for (C;Hj,NOCI), PtCh = 31-64% Pt

« In three experiments, carried out exactly as above, only that
-_- sublimate was not used, the following results were obtained. Here, also,
the alt proved to be pure, giving on ignition a residue of 31:59 per

“ CHOLIN E-PLATIN UM-CHLORIDE
No. of Percentage actually

hours Actual amount found, Theoretical amount, found. in terms of
hydrolysed in grms, calculated from N theoretical amount
of lecithin, in grms.
2 0-2976 0-38250 77-80
3 0-3003 0-38250 78-51
3 0-3031 ‘ 0-38250 79-24

In all these experiments it is seen that not more than from 77 per
= to 79 per cent. of the total nitrogen can be recovered as choline

~ Here it is interesting to note that at the same time as these results
were published, a paper appeared by Moruzzi? describing the results of
hydrolytic decomposition by sulphuric acid. In his experiments the
double salt of cadmium-chloride-lecithin was utilised. The following
a figures taken from his paper show practically the same percentage of

1. Zeits. }. physiol. Chemie, Ba. LV, 8. 363,
2, Loe. cit., 8. 352.

42 BIO-CHEMICAL JOURNAL

choline platinum chloride (average 77'7 per cent.) as was obtained by the
writer with barium hydrate.

Amount of CHOLINE-PLATINUM-CHLORIDE

mium No, of Percentage petanly
Number compound hours Actual amount found, Theoretical amount, found, in terms ¢

used, i hydrolysed in grms. calculated from N theoretical amount
grms. of lecithin, in grms.
1 2-84.50 44 0-6642 0-8687 76-5
2 1-4952 4h 03507 O-4574 76-7 —
3 1-8147 4h 04425 05542 79:8

Hypro.tysis in Watery Sotvurion or Bartum Hypratre

Some experiments were now made in order to observe what results
could be obtained by using a saturated watery solution of barium hydrate
instead of an alcoholic fluid. Five c.c. of the lecithol solution was added to
100 ¢.c. of a saturated solution of barium hydrate in water, and boiled
with a reflux condenser for 2} hours; during the process the flask was
shaken from time to time, especially during the first hour. It was then
allowed to stand, and the precipitate filtered off. After thorough
washing of the residue, the filtrate was freed from barium by means of
CO,; barium carbonate was filtered off, and the filtrate, after the addition
of a little hydrochloric acid, evaporated to dryness. Residue was
extracted with a little absolute alcohol, and after evaporating to small
bulk, was precipitated directly with platinum chloride. Precipitate was
then left to stand till next day, filtered, washed, dried and weighed as
usual, Here the average result obtained was 77°5 per cent. .

In all the above modifications it is seen that the result remains
practically the same, so that not more than about 77 per cent. to 79 per
cent. of the theoretically calculated choline-platinum-chloride is actually
obtainable by experiment from this particular lecithin.

The following table indicates the average results of the different
methods : —

Percentage of Choline-platinum-chloride
obtained, calculated on theoretica

amount
1. Lecithin hydrolysed with saturated solution of
barium hydrate in methyl alcohol, using sub-
limate as intermediate precipitant .. = 17-3 %
2. Same as above, without sublimate oe = 78-5 %
Saturated watery solution of barium hydrate is 77-5 %,
4. Sulphuric acid (Moruzzi) = 77-7 %
Average = 17°75 %,

—NITROGEN-CON'TAINING RADICLE OF PHOSPHATIDES 48

Ow rne Causes or ruts Loss or tHe THEORETICALLY CaLcuLarED
soma CHOLINE

Here we have to deal with a loss of a little over 20 per cent. of the
theory, and in order to elicit its cause the following points were
considered : —

- (1) Is the choline partly destroyed when heated with a saturated

—___ solution of barium hydrate ?

3 Since it is known that the free base decomposes on heating into
trimethylamine, ethylene oxide, and H,O, and since, as first observed by
Heffter, a mixture of lecithin and barium hydrate which has been boiled
for some time has a pronounced smell of trimethylamine, it might be
thought that this decomposition was the cause of the loss. Gulewitsch,!
however, has shown that this destruction of choline is so small as to be

* of little practical importance, and some experiments made by the writer

entirely supported this statement. Lecithin was boiled with barium

hydrate in a flask fitted with a reflux condenser, the latter being connected
with a bottle containing io H,SO,. Through this acid the volatile

decomposition products of lecithin were led by means of a stream of
ammonia-free air, and the amount of acid neutralised estimated by

titration. In various experiments lasting from two to eight hours it was

found that the amount of 0 H,SO, used was very small, amounting,

after long boiling to not more than the equivalent of 1 to 3 per cent. of
the total nitrogen of the lecithin used. From this it is obvious that the
____ loss is not accounted for, or at least only to a very small degree, by the
4 decomposition of the choline.
q (2) Is the lecithin completely split up, and does the filtrate contain
E the total nitrogen of the lecithin used ?
That the lecithin is completely split up would appear from the fact
_ that boiling for ten hours gives no better result than boiling for
two to three hours. As the result of experiment, we may assume that all
the lecithin is entirely split wp after two to three hours or even less. With
regard to the N-content of the filtrate, it is interesting to note, that in
no case was it found to contain all the nitrogen of the saponitied lecithin.
An analysis of the residue after boiling also showed that this residue
always contained nitrogen in a form insoluble in alcohol, as no amount
of washing had any effect in lowering the amount of this residual

1. Zeits. {. physiol, Chemie, Ba, XXIV, 8. 513.

4 BIO-CHEMICAL JOURNAL

nitrogen. This residue was always very carefully washed in the following
manner; after filtration it was returned to the flask and thoroughly
shaken up with about 100 c.c. alcohol; the mixture was then boiled for
five to ten minutes, allowed to stand, and again filtered. This process
was generally repeated three times, but in some experiments as often as
six times; in each case the result was the same. The number of hours
during which the mixture was boiled had also no effect on the result.
The average amount of insoluble nitrogen found in the residue amounted,
as shown by the following table, to 8:5 per cent. of the total nitrogen of
the lecithin.

Total N of N found in Percentage of
Number of hours lecithin used, in residue, in mgrs. total N found in

No boiled mgrs. residue
i 1 1414 1-19 8-4
2 1 14-14 1-13 8-0
3 1} 14-14 0-98 7-0
4 1 14-14 1-26 8-9
5 3 14-14 1-13 8-0
6 3 14-14 1-19 8-4
7 5 14-14 1-33 4
8 5 14-14 1-26 8-9
9 7 14-14 1-26 8-9
10 7 14-14 1-33 9-4
Average 14-14 1-21 8-5

(3) Does the possible presence of traces of impurities (other
decomposition products of lecithin) prevent the complete precipitation of
choline by platinum chloride? _

To test this, some pure choline chloride solution (0°1 to 0°2 per cent.)
in absolute alcohol was taken and divided into equal parts by means of a —
burette. To some of these portions were added glycerophosphoric acid,
glycerine and barium chloride; they were precipitated with platinum
chloride. The other portions were directly precipitated. A comparison
of the results obtained in both cases showed that the presence of these
impurities, while tending to lower the percentage of choline-platinum-
chloride obtained, did not do so to any marked degree. Considering that
only very minute traces of impurities can be present, it is not likely that
this factor is of much importance practically, in preventing complete
precipitation.

(4) Is choline chloride imperfectly precipitated by platinum chloride
in alcoholic solution ?

Gulewitsch made an experiment bearing on this point in the following
manner. He took a 05 per cent. solution of choline chloride in absolute

TTROGEN-CONTAINING RADICLE OF PHOSPHATIDES 45

~aleohol and precipitated it with an alcoholic solution of platinum
chloride. After twenty-four hours the precipitate was filtered off and
‘ washed with absolute alcohol. |The filtrate was evaporated to dryness,
after being decomposed by sulphuretted hydrogen gas. The residue,
which was very small, gave only a slight cloudiness with phosphotungstic
acid and with iodine and potassium iodide solution. From this it was
-eoncluded that platinum chloride precipitates choline chloride
_ quantitatively. Since, however, the quantities obtained in lecithin
oe _experiments are necessarily small, it was thought advisable to weigh
“some choline-platinum-chloride salt; then dissolve it in H,0, decompose
_ __ the solution with sulphuretted hydrogen, evaporate the filtrate to dryness,
____ dissolve out with absolute alcohol and precipitate with platinum chloride.
‘The amount of the latter salt ultimately obtained was compared with the
amount used. In all my experiments it was found that the weight of
choline salt obtained in this way always fell somewhat short of the
: “original amount used. While it is likely that there must be some slight
mechanical loss on account of the necessary manipulation, it would seem
____ that the precipitation is not quite complete, and in this way a certain
amount of the loss of the theoretical choline of lecithin is explained. In
some experiments carried out with erystals that had been several times
3 re-erystallised, and using alcohol that had been treated with barium
oxide immediately before use, I obtained on an average from 93 to 97
per cent. of the original salt after the above manipulations. The
average percentage, when using alcohol that had not been so treated,
_ was somewhat lower. With care this loss is but slight.
J The chief losses, therefore, seem due to part of the nitrogen remaining
in the residue, together with small losses represented by the necessary
: manipulations of the method, combined with the incomplete precipitation
of choline chloride by platinum chloride.
xg _ When we subtract the nitrogen found in the residue from the total
fl nitrogen, we get theoretically the amount present in the filtrate ;
. racially, however, owing to the necessary manipulation, it is obvious
that the ultimate filtrate, when freed from impurities and ready for
precipitation by platinum chloride must contain somewhat less nitrogen.
The actual amount of choline platinum chloride found, when
reckoned on the nitrogen of the lecithin minus the residue nitrogen, was
from 86 to 87 per cent. The relative amount of platinum salt obtained
when the filtrate was treated as described on page (55), was from
91 to 92 per cent. of the total filtrate nitrogen. This difference
as mentioned, is accounted for by small losses during manipulation, for

46 BIO-CHEMICAL JOURNAL

it is obvious that a comparatively swall loss materially influences the
percentage.

Since, therefore, platinum chloride tends to give incomplete precipita-
tion of choline chloride, and since there may be traces of impurities also
present tending to hinder precipitation, and when we consider the difficulty |
of absolutely exact estimation of the nitrogen in these experiments, it
would seem that in this particular lecithin all, or nearly all, the —
of the filtrate is present as choline. ;

Whether the nitrogen found in the residue is derived from the
choline is at present undecided; it will be seen later on that a similar
residual nitrogen is found after saponification of pure heart lecithin.

Lecrruin From Hearrv Mvuscie

In order to compare the choline content of the lecithin of heart
muscle with that obtained from Riedel’s ‘lecithol,’ I prepared a pure
lecithin in the following manner, much in the same way as Erlandsen has
described.!. At the same time the mono-amino-diphosphatide substance
Cuorin was separated, as well as another substance behaving somewhat like
a phosphatide and similar in physical qualities to the substance isolated
by Stern and Thierfelder? from egg yolk, and designated by them
‘ weisse substanz.’

PREPARATION oF Heart Muscie

Oxen hearts were procured as soon as possible after the animals had
been slaughtered, and the fat and fibrous tissue separated off; the
muscular substance was then cut up into small pieces and passed through
a mincing machine. This finely divided material was spread out in a
thin layer on a glass plate, and dried at 30°C. in a current of air, —
generated by a fan arrangement which was worked by a small motor,
From time to time this layer was stirred, and the lumpy parts broken up
into small pieces, and after twelve to eighteen hours it was generally quite
dry. In order to obtain a fine powder suitable for extraction, this
fairly friable dried material was broken down with the hand, and finally
passed through a coffee mill. In this way a very fine powder was
obtained. This was put into a desiccator and preserved in vacuo over
H,SO, until quite dry. |

1. Zeits. f. physiol. Chem., Bd. LI, 8. 87.
2. Loe, cit., Bd, LIT, 8. 370.

SITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 47

EXTRACTION

= About 500 grms. of pints heart substance were taken, and to this

ie air in the upper part of the bottle was eiaced Pies CO,, in » coalie to
prevent as far as possible any tendency there might be for the lecithin to
After thorough shaking it was allowed to stand till next day and
: In order to exclude oxygen during filtration, this process
‘ried out under an atmosphere of CO,. A porcelain filter was
ref fitted into a glass bottle connected ‘eat a suction pump, and
e! at this filter a large glass funnel was placed in an inverted position ;
s funnei was connected by a rubber tube to a cylinder containing CO,,
ad ag filtration a stream of this gas was allowed to pass so that
en was totally excluded. At first the ethereal extract filtered
y well, but after some time it became very slow indeed. The filtrate
' Besta to about 150 c.c., and precipitated by excess of acetone.
‘pit that the dissolved substance should separate out as well as
»ssible it was found best to connect the dish containing the ether-
acetone mixture with an exhaust pump for half an hour or so; the
partial evaporation of the liquid generated sufficient cold to give a fairly
complete precipitate. The fluid part was then poured off, and the solid
substance obtained kneaded together with a fresh portion of acetone.
This plastic mass was then dried in vacuo over H,SO,.
Bach portion was extracted as above described five times; after this
___ it was found that acetone yielded only traces of a precipitate. All the
substance obtained in this way from the different extracts was mixed
r and thoroughly dried. This substance, which represents the
er soluble part of heart phosphatides, contained, as above mentioned,
» ‘lecithin’ as well as other lecithin-like substances‘ Cuorin’ and
ite substance’; impurities such as fat and cholesterin were also

SepaRATION oF ‘ Wuire Sunsstance,’ Far, Erc.

Above mixture of substances was dissolved in ether in a dark
: - seapene faapne bottle, and gave a whitish turbid fluid; it was not expected
that a clear solution would be obtained, for the first 10 to 20 c.c. fluid that
passed during filtration described above was invariably slightly turbid.
_ This fluid was now centrifuged, when a great deal of a whitish substance
was separated. The clear ethereal solution was evaporated to about

W

iF,
a
a

*
=
aa

ial mia tart i " ae a — a
' 7 ae ei
eee he

48 BLO-CHEMICAL JOURNAL

100 ¢.c., and then precipitated by excess of acetone, and the precipitate
treated with a little fresh acetone and kneaded together into a plastic
mass as before. This precipitate was mostly composed of dark brown
masses, but part was also white and flocculent; these floceulent masses
did not fall well, and could not easily be completely separated from the
acetone along with the rest of the precipitate. When as much as possible
was separated, the ether-acetone fluid still contained some floceulent
masses. This mixture was left to stand over night in a closed vessel,
being protected from light and air. Next morning it was invariably
found that all the white masses were precipitated on the floor of the
vessel, and were in all respects similar to the ordinary brownish precipi-
tate first obtained, so that the apparent difference seemed to be only a
physical one. This portion was then added to the first precipitate and —
dried as usual. The dried mass was then dissolved in ether as before,
and the process again repeated. This was done until all the white
substance was got rid of; at the same time fat and other impurities were
separated. As will be seen from the following table it was necessary
to centrifuge six times :—

No. of Amount of Nature of Arrer CENTRIFUGING
times ether used solution
centrifuged in ¢.c. Nature of Relative amount of
solution ‘white substance’ _
l 250 Very turbid and of Deep yellow- Much ‘ white sub- —
whitish appearance brown solution stance ’ From tn
deep in tube of
diameter
2 250 Fairly turbid but Reddish brown About 4 } amount of
much less so than solution white substance in No.1
in No. 1
3 200 Turbid, not markedly Light reddish About }-} amount
80 brown solution found in No. 1
4 200 Slightly turbid i / Trace only
5 160 Slightly turbid ov Fair amount: about
three times as much as
in No. 4
6 160 Almost clear Very light reddish Slight trace of white
solution brown solution substance after long
centrifuging

Ethereal solution was evaporated each time to about 100 c.c, and
precipitated with acetone. After above treatment the substance was
thoroughly dried in vacuo over H,SO,.

The first ether extracts contained a fair amount of fat, but the last
two portions seemed to he quite free from it.

SEPARATION OF SUBSTANCE INTO *Lecrruin’ ann * Cvortn ’

A. substance was now obtained from which the ‘white substance '

was Separated, and which was free from fat and cholesterin. This
DP iahstince which gave a practically (though not absolutely) clear
solution! in ether, contained ‘lecithin ’ which is soluble in alcohol and in
ether, and another phosphatide ‘Cuorin’ which is insoluble in alcohol.
hese substances were now separated as follows :—

xe dried mass was dissolved in 150 ¢.c. ether and to this was added
. absolute alcohol ; the mixture was then placed in the ice cupboard
ty hours. At once a certain amount of precipitate fell, but much
re was evident after some hours, and next day the insoluble part (A)
“apparently completely separated ; the fluid was then filtered off, and
e a rate, which was of a light reddish colour; was partly evaporated
under the pump and then placed in a desiccator in vacuo over
, and evaporated to dryness. When quite dry it was re-dissolved
absolute alcohol; here a small portion remained undissolved, and this
~ us added to the precipitate first obtained. The alcoholic solution was
en evaporated to small bulk, precipitated with acetone and dried as
4 - . It was completely soluble in ether, and appeared on drying as
yellowish orange coloured masses (B).

_——s«sTn this way the substance was divided into two parts : —

: (A) Part insoluble in alcohol = ‘Cuorin’ (impure, containing
oer some * white substance ’).

(B) Part soluble in ether and in alcohol = ‘ Lecithin ’ proper.

Arconot-INsoLtuBLe Portion (A)

The part insoluble in alcohol was thoroughly dried in vacuo and then
dissolved in 150 c.c. ether. Solution was not clear, so centrifuge was

used, and here again a good deal of white substance was separated off.
he clear solution was precipitated with about four times its volume of
solute alcohol, and left to stand over night under CO, in ice. Next
gt alcoholic solution appeared almost colourless. The precipitate
tained consisted of a mixture of white flocculent masses and a resinous

py material. This precipitate was treated with alcohol and left to
in the incubator at 60° ©. for several hours. The white part was
| es now dissolved by the warm alcohol, and a dark brown syrup was left. This
precipitate was again treated as above and a resinous mass again
obtained = ‘Cuorin.’ On cooling the alcoholic solution, the white
i. agen masses separated out and were filtered off = Portion A!.
‘On centrifuging part of this solution no precipitate was obtained.

50 BIO-CHEMICAL JOURNAL

PURIFICATION OF CUORIN

The Cuorin was dissolved in a little ether and gave a fairly but not
quite clear solution; CO. was introduced, and after standing in the ice
cupboard till morning a perfectly clear solution was obtained; a small
amount of sediment was seen on the floor and sides of the glass. Acetone
was now added, but the precipitate did not at first fall well, and an
emulsion-like fluid was obtained. This was left standing im ice under
CO, for sixteen hours; a good precipitate was now obtained as a brownish
sticky mass. Acetone-ether mixture was slightly whitish coloured and
when centrifuged gave a small precipitate of a brownish substance which
was not added to the other portion. After centrifuging, the fluid was
absolutely water clear. * The precipitate, after being dried over H,SO,,
was treated with warm ethyl acetate freshly distilled, and gave a clear
solution ; CO, was now introduced, and fluid was left to stand in ice for
some hours, when precipitate settled down as a resinous mass on the floor
of the flask. The ethyl acetate was somewhat light yellow coloured, and
after separating it from the precipitate, was left to stand in ice till
morning; then a little precipitate was found to have formed on the floor
of the glass consisting of minute small white balls; this was thrown away.
The precipitate was dried and the process repeated: this time the ethyl
acetate after standing overnight remained quite clear. The substance
was now dried, and a small amount dissolved in ether to test its solubility,
when it was found that the solution was not absolutely clear. The whole
precipitate was then dissolved in ether and left to stand under CO, till
morning; fluid was now quite clear, and a little sediment appeared on the
glass. This fluid was then evaporated to dryness and Cuorin obtained
which gave quite a clear solution in ether.

Sunstance Sorvsie rm Arconor at 60° C. (A, Portion)

This, as already mentioned, was obtained when the hot alcoholic
solution was allowed to cool; it was precipitated in white flocctilent masses
and only a small quantity was present; it was filtered off, and re-dissolved
in alcohol at 60°C.; then allowed to stand under CO, in ice, and
re-precipitated; it was then filtered, washed with cold absolute alcohol
and dried over H,SO, in vacuo. This substance seems to be the same
as the ‘white substance’ separated previously by the centrifuge, but
this point is at present being investigated.

ee

NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 51

Ee The following plan shows roughly the general outline of the process
of separation of the diffdrent phosphatides from ethereal extract :—

Portion (A,); dissolves at 60° and
mo | | falls out on cooling : white sub-
stance

i cs (Perper =cut

s we have separated : () Lecithin ;
ae (2) Cuorins
(3) ‘ White substance ’ (by centrifuge) ;
(4) and part, soluble in alcohol at 60°, which seems to be ‘white
substance.’ ;

_ Anarysis or Heart Muscie Lecrruin

n order *e test the purity of this lecithin the following analysis was
ne was estimated according to Neumann’s method, and

after Kjeldahl.
NITROGEN

1. 0-5232 grm. Substance = 6-9 cc. 7 H, 80. <p Vm 1-84 %

2 0-4194 & = 55 % = 1-86 %
r mn | Average = 1°85 %

by PHOSPHORUS © ; '
1. 0-872 grm. Substance = 27:22c.c.5 NaOH = 39%
2 60-2918 a = 21-37 me = 4-05 %

Average = 3-975
N:P =1: 103 %

C ann H ee
1. 01747 ¢grm. == _. 0-4234 grm. CO. = 612%C
dt pate and 0-164] grm. HyO = 10-44% H
gm. = 0-4688 CO. = 66-41%C
and 0-1784 erm. HO Fe

he fale combined with its physical qualities show that this
> is a pure lecithin.
Savonrrication or Heart Lecrrurn

With Methyl Alcohol.—This lecithin was now split up in order to
| compare the choline content with that of Riedel’s lecithin. A carefully
a weighed quantity (from 03 grm. to 07 grm.) was ground up in a mortar

52 BIO-CHEMICAL JOURNAL

with 5 grms. solid barium hydrate. | This was added to 100 ¢.c. methyl
alcohol, and the mixture boiled in a flask fitted with a reflux condenser for
periods varying generally from two to four hours, but sometimes as long
as ten hours. Flask was then allowed to stand for some time when the
fluid above appeared quite clear, and a well-marked sediment separated
out on the floor of the glass. This clear fluid was carefully poured off
and filtered, and from 80 to 100 ¢.c. aleohol added to the flask containing
» the residue; this was boiled for five minutes, left to stand and filtered as
before. This process was usually repeated three times, and afterwards
the residue was poured on to the filter paper, and washed there for some
time. On some occasions, instead of pouring the residue on to the filter
paper, it was thoroughly ground up in a mortar with some alcohol, and
the boiling process repeated as above; thus the residue was sometimes
boiled in all six times. The total filtrates were mixed and evaporated
to about 100 c.c., and the barium precipitated by concentrated HCl; the
mixture was carefully heated, allowed to cool and filtered from barium
salts. The filtrate was evaporated to dryness and dissolved in water;
this was filtered and filtrate again evaporated to dryness. Residue was
now dissolved in a little absolute alcohol, and after being evaporated to
small bulk was precipitated with platinum chloride and allowed to stand
over night; precipitate was then washed, dried and weighed as usual.

Several slight modifications of the above method were from time to
time introduced, but they did not in any way alter the results, and the
above seemed, on the whole, the best.

Here the amount of choline-platinum-chloride obtained was only, on
an average, about 41 per cent. of the theoretical amount when the substance
was boiled for two to four hours. In some experiments in which boiling
was carried out for five to ten hours the results were somewhat lower,
the average being between 37 per cent. and 38 percent. In no case was
I able to obtain a higher percentage than 42°2. The following figures, _
quoted from a series, show generally the results obtained : —

Amount of No. of CHOLIN E-PLATINUM-CHLORIDE
Number lecethin hours |
used, in grms. boiled Found, ingrms. Calculated from Percentage of
‘lecithin N, in grms. theoretical amount
found

1 0-5231 2 0-0879 * 0-2127 413

2 0-4875 2 0-0792 0-1982 40-0

3 0-5916 3 0-1015 02405 42-2

“4 0-4757 3 0-08 10 0°1934 42-0
Average = 414

A mixture of above platinum salts yielded on ignition 31:9 per cent. Pt.

NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 58
Restpve arrer Savontrication witn Ba(OH),

or This : residue, “after being thoroughly washed as above described, was
now examined for nitrogen. As in ‘lecithol’ it was found that this
| psidue pararisbly contained a certain amount of alcohol insoluble

eke the number of times that the substance was washed

“6 not seem to have any appreciable effect on the nitrogen content. That

this residual nitrogen was not present as unchanged lecithin is obvious

from the fact that the lecithin would have dissolved in the alcohol ; also

e number of hours during which the lecithin was boiled made little

e1 in the result. The nitrogen was calculated by Kjeldahl’s
, a blind experiment being made with the chemicals in each case.

No. of times No. of c.c.’s No. of mgrs. No. of mgrs. of — Percentage

sg ashed a : . .
Pilean ached: sae ee me aeet | tn roche
boiled for 5 mins. residue _ (average 1-85 %

each time nitrogen content)
3 0-44 0-616 9-68 6-4
4 0:39 0-546 901 6-0
3 0-45 0-630 10-94 5:8
1 035 0-490 8-8 5-6
2 0-49 0-686 — 1L-50 5-9
5 0-57 0-798 15°37 5-2
3 0-39 0-546 14-09 3-9
3 0-36 0-504 9-64 5-2
3 0-40 0-560 12-05 46
3 0-42 0-588 12-15 48
> ‘ Average = 5-34 %

he ‘Thus it is seen that something over 5 per cent. of the nitrogen of

ithin is found in the residue after boiling; this, however, while rather

© ult to. explain, does not account, to any appreciable extent, for the
ts 1 Loss i in choline-platinum-chloride.

NirroGeN ov FILTRATE

‘The determination of the nitrogen in the filtrate was, of course, of
4 great importance ; if the filtrate did not contain much more nitrogen than

Was represented by the choline-platinum-chloride found, it was likely that
_ the loss was due to volatilisation during boiling. The nitrogen of the
‘= filtrate plus that of the residue when subtracted from the amount actually
_ present in lecithin represents the amount lost through decomposition and
_ volatilisation during boiling. On an average it was found that the

54 BIO-CHEMICAL JOURNAL

iiltrate contained about 80 per cent. of the total nitrogen of the lecithin
used. Thus it will be noticed that the loss due to the formation of
volatile products is greater than that in * lecithol.’

Nrrrocen Founp

No. of —- — TotalN Difference
No. grms. No. of In In Total of between of tota
lecithin hours residue, filtrate, found, lecithin foundand N in
used boiled mgrs. mgrs. mgrs. used calculated, filtrate
in mgrs.
1 0-4393 2 0-58 6-58 7-16 8-12 0-96 8L-0.
2 0-6721 3 0-76 9-81 10-57 12-43 1-86 79-7
3 O-3114 3 0-54 4-69 5-23 5-76 0-53 81-4
4 0-4219 4 0-49 6-02 6-51 78 1-29 770
Average = 79-78%

Since the filtrate contains about 80 per cent. of the total nitrogen of
the lecithin, and since the average amount of choline-platinum-chloride
found corresponds only to about 40 per cent. of the theoretical amount,
it is obvious that the total amount of nitrogen recovered as the double
platinum salt of choline is only about 50 per cent. of the whole. In order
to test this directly without the intervention of filtration and other
mechanical manipulations by which slight losses might be incurred, the
following relative experiments were undertaken. It will be seen that ~
the result is absolutely in accordance with the above, the average’
percentage found being 50°5 of the total N.

Lecithin was boiled as before for some hours with Ba(OH), and the
filtrate evaporated to about 60 c.c.; it was then cooled, and excess of
acetone added, in order to make sure that no unchanged lecithin was
present. A very slight flocculent precipitate formed, which was filtered -
off, after standing for some time in the ice cupboard. The acetone was
then evaporated off and HCl added; BaCl, was filtered off and the
filtrate evaporated to dryness. Residue was dissolved in H,O, filtered,
and filtrate again evaporated to dryness. Residue was now dissolved in
absolute alcohol and filtered; it was then re-evaporated to dryness, and
re-dissolved in absolute alcohol, and ‘evaporated to 5 c.c. or so; to this
solution, which was perfectly clear, about 53 c.c. absolute alcohol was
added, and, after thorough shaking, divided by a burette into two parts
of 26 c.c. each. One part was run directly into a Kjeldahl flask and the
nitrogen estimated; the other was run into a small beaker glass,
evaporated to about 2 c.c., precipitated with platinum chloride, and left to
stand for twenty-four hours. In this way experiments 1 to 5 in the following
table were carried out; experiments 6 and 7 were also done in the same

-NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 55

vay only acetone was not used. In experiments 8 and 9 the filtrate was
- careally evaporated to dryness without the addition of HCl; the residue
_ was then dissolved in absolute alcohol and to this was added a little HCI;
it was then treated as above. This plan was adopted in order to get rid
of traces of glycerophosphoric acid which might possibly interfere with
y precipitation. The results, however, showed, as already found, that this

acid does not materially interfere with the separation of the choline as the
i platinum salt.

25 c.c. for NrrroGEN 25 0.0. FOR CHOLINE-PLATINUM-
CHLORIDE
ay | 4 Nitrogen found, in Choline-platinum- Amount of Choline- Percentage of
th mgrs. chloride, caleu- platinum-chloride _— total N found, as
lated from N actually found, in _— platinum salt of
arent found, in grms. grms. Choline
mis 8 5-60 012300 0-0619 50-3
| ae 7-70 0-1692 0-0864 51-1
m8 588 0-1292 0-0648 50-2
4 8-596 0-1889 0.0983 52-0
5 714 0-1569 0-0786 50-1
pe 5-04 0-1108 0-0566 51.1
uy 574 0-1261 0-0624 49-5
8 3-78 0-0830 "00408 49-2
9 2-80 0-0615 0-0314 51-0
Average = 50-5 %

Analysis of the above salts gave the following percentages of Pt. :—

Mixture of Nos. 1, 2 and 3 31-79 % Platinum
or * 4 and 5 31-93 % »
” a ‘6 and 7 31-79 % Ps

As a result of the above experiments we may assunie that, of the
‘nitrogen contained in the filtrate of this lecithin after saponification with
Ba (OH),, not more than about 50 per cent. can be recovered in the form
of the double platinum salt of choline. As a control, two experiments
with Riedel’s ‘ lecithol’ were carried out as above described, when 92°3
per cent. and 91-2 per cent. of the filtrate nitrogen was recovered as
choline platinum chloride; these results agree well with those mentioned
under ‘ lecithol,’ being, however, a little higher. It is worthy of note
that in almost every case the platinum content of the choline salt is
slightly above the theoretical amount; possibly it is not quite pure, but
contains traces of some other substance. The high content of platinum,
} however, proves that no part of the lecithin can be present as lecithin-
ee platinum-chloride, otherwise the platinum content should be much lower—
in other words the lecithin must be split up fairly completely.

CO

56 BIO-CHEMICAL JOURNAL

SpLirrinGc up or LecrrHin 1N WATERY SOLUTION

Small quantities of the lecithin were thoroughly ground up in a
mortar with barium hydrate, and then boiled for varying periods with
enough water to give a saturated barium solution. The flask, which was
fitted with a reflux condenser, was at first constantly shaken in order to
overcome the marked tendency of the substance to adhere to the sides of
the glass above the level of the fluid. The subsequent treatment was
similar to that described under lecithol, the choline being obtained as the
platinum salt. The following results were obtained by these means; it
will be seen that they are practically the same as those obtained with
methyl alcohol, the amount of choline-platinum-chloride actually found
representing only about 40 per cent. of the total nitrogen of the lecithin
used,

CHOLIN E-PLATINUM-CHLORIDE

Number of Amount of Percentage of
No. hours boiled substance, Found, in Calculated from _ theoretical
in grms. grms. WN of lecithin,ingrms. amount

1 2 0-4227 0-0634 0-1718 37-0

2 4 0-5244 0-0856 0-2132 40-1

3 5 0-7678 0-1330 03121 42-61
4 7 0-6820 0-1085 0-2773 39-13
5 10 0-4132 0-0676 0-1679 40-26

Average = 39-82 %
Resipur

The residue found after boiling was now examined for nitrogen in
the same way as the aleoholic part; here, as before, a certain amount of
nitrogen was always found. When compared with the nitrogen of the
methyl alcohol residue there was a slight increase, but it was by no means .
marked; here it may be mentioned that an absolutely exact quantitative
determination of nitrogen when dealing with these small quantities is
exceedingly difficult, and this may occasionally account for small
differences. In making two parallel blind experiments with chemicals
alone, it is often noticed that despite the most careful precautions, the
results are not absolutely the same; in general the difference is so
exceedingly slight as not to interfere with the result, but in the above
experiments very minute differences interfere to an extent that changes
the result to a slight degree.

EN-CONTAINING RADICLE OF PHOSPHATIDES 57

Bi In these residues the average amount of the total nitrogen found
vas 848 per cent. as shown in the table.

PEE acdes nin Resipue
: ped iper opin Total nitrogen found, Percentage
a co) vidaian leet 986
“ae 70 0-798 8-23
a 631 O48 7
1 8-17 nn 0672 $-29
oi 7-62 0-686 +0
Average =a 8-48 %
- Fivrrare ae

fedt ti,
% htt Owing to lack: of wiatenial only one experiment was lena in aie to
test what percentage of the total nitrogen was present in the filtrate;
after peilias for three hours the result gave 79°9 per cent. of the total
rogen : this agrees with that found in the alcoholic filtrate.
3 Two experiments were made as described on page (54), in which
the concentrated filtrates were divided into two exact parts, the nitrogen
f one ee directly estimated by Kjeldahl’s method, and the choline
. of the other by platinum chloride. Here, as with methyl
) “ a thes over 50 per: cent. of the total nitrogen was recovered as the
‘inum salt.

25 ¢.c. for NITROGEN 25 0c. FOR CHOLINE-PLATINUM-
‘ . CHLORIDE
N found, in Choline-platinum- Amount of Choline- Percen

mgrs. chloride, caleu- platinum-chloride total N as bes

lated from N actually found, in salt

found, in grms. grms,
2-50 0-057 , 0-0295 5L-75
3-22 0-0707 0-0358 50-6
Average = 51-18%,

the above experiments it is seen that in this pure lecithin not
more ‘than about 42 per cent. of the total nitrogen can be recovered as the
double salt of platinum when the substance is boiled with alcohol or water
saturated with barium hydrate : also that not more than about an average
_ of 50 per cent. of the nitrogen in the filtrate can be recovered as choline.
_ A comparison of results obtained by means of similar experiments from
*lecithol * shows that over 77 per cent. of the total nitrogen is recoverable
as choline, while a little over 90 per cent. of the filtrate nitrogen is

[ ee ee

1 ee

58 BIO-CHEMICAL JOURNAL

represented by choline actually obtained as the platinum salt. The
following indicates the general relationship as found by a few
experiments : —

Percentace or Tota N OBTAINED AS PERCENTAGE OF FILTRATE N OBTAINED
CHOLIN E-PLATINU M-CHLORIDE AS CHOLINE-PLATINUM-CHLORIDE
No. Lecithol Heart-muscle Lecithol Heart-muscle
Lecithin Lecithin
l 78°85 41-3 92-3 50-3
2 78-19 42-2 91-2 5l-1
3 77-91 39-1 —— 52-1
4 76-91 42-0 — 49-5

The result of all these experiments indicates that it is probable that
heart muscle lecithin differs in constitution from certain other lecithins
with regard to the manner of combination of its nitrogen; that all the
nitrogen present is not represented by the choline radical, and that this
lecithin contains another nitrogen-containing complex. Investigations
bearing on the nature of this nitrogen are at present being carried out.

CuorINn

Cuorin obtained as above described was also examined with regard to
its nitrogen-containing radical. This seems much more difficult to
hydrolyse than lecithin, but in common with Erlandsen, the writer, after
performing several experiments, is of opinion that the base of Cuorin is
not choline. The results of certain experiments with this and other
substances I hope to give in a later article.

OME OBSERVATIONS ON THE HAEMOLYSIS OF BLOOD
_ BY HYPOSMOTIC AND HYPEROSMOTIC SOLUTIONS OF
SODIUM CHLORIDE

By U. N. BRAHMACHARI, M.A., M.D., Lecturer on Medicine at the
a Cam pbell Medical School and First Physician, Campbell Hospital,
Calcutta.

(Received November 28th, 1908)*

In the Lancet for April 2nd, 1904, Sir A. E. Wright and Kilner, in
4 Wactibing a new method of testing the blood and the urine, state that
‘complete haemolysis takes place when a dark coloration is observed in
a mixture of one volume of suspension of erythrocytes with one volume
of a progressive dilution of a deci-normal sodium chloride solution in a
capillary tube. Later on, Wright and Ross? point out that instead of
making a preparation of the suspension of the red corpuscles, all that is
required is to take a measured volume of the blood and to mix with it two
__ - volumes of the progressive dilution of the deci-normal sodium chloride
fet” solution, and then to observe when the dark coloration takes place.
" It will be seen from the above, that it is assumed, firstly, that it is
possible to bring about complete haemolysis by mixing one volume of
blood with two volumes of a sufficiently dilute solution of sodium chloride,
and secondly, that the point of complete haemolysis can be determined by
letting light fall obliquely upon capillary tubes containing the mixture,
it being supposed to be arrived at when there is a dark coloration of the
___ blood, and there is no bright appearance to be seen in it. In this way,
Wright and Ross conclude that the average European blood haemolyses

eS completely with two parts of a5 sodium chloride solution.

IT am unable to agree with the observations of Wright, Kilner and
- Ross, that complete haemolysis can be brought about in the above way.

By treating normal blood with two volumes of 50 sodium chloride solution

3 ‘as well : as with two volumes of distilled water, q rates succeeded in demon-
strating that in none of these is complete haemolysis obtained.

1 consider that the most accurate conception of complete haemolysis
is that the blood supposed to be completely haemolysed should be perfectly
transparent, or if it is not perfectly transparent, it should give, on centri-
fugalisation, a sediment which, when thoroughly washed with an inactive
fluid should not be red. Further, it should not show the presence of

1. Calcutta postmark, November 12th, 1908.
2. Lancet, October 21st, 1908,

60 BLO-CHEMICAL JOURNAL

haemoglobin-containing erythrocytes, which can be stained with proper
stains. By an inactive fluid is meant a fluid which has no action on the
erythrocytes, and cannot, therefore, dissolve the haemoglobin contained
in them, but can dissolve any free haemogiobin.

To determine whether the sediment is red or not, the supernatant
fluid is to be pipetted off and the sediment treated with a solution of —
sodium chloride which cannot cause any more haemolysis in the blood
under consideration. The mixture is then centrifugalised again and the
sediment separated and treated in the same way as before, and if after a
sufficient number of washings, it is found that the supernatant fluid at
the top is colourless and the sediment is red, then it is evident that
complete haemolysis has not taken place; the sediment may further be
tested for the presence of haemoglobin-containing corpuscles, and stained
with a proper stain to show the presence of stained erythrocytes. If, ou
the other hand, the sediment is colourless, then evidently complete
haemolysis has taken place. !

Under ordinary circumstances, an =) sodium chloride solution will

serve the purpose of the inactive fluid mentioned above. We shall,
however, see, later on, that this solution cannot always be used for the
above purpose, as, for instance, when the blood has been previously treated
with a saturated sodium chloride solution.

We began our investigations by testing different specimens of blood
from the healthy students of the Campbell Medical School, Caleutta.
The blood of a large number of students was examined in the above way,

n : i
100 sodium chloride

solution, generally the latter. In none of these cases did I observe
complete haemolysis conforming to the definition given above. In other
words, I always obtained a red sediment after the blood was treated in the

above way. At the same time the dark coloration described by Wright

n n

and others was generally obtained with two volumes of —— a to a

the diluting fluid being either distilled water or

sodium chloride solution.
That the red sediment obtained in the above experiments contains

undissolved erythrocytes can be shown in the following way :—-
(1) The sediment though insoluble in 0 sodium chloride

solution is dissolved after being repeatedly washed with

mn
100
case may be.

sodium chloride solution or distilled water as the

HAEMOLYSIS OF BLOOD 61

(2) The sediment shows the presence of haemoglobin-containing
oe, _ erythrocytes under the microscope.

= ~ (3) The sediment, when stained with a proper stain, shows the
presence of stained erythrocytes.

In some of my cases I put the mixture of blood with distilled water,
a well as the mixture with sodium chloride solution, for nearly
. » hours in corked tubes, and it was found that complete haemolysis

a fat taken place even after this period, the temperature of the room
ping 29°C. during the day.

‘Phe question now arises as to how many parts of distilled water or
_"

sodium chloride solution can completely haemolyse one part of

= human blood. I have made dilutions of blood several times with one,
a two, and up to nine parts of distilled water, as well as +00 sodium

- chloride solution, and have obtained the red sediment in all of them after
= repeated washing of the sediment with a sodium chloride solution.
_ The sediment also showed the presence of haemoglobin-containing
erythrocytes, which took easily eosin stain. In one case I diluted a
specimen of blood with 40 parts of distilled water, and kept the mixture

for twelve hours in a small tube, and could detect the presence of haemo-
globin-containing erythrocytes, easily taking the eosin stain.

The corpuscles containing haemoglobin, and which are found in the
ee cerment described above will, in future, be called sediment corpuscles.

“4 The sediment corpuscles can be fixed in pure methyl alcohol or
aus alcohol, and stained with a dilute solution of eosin in water, by
_ immersing the slides from twelve to fourteen hours in the solution, or may

‘: be stained by mixing the sediment with a dilute solution of eosin in ——

“0
gaan chloride solution.

a. 2 append here a plate showing the sediment corpuscles after being
: fixed and stained in the above manner, they having been obtained by
a mixing human blood with 2 vols. of 0 ’ sodium chloride solution, and
the mixture left undisturbed for one hour at the temperature of the
room (29°C.).

ee

62 BIO-CHEMICAL JOURNAL

A similar phenomenon is seen when blood is treated with nine volumes
of ia sodium chloride solution, except that the sediment corpuscles are

much fewer in number and the destructive changes noticed in them are
more marked.

The laking of blood by hyposmotic sodium chloride solutions has been
supposed to be due partly to osmosis and partly to the specific sensibility
of the cortical layer of the erythrocytes or the membranes holding the
haemoglobin within the corpuscles. I consider that at least a third
factor is present upon which the above phenomenon is to some extent
dependent. If we examine the sediment corpuscles, it is easily seen
that a large number of them have undergone marked changes in shape,
size and in the amount of contained haemoglobin. Some of them are

Sediment corpuscles obtained after treating
one part of human blood with two parts of

165 sodium chloride solution. (4 oil
immersion. No. 3 eye piece.) Depth of

shading shows amount of haemoglobin.
Drawn by B, L. Doss, Caleutta.

resistant in the sense that they have not at al! discharged their haemo-
globin. But there are others which show marked diminution in the
amount of haemoglobin. Some show marked changes in the distribu-
tion of their contained haemoglobin, as compared with the normal (see
plate). Evidently in these the cortical layer of the envelope has been
ruptured, leading to partial escape of the haemoglobin. What is it,
therefore, that prevents the remaining portion of the haemoglobin from
being completely discharged? The most probable assumption is that the
process is to some extent allied to Mass Action that takes place in
- chemical reactions. In other words, there probably exists a union allied
to chemical combination between the haemoglobin of the erythrocytes
and other portions of their structure, perhaps including the salts. This
combination is probably broken up when water enters their structure,
e.g., when they are treated with hyposmotic sodium chloride solutions, but
the amount of decomposition will depend upon the relative masses of
the interacting compounds.

HAEMOLYSIS OF BLOOD 638

Resistance or tur Eryrurocyres to HAEMOLYSIS UNDER

ABNorMAL CoNDITIONS

If one volume of normal blood is mixed with two volumes =

sodium chloride solution, slight haemolysis is not infrequently observed,

while with —_ it is often distinct or sometimes even marked. In certain
n

forms of anaemia, 3 causes no haemolysis, while go causes very

slight or no haemolysis. In other words, in some forms of anaemia, the
blood resists haemolysis more than normal blood. Captain McCay by
estimating the haemosozic value of serum in certain forms of anaemia,
also arrives at the same conclusion. He thinks that this might be due to

__ the presence of something of the nature of an antihaemolysin.!

Curicat Data or A Case or ANAEMIA Havinc Hieu Resistine

Power to HAEMOLYsIs

Patient (aet. 25) was admitted into my wards on September 16th,
1908. Condition on admission: patient anaemic, slight oedema of the
extremities, no albument in the urine, stools contain ova of ankylostomata.
On September 22nd, 1908, he had red cells—2,700,000, haemoglobin—-20

per cent. One volume of blood plus two volumes of oy sodium
n

chloride—no haemolysis; one volume of blood plus two volumes of sy

sodium chloride—very slight haemolysis. The patient has been treated
with thymol since admission, but. has been worse since coming into
hospital. He is now markedly oedematous, is more anaemic, and his
condition is considered hopeless. On November 10th, 1908, his erythro-

cytes showed more resistance to haemolysis, two volumes of 5 plus

one volume of blood not showing the slightest amount of haemolysis. To
determine whether this resisting power was due to anything present in
the serum, I washed the erythrocytes several times with a deci-normal
sodium chloride solution, till the supernatant fluid obtained on centri-
fugalisation was found to be perfectly free from the slightest trace of
of albumen. One volume of the suspension of the erythrocytes was treated

with two volumes of oe sodium chloride solution, the resulting mixture

1, MeCay, Bio-Chemical Journal, Vol. T11, 1908, p, 97.

64 BLO-CHEMICAL JOURNAL

did not show any haemolysis at all. Salinity was 0585 per cent., and
alkalinity, estimated in the way pointed out by Moore and Wilson,}
was 0095 H,SO,. Red cells—1,770,000; haemoglobin—-13 per cent. It
will thus be seen that the resistance to haemolysis was not due to
anything present in the plasma, as the same resistance was observed when
the serum was replaced by a deci-normal sodium chloride solution.
There was a marked diminution in the alkalinity of the blood, which can
not, however, account for the resistance of the erythrocytes to haemolysis.

BeHAviour or THE Eryrurocytres or MAN AND THE RABBIT TOWARDS
SATURATED SOLUTION OF SopruM CHLORIDE

When one volume of human blood is mixed with fifteen volumes of a
saturated solution of sodium chloride in distilled water the mixture at
once becomes turbid. This turbidity is quickly followed by a marked ~
solution of the erythrocytes, and the mixture at the same time becomes
clear to a great extent. In the rabbit’s blood no such clearing up of the
mixture takes place in a short time, and the fluid remains turbid for a
longer time. If the rabbit's blood, after it has been treated in the above
way, be centrifugalised within ten minutes, it is found that the super-
natant fluid is faintly red, showing that only a slight haemolysis has taken
place by this time, contrary to what is found in the case of human blood
in which the supernatant fluid is found to be markedly red. _ If, however,
the sediment of the rabbit's blood obtained above, is mixed with the super-
natant fluid at its top, it possesses the remarkable property of dissolving
to some extent, showing as it were that some haemoglobin was squeezed

out of the erythrocytes during the process of centrifugalisation. The

n

same sediment, when treated with O° sodium chloride solution, dissolves

to a much greater extent. It is thus evident that the undissolved
erythrocytes are markedly altered in their constitution after the treat-
ment of the blood with saturated sodium chloride solution. Examined ,
under the microscope they are found to be much contracted, but most of
them retain their globular shape and do not look crenated or wrinkled.
The most probable explanation of this haemolysis appears to me to be a
marked change in the outer walls of the erythrocytes brought about by
the sodium chloride of the saturated sodium chloride solution; probably
a sort of combination takes place between the sodium chloride and the
outer layer of the erythrocytes which finally leads to its destruction.
When the blood is mixed with the saturated sodium chloride solution, no
1, Moore and Wilson, Bio-Chemical Journal, Vol. 1, 1906, p. 297.

ae eS eee ee eee
a F ie i ~~ ae

HAEMOLYSIS OF BLOOD 65

| bt water comes out of the erythrocytes by the process of osmosis and
ey accordingly contract; when the sediment from the above is treated

_ with = sodium chloride solution water re-enters their structure, and,

as a result of this, they try to expand and regain their original size.
But either they burst before or as soon as they recover their original
size, or it may be that the water dissolves the compound of sodium
chloride and the outer wall of the erythrocytes and consequently a marked
haemolysis results. ‘The initial turbidity, mentioned above, is probably
due to the production of the compound, which is probably very easily
decomposed. It is evident that osmosis alone cannot explain the
haemolysis of blood by saturated sodium chloride solution. The
remarkable phenomenon of haemoglobin coming out of the corpuscles
during centrifugalisation is probably explained by assuming that the
damaged walls of the erythrocytes allow haemoglobin to pass out of them
2 by a process allied to filtration under very high pressure. As soon as the
unstable compound of the sodium chloride with the outer layer of the

erythrocytes is decomposed, the latter behave like small spheres of sponges

ig containing dissolved colouring matter.

4
4

66

FURTHER OBSERVATIONS ON THE ACTION OF MUSCARIN
AND PILOCARPIN ON THE HEART

By HUGH MacLEAN, M.D., Carnegie Fellow, formerly Lecturer on
Chemical Physiology, in the University of Aberdeen. aw

From the Physiological Laboratory, University of Aberdeen
(Received December 7th, 1908)

In a former paper! I described the parallelism which obtains between
vagus inhibition and the effects of muscarin and pilocarpin on the hearts
of certain vertebrates. This parallelism has two aspects :—

T—Paratier Distrrevtion or Errecrs oN THE Parts oF THE HEART —

All the evidence afforded by my experiments goes to show that in
the vertebrate heart (adult) the action of muscarin and _ pilocarpin
reproduces in a remarkable way the effects of stimulation of the inhibitory
nervous apparatus, the incidence of the effects on the different portions of
the heart being similar in the two cases. Only the parts of any particular
type of heart that are supplied with inhibitory nerves are acted on in the
characteristic fashion by a suitable dose of muscarin or pilocarpin—
causing an arrest which, like that induced by vagus stimulation, is set
aside by atropin.2 Thus the ventricle of the eel and tortoise are not
acted upon, while that of the frog and newt are, the latter very strongly.
It is not simply that the parts endowed with the highest power of
spontaneous rhythm, are more readily acted upon by the drugs*; the case —
of the newt’s ventricle, with little or no spontaneous rhythmic power,
affords important evidence in this connection.

TI—Parattet VartaTion IN EFFrEecTIVENESS OF VaGus INHIBITION
AND THE Two Drvueas

Such variation may be due to different causes, such as :—

(1) Seasonal changes associated with the breeding season.
Inanition may play some part in frogs and other animals
that have been in captivity for considerable periods, but
similar changes were observed in recently caught eels.

1, This Journal, Vol. III, p. 1.

2. The drugs were tried both by dropping on the heart and by intravascular injection-
methods which, as is known, may not in the case of some drugs yield identical results.

8. See Gaskell’s criticism (Journal of Physiology, Vol, VIII, p. 408) of Kobert’s results
(Arch. f. erp. Pathol. u. Pharmakol., Ba. XX, s. 92).

ACTION OF MUSCARIN AND PILOCARPIN 67

- (2) Overdosing with the drugs so that immunity becomes
established.!
(3) The ‘exhaustion’ following prolonged stimulation of the
inhibitory nerves.
The activity of the inhibitory nervous mechanism was tested by : —

(a) Reflex excitation of the vagus.

(b) Faradisation of the nerve in the usual way.

(ce) Faradisation of the sinus in the eel and newt, and of the
sino-auricular junction in the frog (‘posterior white
crescent’); weak and moderate currents were employed, such
as when applied in normal heart give the characteristic
inhibitory effects readily abolished by atropin.

In the course of my work I was quite aware (in 1905) of the fact that
when the usual stimulation of the vagus nerve or the sino-auricular
junction in the amphibian heart failed to inhibit—-whether from absence
of inhibitory power depending on seasonal changes, overdosing with
musearin or pilocarpin, exhaustion of the inhibitory apparatus after
repeated and prolonged stimulation, &e.—it was still possible to arrest the
heart beat for very long periods by running up the secondary coil of the
induction machine with the electrodes applied in a certain manner to the
sino-auricular junction. I did not, however, attach any importance to
these results—obtained by the use of such strong currents and differing
in yarious ways from the phenomena of ordinary vagus inhibition—as
indications of the condition of the inhibitory nervous. apparatus. That
_ faradisation with certain strengths of current can stop the heart in certain
conditions when pilocarpin is ineffective, has very lately been noticed
independently by McQueen.?
_ Some more recent observations I have made entirely confirm the
opinion I then formed in regard to this form of arrest being due to local
effects on the cardiac tissue produced by the powerful current and quite
different in their nature from true vagus inhibition. The arrest is easily
got in the amphibian heart (frog, newt, salamander) when a sufficient
strength of current is employed, more especially when the electrodes
; are made to embrace the sino-auricular junction. Application in the
| usual way (1-2 mm. apart) to the ‘ posterior crescent’ may also produce
4 the phenomenon, but not with so much facility, as a rule. During the
- application of the current there is commonly an acceleration of the heart
% beat, and then, when the current is stopped, a standstill of auricles and

1. See Marshall, Journal of Physiology, Vol. XXX1, p. 129.
2. This Journal, Vol. ILI, p, 402 (Preliminary communication).

— es oe - - ae a a Al ee + ies. 2 ic ° - Ny 2
2", : Bhai tie : re. 7 P ‘ a s ae ot ae “A att aii r es

ed ages, ou oe

68 BLO-CHEMICAL JOURNAL

ventricle of very variable duration—from a few seconds up to eight or ten
minutes or more. Sometimes the period of standstill begins while the
current is still applied. The sinus commonly goes on beating regularly
during the whole period, but its beats fail to be propagated to the auricles
and ventricle, being blocked at the place where the current was applied.
Sometimes the sinus stops also. A single stimulus applied to the ventricle
during the period of standstill gives a single reversed beat of ventricle and
auricles, the contraction failing to pass the blocked area to the sinus.
Again, when recommencement takes place, a condition of partial blocking
at the faradised area often remains evident for some time, only every
second or third beat passing (at first) from sinus to auricle and ventricle.
When the current is kept applied for some little time a naked-eye change
becomes evident in the faradised area—a distinct whitening or opacity
near the electrodes and between them ; a feature noted long ago by Wesley
Mills as a result of the application of very strong currents. The narrow
isthmus of the sino-auricular connection where these blocking effects are
readily induced is, of course, of known structural and physiological
peculiarity and complexity.

When the heart recommences beating after a period of standstill, the
beats are of good strength, as are also beats artificially excited by direct
stimulation of the ventricle during the arrest of the normal rhythm.
This is, of course, what might be expected in a ventricle which only
stopped on account of blocking; there is no sign of the marked depression
of contraction force and excitability such as may be induced by vagus
inhibition.

When the action recommences there is no increase in force beyond
what is shown by the first beat—except such as one may see in the frog-
heart as a result of a prolonged period of quiescence (staircase), e.g.,
resulting from Stannius ligature. Re-application of the current during
standstill may excite beats during its continuance; this obviously arises
from spreading of current to the auricle. There are various other minor -
features in the behaviour of the heart under the influence of strong
currents, applied in various ways, which hardly seem to call for detailed
description—results depending on escape of current to other parts of the
organ, electrolytic and thermal effects, &c. Blocking can, of course, be
induced in this region, as elsewhere, by other and simpler means which
do not involve the same complications.

The same heart may be brought to a standstill répildtedly by applying
the current at intervals. There is not the same susceptibility to ‘ fatigue ’
that is seen in the case of true inhibitory nerve excitation. Again, a

ACTION OF MUSCARIN AND PILOCARPIN 69

weak ill-nourished heart seems to be more easily stopped than a vigorous
- one—in contrast to what has been often noticed in regard to vagus

The arrest occurs almost immediately or after some seconds, or, in the
case of somewhat less powerful currents, after more prolonged and
repeated application. The exact strength of current sufficient to produce
these effects varies according to circumstances—conditions affecting
nt density in the faradised tissue, size and position of electrodes,
988 or emptiness of the organ, the duration of the application of the
ent, the state of the heart, ete.
| With one Daniell cell in the primary circuit and an ordinary

Be is induction machine, the secondary coil at 7-8 em. usually suffices ;
the current is perceptible on the tongue at about 24-25 cm., and causes
cular contraction when applied to a frog’s sciatic nerve at 45-50 cm.
Such a current is strongly felt on the dorsum of the dry hand. With the
small Harvard inductorium the secondary coil has to be moved up to about
4 or 5cm.; the current is perceptible on the tongue with secondary coil at
full distance and inclined to 45°; and stimulates the sciatic nerve with the
secondary coil at its full distance and inclined to 85° or 86°. Under certain
_ conditions as to the mode of applying the electrodes, duration and density
of current, state of heart, etc., currents weaker than these may produce
essentially similar results.

The currents employed to produce the cardiac arrest are quite
sufficient, when applied to the frog’s sciatic nerve, or skeletal muscle, to
_ cause the depression or abolition of excitability, well known as the
Wedensky effect. They are obviously quite unsuitable for testing the
condition of the inhibitory nervous mechanism of the heart. It is clear
that an incautious strengthening of the faradic current beyond certain

a may lead to confusion between two different forms of cardiac
rest :—

(1) Ordinary inhibition due to excitation of the inhibitory
ne nervous apparatus, and

(2) A stoppage depending on the direct effect of the current

on the faradised tissue causing depression, blocking, etc.
This phenomenon may be termed pseudo-inhibition.

It is by no means impossible that such confusion may have sometimes
oceurred in the past; it is often difficult in consulting the writings on the
subject to be quite sure as to the exact strength of current employed, the
duration of its passage, and the precise mode of applying the electrodes ;
a brief application of current is, ceteris paribus, less likely to give the

70 BIO-CHEMICAL JOURNAL

second-mentioned form of arrest. Burdon-Sanderson, in his F Practical
ixercises,’ long ago directed the faradisation to be for a second or less,
the points of the electrodes being not more than a couple of millimetres
apart, and Porter, in his recent * Introduction to Physiology ’ (1906)
specifies faradisation ‘ for a moment.’ ‘Lhe effect of atropin on the arrest

indicates the type to which it belongs.

>

Fic. 1. Faradisation of heart immune to pilocarpin; pseudo inhibition.

Figs. 1 and 2 show tracings of the ventricle of a frog’s heart. Fig. 1
is from a heart which was found to be immune to pilocarpin. Faradisa-
tion of the white crescent in the usual way with the secondary coil at
10-12 cm. (primary cell 09 volt) gave no sign of inhibition. The
secondary coil was then moved up to 6°5 em. and standstill resulted, as

seen in the tracing—beginning when the current was stopped after being

applied for about fifteen seconds. Fig. 2 shows a similar standstill in the
same heart after a large dose of atropin. The sinus action continues in
each case, though much more visible in the tracing in Fig. 1. The time

tracing shows seconds in eat h case,

Fic. 2. Faradisation of the same heart after atropine ; pseudo-inhibition.

The parallelism between the effects of muscarin and pilocarpin and
those of electrical stimulation of the inhibitory nerves is not affected by

the results described above—obviously due as the latter are to the

what holds

= tion of the sinus in the eel and newt or of the sino-auricular junction in

a a a a ad a
< ih ah Bias a Thai
7 — a ee, oe il ‘ ve " '
s —_ i iy ve = oP. i ret
: : eat ee ee, . BA cin 7 = pe “i
‘ * i igs ak
> fice ga! 2 ¢ ? ei the ‘
5 L os : es
y ’ . aN

ACTION OF MUSCARIN AND PILOCARPIN 71

x effects of the excessively strong current on the cardiac tissue
"(causing blocking, etc.) and not affected by atropin—in contrast with
— regard to the inhibitory influence excited by faradisa-

the frog when done in the ordinary way with moderate currents, an
experiment long familiar to physiologists.

It is known that atropin also abolishes the local inhibitory effects
produced by weak faradic currents in the auricle of the tortoise and eel,

a __ and in the auricle and ventricle of the newt. Evidence has been adduced
by Gaskell" in the case of the tortoise’s auricle, and by MacWilliam? in

that of the eel, showing that these local inhibitory effects are due to
excitation of inhibitory fibres in the auricular wall and not to direct effects

on the muscle. The exact place of attack by atropin (whether on

“nerve endings’ or muscle proper) is immaterial in the present
connection ; the essential point is that the influence of inhibitory fibres
(preganglionic or postganglionic) is cut out by atropin. Even strong
currents are then unable to cause the usual inhibitory arrest in a state
of relaxation which is, in ordinary circumstances, easily and strikingly
obtained in the eel’s auricle and newt’s ventricle.

In the light of the evidence available from various sources regarding
the action of muscarin and pilocarpin, it is clear that their effects are
closely bound up with the functional efficiency of the inhibitory nerves,
and their special distribution in the various parts of the vertebrate heart.
The effects of the drugs are, with most probability, to be ascribed to a
* stimulating ’ influence (by chemical interaction, no doubt) on some part
of what, for the sake of brevity, has been termed ‘ nerve endings’ of the
vagus (postganglionic) fibres, i.e., on some part of the linkage between the
nerve fibre and the fundamental contractile mechanism of the muscular
fibre (myoreural junction, ete.).

I have to thank Professor MacWilliam for the facilities he has
kindly afforded me for carrying out my experiments in his laboratory.

1. Sehifer’s Tert-book of Physiology, Vol. TI, p. 208.
2. Journal of Physiology, Vol. VI, p. 228.

72

THE OCCURRENCE AND DISTRIBUTION OF CHOLESTEROL |
AND ALLIED BODIES IN THE ANIMAL KINGDOM —

By CHARLES DOREE, M.A., B.Sc., Lindley Student of the University
of London. ,

From the Physiological Laboratory, University of London
(Received January Ist, 1909)

A large number of observations, accumulated in the course of the past
fifty years, have shown that cholesterol is a constant constituent, so far
as they have been examined, of all animal tissues. The investigations
recorded relate chiefly to man and a few of the more common mammals
and birds, in all of the organs of which, and in most of their fluids and
secretions, cholesterol] has been found. It is evident that the constant
presence of such a substance indicates its great importance from a vital
standpoint, and necessitates its recognition as a primary constituent of
all protoplasm. ‘This conclusion has recently been fully emphasised
by various writers, who have called into question the dictum of Pfliiger
‘Nur das Eiweiss ist lebendig,’ and have assigned to the lipoid class of
bodies, of which cholesterol is one of the most important members,
functions second only in importance to those of the proteins themselves.
What these functions may be in the case of cholesterol we do not know.
They would, one must suppose, be quite different from those of the
proteins, for whereas these bodies are, from a chemical point of view,
unstable, being capable of rapid transformation into innumerable other
substances, more or less complex as required by the life of the organism,
cholesterol is characterised chemically by a remarkable stability. That
it is constantly associated in the cell with lecithin has long been known ;
that lecithin can assist the action of various poisons which act by
producing haemolysis, and that cholesterol, on the other hand, functions .
us an antitoxin in this respect, are facts which have recently come to
light, and in them, possibly, may be found a clue to the imporfant
question of the part played by cholesterol in the life of the organism.

It is a remarkable fact that while cholesterol has been isolated
uniformly from the tissues of mammals and birds, whenever the lipoid
bodies contained in them have been investigated, no other substance
similar to it had, until quite recently, been discovered in animal proto-
plasm.'| From the vegetable kingdom, on the other hand, a large

1. With the exception of the isocholesterol of Schiilze, which will be discussed in the sequel.

Pt ra. ae
¥. 7 “ae an a rag Oe oF

DISTRIBUTION OF CHOLESTEROL 73

Diaibex of bodies, isomeric with, and no doubt closely related to,
cholesterol, have been obtained—the phytosterols. Although recent
_ observation shows that the number of these is not so great as previously
supposed, owing to the fact that many of those described under different
names are mixtures of phytosterol itself, which occurs in a pure form
in wheat germ,! with other allied bodies, still, not only do many different
vegetable cholesterols exist, but, frequently, different forms occur
im the same plant, each associated with one or other of the plant
_ structures. With cholesterol these various phytosterols make up the
cholesterol group. Except in the case of cholesterol itself; we have
_ little or no knowledge of the chemical nature of any of these bodies, but,
: for the present purpose we may consider the cholesterol group as
consisting of :—
ae (a) A number of isomeric secondary alcohols C,,H,,0, which are all
unsaturated, and possess one well-defined double fab: in the molecule.
l= These are all laevorotatory and give the colour tests of Salkowski and

Liebermann.

d : (b) A number of bodies, almost entirely of vegetable origin, which
are undoubtedly similar to those of the preceding class, but which differ
from them in their general properties, and, probably, are not isomeric

with cholesterol. Their relationships to cholesterol and to one another
are entirely unknown.
(ec) The natural derived product coprosterol, with which, for reasons
given below, may be classed isocholesterol.
If, then, cholesterol is a body which is one of the primary constituents
____—s of animal protoplasm, we should expect to find it not only in the highly
; ji ‘organised animals, but throughout the series from Chordata to Protozoa—

_ or if cholesterol itself were not present its place should be filled by other
_ and closely related forms. In the latter case it might be found that
each of the great classes of the animal kingdom was characterised by the
presence of a different member of the cholesterol group. On the other
hand, if cholesterol is not of primary importance to all forms of life,
it is not impossible that animals might be found into the composition of
whose protoplasm it did not enter. In regard to the lipoids contained in
the lower animals our information is very scanty. In the older literature
we find occasional references to cholesterol as a constituent of one or other
of these, but no precise identification of the body was, as a rule, made.
Quite recently the work of Henze? on the Sponge, Suberites domuneula,

1. Burian, Monatshe/te, XVTIT, 553.
2. Zeit. physiol. Chem., 1908, LV, 427.

OES 2. ee y i — a
* a co) ‘

74 BIO-CHLEMICAL JOURNAL

and that of Menozzi and Moreschi! on the pupae of Bombyx mori, have
shown that in the invertebrata there exist cholesterols which differ from
the common cholesterol of the vertebrata. The present writer, on the
other hand, in a preliminary investigation,? found that in two species of

sea anemone (Coelenterata), cholesterol itself was present, and with a

view to throwing more light on the distribution of cholesterol in the
animal series, and to solve, if possible, some of the questions referred to
above, it was decided to examine the cholesterol bodies contained in a
number of animals typical, so far as possible, of each of the great sub-
kingdoms of Animalia. An account of these experiments will be given
in the following pages. tate

CLASSIFICATION AND T'ypES SELECTED

The types selected for experiment may be classified as follows :—*

CHORDATA—

MammMaria Lepus euniculus, the rabbit.

Reprinia Tropidonotus natric, the grass snake.

Pisces Scomber scombrus, the mackerel.
MOLLUSCA-

Gastrrovopa Buccinum undatum, the whelk.
ARTHROPODA—

Crustacea Carcinus menas, the crab.

INSECTA Blatta orientalis, the cockroach.
ANNULATA—

Cua@roropa Launbricus terrestris, the earth worm.
ECHINODERMATA~—

ASTEROIDEA Asterias rubens, the starfish.
COELENTERATA—

Actinozoa —- Tealia crassicornis, |

ake i sea anemones.
Actinia equina, j

PORIFERA
Cliona celata,
E phydatia fluviatilis, |

Spongdla lacustris, eae wise Bete

Atti della R, Accad> dei Lincei [V] XVII, 95.
2. Proc. physiol. Soc., XXXVII, July, 1908.

3. Based upon the system given by Parker and Haswell, T'ext Book of Zoology, Vol. 1,
Macmillan, 1897.

DISTRIBUTION OF CHOLESTEROL 15

Meruop or ExPreriMENT

The animals were, if necessary, killed with chloroform, and then
‘ground up in a mortar with coarse sand, plaster of Paris being added from
time to time to dry up the crushed material. If the tissues were very
tough, as in the case, for instance, of the starfish, they were first passed
- through a mincing machine, all juices expressed being collected in plaster
____ of Paris. The mass so obtained was left till it had become perfectly dry
and hard, after which it was coarsely powdered, and then extracted in a
large Soxhlet apparatus with ether, for periods which varied between
seven and fifteen days. The ether solution so obtained was at once
saponified with a large excess of alcoholic solution of sodium ethylate,
according to the method of Kossel and Obermiiller. To ensure complete
saponification the liquid was allowed to stand at least twelve hours, after
which the precipitated soaps were filtered off and well washed with ether.
The filtrate was then shaken several times with water to remove traces of
soap, the ethereal solution dried over calcium chloride and the ether
distilled off.
‘The crude unsaponifiable residue so obtained is generally described
as cholesterol, and weighed as such. But in the case of animal extracts,
it always contains brownish oily, or resinous substances, which may
constitute as much as three-quarters of the total residue. For the
purpose of the present work it was necessary to isolate the cholesterol
in a pure state so that its identity could be established beyond question.
If the quantity of residue was sufficient it was dissolved in absolute
alcohol, filtered, the hot alcohol diluted to about 90 per cent. strength and
the liquid allowed to crystallise. A microscopic examination at this
point gave valuable indications of the presence of cholesterol, pure or in
an admixed state. After the separation of crops of crystalline matter, the
filtrates, evaporated to dryness (or if very small the original unsaponi-
fiable material), were treated by the following method, in which the
cholesterol is isolated in the form of cholesterol benzoate. ‘The dried
residue was dissolved in pyridine (20 ¢.c. to each gram of substance), and
un excess of benzoy! chloride dissolved in pyridine added and the mixture
allowed to stand overnight. In this way the cholesterol is converted
quantitatively to benzoate.! The pyridine solution was then poured into
water, which precipitated the organic matter, and the whole allowed to
stand, if necessary, until the colloidal solution, which at first formed,
cougulated. This usually happened in a few hours, but very occasionally

1. Dorée and Gardner, Proe. Roy. Soe., Series B, 1908, LXXX, 228.

, 7 oe eS

—_—T- Ss Oe

Rel ie a
ode ay .2°* a a= ‘i

76 BIO-CHEMICAL JOURNAL

salting out was necessary. After washing and drying the precipitate it
was boiled out with absolute alcohol, the liquid allowed to cool, and the
benzoate filtered off. Cholesterol benzoate is very insoluble in alcohol
in the cold-according to a determination made for the author, 100 e.c.
of commercial absolute alcohol dissolve only 0°12 gram at 20° C.—so that
the unchanged oily matters can be completely separated from the
benzoate, and if the quantity of alcohol used is measured, a correetion can
be applied to the quantity of benzoate obtained. The benzoate crystallises
well from ethyl acetate in large rectangular plates.

The formation of a benzoate by this method serves in itself for the ?

recognition of cholesterol, since, so far as the author's investigations at
present carry, vegetable cholesterols do not benzoylate, or if so,
imperfectly under the conditions described. But the characteristic
properties of cholesterol benzoate enable it to be identified with certainty.
It dissolves with difficulty in alcohol, from which it crystallises in square
plates; it melts at 145°C. to a turbid liquid, which clears suddenly at
178°C., and this on cooling, shows a brilliant display of colours, of which a
light blue at the higher temperature followed by a deep violet at the lower
are very characteristic. Some other benzoates of members of this group are
now known to give colours on solidifying. One being the sitosterol of
Burian,! the colour phenomena of which are described by Ritter,? and the
others the new sponge cholesterols described in the present paper. The
colour phenomena of these, however, are quite different from those shown
by cholesterol benzoate.*

A second important method for the isolation and identification of
cholesterol consists in its conversion to a dibromide by the method of
Windaus.* In this process one gram of the substance dissolved in 10 c.c.
of ether is mixed with a solution of 0°5 gram of bromine dissolved in 5 c.e.
of glacial acetic acid. The mixture is allowed to stand at 0° C., when a
crystalline precipitate of cholesterol dibromide forms, which is filtered off

and washed successively with acetic acid, 50 per cent. acetic acid and °

water, after which it is pure. By this means an almost complete
separation of cholesterol from, at any rate, vegetable cholesterols is
possible. Whereas 100c¢.c. of the ether glacial acetic acid mixture
dissolve only 0°6 gram of the dibromide at 20° C., phytosterol dibromide

1. Loc. cit.; from wheat germ. This body should now be called phytosterol.
2. Zeit. physiol. Chem., XXXIV, 431.

3. The colours may be observed very clearly if the benzoate is melted in a thin layer
be —— oe a: plates. As the cooling is comparatively slow the order of the colours can be .
exactly notec

4. Ber., 1906, XX XIX, 518; Chemiker Zeit., 1906, XXX, 1011.

|

“

——

—

Te se

He op Sa

ee ee Se!

DISTRIBUTION OF CHOLESTEROL 77

is very soluble. This reaction, until recently, was characteristic for
cholesterol, but it has lately been found that the bombicesterol of Menozzi
and Moreschi, and the sponge cholesterols described below also give the
reaction in the same way as cholesterol. Since all these dibromides give
the same analytical figures, the melting points become important as a
means of identification. | Windaus gave the melting point of cholesterol
dibromide, prepared and purified by his method as 123°C. Bondzynski
and Humnicki,! who prepared it by the addition of a solution of bromine
in light petroleum to a solution of cholesterol in the same solvent, found
109°C. as the melting point. Menozzi and Moreschi again,? in an
examination of the cholesterol from hen’s eggs (which proved identical
in all respects with the cholesterol from gall stones), found that the
dibromide prepared according to Windaus’ instructions, but subsequently
erystallised from alcohol, melted at 111°C. The figure obtained for
the bromine content of this body agreed closely with that required for
cholesterol dibromide, so that the substance was not altered in constitution
by treatment with alcohol. The present writer, on the other hand, has
found difficulty in obtaining it from alcohol in a crystalline form, but
in order to decide the question of the melting point a sample of cholesterol
was prepared from human gall stones in the usual way and converted to
the dibromide by Windaus’ method, the instructions as to washing, etc.,
being exactly followed. The pure white substance was, without further
purification, dried in vacuo, and found to melt at 123° C., decomposing
at a few degrees higher. Windaus’ statement is thus perfectly correct.
Clionasterol dibromide, prepared and washed in an exactly similar way,
melted sharply at 114°C., and decomposed rapidly between 116° and

120°C. The melting point of bombicesterol dibromide, similarly made,
is stated to be 111° C.

Systematic Examination or Types SELECTED

In the protoplasm of vertebrate animals cholesterol is universally
present. In man it is especially abundant in the brain (2°5 per cent.)
and nervous tissue (1 per cent.), while in the fat it is found to the extent
of 0°35 per cent. and in dry muscle 0°23 per cent. It is also present in
bile, 0°07 per cent., in blood, 0-09 per cent., and in milk, 0°082 per cent.
In herbivorous animals, whose food contains no cholesterol, the figures,
so far as they are available, follow very much in the same order. The

“1. Zeit. physiol. Chem., XXII, 396.
2, Atti della R. Acead. dei Lineei [V] XVI, 91.

78 BIOCHEMICAL JOURNAL

fact that the cholesterol obtained from various animal sources is one and the
same substance, has been demonstrated by Menozzi,! who made a careful
comparison of the physical and chemical properties of the cholesterol
isolated from cow's milk, from horse brain, and from hen’s eggs.
Recently, also, Diels and Linn,? on account of an apparent slight
variation in the chemical behaviour of cholesterol obtained from egg-yelk,
made a comparison of this specimen with others obtained from gall stones
and from brain, respectively, and showed that when properly purified
the melting point and rotation of the cholesterol from each of these
sources was substantially the same. eine
Cholesterol is the only body of its kind so far found in connection
with the higher vertebrate animals, with the exception of the so-called
iso-cholesterol, to which reference has already been made. This has
only been obtained from the wool-fat of the sheep.’ It differs markedly
from cholesterol in its properties, and seeing that it is a product excreted
by the skin, may, perhaps, be classed with coprosterol, which is a
derivative of cholesterol, normally excreted in the faeces by men,* and
by carnivorous animals when fed on a raw brain diet.5 The relation of
coprosterol to cholesterol is at present unknown, but coprosterol, unlike
cholesterol, is a saturated compound, and probably contains two hydrogen
atoms more in the molecule. It is not, however a simple reduction |
product of cholesterol, and, most probably, has a somewhat different
carbon skeleton. But the physical properties of coprosterol stand in
marked agreement with those of iso-cholesterol. Alone among the known
members of the cholesterol group, iso-cholesterol does not give the
characteristic colour-reaction with sulphuric acid and_ chloroform
(Salkowski’s test); coprosterol gives it in a modified way. Both these
bodies too, are, unlike all the others, dextro-rotatory.. An alteration in
rotatory power from negative to positive has been observed generally to
be brought about by the saturation or modification of the side chain of
cholesterol, which contains the double bond. The dextro-rotatory power .
of coprosterol is, no doubt, due to such a modification, but whether that
of iso-cholesterol can be ascribed to a similar change is less probable,
since, according to Darmstidter and Lifschiitz,® it is, like cholesterol,
unsaturated, readily absorbing bromine in chloroform solution, — Tso-
Atti della R. Accad, dei Lincei (V) XVII, 91.
Ber. 1908, XLI, 260.
Schiilze, Ber. V, 1075; VI, 251; XII, 249.
Zeit. physiol. Chem., XXII, 396.

Proc. Roy. Soc., B. LXXX, 228.
Ber., 1898, XXX, 97, 1122.

ee

-~
eo

DISTRIBUTION OF CHOLESTEROL 79

cholesterol is still further distinguished from cholesterol and its isomers
by the melting point and crystalline form of its acetate and benzoate.
While the benzoates of the latter all melt at about 145° C. and crystallise
from alcohol in rectangular plates, the benzoate of iso-cholesterol melts
at 191°C. and crystallises from alcohol in needles. These facts are
collected together in the following table :—~

ACETATE BENZOATE
Crystal form pn YW tay ey See a q
(dilute M.p. {ajo Mp. Crystal M.p. Crystal
alcohol) (ether) form form
Oblong 147° —31° 114° Plates 145° ~—s— Plates
2 Frocks
Tsocholestero! ide ks 137° +60° Below100° Amorphous 191° Needles

100° +24° 88° Needles 122° Plates

__ The presence of cholesterol in the blood and eggs of birds has been
frequently observed. In the case of reptilia its occurrence has not,
apparently been recorded, but it is well known as a constituent of oils
obtained from various species of fish. Dog-fish oil, for example, is said
to contain four to five per cent. of cholesterol. In order to investigate
this point, and to ascertain whether any other member of the cholesterol
group was present in the tissues of reptiles and fishes, an experiment was
carried out with a typical representative of each of these classes. For
purposes of comparison the cholesterol contained in the whole body of a
small mammal (rabbit) was estimated by a similar process.
CHORDATA. Mamaria. Lepus euniculus, the rabbit—A rabbit
weighing 28 kilos. was killed, and the blood, which weighed 75 grams,
was collected separately, mixed with sand and plaster of Paris, and, when
dry, ground to powder; the rest of the animal, including the fur, was
then passed several times through a sausage machine, the minced material
being ground up with coarse sand, and dried with plaster of Paris. The
total mass obtained was extracted for twenty days with ether, and the
solution saponified with sodium ethylate and washed as already described.
The total unsaponifiable matter weighed 6:0 grams. It was at once
dissolved in pyridine and treated with an excess of benzoyl chloride. The
product which was obtained on precipitation with water was boiled out
with absolute alcohol, in which it was very difficultly soluble, and after
re-crystallisation from ethyl acetate appeared in the typical crystalline
form of cholesterol benzoate. In all 4112 grams of benzoate were
obtained, which melted correctly and showed the characteristic colour
play. This corresponds to 3239 grams of cholesterol, or 0117 per cent.
A microscopic examination of the residues soluble in alcohol

Ng ES es ee
ieee! = (i ae eae
’ an ——

80 BIO-CHEMICAL JOURNAL

(consisting of brown oily matter which slowly became resinous) euvetield
no signs of any other crystalline matter.

Rerrmia. Tropidonotus natriz, the grass snake.—Four grass sniadals
weighing 246 grams, were killed, passed through the mincing machine
and ground up with sand and plaster of Paris. The dry mass was
extracted for eight days with ether, and the pale yellow extract saponi-
fied with sodium ethylate. A large quantity of a pale brown soap
separated, which was filtered off and washed. From the filtrate, after
the usual treatment, 0°44 gram of unsaponifiable matter was obtained,
which was usually free from colour. It was dissolved in alcohol and-
crops of crystals weighing 0°12 gram were separated. These, after
re-crystallisation, melted sharply at 145° to 146°C., and under the
microscope showed the characteristic crystal form of cholesterol. On
benzoylation of the residues in pyridine solution 0°09 gram of benzoate
was isolated. This was very difficultly soluble in alcohol, from which it
crystallised in square plates. It melted at 144° to 145°C. to a turbid
liquid, which cleared at 178° C., and on cooling showed the colour play
of cholesterol benzoate. The total yield of cholesterol was thus
0°21 gram, or 0-08 per cent. No other similar body was observed in
the residues.

Pisces. Scomber scombrus, the mackerel.—Five mackerel, weighing
1,452 grams, were ground up in a mortar with coarse sand mixed with
plaster of Paris, and allowed to dry. The mass was then reduced to a
coarse powder and extracted in a Soxhlet apparatus for eight days.
After washing in the manner described above, the ether solution was pale
yellow, and on evaporation left 1:07 grams of brownish crystalline matter.
This was dissolved in 90 per cent. alcohol, and under the microscope
showed perfectly formed, typical, cholesterol crystals, no sign of any
other crystal or mixed form being observed. A small crop, weighing
(08 gram, was isolated, re-crystallized from acetone, and found to melt
at 145° to 146°C. The whole filtrate from this was benzoylated in -
pyridine solution and yielded 0°37 gram of a benzoate, which, after
re-crystallisation from acetic ether, appeared in the form of shining
rectangular plates. These melted at 145°C. to a turbid liquid, which
became clear at 180°C., and showed the colours characteristic of
cholesterol benzoate. The alcoholic filtrate from the crude benzoate
weighed 0:43 gram, and consisted of a brown resin which, on long
standing, only showed minute traces of crystalline matter. The
cholesterol obtained was thus about 0°42 gram, or 0°03 per cent., and no
other similar body was observed.

7 =

DISTRIBUTION OF CHOLESTEROL 81

MOLLUSCA. Gastrropopa. Buceinum undatum, the whelk.—A
number of whelks, weighing 1,179 grams after removal of the shells,

were minced up and treated with sand and plaster of Paris, the mass

extracted for six days with ether, and the extract saponified as described
above. The soaps were considerable in quantity and dark brown in
colour; the filtrate was also dark brown, and on evaporation left 4°5
grams of beautifully crystalline unsaponifiable matter (0°38 per cent.).

This was dissolved in dilute alcohol, and a microscopic examination

showed the presence of typical cholesterol crystals, the same crystalline
forms being observed down to complete dryness. Three crops of white
erystals, weighing 0°75, 0°25 and 0°09 gram were separated and found to
melt at 142° to 143°C. The whole 1:09 grams put together and
re-crystallised from acetone, melted at 144° to 145° C., and appeared as
pure cholesterol. The whole of the residues from these were dissolved
in pyridine and treated with benzoyl chloride in the usual way. By this"
means 0°37 gram of cholesterol benzoate was obtained, which, after
re-crystallisation from ethyl acetate, melted at 145°C. to a turbid liquid,
which became clear at 185° C., and on cooling showed the characteristic
colour play. A sample of the pure cholesterol dissolved in ether was
then treated with a solution of bromine in glacial acetic acid. In a few
minutes the solution set almost solid. The precipitated dibromide, after
washing and drying in the usual way, melted at 118° to 119°C. with
decomposition, in which respect it agreed fairly well with cholesterol
dibromide. The total yield of cholesterol was thus approximately 1-46
grams, or (124 per cent.

The residue left after separation of the benzoate was a clear, brown
oil, which, after standing for six months, contained no trace of
crystalline matter, and showed no signs of solidification.

Crruatopopa.— Henze! has recently shown that the hepato-pancreas
of Octopus vulgaris contains fats and ‘ not inconsiderable quantities’ of
cholesterol.

ARTHROPODA. Crustacea. Carcinus menas, the edible crab.—
A whole crab, weighing 538 grams, was ground up with sand, etc., and
extracted for eight days with ether. On saponification of the extract
a considerable quantity of a stiff reddish coloured soap was obtained. The
filtrate from this, after washing, was pale yellow, and left 1°0 gram, or
19 per cent., of erude residue. This, dissolved in 90 per cent. alcohol,
left a small quantity of brown insoluble substance. A microscopic

1, Zeit, physiol. Chem., 1908, LV, 438.

82 BIO-CHEMICAL JOURNAL

examination of the solution showed abundance of typical cholesterol
crystals, and from it 0°25 gram of nearly pure cholesterol was ultimately
separated. After re-crystallisation from acetone, it was pure white, and
melted sharply at 146° to 147°C. — Its identity with cholesterol was
confirmed by the preparation of the following derivatives :—

(a) The dibromide: 0°12 gram of substance was dissolved in 2 ¢.c. of
ether and mixed with 0°6 c.c. of Windaus’ solution of bromine in glacial
acetic acid. The mixture set almost immediately to a mass of crystals,
which were filtered off and washed. After drying in vacuo, these melted
at 120°C., turning brown and decomposing at 124° to 126°C. The
filtrate on treatment with water deposited a white solid, which contained
bromine, and melted at 100° C., decomposing at 120°C.

(b) The benzoate: This was prepared by heating the dried substance
with benzoyl chloride to 165° ©. for five minutes. The residue was boiled
out with alcohol, and the insoluble white crystalline residue purified by
crystallisation from ethyl acetate, after which it melted at 145°C. to a
turbid liquid, became clear at 178° C., and on cooling showed the colour
play characteristic of cholesterol benzoate. |

The cholesterol isolated was, therefore, about 0°25 gram, or 0048
per cent.

Insecta. Bombyx mori, the silkworm moth.—Owing to the fact that
an oil has been obtained commercially from the pupae of this moth,
several investigations have been made with a view to decide whether the
unsaponifiable residue of the ‘ chrysalis oil’ contains cholesterol—as do
other animal oils—or a phytosterol. Lewkowitsch,! who first examined
the question, came to the conclusion that cholesterol was present in the
oil, since, by the use of Bémer’s acetate method, he was able to isolate
from it an acetate whose melting point, after successive crystallisations,
finally stood at 114°C. M.Tsujimoto,? however, who has recently made
a further investigation, obtained 1°63 per cent. of unsaponifiable residue
from the oil, from which, after repeated purification, he prepared a
substance of M.p. 143° C., which in crystalline form and the meltin
point of the acetate (125° C.) corresponded rather with phytosterol.

Realising that the question of the presence or absence of cholesterol
could be definitely decided by the application of the methods described in
the introduction to this paper, a quantity of the pupae was obtained
from France with a view to the extraction of the cholesterol body from

1. Zeit. fur Nahr. u. Genussmittel, 1907, XIII, 552.
2. Journ. of the Coll. of Engineering, Tokyo, Japan, 1908, TV, 63.

DISTRIBUTION OF CHOLESTEROL 83

_ them on a large scale, when a paper by Menozzi and Moreschi! appeared
which rendered the proposed investigation unnecessary. These authors
showed that chrysalis oil contains 10 per cent. of its weight of unsaponi-
fiable matter, and that in this there are probably at least four different
substances, two of which are paraffin hydrocarbons. One of these hydro-
carbons has apparently the formula C,,H,,, and a melting point of 62°.
The main constituent, however, is a new isomer of cholesterol, to which
the name of bombicesterol has been given, and which, in all its properties,
bears an extraordinarily close resemblance to cholesterol itself. The
melting point and rotation of the two bodies, and of their benzoates
_ correspond exactly with one another, and the melting points of the
____ dibromides (111° C.) are stated to be the same. The crystalline form
-___ of bombicesterol differs somewhat from that of cholesterol, but the most
important difference between the two lies in the melting points of the

formiates and acetates. Bombicesterol formiate melts at 101°C. (as
compared with 96°C.), and the acetate at 129°C. (as compared with
114°C.). The benzoate is said to show on melting the phenomenon of
liquid crystals, but apparently this is not accompanied by a play of
colours, as in the case of cholesterol benzoate.

The authors further mention that from the crude acetate of bombi-
cesterol they obtained, by the method of Windaus and Hauth, two
dibromides. One of these remained in the ether acetic acid solution, and
was thrown out on the addition of water. On reduction it yielded
bombicesterol acetate, melting at 129°C. The other dibromide

erystallised out at once in the ether acetic acid solution, and on reduction
gave an acetate of melting point 114°C., which is the same as that of
cholesterol acetate. The point is apparently still under investigation, but
it is obvious that the possibility of the presence of cholesterol in the pupae
of Bombyx mori is not wholly excluded.

In order to discover whether the larvae of Bombyx mori contained
cholesterol, a quantity of the worms, weighing 102 grams, were kept
without food for three hours and then killed. On grinding them up with
sand a strong leafy smell was observed. The dried mass was extracted
for five days with ether, and the deep green extract saponified. The soap
was moss green in colour, this being, no doubt, due to the presence of
chlorophyll. The green filtrate from the soap, also, on washing, became
pale yellow, all*the green colour going into solution in water. The
unsaponifiable residue weighed 01 gram (or 01 per cent). With this

1. Atti della R. Acead. dei Lincei, (V| XVI, 95.

‘ Sipeor ae id al

B4 BIO-CHEMICAL JOURNAL

small quantity little more than a microscopic examination was possible.
The residue was dissolved in 90 per cent. aleohol, and a drop of the solution
was allowed to crystallise on a slide. At first, long, very narrow plates,
not quite rectangular, were seen, which, although quite different from
those of cholesterol, answered to the description of the crystalline form of
bombicesterol (lamine allungate ed acuminate) given by Menozzi and —
Moreschi. The later crystals were in the form of long, narrow hexagons, —
agreeing in this respect with those of the vegetable cholesterols. They
probably consisted of the phytosterol of the mulberry leaves on which the
worms were fed.

The residue was crystallised from methyl alcohol,! in which it was
very insoluble, and the small crops of white crystalline matter obtained —
melted at 125° C., but not sharply.

The eggs of Bombyx mori were examined in 1885 by Tichomiroff.?
The eggs, which are laid in the summer, develop up to a certain point, pass
the winter in this state, and continue development in the spring.
Tichomiroff, using Hoppe-Seyler’s methods, obtained the following
percentage figures: (A) for eggs which had reached the winter stage;
(B) for eggs on the point of hatching :—

Fat Lecithin Cholesterol
A dew i 8-08 1-04 0-40
B say oe 4-421 1-76 0-35

As, however, no examination was made of the ‘ cholesterol’ obtained,
its identity with either cholesterol or bombicesterol must remain uncertain.

Blatta orientalis, the cockroach.—Cockroaches, weighing 194 grams,
were killed and dried in the steam oven, after which the weight was -
65°5 grams. The solid matter was ground to powder with a little sand,
and extracted with ether for fourteen days. The brownish ether extract
on saponification gave a brownish coloured soap in considerable amount,
which was very solid when dry. The unsaponifiable residue, which was —
very liquid at 100° C., weighed 0°5 gram. It was treated with absolute
alcohol in which a part readily dissolved, leaving an insoluble portion,
which, on warming the liquid to about 40° C., melted, forming heavy oil
drops. By decanting off the alcohol from these and repeating the process
several times, a separation of the insoluble and the soluble parts was —
effected.

A. The part insoluble in aleohol weighed about 0°2 gram. Tt was

1. The methyl alcohol used here and elsewhere was purified, but not absolute.
2. *Chemische Studien iiber die Entwicklung der Insecteneier,’ Zeit. vhysiol. Chem., 1X, 525.

DISTRIBUTION OF CHOLESTEROL 85

tely soluble i in petroleum ether, and Sip so in benzene. From

i eca-arsralline On heating daw ly in a wide <p tube, it
melted at 42°5° to 43° C., and solidified sharply at 40°5° C. When warmed
with pyridine it dissolved, but on cooling, the solution set to an opaque
gelatine-like mass. It did not give Salkowski’s test and did not absorb
ine. This substance, therefore, is very probably a hydrocarbon
‘similar to those found in the pupae of the silkworm moth.
____B. The part easily soluble in alcohol. The alcohol was diluted to
out 90 per cent. strength, and on allowing a drop of the solution to
7 stallise under the microscope, thin plates closely resembling those of
terol were observed. As the substance was somewhat soluble, even
in the diluted alcohol, methyl alcohol was used as a solvent, and from this
after repeated crystallisation, about 0-1 gram of crystals, with a constant
melting point of 139° to 140°. was obtained. That these were not
cholesterol was shown by the following tests:—-(i) When moistened with
lgpkibentrated sulphuric acid the edges of the crystals did not turn red.
(ii) On benzoylation in pyridine solution no action took place, as the
substance easily soluble in alcohol, crystallisimg in oblong plates and
melting sharply at 139° to 140° C., was recovered unaltered.
Pe The body gave Salkowski’s test with chloroform and sulphuric acid
exactly as cholesterol, but owing to the small quantity of the pure
substance that could be isolated, it was not possible to characterise it
_ further. It would, however, appear to be similar in its general behaviour
__ to other members of the cholesterol group.
ANNULATA. Cueroropa. Lumbricus terrestris, the earth-worm.
ee! -worms,! weighing 286 grams, were killed and ground up with
___ sand and plaster of Paris. The dry mass was extracted for five days with
i ether, and the extract, on saponification, gave a considerable quantity of
_ a pale brown, slimy soap. The filtrate from this yielded 0-95 gram of
4 unsaponifiable residue, which was practically solid at L00°C. It was
soluble in 90 per cent. alcohol, with the exception of a trace of brownish
x matter. A microscopic examination showed very badly-formed choles-
s terol plates, together with a few long, blunt-pointed, needle-shaped
__erystals. Crops weighing 0'1 gram and 0°22 gram, respectively, were
. separated, and, after re-crystallisation from acetic ester, melted at 142°
to 148° C., and consisted largely of plates. The 0-31 gram of substance
___ thus obtained was benzoylated in pyridine solution in the usual manner.

AL The worms used were the lob or dew worms of the freshwater fisherman.

86 BIO-CHEMICAL JOURNAL

‘The benzoate obtained was very insoluble in alcohol, and after re-crystal-
lisation from acetic ester, melted at 144° to 145°C. to a turbid liquid,
which became clear at 180° C., and on cooling showed a brilliant colour
play. ‘he filtrate from this was examined under the microscope, when,
beside a few crystals of the benzoate, a number of minute spherular
crystals were observed. .

All the residues, consisting of a dark brown, sticky resin, were put
together, dried, and treated with benzoyl chloride in pyridine solution.
No crystalline matter, however, could be obtained from them. The
total yield of cholesterol was thus about 0°3 gram, or 0'1 per cent. The
tissues of the worm thus contain cholesterol itself as their most important
cholesterol constituent, although some indication of the presence of —
another crystalline body in small amount was observed.

ECHINODERMATA. AsreromeEa. Asterias rubens, the starfish.
~The starfish, which were still living when received, weighed 1,658
grams. ‘They were minced (tlie juices being collected in plaster of Paris),
ground up with sand and plaster of Paris, the mass allowed to dry, and
again reduced to powder. After extraction for twenty-one days with ether
a pale red-brown extract was obtained, which, on saponification gave a
large quantity of a firm porridge-like soap. The red-brown colour of
the filtrate was removed on washing with water, leaving the ether
solution almost colourless. The crude residue, which was yery fluid at
100°, weighed 3°5 grams, and dissolved almost completely in 90 per cent.
alcohol. A microscopic examination showed that a mixture of substances
was present. Plates, closely resembling those of cholesterol, were
seen, together with masses of minute curved needles. After many
attempts it was found possible to effect a partial separation in the
following way. ‘he crude substance was treated with dry pyridine.
A portion proved very insoluble in the cold, but on warming went into
solution coming out immediately on cooling as a jelly of minute flexible .
needles. These were filtered off and treated again with pyridine until
they no longer gave the Salkowski test. The pyridine filtrates were
collected together and treated as described below.

A. The substance insoluble in pyridine was obtained in silvery flakes,
which, after drying in a vacuum, were soft and wax-like to the touch.
Heated slowly in a wide capillary tube it melted at 56° to 57°C. It did
not give Salkowski’s test, and did not absorb bromine in carbon
bisulphide solution. In all about 0°2 gram was separated.

DISTRIBUTION OF CHOLESTEROL 87

On analysis, using a copper boat filled with coarse copper oxide :—

. 0-0987 gave 0-3014 CO, and 0-1301 H,0
eal from which C = 83-28; H = 14-64 per cent.

These figures are difficult to reconcile with any probable formula.
C,,H,,,0 requires C=855; H=14'4, but no C,, formula agrees at all
with the results, which only allow for 2 per cent. of oxygen.

In its properties this body bears a curious resemblance to the little-
known higher aleohols which occur in unsaponifiable residues of animal
and plant tissues. Among these are cetyl alcohol C,,H,,O, of melting
point 50° C., found in spermaceti and the sebaceous glands of geese and
ducks; cery! alcohol, C,,H,,0, of melting point 89°C., and myricyl
alcohol, C,,H,,0, melting point 85° C., which are both constituents of
beeswax; and chortosterol,' C,,H,,O, present to the extent of 0°2 per
cent. in the blades and stalks of the grasses.

B. ‘The portion soluble in pyridine apparently contained a member
of the cholesterol group since it gave Salkowski’s test, and readily
absorbed bromine. It was very difficult, however, to separate this
from small quantities of A. The substance was thrown out of solution
in pyridine by the addition of methyl alcohol, and was then boiled with
very dilute ethyl alcohol. The body A then remained undissolved, and
the solution was poured off from the oil drops and the process repeated.
A small quantity of fairly pure material was finally obtained, which was
instantly soluble in a small quantity of pyridine. From dilute alcohol
it crystallised in needles, which melted at 143° to 144°C. When treated
in chloroform solution with concentrated sulphuric acid, the lower layer
first became cherry red, with a green fluorescence, the upper layer slowly
becoming of the same colour without the fluorescent green. That the
body was not cholesterol was rendered probable by the following experi-
ments. The acetate, made in the usual way, crystallised in plates of
melting point 128° to 130°C. On heating a portion of the substance
with benzoyl chloride for five minutes to 165°C. the melt proved com-
pletely soluble in cold absolute alcohol, so that no cholesterol benzoate
could have been formed.

In the absence of further investigation it can only be stated that the
tissues of the starfish do not, apparently, contain cholesterol, but, instead,
two other bodies, one of which may be a hydrocarbon or a complex
alcohol, while the other shows some points of resemblance to the members
of the cholesterol group.

1. Dorée and Gardner, Proce. Roy. Soc., B, LXXX, 212. Described as Hippocoprosterol by
Bondzynski and Humnicki, Zeit. physiol. Chem., XXII, 306. i

88 BIO-CHEMICAL JOURNAL

COKLENTERATA. <Acrinozoa. Tealia crassicornis and Actina
equina, sea anemones.—About two kilograms of moist sea anemone, con-
sisting of the two species above mentioned in about equal proportions,
were obtained from the Marine Biological Station at Plymouth. They
were ground up with coarse sand, dried in the oven, and the mass
extracted for eight days with ether. On saponification of the ethereal
extract a considerable quantity of a red, slimy soap was precipitated,
which was filtered off and washed with ether. The filtrate, after washing
and drying, left a dark brown, unsaponifiable residue, which was dissolved —
in alcohol and decolourised with animal charcoal. A microscopic examin-
ation of the colourless filtrate showed typical crystals of cholesterol, and
from the solution crops of crystals, weighing 1:0 gram, were separated.
These melted at 143° to 144° C., and after re-crystallisation from acetic
ester, at 145° to 146°C., and in all respects appeared identical with
cholesterol. ‘That this was the case was confirmed by the following
observations and experiments : — |

(a) Determination of rotatory power:

(i) 0-666 gram made up to 25 ¢.c. with chloroform gave (in mean) - 1-03" in a 1 dm. tube
at 20°C. [a] } = -38°.

The rotatory power of cholesterol for the same concentration! would
be — 373°.

(ii) 0-660 gram made up to 25 c.c. with pure acetic ester gave —0-73° in a 1 dm. — at
20°C. [a] P = —27-6°.

The rotatory power of cholesterol (prepared from weiss in this
solvent, is given by Diels and Linn? as — 25°69.

(b) Benzoate: 0°5 gram of the substance was dissolved in pyridine,
and treated with an excess of benzoyl chloride in the usual way. The
benzoate obtained was very insoluble in absolute alcohol, from which it
crystallised in typical square plates. The re-crystallised product —
melted at_ 145°C. to a turbid liquid, which became clear at 175° C.,
and on cooling showed the colour play of cholesterol benzoate.

(ec) Acetate: 0°3 gram of the substance was acetylated by boiling with
acetic anhydride for twenty minutes. The liquid was poured into water,
and the precipitate washed and dried in the usual way. The product,
after re-crystallisation from acetic ester, melted at 114° to 115° C., and

1. Calculated from Bomer’s formula quoted in Lewkowitsch, Chemistry of the oils, fats and

waxes,

2. Ber., 1908, XLI, 286.

“=. ee ae Sere ee
a es ee el
: ae ee te. ;

DISTRIBUTION OF CILOLESTEROL 89

showed opalescent colours on solidifying. It thus agrees exactly with
cholesterol acetate, which is the only acetate among the cholesterol
group which has been observed to give a colour play on solidification.

(d) The purified esters obtained in the above experiments were
dissolved in ether and saponified with sodium ethylate. After washing
with water, the ether solution, on evaporation, deposited pure white
cholesterol, which, after re-crystallisation, melted at 146° C.

(e) The pure cholesterol thus obtained was converted to the dzbromide
by the method of Windaus. The liquid set to a crystalline, semi-solid
mass, and the dibromide thus formed, after washing and drying in
vacuo, melted at 120° ©., decomposing a few degrees higher.

An examination of the residues obtained in these experiments, showed
no trace of any other crystalline substance. The sea anemone,
_ therefore, contains cholesterol identical in all respects with that of the
higher animals.

PORIFERA. — [Suberites domuncula|, Cliona celata, Ephidatia
fluviatilis, sponges.—It being thus established that cholesterol, identical
in all respects with that contained in the tissues of the Vertebrata, occurs
in animals as low down in the scale as the Coelenterata, the investigation
of the cholesterol of the sponges becomes of great interest. Many authors
have regarded the sponges as being very closely related to the Coelenterata,
but apparently the points of resemblance upon which their conclusions
were based are superficial only, and the two groups are now considered to
be markedly distinct. But when the similarity which exists between
the earlier stages of the members of the two groups is taken into account
_it becomes very probable that both were derived from the Protozoa through
a common Metazoan ancestor. But, if the Coelenterata are considerably
higher in the scale than the Porifera, the latter undoubtedly stand nearer
the Protozoa than any other types of the Metazoa. Formerly, the
sponges were regarded as Protozoa, but, ‘a sponge is to be regarded as a
colony of Protozoa only in the sense in which the same may be said of
one of the higher animals. 1t consists of a complex of cells, some of which
have a considerable degree of independence, and some of which have a
close resemblance to certain Protozoa; but the same is true of one of the
higher animals, the difference being one of degree and not of kind.’! So
that the Porifera, if not actually members of the lowest group of animals,
represent the lowest types available for an investigation such as the
present. They are, too, curiously plant-like in the readiness with

1. Parker and Halswell, Text Book of Zoology, 1, 116. Macmillan, 1897.

90 BIO-CHEMICAL JOURNAL

which their form becomes modified by external conditions and environ-
ment—a peculiarity which is shared by none of the higher animals. So
that we have here, possibly, the transition group, from a chemical point
of view, between the Coelenterata which contain cholesterol, and the
plants which contain phytosterols.

The cholesterol of the sponges was first referred ‘5 by Kriikenberg;?
who stated that the red colour of Suberites domuncula was due to the
presence of a lipochrome, which, under the influence of sunlight,
became converted into cholesterol. This remarkable statement was,
apparently confirmed by Cotte? in the course of his work on the Sponges.
In 1904, however, M. Henze* re-examined the question, and showed that
there was present in Suberites domuncula a body of the cholesterol type,
to which he gave the name of spongosterol, with a formula C,,H,,0.
He also showed conclusively that no relation whatever existed between this
substance and the lipochrome of the sponge. Quite recently,4 Henze
has published a further investigation of spongosterol, in which he shows
that the body is a definite substance and not a mixture. Attempts to
resolve it by the preparation of an acetate dibromide, after the method
used by Windaus and Hauth® for the separation of phytosterol and
stigmasterol, showed that it was not apparently unsaturated like the
other cholesterols. Instead of an acetate dibromide a mono-brom-
substitution product was formed. Analyses of this and other derivatives
led Henze to conclude that spongosterol has the formula C,,H,,O, so that
it contains two hydrogen atoms more in the molecule than cholesterol.
Its properties, though they hear a general resemblance to those of
cholesterol, differ considerably in degree from those of that body.
Spongosterol is very insoluble in methyl alcohol, from which it crystallises
in plates, many of which have curiously toothed edges. It melts at
123° ©, and is laevorotatory, having [a], = —19°6° C. It gives Salkowski’s
test in a modified way, but Liebermann’s exactly as cholesterol. The
acetate melts at 124° C., and the benzoate at 128 °C., the latter becoming °
a clear liquid at this temperature and showing no play of colours on
solidification. | When treated with bromine in carbon bisulphide solution
it decolourised the bromine, but no definite addition or substitution product
could be isolated from the solution. In all these respects spongosterol
shows differences from cholesterol, while in some of them it rather

Vergleich. physiol. Studien, Vol. T1, p. 50; Vol. TIT.

Bull. Scientifique de la France et de la Belgique, xxKvint, 509.
Zeit. Physiol. Chem., 1904, XLI, 109.

Tbid., 1908, LV, 427

Ber., 1907, XL, 3681.

ua, wwe
ak ae ad i

DISTRIBUTION OF CHOLESTEROL 91

ae. resembles coprosterol, with which, if Henze’s view is correct, it is
isomeric. But it may be pointed out that the fact of its forming a
-_mono-brom- acetate when that ester is treated with bromine, instead of
an addition product, does not of itself prove the unsaturated nature of the
body. For, although cholesterol acetate, when treated with bromine,
gives a dibromide, the benzoate on the other hand yields, as shown by
Obermiiller,! a crystalline and very stable mono-brom-derivative
C,,H,,0.Br.C,H,0 of melting point 136°C. The reactions of the double
link, which is einai in the end chain of the cholesterol molecule,
| undoubtedly present some peculiarities which are not at present capable
of explanation. Similar causes may be operative in the case of
spongosterol and may mask the presence of an unsaturated linking.
Hausmann,? and Abderhalden and Le Count,* have shown that the anti-
haemolytic action of cholesterol is only exhibited so long as the hydroxy!
group and the double link are intact. If the latter becomes saturated
the anti-toxic power ceases simultaneously. Although in the case of
the lower Invertebrata there is no question of cholesterol playing the
part of an anti-haemolytic agent, yet this anti-haemolytic action no doubt
affords an indication of one at least of its functions in the life processes
of cells. As we have seen, all the animal tissues so far examined contain
an unsaturated isomer of cholesterol, and, apparently, the case is the
same with plant tissues. If the spongosterol of Suberites domuncula,
therefore, does not contain an unsaturated linking, it would point to the
; B fact that the cholesterol function in this, and if the name be any index of
its distribution, in all sponge protoplasm, differs decidedly from that of
, ial of other forms of animal and vegetable life.

In order to throw some further light on these questions, and to
ascertain whether spongosterol was a constituent common to all sponges,
the following experiments were carried out, using, in the first series,
the marine species Cliona celata. The results obtained show that in this
animal there is present apparently one cholesterol body only, which is
easily isolated in a state of purity. This new substance, while quite
different from spongosterol, bears a remarkably close resemblance both
to cholestero] itself and to bombicesterol, with which it is isomeric.
For this new cholesterol the name Clionasterol is proposed, as it indicates,
in some measure, the source from which the substance is derived.

. 1. Zeit. physiol. Chem., 1891, XV, 37.
2. Hofmeister Beitrige, 1905, VI, 567.
3. Zeit. Exp. Path. Ther., 1905, II, 199. b

92 BLO-CHEMICAL JOURNAL

INVESTIGATION OF Cliona celata

The gamboge-yellow coloured sponges were obtained from Plymouth,
und, after very gentle squeezing to remove superfluous water, weighed
1,172 grams. They were minced up, treated with sand aud plaster of
Paris in the usual way, and the dried mass extracted with ether for ten
days. On saponification a small quantity of dark, olive green soap was
obtained. he filtered solution, after washing, was a greenish yellow
in colour, and on evaporation left 3°2 grams of a bright orange yellow
residue, which was just soft at 100°C. This residue dissolved in
methyl alcohol, leaving a small quantity of a brownish red pigment,
which was practically insoluble in the usual solvents, with the exception
of ether. The clionasterol was very difficultly soluble in cold methyl
alcohol, crystallising out immediately on cooling. Examined micro-
scopically the crystals appeared in the form of leaves, the base being
broad and the smooth edges curving to a point. These leaves were
grouped in symmetrical clusters with the pointed ends outward.
Occasionally the edges were strongly notched. From the methyl
alcohol liquors 2°07 grams of white crystals were isolated (or 0'17 per
cent.), and from these, ultimately, 12 grams of highly purified substance
was obtained.

Clionasterol crystallises from absolute alcohol in needles; from
dilute alcohol in plates, some of which closely resemble those of
cholesterol, except that they are less regular in shape. It is diffieultly
soluble in methyl alcohol and in absolute and diluted ethyl alcohol,
easily soluble in petroleum ether, acetic ester and acetone. Its melting
point is 137° to 138°C., and this remained unaltered after repeated
crystallisation. It is laevorotatory, giving figures very near to those of
choltesterol itself. Thus 0442 gram (dried at 100°C.) made up to
25 c.c. with chloroform gave, in a 1 dm. tube at 18°C. (in mean),

a = —0-655°, whence [a) jf = —37°04°.

When treated with the usual reagents it shows the following colour
reactions: (a) When a few drops of concentrated sulphuric acid are
added to a solution of the substance in chloroform the upper layer imme-
diately becomes cherry red, and the lower pale yellow with a green
fluorescence (Salkowski). (6) When strong sulphuric acid is added to a
cold saturated solution of the substance in acetic anhydride the acid
layer becomes first reddish, then violet blue, and, finally, both layers

become green (Liebermann).

DISTRIBUTION OF CHOLESTEROL 93

‘To still further characterise this cholesterol, the following derivatives
were prepared ;—

~~ Chionasterol acetate-—0°5 gram of the substance, mixed with an equal

weight of anhydrous sodium acetate, was boiled for twenty minutes with
an excess of acetic anhydride, and the liquid poured into water and
treated in the usual manner. The acetate is difficultly soluble in
alcohol, from which it crystallises in large wide plates of irregular shape,
and in methyl alcohol from which it appears in the form of oblong
plates, which frequently overlap, forming a saw-like edge. It was
moderately soluble in acetic ester, crystallising out in clusters of very
long, thin, rectangular plates. The melting point is 133° to 134°C.
This substance proved very difficult to burn. A combustion carried out
in the ordinary way gave a carbon percentage three units too low. <A
better result was obtained using a copper boat filled with coarse copper

oxide.

0-1319 gave 0-3884 CO, and 0-1306 H,0
| C = 804; H = 11-0
Cy Hy O, requires C = 81:3; H = 11-2 per cent,

Clionasterol benzoate—06 gram of clionasterol and 0°75 gram of
benzoyl chloride were heated together for twenty minutes at a temperature
of 165°C. After cooling, the mass was boiled out repeatedly with
absolute alcohol, when the benzoate was left as a white crystalline powder.

_ Tt was almost insoluble in boiling absolute alcohol, and, for analysis, a
sample was exhausted with this solvent and then dissolved in acetic ester,

from which it crystallised in beautiful glistening leaves, which, under
the microscope, appeared as rectangular plates, always longer than wide.
In aleohol also it exhibits the same form, whereas cholesterol benzoate
erystallises in perfectly square plates from this solvent. Its behaviour
on heating was very characteristic, and was carefully cbserved. Heated
in a wide capillary tube it shrinks and begins to soften at 141°C.,
becoming a turbid, viscous fluid until it melts at 143° to 144°C. to a
perfectly clear mobile liquid. If the temperature is allowed to rise to
about 160° C., and the tube is then taken from the bath the following play
of colours is observed. A pale green first appears, turning to a greenish
blue, which then abruptly changes to a deep violet colour, which persists
for some time, only gradually fading away. In one or two specimens of
the benzoate the violet tint was noticed to change to an emerald green,
but these specimens were not, perhaps, so pure as those on which the
above observations were made. These colours show slightly during

M4 BIO-CHEMICAL JOURNAL

melting, and may be demonstrated over and over again on the same
specimen.

For analysis the benzoate was dried at 100° C, and burnt in a eopper
boat filled with coarse copper oxide. ;
01527 gave 0-4654 CO, and 0-1399})H,O

Found Calculated for
Cx,H,0.C,H,0
ay ngs, wae 83-20
ms eee 10-20

Clionasterol dibromide.-0'35 gram of the substance dissolved ‘in
35 c.c. of ether were mixed with 2 c.c. of a solution of bromine in
glacial acetic acid, prepared according to the directions given by
Windaus.! The mixture, after standing in ice, deposited a crystalline
precipitate, which, after one hour, was filtered off and washed with dilute
acetic acid and water. The filtrate, on the addition of water, deposited
more solid matter. The precipitate was dried in vacuo and melted
sharply at 114°C., decomposing at 116° to 120°C. A specimen of
cholesterol dibromide, prepared in exactly the same way, and melted at
the same time, showed a ‘melting point of 125°C. A bromine estimation
gave the following figures :—

0-1046 gave 0-0725 AgBr. Found Br
C,H,,0. Br, requires Br

29-4 per cent.
29-3 per cent.

a

INVESTIGATION OF THE FRESH WaTER SPONGE

In view of the discovery thus made that spongosterol is not a con-
stituent of the protoplasm of Cliona celata, it was thought advisable to
extend the enquiry to a further member of the sponge group, and for
this purpose the freshwater sponge was selected. This animal is of

great interest for several reasons. From the point of view of this
investigation it is, perhaps, the lowest animal type that could be obtained
in sufficient bulk for examination. Then, again, it is one of those

species which, while indubitably animal, yet possess chlorophyll granules
in its tissues so that it can elaborate starch from water and carbon-
dioxide by photo-synthetic processes. Although these chlorophyll
granules are alien to the animal, being due to infection by a species of
alga, their presence tends, so to speak, to form a link between the
animal and the plant, and opens up the question whether the plant
activities might not determine the formation of a phytosterol in the cells

l. Ber. 1906, XXX1X, 518

DISTRIBUTION OF CHOLESTEROL 95

of the sponge. On the other hand, we might find the natural cholesterol

pS of the animal side by side with the natural phytosterol of the plant,

although the latter should, on account of the relatively small mass of
_ vegetable substance present with the sponge, be inconsiderable in
quantity, compared gvith the former.

Spongilla lacustris and Ephydatia fluviatilis, freshwater sponges.—
The two common British species of freshwater sponge, though designated
E. fluviatilis and S. lacustris, respectively, commonly occur together in the
same situations. The sponges used in these experiments were obtained
from the Thames, at Oxford, and, so far as a superficial examination
went, consisted chiefly of L. fluviatilis. They were only a pale yellowish
green, whereas S. /Jacustris is usually a dark green. There is
no doubt, however, that both species were present. After removal of
twigs and other débris the moist sponges weighed 1,285 grams. When
dried the sponge sarcode was left as a dusty powder, which was easily
separated from wood, weed and other foreign bodies. It was ground up
with sand and extracted for seven days with ether. The extract was a
deep green in colour, and after saponification with sodium ethylate,
left a considerable quantity of a jelly-like soap, which also was dark
green. As it has been stated that fats are not present in most sponges,
a portion of this soap was treated with water, in which it was soluble,
with the exception ofa trace of greenish pigment. After filtering this off
and acidifying with hydrochloric acid, a white precipitate was formed,
indicating the presence of an organic acid. The green colour was, no
doubt, due to chlorophyll, since, on washing the main ether extract, the
colour dissolved in the water, leaving the ether solution pale yellow.!
After drying and evaporating off the ether 716 grams of gamboge
coloured residue was obtained. This was soluble in alcohol, with the
exception of a small, olive green, sticky residue, which was not further
examined. The alcoholic filtrate was diluted to about 90 per cent.
strength, and a microscopic examination of it showed that two or more
substances were present. Large, badly-formed plates were seen, together
with rosettes of leaves and thin plates. The two bodies were differently
soluble in methyl alcohol, since, on allowing a hot solution of the crude
mixture to crystallise on a microscope slide, long, hexagonal erystals first
formed, and then, as the solvent evaporated, small plates, somewhat
like those of cholesterol, were deposited. Attempts were made to bring
aboqut a separation on these lines, but although a substance of melting

1. The chlorophyll is similarly removed on washing the ether extract of grass or of the
excrement of the herbivora. .

96 BIO-CHEMICAL JOURNAL

point 135° ©. was obtained from the more insoluble part, it was not pure,
and the soluble part, which was liquid at 100° C., was obviously a mixture.
It was finally discovered that the two constituents were very differently
soluble in light petroleum; one, which will be referred to as Spongilla
cholesterol A was readily, and the other, Spongilla cholesterol B, almost
insoluble in that solvent.

Accordingly 755 grams of fresh sponge was obtained and treated as
before, care being taken to remove all foreign vegetable matter. 413
grams of unsaponifiable residue were left, which was dissolved in méthyl
aleohol. On cooling, crops of nearly white crystals, weighing 1:27 gram,
were separated, only a very small quantity of crystalline matter remaining
in the mother liquors. The crystals were dried, powdered and boiled out
several times with petroleum ether. 0°47 gram of spongilla B was left, and
to this was subsequently added 0:19 gram, which had passed into solution
along with A and was recovered from the filtrate. The two bodies A and
B were thus present in approximately equal proportions, 0°66 gram of
each being obtained.

Spongilla A was purified by treating the crude substance with
cold petroleum ether and filtering from any of B left undissolved.
After the separation of B it was dissolved in 90 per cent.
alcohol. A drop of the solution examined microscopically showed
a large number of small oval-shaped leaves, and on allowing it to
erystallise slowly, the substance came out in large glistening leaves or
spangles. After purification and drying at 100° C. it melted at 138° to
139° C. It was diffieultly soluble in methyl aleohol, from which it
crystallised in leaves, closely resembling those of clionasterol, and
irregular plates, many of which had curiously toothed edgés; moderately
soluble in benzene and easily in light petroleum. It gave Salkowski’s test
exactly as cholesterol, and immediately decolourised bromine in carbon
bisulphide solution. It was characterised by the preparation of the
following derivatives : —

(i) The dibromide: 0:12 gram was dissolved in 2 c.c of ether, and
0-6 c.c. of Windaus’ solution of bromine in glacial acetic acid added. In
a few minutes the bromine was absorbed and a crystalline precipitate
formed, causing the whole mass to become semi-solid. After thorough
washing, as previously described, and drying in vacuo, the pure white
substance melted at 112° to 113° C., turning brown and decomposing
violently at 118° C. A qualitative test for bromine was strongly positive.

(ii) The benzoate: 0-12 gram of the substance was heated for five

DISTRIBUTION OF CILOLESTEROL 97

minutes at 165° C. with excess of benzoyl chloride. The melted mass was
boiled out several times with absolute alcohol, the nearly pure benzoate
_ being left. “This ester was practically insoluble in boiling alcohol. The
trace that was dissolved crystallised out, if cooled quickly, in star-like
groups of needles; if slowly, in large, irregular plates. After re-
erystallisation from acetic ester the benzoate, when heated in a wide
capillary tube, began to shrink together at 136° C., and melted at 141°
to 142° ©. to a turbid liquid, which became clear between 185° and
190° C., and on cooling showed a well-marked play of colours, in the
weer following order: greenish blue, dark blue, deep violet, green (transiently
and sometimes not at all), golden brown, finally becoming white. These
were observed between glass plates, and were carefully compared with
those given by other cholesterol benzoates, as will be described below.
‘In the characteristic properties of its benzoate and dibromide,
_ spongilla cholesterol A bears a remarkable resemblance to cholesterol
itself, and must undoubtedly be regarded as a true animal cholesterol.
Spongilla B was easily obtained pure by extraction with light
petroleum, as previously mentioned. It was almost insoluble in methyl
alcohol, and went into solution with difficulty even after the addition of a
little absolute ethy] alcohol. From this mixture it crystallised in long,
well-formed hexagonal plates, which closely resembled those of
phytosterol, the two long sides being parallel and the two short ones at
each end symmetrically placed, one being longer than the other. In
absolute alcohol, it was moderately soluble, crystallising out, on cooling,
| in glistening leaves, which under the microscope were seen to be of a
shorter shape, though generally two opposite corners were truncated,
__ giving the crystals an hexagonal appearance. From acetone it crystallised
F in needles. After purification it melted at 135° to 136°C., and gave
Salkowski’s test in the usual way. When treated in carbon bisulphide
solution, with a solution of bromine in the same solvent, it absorbed the
bromine very slowly and sluggishly. That it was an isomer of cholesterol
was shown by the analysis of the benzoate.
The benzoate was prepared in the same way as that of Spongilla A.
It also was very insoluble in absolute alcohol, from which it crystallised
in large rectangular plates, generally longer than wide, though a few
were square. It was difficultly soluble in petroleum ether, but crystallised
well from acetic ester.
- On heating in a wide capillary tube it melted, showing a bluish
fluoresence, at 135° to 136° C., to a turbid liquid which became perfectly

98 BIO-CHEMICAL JOURNAL

clear and limpid at 139°C. After allowing the temperature to rise to
about 160° C., a beautiful and striking display of opalescent colours is
observed during cooling. A greenish blue first appears, which immediately
turns to reddish violet. This gradually loses the red tint until it has
become a bright peacock blue, which slowly passes to a vivid emerald
green. The latter colour persists for a long time, and is finally sueceeded
by a golden brown. These colours are by far the most brilliant of any given
by the benzoates of the cholesterol group, and the green colour. which is
very characteristic, is given only by this body. Gey

For analysis the benzoate was dried at 100° C. and burnt in a one
boat filled with coarse copper oxide.

0-1573 gave 0-4791 CO, and 0-1448 H,0

Found Calculated for
CyH,,0.00.C,H,
0 os 83-07 83-24
) : Enis 10-22 10-20

Spongilla cholesterol B is thus an isomer of cholesterol with the
formula C,,H,,.0, and, although the quantity of material available did not
permit of the preparation of the dibromide, it was found to absorb
bromine, and therefore in all probability possesses a double link in its
molecule.

The spongilla cholesterols A and B differ from one another in
solubility, in crystalline form and in melting point. The body A absorbs
bromine far more readily than B, and the benzoates of the two substances
show considerable differences in melting point and colour phenomena.
These points are brought out in the following table, in which clionasterol
is also included for 2 tpi tenon of comparison.

Clionastero] Spongilla A Spongilla B
talline f (90%, aleohol Plates Oval leaves Oblong plates
Crystalline form” :methyl aleohol Clusters of leaves Leaves ana notched Heceanall plates —
plates .
Melting point 137-138° 138-139° 135-136°
Solubility absolute alcohol Moderately soluble Moderately soluble Moderately (hot)
na petroleum ether Easily soluble Easily soluble Insoluble

Dibromide, melting point 114° 112-113° —
Benzoate, melting point 141° turbid liquid ; 141-2° turbid liquid;' 135-6° turbid ;

clear 143-144° clear 185-190° -; clear 139° ;

colour play’ colour play} colour play st

It will be noticed that spongilla B differs markedly from both
spongilla A and clionasterol. It bears, however, a certain resemblance
to the phytosterols of the cryptogams, which frequently assume the

a !

DISTRIBUTION OF CUOLESTEROL 99

hexagonal crystalline form (which has not hitherto been noticed among
_ the animal cholesterols). Phytosterol benzoate was found by Ritter! to
melt at 144° to 145° C. to a clear liquid, and to give a colour play on
_ solidification, in which respect, also, spongilla B resembles it. It would,
however, be unjustifiable to attempt to base any conclusion as to the origin
of this body on such slight points of agreement, but it is obvious that
the relatively large proportion in which spongilla B is present (50 per cent.
of the total cholesterols) renders it unlikely that it originates from the
algae which live symbiotically with the sponge. The phytosterols of these
low plants? are believed by Tanret® to be very similar to, if not identical
with, the fongisterol recently obtained by him in a pure form from
spurred rye (Secale cornutum). This body has the formula C,,H,,O, melts
at 144°C., and gives an acetate melting at 158°C. Spongilla B is
; certainly dissimilar to this substance. It is also quite possible that
spongilla B is characteristic of one species of freshwater sponge, and
spongilla A of the other.
q There exists, on the other hand, a fairly close agreement in properties
_ between spongilla A and clionasterol. In melting point, solubility and
erystalline form they show a great similarity, although the characteristic
clusters of leaves in which clionasterol crystallises have not been observed
with spongilla A. The mode of formation and melting points of the
dibromides agree closely, as do those of the benzoates, except that the
turbidity of the clionasterol benzoate clears at 144° C., whereas that of
spongilla B persists up to 185°C. A more extended comparison of
_ chemical and physical properties will obviously be required to decide
definitely on the relationships of these substances. An attempt was made
towards a solution by the following experiments on mixed melting points,
equal quantities of each substance being taken in each case.

(a) The benzoates of clionasterol and spongilla A.—This mixture
melted indefinitely between 135° and 142° C.

(6) The benzoates of clionasterol and spongilla B.—This mixture
shrank at 136° C., melted to a turbid liquid at 139° C., and cleared at
142° C,

(ec) The benzoates of spongilla A and spongilla B.—This mixture
melted completely between 135° and 137° C,

These observations are again inconclusive, as, although it is true the
melting points are indefinite, they show no marked deviation from those

1. Loe. cit,
2. Cf. Gérard, loc. cit.
3. CO. R., 1908, OXLVII, 75.

100 BIO-CHEMICAL JOURNAL

of the constituents of the mixture. In this connection, however, it may

be mentioned that the admixture of even large proportions of a closely

related substance frequently seems to make little impression on the melting
point of the members of the cholesterol group. The phytosterol of
Calabar beans, which was first obtained by Hesse,’ of melting point 133°C.,
has recently been shown by Windaus and Hauth? to be a mixture of the
pure phytosterol of melting point 137° and stigmasterol, an alcohol with
the formula C,,H,,0 and melting point 170°C. The latter body occurs
in the mixture to the extent of 20 per cent., and yet only produces a
depression of four degrees in the melting point of phytosterol. ‘.

The results of this examination of the sponges make it very probable
that each species of sponge is characterised by a different member of the
cholesterol group, so that further investigations may bring to light a
variety of animal cholesterols corresponding with those already known as
constituents of plants. |

SUMMARY AND DISCUSSION OF RESULTS.

1. The results obtained for the distribution of cholesterol and its
allies in the animal series may be summed up as follows :—

CHORDATA— Cholesterol is universally present and is not
accompanied by any closely related body.
MOLLUSCA— Cholesterol is present.
ARTHROPODA—
CRUSTACEA Cholesterol] is present.
InsEcTA Cholesterol may occur in Bombyx mori, of which,

however, the chief~ constituent is bombi-
cesterol, an analogue of cholesterol. In Blatta .

orientalis a body of the cholesterol type is pas

present,
ANNULATA— Cholesterol is present.
ECHINODERMATA—Asterias rubens does not contain cholesterol,
although a body of a similar type is ig
COELENTERATA— Cholesterol is present. ‘

tA

1. Liebig Annalen CXCII, 175.
2. Ber., 1906, XX XIX, 4378.

DISTRIBUTION OF CHOLESTEROL 101

PORIFERA— Suberites domuncula contains spongosterol, which,

=— although probably related to cholesterol,
shows marked differences in properties
(Henze). Cliona celata contains clionasterol,
and the fresh water sponge contains appar-
ently two bodies, all of which bear the closest
resemblance to cholesterol.

a ay 2. In the following table the eee results obtained are
| — together.

Weight i ;
grams Iie nom: Gout. Cholera con
matter Hap ae ms
vas | ees 9800 6-0 0-21 5-24 O17
k ete | 246 O44 0-18 0-20 0-08
ecu, tee | 2458 1-27 0-09 0-32 0-022
iy jenr ~ RETO 4:50 0-38 1-46 0-124
es8 ‘in one 538 10 0-19 0-25 0-048
Pee: 194 0-5 0-25 au oS
eos bee 286 0-95 0-32 0-31 0-10
wpe + ,Jeue 1658 35 0-21 -— --
iy i) Sone: | SOO 2 _ — 1-5 0-07 2
ee ARO ys | 320° 0-27 2-07° 0-17
gee fees 755 413 0-54 1-027* 0-17
" Jan cholesterol itself

ieined, and, therefore, probably of all animals, contains at least one
member of the cholesterol group. This member is not always cholesterol
although it has now been proved that this substance is very widely

4 “distributed throughout the animal kingdom. In the warm-blooded
Vertebrata it is universally present, and is the only body of its type that

has ever been observed in them. The structures of the cold-blooded
_ Vertebrata have also been found to contain cholesterol. In the Invertebrata
_ cholesterol has been recognised as widely, but not uniformly, distributed
_ throughout the series. In the highly organised Mollusca it occurs in

comparatively large quantity. In the phylum Arthropoda the Crustacea
-__ contain it, whereas in Insecta its place is taken by nearly allied substances.
In Annulata it is present, but not apparently in Echinodermata, Its
occurrence, again, in the Coelenterata is significant as showing that the
ae cholesterol function in the protoplasm of these low animals can be carried
out by the same substance as in the case of the most highly developed

: SS) a aaa ™

102 BLO-CHEMICAL JOURNAL

types. Cholesterol itself is not present in the Porifera, and, unfortunately,
no Protozoan species could be obtained in quantity sufficient for
examination.

4. In those species in which cholesterol itself was not found, there
was present in each case as a substitute a body of very similiar properties.
The cholesterol constituent of Asterias rubens, representing Echinodermata,
was not very exactly defined, owing to difficulties which were met with
in effecting its separation from other non-saponifiable substances. It
appeared, however, to be similar to cholesterol in its properties: it was’
unsaturated and gave Salkowski’s test. The researches of Menozzi and
Moreschi on Bombyx mori, and the work described in this paper on the
Porifera, show that the place of cholesterol in these animals is filled by
previously unknown members of the cholesterol group, which in all their
properties are remarkably similar to cholesterol itself. The physical
and chemical properties of cholesterol, of bombicesterol, of clionasterol,
and of the cholesterols of spongilla form a complete parallel. They are
all alcohols of the formula C,,H,,0, contain one well-defined double link
in the molecule, and agree in the behaviour of their derivatives, notably
in the solubility and fusion phenomena shown by their benzoates. They
differ from one another chiefly in their crystalline form and the melting
points of their acetates and dibromides. Among these, also, no sign of an
evolution (if one may use the term) of cholesterol is apparent. Just as
the cholesterol of man is present in the sea anemone, so the bombicesterol
of the highly organised insect is exactly matched by the clionasterol of
the sponge.

5. With the possible exception of the spongosterol of Suberites
domuncula, to which reference has already been made, all the animal
cholesterols so far examined are isomeric with and similar in properties
to cholesterol itself. The unsaturated linking and the hydroxyl group,
which Hausmann and others have proved to be essential to the performance .
of the anti-toxie function of cholesterol, are apparent in all of them. It
may not, therefore, be too much to say that the protoplasm of all animals
contains as one of its essential constituents a cholesterol; that is to say, a
singly unsaturated, monatomic alcohol, isomeric with or very closely
related to cholesterol itself»

6. Can this generalisation be applied to all protoplasm, vegetable
as well as animal? Our knowledge of the vegetable cholesterols is based —
upon a comparatively large number of investigations, which are not,
however, as a rule, of a very precise character. In an Inaugural

DISTRIBUTION OF CILOLESTEROL 103

Dissertation! Hauth has given a list of the various phytosterols which
have been deseribed in the literature under various names. Of these
thirty-one have melting points lying between 132° and 157° C., and closely
resemble cholesterol. They were all obtained directly or indirectly from
the Phanerogams. The work of Windaus and Hauth? has made it
probable that all of these bodies are mixtures of phytosterol with other
alcohols which are capable of forming isomorphous mixtures with it, and
even when present in considerable proportion do not materially lower the
ee ‘melting point of phytosterol. Their separation is extremely difficult, but
Windaus and Hauth succeeded in bringing it about in the case of the
phytosterol of Hesse originally obtained from Calabar beans. This substance
_ was found to contain 20 per cent. of an alcohol, stigmasterol, of melting
point 170° C.; the residue consisted of phytosterol which had previously
been obtained pure by Burian? from wheat germ. This latter body would,
therefore, appear to be typical of the higher plants, just as cholesterol
is of the higher animals. It is an isomer of cholesterol, with the formula
C,,H,,0, and possesses a well-marked double link. It is very similar
in properties to cholesterol, and in all probability possesses a similar

structure in the side chain.*

Our information with regard to the cholesterols of the lower plants is
_ due chiefly to the researches of Gérard and Tanret.6 The former showed
that in yeast and some species of lichen there occurred a body of melting
point 136°C., which resembled the phytosterols in some respects, but
differed from them in possessing a very high laevorotatory power — 105°.
pa similar substance was obtained from Staphylococcus alba and Fucus
_erispus. These bodies show a decided resemblance to the ergosterol of
- Tanret,? which was first obtained by him from Secale cornutum, and which
he has recently® succeeded in separating in a pure form. It is accompanied
_ apparently in spurred rye (Secale cornutum) by fongisterol, which, in

_the opinion of Tanret, is a lower homologue of ergosterol with the formula
C,,H,,0. Ergosterol is apparently isomeric with cholesterol. It melts
at 165° C., and has a rotatory power in ether of — 105°5°.

A large number of other phytosterols have also been described, but
practically nothing is known of their nature, and in all probability they

1. Zur Kenntnis der Phytosterine, Freiburg i. B, 1907.

2. Ber. 1906, XXXIX, 4378.

3. Under the name of sitosterol, Monatshefte, XVIII, 553.

4. Pickard and Yates, J.C.S., 1908, XCITI, 1928.

5&6. Comptes rendus, 114, 1544; 1898, 909. Also Jour, Phar, 18% (6), 1, 601,
7. Ann. de Ch. et de Phys.” XX, 289.

8. Comptes rendus, 1908, CXLVU, 75.

104 BIO-CHEMICAL JOURNAL

are, not single substances. It will be apparent, however, that in the
protoplasm of plants, as well as in that of animals, a ‘ cholesterol ’ appears
to be universally present.

7. The following scheme embodies the results obtained by

Hausmann (loc. cit.) in his investigation of the anti-haemolytie power of

various members of the cholesterol group and their derivatives. It will
be noticed that the naturally occurring alcohols, cholesterol, phytosterol
and parasitosterol, all have a marked anti-haemolytic power. If the
hydroxyl group be replaced by chlorine (as in cholesteryl chloride), or by
hydrogen (as in cholestene), or be esterified (as in phytosterol propionate),
the activity entirely disappears, although the unsaturated linking remains
unaltered. If the double bond be saturated, as in cholesterol dichloride,
or be modified by ring formation, as in the case of cholestanonol and
probably coprosterol, the anti-haemolytic power is reduced to a minimum
although the hydroxyl group is still present. Spongosterol is described
as having a feeble anti-toxie power, and as being similar to coprosterol
in this respect—observations which are in agreement with the observed
chemical reactions of these bodies.

Anti-haemolytic function

Formula towards saponin
CHOLESTEROL Ca, Hy.OH Active
CHOLESTERYL CHLORIDE Ca; Hy. Cl None
CHOLESTENE Ca; Hyg *
CHOLESTEROL ACETATE Cay Hy;. O CgH,O ”
CHOLESTEROL DICHLORIDE Cy; Hy; re Doubtful
CoPpROSTEROL Ce Hy. OH Feeble
CHOLESTANONOL Caz; Hyg O2 Feeble
PHYTOSTEROL - Cy, Hy, OH Active
PHYTOSTEROL PROPIONATE Co, Hy, O. CgH,O f None
PARASITOSTEROL Co; Hy. OH Active
SPONGOSTEROL Cy Hy. OH Feeble

8. In the following table are collected together, for purposes of

reference and comparison, ihe properties of the best known members of
the cholesterol group which are isomeric with cholesterol, and, like it,

unsaturated. Spongosterol, though its similarity in these respects to

cholesterol is doubtful, is also included,

DISTRIBUTION OF CHOLESTEROL 105

Acetate Benzoate Dibromide
Mp. [@)p* Mp. M.p. M.p.

CHoLestEKoL — ieee 114° 145° 123° Generally distributed in the
¢ (colour play) animal kingdom.

Bompiscestero: 148° = — 35" 114° 146° 111° In Bombyx mori.

Crionasterot «138° -3T 133° 143° 114° In Cliona celata,
(colour play)

Sponoiiia A is _ 142° 113° In freshwater sponge.
(colour play)

Sronciiia B 136 = -- 136° _ ‘

; (colour play)

SponcosterRo. 123° —19 124° 128° — In Suberites domuncula.

Puyrosrernor 137 # -—34°t 127° 145° 98° In the Phanerogams.
(colour play)

_Eroosreroi 165° -126° ~—:180° In the Cryptogams.

* In chloroform solution. + In ether solution.

9. The present work has not made clearer the obscure question of
the origin of cholesterol in the animal organism. It is certainly not
without significance that cholesterol never occurs among plant tissues,
and when the great difference in the nutrition of the animal and the
plant is considered, the conclusion seems almost justifiable that the
cholesterol of the animal is derived from the phytosterol of the plant.
For the plant, in virtue of its power of photo-synthesis, can elaborate starch
_ from carbon dioxide and water, and protein from the carbohydrate thus
formed and the nitrogenous food stuffs absorbed from its medium, while
the animal can only utilise the protein, carbohydrate and fat which has
been previously built up from inorganic material by the plant. To these
purely nutritious substances may possibly be added phytosterol elaborated
by the plant. Herbivorous animals, whose tissues and fluids contain
cholesterol, eat only phytosterols; and it has been shown that if fed on
diets free from all members of the cholesterol group, the rabbit, for
example, can absorb cholesterol when given with the food.!| That it can
absorb and utilise phytosterol as such has not yet been experimentally
_ proved, but since it can and does absorb cholesterol, it is obvious that
such absorbed cholesterol is required normally by the organism, and must
usually be derived from the vegetable cholesterols taken with the food.

1. Dorée and Gardner, ‘ The Origin and Destiny of Cholesterol in the Animal Organism,’
Part ILI, loc. cit,

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107

" ALLYL ISOTHIOCYANATE: SOME ASPECTS OF ITS
PHYSIOLOGICAL ACTION

By E. WACE CARLIER, M.D., F.R.S.E.
From the Physiological Department of the University of Birmingham

(Received January 20th, 1909)

There are but two allyl compounds in general use—the sulphide and
the isothiocyanate, and with the latter this communication chiefly deals,
the former having already furnished the theme of a previous research.!

Allyl isothiocyanate, or oil of mustard, is a well-known rubefacient
and counter-irritant when applied to the skin, and in addition is in small

_ doses in constant use internally as a condiment; it appeared, therefore,
desirable to ascertain in what directions its action resembled or differed
from that of its ally, and with this intent a number of experiments were
undertaken upon deeply etherised rabbits, the methods followed being
precisely the same as those indicated in the previous paper.

In the first instance one minim (0°66 c¢.c.) of the pure synthesised

r drug was injected into the jugular vein of a large animal, with the result
g that it died in convulsions a few minutes later, thereby demonstrating
5 how much more deadly the isothiocyanate is than the sulphide, and the
necessity of finding some bland menstruum which would mix with it in all
proportions and form a solution of constant and uniform strength
throughout. Olive oil proved to be the most suitable vehicle for the
____— purpose, because when injected in small quantity into a vein it exhibits no
____ physiological action either on the heart, blood pressure or respiration,
which were the main effects to be attended to in the present research.
+ Sian Ten per cent. and 20 per cent. solutions of oil of mustard in olive oil
were accordingly prepared. For the purposes of this report it will be
sufficient to give in detail the stages in one typical case and to comment
upm others as occasion arises.

a In a rabbit weighing 2,200 grammes, and deeply anaesthetised,
4 1} minims (0°09 ¢.c.) of a 20 per cent. solution of ally] isothiocyanate in
s olive oil were injected into the jugular vein in six seconds, the initial
pressure in whose carotid artery was 72 mm. Hg, with the result that the
pressure began to rise slightly at first, reaching 73 mm. in seven seconds;

1, Bio-Chemical Journal, Vol. 11, pp. 925-8389.

108 BIO-CHEMICAL JOURNAL

this is immediately followed by a rapid fall, reaching its lowest point at
28 mm., twenty-seven seconds after the commencement of the injection ;
a slow rise in pressure follows to 45 mm., which was reached at the sixty-
seventh second, to be followed by a gradual fall to 39 mm. at the 127th
second, when it again began to rise very slowly, only reaching 70 mm.
at the 387th second, after which it again sank almost imperceptibly to

53 mm. at the 950th second, at which moment the record ceases. (Fig. 1.) —

Allyl isothiocyanate, therefore, exerts its maximum depressor effect
almost immediately after administration, followed by rapid partial

recovery; this is succeeded in turn by another fall of blood pressure of —

less intensity but of longer duration, and, like its predecessor, followed

by a slower rise, but only to fall again, and so on. If sufficient time is

given for the drug to be eliminated, chiefly by the lungs, these pressure
waves die out and the normal blood pressure is restored,

This drug, like its congener, has a powerful effect upon the respiratory
system, which lasts for a considerable time, but the disturbance caused by
it is more rapidly recovered from than that produced in the blood pressure.

At first the respiratory movements are diminished in number
and in amplitude, but is soon followed by increased frequence, with
further diminution, the animal’s chest becoming inflated owing to the
inspiratory efforts greatly exceeding the expiratory, all the extraordinary

inspiratory muscles being brought into play; the tracing, therefore, sinks

rapidly and somewhat suddenly at the twelfth second after the commence-
ment of the injection. Soon the respiratory efforts, though still rapid,
increase in amplitude, becoming now and again convulsive, despite which
they show a tendency towards recovery from the fifty-seventh second, which
practically corresponds to the time at which the carotid curve is nearing
its first pressure maximum. ‘Thereafter the movements become less
extensive and less frequent and finally attain their normal rate and
magnitude about the 800th second after injection.

Allyl isothiocyanate, like allyl sulphide, produces a certain measure
of immunity in the animal after it has recovered from a first dose, and —
therefore 3 minims (0°18 c.c.) of the same solution was administered in

seven seconds by injection into the jugular vein of the same animal after
it had completely recovered from the effects of the first, i.e., after half-an-
hour, complete anaesthesia beng maintained during the whole time, —

The carotid pressure at the moment of injection was 78 mm. Hg, but

before the injection was over a slight fall in the tracing was observed,
which was followed at the thirteenth second by a more rapid fall that

oe.

[tO OATIO UT PVUBADOTTZOSI [A[TV JO UOTZNyOS 0%
BO (°9°9D RT ()) surrurar ¢ jo IQqGuA BV JO UIA zejngsnl ety o7Ul womoolu jo sqooyy a a

109

“TTATA ) A me en ame
UO au ~~

JO UOTIBANG ‘ %

*SUOTNRAIASOR

*[t0 @AT[O Ul
PJVUBADOTYIOSI [ATV JO WOTBNOS % OZ B Jo (*o°o 60-0) SUITUTUL G.[ JO ULBA aepnznol ay} O7Ur UoTZOeLUT Jo sq08 [ ‘ola

[SOTHIOCYANATE

ALLYL

110 BIO-CHEMICAL JOURNAL

reached a minimum of 55 mm. Hg at the twenty-sixth second. From
that moment a rapid rise commenced, crossing the normal line at the
thirty-fifth second, and continuing in a series of humped curves to a
maximum of 98 mm. at the seventy-second second, thereby attaining a
height of 20 mm. above the normal. (Such a rise never oceurs with a
first dose.) This rise is followed by a very slow fall to 92 mm. at the
290th second, at which point there was a marked change in the
respiration, accompanied by an abrupt fall in pressure to 83 mm. With
the exception of a slight rise. followed by a slight fall, the pressure
gradually sank to 82 mm. at the 800th second, reaching the normal as
the effects of the drug slowly passed away. (Fig. 2.)

With this dose, which would have been fatal to a fresh rabbit, the
breathing became quicker and shorter from the moment the carotid
pressure commenced to fall, the chest very rapidly became inflated and the
tracing falling almost suddenly, the respiratory movements increasing in
amplitude, despite their rapidity, as the bottom of the curve was reached ;
the tracing then rose somewhat, but at the fiftieth second spasms moderate
in force and duration supervened, and were succeeded by a few small
respiratory efforts that quickly grew in amplitude, the respiration finally
settling down to a steady and regular rhythm, about three times less rapid
than before the injection, passing into normal about the 320th second.

The animal having again recovered, a dose of 6 minims (0°36 ¢.c.) was
administered in ten seconds in the same manner as before. This produced
a fall of 7 mm. in the pressure curve in twenty seconds, followed by a rise
to 16 mm. above the initial pressure some eighty-five seconds after the
injection, from which point it began to sink until the animal died. |

On the respiratory mechanism it produced first a quickening of the
breathing with diminished amplitude, and at the twentieth second the
usual sudden fall in the trace occurred followed by convulsions, in which
the whole body participated. Tremors first appear in the abdomen and
thorax, then in the hind limbs, which become rigid, with the toes widely
separated and fully extended; the muscles of the spinal column are next
attacked and lift the animal quite off the table in rigid opisthotinos, finally
the fore limbs become affected, but the spasm in them is less marked.

At this stage artificial respiration was had recourse to, but some little
time elapsed before the chest became sufficiently flaccid for it to be
effective. During the whole spasm the heart was beating strongly, and the
blood pressure kept up, reaching its highest point just after artificial
respiration became effective, at which time the spasm also began to subside

ALLYL ISOTHIOCYANATE 111

in the hind limbs, though it continued in the paws and passed to the head
and neck, producing convulsive movements of the jaw muscles and wheel-
like movements of the eyeballs.

The animal was now killed by stopping the respiratory pump and
opening the thorax, having been kept in the deepest anaesthesia during
the whole course of the experiment.

The heart when exposed was beating well, the drug apparently in no
way affected its muscle fibres, though this large dose of the drug produced
violent and convulsive contractions of the skeletal muscles, the extensors
being chiefly affected by it.

Allyl isothiocyanate is, therefore, a much more powerful poison than
allyl sulphide. It attacks the respiratory system more powerfully than
the vaso-motor, though its effects pass off more quickly from the former
than from the latter. Poisonous doses kill by paralysing the respiratory
_ centre, as with allyl sulphide. The expiratory centre seems more affected

than the inspiratory, resulting in considerable chest inflation.

A tolerance is set up for the drug on the administration of repeated
doses of the same strength, so that a stronger dose has to be given each
time to produce an equal effect. By the first dose the vaso-motor centre is
depressed and recovers but slowly, but from subsequent doses it not only
recovers more quickly, but is less affected and becomes stimulated, so that
the blood pressure rises high above the normal. This may be due partly to
the muscular tremors that invariably occur with moderate doses, causing a
more rapid return of the blood from the compressed veins towards the
heart, but that is not the only cause, for considerable rises in pressure can
be obtained by administering repeated small doses that do not produce
visible tremors. With small doses the depressor effect alone is marked,
with larger ones a pressor effect is subsequently developed, and with large,
i.e., fatal doses, a pressor effect alone may be registered. The vaso-
motor centre seems more sensitive than the respiratory centre to smal]
doses, though with medium and large doses the reverse is the rule. (Fig. 3.)

With a small dose the respirations may be only quickened and some-
what reduced in excursion without any chest inflation, which is such a
constant occurrence with medium ones.

The minimum fatal dose was not accurately determined, but it
approximates to 1 minim (0°06 c.c.) of a 20 per cent. solution per kilo. of
body weight, i.e., one-fifth minim (0°012 c.c.) of normal oil, and therefore
this drug is two and a half times more fatal than allyl sulphide.

Even with fatal doses, if the animal be kept alive by artificial
respiration, the vaso-motor system remains efficient, because every

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ALLYL ISOTHIOCYANATE 118

cessation of the ventilation is followed immediately by a rise in blood
‘pressure, which passes off immediately the air-pump is restarted, and

because when the depressor nerve was stimulated electrically in the only
___ ease in which its efficiency was tested, there was a slow fall of pressure

succeeded by a slow rise after cessation of its excitation. (Fig. 4.)

With the vagus nerve things are different, it loses its efficiency upon
the heart as the strength of the dose increases, failing to reduce the blood
pressure, though it can still impede the heart beat to some extent; in this
the action of the isothiocyanate closely resembles that of allyl sulphide.
(Fig. 5.) |
- On skeletal muscles the drug acts powerfully, producing spasm, the
extensor groups being more affected than the flexor. Its action in small
doses on cardiac muscles is slight and of short duration; beginning early,
it passes off in about 150 seconds and amounts to no more than a slight

decrease in the rate of the heart beats, with lengthening of the systole.

With the frog’s heart phenomena occasionally seen with allyl sulphide

es are well marked with the isothiocyanate, more especially so with winter

frogs. In all cases pithed and decerebrated frogs were used, and the
records obtained by placing the frog on a Pembry myograph, exposing
the heart, inserting a hook in the apex of the ventricle, from which a
thread passed to the lever of the apparatus. The drug, either pure or
diluted with olive oil, was passed directly into an auricle by means of a
hypodermic syringe.

A dose of 1 minim (0°06 c.c.) of the pure drug stops the heart at

once, but a dose of half that quantity only takes effect after thirty
seconds, the ventricle muscle passing into delirium before finally ceasing
to beat. The auricles, however, continued to beat synchronously, though
jerkily, for a considerable time longer.
‘ One minim (0°06 c.c.) of a 33 per cent. solution produces some
diminution both in amplitude and rate, followed suddenly some sixty
seconds later by a marked change in speed with increase of amplitude,
which gradually declines as the speed again increases for a while This is
followed by marked slowing just before the sudden arrest of ventricle
comes on, some 280 seconds after the injection. The auricles beat
normally throughout, and continue so doing for another seventy seconds
at least, after which their speed very gradually diminishes. Though
arrested, the ventricle is not permanently stopped but resumes beating
again with occasional stops, that become shorter as time wears on, until,
by the end of fifieen minutes, the whole heart so far recovers as to beat
quite rhythmically though slowly and with diminished force.

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116 BIO-CHEMICAL JOURNAL

With this dose the results naturally vary somewhat with the size and
vigour of the frog used; with small weak frogs the heart is permanently
arrested, the auricles always outlasting the ventricle for a considerable
time; with large and vigorous frogs partial or complete recovery in the
rhythmic sequence is customary; the speed, however, seems always
permanently diminished.

I have obtained the best results with an injection of 1 minim
(0°06 c.c.) of a 20 per cent. solution into the auricle, as may be seen from
the tracing here given, in which the production of heart block, with
gradual recovery therefrom, is better illustrated than is possible by mere
description. (Fig. 6.)

When frogs, taken fresh from the fields in summer, are treated as
above, heart block rarely supervenes, the heart either stopping suddenly
as a whole with large doses, or becoming gradually slower and feebler
with smaller ones, until it finally ceases to beat altogether.

The effect of the drug upon the mammalian heart may be studied
in the rabbit, provided artificial respiration is had recourse to, because
the large doses required are very much more than sufficient to paralyse
the respiratory mechanism. In all cases the ventricles are more
profoundly affected than the auricles; with a dose of 7} minims (0°45 c.c.)
of a 20 per cent. solution injected into the jugular vein the amplitude of
the heart beat is first lessened, this is followed by diminished speed with
some increase-in amplitude, but the heart is finally paralysed in about
twenty minutes. (Fig. 7.) Only in one case was some blocking noticed,
towards the end of the experiment and after the animal had received
several consecutive and increasingly larger doses of the drug. In this case
three auricular beats corresponded with one of the ventricles. With these
large doses the auricles occasionally fail a fow seconds before the
ventricles, but this is not the general rule.

ConcLusION

Allyl sulphide and allyl isothiocyanate act in a similar manner upon
the organism, the latter drug being the more powerful; both paralyse the
respiratory and vaso-motor centres, both produce muscular spasms and
affect the heart beat, and both lower the body temperature, as most |
essential oils do. Neither can be recommended for internal adminis-
tration, despite the fact that they are commonly taken in minute quantity
with food.

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117

- CHOLINE IN ANIMAL TISSUES AND FLUIDS

By W. WEBSTER, M.D., C.M., Demonstrator of Physiology in the
University of Manitoba, Winnipeg, Canada.

From the Physiological Laboratory, University of Manitoba
Communicated by Professor Swale Vincent

(Received February 4th, 1909)

IntTRopuctToRY AND HistroricaL

_ Although the morphological changes accompanying nerve
degeneration have been very minutely studied and certain definite results
obtained, the study of the corresponding chemical changes has not, so far,
thrown any light on the metabolism of the nervous tissues. Gulewitsch!

was the first to isolate free choline, one of the substances entering into the

constitution of nerve tissue, from extracts of ox-brain. Vincent and
Cramer? isolated very small amounts of a similar substance by a process
less likely to produce decomposition. These observations constitute the only
direct chemical evidence of metabolic processes in nervous tissues. But it
must be pointed out that the possibility of post-mortem changes, or of
changes occurring during the manipulations involved in the isolation,
cannot be altogether excluded. This latter objection seems, however, to
lose its weight in view of the observations of Gumprecht,? who found by a
micro-chemical test that a substance giving the reactions of choline is
present in the normal cerebro-spinal fluid of healthy animals and of

patients suffering from diseases other than nervous.

Another fact pointing to the existence of metabolic processes in
nervous tissue has been brought forward by Waller,4 who suggested that
the increase in electrical response after repeated excitation of nerve is
due to carbonic acid having been evolved in consequence of the activity
of the nerve,

The evidence put forward by Halliburton for the presence of choline

1. Zeitachr. {. physiol. Chem., XXTV, 8. 513, 1898.
* 2. Journ. of Physiol., Vol. XXX, p. 149, 1904.
3. Verhandl. d. Congress /. innere Med. Wiesbaden, s. 326, 1900.
4. Centralbl. {. Physiol., XII, s. 745, 1899.'
5. Journ. of Physiol., Vol. XXVI, 1900-1, and numerous other publications.

118 BIO-CHEMICAL JOURNAL

in saline extracts of nervous tissue can not, in our opinion, be accepted.
He believes that the effect on the blood pressure of nervous tissue extracts,
observed by himself and by Osborne and Vincent,® is due to the presence
of choline, and that by means of this ‘ Physiological test ’ the presence of
choline in animal extracts and fluids can be detected. This was disputed
by Osborne and Vincent,’ Vincent and Sheen,* and Vincent and Cramer,®
who pointed out the difference in behaviour between brain extracts and
choline after the administration of atropine, and demonstrated that the
depressor effects are due to substances other than choline.

The chemical evidence submitted by Halliburton, which is based on
the appearance of crystals of a double salt of platinum chloride in brain
extracts, has been shown to be unsound. Vincent and Cramer, and
independently Mansfeld,'° showed that these crystals consist almost
entirely of inorganic salts, from which it is impossible to free the extracts
even after repeated extraction with absolute alcohol. These results were
confirmed by French and Allen;!! and Bayliss '? in his investigations on
adsorption, showed that this phenomenon is due to the adsorption of
inorganic electrolytes by organic colloids.

The chemical evidence as to changes in pathological conditions of the
nervous system is of a more definite character. Mott and Barrat!* were
the first to show that degeneration in the spinal cord leads to a diminution
of the phosphorus contents. Noll!4 found that the protagon diminishes
after nerve section, and Halliburton!5 also showed that a diminution of
phosphorus occurs under these conditions.

These results led Mott and Halliburton!® to seek for choline in the
blood in conditions of nerve degeneration. In a series of experiments on
cats the sciatic nerves were divided, and the physiological and chemical
tests for choline were applied to the blood of these animals. The results
showed a striking parallelism between the effect on the blood pressure and

6. Journ. of Physiol., Vol. XXV, 1900.

7. Loe. cit.

8. Journ. of Physiol., XXIX, p. 242, 1903.

9. Loc. cit.

10. Zeitschr. f. physiol. Chem., Bd. XLII, 1904.

ll. Proc. Physiol. Soc., p. 29, 1903; Journ. of Physiol., p. 30.

12. Bio-Chemical Journ., Vol. I, p. 209, 1906.

13. Proc. Physiol. Soc., p. 3; Journ. of Physiol., Vol. XXIV, 1899.
14. Zeitschr. {. physiol. Chem., XXVII, p. 370, 1899.

15. Croonian Lectures, 1901.

i ae Eee. Physiol. Soc., Feb., 1898; Journ. of Physiol., Vols. XXI, XXII, and XXIV,
eb., 3

CHOLINE IN ANIMAL TISSUES AND FLUIDS 119

the number of crystals obtained. These results constitute the only

experimental basis for the so-called ‘ Choline hypothesis,’ that degenerative
processes in nervous tissues can be detected by the appearance of choline in
the blood. Although it is now generally admitted that the tests employed
in these experiments are fallacious, the question has not, up to the present,
been re-investigated in experiments in which more reliable tests for choline
have been employed. Such tests are recommended by Gumprecht,’’
French and Allen,'® Donath,!® and Rosenheim,?® but have only been
applied to the cerebro-spinal fluid in the case of patients suffering from
nervous diseases. With the exception of Gumprecht’s observations, no
controls have been made upon patients suffering from other kinds of
diseases, although the facts given above as to the presence of a substance
giving the reactions for choline in normal cerebro-spinal fluid would appear
to have rendered some such control imperative. The attempt to find choline
in the blood in cases of nerve degeneration has now been almost completely
abandoned. The results of clinical investigation by various observers are
very conflicting, and are explained by each author as being due to the
inferiority of the tests employed by the others.

_

EXPERIMENTAL

In view of the conflicting nature of the evidence before us, it seemed
to me very desirable to test the choline hypothesis by means of a series of
experimental lesions in animals. My observations have so far been
restricted to testing for choline in the blood of those animals where choline
is normally not present. The fact that choline is found in normal cerebro-
spinal fluid would necessitate a quantitative estimation of choline in this
fluid under various conditions, a proceeding which appears to present very
considerable difficulties.

In my experiments I have employed seven dogs and one cat. In six
dogs and in the cat both sciatic nerves were divided and a long piece
resected; the blood was taken a variable number of days after the
operation. The results are put together in the following table :—

17. Loe. cit.
" 18. Loe. eit,
19. Loe, cit,

20. Journ. of Physiol., Vol. XXXIII, p. 220, 1905-6,

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CHOLINE IN ANIMAL TISSUES AND FLUIDS 121

cf It may be mentioned that because dogs were employed instead of cats

7 in these experiments the precaution was taken to compensate for their

larger size by injecting an extract from a large quantity of blood four to
five times as much as the quantity employed by Halliburton.??

In order to produce even more extensive degeneration, in one dog a
portion of the dorsal spinal cord about an inch in length was excised.
After eight days the animal was killed, and its blood subjected to the
physiological test. The effect produced upon the blood pressure was
identical with that produced by an extract of an equal quantity of normal
blood.

It will be seen that there is no progressive increase in the depressor
effect such as was described by Halliburton. In fact, in no case did the
extract of pathological blood give a fall greater than that obtained by the
extract from a normal animal. Further, the effects were precisely the
same after the administration of atropine as before.

This result completely confirms the observations of Vincent and
Cramer, who arrived at the conclusion that the substance present in the

_ pathological blood which gave a fall of blood pressure in Halliburton’s

experiments was not choline, nor indeed anything arising from morbid
processes, but some substance common to normal blood and to all animal
tissues.

CHEMICAL

I have applied the chemical test as recommended by Rosenheim to
the blood of animals operated upon as described above. Using a pure
choline solution, the formation of dark brown crystals can be easily
verified. They are very few in number, and small when a solution of
choline of 1-200000 is used. I have, however, been unable to find any
similar crystals in the blood of any of the animals experimented upon.
An abundant crop of the platinum-chloride crystals can be obtained from
both normal and pathological blood if the old test originally recommended
by Halliburton is used. This, again, is confirmatory of the result

obtained by Vincent and Cramer, and renders very doubtful the present

claim of Halliburton that ‘the obtaining of a large crop of crystals,
whether they be those of the choline salt or a mixture of the potassium and
choline salts, is diagnostic of an extensive breakdown in nervous tissues.’23

.22. Such a precaution is really not necessary, because, together with the increase in size,
there is an increase in the weight of the sciatic nerves, and, ecaalnar F of the amount of
nervous tissue degenerating after section of the nerve.

23. ‘Oliver Sharpey Lectures,’ Brit, Med, Jowrn., May 4th, 1907.

122 BIO-CHEMICAL JOURNAL

The addition of iodine solution to these crystals does not cause the
formation of choline pericodide. Some brown crystals are formed,
irregular in shape and varying in size, but none are oblong—the
characteristic shape of the periodide crystals—neither do any of them
resolve themselves into ‘oily droplets’ on drying of the solution, but
retain their form days or weeks after drying has occurred.

I have been greatly disappointed to find that none of the statements
which constitute the basis of the choline hypothesis can be verified. The
observations upon which the hypothesis rests are the outcome of investi-
gations which have not been sufficiently controlled by the examination of
normal tissues and fluids, since all the effects alleged to be characteristic
of pathological conditions can be obtained in the normal state.

That my results are not due to faulty technique can be proved by the
following calculation :— .

Taking the total weight of the central nervous system as 1,400
grammes, it can be calculated on the basis of our present knowledge of the
chemistry of nervous tissue that about 8°5 grammes of choline are contained
therein. This follows from the observations of Coriat,24 who found that 10
grammes of moist brain tissue yield 0°547 gramme of choline, as against the
theoretical amount of 0°0584 gramme of choline calculated from Koch’s
figures. It follows then that 1 gramme of moist brain substance contains 0:006
gramme of choline. The average volume of total blood is 4,700 c.c. After
a sudden destruction of 1 gramme of brain substance the blood of a patient
would contain 0°006 gramme of choline. This corresponds to a dilution
of 1 in 800,000, therefore 20 c.c., the volume recommended and generally
employed by Mott and Halliburton, would contain 0:000025 gramme of
choline = one-fortieth of a milligramme. This only holds good if
1 gramme of brain is destroyed suddenly, as choline is oxidised in the
organism. Asa rule, degenerative processes destroying 1 gramme of brain
substance would be spread over a considerable time, so that the amount of .
choline at any particular moment would be much smaller than is given —
above.

Now, none of the tests hitherto employed are sufficiently delicate to
indicate choline in such degrees of dilution.25 Any statement hitherto
made to the effect that choline has been demonstrated in the blood may
therefore be taken as showing either that the test by which it has been

24. American Journ. of Physiol., Vol. XII, p. 353, 1905.

25. Some very delicate tests have been devised by Gumprecht and by Reid Hunt, but
these tests have as yet not been applied to the investigation of this q

CHOLINE IN ANIMAL TISSUES AND FLUIDS 128

recognised is fallacious, or if that can be excluded, that the choline which
| is present is not derived from the degeneration of nervous tissues.
It is interesting to compare these figures with the amount of choline
found i in the blood by Halliburton and by Donath. The former observer
_ found in the blood of cats he experimented upon choline up to the amount

of 0°0052 to 0-0078 per cent. at a time when the degeneration was stated
by him to be at its height. Taking the volume of the blood of a cat at
150 e.c., these figures would correspond to a sudden destruction of more
than 1 gramme of nervous tissue at the time when the animal, a cat, was
killed. As choline is stated to be abundant from the eighth to the
thirteenth day after section of the nerves, the amount of nervous tissue
destroyed in order to account for the amount of choline alleged to be
present would be enormous.

With regard to the cerebro-spinal fluid, the physiological effect on
_ the blood pressure produced by injections of 10 c.c. of cerebro-spinal fluid
from cases of general paralysis is compared with the effect of injections of
1 to 5 c.c. of a 0:2 per cent. solution of pure choline. The amount of choline

injected in the latter case is 0:002 to 0°01 gramme, and is, according to
~ Halliburton,2¢ comparable to the amount of the base presumably present
7 _ in pathological cerebro-spinal fluid. We have seen that (if caleulated for
| the total cerebro-spinal fluid = 100 ¢.c.) such an amount could be produced
only by the sudden destruction of about 3 to 15 grammes of nervous tissue.
These considerations completely justify the statement with which
Vincent and Cramer concluded their paper, and to which Halliburton has
taken exception. Indeed, it appears, a priori, improbable that the
substance in these pathological specimens of blood should be choline
: derived from nervous tissue. That such comparatively slight destruction
___ of nervous elements as takes place even in extensive disease should supply
___—_—_—s sufficient choline to the blood to give the physiological test seems scarcely
———s goneeivable, especially when we remember that choline is not a very
: powerful depressor substance.

Donath?’ determined the amount of choline in the cerebro-spinal fluid
of patients. Taking the volume of this fluid at the minimal value of
100 ¢.c., his results indicate the presence of from 20 to 40 milligrammes of
oe choline. This would correspond to a sudden destruction of 3 to 7 grammes
of nerve tissue, and that in cases of epilepsy, both Jacksonian and
of idiopathic, and of neurasthenia.

26. Croonian Lectures, p. 51, 1901,
27. Deutache Zeitachrift fiir Nervenheitkunde, Vol, XXVU, p. 71, 1904,

124 BIO-CHEMICAL JOURNAL

Quite recently Halliburton, although admitting that the platino-
chloride crystals obtained from blood are due partly to the presence of
potassium, makes the claim?* that the obtaining of a large crop of erystals,
‘whether they be of the choline salt or a mixture of the potassium and
choline salts, is diagnostic of extensive breakdown in nervous tissues.
The contrast between such cases and the insignificant yield from normal
blood is most striking. This is quite intelligible, when we take into
account the very high percentage of potassium that nervous tissues
contain.’ If the presence of potassium in the blood leads to the formation
of these crystals, it may be asked why the considerable amount of
potassium which is known to be present in normal serum should give an
insignificant yield of crystals, while an increase which must necessarily be
very limited so long as the kidneys are performing their work, should
produce an abundant crop. Asa matter of fact, I have never been able to
find such a difference, having obtained as abundant a crop of crystals
from normal blood as from the blood of animals with recent nerve lesions.
The idea that degenerative processes in the nervous system should lead
to such an increase in the amount of potassium in the blood that it ean
be recognised qualitatively by the yield of the platino chloride crystals
is, as in the case of the choline hypothesis, based on a complete
misconception of the quantitative relationships. Taking the minimal
values given?? for the amount of potassium in the central nervous tissues
as 17 per mille, it follows that 1 gramme of brain contains 0°0017 gramme
K,0; taking the minimal value of K,O content of blood as 0°2 per mille
and the total volume again as 4,700 c.c., we find that in man a sudden
destruction of 1 gramme of nervous tissue would add 00017 gramme
K,O to 09 gramme K,O in the total blood. This is a difference smaller
than that which appears in Abderhalden’s* very careful analyses of samples
of blood from two different animals of the same species. In 20 c.c. of
blood the amount of potassium would be increased by 0°000007 gramme,
or less than 1/100th of a milligramme.

In smaller animals, e.g., in cats, the proportion between the astioaill
of potassium normally present in the blood.and the amount which may be
set free at any given moment by degenerative processes in nervous tissue
remains of course essentially the same, since with a diminished volume of
blood we have also a diminished amount of nervous tissue.

28. ‘Oliver Sharpey Lectures,’ Brit. Med. Journ., May 5th, 1907.
29. See, for instance, Hammarsten, Text Book of Physiological Chemistry, p. 414, 1904,
30. Zeitachrijft. |. physiolog. Chem., XXV, p. 65, 1898.

CHOLINE IN ANIMAL TISSUES AND FLUIDS 125

ay It is clear that even the most extensive degeneration which can
possibly oeeur is quite incapable of yielding sufficient choline and
potassium to account for the striking contrast in the yield of crystals to
which Halliburton refers. If the preliminary statement by the same
author is correct, that actual estimations of the potassium content of the
blood show an increase in some cases of acute degenerative diseases, it is
te ually clear that this increase must be due to other factors than the
ration of potassium by the degenerating nervous tissue.

_ The smaller volume of cerebro-spinal fluid makes this a more likely
place wherein to find the products of degeneration of nervous tissue. But
from the data given above it follows that the destruction of nervous tissue
would have to be very considerable, 1 gramme at least, before one would
be able to detect choline in the cerebro-spinal fluid by means of
~_ Rosenheim’s modification of the iodine test, which is stated to indicate
ae choline in a dilution of 1 : 20000. Kaufmann,*! who collected a litre of
cerebro-spinal fluid from various cases of nervous diseases’ and who was

ius in a position to investigate the subject by exact cheinical methods
G instead of relying upon micro-chemical tests, was unable to isolate choline
from the cerebro-spinal fluid. The fact that Kaufmann found another
organic base to be present which, although having many reactions in
. common with choline, proved on more detailed chemical examination to
S be altogether different from choline, detracts from the value of observations
.; based upon micro-chemical reactions for the detection of a substance
‘7 which, if it is present at all, is present only in fractions of a milligramme.

Those who first advanced the choline hypothesis were under the
__ impression that the amount of choline liberated in diseases could be
measured by multiples of milligrammes; and the relatively great
quantities of choline liberated, which their experiments appeared to
_ indicate, was one of the strongest arguments in favour of their hypothesis.
We know now that the amounts which can possibly appear as the result of
degenerative processes in nervous tissue are exceedingly minute, and the
question arises whether such small amounts may not be derived from other
tissues. Lecithin is a constituent common to all cells. A destruction of
a great number of white or red blood corpuscles may just as well lead to
choline being set free. Thus the leucocytosis occurring in the cerebro-
spinal fluid in some diseases, or the degeneration of a mass of red blood
corpuscles after a haemorrhage may be a source of choline.

31. Neurologisches Contralblatt, Vol. XXVII, p. 260 1908

ti =” A ae ee ee Se a
a ee ee ee eee

126 BIO-CHEMICAL JOURNAL

SuMMARY

1. No choline can be detected in normal blood, provided that the
decomposition of the lecithin be prevented in the methods employed. Nor
can choline be detected in blood of animals after extensive lesions of the
central or peripheral nervous system. :

2. The maximal quantity of choline which would be set free in oe |

processes of degeneration is too small to be detected in the blood by any of
the methods hitherto employed. The same holds good for potassium.

3. The physiological and chemical tests given by pathological blood,
and alleged to be characteristic of choline, are exhibited in exactly the
same manner and to the same degree by normal blood.

4. The micro-chemical reactions recommended for the detection of
choline are given irregularly both by normal and pathological cerebro-
spinal fluid. The presence in the cerebro-spinal fluid of a substance giving
the micro-chemical reactions of choline cannot, therefore, be considered
as indicative of degenerative changes in the nervous system; and in view
of Kaufmann’s results it may even be doubted whether any of the micro-
chemical tests at present in use are specific for choline. Whether such
changes increase the amount of choline in the cerebro-spinal fiuid by
fractions of a milligramme, and whether the same effect may not
be produced by the disintegration of tissue other than nervous, will have
to be determined by further experimental investigation.

127

+ |
; THE BIURET REACTION AND THE COLD NITRIC ACID

"TEST IN THE RECOGNITION OF PROTEIN

By KARL Hl. VAN NORMAN, M.B. (Loronto).

Communicated by Francis M. Goodbody, M.D., M.R.C.P.
From the Pathological Chemistry Department, University College, London.
(Received February 9th, 1909)

In all books on physiological chemistry and clinical diagnosis one is
recommended to place great reliance on the biuret reaction. In the
course of some investigations on the comparative reliability of various
tests for serum albumen I found that I obtained very contradictory
results with this test, and therefore I think it would be of interest to give

the results which I obtained in an exhaustive series of tests.

The usual method given in books for carrying out this test is: that

one adds to the albumen solution some soda solution and then, drop by

drop, a very dilute solution of copper sulphate, a blue precipitate
appearing which, on shaking, dissolves with a pink tinge, finally changing
to a reddish violet." But on doing this I found that one could not be
certain of obtaining the reaction in a urine which by other tests, such as
cold nitric acid, acetic acid and heat, and potassium ferrocyanide and
acetic acid, gave clear indications of the presence of albumen.

It was therefore necessary to make up a solution containing a definite
quantity of serum albumen and to repeat the tests. On doing this I found
that it was impossible to make certain of obtaining constant results unless
one used solutions of soda and copper sulphate of definite strength, for the

addition of the soda solution in varying amounts made a great difference

in the result of the test.

It is generally recognised that it is necessary to add a dilute solution
of copper sulphate, and I found that, although with a high percentage
of albumen a 2} per cent. solution of copper sulphate may be used, yet
for all practical purposes the solution of copper sulphate should not be
stronger than 1 per cent.

Another point on which the various text-books, which I have had an
epportunity of consulting, lay little stress is on that of heating, and

1. Some text books advise adding the copper sulphate solution before the soda solution.

128 BLO-CHEMICAL JOURNAL

although one can obtain a more or less definite result in the cold, I found
that the reaction is much more marked on heating. In fact, in a very
weak solution of albumen the violet is not perceptible until the solution
is boiled, Pe
In consequence of these difficulties I decided to work with solutions
of albumen, soda and copper sulphate of definite strength, and the original —
solutions which I made up were as follows :—
I. Albumen solution. Distilled water containing 0°2 per cent. of —
albumen,
If. A solution containing sodium hydrate 10 grammes and distilled
water to 100 c.c.
III. A solution containing re-crystallised copper sulphate 5 grammes —
and distilled water to 100 c.c.

Solution I.—Beginning with the original solution of 0°2 per cent. of
albumen, I made an exhaustive series of dilutions with distilled water,
these diluted solutions varying in strength from 0°05 per cent. to 0°00033
per cent. of albumen. A number of these dilutions are referred to in
Tables I and II.

Solution IJ.—As a result of many tests in which I used varying
perceniages of sodium hydrate, in albumen solutions of different strengths, —
I found that a 10 per cent. solution of sodium hydrate gave the most
constant results. The quantity of this soda solution used in each bettie:
final result—is given in Tables I and II.

Solution I1I.-The 5 per cent. solution of copper cciphdtom was too |
strong, for even in the original solution of albumen (Solution I) a brown
coloration with a white flocculent precipitate was obtained on the addition
of one drop of this copper solution with soda. I therefore diluted to a
2} per cent. solution, but, after three or four estimations, I found this in
turn too strong, and it was necessary to dilute to a 1 per cent. solution.

Table I shows results on using a 2} per cent. solution.
Table II 9 ” 1 oe) oe)

For adding the copper sulphate and soda solutions drop i drop, I
used an ordinary glass pipette, that is, ordinary glass tubing, drawn toa
small calibre at one end.

It was necessary to do a great many tests before being able to
determine the best method of performing the test. I found as follows :- —

(a) 10 c.c. of the albumen solution is the best amount to use.

(6) In all the albumen solutions of from 0°004 per cent. to the limit

| oe oe eS
bi ig ak late

THE BLIURET REACTION 129

sodium trydrate (10 per cent.) gave the best results.

- In the albumen solutions stronger than 0°004 per cent. two to ten
drops of copper sulphate (1 per cent.) can be added, according to the

strength of the solution of albumen.

In the solutions from 0°05 per cent. to 0°012 per cent. of albumen I
used 2} per cent. copper sulphate solution. (See Tables I and II.)

; : _ (e) The copper sulphate should be added first, then the soda solution.
‘The contents of the test tube should then be mixed by inverting twice.

(d) After adding the copper and soda the solution should be heated
to boiling, when the violet is intensified.

mw All text-books I have consulted mention that the colour obtained
4 _ in the biuret reaction is pink or red, changing to violet. I was unable
to obtain this play of colours, as all my positive tests gave the violet colour
at once. I found that the stronger the solution of albumen the more
marked was the violet colour—gradually diminishing with dilution—and,
also, that in strong solutions of albumen the violet is intensified by the
_ addition of copper sulphate solution drop by drop, boiling between drops.

In the very dilute solutions of albumen there is some difficulty in
_ recognising the violet colour. I found that the colour was best seen as
follows :---

1. In the meniscus.
2. On looking down the test tube—against white.

Re 3. On comparing the albumen solution with a test tube containing
an equal quantity of distilled water. The two tubes should be held side

Ta _ by side and compared : —

(a) On looking down both—against white.
(6) In the meniscus—-

The best light is that which strikes from above. Both tubes should
be slanted towards the operator at about 35°. One should hold the tubes
above the head and look up through the meniscus from below, against a
dark background.

sataph Taste I .
Albumen CuS0, Solution NaOH Solutioi

per cent. 24 per cent. 10 per cent, Result Remarks
S080 3 dro 5 dro Violet Reaction well marked
0-016 my on a se ahett ta
” ws oo fs Reac
0-012 1 drop ee . ee

130 BIO-CHEMICAL JOURNAL

In the next dilution—solution of 0°008 per cent. of albumen—one drop
of copper sulphate solution (2) per cent.) and five drops of sodium hydrate
solution (10 per cent.) were added, and the solution at once turned brown.
On boiling, the brown colour deepened with the formation of a whip
flocculent precipitate. The result might be shown thus :—

Albumen CuSO, Solution NaOH Solution pect Remarks
per cent. 2) per cent. 10 per cent.
0-008 1 drop 5 drops No violet Solution at once turns brown

In all the succeeding tests I used al per cent. solution of copper
sulphate.

Tanie IL
Albumen CuSO, Solution NaOH Solution Result Bisiarks
per cent. 1 per cent. 10 per cent. i

0-0080 2 drops 5 drops Violet Colour deepened on addition of one
more drop CuSO, solution.

0-0040 1 drop - ir ;

0-0026 ” ’ *s

0-0020 oy ”

0-0016 bs “F aS

00013 ” bb] ?

0-O011 a sg su

0-0010 ” ” ”

0-00088 9 ” ” Violet faint before boiling.

0-00080 ” ” :

0-00072 o ‘9 ” |

0-00066 ” ” a Violet very faint before boiling. —

0-00061 ” ” ” llea comparison,

0-00056 re a ” distilled water as
gern oy is just perceptible before
boiling.

0-00053 ” ” ”

0-00050 ” ” ”

0-00047 a mn ‘i Violet imperceptible | before boiling

a on comparison with dis-
water.

0-00044 ” ” ”

0-00042 ”? ” ”

0-00040 af - Violet imperceptible be before pie
ied voles, cal joo pean
tilled water, jus
after boiling when compared
with distilled water.

0-00038 Er $d Z On standing for two minutes solu-
tion turns brown.

0-00036 0 ” - On standing for one minute solu-—
tion turns brown.

0-00034 ” ” » On standing for twenty seconds

, solution turns brown.
0-00033 a a No violet Solution at once turns brown, with

the formation of a white floceu-
lent precipitate.

From the results which I obtained in a watery solution of albumen,
I decided to make further tests, using as my original solution urine and
albumen. TI therefore took a urine free from albumen and added albumen
to make a 2 per cent. solution.

4 ee
a ee

Oa yh
az. i ee a

THE BIURET REACTION 131

, _ As in the previous tests, I made a great many dilutions with distilled
ed out investigations as before. The more or less abridged
Its are seen in Table ITI.

In these reactions I used sodium hydrate (10 per cent.) and copper

Walt Ll.
‘ BD toe
‘

lrop sed copper sulphate solution was used, but in 0°04 per cent. and all
he succeeding solutions to the limit of delicacy I found that one drop of
‘copper sulphate solution gave the best results. In all cases five drops of
sodium hydrate gave most constant results.

On account of the added colour of the urine, I found it more difficult
to recognise the violet colour in this than in the first series of tests.

Tansie UL
- CuSO; Beanies NaOH Solution Result Remarks
1 per cent. 10 per cent.
Five drops Five drops Violet Colour deepened to maximum on
addition of ten more drops of
Cu80,—drop by drop—boiling
between drops.
me as -s Colour deepened to maximum on
addition of five more drops of
CuSO,—drop by drop—boiling
between drops.
One drop Five drops Violet Reaction good.
” ” ”
i x Violet faint before boiling.
‘i 2 Violet very faint before ee
si Z ns Violet perceptible before boiling
only on comparison with dis-
tilled water.
a ” - ”
a tbe i $iggr: a gy ible before boil-
ter boiling violet just

percept on comparison with
tilled water,

” ” ” But changing almost at once to
; brown, with the formation of a
white flocculent precipitate.

albumen, and in a urine containing albumen diluted with water, I next
tried to obtain the limit of delicacy in a urine containing albumen when
urine free from albumen was used for dilution.

ye Having determined the delicacy of the test in a watery solution of —

132 BLO-CHEMICAL JOURNAL

According to Hammarsten,' an excess of creatinin reduces copper
sulphate, and Kellas and Wethered? have shown that an excess of uric
acid and urates, as well as creatinin, causes a reduction of copper sulphate.

The first urine which I used for diluting contained an excess of uric
acid, and in these solutions I was unable to obtain the violet colour
except in the stronger solutions of albumen—0°2 per cent. and stronger. :

I then did further tests with solutions made by diluting the
albuminous urine with urines containing an excess of urates and
creatinin, but was unable to obtain positive results except in the stronger
solutions of albumen—0O'2 per cent. and stronger.

While working on the biuret reaction it occurred to me that it would
be of interest to determine the delicacy of the cold nitric acid test for
albumen, as this test is so well known and so commonly used.

The limit of delicacy of this test given in text-books is 0°002 per cent.,
but I was able to obtain positive results in very much weaker solutions,
as will be seen in Table IV.

To obtain satisfactory results in doing the test the following points
should be noted :—

(a) 12 c.c. of albumen solution should be used.

(b) 2 c.c. of nitric acid should be used.

(c) A pipette should be used for adding the nitric acid to the albumen
solution. This pipette is made as follows:—One end of an ordinary piece
of glass tubing, 5 mm. diameter, is drawn to a calibre which allows the
nitric acid to escape from the pipette by the drop and not in a continuous
stream, when the finger is removed from the large end of the pipette.

The pipette should be not less than 28 em. in length.

Technique of the Test-—-Take 12 c.c. of albumen solution in a test
tube. Draw up 2 c.c. of nitric acid in the pipette, place the finger over
the end and carefully lower the pipette into the albumen solution until
the point of the pipette is on the bottom of the test tube. Remove the
finger from the pipette and allow the nitric acid to run in. When no more
nitric acid runs in place the finger over the end of the pipette and
carefully withdraw it. Care must be taken not to shake the test tube
during the test.

By this method the line of demarcation between the albumen solution
and the nitric acid is narrow and very distinct, thus facilitating the
recognition of the albumen ring as it forms.

1. Physiologischen Chemie.
2. The Lancet, October, 1906.

ee .
wae “Ccles ane 38 * “sf . . "a ee. h
a THE BIURET REACTION 138
*% My first series of tests was done with diluted solutions of my original
albu ation, that is, distilled water containing 02 per cent. of
bumen. The Beeslic in a number of these solutions are given in
One finds in text-books the statement that, for the cold nitric acid
r albumen to be positive, the albumen ring should occur within
minutes, but on referring to Table IV it will be seen that in very
lut ‘solutions of albumen the ring did not occur until as long as fifteen
utes, and reached its maximum density in one hour after adding the
Taste IV
Vere Result-- Time
2 c.c Positive In 20 seconds
soy bi ST] maliawte
bed ” ” 1 29
” ” ” 1} minutes
bid ” ” 2 b 2
” bb 7? 2 ”
” ” ” 23 ”
” ” s”? 7 ”
2 tite
” ” bd 4 Phd
” ” ” 4) ”
” bb ” 5 °°
” ” ” 5 ”
” ” ” 5S ”
” ” bed 6 ”
” ” ” 6) ”
” ” ” 7 bed
” ” ” a ”
. : 9 is Ring most marked in 15 minutes
” ” ” 10 ” ” ” 20 ”
” ” ” ll ” °° ’ 25 ”
” ” » 12 ” ” ” 30 ”
” ” ” 13 ” ” ” 35 ”
” ” ” a ” ” ” 40 ” X
ae ll + aaah oe oe
” ” ” 15 ” ” ” 1 hour
” _ aw ” ” ” 1 hour. Very

faint

a For my next series of tests I used urine containing 2 per cent. of
_ albumen—made by taking a urine free from albumen and adding albumen

to make 2 per cent. Taking this as my original solution I made a. large

number of dilutions, using for dilution a urine free from albumen.

The results in a number of these solutions are given in Table VY.

¥ _. <1 Q « a

134 BIO-CHEMICAL JOURNAL

In doing the cold nitrie acid test in albuminous urine containing an
excess of uric acid, after adding the nitric acid a cloudiness appears in a
few minutes, gradually increasing in density all through the urine from
the top of the solution down to the nitric acid ring. To differentiate
between this cloudiness, due to uric acid, and the albumen ring, the urine
should be heated gently just above the nitric acid ring. The cloudiness
due to uric acid disappears and the albumen ring remains. There will
then be seen in such cases, in order from below, the nitric acid ring, the
white albumen ring, a clear space, and above this more or less cloudiness.

It must be remembered in this connection that albumose may be _
present also. If so, on gently heating just above the nitric acid ring, the —
depth of the white precipitate will diminish, since the albumose is
dissolved and the albumen is left. By this means one can obtain a rough
idea as to the quantity of albumose present.

In Table V it will be seen that in very weak solutions of albumen
the albumen ring did not enn until ten minutes after the addition of
the nitrie acid.

TABLE V

Albumen Nitric Result Time

per cent. Acid
0-04 2 c.c. Positive In 1 minute
0-0040 ” ra + 1} minutes
0-0020 ” ” s¢ 2 ss
00013 ys a8 a | a
0-0010 a ” » 3 ”
0-00080 oe - eer | Pr
0-00060 an s9 pe ”
0-00057 . ° 99 43 9
0-00050 me o ae »»
0-00044 ” ” 9? 5} ”?
0-00040 Ld 7” s¢ 6 ”
0-00036 os ‘ 64 ”
0-00033 ; . 7
0-00030 * “a
0-00028 , * 8
0-00026 ” ? ’ 8}
0-00025 : 9 ;
0-00023 = of
0-00022 ae ae 9 Faint
0-00021 i a ae a
0-00020 * * » 10 9% Very faint

CONCLUSIONS

A. Biuret Reaction.—Limit of Delicacy.

I. Ina watery solution of albumen is 0°0004 per cent. or four parts of
albumen in 1,000,000 parts of distilled water.

THE BIURET REACTION 135

io” II. In albuminous urine diluted with distilled water is 0°001 per
am ‘cent, or one part of albumen in 100,000 parts of urine and distilled water.

Ill. In albuminous urine diluted with urine free from albumen.

The biuret reaction is very difficult to obtain in concentrated urines
containing an excess of uric acid, urates or creatinin. In such cases it is
only in the stronger solutions—0'2 per cent.—that the reaction is good.
In the weaker solutions of albumen the violet colour may be obtained,
but almost at once the colour changes to brown.

In most cases no violet colour is obtained, the solution at once turning
brown with the formation of a white flocculent precipitate.

B. Cold Nitric Acid Test.Limit of Delicacy.

____-L.__ Ina watery solution of albumen is 0°00006 per cent. or six parts
of albumen in 10,000,000 parts of distilled water.

a II. In albuminous urine diluted with urine free from albumen is
a ca per cent. or two parts of albumen in 1,000,000 parts of urine.

ee tiny best thanks are due to Professor Harley and Dr. Goodbody for
much kind assistance and advice.

136

THE PROPERTIES AND CLASSIFICATION OF THE

OXIDIZING ENZYMES, AND ANALOGIES BETWEEN

ENZYMIC ACTIVITY AND THE EFFECTS OF
IMMUNE BODIES AND COMPLEMENTS yee

By BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio-
chemistry, University of Liverpool, and EDWARD WHITLEY, M.A.
(Owon.). Lea

(Received March 15th, 1909)

- po

The presence in plant and animal juices of bodies possessing the
properties of ferments which act as oxidizing agents for unstable bodies,
such as the guaiaconic acid of guaiacum resin, was first demonstrated by
Schénbein! in 1856. .

The action of these bodies was studied in greater detail by Bertrand?
in a series of papers beginning in 1894. Bertrand first showed that the
formation of Japanese lacquer was due to the oxidation of a substance
present in the plant juice of certain species of Rhus by a ferment which he
termed /accase. The browning and blackening of the cut surfaces of
fruits and other parts of vegetable tissues is also due to the action of
oxidizing ferments.

From the juice of the potato an oxidizing ferment termed tyrosinase
was obtained, which acted vigorously upon the isolated tyrosin, yielding
a dark brown substance, richer in oxygen than the tyrosin from which
it was formed.

These browning or blackening ferments are distinct in nature from
the oxidizing ferments present in most fresh plant juices, and also in pus,
milk, blood, and extracts of some animal organs, which possess the
property of turning tincture of guaiacum blue, of oxidizing and thereby
rendering coloured certain phenolic bodies, or of setting free iodine from
a solution of hydriodic acid.

Bertrand showed that this latter class of oxidizing ferments was
present in many different types of plant, and he expressed the view that
they were universally present in all plants.

It is, however, certain that they are not present in all plant juices,
although they may be present in other parts of the same plant. Our own
experiments recorded below show, for example, that the class of oxidizing

1. Zeitasch. f. Biol., Bd. TI, 8. 325.
2. Compt. rend., p. 1215, 1894.

Bi
r. ;
-

Pe

PROPERTIES OF OXIDIZING ENZYMES 137

- ferments is absent in the fresh juice of the fruit of the lime, lemon, and

orange, but present in the crushed seeds of lemon and orange. Other
experiments have demonstrated to us that their distribution in different
parts of a root or fruit varies widely, and that they are present in greatest
quantity in the region containing the greatest abundance of respiratory
vessels,

Thus a section across a carrot stains almost at once in the protoxylem,

and the staining radiates out from this along the path of vessels, while
internally to the protoxylem there is very little staining (see Expt. XIX).

It was found by many observers that the guaiacum test for these
oxidizing ferments often failed, especially in fluids of animal origin and
when freshly made tincture of guaiacum was used for testing.

Further, it was found that in such cases the blueing could still be
obtained, provided either hydrogen peroxide were added or some organic
form of peroxide, containing oxygen linked in the peroxide form.

A similar oxidation is seen in the well-known test for traces of blood
in urine or other fluids in which tincture of guaiacum and ozonic ether (a

ae form of peroxide) are added to the suspected fluid. This test also

demonstrates that bodies other than oxidizing ferments occurring in living
tissues are capable of giving the guaiacum blueing. Many inorganic
and organic bodies also give it, such as sulphurous and nitrous acids,
ferrous salts, and potassium permanganate.

The substances causing this reaction may be either oxidizing or
reducing bodies, so long as they can act as oxygen carriers.

In this connection it may also be pointed out, as has been done by
Bach,’ that all the oxidations shown to occur with this class of oxidizing
enzymes, viz., oxidation of hydriodic acid, of the aromatic amines and

phenols, and of the guaiaconic acid of the guaiacum resin, possess the
_ eommon factor of a moveable or dynamic hydrogen atom in the molecule.
Between this hydrogen atom and the atom of the oxygen in the peroxide

form there is already a strong tendency to reaction, and the catalyst
simply increases this tendency.

On account of the too great readiness with which the guaiacum
underwent the oxidation change giving the characteristic blueing, and
also on aceount of the fact that blood, and other oxidizing bodies not
ferments, readily yielded the guaiacum test, other more definite tests were
sought out. A considerable number of tests have been described in which
phenols or amido-phenols are oxidized to bodies which possess various

3. Berichte d. deut, Chem, Gesellach., Vol. XL, p. 230," 1907.

SS Le ee ee a

138 BIO-CHEMICAL JOURNAL

colours. These colour tests for oxidizing enzymes are all very similar, and
when one of them has been obtained, or not obtained, in any given case, a
similar result is usually obtained with all the others, probably because
the potential of chemical energy required for oxidation lies at about the
same level for all of them.*

The results of carrying out a number of these colour tests will be —

given later, but one important difference which we have found may be
stated here in general terms, since, in our opinion, it leads to certain
important conclusions regarding the classification and mode of action of
this class of oxidizing enzymes different from those generally accepted at

the present time. This result is that none of the colour tests, with the

exception of the guaiacum test, are appreciably increased in velocity, or
catalysed, by the solution of the oxidizing ferment alone, except when the
fresh juice is taken immediately after its preparation (see Expt. VII), and
are only so catalysed when hydrogen peroxide or some other form of
peroxide is added at the same time.

The reasons why the guaiacum test is often positive when the others
are negative are (1) the presence of organic peroxide in the guaiacum itself,
and (2) that the oxidation occurs much more readily than with the other
colour tests with the oxy- and amido-phenols, and such like bodies, and

so the naturally occurring organic peroxide of the juice or of the reagent

is sufficient to give an oxidation. Whereas the more stable amino-acids
only break up with appreciable velocity in presence of the more readily
decomposed, and hence more powerful, free hydrogen peroxide, which has
been artifically added.

No attempt will be made in this paper to give a full account of the
very wealthy literature of the oxidizing ferments,® sincé accounts in
English have recently appeared in ‘The Nature of Enzyme Action,’ by
Bayliss,® and the ‘ Intracellular Ferments’* of Vernon;‘ but some outline
of the classification introduced by Bach and Chodat, and of the basis for
that classification, must be given in order that our own views and
experiments may be more easily followed. It may be added that this
classification has been almost unanimously adopted by subsequent writers.

4. Minor differences in reactivity of the different colour reagents are given in the experi-
mental part of the paper.

5.” Citations of the literature are also to be found in the comprehensive review by Bach.
and Chodat, Biochemischen Centralblatt, 1903; in ‘* Uber tierische Peroxydasen,’ Ernst von

Czyhlarz u. Otto von Fiirth, Beitrage z. Chem. Physiol. u. Path., Zeitsch. {. Biochemie, Ba. X,

8. 358, 1907; Bach, Berichte, 1904 to date ; Spence, this Journal, Vol. III, p. 165, 1908.

6. Monographs on Bio-Chemistry. Edited by Aders-Plimmer and Hopkins. Longmans, —

yreen and Co., 1908.
7. Published by John Murray for Physiological Laboratory, University of London.

PROPERTIES OF OXIDIZING ENZYMES 139

The experimental work of Bach and Chodat, especially that of a
= quantitative nature on the action of these oxidizing ferments, is, in our
- opinion, sound, and forms an excellent basis for further work; but we
- eannot agree that their experimental observations give sufficient ground
for the classification they have adopted nor for belief in two classes of
oxidizing ferments, the owygenases and the perowidases, which they
postulate, forming together a mixture of ferments corresponding to the
In our opinion there is but one class of such enzymes concerned,
which, since they act only in presence of oxygen linked as a peroxide,
might still be known as the peroxidases. The oxygenases do not exist as
____ ferments at all; there is no use for such a term, and it might be allowed
to drop out. The oxygenases are simply preformed peroxides in juice or
reagent, and not in any sense ferments.
___. + The experimental basis, and as far as we can discover the only one,
for a belief in the oxygenases is that certain juices, such as that of the
potato, give at once a blue colour with fresh guaiacum alone without
added hydrogen peroxide, while other fresh juices, such as those of
‘Tadish and cucumber,® give absolutely no blue coloration with the fresh
- guaiacum tincture until hydrogen peroxide, or some other peroxide, is
added, when almost at once a strong blue is obtained.

Further, if the potato juice be heated for some hours to about 60° C.

_ it in most cases loses the power of blueing spontaneously, and now only
gives the blue colour when a peroxide is added.?

The explanation of this is easy on Bach and Chodat’s classification,
which represents that there are two enzymes present in the juices, viz.,
oxygenase which manufactures peroxides from the material at hand in

_ the juice, and a peroxidase which then activates this peroxide and causes
it to attack the oxidizable body yielding the colour test and oxidine it,
‘so producing the colour.

_ We submit that there is no proof that there is an enzyme or enzymes
forming the class of the oxygenases and producing organic peroxides, and
that the whole difference between the two classes of juices is simply that

8. Bach and Chodat, loc. cit.

This destruction of peroxides (or of o oops tegen caper sp):
lla ge cv wh t is usually described, for potato juice may be briskly boiled in
a test-tube for half a minute without cutiexinn completely all the store of peroxide. If it is
again boiled, ea ag or if it be thoroughly boiled for some minutes in a beaker so that all
ta age Bee iled, the peroxide is completely destroyed. This refractiveness was discovered by

Cal Cr U.S. Dept. of Agriculture, No. 18, p. 17) in the case of the tobacco oxidase, and
by him attributed to part of the oxidase existing as a zymogen. There is little doubt, however,

t the result is due to incomplete destruction of the peroxide and peroxidase,

140 BIO-CHEMICAL JOURNAL

one has a store of peroxides and the other has not, and, further, that
these peroxides are thermally unstable and are destroyed by heat.

After we have put forward the evidence in favour of this view, we
shall draw attention to the similarity between this simpler scheme for
the activity of the oxidizing ferments, and that for the action of ‘ immune’
body and ‘complement’ in the case of haemolytic and other cytolytie
sera and the cells attacked; and also for the action of ordinary hydrolitie
enzymes upon their substrates.

In all the above cases three things are required, viz.:—({1) a body of
a ferment nature, (2) a substrate on which it can act, and (3) a body ~
which enables the ferment to act upon the substrate so as to cause
hydrolysis, oxidation, or some other type of chemical reaction. Also,
the ferment and the substrate are usually much more specifie to each other
than is the third body, which is simpler in nature, such as an alkali or
acid or a peroxide, and we suggest that complement is an activating
substance of this kind.

Returning to the oxidizing ferments, we may now quote the classi-—
fication given by Bach and Chodat. In their general review of the subject
of oxidizing ferments, published in 1903, these authors give the following
list :-—

‘I. Ovygenases. Protein-like bodies which take up molecular
oxygen with peroxide formation.

‘II. Perowidases, which enormously raise the oxidation power
of the peroxides which by themselves oxidize very sluggishly, at
the dilutions in question.

‘IIL. Katalases, which destroy peroxides with evolution of
oxygen.’

In our opinion, all these three names are ill-chosen, because they
break the general law that the name should, as, for example, in lactase,
tyrosinase and lipase, indicate by the root of the word the substrate acted
upon, and by the termination ‘ase’ the fact that the body designated is
an enzyme. Now, in the first two classes named above, oxygen and
peroxide respectively are not the substrates, but the bodies which the
particular enzymes bring into action upon the substrates, and much more
resemble the alkali in trypsin action or the acid in pepsin action. Further,
in the third class, the ‘ katalases,’ there is no reliable evidence of this
destructive action upon hydrogen peroxide being due to an enzyme at all.
Although it is clearly a distinct activity from that of the oxidizing
ferments, yet it is not an oxidation, and it is not specific, occurring, as it
does, with every ferment solution of whatever type, with nearly all animal

PROPERTIES OF OXIDIZING ENZYMES 141

or vegetable fluids, and with numberless inorganic catalysts. In any
_ ease, it is absurd to give it a name which belongs to or includes the whole
yast range of catalytic actions. Every true enzyme is a ‘katalase’ in
the sense that it acts catalytically, and why a catalytic agent, which
happens to act upon hydrogen peroxide, and of which it has never been
ae, clearly shown that it is a specific enzyme, should be dignified with the
a name of ‘ katalase’ it is difficult to conceive. Whatever the body is (or
; the large number of bodies), it is quite certain no oxidizing enzyme is in
= question, since nothing is oxidized and the oxygen is simply discharged as
molecular oxygen. 3
‘The Deiter which is made by Bach and Chodat!® that the so-called
oxydases are mixtures of peroxide bodies and peroxydases, appears to have
led some of their English reviewers to entertain the view that they regard
_ the oxygenases as unstable peroxides rather than as true enzymes
producing peroxides. But that this is an error is shown by the above
classification, as also by the repeated statement in their works that the
oxygenases are ferments which produce the organic peroxides, and that it
is only in the presence of the peroxides that the peroxidases have any
action whatever.
i Thus the rationale of the complete reaction of oxidation by these
ferments, according to Bach and Chodat, is as follows :—

First an enzyme, called oxygenase, acts upon certain substances
present in the plant and forms organic peroxides; and, secondly, another
and distinct enzyme, peroridase, which is entirely unable to act in absence
of formed peroxide now comes into activity and causes a reaction which
_ transfers the oxygen, previously attached to the peroxide by the

oxygenase, to the oxidizable substrate, which may be one of several
substances.
ul ‘We have also looked carefully through the later papers by these
authors, and have not been able to find any abandonment of the position
that the oxygenases are peroxide-producing enzymes.

Thus, Bach! states the oxydases are nothing else than mixtures of

roxy and oxygenases, that is, of peroxide-activating and of

peroxide-building enzymes. In this paper, because he obtained by alcohol
precipitation, and re-dissolving in water, a solution which alone only
slowly attacked tyrosin, but was strongly activated towards tyrosin by
addition of small amounts of hydrogen peroxide, Bach claims to have
10. Bio-chemisches Centralblatt, 1903, and elsewhere.

"IL. Berithte, Vol. XXXIX, No. 10, p. 2126, 1906,

142 BIO-CHEMICAL JOURNAL

shown that the usual tyrosinase which attacks tyrosin at once is also a
mixture of an oxygenase and of a peroxidase which is specifie for tyrosin.
No further proof is here given of the enzymic nature of the supposed
oxygenase of the tyrosinase.

Again, in 1908, in a polemical paper against Chodat, who had been
unable to repeat his observations as to the activation of tyrosinase by
hydrogen peroxide, Bach'? states that the failure of Chodat to repeat his
results was due to the use of too high concentration of hydrogen peroxide,
and in this paper he once more enunciates that ‘tyrosinase, like the
‘ordinary oxydases, is composed of an oxygenase, that is, a body which
‘ produces (“ bildet”) peroxide accompanied by uptake of oxygen, and is
‘replaceable by hydrogen peroxide, and of a peroxidase, which activates
‘the so-formed peroxide, or the added hydrogen peroxide.’

There is proof given by Bach and Chodat that peroxide is present in
fresh plant juice which has been treated with air,!* but none that this
peroxide is produced by an enzyme. The proof as to the presence of
peroxide, which is not entirely free from experimental suspicion, was
obtained as follows. Air was passed through a sample of fresh juice from
Lathraea squamaria containing oxydase, and at the same time a 1 per cent.
solution of barium hydrate was slowly dropped into the juice. A barium
precipitate was obtained which did not give the hydrogen peroxide reaction
with the titanium reagent, after washing and decomposing with dilute
sulphuric acid. The solution did, however, intensely blue potassium
iodide and starch, and as the absence of nitrate was shown otherwise, it
was assumed that an acylated hydroperoxide was present.

It does not appear to us that much that is new is proven by this
experiment, since the oxidase known to be present in the original juice
would have similarly acted on iodide and starch solution, and there is no
reason why it should not be precipitated unchanged by the baryta.

On other grounds it is highly probable that traces of organic peroxides
are present in most fresh plant juices. (See Expt. XX.)

This does not, however, prove that they are formed either in the plant
or after separating the juice by an enzyme such as the postulated
‘oxygenase’ of Bach and Chodat, and all experimental proof of such
enzymic origin is hitherto lacking. |

12. Berichte, Vol. XLI, p. 216, 1908.

13. We shall show in our own experiments later that excess of air or ae as by ]
in shallow layers in open vessels, again destroys the peroxide first formed, so that once more
no result is obtained without added peroxide.

Red See Fo ee

PROPERTIES OF OXIDIZING ENZYMES 148

_ Again, the presence of the elusive oxygenase is not proven by the
thermal. instability of the spontaneous blueing by guaiacum. For the
peroxides are also very unstable bodies thermally, and the failure to obtain
a blue with juice and guaiacum alone after heating to 50°— 60° C., and
then obtaining it on adding hydrogen peroxide, may easily be due to the
destruction of the thermally unstable organic peroxides, and not to that
of an enzyme which produces such peroxides.

In fact, the failure of the blueing after heating is all in favour of the
simple presence of unstable peroxides. Because, if it were an enzyme
(oxygenase) only that were destroyed by the heat, then this enzyme
previously to the process of heating would have had ample opportunity to
preform a good supply of organic peroxide, and although the enzyme
were destroyed on heating to 50°—60°C., there would be enough pre-
formed peroxide to still give the blueing afterwards.

The simplest hypothesis, accordingly, is that the heating merely
destroys the organic peroxides which are essential to the action of the
only ferment required, viz., the peroxidase, and on now replacing this
loss of organic peroxide by the simple hydrogen peroxide, the blueing is
obtaimed because the essential chemical linkages are present for the
peroxidase to act.

In regard to the experimental fact discovered by Bach and Chodat
that the juices of certain plants, such as cucumber and radish, do not
give a blue with guaiacum alone, but at once give a fine blue on addition
of traces of hydrogen peroxide, while other juices, such as those of potato
and carrot, give a blue at once, with guaiacum alone, we can completely
confirm the experimental observation, but believe it is susceptible of a
much simpler explanation than that the potato and carrot contain two
ferments, oxygenase and peroxidase, which act as above described, while

‘im cucumber and radish the oxygenase is absent and peroxidase only

present.

The oxidation in presence of added hydrogen peroxide is easily
demonstrated by means of any of the colour tests, in the many vegetable
juices or animal extracts or secretions which contain an oxidizing ferment.
But it is an exceedingly difficult matter to trace to their true causes those
cases in which a positive result is obtained without the addition of
hydrogen peroxide,

Such a positive result is more often obtained when the guaiacum test
is employed than with the other reagents we have used for testing. This
very frequent positive result is not always due to development of peroxide

144 BIO-CHEMICAL JOURNAL ©

in the reagent on standing, as is usually said to be the case, but is present

or absent in a most capricious way. In our opinion, the variation in

result is due to the presence or absence of preformed peroxide of organic
nature in the particular pieces of resin from which the tincture was made,

although no doubt there may be a tendency for the amount of this pe ro

to increase as the reagent grows older.

The only similar variation with the other test substances we have
observed was in the behaviour of potato juice to p. phenylene-di-amine
in giving the characteristic green without added peroxide of hydrogen

when the potato juice was tested immediately after grating the potatoes.

Within half an hour the same juice gave not a trace of green until
hydrogen peroxide had first been added. Further, even on adding
hydrogen peroxide to its juice, if the mixture was left for some few minutes
before the p. phenylene-di-amine was added no green was obtained. Thus,
the peroxide had been destroyed in the meantime, instead of any having
been formed as would have occurred if an oxygenase or peroxide-forming
ferment had been present.

In this same potato juice, even when quite fresh and giving the
p. phenylene-di-amine reaction, the positive result of an amethyst colour
with a-naphthol could not be obtained until hydrogen peroxide had also
been added. .

With this one exception of the potato juice immediately on
preparation, all the colour reagents except guaiacum invariably gave us
a negative result until hydrogen peroxide was added. The guaiacum gave
us a positive result in the capricious way described above in many
instances, such as potato, carrot, wheat, oats, apple, banana; and several
species of nuts.

The causes of the variations with the guaiacum will presently be

traced out as we followed them up experimentally, but before taking this’

up we would like to point out that in order to justify the views of Bach
and Chodat, that there are two enzymes present, one of which produces
peroxides, while the other activates reaction between such peroxides and
the oxidizable chromogenic body, the evidence obtainable from the use of
different colour-reagents must be consistent and concordant. That is to
say, in the same juice one reagent must not indicate the presence of
peroxidase only and absence of oxygenase, and another reagent indicate
both oxygenase and peroxidase.

Now this is precisely what cecurs experimentally, for with the
exception of .the freshly made potato’ juice reacting positively to

i i!

PROPERTIES OF OXIDIZING ENZYMES 145

p- phenylene-di-amine at the same time that it reacted negatively to
‘naphtha, the evidence was conclusively negative in regard to
‘oxygenase’ throughout, save for the capricious results with guaiacum
only. If now it be admitted that these variable guaiacum results were
due to varying amounts of peroxide in the guaiacum, and hence afforded
no evidence of ‘ oxygenase’ in the juice being tested, then all evidence for
the existence of oxygenase disappears, and we are left with one enzyme,
or type of enzyme only, which produces ifs effect by activating towards
each other peroxide and the substrate to be oxidized.

In support of the view that the different behaviour of guaiacum
tincture was due to varying amounts of peroxide in the tincture itself, we
submit the results of Expt. XX detailed later (see p. 158), which show
_ that when means are taken to exclude peroxide from both guaiacum and
_ juice being tested, a negative result is always obtained until peroxide has
been added from outside.

. But if this be accepted we are left only with the positive result with
perfectly fresh potato juice and p. phenylene-di-amine, and since this

rapidly disappeared as the juice stood, and, further, as traces of added

os hydrogen peroxide similarly disappeared on standing, we are disposed
to attribute this effect to traces of peroxide in the fresh juice which
disappeared on standing.

Tf such a ferment as the postulated ‘ oxygenase ’ had really existed
.. in the potato juice, then the amount of peroxide would not have decreased
a on standing, but, on the contrary, there would have been an increase all
é the time, and the test would have grown stronger instead of disappearing.
_ The disappearance was probably due, in part, to the substances

_(‘katalases’) present in all such juices which decompose peroxides after
the juice is shed from the cells, and in part due to the using up of peroxide
in the oxidations brought about by the tyrosinase and other oxidizing
-ferments which we have been calling peroxidases.

That the rate of disappearance of peroxide is in some way connected
with exposure is shown by the fact that the positive reaction with
p- phenylene-di-amine is lost in a very short time (inside half an hour)
when exposed to the air in a thin layer in a flat-bottomed glass dish, while
it is still present after several hours in a sample of the same juice preserved
in a tightly corked flask which has been completely filled. A positive
reaction to a sample of guaiacum tincture which gives no colour with
carrot juice is still given by this potato juice, for long after it has ceased
to react with p. phenylene-di-amine. This is probably due to two reasons :

146 BIO-CHEMICAL JOURNAL

first, that the guaiaconic acid is. more readily oxidized (or at a lower
chemical potential) than the p. phenylene-di-amine, and secondly, that
there is a conjoined effect of the minimal traces of peroxide in both
reagent and juice in one case, and of that in juice only in the other.

It would thus appear that all those cases in which a colour test of
oxidation is obtained without adding hydrogen peroxide are due to
peroxide already present in traces either in the juice or in the reagent.
Also, that the amount of peroxide in the juice decreases instead of
increasing on standing, and that the peroxide which is specially present
in varying amount in the guaiacum reagent exists in the resin and its
products quite at the beginning, and so may be present in absolutely
freshly made tinctures, from which it may be removed by treatment with
charcoal, ig it:

It was further found that a mere trace of a reducing agent, such as a
drop of dilute ammonium sulphide, or sulphuretted hydrogen water added
to half a test tube full of a fresh juice showing the guaiacum test without
addition of hydrogen peroxide, was quite sufficient to permanently destroy
all the trace of organic peroxide present, so that now the reaction became
negative,

RECORD OF EXPERIMENTS

The chief substances used by us for demonstrating oxidation were
(1) Guaiacum in fresh 10 per cent. tincture made from the resin.
(2) A 1 per cent. solution of ». phenylene-di-amine in distilled water.
This gives on oxidation a fine green colour, which usually strikes out
quite suddenly after a pause of several seconds; it occurred positively
only after addition of hydrogen peroxide, saving the exception above
mentioned of potato juice, where it is obtainable without added peroxide
for a period of about half an hour in the fresh juice; after that it is only
obtainable when hydrogen peroxide is also added. (3) A 1 per cent.
solution of a naphthol in 50 per cent. alcohol. This reagent never gave
a positive result with any of the juices, however fresh, until hydrogen
peroxide was also added; it strikes a fine amethyst colour in presence of
the peroxidase and peroxide; the reagent turns slowly amethyst coloured
on standing, and should be made fresh. (4) Indo-phenol or Spitzer's
reagent, which we prepared fresh in’all cases by mixing equal parts of the
two previous solutions of p. phenylene-di-amine and @ naphthol, and of a
2 per cent. solution of sodium carbonate. This on oxidation yields a fine

PROPERTIES OF OXIDIZING ENZYMES 147

purple; the test never gave a positive result unless hydrogen peroxide was
ad ded, the development of colour being no greater than occurs
spontaneously in the diluted reagent or in boiled juice of equal
eoncentration, to which the reagent has been added in equal quantity.
5) Hydrochinon in 1 per cent. solution in water. (6) Synthesized
guajacol’ (Merck). This behaves quite differently from the natural
um resin, giving a strong brown colour, and on standing a brown

ipit te, obtainable only in presence of peroxide. ” Guaiaconic

ni In ‘making comparative tests care was taken to use as nearly as
possible corresponding amounts of juice equally diluted, of reagent, and |
Se pct oe peroxide where that reagent was added. In the earlier
ex) ents a solution of hydrogen peroxide, the usual laboratory
pe ng rth (10 vols. of HO, per cent.) ten times diluted was employed,
and in the later experiments the pure perhydrol of Merck was used, first
uted ten-fold, as a stock solution made up in small volumes at a time,
1 this was then again ten times diluted immediately before use.

Asa general rule, about 5 c.c. of juice was taken or a given dilution
(ue eee with distilled water, and to this about 1 ¢.c. of the diluted
peroxide was added and 1 c.c. of the reagent being used.

“Where necessary three tubes were used, of which one contained the
juice. after boiling, another the juice without boiling, with reagent only
added, and a third, unboiled also, to which both reagent and hydrogen

In other cases ere the juice was of known character and the
» of peroxide was not being proven, but rather the effect of
nts upon its activity, this procedure was not necessary, and the
‘imentation was modified accordingly.

t I.—-Testing of wheat for oxidizing ferments. One part of
y weight, extracted with three parts by volume of distilled water
, filtered to an almost clear filtrate—water in contact with the
dered Bain for about one hour.

Reagent 1. Boiled 2. Unboiled extract 3. Unboiled
extract plus hydrogen extract
: alone peroxide alone
1. Guaiacum Blue* Blue Blue
2. (eager a some Nil _ ,, Green ; Nil
3. a@ naphthol Nit © - © ~~ Amethyst Nil
| 4. -Hydrochinon Nil Brown ©) © >) Nil

* This result was afterwards shown to be due to insufficient boiling of the extract.

148 BIO-CHEMICAL JOURNAL

Experiment II.—Similar experiment with powdered and extracted
oats, giving exactly similar results.

Experiments IIT and IV.—Swiss Condensed Milk and a proprietory
food called Glaxo gave negative results on testing with all the usual
reagents. This is to be expected, since all such foods and also canned
fruits and patent foods for children and invalids are sterilized by boiling.
It is a point worth bearing in mind that this absence of oxidizing ferments
distinguishes all preserved foods from fresh foods.

Experiment V.—Fresh milk, tested as above to guaiacum, p. pheny-
lene-di-amine, a naphthol, and hydrochinon, gave a reaction only in
presence of added hydrogen peroxide, except in the case of guaiacum,
where a slow blueing occurred without the peroxide.

Experiment VI.—Fresh carrot juice was positive to guaiacum with
and without addition of peroxide, but positive to other reagents only after
peroxides. In latter experiments with a guaiacum free from peroxide
carrot juice was often found negative, especially after standing in air for
some time.

Experiment VII.—Fresh potato juice was found the most strongly
positive of all the juices tested towards guaiacum; no preparation of
guaiacum was used throughout which did not give a blue with it, but
when both potato juice and guaiacum were deoxidized as much as possible,
the blueing effect practically disappeared. Potato juice when just drawn
oft is slightly positive to p. phenylene-di-amine but negative to the other
tests, and turns negative to the p. phenylene-di-amine also after about
half an hour. :

Experiment VIII.—Potato juice was dried in an air oven at about
50 to 55° C. and then extracted with water at 48° C. for forty-eight hours.
This very materially reduced the oxidizing power, a faint blueing was
still obtainable both with and without added peroxide, but stronger with;
hydrochinon both negative; a naphthol negative without, slightly -
positive with; p. phenylene-di-amine, both negative.

Experiment I1X.—Wheaten flour gave strong plus to guaiacum with
and without peroxide; faint reactions with other reagents and only in
presence of peroxide in each case.

Experiment X.—Serum of pig’s blood, three days old, gave negative
to guaiacum both in presence and absence of hydrogen peroxide; to
p- phenylene-di-amine slight reaction without and strong reaction with
peroxide; to a naphthol, peroxide tube strongly positive, doubtful without
peroxide.

PROPERTIES OF OXIDIZING ENZYMES 149

: BE Baperiment XJ.—Effect of minute amounts of acid and alkali and of
id and alkaline phosphates on the oxidizing reactions. This was tested

follows, using guaiacum as reagent :—

~The acid stops in minute amounts; much more alkali is required to
p the oxidation, but the colour changes from blue to a 265 aa green.

e were carried out with potato and carrot juices :—

Fresh potato juice diluted ten-fold with distilled ete: and 5 e.c. of

3 e taken to quantity named below of reagent and then 0°5 c.c. of tincture
of guaiacum added.

. WM Normal control Blue at once.
ike 2, Added 0-1 c.c. of iy Ha (= 359 approximately). Faint blue, very slowly deepening.
Added 01 cc. of Sf NaOH. As blue as contro.

| A Added 0-5 c.c. of 5 per cent. NaH,PO, (=

5 Added 0-5 c.c. of 5 per cent. Na,HPO, Blue much weakened.

a Y 3

eh a _ 1. This reversed behaviour of the phosphatic solutions as compared to the acid and alkali
isv -y peculiar, and difficult to understand, but it was several times observed.
ai x ui fh RS * |

approximately) As blue as control.!

rot juice treated exactly the same way gave :—

2 “Added 0-1 0. of 2 Htc No bleting,

14 Added 0-1 c.c. of 7 NaOH. Strong blueing.

| a ao 4 Added 0-5 c.c. of Fan ii NaH,PO,. Strong blueing.

“a “Added 0-5 c.c. of 5 per cent. Na,HPO, Weak blueing.

_ EBaperiment XII.—¥resh juice from grated radishes was taken

and tested to (1) guaiacum, (2) p. phenylene-di-amine, (3) a ey agen

4) pyrogallol, (5) indo-phenol (Spitzer’s reagent).

The same result was obtained throughout, viz., negative in absence

ia ; hee

‘of hydrogen peroxide; positive in presence of the peroxide. There is

absolutely no blueing with guaiacum alone, even with guaiacum which

alone gives a good blue with potato juice. On standing over night in a

. stoppered vessel the same radish juice now gives a fair blue with the same

_ guaiacum. The remaining portion of the radish juice was centrifuged,

first alone and then after fractional precipation, with alcohol added up to
25 per cent. in the mixture. It was found that both sediments were very
_ strongly active, much more so than the supernatant liquor.

. Experiment AUT. —This experiment was made with carrot juice and

with apple juice. In each case the fresh juice got, as usual, by grating

~

"lass

150 BIO-CHEMICAL JOURNAL

and filtering was tested alongside an extract made by drying the grated
mass at 45° C. for some days. ‘The carrot gratings had been in the oven
for three days and the apple gratings for eight days.

The contrast in the filtrates from the fresh and the dried preparations
is most striking. The dried is throughout completely negative, and the
fresh positive. With guaiacum alone there is little blueing, even in the
case of the fresh juices, but immediate effect with addition of peroxide;
in neither case is there an effect with the filtrates from the dried materials.
With p. phenylene-di-amine, a naphthol, and indo-phenol there is a very
strong reaction, but only in presence of hydrogen peroxide with the fresh
preparations, and nothing with preparations from the dried material.

Experiment XIV.—Effeets of partial or fractional precipitation of
potato juice with alcohol. The properties of the different alcohol pre-
cipitates, and results of combustion of the dried alcoholic precipitates.

A quantity of 320 c.c. of fresh potato juice was taken and thoroughly
centrifuged. The deposit consisted of starch underneath with a thinner
greenish brown layer on top like very fine mud, or ooze. The upper
layer could be easily washed off from the strongly impacted starch granules
underneath,

It was so removed, shaken up with distilled water, re-centrifuged,
and separated again from the small amount of starch mechanically
removed with it at the first separation. It was once again shaken up with
distilled water, and the brownish colloidal solution or suspension which
frothed strongly was found to be strongly active, giving a good blue with
guaiacum alone. Examined under the microscope it shows a field
crowded with exceedingly minute particles much less than 1» in active
Brownian movement,

This deposit is like an excessively fine mud, which readily passes into
suspension; it closely resembles the different fractional precipitates with
alcohol, about to be described.

The supernatant fluid after centrifuging was still opalescent and gave
a strong blue with guaiacum, to this one-quarter of its volume of absolute
alcohol was added, making a 20 per cent. alcoholic solution, in which a
copious greyish brown precipitate appeared.

Throughout it was found difficult and tedious to nncenl this
exceedingly fine precipitate by filter and pump, and that they settle
excellently and quickly into a compact mass with the centrifuge so that
they can readily be separated by decantation.

The precipitate so separated from 20 per cent. aleohol on shalsual up

a}

<a 7 a eee 2 .

PROPERTIES OF OXIDIZING ENZYMES 151

_ with a considerable quantity of water gives a fine and durable suspension,
_ which gives-a fine blue with guaiacum alone and strongly positive tests with

x the other reagents after addition of hydrogen peroxide. The supernatant

fluid is still opalescent and still gives a good blue with guaiacum. A

quantity amounting to 350 ¢.c. of this supernatant fluid was taken, and to

it 88 c.c. of absolute alcohol was added, bringing up the alcohol concentra-

; tion to 36 per cent., giving rise to a considerable amount of a second

aleoholie precipitate, which, on separation and shaking up with water,
was likewise found to be strongly positive. Separated supernatant fluid

2 again taken and treated with one-fourth its volume of absolute alcohol,

so bringing strength up to approximately 49 per cent. by volume; the
third alcoholic precipitate was obtained, also giving fine blue with
guaiacum alone and positive result with other reagents plus hydrogen
peroxide. Again added one-fourth volume of absolute alcohol to super-
natant fluid, bringing percentage of alcohol up to 60 per cent., and giving

a fourth alcohol precipitate, which also gives strong positive tests.

Finally, this supernatant fluid, which was now quite clear of
f suspended particles, had an equal volume of absolute alcohol added to it,
_ 80 bringing the alcoholic content up to 80 per cent. by volume. A heavy
floeeulent precipitate was so obtained and separated by centrifuging and
decanting. The supernatant liquid now gave no positive tests, but the
fifth and last precipitate did give such positive tests after suspending by
shaking in water. Bs

All the different precipitates caused by successive additions of alcohol
are alike in appearance, all contain oxidizing enzyme, and all give similar
composition on combustion; they are, moreover, exactly like the
: Suspension naturally present in the potato juice and partially thrown out
_ by the centrifuge. All the precipitates have the appearance of a fine
alluvial mud, and pass up into a fine suspension with fine particles in
_ Brownian motion when worked up into water. This suspension remains

‘permanent over night, and settles very slowly. Notwithstanding this,

quite clear solutions yet exceedingly active as oxidizers are obtainable
when fresh potato juice is allowed to stand untouched for twenty-four
hours, so that there must also be a portion. of oxidizing ferment in true
solution. It is probable that a part of the fine deposit can pass into
solution and show oxidizing properties, for even after thorough washing
the suspensions show strong oxidizing properties.

‘An explanation of the peroxidase results obtainable both with the
water clear juice, and with the thoroughly washed colloidal suspension

wea eee ye ee eee

152 BIO-CHEMICAL JOURNAL

of the same juice may possibly be that the oxidizing ferment exists in two
phases, a portion being in true solution and a portion in excess as colloidal
particles in fine suspension. .

Iodine gives no distinctive colour, although the suspensions show
marked reduction with Fehling’s solution.

When the neutral suspensions are boiled there is very marked
frothing, the test-tube filling with froth. Part of the emulsion breaks

down on boiling, but a good deal still remains as a milky suspension.
After just boiling, there is a very slow blueing with guaiacum alone, much

increased on adding hydrogen peroxide, and on further boiling for about
one minute, all oxidizing power is lost in either presence or absence of

peroxide.

Addition of a trace of hydrochloric acid causes no immediate effect,
but on standing for a few minutes the emulsion settles completely and the
supernatant fluid has lost all trace of oxidizing properties with or without
peroxide,
A trace of alkali does not cause loss of oxidizing properties, but a
greenish yellow shade appears in the blue colour produced, more alkali
inhibits the activity and destroys the ferment.

In a second experiment with potato juice a similar fractional
precipitation with alcohol was carried out after all starch had been
thoroughly thrown out by prolonged centrifugalization, and then, instead
of taking up in water, the several precipitates were dried in a desiccator
over sulphuric acid to a constant weight, and the dried scale preparations
so obtained were first examined qualitatively by the protein tests, tested

for phosphorous and manganese, and a portion was combusted of each in —

order to ascertain if the composition varied as the percentage of alcohol
was increased. Similar testing was also carried out with similar pre-
cipitates from turnip and carrot juices.

The following table shows the ultimate By teu of the precipitates _
caused by the alcohol in the potato juice after drying to constant weight;
the first precipitate, with 20 per cent. of alcohol, was too small for
analysis :— 3

CoMpPosiITION OF DIFFERENT OXIDIZING PRECIPITATES THROWN Our BY “THE

Given PERCENTAGES OF ALCOHOL FROM FresH Potato JUICE tislde

No. of Pp. and percentage of alcohol by Ash Percentages reckoned in ash-free

olume in fluid from which Percentage substance of— saat

thrown out Cc H N O, ete. —

No. 2. pp. from 36 per cent. of alcohol 707 48-9 73 10-2 33-6
o & Pp» ” 4-71 46-9 70 15 Ries 5)

” = pp- ” be ” ” 10-96 : 463 7-2 12-2 3435 Ped

” willy ” 29-89 45:1 6-9 9-9 38-1 7

Average for organic io canceled _ 46-8 71 10:8 35-3

—

.
.»

PROPERTIES OF OXIDIZING ENZYMES 158

De aa It is to be observed that while the ash varies considerably, the
composition of the ash free organic matter remains fairly constant. The
position approaches more nearly that of the mucins or gluco-proteins
n that of any other class of bio-chemical bodies. In this connection
noteworthy that the suspensions in water of all the precipitates very
ongly reduce Fehling, and they are also thrown down as above stated
svery dilute acids, and even the less strongly ionized organic acids,
which destroy their oxidizing activities. On the other hand, as we shall
t out, the precipitates either fail altogether with certain of the protein
s, or in other cases indicate traces only of proteins.
ae The ash always contained manganese, as shown by Bertrand, and iron
- in small amount, and phosphates i in fair quantity were invariably present

As a general rule, a biuret test could only be obtained after boiling,
d then only a trifling show of colour, nothing whatever of the colour
ven by free protein being seen in the cold. The xanthoproteic test was
gative in the cold, and after boiling only the palest yellow was
btainable, but a certain amount of deepening towards an orange colour
ae ; obtained on adding ammonia in excess. The Millon’s reagent gave
quite negative results throughout.
_ Our general impression from the qualitative protein tests is that
wrotein is absent in these highly active precipitates, although there is a
i nitrogen percentage, as shown by the above figures, which indicate
a carbohydrate moiety joined on to a radicle rich in nitrogen but not
containing the groups which are present in ordinary protein and give the
tests.

In order to find out whether prolonged drying to a constant weight
“Dyaie acid in the desiccator had any effect upon the oxidizing
, the remaining portions of the four dried precipitates which yielded
malytical results given above, and which had been kept dry from
mber 11th till November 26th (fifteen days), were taken and ground

ip m ¢ istilled water. All yielded again very fine emulsions like those
originally obtained, showing exceedingly minute particles, under the one-
sixth objective, in active Brownian movement. The only change from the
i condition is that now scarcely any blueing is obtained with the

- guaiacum test until hydrogen peroxide also is added, when a fine blue is
obtained ; this shows that all the preformed peroxide of the fresh juice
has disappeared, but that the peroxidase enzyme is still present and active
and ready to induce interaction between substrate and peroxide. The

154 BIO-CHEMICAL JOURNAL

other colour tests are, as usual, completely negative in absence of
hydrogen peroxide, and strongly positive in its presence.

Carrot juice behaves much like potato juice in regard to fractional
precipitation by alcohol, all the precipitates being less in amount from
the same amount of juice, and all being active; the 80 per cent. alcohol
finally left after precipitation is inactive. With turnip juice nearly all
the oxidizing ferment comes down in the third fraction (57 per cent. by
volume), and the 80 per cent. supernatant fluid is clear and free from
oxidizing action, The precipitates by showing a reducing action on
Fehling, and by failure to give protein colour tests, behave one
identically with the potato juice precipitates.

Experiment XV.—Effects of exposure to oxygen, and of shutting off
from air, or of exposing to air in shallow layers respectively.

Here we came across one of the most puzzling and difficult to
understand of all our results. So faras we are able to follow them, there
appear to be two opposed effects, one of a more rapid formation of peroxide
in very small amount from some precursor in the very fresh juice, and the
other of a slow breaking up of the preformed peroxide which is slowed
or avoided in tightly sealed up full vessels.

For example, a freshly cut surface of cucumber, radish, carrot, or
certain other vegetable tissues shows no blueing with a guaiacum prepara-
tion which is itself free from peroxide either naturally or by special
treatment. (See Expt. XX.) But merely grating the cucumber to get
the juice and filtering from débris of tissue through a clean cloth is
enough to give origin to a certain amount of blueing power in absence of
added hydrogen peroxide Further, vigorous shaking up of this juice
’ with air, or bubbling oxygen through it for some minutes, materially
increases the amount of natural peroxide, and hence the blueing effect
with guaiacum alone. Now, if the juice be separated into two parts, one
of which is used to fill a small bottle, which is then tightly corked, and

the other is spread out in a thin layer on the bottom of a Petri dish,

then, entirely contrary to what might have been anticipated, the tightly
corked up sample juice will be found after a few hours, or even after a

couple of days, to give a good blue with guaiacum alone, while the juice —

exposed to the air will probably have lost all blueing power except on
addition of hydrogen peroxide. This change occurs probably in a few
hours, certainly within twenty-four hours. Hence, in the early stages
shaking with air develops peroxide, but the peroxide formed is unstable.
in presence of atmospheric oxygen, which ultimately robs the juice of all
its small store of peroxide.

Pe

PROPERTIES OF OXIDIZING ENZYMES 15!

eT
ot

Not only can the exposure destroy the natural peroxide, it (or some
oxide-destroying enzyme such as catalase present in common solution)
> destroy added hydrogen peroxide, for if to a juice which is not
1 i y an oxidizing reaction with guaiacum alone one adds a minute
ount of hydrogen peroxide, sufficient to give a good immediate blue on
‘urther addition of the guaiacum reagent, and if now the juice so treated
3¢ divided into two equal parts, to one of which guaiacum is added, at
e giving a good blue, and the other allowed to stand for five minutes,
per ‘agi is found that no 0 colour i is given by the latter owing to destruction

Phau “That this is the true ee a is shown by the immediate blueing
- obtained on adding more peroxide after the guaiacum has failed to give
the colour in the second portion.
_ By taking a large quantity of juice, adding small quantities of
drogen peroxide at a time, and testing small portions at intervals after
each addition of peroxide, this peroxide destruction may be followed out
f aften as desired.
__ This experiment further demonstrates that there is no firm linkage
etween ferment and peroxide such as can prevent the latier from
truction, for then enough peroxide would be permanently preserved
| on to the ferment to give a blue colour at once when the substrate
- (guaiacum) was added even after a time interval.
Fac Experiment XVI.—Effect of reducing agents upon ferment and
- owidizing reactions.
: i _ The oxidizing ferment is extremely susceptible to the merest traces
_ of reducing agents, such as sulphuretted hydrogen or ammonium sulphide,
for the addition of a drop of diluted ammonium sulphide or sulphuretted
hydrogen water to a half test tube full of potato juice will not only stop
lueing with guaiacum alone, but will prevent it even in presence of a
vess of hydrogen peroxide. The ferment appears to be completely
coverably destroyed, either by molecular change or by firm
anchora ze of the sulphide. At any rate, we have never been able to restore
activity once the reducer has been added.

_ Experiment XVII.—T hermo-labile nature of the perowides.
7 Except that we prefer to call it peroxide instead of oxygenase, we
ean experimentally confirm the statements of Bach and Chodat, that the
: substance giving oxidizing reactions without added hydrogen peroxide is
a ~ Tess. stable than the ferment or peroxidase which gives rise to the oxidation
__ in presence of peroxide. We have pointed out that our dried precipitates

156 BIO-CHEMICAL JOURNAL

from alcohol had lost practically all their peroxide, although they had
plenty when freshly thrown down and at once taken up in water. Heating
to about 55° C. destroys the unstable peroxide bit leaves the peroxidase ;
this occurs more quickly in some juices than in others. Thus, potato
juice takes some hours, and even then although the amount is very
greatly diminished a slight trace remains which is most difficult to get rid
of. For example, a portion of freshly prepared potato juice was divided
into eight similar portions, which were placed in an air oven kept at
55°C. on February 7th, 1909, at 12-20 p.m., and the tubes were tested
one at a time at the following intervals afterwards:—Tube 1, tested at
12-45, still gives intense blue immediately; tube 2, at 1-20 p.m., blue not
so intense and comes more slowly; tube 3, at 2-20, more slowly still ;
tube 4, at 3-50 p.m., still a good deal of blue, partial precipitation by
the heat; tube 5, at 4-30 p.m., still blue; tube 6, at 11-40 a.m. (February
8th), very faint blue only coming after prolonged shaking; tube 7, at
3-40 p.m., no longer gives blue until hydrogen peroxide has been added.
Thus a period of about twenty-seven hours was required to destroy all the
natural peroxide at 55° C.

The same result occurs more slowly, as pointed out, on standing in
air at ordinary temperatures, and here again potato requires longer than
any other juice we have experimented with, taking several days, while
carrot juice will be almost free within twenty-four hours. We have not
investigated whether this is due to a larger original supply, or to slower
reduction or greater resistance of the natural potato peroxide. Great
variations are experienced of a perplexing nature both as to the rate at

which the maximum amount of peroxide is developed, and the rate at

which it disappears, for which we are quite unable to account.

Experiment XVIII.—Effeets of germination on distribution of
oxidizing ferment and peroxide.

A small quantity of oats, from the same sample as the grain used
in Experiment II, were sown on moist cotton wool, and left in the
incubator to germinate from November 4th till November 16th, being
kept moist during the period and at a temperature of 32°C. At the

end of the period the sprouts were about 10 centimetres long. They were —

pulled out from the oats, and the sprouts and residues of seeds were tested
for oxidizing ferment with the following results :—

Both gave a positive result with ordinary guaiacum tincture alone;
both were quite negative to all the other coloured indicators for oxidation
until hydrogen peroxide had also been added, and both contained

PROPERTIES OF OXIDIZING ENZYMES 157

bd

se,’ as shown by vigorous discharge of oxygen when added to
peroxide (5 vols. per cent.).
Experiment XIX.—Absence of oxidizing enzymes in the fresh juice of
lemon, and orange ; but presence of such in seeds of lemon and orange.
One of our incentives in commencing this research on oxidases was
wry absence of oxidizing enzymes by reason of the sterilization,
other form of preparation, from all preserved foods, vegetables, and
, as also from sterilized milk and milk substitutes, such as children’s
infant food. While such enzymes are present, and in considerable
‘ity, in fresh vegetables and juices, in fresh fruits eaten uncooked,
This is a difference of a tangible nature in the chemistry of food, of
_ these two classes, and it appeared possible that it might give some basis
( r a a better understanding of the etiology of such diseases as rickets or
scurvy, the former of which is said to be associated with exclusive use of
boil aaa proprietary foods, while the latter, which is, however, a distinct
ondition, appears to arise from prolonged abstention from fresh vegetable
oe | a I
It was these considerations which led us to try those antiscorbutie
vegetable juices which are accredited with the most powerful properties,
such as lime juice, lemon juice, and orange juice.
‘We were greatly surprised to find that this group amongst the many
_ which we tested was the only one which did not yield good oxidase
_ reactions either in presence or absence of peroxide. Entirely negative
results were given both by juice and rind, and the crushed seeds only in
the case of lemon and orange (we were unable from lack of material to test
_ the lime seeds) gave a somewhat feeble positive effect.
a _ The absence of oxidase in the juice of the fruit of this group is very
interesting, although we are at a loss at present to account for it. The
is not simply concealed by the somewhat high acidity of the
_ juices, for no more success is obtained in the testing on neutralizing the
_ juice. It may be that the organic acids present inhibit the production of
the ferment, as they certainly inhibit its action when added from without,
and tend to destroy it. Thus, if a quantity of potato juice be mixed with
the acid (or even almost neutralized but still faintly acid) lemon juice, on —
now adding guaiacum a negative result is obtained; but if the acid be
completely neutralized the usual blueing follows, especially on adding
hydrogen peroxide.

In testing the lime juice care must be taken to use a sample which

158 BIO-CHEMICAL JOURNAL

has not any sulphurous acid as preservative, as this gives a transient
oxidation and blueing with guaiacum even in minimal amounts.

The crude juice gives not a trace of effect with any of the tests for
oxidizing enzymes.

Experiment XX.—Direct application of guaiacum test for pn
and perowide, to fresh cut surface of vegetables.
This was carried out by slicing, after thorough washing of the outer

surface, and then applying an ‘old’ solution of guaiacum to one surface

and a ‘new’ solution of guaiacum to the other surface. The test is a
convenient one for peroxide and peroxidase distribution in different parts
of the tissue, and also demonstrates that even in those plants which yield
most easily the reaction with guaiacum alone, such as the potato and carrot,
the natural organic peroxide is not present as such in the plant tissue,
but is formed in the first few minutes after cutting from some precursor
in the plant juices.

It may be pointed out that although the terms ‘old’ and ‘ new’ are
used in describing the results of this experiment, this is done because at
the time we thought the difference was due to age of the two tinctures.
Later we found (see Expt. XXI) that the amount of peroxide present in
any given sample of guaiacum was more an accident of amount of
impurities of vegetable origin in the piece of resin from which it was
originally made up. Accordingly ‘old’ means containing more peroxide
and sufficient to give direct test in presence of peroxidase, and ‘new’
means comparatively peroxide-free, and hence enables to give blueing
with peroxidase in absence of added hydrogen peroxide.

A potato was taken, washed thoroughly, dried, and sliced through
with a clean knife. At once tincture of guaiacum was placed on the two
cut surfaces; ‘old’ guaiacum on one, and ‘ new’ guaiacum on the other.
The surface exposed to the ‘ old’ guaiacum blues instantly all over; that

with the ‘new’ guaicum only at one or two spots and much more faintly. «

The blueing with the ‘new’ tincture occurs more especially at a bruised
spot or just close to the epidermis. On cutting away about a centimetre
all around the peel to remove any injured portions, only a very slight
blueing is obtained, for a few minutes, but comes on standing.

In order to test whether the residual amount of blueing in the case
of the potato were due to age, as the potato had been removed from the
earth for some days or weeks, and since quite negative results had
previously been got with fresh radish and fresh cucumber treated with

‘new’ guaiacum tincture, the following similar experiment was next
tried.

Pe ee ee eT ete ee © aE ht hy eee RT

PROPERTIES OF OXIDIZING ENZYMES 159

| ha fresh clean carrot was taken, a sectional cut made across it, and
m cut t surfaces were treated one with ‘old’ and the other with

1 for about a zone of half a centimetre thickness round the outside,
1 again most marked in the protoxylem, the central part is less
ter stained, but the whole surface is still distinctly blue.
= - The ‘new’ tincture, on the contrary, gives for over ten minutes’ time
anid application no blueing except at ~ minute spots where there
happen to be old bruises just under the skin; “ at about the expiry of ten
i a a faint blue appears as a ring sending out radiating branches,
s distinctly marking out the protoxylem. It is noteworthy that
5 is part of the section where most oxygen would probably be
pr in the plant tissues.
On adding to the section of fresh carrot treated with ‘new’ guaiacum
neture, a minute trace of hydrogen peroxide, by touching with a fine
ass rod moistened with the diluted peroxide, there is produced at once
intense blueing, showing the failure to obtain blueing initially is due
: to absence of peroxide and not to absence of ferment.
Beperiment XXI——Effects of destruction of peroxide in both reagent
is (geaiaen tincture) and vegetable juice by treatment with animal charcoal
‘This experiment was conducted in order to make a closer study of
certain results of the previous experiments which show rather perplexing
e variations i in behaviour towards the guaiacum test (a) with different types
of vegetable juice, or the juice of the same origin with the age of the
juice, and (5) taking the same juice and at the same time, variations
according to whether one or another tincture of guaiacum made from the
“ ay stock of the resin was employed for the test.
_--———s« ith the single exception of potato juice, for an hour or so after it is
ke made, giving a positive result with p. phenylene-di-amine without addition
be ‘also of hydrogen peroxide, all the oxy- and amido-phenol coloured
indicators of oxidation had concordantly given that a ferment peroxidase
r alone was present, which required to have peroxide added in order that it
might act.
___-‘The guaiacum test, on the other hand, as above indicated, gave most

-

_ n to a section of an showing a bruise did not, however, demon-

Bear ke anes acted viesing to the aught reed of thet injured and browned cells. Still,

‘i the naturally occurring must soa an oxidizing effect upon tyrosin or similar chromo-
genie substances giving colour on oxidizing.

160 BIO-CHEMICAL JOURNAL

perplexing variations. Thus, even with fresh guaiacum and potato juice
as fresh as it can be separated from the grated tuber, -a considerable
blueing was obtained; fresh carrot under like conditions sometimes gaye
a blue fainter than that with the potato, sometimes nothing; and fresh
cucumber or radish juice, even with moderately stale guaiacum tincture,
gave but a poor effect and nothing with fresh tincture.

The most feasible explanation of these peculiar variations which
occurred to us, and that which suggested the following experiments, was
that there are two natural sources of peroxide, viz.:—-(a) the plant juice,
and (6) the tincture of guaiacum, The summation of these two effects causes
the guaiacum test to be positive, especially with a strongly peroxide
containing tincture, in cases where the small amount of peroxide in the
juice is insufficient yet to start the oxidation of the oxy- and amino-
phenols. In further support of this, the potato juice, which of all the
juices we examined is most strongly positive to guaiacum alone, reacts
positively, when it is quite new and highest in its content of natural
peroxide, to p. phenylene-di-amine, but just fails to appreciably quicken
the oxidation of a naphthol, unless hydrogen peroxide be added,

We hence had to examine separately the tincture and the vegetable
juices, using mildly destructive agents for peroxides which would not
destroy the ferment also. Direct reducing agents, such as sulphuretted
hydrogen or ammonium sulphide could not be employed, since, as above
stated, they appear permanently to destroy the ferment also, either by
altering its constitution or by firmly linking on to it.

An attempt to clear the guaiacum of colour by means of animal
charcoal although it failed in its immediate objective, giving a much
deeper green coloured solution, happily had the advantage of destroying
the peroxide of the tincture, and yielding a clear filtrate which had no
power of blueing until hydrogen peroxide was also added.

The usual explanation of the variations in peroxide content of
guaiacum tincture is that the peroxide is formed as the tincture stands,
and hence, for purposes of the test, that freshly made tinctures must
always be employed. ;

While we are not prepared to deny that peroxide may be so formed,
as the tincture stands and darkens in colour in the course of several weeks,
we have clear evidence that this is not the main source of the variations,
and that a quite freshly made tincture just filtered off may give marked
blueing, while another sample of tincture a week old may give quite a
negative result, although it was made from some pieces of the same stock
of resin.

PROPERTIES OF OXIDIZING ENZYMES 161

a nade-with the same absolute alcohol in 10 per cent. solution from
Pia - same stock of guaiacum resin, but one was made about ten weeks
previously and the other four days previously. The ‘old’ tincture gave
_ fine blue with all our vegetables on the cut surfaces, as detailed in
a Experiment XIX, and with the separated juices, while our ‘ new ’ tincture
_ gave scarcely a trace of effect. In order to make sure that it was a
_ question of ageing and accompanying peroxide formation, we determined
upon making an absolutely fresh preparation, and for this purpose we
some pieces of the guaiacum resin, and washed the outer surface
ily with alcohol in order to remove a green powder which forms on
ther surface of the broken guaiacum, and which we thought might
be oxidized. These washed pieces were dissolved as far as they would
_ dissolve to make a 10 per cent. tincture in previously boiled absolute
aleohol, and a clear filtrate was obtained and used immediately for testing.
_ To our great surprise, this gave at once a fine blue immediately with
the sections of vegetables and juices without adding any peroxide what-

We therefore had now a tincture just made which reacted positively
_ without added peroxide, a four days old tincture made without any special
precaution which acted negatively under like conditions, and a ten weeks
old tincture which behaved exactly like the just made one.

ig Further, a tincture made from the green powder on the outside of
the pieces of guaiacum gave, like the four days old tincture, a negative
result.

| Tt was also nctiond that while the ten weeks old tincture and the
‘new’ tincture possessed the deep yellow brown colour of guaiacum
tincture, the four days old tincture was much paler i in colour, and the
_ tineture made from the surface powder was green in colour. This led us

_ to boil up some of the ‘ old’ tincture with animal charcoal, in order to try
to decolourize it, when we found that instead of decolourizing it went
darker, and the filtrate had a green colour.

- This filtrate tested now upon carrot juice gave no blueing until
hydrogen peroxide was added, showing that its peroxide had been removed
by the charcoal; it still, however, gave a blue with potato juice alone.
____ Boiled up once more with charcoal, and again added to potato juice, it
still caused blueing, but only slowly and less intensely. This final amount
of blueing was probably due to the peroxide in the potato juice itself
(vide infra).

Cee a
i 5 a

162 BIO-CHEMICAL JOURNAL

On examining now the residue left behind on the filter paper in
filtering the alcohol extract of the guaiacum resin before the addition
of animal charcoal, the chief source of the peroxide of the —
tincture, and the cause of the variations was discovered,

The greater part of this residue consisted of broken seeds and tests
of seeds, and other vegetable material. These impurities evidently had
been collected with the resin, and since, like all such fresh vegetable
material they would contain peroxides, it can easily be understood that —
they would yield peroxide to the tincture. Also, in making up tinctures
from the same stock, the amount of such impurities varying in different
pieces, would explain the variations in degree of spontaneous oxidizing
power shown by the different tinctures in absence of added peroxide.
This all the more so because the pieces of seed are quite large, and whole
seeds even were seen, much larger than barley seed. It was on this
account, in all probability, that the green outside powder was free from
peroxide, and by accident, probably, some pieces more free than usual
had been picked up for the preparation of the four days old (‘ new’)
tincture.

We would accordingly recommend that in preparing a guaiacum
tincture for peroxidase experiments this débris be avoided, and that the
filtrate be thoroughly boiled with animal charcoal and filtered. Under
such conditions the filtrate can be safely used for several days at least,
and any spontaneous blueing observed may be certainly set down to the
action of peroxide in the juice or vegetable tissue being tested, and not
in the guaiacum itself.

The view that the animal charcoal acts by destroying peroxide in
the guaiacum is supported by the fact that animal charcoal added in small
amount to diluted (1 in 10) hydrogen peroxide solution causes an
immediate effervescence.

Having ascertained that the peroxide could be discharged from:
guaiacum tincture by animal charcoal, we turned the method upon those
vegetable juices giving blueing alone, such as potato and carrot, and found
that here the peroxide could also be removed by treating in the cold with
animal charcoal and filtering.

Carrot juice, potato juice, and a sample of tincture of guaiacum which
gave a good blue with each of them without hydrogen peroxide, were
severally treated at laboratory temperature with animal charcoal and left
to stand for one and a half to two hours, then they were filtered, the —
guaiacum having turned green in colour.

PROPERTIES OF OXIDIZING ENZYMES 163

The carrot juice so treated when now tested with the ordinary
untreated-guaiacum gives a dirty greyish green instead of the previous
deep blue, whereas with the charcoal treated guaiacum it no longer gives
_ a trace of blue or green. Here peroxide has been completely arpa
_ both from juice and reagent.
The potato juice which before gave an intense blue when now, after
charcoal treatment, tested with the ordinary untreated guaiacum gives a
dirty greyish blue, and when both charcoal treated potato juice and
; arcoal treated guaiacum are used only a grey with the faintest
fpeeeetion of blue is obtainable.
This experiment, therefore, supports the view that a positive result
wit! th the guaiacum test in the absence of added hydrogen peroxide,
s traces of peroxide either in the guaiacum or in the plant or other
ee being tested. By the charcoal treatment the guaiacum can be freed
peroxide, and then the test may be utilised as a fairly delicate one for
oxides naturally occurring in plants, or formed soon after the juice is

Experiment XXI1—Dialysis in parchment paper.
__ A quantity of 500 c.c. of fresh carrot juice was placed in a sausage
gi tube of parchment paper, and dialysed against 1,500 c.c. of distilled water
in a tall cylinder. Next morning the outer fluid gave with guaiacum
(ordinary) alone a slow but distinct blueing. This subject, however,
- ae further investigation.
Experiment XXIII—Supplementary experiment on presence of
, ses in bananas, in certain species of nuts, viz., Spanish chestnut,
‘almond, filbert, walnut and Brazil nut and in hyacinths.
_ None of these gave positive results with the phenol or amido-phenol
derivatives, or with guaiacum free from peroxide, except when hydrogen
_ peroxide was added, when they reacted in varying degree.
In the case of the banana, the inner surface of the peel rapidly blues
a with peroxide containing guaiacum, but the pulp only blues very slowly,
and the reagent applied to the cut surface shows marked veining
a accompanying the course of vessels.
-___ Cross sections of a Spanish chestnut, tested at once with both peroxide
iz containing and peroxide free guaiacum, show blueing with the former at
- once, but no blueing with the latter, even after ten minutes. The blue
first produced by the peroxide containing guaiacum bleaches in a few
minutes, but can be brought out again even more strongly by re-applying
more of the reagent; this is repeated several times. On touching a spot

164 BIO-CHEMICAL JOURNAL

on one of the sections treated with peroxide free guaiacum with a glass
rod previously dipped in dilute hydrogen peroxide solution, there is an
instant blueing at this spot.

Brazil nut gives exactly similar results. Almond, filbert and walnut
give no colour with peroxide free guaiacum. Walnut and almond give a
slight colour, filbert rather more colour, with peroxide containing
guaiacum. Haricot beans, old and very dry, gave no colour either with |
peroxide containing or peroxide free guaiacum, but an extract gave a
feeble positive effect with ordinary guaiacum alone after vigorous shaking,
heightened by adding hydrogen peroxide.

The green leaves, bulb, and rootlets of a hyacinth, taken fresh folk
the garden were examined with peroxide free guaiacum, and only the
rootlets gave a rather slow blueing with this alone, but all three parts
gave a fine blue on also adding hydrogen peroxide.

Experiment XXIV.—Effect of synthetic guaiacol (Merck).

This reagent behaves quite differently from the natural resin, giving
a deep brown colour in oxidation which is very distinctive. Tested with
fresh potato and carrot juice it gives no effect until hydrogen peroxide is
also added, when the mixture at once turns brown, which deepens in
colour to reddish brown, and a reddish brown precipitate is thrown down.

Experiment XXV .—Effect of gquaiaconic acid (Merck).

This substance is contained in guaiacum resin and turns blue on
oxidation. A supply obtained from Merck was found to dissolve
completely to a brown solution in absolute alcohol. A 2 per cent.
solution was used for testing the fresh juice of potato, carrot, apple, and
cucumber. The colour obtained was identical with that given by the
tincture of the resin, and, as the following results show, it contained a
trace of peroxide, about equal to that in the best of the tinctures made
from the resin, and distinctly greater than the tincture when freed from.
peroxide by thorough treatment with animal charcoal.

Potato juice gave with guaiaconic acid alone a greyish blue, deopantiig
on standing; on the addition of a few drops of diluted
hydrogen peroxide an immediate blue was obtained, rapidly
deepening on standing.

Carrot juice gave practically a negative result with the guaiaconie acid
alone, there being only a slight dulling of the natural carrot
colour and no trace of blue in fifteen minutes; addition of a
few drops of dilute hydrogen peroxide to half of the test gave
an ‘instantaneous deep blue.

Bi)
ae Fa 5 a

PROPERTIES OF OXIDIZING ENZYMES 165

; ae ___..-bhueing on standing; on adding peroxide also the usual deep
~ blue was obtained,

increasing and showing just a shade of blue on standing;

addition of hydrogen peroxide also caused a deep blueing at
fiat once,

Sth THE PARALLELISM IN MODE OF ACTION BETWEEN

DROLYTIC ENZYMES, OXIDIZING ENZYMES, AND THE
ACTIVE BODIES DEVELOPED IN IMMUNE SERA

4 “The experiments recorded in the previous section appear to us to

demonstrate clearly that the whole difference between the various juices
_ and other fluids showing an oxidizing action consists in the presence of
4 a variable small amount of peroxide which is chemically unstable and
i. ee by the agencies recorded.
a _ All the juices showing oxidizing properties possess one type of ferment

ich, since it acts only in presence of either naturally present or
+ antifically added peroxide, may provisionally be styled a peroxidase, and
there is no proof of the existence of any other type of enzyme engaged
in oxidation processes.

This not only materially simplifies our conceptions regarding the

_ oxidizing ferments, but, in our opinion, brings the class into line both
with the great division of hydrolytic ferments engaged in the processes
nf of digestion and metabolism and with the active bodies in the natural
me and i immune sera which combat and immunize against disease. So that
“the oxidizing enzymes form a connecting link between the two classes.
ah Th all three classes of enzymic action it is to be observed that three
interacting bodies are required. These three are (1) the substrate on
Emr whic h the ferment is to act, (2) the body which is to be combined directly
or indirectly with the substrate and alter its chemical and physiological
properties, a and (3) the enzyme or ferment which is to activate the reaction.
In the case of the ordinary hydrolytic enzymes these are as follows :—
Substrate, the foodstuffs, protein, carbohydrate, or fat; the Combining
Body, % the elements of water finally, intermediately the acid or alkali,
in presence of which alone the ferment is active; the Catalyst, one of the
digestive or other hydrolytic ferments, such as pepsin, trypsin, diastase,
zymase, lipase, &e.

15. No generic name, so for as we are aware, has yet been given to this substance usually

ture than the substrate, which ix added to or taken away from the substrate
i . Dasivtie actin. We suggest that it might conveniently be called the Combinate.

3 tea

166 BIO-CHEMICAL JOURNAL

In the case of the oxidizing ferments: Substrate, the oxidizable
substance, such as tyrosin, naturally occurring phenols in the plant, or
the chromogenic indicators used in the preceding section; the Combining
Body, oxygen yielded by peroxide bodies present in some form, either as
simple hydrogen peroxide or as organic peroxides; the Catalyst, the
enzymes, such as tyrosinase, and the peroxidase experimented with in the
preceding section.

In the case of the immune sera, cytolysins, etc.: Substrate, the cell
or bacterium to be dissolved or the toxic or foreign substance in the serum
to be attacked and rendered inert; the Combining Body, the complement,
or thermo-labile substance, in the absence of which the reaction cannot
proceed; the Catalyst, the specific immune body or anti-body which
attacks and disintegrates the foreign cell or ‘neutralizes’ the toxic
substance.

Between two of these three reacting substances, viz., the substrate and
catalyst, there exists usually a considerable amount of specific relationship.
This specific relationship is in most cases not narrowed to a single chemical
substance, but is closely confined to the members of a class of bodies
possessing in their molecular constitution a certain definite grouping.
Even very nearly allied groupings are quite inert to the particular
catalyst, as, for example, the fermentation by organisms of certain sugars
or other substances, which are stereo-isomers of others which are not
attacked in the least. But given the zdentical molecular conformation at
a certain portion of the molecule, there may be, and will be, attack and
chemical action, although the molecular structure at other parts may be
such as to render the two bodies in other respects very different physically
and chemically.

For example, pepsin and trypsin attack all classes of proteins, down
to certain well-marked stages of hydrolytic cleavage so long as certain —
connections in the molecular aggregate exist, although in physical
properties, in reactions to precipitates and indicators, and even in
ultimate chemical composition, these proteins are very distinct from one
another.

Similarly, the peroxidase ferment in presence of peroxides attacks a
large number of oxidizable substances, such as those experimented with
in the preceding section, but leaves other classes of oxidizable substances,
both those which are more readily and those which are more ree
oxidizable, unattacked.

Turning to the third member of the group of three, represented Se

PROPERTIES OF OXIDIZING ENZYMES 167

. : B the oxidising forments and by the complement in the immune sera,
we find that this is much less specific in character. Thus, an immune
dy or cytolysin of very specific character may be activated by practically
yy serum, including the natural serum of the same species as the animal
‘ich had been immunized. Similarly, any body containing a peroxide
\kage, organic or inorganic, will activate a peroxidase, and any type of
id or alkali which increases hydrogen or hydroxy] ion concentration,
spectively, will activate a hydrolytic enzyme.

_ The ferment character of the reaction in the case of immune sera is
@ o shown by the disproportionately large amount of complement from

. sp normal serum which can be bound to lipéid, and so rendered inert
7 ad haemolysis in the second stage of the Wasserman reaction, by very
small amounts of syphilitic * immune’ body. In this reaction it is clear
that an active principle in the syphilitic serum acts as a catalyst or
ferment, the lipéid from liver or elsewhere as a substrate, and the
- complement from any serum as a combining body with the substrate under
_ the influence of the catalyst.

Finally, attention may be drawn to the similarity between the
oxidizing ferments and immune sera in regard to thermo-stability. When

an immune serum is heated for some time at 55°C. the complement is
be destroyed, but the ‘immune’ body remains untouched; the heated serum,
ae — is inactive until complement is added by mixing with some
unheated serum which may be drawn from any animal.

Quite similarly, when a vegetable juice is heated to 55°C. the
_ peroxide is destroyed, but the ferment or peroxidase is untouched, and
ough the heated juice is inactive as an oxidizing agent it is at once
ted on adding hydrogen peroxide or other form of peroxide.

.. 7,

aki if a vegetable j juice be kept it loses gradually its siluatiaieoes
: — of oxidizing, but regains it as soon as a peroxide is added.

Tian

bn abe |

168

ON THE OCCURRENCE OF A MON-AMINO-DIPHOSPHATIDE
LECITHIN-LIKE BODY IN EGG YOLK

By HUGH MacLEAN, M.D., Carnegie Research Fellow, University "
Aberdeen.

From the Department of Physiological Chemistry, Institute of
Physiology, Berlin

(Received March 17th, 1909)

In making some investigations on the ethereal extract of egg yolk, I
succeeded in separating a body of the general nature of a lecithin, but
containing in its molecule two parts of phosphorus to one part of nitrogen.

This compound is of the same type as that separated from heart
muscle by Erlandsen,'! and called by him ‘Cuorin,’ though, as shown
below, it differs in certain respects from this substance. A preliminary
note giving the N and P content which characterise it as a mon-amino-
diphosphatide was published some time ago,? but as this portion was
prepared from a comparatively small number of eggs, no account at its
properties or probable elementary composition was given.

During this winter, however, I have isolated the substance Pic a
large quantity of egg yolk, and having used certain modifications in the —
preparation of different portions, will now proceed to give a short nenoumt
of its isolation, composition and chief properties.

PREPARATION

The egg yolk of fresh eggs was separated from the white portion, and
after being spread out in thin layers on a glass plate was dried by means
of a current of air generated by a fan-like arrangement attached to a —
motor. When thoroughly dry the mass was broken up into small pieces; —
and finally passed through a coffee mill. In this way a very fine powder
was obtained, which was carefully extracted five times with ether. The
ethereal extracts were mixed together, evaporated to a fairly small volume,
and treated with acetone. The precipitate was dried in vacuo over H,SO,
and divided into two parts. Portion A was treated by a combination of
the methods used by Stern and Thierfelder? for purifying lecithin, and by

1. Zeitschrift f. physiol. Chemie. Ba. LI, 8. 92.
2. Ibid., Bd. LVI, 8. 304.
3. Ibid., Ba. LITL, 8. 370.

- MON-AMINO-DIPHOSPHATIDE LECITHIN-LIKE BODY 169

Erlandsen* for the isolation of cuorin; as will be seen, this combination
____ is rather-tedious in carrying out.

* Portion B was treated by a much simpler method, which was found
exceedingly easy to carry out, and capable of giving quite a pure
substance.

TREATMENT OF Portion A

___ This portion was again dissolved in ether and gave a markedly turbid
solution. By means of the centrifuge a perfectly clear fluid was obtained,
the residue in the centrifuge tubes being of a whitish colour, and with
difficulty soluble in ether. The clear solution was again precipitated with
acetone and dried. This process of purification was repeated five times,
and by this means impurities such as cholesterin and fat were in great
part got rid of. The final material gave a perfectly clear solution in
ether, but only after standing for some time, being at first slightly turbid.
The solution was now treated with about four times its volume
of absolute alcohol, and left to stand under CO, in a closed vessel
for twenty-four hours; an alcohol insoluble residue remained,
which was separated by filtration. This substance was washed with cold
: alcohol, and the united filtrates evaporated in vacuo to a small bulk,
i precipitated with acetone, and dried as usual. This precipitate was now
= treated with absolute alcohol, when a small portion remained behind,
F ft which was added to the above alcohol insoluble part. Thus, the ethereal
solution was divided into two parts—the part soluble in cold alcohol
consisting of ‘ lecithin.’
The alcohol insoluble part was now placed in an incubator and
heated for a fairly long time with alcohol at 65° to 70°C. By this means
ee part of the substance went into solution, but separated again on the
-___ aleohol cooling. This process was repeated three times, and finally boiling
aleohol was used in an attempt to obtain an alcoholic solution that would
remain quite clear after cooling. In every case, however, the alcoholic
filtrate on cooling became slightly opalescent, though only faintly so.
By this procedure the part insoluble in cold alcohol was divided into two
parts, one of which (portion a) was soluble, the other (portion 4) insoluble
in hot alcohol.
Portion (a) was thoroughly washed with cold alcohol and treated as
3 described later.
ihe _ Portion (6) was dissolved in ether and left to stand over night under
CO,; next day a slight precipitate had settled out, the solution itself being

4. Loe cit,

i i 2

ON eee eee
ae

170 BIO-CLLEMICAL JOURNAL

almost clear. On centrifuging, the perfectly clear solution obtained was
precipitated by acetone; on again dissolving in ether it gave practically a
clear solution. This solution being again precipitated, the substance was
dissolved in hot ethyl acetate, out of which it separated on cooling. It was
then filtered, dried and analysed.

TREATMENT OF Portion B

This portion was thoroughly extracted with cold absolute alcohol,
whereby the greater part went into solution. This solution was
evaporated to a small bulk and the syrupy residue dissolved in ether;
the ethereal solution was then precipitated by acetone. The precipitate
was dried, redissolved in ether and reprecipitated by acetone. By this
means ordinary ‘ lecithin * was obtained in a pure condition, the ordinary
contaminating substances present in the raw ethereal extract mass being
relatively insoluble in cold alcohol.

Residue insoluble in cold alcohol was now treated with hot alcohol
exactly as described under Portion A. By this means impurities such as
cholesterin and fat went into solution. This treatment with hot alcohol
was repeated four times, boiling alcohol being ultimately used. The
residue was then further purified by dissolving in ether, precipitating with
acetone, and finally dissolving in hot ethyl acetate, as described above; it
was then dried and analysed.

The hot alcoholic solution on cooling deposited a voluminous
precipitate of floccular masses. This was obviously composed in great
part of fat and cholesterin, and was not examined.

This method of separating the raw material is exceedingly simple and
quite efficient. It is much more quickly carried out than the method
adopted under Portion A, and can be recommended as an easy means of
separation of the mono- and di-phosphatides present in the ethereal extract
of egg yolk, heart muscle, and probably in other substances.

In the following analyses Parts I and II were prepared as described —
under Portion A. Part III was prepared as mentioned under Portion B.

ANALYSIS
I
Nitrogen ( Kjeldahl)
can substance used 1-5 c.c. yy H,SO, = 0-81 %
0-3122 , - » 2826.0. a = 0-82 9

Phosphorus (Neumann)

0-2411 gm. substance used 15-66 ¢.c. } NaOH = 3-60 %
0165, “» ww» 10Bex. , = 364%
Elementary Analysis
0-1625 gm. substance gave 0-3527 gm. CO, = 59-19,% C

and 0-1388 gm. H,0 = 9-56 % H

e} Nit

03140 gm. substance used 1-78 ¢.c. J, H,SO, = 0-794

CMe don coed
Phosphorus

0-4139 gm. substance used 26-55 c.c. } NaOH = 3-55 %
02729, » » ITBbec. ., = 356%
Elementary Analysis

0-1541 gm. substance gave 0-3329 gm. CO, = 58-92 % C
and 0-1296 ,, H,O = O-dl 92 H
01701, », » 03686 ,. 0b, = 59-00 C
and 0-1413 ,, mp = 929%H
, 01448 ,, % 0-3140 ,, == ae Cc
“and 0-1217 H, H
Iodine Number
0-1691 gm. substance pend Ca Iodine = 76-8
02254 ” ” ” ” ” = 77:1
Il
Nitrogen
0-4836 gm. substance used 2-85 c.c. to H,80, = 0-83 %
Phosphorus
_ 0/2132 gm. substance used 13-82 ¢.c. $ NaOH = 3:59 %
Elementary Analysis

otis substance gave 0-2453 gm. CO, = 59-31 % C
and 0-0963 ,, H,O = 955% H

a Portion F Foriien: II Portion III Average of
Cuorin

| percent per cent. per cent. per hai per cent. per cent. tes (Erlandsen)
- 219 — 58-92, 59:09 59-14 59-31 59-12 61-63
9-56 — | &él 9-29 94 9-55 O44 9-03
O81 0-82, _— 0-794 0-806. 0-83 O812 LO
3-60 3-64 = 355 3-56 3-59 3-59 4-46

_ ied et es 27-048 a

' ser , a comparison of the two substances is of some interest. A
» to the analyses shows that they are not identical. If, for

ut the difference is relatively high. Again, the O content of my
is higher than that of cuorin. At first it was thought that this
e in percentage composition might possibly be dependent on the
ence of a certain amount of oxidation, prior to, or during, the

‘ion of the substance, and the extreme facility with which cuorin
oxidation lent colour to this view. An examination of the

172 BIO-CHEMICAL JOURNAL

figures, however, shows that possible oxidation does not account for the
difference ; a priori the probability of any marked oxidation was unlikely,
since all manipulations were so conducted so as to exclude as far as possible
the presence of oxygen.

Since cuorin contains a relatively higher percentage of N and P than
this substance, it is obvious that the addition of more O would lower the
N and P percentage, and so tend to lessen the difference in composition.

For a specimen of oxidised cuorin, Erlandsen gives the following
figures: —O 30°72, N 0°92, P 4°06 per cent.; while my substance gives
O 27, N 0°812, P 3°59 per cent.

If we consider cuorin oxidised only to the extent of 27 per cent.
(instead of 30 per cent. as given), this portion must contain something

over 0°92 per cent. N and something over 4°06 per cent. P. Since my

substance, oxidized to 27 per cent., contains only 0°812 per cent. N and
3°59 per cent. P, it is clear that possible oxidation is not the cause of the
difference. It is probable that this is really accounted for by a difference
in the fatty acids of the two substances,

Again, though the substances differ somewhat in their iodine figures,
this difference is not great enough to satisfy the assumption that the one
is but an oxidized form of the other.

Whether the somewhat prolonged treatment with hot alcohol
necessary for the isolation of the substance in a pure condition has any
action in causing some decomposition, and so a slight change in the
ultimate formula of the phosphatide is perhaps worth consideration ;
if this is so, it is possible that the exact percentage composition may vary
slightly, depending on the amount of hot alcohol treatment. Since,
however, the different portions mentioned above yielded practically the
same results, this is not very probable; in any case, it does not affect the
fact that in egg yolk there is contained a well defined lecithin-like
substance containing N : P in the proportion of 1 : 2.

PROPERTIES

This substance is obtained as a yellowish brown brittle substance,

which, after being carefully dried, is easily ground to an exceedingly fine
powder; it is much more brittle than cuorin, and though hygroscopic, is
by no means markedly so in comparison with certain other phosphatides.
In common with all lecithins it undergoes oxidation on exposure to air.

It is insoluble in cold aleohol but somewhat soluble in boiling alcohol, —

and thus differs from cuorin, which is said to be insoluble in boiling

. ~ MON-AMINO-DIPHOSPHATIDE LECITHIN-LIKE BODY 173

alc leohol ; in chloroform, ether and petroleum ether it dissolves easily at
the the ordinary temperature. In ethyl acetate it dissolves on heating, to be
thrown out on cooling. From its ethereal solution it is precipitated by
acetone. With water it forms an emulsion. With platinum chloride it
gives double salts, and by hydrolysis gives fatty acids and glycero-
_ phosphoric acid; no base of the nature of choline could be obtained.
When heated to 90° to 100°C. it changes colour, but an exact melting
~ t could not be determined. After hydrolysis with weak hydrochloric
acid no reducing carbohydrate was obtained. All its general properties
_ show that here we have a typical phosphatide substance.

| Warr Supstance Sotvusie 1x Hor Atconor AnD ieee Ovt on
CooLInG

This substance (Portion a, page 169) was dissolved in hot alcohol,

filtered hot, allowed to cool, and again filtered. By this means a
voluminous precipitate of whitish snowflake-like material was obtained,
tich on drying shrunk to a very small volume. This process was

peated four times. By this means a fine whitish granular powder was

obtained, which was completely soluble in ether, chloroform and benzene.
On the addition of acetone to any of these solutions it was, after a little
time, but not immediately, precipitated.
As some doubt exists as to the composition of this ‘ white substance,’
Erlandsen having found N but no P in apparently similar material, while
_ Thierfelder and Stern found both N and P, I made some further
investigations with the following results.
_____In this particular preparation there was neither N nor P present.

Melting point was easily observed being 62° to 63°C. When burned on
platinum foil it left no inorganic residue. After boiling for some time
rith HCl no substances of a reducing nature were obtained. Saponification
with Ba(OH), failed to give any substances of basic nature precipitated
by platinum chloride. From these observations it was apparent that this
substance was probably of the nature of a fat. A combustion experiment
_ showed this fat to be tripalmitin.

Analysis
0-1068 gm. substance gave 02964 gm. CO, = 75-70 % C
and 0-1179 ,, H,O = 12:35 % H
Calculated jor Tripalmitin

CuHy,04 = 75°86 % C
and 12-24 % H

I would here noel ike no ional of
in purifying the above substance was made. This
somewhat soluble in ‘lecithin,’ so it is doubtful if,
‘lecithin ’ ariel free from fat.

” Reale

‘lecithin’ with N : P as 1: 1 another lecithin
amino-diphosphatide body—with N : P as 1: 2.

This substance, as above obtained, differs somewhat in
and properties from the substance, cuorin, prepared from h
the difference being probably a on the presence of d
acids.

The other substance in the ethereal extract whi dissol
alcohd] and ey ont on wae was bapa to be pure t

wren 2
wey.

175

10 : )D DO-EOSIN: AS A TEST FOR FREE ALKALI IN DRIED-UP
a = ANT TISSUES

By A. C. HOF, Héchst a. Main.

a Ciinptediented by Péafeswe P.Bhelich

i (Received March 24th, 1909)

a ay free dye-acid of iodo-eosin has been employed by Professor

lant tissues.”
Sy The dye used is iodo-eosin, the potassium salt of tetraiodo-fluorescein.

soluble in ether or any other organic solvent. The free dye-acid,
e * obtained as a yellow precipitate from the alkaline solution of
eosin by adding hydrochloric acid in excess, dissolves easily in ether
in ai any other organic solvents, but is insoluble in water.
It is for this reason that the reaction can be made use of for testing
free alkali in a perfectly dry tissue.
Suppose we have a free hand transverse section of a dry twig of a
eommon forest-tree, for example, a spruce fir, and put the section into
a solution of the dye-acid of iodo-eosin that has been dissolved in ether,
leave it there for some minutes, then wash out the section thoroughly
with ether. The preparation brought into xylene should now be

‘amined under a low power of the microscope, when some of the

istologically differentiated elements of the tissue will be stained
d y red. In our section, for example, the cambium, the resin
ug ‘canals and the secreting cells lining the cavity appear plainly red-
24 Iti is easy to see that these red-stained elements of the tissue must be
those that contain free alkali, and, further, that their red colour is due
to the red alkali salts formed.

We are justified in supposing that the free alkali located in these
_ tissues is acting as a dye-base as soon as the dye-acid of iodo-eosin is

1. Ehrilich-Lazarus, Die Anaemie, Vienna, 1898.
9. A. C. Hof, Botanisches Centralblatt, UX XXIII, 1900.

176 BIO-CHEMICAL JOURNAL

added, yielding immediately the characteristic red dye-salt. If there is
no water present the red colour remains in the tissue fixed at the exact
place where it has been formed. Thus we get an exact idea of the
distribution of free alkali in dry tissues.

Mernops

Preparation of the dye-acid of iodo-eosin.—Dissolve 1 gramme iodo-
eosin, the commercial dye, in 1 per cent. potassium hydroxide, add
hydrochloric acid in excess. The dye-acid precipitates at once. Filter
and wash the precipitate with hot water thoroughly till the filtrate is
absolutely free from hydrochloric acid. Dry the precipitate of dye-acid
and dissolve it in 100 c.cm.-ether.

Staining of sections—Bring the sections, either sectioned free hand
or in a microtome, into the above solution of dye-acid, leave them in it
for some minutes, wash out the sections carefully with ether, transfer to
xylene, and seal preparation in Canada-balsam. For preserving the
sections distinctly stained it is absolutely essential that the balsam used
is of perfectly neutral reaction. The ordinary balsam bought from dealers
in microscope supplies often reduces the dye-salts immediately. By this —
reduction of the dye the colourless compound—the leuco-base or leuco-
compound—is formed.*

I venture to hope that the foregoing reaction, simple, effective and
feasible as it is in the case of almost any drug, may be of some use to
students of pharmacognosy.

3. P.G. Unna, Centralblatt fiir Bakteriologie und Parasitenkunde, Vol. ILL, 1888,

7

HE GROWTH OF THE BACILLUS TUBERCULOSIS AND
-——~OTHER MICRO-ORGANISMS IN DIFFERENT
PERCENTAGES OF OXYGEN

: vas BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio-
Chemistry, University. of Liverpool, axp R. STENHOUSE
WILLIAMS, M.B., D.P.H., Lecturer on Public Health Bacteriology,
University of Liverpool.

From the Departments of Bio-Chemistry and of Bacteriology, University
“a of Liverpool

(Received March 31st, 1909)

‘The experiments here described were suggested by the seats of growth
of the Bacillus tuberculosis in the body corresponding as they do to
- situations where there is a high pressure of carbon dioxide and a low
_ pressure of oxygen. It was thought from this that the bacillus might
either require a certain definite percentage of carbon dioxide in the air,

or might be very sensitive to high pressures of oxygen, and only grow,
as in the lungs or elsewhere, where the partial pressure of the oxygen is
normally much lower than in the atmosphere.
The experiments did not quite justify the theory; but the theory
served the most essential purpose of a theory in leading to experiment,
and certain of the results have proved in some respects sufficiently
interesting to warrant description.
ly These experiments are still being continued, but as far as they have
——_ gone their chief results may be briefly put as follows:—-The Bacillus
tuberculosis either does not grow at all, or grows very badly in the entire
sence of oxygen, or in presence of a partial pressure of oxygen
amor ting to 80 to 90 per cent. of an atmosphere.
A number of other organisms have also been tested, and certain of
these, like the Bacillus tuberculosis, cease to grow in the higher oxygen
percentages, while others appeared to be unaffected by the variations in
oxygen. The experiments with the Bacillus tuberculosis will first be
described, and afterwards those with the other organisms.

Experiment I.—Growth in large tubes with stopcocks, and analyses of
the gases in the tubes after growth.

Since carbon dioxide is such a heavy gas, it was thought that under
the ordinary condition of culture in test tubes with cotion-wool stoppers

ee a ee oe
a ee

178 BIO-CHEMICAL JOURNAL

there might be an accumulation of carbon dioxide in the culture tubes in
which the bacilli might grow, as in the lung. Accordingly two extra
large tubes of about 120 c.c. capacity were made, each with a draw-off
tube for analysing the tube gases, closed by a small glass tap near the
bottom; and one tube was plugged with cotton-wool in the usual way,

while the other was closed above with a rubber cork, through which passed =

a small glass tube, also closed by a glass tap. Each tube was inoculated
on a slant surface of glycerine agar! with a strain of Avian tubercle, and
the two tubes were incubated alongside of each other for fourteen days at
36° C.

Then the gases in each tube were analysed by drawing samples off
into a Hempel gas burette in the usual way. The tube plugged with
cotton-wool showed 19°4 per cent. of oxygen and no carbon dioxide; while
that closed air-tight by the rubber cork gave 18°7 per cent. of carbon
dioxide and no oxygen. Hence the reply of the experiment is that there
is no accumulation of carbon dioxide in growing in the ordinary way with
wool-stoppered tubes. There was a good growth of the bacillus in both

tubes. This appears to be contradictory to the result of some of the later ~

experiments, which show no growth in hermetically sealed tubes in
absence of oxygen; but it is to be remembered that in the later
experiments ordinary small test tubes were used, in which the supply of
oxygen would soon be exhausted, while here, in order to get sufficient gas
samples, large tubes of about 120 c.c. capacity were used, and there would
be a sufficient amount of oxygen to allow of a good growth before the
oxygen became exhausted.

Experiment I1.—Effect of high partial pressure of oayyen.
This experiment and some of the later ones were carried out in a

small copper autoclave, kept in an incubator at 36 to 37°C., and filled
with the desired oxygen mixture, while the controls were grown in the

same incubator outside the autoclave but alongside it. The autoclave was ~

used because it was intended to pass on later to oxygen pressures higher
than atmospheric, but when it was found that growth was stopped at
80 to 90 per cent. of oxygen at ordinary atmospheric total pressure, the
autoclave was not found to be necessary, and hence in later experiments a
glass desiccator on a ground base with a mercury seal all round outside
the glass junction was used, as subsequently described.

Six tubes containing cultures on glycerine agar of Bacillus tuberculosis

(Avian) were used for the experiment, four being placed in the autoclave

1. Veal broth agar containing 5% glycerine, and 2% peptone, and acidity iw to phenol ,

phthalein.

ae

GROWTH OF THE BACILLUS TUBERCULOSIS — 179

‘ith a vessel containing soda lime to absorb any carbon dioxide set free,
_ while the other two were grown in air as controls outside the autoclave in
- the same incubator. The autoclave was exhausted and then joined up to
a gasometer holding oxygen, prepared carefully in the laboratory from
potassium permanganate.

The exhaustion and filling up was repeated four times, and at the
| end the autoclave atmosphere was found by analysis to contain 87°5 per
gent. of oxygen. The experiment was begun on June 24th, 1908, and the
autoclave was opened and results noted and compared with control on
‘Fuly 8th, 1908.

_--—~—sS'‘The following show the percentages of oxygen in the autoclave on
q ‘different days: —June 24th, 87°5 per cent.; June 26th, 85°77 per cent.;
July Ast, 85°1 per cent.; July 4th, 84 per cent.; July 8th, 63°2 per cent.
re was always an analysis made for carbon dioxide, but none was found,
owing that the soda lime was quite effective. The drop in oxygen in
e last analysis indicates the starting of a very slow leak around the rim
f the autoclave; but there was never less than sixty-three per cent. of

_ oxygen, and in the earlier part the percentage was nearly ninety,

4 a On opening the autoclave it was found that the four tubes grown
i inside showed practically no growth at all, while the two controls grown
in air showed a moderate growth.

. Experiment I11.—Growth of Bacillus tuberculosis (Avian) in ordinary
q small test tubes (a) hermetically sealed, (b) stoppered with rubber corks, and
(ei in controls grown in the ordinary fashion with cotton-wool stoppers.
fe Eight test tubes containing the glycerine agar culture medium were
inoculated with Avian tubercle on June 17th, 1908; four of these were
hermetically sealed off at the upper end, avoiding any injury to the
edium; two were stoppered with ordinary rubber corks; and two were
sed with cotton-wool in the usual fashion. The whole eight tubes were
| then incubated alongside of one another in the same incubator for twenty-
one days (till July 8th, 1908), when the condition of growth was noted in
each set, and the gases analysed in the hermetically sealed and in the
rubber closed tubes respectively.

The two control tubes showed a good growth, the four hermetically
sealed tubes showed very slight growth, and the two rubber stoppered
tubes showed slightly more growth than. the sealed tubes, but much less
than the controls.

The glass point of one of the hermetically sealed tubes being broken
under water, a negative pressure was shown by an in-rush of water. The

=

180 BIO-CHEMICAL JOURNAL

free capacity of the tube was about 40 c.c. and 26°8 c.c. of residual gas was
obtained. This contained 07 c.c. of carbon dioxide (2°6 per cent.) and no
measurable amount of oxygen, so that practically all the residual gas was
nitrogen. A similar analysis in one of the rubber stoppered tubes gave
29°6 c.c. of total gas, containing 3 per cent, of carbon dioxide, no oxygen,

and the balance nitrogen. ;

Experiment IV.—Growth of Bacillus tuberculosis (Avian) in high
partial pressure of oxygen.

Six tubes of glycerine agar, plugged in the usual fashion with cotton-
wool and equally inoculated with Avian tubercle, were taken (July 15th,
1908), and of these four were grown in the autoclave in increased oxygen,
while the other two were grown in air alongside in the same incubator.
The autoclave after receiving the four tubes, and also an open vessel
containing 25 grammes of soda lime, was screwed up, exhausted by a
water pump, and allowed to suck in oxygen from a reservoir, the oxygen
being made previously, as in all the experiments, from potassium
permanganate. The exhaustion and filling was repeated five times, and
the final percentage of oxygen in the autoclave was 82°6. The experiment
was run for twenty-one days, viz., from July 15th to August 5th, 1908,
and analyses of the autoclave atmosphere at intervals gave the following
results :—July 18th, 75°5 per cent.; July 22nd, 70°9 per cent.; July 26th,
57-9 per cent.; August 5th, 50°3 per cent.

There is here again a slow leak in the autoclave packing, and an
oxygen percentage varying from 82°6 at the beginning to 50°3 at the close.
There was no appreciable amount of carbon dioxide present throughout.

Examined at the end the two control tubes show a fair growth, while
the four tubes grown in the increased oxygen show practically no growth.

Experiment V.—Growth in stoppered large tubes alongside cotton
stoppered similar tubes.

In order to examine more fully the gaseous exchanges in growth in»
sealed tubes where the growth was finally inhibited, and also to examine
the effect of the medium alone upon the air in the sealed tubes, the —
following experiments were carried out in larger tubes than usual, the
capacity of each tube being about 120 ¢c.c. Six culture tubes were taken
for the experiment and treated as follows : —

A.—Control. Glycerine agar inoculated with Avian tubercle
and plugged with cotton-wool in the usual manner. ©
B.—Same as A, but plugged air-tight with solid rubber stopper.
C.—Glycerine agar, not inoculated, but plugged exactly like B.

GROWTH OF THE BACILLUS TUBERCULOSIS 181

' D and E.—Same as B, and inoculated, but with a glass tube
_.-~sealed at outer end inserted through rubber cork so as to

be easily broken afterwards in rubber tubing to allow
sample of gases to be taken for analysis.

F.—Glycerine agar, not inoculated, arranged as in D and E.

The six tubes were grown together in the same incubator from July

28rd till August 21st, 1908 = twenty-nine days.

_ The final result was as follows :—

Amount of Growth Analysis of Residual Gases

Ry i “The whole surface of the medium Open to air; therefore no analysis.
—s eovered with 7. B. growth.

fifth of surface covered carbon dioxide 5-7 per cent.
oxygen; no carbon dioxide.
cent. carbon dioxide.
more than D. cent. carbon dioxide.

no carbon dioxide

This ee shows that the control grown under atmospheric

_ oxygen gives by far the best growth; that there is an up-take of oxygen

by the medium alone but no output of carbon dioxide, about half the
available oxygen being so used up in the time; and that in the inoculated

_and stoppered tubes all the oxygen disappears, but only about one-third

u of the corresponding amount of carbon dioxide appears.

Experiment VI.-Growth of strains of Bacillus tuberculosis of Avian,

vine and Human origin, in inereased percentage of oxygen.

This experiment, instead of being carried out in the autoclave, was

done in a large desiccator or bell-jar, resting on a well-fitting ground
glass base, and having a glass tube and well-fitting tap above fixed in a

-mereury seal was arranged all round the base by constructing a circular
mound of putty all round about a centimetre high and of about two

centimetres greater diameter than the rim of the bell-jar. In this way
_ the slow leak of the autoclave experiments was avoided, and, as shown
by’ the analyses in this and and the succeeding experiments, the per-
centage of oxygen was kept high and very nearly constant throughout
the necessarily somewhat prolonged experiments.

_ Fair growth, roughly about one- Total volume 83-4 c.c.; no oxygen;
Not inoculated; no growth. Total volume 846; 12-4 per cent. of
Hardly any growth. _ Total volume 74°8; no oxygen ; 5-0 per
Moderate growth; less than B, Total volume 82°9; no oxygen; 6°7 per

No inoculation ; no growth. Total volume 80; 11-5 per cent. oxygen ;

_ rubber cork in a wide tubulure. In order to keep a quite tight joint below, a

Se eo eee ee a |

il) i iia a

192 BIO-CHEMICAL JOURNAL

Two tubes each were inoculated on glycerine agar with tubercle
bacilli of avian, bovine and human origin, and plugged in the ordinary
way with cotton-wool. These were placed in the bell-jar above deseribed
along with 20 grammes of soda lime in an open flat vessel. One tube of
each strain was inoculated and grown in air outside the bell-jar, but close
alongside it in the same incubator, to serve as a control.

The experiment was commenced on the 2nd December, 1908, and
concluded on the 24th December, 1908 = twenty-two days. The per-
centage of oxygen was got up at the commencement in the usual way by —
exhausting and connecting with a reservoir containing permanganate —
oxygen. The initial value for the oxygen percentage was 773 on
December 2nd and 76°53 on December 24th, showing that there was no
appreciable leak.

Examination of the nine tubes at the later date shows in every case
that the growth is stronger and thicker in the controls than in those
grown in the oxygen. This is particularly well shown in the human
strain, in which one of the two tubes shows hardly any growth and the
other a very scanty growth, while there is a much better growth in the
control. :

The same holds for the bovine growths, but all three tubes are some-
what further advanced.

In the avian the growth is considerable in both oxygen grown tubes
and controls, but the control is thicker and better grown.

Experiment VII.—Growth of Bacillus tuberculosis (Human) in *
increased oxygen percentage. ;

Three tubes were used of human strain, grown as before on glycerine
agar. Two were grown in the bell-jar with increased oxygen, one as
control in air outside and close to the bell-jar in the same incubator.
Bell-jar exhausted and refilled six times; final oxygen percentage on
January Ist, 1909, was 90°5. Experiment continued till January 27th =
twenty-six days, when percentage of oxygen was 889; no carbon dioxide
present, . rs

Examination showed that there had been no growth in the two tubes
kept in the oxygen, and a moderate but quite obvious growth on the air
grown control tube. As the control had dried somewhat during the
twenty-six days of the experiment, in the succeeding experiment this was
obviated by placing the control also under a similar bell-jar and in all
respects in similar condition to the other set of tubes, except that the

growth was made in air instead of in a high percentage of oxygen. But

VT a

GROWTH OF THE BACILLUS TUBERCULOSIS 183

it may be pointed out in regard to this present experiment, that any

drying would militate against growth rather than favour it, yet the control

had obviously grown, while the oxygen grown tubes had not grown.

Experiment VIII—Growth of Bacillus tuberculosis (Human and

Re: Bovine) in increased oxygen percentage.

Four culture tubes each, of human and of bovine strains were made,

“and eee ornare’ 5 in the usual manner.

3 Two tubes of each strain were placed in the oxygen bell-jar, which
was exhausted and refilled in the usual way five times, the final per-

centage of oxygen obtained being 87°6. Alongside this was placed another

_ similar bell-jar, fitted up in the same way but filled with atmospheric

air, and in this likewise two tubes of each strain were placed.

The experiment was commenced on February 4th, 1909, and on

_ February 24th and from that date onward a most marked and increasing
difference was observable in the two sets of tubes; those in the oxygen
had not appreciably grown, while the surfaces of the air cultures were
‘doited over with vigorous colonies of growth. The experiment was
‘ discontinued on March 3rd, when the percentage of oxygen was found

3 to be 75.

Fe The photographs shown in fig. 1 were made from this experiment.

In the photograph the four upper tubes were those grown in the
oxygen, and the four lower tubes were those grown in air. In each
series the two tubes on the left-hand side are bovine tubercle, and the two
tubes on the right are human tubercle.

In order to test whether the growth of the bacillus was only inhibited
_ by the oxygen, the organisms still remaining alive, or whether they had
bs been permanently destroyed by their stay in the higher oxygen per-
“centage, sub-cultures were made of both bovine and human, from both
| “the tubes in air and those in oxygen. The results showed a good growth
= about fourteen days on the sub-cultures from the air grown tubes,
while the sub-cultures from the previously oxygen grown tubes showed
no growth, but turned brown where there were specks of the inoculated
material on the agar. These sub-cultures were, of course, in both cases
grown in air. The experiment, therefore, shows that in this case the
oxygen had killed the bacteria; but the experiment requires repetition.
Experiment 1X.—Growth of Bacillus tuberculosis (Human and
Bovine) in increased oxygen percentage.

In this experiment other micro-organisms were cultured alongside
the B. tuberculosis under the same oxygen and air bell-jars, but the

184 BIO-CHEMICAL JOURNAL

'
; ‘ m 7 j
Bacillu uberculosis (Bovine). . Bacillus tuberculosis (Human),
’ ; ; , ° . ) ; ; ’ “
Bacillus tuberculosis (Rovine). 4. Bacillus tuberculosis (Human).

GROWN IN AIR

2. i

| Bacillus tuberculosis (Bovine) ; Bacillus tuberculosis (Human).
Bacillus tubercul (Bovine). 4. Bacillus tuberculosis (Human).

Kia. | [uBERCLE BacitLus GROWN IN AIR AND IN OXYGEN.

GROWTH OF THE BACILLUS TUBERCULOSIS — 185

growth of the tubercle bacillus is so slow that the tubes containing the

tubercle bacilli had to be kept in the bell-jar during more than one
a experiment on the other organisms, which grew abundantly in two or
_ three days. For clearness of description the effects on the tubercle bacilli
___ are described separately, but the opening of the desiccator to remove and
e — replace other organisms is noted, and the oxygen percentage at each
period in the experiment was usually determined each time the atmosphere
was changed.

March 5th, 1909. Two tubes of Bacillus tuberculosis (human) and
two of B. tuberculosis (bovine), which had been inoculated March 2nd,
were placed in the oxygen bell-jar containing 90°0 per cent. of oxygen.

a Four similar tubes, two of each strain, inoculated at the same time
___ and grown in the interval alongside in the same incubator, were placed
in the similar control bell-jar in atmospheric air.

‘The two sets of tubes show a commencing growth in each case, but
slightly advanced.

‘The two bell-jars placed alongside of each other in the incubator at
630 p-m., March 5th, the eight tubes being very similar as to growth.
“The two bell-jars were opened on March 9th to remove the tubes with

* étlae micro-organisms described in the next experiment (see Expt. X).

The growths of the tubercle bacillus had not yet advanced far enough
for comparison, so they were replaced and the oxygen bell-jar
again charged by six exhaustions and refilling with oxygen
from the reservoir? The oxygen percentage at this second
charging was 91°7 per cent., and the tubes were not again disturbed till
March 11th, when they were reopened to remove the other organisms
grown in tubes alongside. The tubes were again replaced in their
‘Tespective bell-jars, and the oxygen percentage raised in the oxygen bell-
ar to 88°8.
‘The bell-jars were again opened temporarily and at once , restarted
on March 12th, March 16th, and finally for examination at the conclusion
of the experiment on March 17th, after twelve days’ growth in the
oxygen, and in the air alongside for the controls.

The oxygen percentage on March 16th was 90°8 per cent.; it was not
determined on March 12th or 17th.

All four tubes grown in air showed good growths at the end of the

‘2. That the failure of the Bacilli to grow was not due to the f exha
bell-ja¥, is shown by the vigorous growth under such conditi f B co Deke x She
grown alongside, and also in subsequent experiments. ae the vs arama

186 BIO-CHEMICAL JOURNAL

period; the human and bovine strains in this experiment grew at about
the same rate.

None of the four tubes grown in oxygen showed any growth whibiins,
and the small amount of growth which had occurred. in air during the
period from March 2nd till March 5th, when they were placed in the higher
oxygen percentage, had turned dark brown in colour, the cultures being
obviously dead.

Experiment X.—Growth of other organisms—Staphylococcus albus, and—
Bacillus coli—in increased oxygen percentage.

March 5th, at 6-30 p.m. Two tubes of Staph. albus and two tubes of
B. coli immediately after inoculation were placed in the oxygen bell-jar
and grown in 90 per cent, oxygen. As controls, one tube of each a
was grown in the air bell-jar alongside.

March 9th, 2-30 p.m. The two bell-jars opened and growths
compared.

The two tubes of B. coli grown in the oxygen showed a growth quite
equal to that of the tube grown in air. (See fig. 2.)

On the other hand neither of the two tubes of Staph. albus grown in
the oxygen showed any appreciable growth, while the control tube grown
in air had a very good growth, the whole surface of the median being
covered. (See fig. 3.)

The six tubes were at once photographed, and the results are As
in figs. 2 and 3.

Experiment XI.—Growth in fiicreased oxygen percentage of Staphy-
lococeus aureus, Bacillus coli, and Bacillus typhosus.

March 9th till March 11th, 1909. Two tubes of each organism ‘were
grown in the oxygen and in the air bell-jars respectively. Growth was
commenced on afternoon of March 9th. When examined without
opening on March 10th, afternoon, all six control tubes in air show good
growths; in the orygen the B. coli and B. typhosus show good growths, ©
while the Staph. aureus shows no growth at all. :

March 11th. Both bell-jars opened and tubes examined.

All four B. coli tubes show full normal growth.

All four B. typhosus tubes show full normal growth.

The two Staph. aureus grown in the air bell-jar showed full sicsiad
growth; but the two grown in oxygen showed the merest trace of growth.

Experiment XII.—Growth in increased oaygen percentage of Bacillus
pyocyaneus, Vibrio cholerae, Bacillus dysenteriae (Shiga), Batillus

dysenteriae (Flexner), and Staphylococcus citreus.

GROWTH

OF

THE

SACTLLUS

TUBERCULOSIS

:
f
i
%

Ark AND IN OXYGEN

187

188 BIO-CHEMICAL JOURNAL

Experiment commenced at 4 p.m., March 11th; growths examined
March 12th at 4 p.m.; oxygen percentage = 88°8. Two tubes of each
organism grown in air and two in oxygen.

All ten tubes grown in air show a good full growth. Of the tubes
grown in oxygen—

Vibrio cholerae shows good growth, as good as control.

B. dysenteriae (Flexner), growth as good as control.

B. pyocyaneus, growth as good as control.

B. dysenteriae (Shiga) shows no growth. (See, however, Expt. XV.)

Staph. citreus. Both the oxygen tubes show an appreciable growth,
but much less than controls.

Experiment XIT1.—Growth in increased oxygen percentage of Bacillus
diphtheriae, Staphylococcus citreus, Staphylococcus aureus, Staphylococcus
albus, Bacillus dysenteriae (Shiga), and Bacillus dysenteriae (Flexner).

Examined after twenty-four hours, the two Staph. citreus tubes grown
in oxygen show a quite perceptible growth, rather more than either Staph.
albus or Staph. aureus, but nothing like the growth of the controls grown
in air. The two tubes of Staph. albus and Staph. aureus respectively from
oxygen bell-jar show just the merest trace of growth, practically _ no
growth, while the four air grown tubes show’a good growth.

The two B. dysenteriae (Shiga) tubes from the oxygen show no greath
at all; the two from the air are well grown (see Expt. XV). All four
from B. dysenteriae (Flexner) tubes, on the other hand, show good growths
both in oxygen and in air, there being no appreciable difference.

None of the four B. diptheriae tubes show a good growth, but the
oxygen tubes appear to be a little less than the air grown tubes.

Experiment XIV.—Growth in increased oxygen percentage of Staphy-
lococeus albus, Staphylococcus aureus, Staphylococcus citreus, Bacillus
dysenteriae (Shiga), and Bacillus dysenteriae (Kruse).

Four tubes of each of the above organisms were cultivated from March
19th (afternoon) till March 22nd (afternoon). Two of each in air and two
in oxygen of 90 per cent. .

All the tubes grown in the air showed good growths.

Grown in the oxygen. B. coli, good growth, quite as good as controls.
Staph. albus, fair growth, but not nearly so good as controls. Staph.
aureus, poor growth, less than Staph. albus and not nearly so good as

a i, ee Deas

GROWTH OF THE BACILLUS TUBERCULOSIS — 189

7 “eontrols. Staph. citreus, fairly good growth, much better than the two
_ previous, and approximately half its own controls. B. typhosus, a very
. - sal growth, perhaps just a little poorer than the control. 2. dysenteriae
_ (Shiga), a very poor growth, not to be compared to the control.
4 _ B. dysenteriae (Kruse), poor growth, less than one-third that of control.

In this experiment, which was of longer duration than the previous
ones with the more rapidly growing organisms, there was distinctly more
h in the oxygen grown cultures than in the shorter experiments,
but, at the same time, the inhibition of certain of the organisms was
es undoubted. It was also particularly noticeable here, as was more or less
- obvious throughout the whole series of experiments, that in those cases
_ where the bacteria grew in a suppressed fashion, under the inhibition
“Ss athe oxygen, that the cultures, instead of forming a more or less uniform
__ mass or smear over the surface of the culture medium, consisted of a
~ number of very marked round colonies heaped up.

In several cases where tubes of staphyloccus which had not appreci-
ly grown while in the oxygen had been left on longer in air in the
ou nator, it was noticed that these recovered and grew fairly well in a
rather spotted and heaped up fashion. So that the shorter stay in oxygen
does not appear to kill the more rapidly growing organisms in the same
manner as the prolonged stay 1 in oxygen appears to kill the tubercle
bacillus.

To test the effects of a more prolonged stay in oxygen Experiment XV
was carried out.

oe Experiment XV.—More iota growth in inereased oxygen per-
centage of Bacillus coli, Staphylococcus citreus, Staphylococeus aureus,
roves albus, Bacillus diphtheriae, Bacillus typhosus, Bacillus
86 ae (Flexner), Bacillus dysenteriae (Kruse), and Bacillus dysenteriae
a),

_ Two tubes of each of these organisms were grown in oxygen of 90°5
per cent. and in air respectively, the experiment being continued from the
afternoon of March 25th to that of March 30th.

The following is the comparison of the two sets of growths :—

B. coli, typhosus, diphtheriae show no difference in air or oxygen of

any great magnitude, the air tubes perhaps slightly better grown, but
difference very slight.

Staph. aureus shows a good growth in air, none in oxygen.

surface. eR: 2 sit ‘
Staph. alls shower a would growth in’ “air 4} re
oxygen. Bay 5

B. dysenteriae (Shiga) shows romth in Doth,
oxygen.

oxygen.
B. dysenteriae (Kruse) was nearly caval grown jin both

pak!

2 TE iris 5s

We desire to express our itd to as; Arthur Witenes
valuable assistance in the sare ancen gated work meh sep just

191

_ THE ELECTRICAL FORCES OF MITOSIS AND THE
ORIGIN OF CANCER

By A. E. ann A. C. JESSUP, E. C. C. BALY, F.RS., Fellow of
University College, London, F. W. GOODBODY, M.D., M.R.C.P.,
anp E. PRIDEAUX, M.R.C.S., L.R.C.P.

(Received March 21st, 1909)

Professor Hartog has recently brought forward the interesting
suggestion that the phenomena of mitosis, that is to say the well-known
mitotic figures, are due to the existence of a dual force, as for example, a
magnetic or, better, electrical type. Without pre-judging its nature, he
al this force mitokinetism. He introduces the conception of relative
8! permeability in elucidating the behaviour of this dual force in comparison

with the phenomena of magnetism. He goes on to say that as the cell

_ structures are all material the conception cf geometrical lines of force is
adequate to explain them. He says that the effect of stresses within a
mixture of substances which are of different permeability and free to
arrange themselves will be to segregate out the more permeable material
in strands along the lines of force. While the idea of an opposite polarity
is reasonable, it is difficult to accept the polarity as being magnetic in
any way, because there does not seem to be present any mechanism
whereby magnetic stresses are to be produced. On the other hand, the
substitution of an electrostatic difference of potential for the magnetic
removes these difficulties, for it would appear that in the configuration
of the protein material we have at hand all the necessary conditions for
the establishment of such charges. The - NH - CO- linking which, from
a physico-chemical point of view, is both acid and basic in character, or,
as usually called, amphoteric, possesses residual affinity of two opposite
types, and it is in the existence of these two types that we can obtain the
mechanism for the establishment of electrostatic difference of potential.
Although in the cytoplasm and centrosome we can find an analogy with
the solvent and the dissolved and ionised salt in inorganic chemistry, yet
it must be remembered that in the organic cell the phenomena must be
those of colloids, and for this reason we are somewhat hampered by
ignorance of the nature of colloidal substances. It is possible, however,
to develop a theory that electrically charged colloids play a very important
role in mitosis, a theory which leads to some very interesting results.
When a crystalline salt, as, for example, sodium chloride, is dissolved in

192 BIO-CHEMICAL JOURNAL

water the residual affinity of the water molecules causes them to condense
round and form loose hydrate compounds with one or both of the sodium
and chlorine portions. The lines of force due to the chemical combination
of the sodium and chlorine are thereby weakened, and by virtue of their
velocity of movement by diffusion the two ions get separated, becoming
at the same time seats of positive and negative charges respectively. In —
an analogous fashion the cytoplasm can resolve a discrete molecule or
complex of molecules of amphoteric type into two oppositely charged
portions. The cytoplasmic mass can, by virtue of its two types of residual
affinity, form loose compounds with the two portions of the simpler
compound. The lines of force between the two will be weakened, and the
two portions can be separated and become seats of positive and negative
charges respectively. It appears, therefore, a justifiable assumption that
electrostatic differences of potential are established during mitosis and
that the two centrosomes represent the location of two of these charges.
Very much the same reasoning may be applied to the chromosomes or
chromatin granules; these, by the same process as detailed above, can
become resolved into two oppositely charged portions. Without in any
way assuming that these differences of potential cause the phenomena of
mitosis to occur, it is very difficult to believe that they are not produced
when mitosis does occur. How far they act as the causae causantes is not
determinable with any certainty in the present state of our knowledge of
the vital processes, but our knowledge of the chemical and physical
properties of the protein configuration leads us to the standpoint that the
resolution of the centrosomes and chromatin granules must be accompanied
by the establishment of definite electrostatic differences of potential.

If now we consider the probable influence of the lines of force between
two oppositely charged bodies upon a colloidal mass it will be seen that
the colloid will tend to coagulate. The well-known coagulation of
colloids in the presence of an ionised salt is now attributed to the
alteration in their surface tension by the lines of force between the ions
passing through the surface. If the view be accepted that a colloidal
solution is due to the existence of a negative surface tension, that is to
say, a tendency to form as great a surface as possible, the penetration of
that surface by the lines of force between the ions becomes at once
comprehensible when a colloidal and ionised solution are mixed together.
Applying this view to the prophase of mitosis, the penetration of the
nucleus by the lines of force between the centrosomes will cause the

coagulation or partial coagulation of the chromatin in the chromosomes

THE ELECTRICAL FORCES OF MITOSIS 193

a
ll

oagulation so will increase, the greater the number the lines of
ree whicl s through the nucleus. Thig perhaps gives a reason for
: Ts denention of the reticular structure in the chromosomes in the
arliest prophase of mitosis.

fis To deal next with the chromosomes themselves. There is little doubt
it these consist of discrete particles or granules of chromatin, and, in
| probability, each of these is resolved into two daughter particles
_ charged with positive and negative electricity respectively. The
_ chromosome, by virtue of the splitting of each of its particles into two,
forms two daughter chromosomes, one charged with negative and the
% — positive electricity. It may be argued here that the splitting
» chromosomes occurs at a much later stage than the commencement
ration of the centrosomes. ‘This, however, would be the result
fact that during the earlier phases the chromosomes are
rulating; for it is not likely that the resolution of the material can
occur until the coagulation of the granule into a discrete particle with a
finite surface has taken place.

‘ So ae vt

it

eel Se

; - “The first stage of somatic mitosis would, therefore, appear to be the
resolution and separation of the centrosomes with their definite, equal
| ae opposite charges of electricity. The lines of force between the two
itrosomes as these lines penetrate the nucleus cause the coagulation or
x n of the chromosomes. ‘The next stage is the resolution of the
ivcmese granules, each into two daughter particles of equal and

_ Opposite charge, and this is eventually followed by the splitting of the
chromosomes. The first evidence of an incipient splitting of the chromo-
4 somes will be their polarisation; that is to say, each chromosome will be
half positively and half negatively electrified and will tend, therefore,
to move and take up its — in the equatorial plane of the mitotic

figure (as in fig. 1).
The next change in the mitosis will be the actual parting of the

Les

194 BIO-CHEMICAL JOURNAL

chromosomes into two daughter chromosomes, which by reason of the
electrostatic attraction will migrate to the oppositely charged centrosome.
When the daughter chromosomes arrive at the centrosomes the electrical
charges will be neutralised. It is necessary to assume that the charges are

entirely neutralised, for if there remain a balance of*positive or negative —

electricity, this will tend to mount up in successive ‘divisions—a condition
which it is quite impossible to accept. We are, therefore, bound to take
the view that the sum of all the charges on one set of daughter chromo-
somes is equal and opposite to that upon one centrosome, and that perfect
neutrality of charge is established at the end of each somatic mitosis.
When the lines of force cease to exist, each chromosome tends to become
again de-coagulated. It at once begins to increase its surface, which it
does by means of growing processes which extend until the whole nucleus
appears to all intents and purposes structureless. The chromosomes,
however, must preserve their individuality, although their processes
appear inextricably intermingled. The application of the lines of force
due to the commencement of a new mitosis will at once cause each
chromosome to contract and condense until it exists once again as a dense
individual, capable of being highly stained.

. ++ ee pe

ed

We have not considered as yet several of the attendant phenomena ~

of mitosis; for example, no reason has been advanced for the separation
of the centrosomes, the formation of the spindle figure, and also the
disappearance and the reappearance of the nuclear wall.

Considering first the separation of the centrosomes, which appears to
us to be the most important feature of mitosis, it must be remembered that

in the analogous case of ion formation in aqueous solution the separation of |

the ions is due to diffusion. But this is quite inapplicable to the present
case. It is equally necessary, however, to postulate some definite influence

which separates the centrosomes—an influence which is stronger than —

and quite apart from the electrical forces: for these would naturally

tend to draw the two centrosomes together. In other words, the electrical _ >

force cannot be the causa causans of mitosis, but must be a concomitant
phenomenon. This fact cannot be too strongly insisted upon, for although ~
the conditions of a living cell would seem to be such that the vital units

must become resolved into two oppositely charged masses, yet unless some

definite and separate stress were present, the two oppositely charged

masses would lie side by side without any electrical influence on their
surroundings. As stated above, the resolution into oppositely charged

masses is the natural result of loose combination between the vital unit

THE ELECTRICAL FORCES OF MITOSIS 195

‘ } | surrounding medium, and, again, these loose combinations are
tl resthe result of the chemical structure of the cell materials.
Finns compounds must play a very important part in the general
"fife of the cell, as, for example, in the growth of the chromatin. There
E ‘is no doubt a strong separative force at work—a force which would appear
3 4 0 to be connected with the mass division of the cytoplasm. It would seem
a this mass division is an inherent vital property of living cytoplasm ;
; = we incline to the view that it is the real causa causans of mitosis, and
_ that the phenomena described above are produced by the mass division
_ taking place. There is a sound foundation for our view, for it has been
‘shown that the periodic activity of cytoplasm is independent of both
1 and centrosome. For example, in the case of a fertilized egg
ded into two portions, one of which contains the nucleus and the other
10 pte behaviour of the enucleated portion is most remarkable. It
s three times in succession a polar lobe at the same time that the
half is dividing, becoming spherical after each period without
_ At the fourth cleavage a fourth lobe is formed, which is not
a ed but grows steadily larger, so that the fragment appears finally
be divided into two. The activity of the enucleated half is thus not
ly rhythmic in character but changes in character at the fourth
cleavage when in normal development the polar lobe no longer forms a
temporary structure but is permanently cut off by cell division. The
_ eytoplasm, therefore, would seem to possess a power of mass division—a
power which is also periodic in its action, its periodicity no doubt
depending upon the cytoplasm reaching a certain stage of development
during the vegetative period of the cell. The existence of this power of
a — division possessed by the cytoplasm gives a reasonable explanation
r the separation of the centrosomes. When the period has been reached,
‘the first feature of the phenomenon is the resolution of the centrosome
om the previous division into two oppositely charged centrosomes.
When the mass division begins the axis’ is determined by the position of
_ the centrosomes, so that they are drawn apart. The electrical forces
_ brought into play cause the condensation or coagulation of the chromo-
_ somes, their resolution and migration, and, finally, when the mass division
of the cytoplasm is finished the formation of two daughter cells. By the
statement that the axis of mass division is determined by the centrosomes
we mean that the two daughter centrosomes are separated, because one
muist be in each daughter mass of cytoplasm. This fact cannot be a
matter of chance, for if it were so, many more divisions of cytoplasm

196 BIO-CHEMICAL JOURNAL

would occur than mitoses of the nucleus, which is absurd. It is a natural
sequence that the line of cleavage of the cytoplasm is determined by the
centrosomes. This would seem to be the normal order of events in any
mitosis, but there is no reason why certain minor details should not be

altered either as regards their character or their position in the scale of
operations. Such variations need not, and do not, militate against the

electrical theory in any way. For example, in many cases the centrosome
in the daughter cell divides immediately after the mitosis is finished in

readiness for the next division. All that the theory demands is that the

centrosome divide into two; the period at which this occurs is not of any
moment, but the fact that is of the greatest importance is that the two
new centrosomes never get separated except during mitosis, and then only
with the formation of the spindle figure. |

In reference to the statement that the axis of the mags division is
determined by the centrosomes, 0. and R. Hertwig! and also Roux? have
noticed that as a rule the plane of division of a non-spherical cell is at
right angles with the direction of the greatest diameter or extension, and
Driesch has shown that if the newly fertilized egg of the sea urchin be
gently pressed under a cover glass, so that it is slightly flattened, the

plane of division is at right angles to the slide. The position of the plane

of cleavage is determined by the position of the nuclear spindle, and this
depends upon the position of the centrosomes. Moreover, in the process
of cell division the egg of some animals becomes elliptie with the long
axis falling in the direction of the common diameter of the amphiaster.
This has given rise to the idea that it is the spindle itself which elongates

the egg, but Loeb? has often noticed that the elongation, though in the ~

direction of the spindle, always occurs immediately befase the cell
division.
Again, the possession by the cytoplasm of a power of mass division

will give an explanation of the phenomenon of streaming which is observed,

during mitosis, for the streaming will merely be the flowing of the

cytoplasm from the cleavage plane into two daughter masses. Inasmuch — :

as the sum of the masses of the cytoplasm of the two daughter cells is less

than that of the mother cell, it at once suggests itself that the mass
division is caused in the first place by a loss of water from the periphery -

of the cell by osmosis.

1. Untersuch. zur Morphol. wnd Physiol. der Zelle, V, 1887.
2. Breslawer Arzt. Zeit., 1885.
8. Loeb, Dynamics of Living Matter, p. 64. Columbia University Press, 1906.

i. oe

THE ELECTRICAL FORCES OF MITOSIS 197

To give an explanation of the formation of the asters and spindle
fig: »-of mitosis is not easy. The natural view to take would be that
' these are due to the coagulation or condensation of the cytoplasm along
_ paths parallel to the lines of force, using the same argument as in the
ease of the chromosomes above. Furthermore, it is quite true that in
- many cases the mitotic diagrams present great similarity with the lines of
force between oppositely charged bodies. This view has been previously
advanced by A. Fischer.' In certain cases, however, the rays from the
two asters appear to cross one another—an effect which is impossible if
they are simply due to lines of force. But it may be that this is only an
apparent effect due to the point of view of the phenomenon. If this be a
real crossing it is clear that the rays cannot be due to threads of coagulated
cytoplasm; although it might be possible to look upon them as direct
growths from the centrosomes, yet the former explanation would seem
far more reasonable, provided that the difficulty as regards their crossing
one another be surmounted. Of course it must not be forgotten that the
separation of the centrosomes by the mass division of the cytoplasm will
induce stresses which may disturb the position of the threads. On the
whole the evidence would appear to support the view that the mitotic
figures are due to the coagulation of the cytoplasm under the influence
of the lines of force.
In the present state of our knowledge of the chemistry of cell
physiology it is impossible to account for the disappearance of the nuclear
membrane during mitosis and its reappearance after the process is finished.
We might say that the membrane is due to a definite chemical reaction
between the nucleic acid and the cytoplasm, and that this reaction is
reversed under the stimulus of the lines of force, so that the membrane
disappears only to reappear when the force lines die away; but this can
only be pure hypothesis.
_ We may next turn our attention to the maturation divisions of the
germ cells, and investigate the relations which exist between the electric
charges in these cases. The results obtained are peculiarly interesting,
inasmuch as it seems absolutely certain that complete electrical neutrality
does not, and cannot, result from these divisions.

The first fact which we are met with in these divisions is the fusion
together of the chromosomes in pairs to give the meiotic gemini. In
order to account for this and bring it into line with the other phenomena
it is necessary to assume the existence of some form of polarity difference

1. Fixierung, Farbung und Bau des Protoplasmas, 1899,

198 BIO-CHEMICAL JOURNAL

between each of the two individual chromosomes, which fuse together to
form the gemini. All recent cytological research leads to the view
that in each case it is one paternal and one maternal chromosome which —
fuse together, producing a bi-polar twin; and, moreover, that it is not

any chance pair which fuse together, but there exists some type of — .

selective pairing between paternal and maternal chromosomes. For this —
pairing there must exist in the cell some opposite polarity between the
two members of each twin-—a polarity which miy well be of electrical
type. This would point to the existence of some difference in electrical
charge between the paternal and maternal chromosomes which comes into
play during the long resting period of the germ cell. That an electrical
attraction can be produced between paternal and maternal chromatin in
the germ cell follows readily from the general theory, but its consideration
may be postponed for the moment. It must be confessed that the phases
of the phenomena of maturation divisions differ so much according to
various observers that it is impossible to deal with more than what appear
to be the most typical cases; and we must content ourselves with pointing
out how the general relation between the charges is not altered in ms
of the cases observed.

The simplest case to deal with is when no tetrads are formed and
when the first maturation division takes place between the split halves
of the meiotic gemini, while the second division is a somatic division of
the reduced number of chromosomes. It is necessary, in order to follow |
out the distribution of the charges in the maturation divisions, that the
relative values of the charges upon the centrosome and chromosome be
considered. Beyond the bare statement that it is essential that at the
end of each somatic mitosis the charges upon the centrosomes must be
neutralised by the chromosomes nothing has been stated as to the relative
values of the charges. The establishment of electrical neutrality at the
close of each somatic mitosis is of a very great importance, for if by some
means or other the charges were not neutralised entirely and a small
amount were left over in the daughter ce qes ly this would go on
mounting up | steadily in-suecessive divisions wiSa “98« apparent limit.
This _is 18,1 CC course, impossible of acceptance, and therefore we are driven
to the bohal gio which is indeed the simplest, that complete neutrality
obtains at the end of every somatic mitosis. We must therefore equate
the charge upon each centrosome to the sum of the charges upon the
daughter chromosomes, and in doing this it will be convenient to speak
of the charge upon each centrosome as a unit-charge of positive or

THE ELECTRICAL FORCES OF MITOSIS 199

ative aw respectively. In the case, for example, of an individual
with fou atic chromosomes, the sum of the charges upon the four
“a daughter ey which migrate to one daughter cell must equal the
F Ze charge upon the corresponding centrosome. We therefore in this
would expect the average charge upon each of the chromosomes to be one
quarter of the unit charge, but it may be pointed out that there is no
_ @ priori reason why the charges on all the chromosomes should be equal
a _ in amount, the essential condition only being that the sum of the charges
on them be equal to the unit charge.

a atering now to the germ cells we may cay; as above, that a definite
erence of potential is developed between paternal and maternal
p | on osomes; and let us say, merely for purposes of argument, that this
difference of potential is half that carried by the chromosomes in somatic
tosis. If we continue to deal with the case of an individual with four
tic chromosomes, then we will assume the difference between paternal

i c 1 end a one-eighth unit-charge of positive and snantivs electricity
- respectively The result will be that the gemini will carry themselves

+ iste

Fic. 2.

_ When the gemini split again, and the two chromosomes migrate to
_ the centrosomes, the charges will not be neutralised, but in each daughter
cell there will be left a residue of electricity. When the meiotic gemini
split they each give two halves carrying an eighth positive and negative
charge respectively, and in fig. 2 the two positive halves migrate to the
centrosome on the right and the two negative ones migrate to the
es centrosome on the left. When the two positive halves arrive at the
a negative centrosome they each bring a one-eighth unit, that is to say,
one-quarter unit positive electricity altogether. Since the centrosome

we
ee

eo, ee ae

200 BIO-CHEMICAL JOURNAL

carries a whole unit of negative electricity, we have one unit negative and
one-quarter unit of positive electricity, which leaves a balance of three-
quarter positive electricity. Similarly at the other side there will be left
a residue of three-quarter unit negative electricity. On these lines it is
clearly impossible for the charges to be neutralised in the daughter cells,
for the only condition under which this could be secured is that the

difference of potential between the paternal and maternal chromosomes —

was half a unit-charge, or twice as much as between the daughter
chromosomes in the previous somatic mitoses. Such a large difference of
potential is out of the question, for it would be impossible for such a
charge to lie dormant through the various somatic divisions which
occurred previously to the maturation divisions. We must conclude,
therefore, that whatever may be the real value of the potential difference
between paternal and maternal chromosomes, a residual charge is left in
the daughter cells of the first maturation division, and at the same time
point out that the smaller we assess the potential difference between
paternal and maternal chromosomes in relation to the normal somatic
charge, the greater will be the residual charge.

The next division is of the ordinary somatic type, but with this
difference—that there are now only half the somatic number of chromo-
somes, and therefore only half the number of chromatin granules in each
cell. It is quite evident, therefore, that in this somatic division the

respective charges cannot be neutralised, for we have the whole mechanism ~

of mitosis but only half the proper number of chromatin granules.
Following out the case above, the reduced number of chromosomes is two,
and each will give two daughter chromosomes, carrying one-quarter
positive and negative charges respectively, which are the normal somatic
charges for the individual in question. At the end of this mitosis there
will be again a balance of charge left over, for in one daughter cell there
is one unit positive charge and two one-quarter unit negative charges,
leaving a balance of a half-unit positive charge; similarly, in the other
daughter cell there is a balance of a half-unit negative charge.

A second case, which frequently occurs, is the formation of the
tetrads, and in this case, as in the previous, the result is the same—a
balance of charge must be left in the daughter cells. The formation of the
tetrads is due, no doubt, to the somatic division of the chromosomes taking
place before the first division has proceeded very far. Assuming, as
before, the existence of one-eighth unit-charge upon paternal and
maternal chromosomes respectively, the first stage will be the formation

+ Se:

_-——s THE: «ELECTRICAL FORCES OF MITOSIS 201

the bi-polar gemini. These gemini will again arrange themselves
- equatorial plate of the mitotic figure and then will begin to
- undergo the somatie resolution in readiness for the second division.
Owing to the bi-polarity being most pronounced at each end of the twin
chromosomes this resolution, under normal circumstances, should begin
i in the centre of each twin, which thus forms a ring, the procedure being
of the true hetero-typical kind. This resolution, however, will be
accompanied by the establishment of a positive and negative charge
respectively on each side of the ring; and, in our case of four somatic
_ ¢hromosomes, this charge will in each case amount to one-quarter unit-

_ eharge. This ring formation is followed by the completion of the somatic
_ splitting and the resolution of the gemini back again along the lines of
the preliminary fusion. Each ring thereby breaks into four portions,
which form the tetrads, and the two cell divisions rapidly take place in
‘succession. The distribution and balance of charges follow exactly the
_ same lines as before, with the establishment of three-quarter unit positive

and negative charges in the two first daughter cells and the further
establishment of a residual half positive and negative charges alternately
1 the grand-daughter cells. The only proviso that we must make as
regards maturation by tetrads is that the two divisions follow one another
in rapid succession with no intervening resting stage. This, however,
seems quite a reasonable position to take up, viz., that when the somatic
division of chromosomes takes place with the formation of tetrads during
the first maturation division, the second maturation division must follow
the first at once. If the somatic resolution does not take place, or only takes
place incompletely, then the charges will be the same whether there is a
resting period betwen the two or not.

_ Before discussing the result of the establishment of the residual
electric charges in maturation, it may be pointed out that the hetero-
typical resolution of the meiotic gemini with the formation of rings will
only take place when all the chromatin granules in each twin are perfectly
‘uniform. We might readily imagine that the chromatin granules in one
paternal chromosome, for example, are weaker in character than those
in the maternal chromosome with which it fuses to form a twin. In this
special case the ring would not necessarily be formed when the somatic
split took place, but rather, a body of the form :

<> or aS

eee Sk ee ey ee ee ee
z * : ee, es meta yt j s ‘ee
|

202 BIO-CHEMICAL JOURNAL

Moore and Arnold have described various forms of these gemini in
the meiotic phases of many germ cells, and it would seem that if their
existence be confirmed they can be explained by certain distributions of
activity in the chromatin granules in the twins. They are, therefore,
only of secondary importance as far as regards the phenomena - under
consideration.

It may be argued that the formation of residual charges might be
prevented by the cytoplasm providing in each division centrosomes of
just sufficient charge to meet the needs of such division. This, however,
seems impossible of belief, for it would mean a variation in the power of
the cytoplasm between very large limits in a very short space of time.
Against this view, we would point out that the cytoplasmic activity of the
germ cells at the time of maturation is exceedingly great, and, therefore,
it seems in the highest degree unlikely that the resolving power should
become half the normal value or even less, and, further, that it should
vary. Although it may be said that we have arbitrarily assumed a
difference of potential between paternal and maternal chromosomes and
fixed it at one-eighth of a unit-charge, it must be remembered that
whatever be the view taken of it, the establishment of residual charges is
necessary, for only one somatic division of the chromatin granules occurs
and twice the somatic number of centrosomes are brought into play. On
these grounds alone, without making any assumption whatsoever as regards
the existence of the potential difference between paternal and maternal
chromatin being necessary to cause the preliminary fusion in pairs to
form the meiotic gemini, it appears absolutely impossible for the residual
charges not to be established in the maturation divisions. At the same
time, as we have already shown, it is necessary to assume some type of
difference of potential between paternal and maternal chromatin, and the
existence of one of electrical type, as we shall presently show, is quite
easy of acceptance, although its value cannot be directly estimated in the
present state of our knowledge. If we, therefore, put this at half
the somatic charge on the daughter chromosomes, that is to say, in our
example of four somatic chromosomes, one-eighth of a unit-charge of
positive and negative electricity on paternal and maternal chromosomes
respectively, and follow out the mounting up of the residual charges 1 in
the maturation divisions, we arrive at the following values : —

In the first, or meiotic, division, the residual charges will be three-
quarters of a unit positive and negative charge respectively, for the
centrosomes each carry one unit-charge, and there are two chromosomes

i” Of a re it <a bead

THE ELECTRICAL FORCES OF MITOSIS 208

i with one-eighth charge on each. In one daughter cell we have,
_ therefore: "~

a +1+(-t-t)=+?

and in the other we have :—

—-1+(4+#)=—3

~The second, or post-meiotic, division proceeds naturally quite
independently of these charges, and as we have before shown, owing to
there being only half the number of somatic granules, a further charge
is established in each grand-daughter cell of half-unit positive or half-
unit negative charge. To determine the total residual charges in the
grand-daughter cells it is necessary to add the pre-existing residual
charges in the daughter cells to those formed in the grand-daughter cells.
_ To one pair of the latter, which carry a half-positive and negative charge
respectively, we must add the three-quarter positive charge. To the other
pair we must add the three-quarter negative charge, so that the total
__ residual charges in each set of four grand-daughter cells are one-and-a-
_ quarter positive, one-quarter positive, one-quarter negative, and one-and-
a-quarter negative respectively. This is perhaps shown more clearly in

fig. 3.

Mother Cell { Neutral}

Fic. 3.

It may be pointed out here that the amount of the residual charges
is entirely independent of the somatic number of chromosomes. We have,
purely for purposes of argument, considered the special case of four
a3 somatic chromosomes. Exactly the same results and the same quantities
Re of residual charges are obtained, as may readily be seen, whatever be the
ae ‘number of somatic chromosomes.

Now, in the case of the maturation of the ovum, only one of the

204 BLO-CHEMICAL JOURNAL

. grand-daughter cells is utilised, the other three being rejected as the polar
bodies; but in spermatogenesis each of the four grand-daughter cells

produces a functional spermatozoon, so that we arrive at the interesting
result that one of the spermatozoa is of the same type as the ovum and one —
of the exactly opposite type; while the other two are of an intermediary

type.

| Herein, in all probability, lies the secret of sex production and the
explanation of Mendelism, for it would seem perfectly natural that if the
ovum be fertilised with the spermatozoon of the same type, a female
embryo will be produced; if with spermatozoa of opposite type, male;
and if with either of the two intermediary spermatozoa, a heterozygote
will be formed. We use the word heterozygote in the Mendelian sense,
and would mean either a male embryo, which is not entirely male, but
one with male characteristics predominating over the female; or female
embryo in which female predominates over male. On the above lines, it
is evident that the essential of sex is primarily established in the meiotic
division, for it is here that the first residual charges are established. The
post-meiotic, or somatic division, merely alters the proportion in which
the male and female characters predominate.

These results afford a simple explanation of Wilson’s! experiments
upon the insect Protenor belfragi, without any assumption of selective
fertilisation. The male Protenor possesses five chromosomes in its
somatic cells, while the female has six, and as there occurs an irregular
distribution of chromosomes in the spermatogenesis of the male, it is
possible to follow the results of fertilisation with greater accuracy than
in an ordinary case. Wilson finds that in the spermatogenesis the odd, or
hetero-tropic, chromosome does not fuse with any of the other chromo-
somes, but passes bodily over into one of the daughter cells of the meiotic
division. In the post-meiotie division this hetero-tropie chromosome
divides as usual, so that of the four grand-daughter cells two give
spermatozoa with three chromosomes and two give spermatozoa with two
chromosomes. In the case of the oogenesis of the female, there is no
irregularity, and the ovum possesses naturally three chromosomes. When
the ovum is fertilised by a spermatozoon containing the hetero-tropie
chromosomes a female is always produced, while if the fertilisation take
place by one of the spermatozoa containing two chromosomes a male is
always produced. Now, on the theory which we put forward, the male
embryo receives two chromosomes from the spermatozoa and three from

1. Science, N.S., XXIII, p. 500 (1905).

“THE ELECTRICAL FORCES OF MITOSIS 205

i» and when, therefore, the spermatogenesis of this individual
jee, the two paternal chromosomes fuse with two of the maternal
®mes, giving meiotic gemini, leaving the third maternal chromo-
‘over as the hetero-tropic one. When the splitting of the meiotic
fi takes place the two maternal portions go to one daughter cell and
wo paternal portions to the other. The hetero-tropic chromosome
g°of maternal origin will go along with the two maternal halves of
“fhe gemini into the same daughter cell. It is this cell which, by virtue
0 of its residual charge, has established in the female predominance, so that
~~ when one of its daughter cells fertilises the ovum the female will always
ibe produced. Similarly, the other daughter cell of the meiotic division
has male characteristics formed on it by its residual charge, so that its
- daughter cells always give male insects in fertilisation. Wilson attempted
rea i o explain these facts by means of attributing a female determinancy to
Lf hetero-tropic chromosome, but owing to the thereby necessary
existence of the male determinant, it became necessary to assume a
selective fertilisation ; this assumption, which, from first principles, seems
improbable, is therefore rendered entirely unnecessary.

Tt is only right to point out that McLung! was the first to suggest
from the investigations upon the accessory chromosome that the sex
determinancy lies in the spermatozoa. To quote McLung’s own words :—
*T must here also point out a fact that does not seem to have the
recognition it deserves, viz., that if there is a cross division of the
chromosomes in the maturation mitosis, there must be two kinds of
spermatozoa regardless of the presence of the accessory chromosome. It
is thus possible that even in the absence of any specialised element a
2 preponderant maleness would attach to one-half of the spermatozoa, due
___ to the qualitative division of the tetrads.’

The differentiation between the cells produced in the maturation
division appears to give also the key to the problems of parthenogenesis.
In the first type of parthenogenesis, as in Aphis, when only one polar
body is formed the female only is produced. As we have already shown,
the meiotic division establishes the sex of the cell, so that in Aphis the
cell with the male character, established in it by virtue of its residual
charge, is rejected as the polar body; the nucleus remaining contains
the female character established in it, so that when this egg develops
parthenogenetically a female insect must be produced.

©, E, MeLung, ‘The Accessory Chromosome: Sex Determinant?’ Biological
Bulletin: IIT, p. 48, 1902. "

206 BIO-CHEMICAL JOURNAL

The second type of parthenogenesis, as occurs for exa
honey-bee, where fertilisation takes place by means of the
body, the egg always produces a male, while the eggs fertili
males always produce workers, which are, of course, female, bu
develop into queens unless specially fed during their early s
development. On the theory of the differentiation between the
grand-daughter cells of the maturation division, the fertilisation of
ovum by the second polar body becomes at once comprehensible, for
polar body bears exactly the same relation, as regards its residual charge
to the ovum, as does one of the spermatozoa; indeed, the polar body must
be of male character towards the ovum, so that inasmuch as the
fertilisation takes place within the one individual a male embryo is
produced. This means that the female characteristic established by the —
first or meiotic division does not alter the actual relations between the
polar body and the ovum, a view which is supported by the fact that the
cleavage centrosomes arise from the cytoplasm of the ovum. Further-
more, inasmuch as the fertilisation is carried out under the aegis, so to
speak, of the female dominance, the male produced will undoubtedly be
heterozygote, moreover, heterozygote with an unusually large presence !
of female character. The spermatozoa eventually produced by a male of q
this type will clearly be of different character from those of the
hypothetically true homozygote male. So far is the female character
present that he cannot beget any but female offspring—a fact not at all
incomprehensible if it be remembered that the queen bee is an unusually
highly developed female. It is not surprising, therefore, that the females
normally produced from the fertilised egg are undeveloped, and it at once
raises the question whether they are all exactly the same. Asa result of
this explanation, it would be expected that the worker bees possess among
themselves differences in their potential powers of development into a
queens. Indeed, it might be expected that the special feeding might fail ;
in developing queens from a small percentage of the worker larvae. The
important point, however, for the present purpose is the fact that the
present theory gives a reasonable explanation of the fertilisation by the

i ii. ae

second polar body. 7
' The point was raised above as regards the sex characters of the

spermatozoa of the heterozygote male, and it was stated that the relations
between the charges of the spermatids in each quartette would be altered.
This deduction follows reasonably from the general theory, and if, for
example, a male was heterozygote to the extent of three parts male and

THE ELECTRICAL FORCES OF MITOSIS 207

_ two parts female, it is probable that the intermediary spermatozoa would
e altered: It is quite conceivable, for example, that the spermatozoon,
ich in a normal case would give a heterozygote male, might be so far
‘red by being itself produced from a heterozygote as to become
entially female in character, so that, on fertilisation, a heterozygote
e would be produced. On the average, a homozygote male and
nale would produce equal numbers of male and female offspring, but a
_Reterozygote male would beget a greater proportion of female offspring.
as _ Amongst the human race this may possibly account for the large
red pminance of male or female children which frequently occurs in certain
nilies. Again, it is not impossible that the condition of health that
4 all may be in at the time of maturation may influence the value
the residual charges in the grand-daughter cells. If the vitality were
is r; ‘it might readily follow that the residual charges be reduced so far
that the relative dominance be affected. This would account for the
results of the experiments upon the feeding of one parent and starving
» other in the case of certain amphibia, etc., when it was found that
s sex of the stronger parent predominated in the offspring.
% - We have taken for granted in the case of the oogenesis of the female
P tet the ovum is the one grand-daughter cell of the mother cell, which is
the true homozygote female. Our reason for this is simply that it seems
_ the most likely condition to occur, though there seems no valid reason
why the other conditions should not obtain. Even if one of the other
conditions were to take place, it would not materially alter any of the
conclusions drawn above.
_--——s«sTt must be remembered that the post-meiotic division tends to increase
___ the residual charges in the resulting cells, and therefore it would follow
that the fact of there being no further divisions after the post-meiotic
me is due to the residual charges having already reached the limiting
value. There naturally must be a limit to the power of the cytoplasm and
_ chromatin i in building up these residual charges, and, no doubt, this limit
_ is reached in the post-meiotic division.
While it is by no means necessary, therefore, that the charges are
a of the actual value we have arrived at, yet the establishment of some type
i | of residual charges seems an essential consequence of an electrical theory
“aN of mitosis, and moreover these charges can, in a satisfactory manner, give
an explanation of the phenomenon of sex production.
' Although it is probable in the main that the maternal chromosomes
pass during the meiotic division into the female daughter cell, yet there

208 BIO-CHEMICAL JOURNAL

is no doubt that during the existence of the meiotic gemini, some
re-distribution of the chromatin granules takes place. In order to account
for the Mendelian distribution of hereditary characteristics, it is essential
that the chromosomes formed by the splitting of the meiotic gemini be

not necessarily the same individuals as originally fused together; there —

must be an interchange of the granules during the time of fusion,
according to Dr. Vries’s theory. This, however, does not concern the
present purpose very much, for whether this takes place or not, no
fundamental change in the relative charges will occur. It must be
remembered, however, that any re-arrangement of the granules in the
meiotic gemini will tend to decrease the potential difference between the
maternal and paternal portions, and, therefore, the potential difference
between the two chromosomes obtained when the gemini are split. From
a consideration of the residual charges obtained in the first maturation
or meiotic division, it is evident that the smaller the potential difference
between the split chromosomes of the gemini, the greater the residual
charge or sex determinance in the resulting daughter cell. This affords
a reasonable explanation of the De Vries re-arrangement of the chromatin
granules, viz., the decrease in the potential difference between the two
halves of the gemini. For there is no doubt that the re-arrangement

will take place, since such re-arrangement tends to decrease the potential —
difference. We may say, therefore, that the De Vries’ re-arrangement of

the granules is not a matter of chance; it is a direct consequence of the
potential gradient existing within the bi-polar twin, and the number of
individual granules which interchange will be determined by the steepness
of this potential gradient. In other words, the greater the potential
difference between the paternal and maternal chromatin, the greater the
number of interchanges among the granules, and hence, also, the more
pronounced become the sex characteristics of the grand-daughter cells.

Now there remain certain other facts in connection with fertilisation

which are brought into co-ordination by this theory. In the first place we

have the very profound changes which are induced in the cytoplasm of the

ovum at the time of fertilisation. Space will not permit of our dealing
with these in detail, but they can, in general, be explained by chemical
and physical reactions taking place when the charge on the spermatozoon
enters the ovum. It must be remembered that the density of the electric
charge on the spermatozoon is very much higher than that on the ovum,
owing to its being so excessively minute compared with the ovum, and the

fact of its sudden introduction will be to cause a wave of energy to travel —

f

THE ELECTRICAL FORCES OF MITOSIS 209

rough the cytoplasm. Again, the difference in polarity between the
wum and the spermatozoa nuclei will account for the different staining
r ; which have at times been observed between the two, for a charge
i positive electricity on a mass of protein materia] will increase its

: vote while its acidity will be increased by a negative charge. After
_ fertilisation, as the charges become equally distributed, the staining
reactions of the two nuclei will approach more and more nearly together.

Perhaps one of the most important results of this theory is that it
leads to the view that the ovum and spermatozoon are both cells with
ial charges established in them, and which cannot reproduce them-
ves of their own power, owing to these charges. The ovum, moreover,
: + not possess any material wherewith to form centrosomes and so start
mi to ‘is, while the spermatozoon, although it possesses a centrosome in the
idle piece, has no cytoplasm to start the mitotic machine; when the
vo are brought together, the machinery is completed and mitosis can
% t, provided, of course, that the temperature is sufficient. We are,

therefore brought to the conclusion that the chromosomes are individual
tities, preserving their individuality all through each cycle, and that

Tiesizesanace are of paternal origin. What. the latter consist of, it is
idle to speculate, beyond saying that they are, in all probability, discrete
granules of protein, considerably simpler in character than the cytoplasm,
_ which can be resolved by the cytoplasm into two parts of basic and acid
| ip with a positive and negative charge respectively.

It follows that the fertilisation of the enucleated ovum will give rise
+ ican embryo (with, of course, only half the normal number of chromo-
: somes), this being the natural result of the theory. There is no essential
need for both nuclei to be present, since the machinery is complete for

is when the spermatozoon enters the cytoplasm of the ovum. The
verse of this, or the artificial fertilisation of an ovum, also follows,
+ equally easy of explanation. The most striking case is that of

Loeb, who succeeded in causing sea-urchin eggs to develop partheno-
genetically by placing them in a solution of magnesium chloride. If a
olloid mass be placed in a solution of an ionised salt, the osmotic
pressure of the ions will be exerted upon the surface of the colloid. In
_ time, as the water penetrates a little into the colloid, the ions, owing to
their diffusion velocities, will tend to penetrate the surface. The
_ velocities of the two ions, however, are different, so that one will always be
very slightly ahead of the other, with the result that the surface of the
___ colloid will tend to become electrified. Thus, when the eggs are put into

210 BIO-CHEMICAL JOURNAL

magnesium chloride solution the outer layers become electrified, and even
though this effect be produced at only one small portion of the surface,
yet, with the previously existing charge of the egg, it is enough to cause
parthenogenetic development. The first result will be much the same as
when the spermatozoon normally enters, viz., the same modification of the —
cytoplasm, which is doubtless due to the re-distribution of the charges.
The production of the centrosomes would appear to arise from some portion
of the cytoplasm which is coagulated, and, possibly, hydrolysed to a
certain extent by the electrical and chemical stimulus given by the ions.
The parthenogenetic development of the eggs of the silk worm noticed by
Tichomiroft! on gently rubbing them with a brush is capable of the same
explanation, namely, the electrification of the surface layers of the
cytoplasm. LLL

The question may be raised here as regards the continuity of the
centrosomes through the life of any individual, and it may be pointed out
that from the point of view of the electrical theory, there is no prima
facie evidence why they should possess any continuity. The disappearance
of the old centrosomes and the appearance de novo of another set is
perfectly possible. They may arise from the old spermatid centrosome in-
the middle piece of the spermatozoon, or from one of the vesicles of the
archoplasm. The same centrosome may go on dividing itself up
continuously all through life, or at any period a new set may arise as far
as the theory is concerned. It is not probable that any change in the
centrosomes will arise after several generations of cell division have taken
place, for the number of available particles introduced by the sperma-
tozoon get fewer owing to their absorption or distribution. In the first
few generations a new set of centrosomes might easily arise and, indeed,
have been seen to do so.”

There is another point which may be mentioned, and which gives

some support to this theory, namely, that the second polar body is some-

times not formed in the ovum until after the entrance of the spermatozoon,

and, indeed, in Chaetopterus® the first polar mitosis stops at the anaphase

until the sperm has entered, when the mitosis is resumed and both polar

bodies are formed. The explanation of these facts on the electrical theory

is that the cytoplasmic power is not sufficient to carry out the maturation

divisions and establish the residuai charges. When the tension is relieved
1. Loeb, loc. cit., p. 165,

2. Morgan Rept. of the Amer. Morph. Soc. Science, 111 (1896).
3. Med. Journ, Micros., X, p. 1 (1895).

THE ELECTRICAL FORCES OF MITOSIS 211

* the entrance of the spermatozoon then the division proceeds normally.
Th is a strong support of the view that polarity or stress is established
the maturation division and that it is relieved on the entrance of the
_ spermatozoon ' Mead has noticed that the eggs of Chaetopterus throw out
their polar bodies if a little iodide of potassium be added to the sea-water.
This is quite analogous to the artificial parthenogenesis discussed above,
for the influence of the potassium ions relieves the tension set up by the
_ maturation divisions in the same way as does the spermatozoon, and the
7 two divisions can complete themselves in the normal way.
_ §$trong support for the electrical theory is also to be found in the
___ faet that both males and females are rendered completely sterile by
“X-rays. The action of X-rays in breaking down the di-electric is well
nown, and in these cases of sterilisation by X-rays the cell material
comes a conductor and the residual charges are dissipated, with the
sult that both spermatozoa and ova are defunctionated. These facts
fer the most striking support to the theory.
There remains now only to be considered the assumption made in the
section dealing with maturation that a definite potential difference exists
in the germ cells between paternal and maternal chromatin. As a matter
of fact, there is no doubt that there is a considerable difference between
the paternal and maternal chromatin, which may very possibly be
__ established by the residual charges in the ovum and spermatozoon, for
whatever may be the actual value of the residual charges produced in
- maturation, the greater density of charge on the spermatozoon relatively
to that on the ovum must be borne in mind—a result which is due to the
relatively minute size of the spermatozoon. It is possible that this
difference may be the origin of the potential difference between the
sa ernal and maternal chromatin in the germ cells, for though there is
=A 1 equal distribution of the electricity tending towards neutrality at
a fertilisation, yet it is quite reasonable to suppose that perfect neutrality
is not at once established, or, in other words, that paternal and maternal
chromatin do not at once become identical. ‘That it becomes so eventually
in the somatic cells must indeed be the case, while it is not a great
assumption to make that the potential difference is preserved in those cells
destined to become germ cells. If this were the case, it is evident that
there should be some differentiation between the somatic cell ancestors
and the germ cell ancestors from the very beginning of the cleavage

“1. Lectures at Wood's Hole, Boston, 1898.

212 BIO-CHEMICAL JOURNAL

nucleus. Indeed, Boveri! has found such a differentiation in Ascaris at
the two-cell stage, for in the cell destined to give rise to the somatic cells
there is evidenced a definite rejection at each division of some of the
chromatin, while in those cells destined to form the germ cells this does
not occur. It would appear from this that the casting out of some of the

chromatin in the early somatic cells is the means adopted to establish the ms

necessary complete neutrality between paternal and maternal chromo-
somes, while in the germ cells this neutrality is not established ‘and the
potential difference remains, but of too small an amount to make itself
felt. As the individual grows, and the sex becomes established, the
originally existing difference of potential would tend to become
emphasised until it becomes sufficiently large to interfere with normal
mitosis; the cells then enter their long resting stage only to emerge with
the formation of the meiotic gemini.
An interesting point arises in connection with the fact that in the
animal kingdom only one somatic division occurs after the reducing
division. It is evident from the theory put forward of the electric
residual charges that the establishment of these charges means a certain
condition of stress, and it therefore follows that the work done by the
organism in carrying out a maturation division must be greater than that
in a somatic division. If we consider for a moment the driving force
which works the somatic machine, we can clearly equate this to the
integration or sum of all the small amounts of work done in each cell
division. The energy used and the work done are undoubtedly commen-
surable and capable of expression in terms of some simple unit. There is

no doubt, therefore, that the force available for any one cell division is -

limited, and it would appear that the limit is reached at the end of the
post-meiotic division. The available force is not sufficient to carry out
any further divisions resulting as they would in still further enhanced
values of the residual charges.

It is no essential part of the theory here put forward that there should —

be one, and only one, division of the somatic type after the meiotic or
reducing division. As we pointed out above, when this state of things
occurs it would mean that the available energy is not sufficient to carry
out another division—the limit has been reached. If, however, the
stresses set up by the two maturation divisions were smaller in proportion
to the driving force then the limit would not necessarily be reached at

1, Boveri, ‘ Befruchtung,’ Merkel und Bonnet’s Ergebnisse, 1891.

THE ELECTRICAL FORCES OF MITOSIS 213

a » end of the post-meiotic division, and thus further divisions would
ensue until the-limit is reached. The number of divisions following the
eins division should be proportional to the ratio of the driving power

to the stresses set up in the reducing division.
In advancing our theory of the electrical forces of mitosis we have
discussed the phenomena as present themselves in the higher animals
wherein the sex characteristics are as marked and as differentiated as
possible. Clearly we should find a completely graduated scale down from
the animals with complete sex differentiation to the lowest organism
_ where no sex differentiation exists at all. Following on the lines laid
down in this paper we would say that the complete establishment of sex
ferentiation means the establishment of certain definite electric charges
om germ cells during maturation, and that the magnitude of these
yes is such that only one post-meiotic division can occur. If the
sex characteristics are not so strongly developed then the charges
Eiblished ; in maturation will not be so great in relation to the driving
force, with the result that more divisions will occur before the limit is

ery

_ ~=We may form some idea of the evolution of sexual differences from
_ the standpoint of the electrical theory without any assumption beyond
those already made. The essential point is, of course, the first inception
of the reducing division, why it first occurred and why the fusion of. two
cells carrying the reduced number of chromosomes takes place. If we
consider a single living cell such as that of a unicellular organism with
no very well defined characteristics (using the word in the hereditary
sense), it is natural to imagine that this organism can reproduce itself by
____ ordinary mitosis without any of the sexual phases. When, however, in
_ the process of evolution more characteristics are acquired we at once
expect a break in the continuity of reproduction. When an organism
possesses characteristics of opposite polarity it is an absolute necessity that
_ @ reducing division shall sooner or later take place.
7 As long as the acquired characteristics are of the same type an
organism can reproduce itself mitotically without difficulty, but when the
chromatin granules or chromosomes carrying the characteristics show
opposite polarity, a reducing division must occur, because sooner or later
the difference of polarity between the chromosomes will reach a value
sufficiently large to cause the fusion of them together to form the bi-polar
twin. This will be followed by the so-called reducing division. Now

214 BIO-CHEMICAL JOURNAL

this division sets up the residual charges, however small they may be, and
these will go on slowly mounting up in the following divisions until they
are of sufficient size to cause the fusion or union of two cells (of opposite
charge) with the restoration of the original number of chromosomes.

During the process of evolution, as the acquired characteristics

become more numerous, they will by virtue of their different polarity be
segregated more and more on the one side and the other until finally we
have two different individuals—the male and female. .

It is interesting to follow out the cell divisions as might be expat

to occur in cases where the sex differentiation is small. We may consider
an organism with 2n chromosomes which carry characteristics of not

very marked opposite polarity. The cells will go on dividing normally —

until the opposite polarity is sufficiently great to cause the fusion of the
chromosomes with formation of the bi-polar gemini followed by the
reducing division. The new cells will now have n chromosomes, “and
these will go on dividing with a balance of charge left at each division.
If now the balance of charge left in each daughter cell at each division
for purposes of argument be put at }+ and }— respectively, then the
following values will be obtained :—

OORIGINAL CELL ( 2N chromosomes)
Reaucing Division

Sto kn—(N Chromosomes
/+ ° QWeulra/ Neutral ; 5 Jan
/5* R4* 5 J 4= tg . e 4> CO 4> rs Em (
CJ U o <. J 7 *® ) + ° se ) a + * , e
4+ /+ % /+ e 54 4- ft ae Te Tiny. Tu /- /- 2
& ee & a Sw ss é ;

As can be seen from the diagram, there will be, after the third post-
meiotic division, sixteen cells, each carrying x chromosomes, and of these
six will be neutral, four will have a unit positive charge, four a unit

negative charge, one 2 units positive and one 2 units negative electricity —

a | OR eee
bs Je, eee A sr

THE ELECTRICAL FORCES OF MITOSIS 215

‘respectively.! If the distribution of charges be followed out for the
ensuing divisions, it will be found that two of the cells formed always
carry equal maxima of positive and negative electricity respectively, and
there will continually be produced a constant ratio of neutral cells and cells
with intermediate values of charge. Sooner or later the magnitude of the
charges produced will cause the maximum charged cells to fuse together
_ with the formation of new cells carrying 2n chromosomes, when, of course,
the cycle begins again. The organism, therefore, will present the
_ appearance of having two types of cell, one carrying 2 and the other
chromosomes. Some of the latter will from time to time fuse together
to form new cells with 2n chormosomes, and so the cycle is complete.
_ This state of affairs will be the natural result of the sex differentiation
being very incomplete, or, in other words, when the hereditary character-
lies have not developed the maximum possible difference in polarity.
- When this occurs we find the condition that, owing to the magnitude of
the charges involved, only one post-meiotic division takes place, the cell
_ being at once sufficiently charged for fusion or fertilisation by one of
a opposite type. The whole difference between the various types of re-
_ production may therefore be summed up in the conception of. sex
_ differentiation, or of opposite polarity in the hereditary characteristics.
It is also of some importance to note that as sex differentiation is
increased the relative size of egg to sperm is increased. When the sex
differentiation is small, or as we might say merely embryonic, there will
be no difference in size or visible character of male and female cells.
_ When the male and female chromatin begin to differ materially then we
find a difference in the development of the two, the male becoming smaller
than the female, until finally we arrive at the spermatozoon and ovum,
_ where the ratio of size is very marked indeed.
In the development of the above argument we have attributed the
variation in the number of post-meiotic divisions, i.e., divisions between
the reduction and the ensuing fusion, to the ratio between the driving
force and residual charges established; the greater this is, the greater the
number of post-meiotic divisions. We have introduced the conception of
driving force merely to illustrate our point, and would now deal with this
conception in more detail and consider the relation of the chromatin to
the surrounding nucleic acid. There is no doubt that during the resting
period between two successive divisions the chromatin must be growing

- 1. Exactly the same relative values are obtained, whatever be the size of the residual

216 BIO-CHEMICAL JOURNAL ~

by means of chemical reactions between itself and the surrounding nucleic
acid. Indeed, we may go so far as to say that unless this growth occurs
any somatic division is not possible, for such division would result in a
decrease in the active mass of chromatin in the daughter cells, a
consequence impossible to accept. Now from a physico-chemical point of
view it is evident that any reaction between chromatin and nucleic acid
must be based upon some essential points of similarity in structure between
the two, and it would seem a natural deduction to make from this stand-
point that any influence tending to decrease the similarity between the
two would retard the growth of the chromatin at the expense of the

nucleic acid. Hence it is quite a natural sequence that a decrease in the

similarity would tend to act as a deterrent to mitosis. The establishment
of the residual charges in the maturation divisions, of course, means a
considerable modification in the chromatin of the daughter cells, i.e., a
considerable decrease in similarity between the chromatin and nucleic
acid, so that we finally arrive at the conclusion that the establishment
of the residual charges is a direct deterrent to mitosis. Hence the failure
in the animal kingdom of the daughter cells of the post-meiotie division
to undergo further divisions may be explained at once by the fact that
the residual charges prevent the growth of the resulting chromatin, so
that no reason or scope for division exists.

We arrive, therefore, in this way at a very definite foundation for the
assumption made previously that the number of post-meiotic divisions
depends upon the ratio of driving force to amount of residual charges
established, for we find that the driving force is essentially determined

by the growth of the chromatin during the so-called resting period. The —

influence of the amount of differentiation between male and female
characteristics upon the number of post-meiotic divisions follows very
clearly from this view, for, as several times pointed out, the amount of
residual charge in the meiotic division depends directly upon this

differentiation, or, as we would now state the law: The greater the ‘

number of different hereditary characteristics of opposite type the greater
is the dissimilarity between the chromatin and nucleic acid in the grand-
daughter cells of the reducing divisions, and hence the fewer the number
of post-meiotie divisions.

It may be stated here, parenthetically, that whatever view be taken of
the method by which the chromatin grows or the period at which the
vrowth occurs, the same conclusion is arrived at: that if the growth be
stopped then mitosis cannot occur.

THE ELECTRICAL FORCES OF MITOSIS 217

a It is impossible to enter fully into the various types of reproduction
which are known to oceur, especially in the vegetable kingdom, but there
seems no doubt that they all are capable of explanation on the theory here
put forward. We have dealt with the ease of the unicellular organism
with no sex characteristics, those cases in the vegetable kingdom where
incomplete sex differentiation exists, and the zoological cases where the
sex differentiation is complete with the two sexes as distinct as possible.
All other cases seem only to be intermediate between the first and last.
A very important corollary must be added to what has been said
above upon the reducing divisions. At the completion of this division
there is established a definite amount of residual charge in the daughter
-eells. In other words, a certain amount of energy is stored in the cells,
and this must result in the vitality of the cell being increased. This fact
____ is very important, for it would seem that the reducing division forms a
-_ means by which the vitality or activity of the cells is renewed, for there is
“no doubt that a considerable amount of energy is absorbed at the time.
_ _ Hitherto we have made the tacit assumption that the phenomena
_ described are those occurring in the mitosis of normal healthy cells. The
_ question now arises as to what would happen if the electric charges in the
somatic cells were disturbed by some means, and the equilibrium between
them upset. It is very evident that important changes in the phenomena
might take place, and it has occurred to us that the various types of
malignant growth might very readily be explicable by their being due to
the derangement of the electrical forces present during mitosis. On
studying the experimental results obtained in this field we were very
_ foreibly struck by the support given to our idea, and we feel more than
BE justified in offering this as a reasonable explanation of malignant growths,
_viz., that they are due to abnormal cell reproduction arising from a
disturbance of the electrical equilibrium of mitosis.
_ - One of the simplest methods of causing a disturbance of the electrical
equilibrium in the cell is by the external application of an electrical
stimulus. . If, for example, a somatic cell was given an added charge,
ie., if the cell wall by some means were electrified, a natural sequence
would be an artificially produced multi-polar mitosis as already described
in the fertilisation experiments of Boveri and Loeb above. Furthermore,
the daughter cells produced as a result of such mitosis will naturally
have a balance of positive or negative electricity left in them, owing to
the result of the asymmetric distribution of the chromosomes. Since
there is no means of dissipating these residual charges, the effect of a

218 BLO-CHEMICAL JOURNAL

single initial external stimulus will be handed on from generation to
generation of daughter cells. There is no doubt that the periodic
activity of the cytoplasm resulting in its mass division would not be
interfered with by the small external stimuli referred to. The mechanism

of the cell mitosis would be the same in the main, but owing to the excess

of electrical energy multi-polar and asymmetric mitoses must result.

Now, multi-polar and asymmetric mitoses are frequently observed
in malignant growths, and, indeed, Galeotti has artificially produced
asymmetric mitoses in Salamander cells by treating them with certain
chemical substances.!| These substances undoubtedly acted as a stimulant
to the cell either by electrification of the cell walls by virtue of the
different velocities of the ions, as already shown in the case of Loeb’s
experiments, or by more purely chemical means. That the action of an
external stimulus is capable of producing these two pathological mitoses
in cancer, by which term we understand all malignant neoplasms, is thus
evident. Waller? and others have shown that electrostatic differences of
potertial are a normal result of any external stimulus being applied to
healthy tissue, and whether we accept Loeb’s* explanation or not that the
effect is due to the migration of hydrogen ions, still the fact of sufficient
potential difference being set up to deflect a galvanometer is completely
established. It is possible, therefore, that an external stimulus, as for an_
example a bruise or blow, could induce sufficient electricity to derange all
the neighbouring cells, that is to say, a sufficient electric stimulus could
be established to start pathological mitoses. It would thus appear that,
provided the necessary conditions were existent, a blow or bruise could
give rise to a malignant growth. It stands to reason that the more
healthy is a cell and the stronger its vitality, the greater will be its .
resisting power against the effect of an external stimulus. Conversely,
the lower the vitality of the cell the more liable it becomes to derangement.
There is no doubt that, speaking generally, the vitality of cells must
decrease with their age, so we would expect the tendency to malignant _ :
growths to increase with age; in other words, we have herein a direct
explanation of the age incidence of cancer. The actual change which
takes place, and which we have spoken of as a decrease in vitality, would
be due partly to a decrease in the active growth of the cytoplasm and
partly to a decrease in the active growth of the vital units or chromatin

1. Beib. 3, Path. Anat., XIV, 2 (1893).

2. Waller, Signs of Life, p. 143.
3. Loeb, loc. cit., p. 68.

THE ELECTRICAL FORCES OF MITOSIS 219

_ granules. Whereas in a healthy cell the mitotic phenomena are due to
forces which-are periodically brought into play, so a decrease in these
73 forces will tend to weaken the vitality of the cell. We have shown, also,
that the possibility of mitosis is dependent upon the growth of the
- __ ¢hromatin, and as this certainly will decrease with the age and differentia-
___ tion of the cell, so the vitality of tlie cell will decrease with age. We
____ therefore have two causes for the decrease in the vitality of a cell.
a It is not possible in the present state of our knowledge to advance
any definite chemical theory for the change which is developed with age,
= but it is fairly certain from cytological investigations that a stage is
reached when the cytoplasmic power is not sufficient to resolve the centro-
somes and chromosomes with the formation of the mitotic figures, and
en amitotic division may and does frequently occur. It may, however,
e concluded that the occurrence of amitosis is due to the fact that the
active growth of the chromatin has fallen to a value below the limiting
value for mitotic resolution of the chromatin granules while the
cytoplasmic activity is sufficiently great to cause the mass division to take
place. We have already directly connected the decrease in vitality of
the cell with the occurrence of cancer, so the ocurrence of the amitotic
divisions of cancer cells is easy to understand. As already pointed out,
the age incidence of malignant growths is the natural sequence of the fact
that the somatic cells must reach a certain critical minimum of vitality
before they can be disturbed by any external stimulus. We may for the
; present purpose give the name of the nth generation to that generation
at which the cells reach the critical minimum, i.e., when their vitality has
fallen low enough to be susceptible of disturbance by the external
stimulus. It is of some importance at this point to notice that when any
tissue is subject to continually repeated abrasion or irritation, the
resulting continuous renewal of tissue will cause the nth generation to be
arrived at somewhat earlier than would otherwise be the case. From
_ Waller's experiments it is a natural sequence that any irritation or stress
should tend to produce an electrical difference of potential. This would
seem at once to account for the occurrence of malignant growths in those
parts of the body which are subject to such stresses.

Hitherto we have been considering the derangement of a cell by
external stimulus, but it is also possible that derangement may occur
internally.

If we consider the case of a cell which is highly differentiated, and,
moreover, one the vitality of which has sunk very low, it is evident on

Re oe ae - a at es a a Le ee a,

220 BIO-CHEMICAL JOURNAL

first principles that the potential difference, actually set up when the vital —
units are resolved, has fallen to a very small value. This decrease in—
activity, sooner or later, as we have before pointed out, results in amitotie —
division. This oceurrence of amitosis, however, demands. that all the =
chromatin granules nust be at a low ebb of vitality. The formation of | 2
the loose compounds between the nucleic acid and the chromatin granules
must, to all intents and purposes, have ceased, or, in other words, the
growth of the vital units has become very slow. It is not essentially
necessary, however, that every single chromatin granule of the cell
decrease in vitality at the same rate, and the question arises as to what
would happen if the vitality of the various granules differ considerably
within a single cell. If we consider for a moment the effect of inbreeding
and hybridisation, it is perfectly evident that by far the most probable —
condition is the one specified, namely, a considerable variation in vitality
among the granules of chromatin within each cell. Clearly this gradation
of vitality among the chromatin granules can give rise to a derangement
of the electrical equilibrium in mitosis, as can be seen from the following.
Whereas in the normal mitosis of a healthy somatic cell the chromosomes
give daughter chromosomes of equal and opposite charge, and as in true
amitosis the chromosomes are not resolved at all, in the present case a
condition may arise when some of the chromosomes are resolved while
the remainder are not so resolved, owing to the vitality of the granules of
the latter being very much lower than that of the granules of the former.
These latter chromosomes would only be polarised, and therefore would
not migrate to either centrosome; a fusion of the electrified daughter
chromosomes with the polarised chromosomes would naturally ensue, and
an asymmetrical mitosis would take place, with a reduction in the number
of chromosomes. This would again cause a balance of residual electric
charge in each daughter cell, exactly in the same way as occurred in the
case of the external stimulus described above.

Further, it must be pointed out that the age incidence would apply
to this condition just as much as to an ordinary cell, for the derangement
is caused by the vitality of the weakest granules falling below the
minimum value and also by a general decrease in the activity, both being
the normal results of the age of the cell. To assume for the moment that
this condition of chromatin granules is possible, we may now re-state our
case as it stands. An electrical theory of mitosis at once renders possible
abnormal mitosis by reason of the electrical charges becoming disturbed.
The electrical theory of mitosis in itself receives very considerable support

tre 9 8 8 ee ee
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_
-

—

THE ELECTRICAL FORCES OF MITOSIS 221

‘from experiments upon artificial fertilisation, from parthenogenesis, from
sterilisation of the ova and spermatozoa by X-rays, etc. The disturbance
of the balance of electrical quantities in the cell may arise from the
_ application of an external electrical stimulus arising from a blow or from
- considerable and repeated irritation or stress, It may also arise internally
in the cell from a certain number of the chromatin granules being of
_ such a character that they do not give daughter granules with sufficiently
different charges. In both cases the result is the same. The normal
equilibrium between ihe charges is disturbed and asymmetric and multi-
polar mitoses occur, producing daughter cells with a reduced number of
chromosomes and a residual positive and negative electrical charge. The
tendency to both these disturbances is increased with the age of the cell.
* i: » The main point we have arrived at is that when derangement of the
¢ occurs, the daughter cells are produced with a balance of positive
‘negative electricity and that this is accompanied by a reduction in the
number of chromosomes. It is, therefore, extremely interesting that
Farmer, Moore and Walker! claim to have discovered that a true reducing
division occurs in malignant growths; that is to say, a division of the
same type as the first maturation division of the germ cells. Although
‘these results are still sub judice, yet the existence of cells with: fewer
chromosomes than the normal somatic number seems faily well established.
The condition of chromatin granules such as we have postulated would
give rise to cells containing any number of chromosomes between the
somatic number and half that number, and would not necessitate a true
reducing division. For this reason we would give the name of pseudo-
reduction to this process.

As was pointed out before, in dealing with the reducing division in
the vegetable kingdom (that is to say, the cases where the sex differen-
tiation is incomplete) the operation leads to an increase in the vitality
of the daughter cells. When, owing to the decrease in the activity of a
cell, the pseudo-reducing division occurs, the resulting daughter cells
carry a certain amount of residual charges, that is to say, a definite amount
of fresh energy has been absorbed by the chromatin. This clearly will
enhance the activity or vitality of the cells considered as a whole, and
therefore we are met with the condition of a new growth of cells of
greatly increased vitality in a tissue of cells of low vitality. It stands
to reason that the new cells will multiply with great rapidity and will be
out.of co-ordination with the soma. Their growth will depend entirely

1. Proc. Roy. Soe., Vol. LXXIT (1903).

222 BIO-CHEMICAL JOURNAL

upon the new lease of vitality which they have received from the reducing %.
division, that is to say, it will be inversely proportional to the age sce ~~
cells before the derangement.
The course followed by these new cells should be very femdi on ‘aie
same lines as that given in detail upon page 214 for the cells obtained: “by "
the true reducing division in the ease of an organism with small sex
differentiation. The descendants of the pseudo-reducing division of
cancer will produce a certain number of neutral cells, a few wile
charged cells both with positive and negative electricity, and the — é
remainder with intermediate charges. It is impossible to assess the
relative number of the three types, since the original reducing division —
was not a true reducing one, but only a pseudo one with an indefinite
reduction. The maximum charged cells will soon reach the limit, which
cannot be passed owing to the magnitude of the charges involved, while
the remainder sub-divide indefinitely. When the maximum charged cells
have reached the limit the question at once arises as to their future
behaviour. The normal course would be for them to fuse together in
pairs of opposite charges, with production of new cells with an increased =~
number of chromosomes. A new generation of highly active cells would
thus be: formed, and thus the cycle would be complete, just as in the —
botanical case detailed before. It must also be remembered that under —
the peculiar circumstances owing to the reaction of the soma there isa
supply of leucocytes continuously made. These leucocytes being neutral
and active cells would be expected to conjugate with the maximum —
charged ‘reduced’ cancer cells, since the electric potential would in this
way be reduced. Now Farmer, Moore and Walker! have observed such —
conjugation between leucocytes and cancer cells—an observation which
strikingly confirms the theory. No doubt conjugation also occurs with
the cells of the surrounding tissue, to which may partly be due the
infiltration of the malignant growths. .
This seems to us to account fully for the cyclical form of growth of |
cancer in mice as demonstrated by the Imperial Cancer Research (No. 2,
Pt. 2, 1905), but the space at our disposal prevents us from producing .
further proofs for our contention. tel,
An important point arises here in relation to the age and differen-
tiation of the cell. It would be readily understood that the greater the
vitality of any cell, the greater will be its activity in proliferation when
the malignant diathesis has once been established. If we suppose, for

1. Proc. Roy. Soc., Vol. LXXII, 1903.

THE ELECTRICAL FORCES OF MITOSIS 223

a ple, that either owing to the abnormal condition of the chromatin
the presence of an external electrical stimulus, the electrical
equilibrium is disturbed when the cytoplasmic activity is still very
considerable, it will be evident that the rate of proliferation will be
greater than would be the case had the cytoplasmic activity been less.
In other words, the greater the amount of the activity the more rapid
the growth of the tumour, the more highly differentiated the cell and
the older it is before the taint is established, the less rapid will be the
owth of the neoplasm. As the age of the host is increased, therefore,
ss potent becomes the taint. If the development and differentiation
the cell has proceeded sufficiently far before the cancerous diathesis is
stablished, the smaller will be the potential differences established by the
eduction in the number of chromosomes. The tumour then loses its
mancy to a certain extent, and a cancer of slow growth, such as an
ic scirrhus, is established. There can be no doubt that the growth
of the tumour by reduction in the number of chromosomes produces, as
before stated, daughter cells which are out of somatic co-ordination with
he host, and the tumour grows parasitically, feeding upon its host.
Resulting from this condition of parasitic growth of cells out of somatic
_ ¢o-ordination, the wandering of some of the active cells from the seat of
the tumour may occur. When one of these charged cells comes to rest,
it possesses potential probability of reproducing itself, resulting in the
growth of a secondary tumour of the same type as the primary one. The
tendency to the formation of secondary growths must, therefore, depend
_ directly upon the activity of the cells when the taint is first established,
because, as before said, the greater the activity the greater the activity
of the daughter cells of the pseudo-reducing division.
_ With reference to this subject we are at once struck with the
narkable facts which have accumulated during recent years with regard
ie transplantation of cancerous tissue. As the Imperial Cancer
Research have indicated, it is much easier to transmit any malignant
“E “growth from a mouse of one locality to a mouse of the same locality than
to a mouse of another locality; moreover, there is a general concensus
of opinion that cancer can in no way be transmitted from an animal of one
species to an animal of another. This is the natural outcome of our view
on the cytoplasmic activity.
_ For the more different the species of the animals in question, the
more dissimilar would be their respective cytoplasms; and at a certain
limit of difference, conjugation either with leucocytes or normal tissue

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224 BIO-CHEMICAL JOURNAL ©

cells would be impossible, and the engrafted tumour would not be able
to survive the reaction of the connective tissue stroma.

In order that conjugation may take place between a charged nucleus
and a neutral nucleus or between two oppositely charged nuclei, it is

essential that the limiting surfaces of the two masses of cytoplasm be

eliminated at any rate during the process. This elimination of the surface
layer can only occur if the two cytoplasmic masses be of specifically the
same chemical nature. If, however, as a result of the two individuals
having lived in different localities or having been fed with different food,
their cytoplasmic material be of different chemical character, conjugation
between the nuclei will be impossible owing to the difficulty of ‘ae
the limiting layers of the cytoplasm of the adjacent cells.

The malignancy of new growths varies generally indirectly with the
age of the host. As a corollary to this it may be added that the condition
of the chromatin granules may be such that the electrical disturbance
occur at a very early age, e.g., in the embryo. The occurrence of sarcoma
in utero is doubtless due to this condition, and such cases should be, as
they undoubtedly are, exceptionally malignant.

We have hitherto confined ourselves to the mere statement
that the existence of chromatin granules of such a type that the daughter
particles produced in mitosis have a very small difference of charge will
tend to cause electrical disturbances, such as seem to occur in cancet.
We may now turn our attention to the investigation of this possible
condition, for if this possibility be established it will afford an explana-
tion of the true origin of cancer. In any healthy race with no close
inter-marriages, it would appear from first principles that the chromatin
granules would be perfectly normal, if healthy female ova are fertilised
with spermatozoa of a different type; that is to say, as long as father
and mother are sufficiently differentiated and both healthy, the chromo-
somes of their children and the chromatin granules of their descendants
will be normal. If, on the other hand, close inbreeding takes place

through several generations, then the chromatin granules will tend to

become more and more uniform, so that they give in mitosis daughter
particles of smaller and smaller potential difference. The tendency to
abnormal mitosis is thereby progressively increased until a stage is
reached when the incidence of the malignant growth is markedly
intensified.

Up to the present we have not taken into consideration the chemical

side of the problem, and although the action of certain chemical

2 nM).

THE ELECTRICAL FORCES OF MITOSIS 225

4 ‘substances has been instanced, as, for example, the production of
asymmetric mitoses by antipyrine as observed by Galeotti, the influence
of the metabolic products of the cells has not been discussed. We have
dealt with the possibility of electrical stimulus and the disorganisation of
cells by means of a bruise or blow, and it is a natural sequence of the
electrical theory that disorganisation could be produced by a purely
chemical stimulus apart from that arising electrically by the different
velocities of two ions. Owing to the peculiar configuration of the protein
molecule it must be sensitive to both bases and acids, and the influence of
these two reactions seems of considerable importance.
_ As far as is known at the present time the normal cells seem to be
strongly influenced by both bases and acids, and the influence of the
former seems to be one of stimulation, while acids seem to produce the
opposite effect.! For example the well-known case of a nerve fibre may
be quoted: if two needles be inserted into the fibre andone be connected
with the copper of a Daniel cell and the other to the zinc, the nerve cells
are stimulated around the latter needle and depressed near the former.
It is evident that around the latter there is an excess of alkalinity and
an excess of acidity around the former. Whiile several experiments giving
similar results might be quoted, the above shows clearly that cells are
stimulated by alkali. From a physico-chemical point of view this action
of alkali is explicable if the tautomerism or dynamic isomerism of the
protein molecule be considered. Recent work has shown that there is a
distinct and definite tendency on the part of a - NH - CO - grouping not to

exist in either of the two possible desmotropic forms - NH - C - and- N =C -

I
a ti 0 OH
but rather as a mixture of the two in dynamic equilibrium with
one another. Moreover, the chemical activity of this group as concerns
either the nitrogen atom or CO group is determined entirely by this
_ dynamic condition, for in a great number of cases it has been proved
that a molecule in a static quiescence is singularly inactive to all
| chemical reagents. In order that a molecule should be reactive it is
+ necessary that a certain amount of dynamic oscillation between the
t residual affinities be present, and the reactivity is proportional to the
amount of dynamic isomerism which exists. In the case of the - NH - CO -
grouping in question the phenomenon seems to be connected with the

"1. Moor) Roaf, and Whitley, Proc. Roy. Soc., B. 77, p. 102, ete., 1905, Moore and
Wilson, Bio-Chemical Journal, Vol. 1, p. 297, 1906. Moore, Roaf, and Knowles, thid,,
Vol. LI, p. 279, 1908.

226 . BIO-CHEMICAL JOURNAL

wandering of the hydrogen atom from the nitrogen to the oxygen. Now
in all such cases the dynamic isomerism is increased by the addition of
alkali and decreased by the addition of acid. Since the chemical
reactivity of a substance depends upon the amount of dynamic isomerism
present, so the reactivity of protein must be enhanced by the addition of
alkali and depressed by the addition of acid. The activity of a eell —
must essentially be determined by the chemical reactivity of its
components, so it is a natural sequence that alkali will stimulate and
acid depress the normal functions of a cell. If we apply this
argument to the special case already considered of a somatic cell whose
chromatin granules vary very much in activity, it leads to an interesting
conclusion. It was previously shown that owing to the potential gradient
arising from the variation in the activity of the chromf’tin, a pseudo-
reducing division can occur with production of cells possessing
enhanced activity, or, in other words, a new growth is started. The
application of alkali to a cell of the above type will stimulate the activity
of all the granules present, and will consequently increase the potential
gradient in the chromatin. The application of alkali would tend, there-
fore, to increase the probability of the pseudo-reducing division, that is to
say alkali would tend to act as a direct cancer irritant when the necessary
conditions are present, the necessary conditions being the existence of a _
gradation in the vitality or activity of the chromatin granules. ;

The term alkali has been used in the broad sense of any baile
substance, and there seems no reason to doubt the direct connection
between cancerous growth and irritation by basic substance. Two of the
best known examples need only be quoted, namely, chimney-sweep’s
cancer, where the soot is the irritant, and, further, the undoubted
connection between tobacco smoke and cancer of the lip and tongue.
Both soot and the distillation products of tobacco are strongly alkaline
substances, since they contain nitrogenous bases.

When once the new growth has started, the metabolism of the new
cells comes into action, and it is not improbable that a chemical stimulus
may arise from the hydrolytic and degradation products therefrom. It is
conceivable that these products may themselves act upon the surrounding
tissue cells and cause them to undergo the pseudo-reducing division. For
this reason we do not wish to restrict ourselves to the statement that
infiltration is due entirely to conjugation of the maximum charged cells
with neutral tissue cells. The chemical stimulus arising from the
hydrolytic and degradation products of the cancer cell métabolism can,

THE ELECTRICAL FORCES OF MITOSIS 227

and doubtless does, disorganise the surrounding tissue cells, causing them
to undergo-the pseudo-reducing division, thus infecting them with the
cancer taint,

The variations in the type of initial stimulus account readily enough
for the various types of cancer which are known to arise in the same type
of tissue. While it should not be possible from a given stimulus to

_ develop more than one type of cancer cell, yet the possibility is by no
means precluded of producing a new type of cancer in experimental
transplantation. If a tumour be ingrafted on to a new host and if the

new host were closely similar in every way fo the first host, the new
tumour would grow and infiltrate without great difficulty. A slight
- difference: between the two hosts would tend to increase the resistance of

te _ the second to the ingrafted tumour, with the result it would become

a
3
a
‘

eneapsuled. On the other hand, it should be possible to produce a new
. infection by virtue of the chemical stimulus arising from the
degradation products of the cell metabolism of the ingrafted tumour.
‘This new infection might give rise to a cancer of the same type as the
_ original tumour, but it might also, if slightly differently differentiated
cells were affected by the chemical stimulus, give rise to a different type
of new growth altogether.
Although the connection between cancer incidence, inbreeding, and
hybridisation follows quite naturally from the theory of cytological
processes advanced above, yet we feel the importance of entering more
fully into the detail of a subject which, taken as a whole, would seem to
open up a new field of research, namely Mendelism and environment as
explained by electro-cytology. To turn to the major factor of the
7 equation, in Mendelism we see the synthetical links which bind together
____ the variations in chromatin distribution with racial index of cancer
incidence. We have already pointed out that one of the fertile sources
of cancer lies in the existence in the chromosomes of an individual of a
certain number of chromatin granules of a poor or weakly type which
tend to cause fusion of the chromosomes in mitosis with the production
of daughter cells with a reduced number of chromosomes. The effect of
this will be most marked when the segregation of these chromatin
granules into one or other of the paternal or maternal sets of chromosomes
ee oceurs. However the Mendelian segregation of these granules takes place
in the maturation division of the germ cells of the mothers, their ova
will on the average contain chromosomes possessing a definite number of
these granules. These ova, when fertilised by spermatozoa from men of

228 BIO-CHEMICAL JOURNAL

lower cancer incidence, will give rise to individuals having the same
cancer incidence as the mothers, because the cancer incidence depends
upon the presence of the weak granules in the one set (maternal) of
chromosomes. A further reason for the maintenance of a mother’s cancer
incidence in her children is the fact that the cytoplasm of their cells is
entirely derived from the maternal side. The first generation resulting
from hybridisation, therefore, must preserve the cancer incidence of the
mothers, although the fathers have a smaller incidence. The second
generation of hybridisation will, however, decrease the cancer incidence,
as can readily be seen. The cells of the first generation possess two sets of
chromosomes: the maternal with their weakly granules and the paternal
without them. When the meiotic gemini are formed and the De Vries
re-arrangement occurs the weakly granules distribute themselves, and on
the average the chromosomes formed by the splitting of the gemini will
have fewer of the weakly granules. The children produced from these
gametes with gametes of the new stock will of necessity have a lower
eancer incidence. We have, therefore, a direct connection between the
Mendelian segregation of the weaker chromatin granules and cancer
incidence, a fact which explains the different incidence in the children of
one family, and also the frequently observed skipping of a generation by
the disease.

It would appear from what has already been said that cancer as a
disease cannot be inherited—it is only the predisposition to the disease
which is inherited, and we have shown that this predisposition must be
influenced by inbreeding and hybridisation. We have emphasised the
fact that for inbreeding to have any eyil effects, it must occur throughout
more than one generation; it is the inbreeding of a stock already inbred
that will lead to deterioration.

Again, it is obvious that hybridisation in one generation cannot
prevent the diathesis being handed down to the descendants, if the
offspring of the first hybridisation be again inbred, for whether the pre-
disposition in the inbred race is dominant or recessive to the hybrid, on
again inbreeding there must be produced some pure dominants or
recessives, as the case may be; on the other hand, every successive
generation of hybridisation increases the immunity to the disease.

After consideration for some years past of the prevailing views
concerning the aetiology of cancer, we are forced to conelude that the
explanation is to be found in a study of cytology and cytopathology. We
put forward in this paper the view that all malignant growths are due

THE ELECTRICAL FORCES OF MITOSIS 229

to a derangement of the electrostatic forces normally present in somatic
mitosis, which is initiated in the first place by a definite stimulus, internal
___ or external, physical or chemical.
We have shown that the susceptibility of the cell to this derangement
will be increased with decreasing vitality, such as occurs with age and as
the result of inbreeding. Though fully cognizant of the fallacies inherent
in statistics, we venture to refer to the results of a study made by us of the
geographical distribution of cancer—results which confirm the important
role played by inbreeding, hybridisation, and racial immunity. We
propose to publish these statistics in detail elsewhere, and will at this time
only very briefly deal with the more important facts which have come to
ee t.
esse as the highest cancer incidence known is to be found in
_ Switzerland, we have paid special attention to this country, and have
made an exhaustive investigation. The data have been corrected for age
* and sex constitution of the population, and the results obtained
_ demonstrate clearly that it is in the isolated communities, which have
been created by their geographical positions and the influence of religious
antagonism, we meet with the highest cancer incidence, while in the
"Passes that have served since the time of the Romans as the highways of
invasion and commerce from Northern and Central Europe into Italy, we
meet with the lowest.
Thus the cancer incidence (54°77 per 10,000 persons living, aged 30
and over) is highest in the canton of Appenzell in Rhoden, a Catholic
canton, wherein until 1848 no Protestant or even Catholic aliens were
allowed to settle. It falls to 27°70 in the neighbouring district of Ober-
_ Rheintal, which lies in the valley of the Rhine, in the pass which leads
ay to Chur, and from thence by the Spliigen to Italy.
q Z _ In the canton of Graubunden the lowest cancer incidences are in the
“T passes, viz. :—-Bernina (23°56), Munstherthal (24°01), and Maloja (22°87).
The cantons of Ticino and Valais have a remarkably low cancer
incidence, 19°34 and 11°61 respectively. In Ticino those districts bordering
< on the St. Gothard Pass (Bellinzona 12°28 and Blenio 12°60) have the
wee lowest incidence, which gradually rises to its maximum in districts most
remote from the St. Gothard. The same occurs in Valais, and it is the
___ district of Entremont (4°11) traversed by the Great St. Bernard Pass,
which has the lowest cancer incidence for the whole of Switzerland.
‘The immunity produced by hybridisation is fully borne out by
the low cancer incidence amongst the Eurasians, as reported by Dr.

a

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sie s
=
4

a

230 BIO-CHEMICAL JOURNAL

Sutherland, of the Mayo Hospital, in the Third Report of the Cancer
Research Laboratories of Middlesex Hospital (p. 87), wherein he says :—

‘A striking fact is the small number of cases amongst
Eurasians, who make up a large proportion of the in-patients in
the Albert Victor wing of the Mayo Hospital. Only one case of
carcinoma and one abdominal growth occurred out of 790
admissions for malignant disease.’

We have also made investigations as to the local origin of cancer,
and we find, in comparing the cancer incidence in the various organs
between the sexes and different races, that wherever any organ is specially
liable to stress or excessive irritation, there is an increase in the number
of malignant growths of that organ in the sex or race under consideration.

Although exception may be taken to any conclusions which are
drawn from purely statistical data, yet it would appear that the evidence
so arrived at is overwhelmingly in support of the fact that one of the
major predisposing causes of cancer is to be found in close inbreeding.
Moreover, it would also appear from a detailed comparison of the organs
attacked, that those organs which are subjected to stresses and irritation —
are most liable to develop malignant growth. Both of these conclusions
are in close agreement with the theory of electrocytology put forward in
this paper.

CONCLUSIONS

The following conclusions are arrived at in this paper :—

1. The phenomena of somatic mitosis are readily susceptible of
explanation by a simple theory of electrostatically charged colloids.

2. The simplest possible case is that when there are present in the
cell hereditary characteristics of only one type, as exists in unicellular
organisms.

3. When hereditary characteristics of two types occur, a reducing
division is bound to occur at some period owing to the fusion of the
chromosomes of opposite type. This reducing division is the forerunner
of the maturation divisions of the more highly developed species. —

4. The reducing division establishes residual charges of electricity
in the daughter cells, the amount of charge depending upon the amount
of differentiation between the opposite types of the characteristics.

5. In the animal kingdom where the sex differentiation is complete,
the reducing division only occurs normally in the germ cells, and this

a rere eee eee, gS QS
a , bad i es

THE ELECTRICAL FORCES OF MITOSIS 231

: icin is followed by one somatic division. After this second division
F Speecarther- divisions can take place owing to the magnitude of the residual
& developed.
6. In the lower types, such as occur in the vegetable kingdom where
the sex differentiation is incomplete, the reducing division is not confined
to the germ cells, but all the cells undergo it at some period of their
‘development. The daughter cells of the reducing division give rise to an
indefinite number of cells with the reduced number of chromosomes. Of
these cells, in any one generation two have maximum residual charges
of positive and negative electricity respectively, a fixed number are
_ neutral and the remainder carry intermediate charges. The highest
ged cells fuse together with restoration of the original number of
re mosomes, thus completing the cycle.- The occurrence of the reducing
vision endows the daughter cells with renewed vitality.
fe 7. In the animal kingdom the four spermatozoa all carry different
ges, one a charge equal to that of the ovum, one an equal and opposite .
rg », while the other two have intermediate charges.
$8. The phenomenon of sex production may be attributed to these
residual charges; all the phenomena of parthenogenesis, artificial fertili-
zation and sterilization by X-rays are explained by the same theory.

_ 9. The distribution of chromatin granules, required by the De Vries
theory, is established as a necessary consequence of the maturation
division.

10. The occurrence of pathological mitoses, as the result of external

—- stimulus or internal stress, is established provided that the inherent

precancerous condition be already present.

AL. ‘The possibility of a pseudo-reducing division of somatic cells is

accounted for.

__—*12. ‘These pathological mitoses result in the establishment of residual

charges in the daughter cells similar to those of the maturation division.
18. The daughter cells of the pseudo-reducing division possess

renewed activity. They possess potential probability of conjugation with

leucocytes and normal tissue cells.

14. The direct stimulation by alkali and bases generally is found to
be a normal action, and it would seem that in certain cases alkali can act
as a direct cancer irritant.

.15. The stimulation of the surrounding tissue cells by the degradation
products of the cancer cell metabolism is possible, and to this and the

232 BLO-CHEMICAL JOURNAL

facts mentioned in 14 is attributed the formation of a neoplasm with power
of continuous growth.

1€ The susceptibility of the cell to these derangements is increamel
with decreasing vitality, such as occurs with age, and as the result of
in-breeding.

17. In close in-breeding through several generations the chromatin
granules become more and more uniform so that they give in mitosis
daughter particles of smaller and smaller potential difference, which
markedly increases the tendency to abnormal mitosis; conversely hybridi-
sation produces a maximum of cell stability and an individual with all its
Mendelian allelomorphs as differentiated as possible.

18. The rate of proliferation depends upon the activity of the
cytoplasm; the greater the activity the more rapid the growth, while the
more highly differentiated the cell and the older it is the less rapid the
rate of proliferation.

19. Age incidence, local origin, infiltration, metastases, transmission
with all its limitations, and power of continuous growth are the natural
outcome of abnormal cell proliferation induced by a disturbance of the
electrostatic forces present in normal mitosis.

| THE ESTIMATION OF PHOSPHORUS IN URINE

_ By G. C. MATHISON, M.B., B.S. (Mexs.), Sharpey Scholar.

From the Physiological Laboratory, University College, London
(Received April 9th, 1909)

_ As a preliminary to some investigations on the metabolism of
orus, an examination of some of the methods that have been.
employed for the estimation of P,O, in urine was carried out. In view
of the probable presence of organic phosphorus compounds, Neumann’s
method, as modified by Plimmer and Bayliss (1), was also*employed.

First Neumann’s method, precipitation with magnesia mixture and
‘precipitation with magnesium citrate mixture,! were compared on a
solution of K,HPO,, and were found to give results concordant within
one per cent. For Neumann’s method 10 c.c. of urine are combusted with
10 c.c. of sulphuric acid, repeated small amounts of nitric acid being
added towards the end of the combustion. The rest of the procedure is
that described by Plimmer and Bayliss.

_ For imorganie phosphates, about 4¢.c. of the magnesium citrate
mixture are added to 10 c.c. of urine, and enough ammonia to make the
mixture smell distinctly of ammonia. The mixture is well stirred and
ee let stand over night, and is then filtered through an ash free paper. The
_ precipitate is dried and ashed; P,O, is calculated from the ash, Mg,P,0,.
Since the magnesia mixture method was found unreliable, it is not
described in detail.

When the three methods mentioned were applied to urine, the highest

_-_—s values were given by Neumann’s method, while magnesia mixture gave
: higher values than magnesium citrate. Neumann’s method or magnesium

1. This reagent is prepared as follows :—Dissolve 40 grams of citric acid in 500 ¢.c, of water,
add to the hot solution 20 grams of light magnesium oxide. Cool ; add 400 c.c. of 0-880 ammonia
and water to 1500 c.c. Let stand twelve to twenty-four hours, and then filter.

234 BIO-CHEMICAL JOURNAL

citrate gave practically constant results, but those given by magnesia
mixture showed considerable variations. The P,O, in the filtrates after
precipitation with magnesia mixture or magnesium citrate was determined
by Neumann’s method.

Taste I—Comrartson or NEUMANN, MaGNestum CirraTE AND MAGNESIA

Mixture MrtTuops

Grams P,O, in 100 c.c. Urine

Magnesium Citrate Magnesia Mixture
Neumann Ppt. Filt. Ppt. o Filt.

I (0-153 (0-144 —_ (0-151 {0-005
3 (0-156 (0-143 0-011 (0-156 (0-004
ae ( 0-223 0-013 7+ _

I (0-224 (0-204 (0-017 {0-228 (0-006
¥* (0-230 {0-207 (0-019 (0-242 (0-012
IV (0-160 {0-153 0-016 (0-160 {0-004
me ( 0-166 (0-151 — ( 0-164 (0-007

The sum of the magnesium citrate precipitate and filtrate values
corresponds fairly with the Neumann value; but the sum of the magnesia
mixture precipitate and filtrate values is considerably higher. This
discrepancy will later be shown to be due to errors inherent in the
magnesia mixture method.

The magnesium citrate results are very constant, even when quantities
varying from 2c.c. to 10 c¢.c. of the solution are used to 10 c.c. of urine.
The precipitate is insoluble in 1: 3 ammonia, for the results are not
affected by prolonged and frequent washing.

To obtain evidence of complete precipitation of phosphates, Scott’s
reagent (2), which is capable of detecting 0°005 milligram inorganic P,O,,
was applied to the filtrate after separation of the citrate precipitate. No
reaction was obtained. It was found, however, that in the presence of
citrates, Scott’s reagent was far from delicate, so that the desired evidence
had to be obtained in a less direct way, which will be described later.

The non-agreement between magnesium citrate and magnesia mixture
values required some explanation. Neumann’s method, applied to the
ash from citrate precipitate, showed the theoretical amount of P,O, to be
present; applied to the ash from magnesia mixture precipitate, it showes
less PO, than the theoretical, the amount being variable.

THE ESTIMATION OF PHOSPHORUS IN URINE — 235

p TI—Estneation GRAVIMETRICALLY AND BY NeuMann’s MEeruop oF

SAREE

y Grams P,O; in 100 e.c. Urine
Megeeses. Oyen Citrate Magnesia Mixture

et ee,

Gravimetric Neumann Gravimetric Neumann

I 0-151 0-149 0-160 0-154
u 0-144 0-143 0-153 0-148
at (0-207 - — 50-228 {0-217
"(0-204 0-204 (0-224 — (0211

IV 0-194 oo 0-270 0-211
vi 0-226 0-222 0-235 0-227

_ If the magnesia mixture ash is dissolved in acetic acid, the addition

of potassium oxalate shows the presence of calcium, which accounts for
art of the error.

_ By adding magnesia mixture to the filtrate after precipitation with

gnesium citrate, a further precipitate is obtained. This contains a

le calcium, and also some phosphorus. The P,O, was estimated in

sh gravimetrically and by Neumann’s method.

: I1l—Torat P,O,, Ivoreantc P,O, ann PO, PRESENT IN THE
*AppitionaAL’ MaGnesta Mixtcre PRrecrprrate
Grams P,O, in 100 c.c. Urine

‘ Additional ’ Ppt. with
| wi aceon Oe PN
Total Inorganic Gravimetric Neumann by difference
. (0-224 | 0-204 (0-025 (0-016 0-003
(0-230 (0-207 (0-020 (0-012 0-011
ir 0-220 (0-194 0025 0018 0-008
0-218 (0194 is ot
snilgey 0-244 —- (0-226 (0-028 (0-010 (0-008
0-241 0-222 (0-024 (0-011 (0-009
IV... 0222 «=—-0-208 0-014 0-006 loo1s

This additional precipitation of P-containing substances suggested
that magnesium citrate had failed to precipitate all the inorganic
phosphates. Seott’s reagent was applied to about 50 milligrams of the
additional precipitate, and failed to give any reaction. Thus evidence
was afforded of the complete precipitation of inorganic phosphates by
magnesium citrate. The inaccuracy of the magnesia mixture method is
thus due to two factors, first, that some calcium is precipitated; second,
that some of the organie phosphorus is precipitated.

236 BIO-CHEMICAL JOURNAL

Ammoniacal solutions of barium chloride and caleium chloride have
been used to precipitate phosphates, Since these reagents also precipitate
sulphates, they are not convenient for quantitative estimations in urine.
But in the filtrate from these the organic phosphorus of urine can readily
be determined by Neumann’s method. Indeed, this direct determination
is much more readily performed in the barium chloride filtrate than in
that from magnesium citrate, since the combustion of the latter is a
matter of considerable difficulty.

Taste [V—Comparison or Orcanic PO, Vatves py Dirrerent METHODS
Grams P,O; in 100 c.c. Urine
Tote!’ Tnorgaai ——

(etidaind (Citrate) Total, minus _Citrate BaCl,

Inorganic Filtrate Filtrate

ee 0-222 0-020 0-018 0-016

fn (a) oe 0-148 0-013 0-011 0-011

Il... = 142 0-127 0-015 0-012 0-013

IV... 0180 0-170 0-009 0-009 0-011

V 0-196 0-183 0-010 0-015 0-011

The ies Bet, agreement of these methods affords further oxide
of the accuracy of the citrate method for inorganic phosphates. The
difference between Neumann and magnesium citrate values represents
the amount of phosphorus present in organic combination.

The uranium acetate method was found useless for accurate work.

Répiton (3) has shown that uranium acetate solutions must be
standardized for the particular phosphate which is being estimated.
Thus the method is inaccurate in a solution of mixed phosphates.

Taste V—Comparison oF NEuMANN, MaGnesitum CITRATE AND URANIUM
AceTATE! VALUES

Tincture of Cochineal as a warning and Potassium Ferrocyanide as final indicator.
Grams P,O, in 100 c.c. Urine

Neumann Mag. Cit. | Uran. Acet,
I {0-229 -- {0-213
| 0-233 0-205 (0-217
I 0-201 0-187 {Oro
ie 0-166 0-156 0-166
IV 0-204 0-194 {Cite

ze — 0-

y (0-196 0-183 (0-192
ett (0-198 _ (0-186
Wis bax 0-154 0-142 0-160

1. This uranium acetate was standardized against the K,HPO, solution originally employed

to test the different methods.

THE ESTIMATION OF PHOSPHORUS IN URINE — 287

The uranium acetate values are usually somewhere between the total
and the inorganic values, but may be above the total. Any attempts to

_ determine organic P,O, by methods involving the use of uranium acetate
must give incorrect results.

Tue DISAPPEARANCE OF ORGANIC PHOSPHORUS

In the course of the investigation it was several times noticed that

a | duplicate samples left some days before being analysed, gave lower values
____ for organic phosphorus than samples analysed immediately; in some
a __ @ases no organic phosphorus was found. It was thought that this might

_--——ibe due to the destruction of organic phosphorus compounds, perhaps by
____ the enzymes present in the urine.

# Samples of urine were kept in aseptic flasks, in an incubator, at
87° C., a little toluol being added. Small quantities were removed at
Haletvals of a few days, care being taken to cool the flask so as to obtain
, the correct volume, and estimations of inorganic P,O, made. In other

eases ammonia was added to the urine, which was ‘giailaly incubated.

_ Owing to the precipitation of phosphates it was impossible to obtain

uniform samples, so the organic P,O, was estimated in the filtrate from
50 c.c. of urine after treatment with magnesium citrate.

The following are a few of the results : —

— —«T:ss Jan. 12. 250.0. Urine + toluol at 37°C. Total P,O, = 0-196 grams in 100 c.c. Urine
a Inorganic P,O, = 0-183 a i

Total P,O, = 0-282 grams
Inorganic ,, = 0-255 ”
20. Organic P,O, = 0-007 ,,
Feb. 27. No trace of Organic P,O, present

» 15. Inorganic PO; = 0-187 grams in 100 c.c. Urine
» 23. i » 20-194 o ”
on SO » = 0-194, * ta No phosphorus present in filtrate
Il Feb. 13. 250 o.¢. Urine + toluol, at 37°C. Total P,O, = 0-247 grams in 100 c.c. Urine
Inorganic P,O; = 0-230 __,,
Feb. 27. Organic P,O; = 0-009 grams in 100 c.c. Urine
Mar. 25. can + ae OO0T Fi,
If Feb. 13. 2500.0. Urine + 10¢.c. Ammonia at ‘3T°C.
Feb.

These results show that the organic phosphorus compounds are
partially or completely broken down when urine is kept at body
temperature. This decomposition is accelerated by ammonia. It is,
therefore, important not to leave urine standing too long after adding
the magnesium citrate mixture, and also to make estimations of organic
P.O, in fresh urine.

238 BIO-CHEMICAL JOURNAL

As it has been suggested, though without adequate proof, that
glycerophosphoric acid is the form in which organic phosphorus is present
in urine (4), 10 c.c., containing 1°2 grams organic PO, were neutralized
and added to urine containing a known amount of organic P,O,. “The
urine was left in the incubator for a month. The increase of inorganic
phosphates was no greater than could be accounted for by the breaking
down of the pre-existing organic phosphorus compounds; the glycero-

phosphoric acid escaped destruction.
eb

Tue Resvuvts or DiArysis

It was thought that by dialysis it might be possible to remove > the
inorganic phosphates and leave organic phosphorus behind. Urine in
quantities of 200 c.c, was dialysed for some days, the dialysing fluid being
changed three times a day. In some cases continuous dialysation was
employed during the last twenty-four hours. Nearly all the phosphorus
dialysed out in the first twenty-four hours; at the end of four days no
phosphorus could be detected in the residual fluid. By the addition of
Folin’s ammonium sulphate and uranium acetate reagent to this fluid
a nitrogen-containing substance was precipitated, but it contained ‘no
phosphorus. Filtrates obtained after precipitating urine with
magnesium citrate were similarly dialysed, but no phosphorus-containing
substances remained in the tube. The addition of Hedin’s tannic acid
reagent to urine occasionally produced a slight precipitate, but no
phosphorus could be found in this. The organic phosphorus compound
of urine is thus not identical with the protein-like material ‘sometimes
present.

SuMMARY

1. The estimation of total phosphorus in urine is most conveniently
carried out by Neumann’s method as modified by Plimmer and Bayliss.

2. Inorganic P.O, is best estimated by precipitation with
magnesium citrate mixture, incineration and calculation from the weight
of the ash, Mg,P,0,. This method is shown to precipitate inorganic
phosphates completely.

3. Magnesia mixture gives incorrect and variable results, partly
owing to the precipitation of calcium, partly to precipitation of a portion
of the organic phosphorus.

THE ESTIMATION OF PHOSPHORUS IN URINE 239

4, Organic P,O, can be determined either by subtracting the
_inorg vnic from the total P,O,, or directly by applying Neumann’s method
) the iltrate after precipitation of inorganic phosphates by magnesium
trate or barium chloride.

5. The uranium acetate method is unsuitable for accurate work.
atements as to the existence or non-existence of organic phosphorus in
e urine based on uranium acetate estimations are valueless.

7. It is important to make the determinations of organic P,O, in
h samples of urine, as the organic compound is partially or
pletely decomposed in the course of a few weeks, or, if ammonia be

in a few days.

i. _ The organic phosphorus compound is readily dialysable, and is

a“

ot prec’ aihgas by sian that precipitate traces of protein.
‘is

~s it is with ses pleasure that I acknowledge my indebtedness to Dr. :
rs Plimmer for his freely given help and suggestions as to methods

I i is investigation.

240

ON THE NITROGEN-CONTAINING RADICLE OF LECITHIN
AND OTHER PHOSPHATIDES

By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of
Aberdeen.

From the Department of Physiological Chemistry, Institute of
Physiology, Berlin
Part II
(Received April 13th, 1909)

In a former article! it was shown that only about 42 per cent. of the
nitrogen of heart muscle lecithin could be accounted for when estimated
in the form of the choline-platinum-chloride salt. When a sample of
trade lecithin was treated in a similar manner it was found that the yield
was considerably higher, being equivalent on an average to about 76 per
- cent. of the total nitrogen of the substance.

The comparatively low choline content of heart lecithin suggested the
probability of part of the nitrogen being represented by some other
N-complex differing from choline in its precipitation properties. As the
method of preparation of the trade lecithin could not with certainty be
ascertained, lecithin was prepared from egg yolk; an analysis of this
lecithin agreed in general with that obtained from the heart, but, as will
be shown below, the amount of nitrogen that could be accounted for as
above described was considerably greater than in heart lecithin, and a
good deal lower than in trade lecithin.

This curious result seemed to point to the probability of these different
lecithins being really different bodies, at least with regard to the manner
of combination of their nitrogen, and in order to test this a long series of
experiments was made.

In this paper it is proposed to deal with the results of hydrolysis of
egg lecithin, and at the same time describe certain experiments made
with a view to ascertaining whether possibly any circumstances were
present that might reduce the final output of choline, even assuming that
all the nitrogen was present as this base, in accordance with the ordinary
formula for lecithin.

PREPARATION OF EaG LEciTHIN

Lecithin was prepared from three different portions of eggs. The
method adopted was essentially the same as that described in my former
papers,” and need not be repeated; in every case (with one exteption)

1. This Journal, Vol. IV, p. 38.
2. This Journal, Vol. [V, pp. 47 and 168.

er:
oa
Be,
“
2
Beat
4 a,

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 241

I used the lecithin obtained from the ethereal extract of egg yolk, the usual]
precautions being adopted to exclude, as far as possible, air and light
during the process of preparation.

In one set of eggs a curious state of affairs was noticed; these eggs
(100) seemed to be perfectly fresh and were all rather large, it being
naturally thought that this would ensure a greater yield of lecithin. On
extraction, however, it was found that the ether showed quite an abnormal
increase of fatty matter, but only a trifling amount of substance
precipitated by acetone; after treatment with five consecutive portions of
fresh ether, the extract still contained much fat, and the combined yield
of lecithin was so small that after purification the total amounted only to
a few grammes. On subsequent extraction with alcohol the lecithin yield

ieee was also very much below the ordinary average.

In this particular case it would seem as if there was a great increase
of fatty matter at the expense of the lecithin; unfortunately, I had not
an opportunity of further investigating the nature of this substance, but

a comparison of the relationship of fat to lecithin in the egg, and perhaps
in other organs, together with an examination into the nature of this fat,

would, in the light of the above observation, probably be of some interest.

A curious point with regard to the lecithin here obtained was that it
differed greatly in general appearance from lecithin obtained from other
eggs; it was from the beginning quite dark brown in appearance and not
so plastic as is generally the case, despite the fact that the greatest
precautions were taken to prevent oxidation; in general it looked more
like a specimen that had been exposed for some time to air than the
freshly extracted material, which is usually precipitated as a plastic,
more or less whitish mass with a slight brown tinge. The amount of
lecithin here obtained was so small that it was not made use of for this
investigation.

The first sample purified gave the following figures on analysis;
comparison with heart muscle lecithin shows a marked similarity in
elementary composition :

Egg Lecithin Heart Lecithin
N (3 experiments) Average 1-876 %, 1-87 %
P32 ” és 3-95 % 3-95 %
© 1 experiment » 818% 66:29%
Hl ” » 106% 10-17 %,

: N:P = 105:1

242 BIO-CHEMICAL JOURNAL

Hyprotysts or LEecrrnHin ae |

(a) In alcoholic solution of barium hydrate-—Uere about 1 gramme —

lecithin was boiled for varying periods of from two to six hours with
100 c.c, methyl aleohol containing 5 grammes Ba(OH),. After separating
certain products of decomposition, as formerly described, the purified
aleoholic extract was treated with alcoholie-platinum-chloride solution,
and the precipitate washed, dried and weighed as usual.

Two experiments taken at random from a long series gave the
following results; percentages are expressed in terms of actual amount
obtained, as against theoretical amount calculated on N present :—

1-0268 gm. Lecithin boiled 3 hours = 0°2766 gm. Choline-platinum-chloride = 65-4 %
0-8989 ” a) > 6 ” = 0°2448 . ” ” = 66%,

Here, as in experiments on heart lecithin, it was invariably found
that the residue obtained after hydrolysis contained nitrogen, despite the
most prolonged and careful washing; experiments described later on
suggest that this nitrogen is probably not of choline nature; in the two
experiments given above, this insoluble N amounted to 6°08 per cent.
and 913 per cent. respectively of the total nitrogen contained in the
amount of lecithin used. In six experiments in which this residual

nitrogen was estimated, the average percentage of the total lecithin.

nitrogen found in the residue was 6°74 per cent.
(b) In watery solution of Ba(QH),.—In this case the bydrolaale
was carried out in general as above described, certain modifications
being adopted in order to obtain the end alcoholic solution of
choline as free as possible from impurities. The time during which
boiling was continued varied from three to five hours, but in one experi-
ment this was extended to eighteen hours. A reference to the result shows
that this prolonged heating had a comparatively trifling effect in
destroying choline, and thereby lowering the percentage figures. In
general, the results stand in accord with those obtained by the use of
alcoholic solution; the following three experiments suffice to show this : —
0-8403 gm. Lecithin boiled 3 hours = 0-2246 gm. Choline-platinum-chloride = 64-8 %
07821 ,, —s, + 2. eee .. e: # = 65-2 %,
O7411 ,, ” » 18 ,, = 0-1920 ,, a» 9 = 63%
Thus, it is seen that this specimen of lecithin when hydrolysed in a
watery or alcoholic solution of Ba(OH), gave practically similar results,
varying on an average from 65 to 66 per cent. of the theoretical amount.
Here, as with alcohol, the residue obtained after filtration of the hydrolysed
solution invariably contained some nitrogen.

a RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 243

In a few experiments a special attempt was made to render this
residue free- from nitrogen, but without avail. After filtration the
2 3 ~ substance was thoroughly broken down, returned to the flask, and boiled
_ for fifteen minutes with 50 to 80 ¢.c. H,O. Again it was broken down and
_ treated as before, this process being repeated three times; afterwards it
__was carefully washed on the filter with water almost at boiling temperature.
. Despite this prolonged washing, examination in every case revealed the
; * of a distinct amount of nitrogen in the residue.

fe) In mineral acid solution.—In almost all the text-books, both
. Sela: and modern, there appears the statement that lecithin while easily
- uit up by the action of an alkali, is but very slowly attacked by acids. In
_ testing this, however, it was found that such is not the case, at any rate
* th regard to the splitting off of choline. After boiling with a weak acid
¢ a comparatively short time, choline seemed to be completely separated
off, and as the use of acid in this connection possessed certain advantages
some experiments were carried out with it. At first sulphuric acid was
used, but the difficulties experienced in getting rid of it after hydrolysis
was completed rendered the method somewhat cumbersome, and instead of
sulphuric acid use was made of hydrochloric acid. Here it was thought
_ that removal of the acid previous to precipitation by platinum chloride
_ would not be necessary, though after evaporation of the watery part of the
solution it was obviously present in fair concentration, and some experi-

ments (described later) showed that this assumption was correct.
In all these hydrolytic processes it is an advantage to avoid if possible
the use of barium, owing to the great difficulty of getting rid of it
_ completely afterwards. With an alcoholic solution this is a matter of
___— very great difficulty, and even with water, from which separation is much
—— more easily obtained, it is sometimes found to be present in traces towards
__ the end of the operation of purifying. The danger in the presence of
barium results from the fact that this substance forms a double salt with
platinum chloride, which may be thrown down with choline-platinum-
chloride. It is true that this barium salt is fairly easily soluble in
alcohol, but in spite of this fact, great care must be taken to wash the
platinum chloride precipitate thoroughly with cold aleohol when there is
is any chance of barium being present. If this is not attended to somewhat
pre variable results, difficult to account for, may be obtained. In all my
ey experiments in which Ba(OH), was used, particular care was taken to
ensure the thorough washing of the platinum chloride precipitate in order
bes: to dissolve out any traces of the double barium salt that might be present.

244 BIO-CHEMICAL JOURNAL

When this precaution is neglected, it is quite possible that traces of barium
may sometimes account for the apparent percentage of platinum being
somewhat above the theoretical amount when the salt of choline-platinum-
chloride is ignited.

With HCl, however, the separation of barium is got rid of, and, so far,
a great simplification introduced. Experiment showed that all that is
necessary is to boil the lecithin for some time with a weak HCl solution,
filter, evaporate to dryness, dissolve in absolute alcohol and precipitate
directly with platinum chloride. This method takes up very much less
time than when done with Ba(OH),, and is to be recommended when
estimating the choline of lecithin; here the choline is present from the
beginning in the form of tlie chloride compound, and as such is less likely
to undergo decomposition than when present in the free state in alkaline
solution.
As pointed out above, however, the action of Ba(OH), on choline in
saturated watery or alcoholic solution is not of much importance, the loss
in a sample boiled for eighteen hours with 5 per cent. Ba(OH), in H,O
amounting only to about 2 per cent. of the total choline present. Im my
experiments the HCl hydrolysis was performed as follows :—

About 1 gramme substance was taken and boiled for varying periods
with 100 c.c. of a 10 per cent. watery solution of hydrochloric acid
(10 c.c. HCl sp. gr. 1°81 + 90 c.c. HO), boiling being continued with the
aid of a reflux condenser for from two to five hours. The fatty material
separated out as an oily mixture, and could not be conveniently filtered
off while the solution was hot, but on cooling formed a solid mass, which
was easily separated from the liquid. This fatty residue was returned to
the boiling flask, and again boiled with 100 c.c. H,O for ten to fifteen
minutes; the mixture was then allowed to cool and filtered as before, the
process being in many cases repeated three times. The total filtrates
mixed together were now evaporated to dryness on the water bath, and
the residue extracted with absolute alcohol, filtered, evaporated to small
bulk and precipitated directly with platinum chloride. It was then left
to stand till next day, and the precipitate then filtered, washed, dried and
weighed as usual. .

The obvious object of the above prolonged washing of the fatty
residue was, if possible, to render it nitrogen free. This, however, could
not be accomplished, and it was found quite unnecessary to return the
residue to the flask more than once, as further washing did not lessen the

nitrogen content.

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 245

| lt is noteworthy that in whatever manner lecithin is split up the
residue always contains a certain amount of nitrogen, and since this
residue is composed practically of fatty matter, it suggests the probability
of this nitrogen being in close relationship with the fatty acid radicle of
the lecithin body.

The following is an example of the figures obtained with HCl :—

0-7601 gm. Lecithin boiled 1} hours = 0-2012 gm. Choline-platinum-chloride = 64-2 %

IE ge ip . 3 » = O2222 ,, a S = 65-1 %,

O91 , * oa = 02425 ,, ” ” = 645%

The its are practically identical with those obtained when barium

eeeinte was used, and other samples of egg lecithin gave almost similar
_ results. One sample, however, differed somewhat from the above in its
ij nitrogen percentage,' and in this case a slightly larger yield of choline was
obtained when calculated on the total nitrogen present, though when
: “calculated on the weight of substance it was precieatty 3 in agreement with
the above sample.

A trade lecithin, having a nitrogen percentage of 177 and a N : P
_ ratio of almost exactly 1 : 1, was now taken, and samples treated along-
side of the egg lecithin as follows:—About 1 gramme of this lecithin
and a similar amount of egg lecithin were hydrolysed under exactly similar
conditions as parallel experiments; they were then treated as nearly as
possible in the same manner, the same amount of fluid being used to wash
the residue, &e., and the choline precipitated in the usual way.

_ One experiment was done using alcoholic Ba(OH),, another with
watery Ba(OH),, and a third with HCl. The following results were
obtained, and clearly prove that the two lecithins, though having a fairly
similar elementary composition, are certainly not identical substances
ie rite regard to their nitrogen complex :—

Fluid used for Percentage of choline Percentage of choline Difference
hydrolysis ofegglecithinfound oftradelecithinfound in percentage

Alcoholic Ba(OH), 65-8 80 14-2
Watery Ba(OH), 64-6 79-6 15
| ow) 65-3 80 14-7
na Again, heart lecithin differs from these results in a greater degree

than they differ from each other; a comparison of the three substances
Me gives the following figures with regard to choline recovered as the double
ae platinum salt :—
ene Heart lecithin = 42 %,
Egg lecithin = 65 %
Trade lecithin = 80 %

1. This sample gave an average of 1-77 %(, N.

246 BIO-CHEMICAL JOURNAL

From these results it is obvious that so-called lecithins are really
bodies of different composition, despite their general agreement when
viewed from the results of elementary analyses.

Retation or N or Env Fiurrate to CHo.ine-PLATINUM-CHLORIDE
ACTUALLY OBTAINED

As in the case of heart muscle lecithin, some experiments were done
in order to test directly what proportion of the nitrogen in the end filtrate
—i.e., the end alcoholic solution obtained after hydrolysis, and purified as
much as possible from other decomposition products and ready for
precipitation with platinum chloride—could be obtained as the double
platinum salt. In some respects this direct estimation gives more definite
results than the ordinary lecithin estimations described above, for it is
known exactly what amount of the nitrogen appearing in the end solution
is actually precipitated as choline. To determine this, hydrolysis was
performed as before, and the alcoholic solution ultimately obtained divided
into two equal parts. One part was used for N estimation, and the other
precipitated directly with platinum chloride. In three experiments it
was found that 23 per cent., 24 per cent. and 26 per cent., respectively, of
the N actually present was not recoverable as choline-platinum-chloride. —

All these results prove beyond any doubt that the nitrogen of the end
filtrate is not present as choline, and suggests strongly that in lecithin
the generally accepted formula is insufficient for the facts obtained.

In the light of the above results, two considerations present
themselves—

(1) That the nitrogen of the lecithin is really not all present in the
form of choline or similar basic compound; or

(2) That all the nitrogen is really present as choline, but that some
interaction with the chemicals employed during hydrolysis, or with some
of the substances of hydrolytic decomposition, gives rise to some unknown
nitrogenous complex which is not precipitated by platinum chloride.

In order, therefore, to strengthen the probability that lecithin N is
not all present in the form of choline, it was necessary to thoroughly
investigate this point; for this purpose pure choline chloride was used,
and the following experiments performed : —

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 247

Exrertments wirn CHoLine CHrormpe

Samples of the choline-platinum-chloride salt obtained in the above
ey ents were mixed together and dissolved in water, filtered and
re-crystallised. The typical crystals obtained were again dissolved in
‘a f, and the process of re-crystallisation twice repeated. On ignition
ly the theoretical amount of platinum was obtained. A strong
: Watery solution of the crystals was then treated with HS to remove the
_ platinum, the fluid being heated during the passage of the gas in order
Ba to ensure complete separation. The mixture was then freed from platinum
| sulphide by filtration, and the filtrate, which was water clear, evaporated
; ‘dryness on the water bath. Residue was then dissolved in absolute
cohol, and this solution used in the experiments.

_ The object of these experiments was to find out what percentage of
ie choline chloride actually present in the alcoholic solution could be
vovered as the double platinum salt after the solution had been treated
th Ba(OH), in alcoholic, and in watery solution, and with HCl, exactly
in the hydrolysis of lecithin. :

: For each experiment an equal amount of the above solution was
and the following tests performed : —

A. For Nitrogen

| 5 c.c. choline solution was directly run in to a Kjeldahl flask and the N-content estimated
with the following results :—

(1) Gave 9-24 mgr. N equivalent to 0-2031 grm. Choline-platinum-chloride

@ . o%.,, Re » ©2081 ,, ” *

_ Here in both experiments the results were absolutely identical.

; B. For Choline-platinum-chloride by Direct Precipitation
5 c.c. directly precipitated by 10% alcoholic platinum chloride solution, allowed to stand

Result = 0-1961 grm. Choliae-platinum-chloride
Se but 5 ¢.e. evaporated to about 2 ¢.c. before precipitated with platinum
chloride :—

Result = 0-1968 gm. Choline-platinum-chloride
From the above it is seen that the amount of choline actually present
in the solution was, on the nitrogen calculation, equivalent to 0°2081
gramme of the double platinum salt, and calculated on direct precipitation
- about 01964 gramme, taking the average of the two experiments. This
slight difference may be accounted for by the difficulty of absolute
exactness in allowing for the necessary reduction of nitrogen formed from

oe
r.

248 BIO-CHEMICAL JOURNAL

the chemicals used in the Kjeldahl estimation, coupled with the ordinary
experimental errors and perhaps a slight loss due to traces of the choline
remaining in solution, It would seem that evaporation of the choline
containing fluid to very small bulk is not necessary, as the above results
with volumes of 5 c.c. and 2 c.c., respectively, are practically the same.
As a result of all these figures, it is clear that 5 c.c, of this solution ought
to yield at least 0°1960 gramme of the double salt when precipitated by
platinum chloride.

C. Choline Solution Boiled with Different Reagents to Imitate Hydrolysis
of Lecithin

(1) Watery solution of Ba(OH),.—To 100 c.c. of a 5 per cent. solution
of Ba(OH), in water, 5 c.c. of choline solution was added and the mixture
boiled for three hours, a reflux condenser being used. After cooling the
solution was filtered, the filter paper thoroughly washed, and the combined
filtrates treated with CO, to precipitate the barium. Mixture was now
filtered and the barium carbonate residue thoroughly washed with hot
water. Filtrates were united, a few drops HCl added, and evaporated to
dryness. Residue was then dissolved in alcohol, evaporated to small bulk
and precipitated as before.

Gave 0-1963 gm. Choline-platinum-chloride
9° 0- 1 959 bad ” °° °

(2) Methylic alcohol solution of Ba(OH),.—This was carried out as
above, with a few modifications mentioned in a former article, to ensure

the removal of the barium. Owing to an accident, only one experiment
was completed; it gave—

0-1957 gm. Choline-platinum-chloride

(3) Hydrochloric acid.—(a) To 100 ¢.c. of a 10 per cent. watery
solution of HCl, 5 c.c. choline chloride was added and the mixture boiled
for two hours; it was then filtered and the filter paper thoroughly washed.
Filtrate was then evaporated to dryness over the water bath, residue
dissolved in absolute alcohol, filtered, evaporated to a few c.c.’s and
precipitated as usual.

(6) Here a 5 per cent. HCl solution was used, and boiling was
continued for one and a half hours; otherwise it was identical with above.

(a) Gave 0°1970 g.m. Choline-platinum-chloride
(d) ” 0-1951 ” ” ”

~ ee eee oe ae Bb) ve
= =f}

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 249

; It is thus obvious that HC] when used as above does not interfere to
_ any extent with choline chloride. The only point in the manipulation at
which some action might be expected is during evaporation to dryness of
‘the dilute acid solution, for it is obvious that towards the end the
concentration of HCl is very great; however, this apparently exerts no
destructive action.

_ The following gives an indication in tabular form of the results
obtained :—

ai CHoLIne-PLatTiIncM-CHLORIDE IN GRAMMES

_ Galeulated from Found by direct Found, using Found, using Found, using

__N found precipitation HCl alcoholic Ba(OH), watery Ba(OH),
09031 0-1961 0-1970 0-1957 0-1963
0-203! 0-1968 0-1951 sae 0-1959

The above experiments were carried out with every possible
ae precaution in order to ensure parallel results, and a comparison of the
figures shows that the greatest difference between any two amounts only
a to 0-008 gramme, the average difference being very slight indeed. When
_-__ it is considered that a rather long process of manipulation, including
. several filtrations and the necessity of changing from vessel to vessel
(evaporation, &c.), was involved the results agree remarkably well, and
show conclusively that the loss of choline-platinum-chloride in the experi-
ments on lecithin is not due to any destructive action on the part of the
hydrolysing agenis; further, they indicate that the necessary manipu-

Pea. lations can be conducted with little or no loss, though in doing this the
ie a greatest care is necessary.

-_
a
#
~
a

In view of the above results there remained only the possibility,
already mentioned, that some inter-action between choline and some other
product of lecithin decomposition might ensue, and so yield a nitrogenous
complex of obscure nature which was not precipitated by platinum
chloride. In order to test this a similar solution to the above was used,
but to the Ba(OH), or acid mixture some of the known products of lecithin
decomposition were added before boiling. From the results obtained it is
obvious that no such inter-action takes place. The experiments were
carried out as follows: —

250 BIO-CHEMICAL JOURNAL

ExrPEerRIMENtTs with Lecirnin Decomposition Propucrs

For these experiments a solution of choline chloride similar to above
was used, but of somewhat different strength; 5 c.c. gave on direct
precipitation 0°2200 gramme of the double platinum salt.

(a) 5 c.c. of this solution was added to 100 c.c. water containing 2 c.e.
glycerophosphoric acid. This mixture was boiled for half an hour and
5 grammes Ba(OH), added; boiling was continued for two hours, and
after cooling a whitish residue was obtained on filtration. This residue
was thoroughly washed three times with 100 c.c. boiling water, being each
time returned to the flask and boiled for ten minutes; finally it was washed
with hot water on the filter. Filtrates were then treated with CO,, and
the usual manipulations performed.

(b) This experiment was conducted exactly as above, only that
Ba(OH), was present at the beginning of boiling. Results were as
follows :

(a) Gave 0-2173 gm. Choline-platinum-chloride
(b) ” 0-2161 ” ” ”

A number of experiments were now made with different amounts of
other known decomposition products, such as glycerine, phosphoric acid,
oleic acid, &e. The average results obtained showed that only a trifling
loss was accounted for by the presence of these materials, and as their
introduction gave rise to marked difficulties in the way of filtration and
washing of residues, this small loss cannot be held to account for the great
loss in lecithin experiments; in any case this loss did not amount to more
than 6 per cent. of the total, and since much greater quantities of above
products than would ever be obtained as the result of the hydrolytic
decomposition of lecithin containing a similar amount of choline
(equivalent to 0°2200 gramme platinum salt) were used, much more
difficulty was experienced in filtering than is the case with lecithin.

The following experiment is sufficient to show that, even when
excessive quantities of substances representing lecithin decomposition
products are used, the final yield of lecithin is not materially decreased.

5 c.c. choline solution was mixed with the following substances :—

Glycerine iy Tee Ys she L2C,0,
Glycerophosphoric Acid ses Ses in) TSS,
Phosphoric Acid ay ii ‘us .. O05 grm,
Stearic Acid ... os ety sas «» O05 grm.

Oleic Acid... es oad i .. O05 e.c,

‘ RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 251

and the mixture boiled in a 5 per cent. watery solution of Ba(OH), for
_ four hours; it was then treated in the ordinary way to remove impurities.
‘a . In this case the difficulties in filtration were very great, the experiment
taking a very long time owing to the fatty nature of the residues obtained.
_ The final yield, however, gave 0°2110 gramme choline-platinum-chloride,
or about 95°5 per cent. of the total obtained on direct precipitation, showing
_ definitely that when choline is really present as such at the beginning,
such treatment does not materially lessen the final amount obtained.

Here again it is seen that the residue obtained on boiling choline with
ma: ‘Ba(OH), and other substances such as the above products gives a residue
whieh can be rendered N-free. From this it must be assumed that the
“nitrogen remaining in the first and other residues in the hydrolysis of
ecithin does really not represent nitrogen of choline, but is present in the
rm of another nitrogen complex.

; The result of all the above experiments seems to show definitely that
4 if choline were really present in lecithin to the amount represented by the
nitrogen content, a much greater yield would necessarily be obtained on
eo and precipitation with platinum chloride.

Since little or no evidence of the breaking down of choline can be
Gitined experimentally under the conditions present in the hydrolysis of
lecithin, it is but fair to assume that a similar state of matters holds good
for lecithin itself, and that the results actually obtained gave a very fair
indication (allowance being made for slight losses due to possible defects
of the method) of the amount of choline actually present as such in the
original substance. A consideration of these facts shows the absolute
: _ futility of endeavours made at one time to estimate the amount of lecithin
__ present in an organ in terms of the yield of choline obtained. It is obvious
____ that this method would give widely divergent results when applied to, say,
ee heart lecithin and egg lecithin respectively.

a:

Thus the accepted formula for lecithin does not account for the facts,
and in view of the differences existing between apparently similar
lecithins but derived from different sources, can hardly be taken as a
representation of any lecithin as obtained by the best methods at our
disposal at present.

It was next thought that an investigation of some of the salts of
lecithin such as the cadmium chloride compound might yield some
information; the results obtained strengthen the above view.

252 BIO-CHEMICAL JOURNAL

CapmiuM-CHLORIDE-LECITHIN

82°53 grammes lecithin were dissolved in absolute alcohol, and to
this an alcoholic solution of cadmium chloride was added till no more
precipitation occurred. After standing for eighteen hours precipitate was
filtered off and thoroughly washed with cold alcohol, filtrate and wash
alcohol being preserved. It was then dried and weighed, and yielded
36°41 grammes lecithin-cadmium-chloride.

Filtrate and wash alcohol together was now carefully evaporated to
dryness and gave a total residue of 2°64 grammes, part of this obviously
consisting of cadmium chloride.

This residue (which may be termed Residue A) was now treated with
water in order to dissolve out the cadmium chloride, and was extracted till
the solution gave no evidence of the presence of cadmium on the passage
of H,S. .

On evaporating down this wash water it was tested and found to
contain a good deal of nitrogen. A mere trace of phosphorus could be
detected, the relation of N : P standing as 141 : 1.

Here it was obvious that some nitrogen must have been split off from
the lecithin; on boiling this watery solution with HCl and treating in the
usual way with platinum chloride, no precipitate could be obtained,
indicating that this nitrogen was not present in the form of choline.

After this extraction Residue A weighed only 1:16 grammes, so that
1-48 grammes must have gone into solution, the greater part of this being
eadmium chloride. Thus, the total amount of substance obtained from —
32°53 grammes lecithin after removal of excess of cadmium chloride was
36°41 grammes + 116 grammes = 37°57 grammes. The remaining
portion (1:16 grammes) was now thoroughly dried and analysed, with the
following results : —

A (1°73 9
Nitrogen i ay $I average 1-75 %

Phosphorus 2-18 %,

On treating in the usual way for choline, only about 20 per cent. of
the theoretical yield was obtained, but the small amount of substance
rendered it difficult to get quite an accurate result.

Since it is well known that lecithin is not entirely precipitated out of
alcoholic solution by cadmium chloride, this residue might be expected to
be composed of lecithin-cadmium-chloride; the analysis shows plainly,

4 RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 253

however, that something more containing a higher percentage of nitrogen
and a lower percentage of phosphorous than this compound must have been
BZ present as well. The choline-platinum-chloride found probably represents
the choline of the double lecithin salt, while the remaining nitrogen was
nt in some other form. All these points go to strengthen the
conclusion hitherto suggested, that the nitrogen of lecithin is not all
represented by choline or such basic body.

Awanysis or Lecrrury-CapMiuM-CHLORIDE

In the light of the above results it was of interest to ascertain what
percentage of the nitrogen in the double cadmium salt could be recovered
‘in the form of choline-platinum- -chloride. Since, in the manipulation
described, nitrogen was found which did not seem to represent choline
oh uitrogen, it was naturally concluded that the percentage of nitrogen
actually representing choline in the double cadmium salt ought to be
wah somewhat higher than in lecithin. This was found to be the case.
- The following figures for N and P were obtained.

Nitrogen {iaie &f average 1-415 %
; Phosphorus : 3104 | average 3-095 %
q N:P 21:1

From these figures it is, of course, easy to deduce the theoretical
yield of cadmium-chloride-lecithin that should be obtained from a given
quantity of lecithin. In the above sample the average nitrogen content

____ of the lecithin used was 1-876 per cent., and of the cadmium salt 1°45 per
went. If all the nitrogen of the lecithin were contained in the cadmium
____ salt a simple calculation shows that 1 gramme lecithin contains sufficient
nitrogen to yield 1°294 grammes of the cadmium salt, and, therefore, the
quantity used (52°53 grammes lecithin) ought to furnish 42-093 grammes
lecithin-cadmium-chloride. The amount actually obtained after removal
of excess cadmium chloride was, as mentioned, only 37°57 grammes—
another préof that all the nitrogen present in the lecithin did not go to
‘i form the double cadmium salt.
e Although, as shown, a certain amount of the lecithin-cadmium-
f chloride remained in solution, this was much too small to account for the
loss of about 45 grammes calculated on the theoretical amount, using
nitrogen as a basis.

254 BIO-CHEMICAL JOURNAL

Hyprotysis or Lecrrarn-CapMiuM-CHLORIDE

The salt was treated with watery solution of Ba(OH), in the usual
way, and the choline calculated as choline-platinum-chloride with the
following results : — a

0-6348 gm. Salt = 0-1470 gm. Choline-platinum-chloride = 74:5 %
05587 ” Sa cd O17T77 ” ” ” = 75%

These figures show that the cadmium salt of lecithin yields about
10 per cent more choline-platinum-chloride than lecithin itself does,
results being in both cases calculated on the original N-percentage of the
substance. This again shows that some nitrogen, which was not present
as choline, must have been thrown off from the lecithin, otherwise the
results cannot be explained. On the other hand, it is obvious that this
lecithin salt must, like lecithin itself, contain a good deal of nitrogen
which is not present as choline.

It is intended to again recover this lecithin from the salt, and to
estimate its choline content; then to repeat the process of precipitation
and analysis in order to ascertain whether this splitting off of nitrogen
would be in evidence on a second precipitation.

It is quite possible that this particular part of the nitrogen may be
present as an impurity in the form of some other complex not really
belonging to the lecithin molecule; if so, it must have the same general
properties as the lecithin itself, both in regard to solubility in ether and
precipitation by acetone, and is likely present in many so-called lecithins.
Of course it represents only a comparatively small part of the total
lecithin nitrogen.

In the hydrolysis experiments carried out with lecithin-cadmium-
chloride mentioned above, it was found that the residue obtained gave, as
usual, a distinct indication of the presence of nitrogen; as before it was
found quite impossible to get rid of this however prolonged washing was
attempted; as in lecithin, it seems certain that this residual insoluble
nitrogen has nothing to do with choline.

Since it has been shown that on the hydrolysis of lecithin a
considerable percentage of nitrogen actually present in the end alcoholic
solution is not precipitated as choline-platinum chloride, thtis nitrogen
ought to be present in the filtrate after the separation of the platinum
chloride precipitate. That the substance obtained was really the double
platinum salt of choline is indicated by the results of ignition experi-
ments, which generally gave a residue of platinum but slightly wide of
the theoretical amount; it is, of course, quite likely that in some cases,

fa
=
~

x

Se z

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 255

at any rate, the salt was not of absolute purity, but any admixture of other

material must have been so slight as to be of little or no importance in

judging of the general results. ‘To substantiate this some experiments
were made in order to ascertain if the amount of nitrogen used up
actually corresponded to the amount necessary for the quantity of
choline-platinum-chloride isolated. This was found to be so, the
remainder of the nitrogen being present in the filtrate.

A number of these final filtrates were now united, some water added,
and the excess of platinum separated by means of H,S, the solution being

_ heated during the passage of the gas. It was then filtered from platinum

sulphide and the clear filtrate evaporated to dryness; residue was then
dissolved in a smal] quantity of absolute alcohol and again treated with
platinum chloride; in each case a slight precipitate was obtained, but

it invariably proved to consist of a mixture of a small amount of

choline-platinum-chloride with some of the barium salt of platinum
chloride. This was in turn filtered off and filtrate again treated with
HS, as above. The final solution obtained was then treated with various
general precipitating reagents, but no definite substance could be isolated

_ in this way. Experiments in this direction are at present being carried
out, but it seems certain that the excess nitrogen of lecithin is not present

as an ordinary basic compound ; on the other hand there is some evidence
that part of it, at any rate, is present in the form of an amino-acid, but
this will be entered into in a later communication. The purpose of the
present investigation was to ascertain the reason why in the hydrolysis of
lecithin the actual amount of choline isolated should invariably fall so
far short of the theoretical amount, and also to obtain some definite

evidence with regard to the correctness or otherwise of the generally
_ accepted formula with regard to its nitrogen distribution,

In the light of the above experiments it seems to me that the reason

for the discrepancies mentioned depend on the fact that different lecithins

contain varying amounts of choline; also that the ordinary formula
cannot be accepted. Again, it is likely that many of the specimens
formerly examined contained mixtures of other phosphatides besides

_ lecithin, and so gave even lower results than recorded above.

With regard to other phosphatides examined mention may be made
of two—the mon-amino-diphosphatide isolated from the heart muscle by
Erlandsen, and from eggs by the writer. As mentioned in a former
paper these two substances differ somewhat in their general analyses, but
im each case no definite evidence of choline or any other substance of
basic nature could be obtained. On hydrolysis in the usual way, platinum

256 BIO-CHEMICAL JOURNAL

chloride may give a slight often ill-defined precipitate, but the amount
is so exceedingly small even when a large quantity of the substance is
used, as to make it impossible to say what it really consists of. It would
seem to be more of the nature of an impurity, and is in no way equivalent
to the amount of nitrogen present. With the comparatively small amounts
of substance at my disposal it is difficult to make any definite statement,
but so far, in the case of the egg phosphatide, I have failed to obtain any
substance of a basic nature in the ordinary sense though many
experiments in this direction have been made. At present a large supply
of heart ‘cuorin’ has been prepared, and it is hoped that some future
experiments with large amounts of substance may settle the point.

The general results of the above investigation and the chief con-
clusions inferred in the light of these results may be shortly summed
up as follows :—

SUMMARY

From different lecithins (heart, egg, etc.) prepared with the greatest
care, and having a ratio of N : P as almost 1 : 1, different amounts
of choline are obtained under similar conditions. On the other hand the
amount obtained from any given specimen is practically constant, the
result being the same whether watery or alcoholic solution of Ba(OH),,
or watery solution of hydrochloric acid be employed. From this it is
obvious that these different lecithins, though showing somewhat similar
figures as the result of elementary analyses, are not identical in
composition, particularly with regard to their nitrogen distribution.

A long series of experiments with choline chloride has shown that
if choline is really present at the beginning of hydrolysis, a very large
percentage of it can be ultimately recovered as the double platinum salt,
and the presence of substances corresponding to the known decomposition
products of lecithin do not, by means of any more or less obscure reaction
with the choline present, prevent this base being ultimately recovered
as the double platinum salt; in one experiment containing excessive
amounts of all the known products of hydrolytic*decomposition of lecithin,
there was a loss of only 5 per cent. of the choline, obviously due in great
part to the difficulty experienced in thoroughly washing the bulky
residues present. .

Since these insignificant losses entirely fail to account for the small
yield of choline obtained from lecithin, it is necessary to assume that the
whole of the nitrogen of lecithin is not represented by choline.

In some lecithins, however, more of the nitrogen present is actually

|) Tee Ne ee

RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 257

represented by choline than is the case in other lecithins, a comparison
_ of the amount of choline-platinum-chloride salt obtained from heart and
egg lecithin, respectively being roughly 42 per cent. and 65 per cent,
while from a trade lecithin 80 per cent. was obtained.

Results substantiating the above were obtained on precipitating egg
lecithin with cadmium chloride and examining the filtrate and salt;
in the sample examined some nitrogen was split off by CdCl, in a form
soluble in H,O; this nitrogen did not represent choline. The cadmium-
leeithin-chloride salt contained 10 per cent. more choline (based on its
N-content) than the lecithin from which it was obtained. This
practically proves that some of the nitrogen originally present in the
lecithin was not present as choline.
Other evidence with regard to direct estimation of nitrogen and
m4 in different filtrates all points in the same direction, and lends
: additional weight to the inferences derived from a consideration of the
above experiments. Whether lecithin is really a chemical unit, or a
mixture of two or more substances having the same properties does not
affect these facts.

CoNcLUSIONS

1. In the light of the results obtained, it is the opinion of the writer
that the generally accepted lecithin formula, i.e.—

i O« (acid radicle)
CH - O° (acid radicle)

CH,
OH— > PO
(N. in form of choline or possibly other base) - O

in which the whole of the nitrogen present is represented by a base—
. choline—is incorrect, and can no longer be accepted; probably part of this
‘ nitrogen at any rate is present in the form of amino-acid.

2. Lecithins obtained from different sources differ, often to a great
extent, with regard to the percentage of nitrogen actually represented
by choline, and though often giving somewhat similar results on
elementary analysis, cannot be regarded as identical chemical substances.
2 3. These results fully explain the general failure of former
j investigators to obtain anything corresponding to the theoretical yield
: ; of choline from lecithin.

258

A POLARIMETRIC STUDY OF THE SUCROCLASTIC
ENZYMES IN BETA VULGARIS

By R, A. ROBERTSON, M.A., JAMES COLQUHOUN IRVINE,
D.Sc., Pu.D., axnp MILDRED E. DOBSON, M.A., BSe.,
Carnegie Scholar.

From the Chemical and Botanical Research Laboratories, United College,
University of St. Andrews ;

(Received April 16th, 1909)

Notwithstanding the large number of exhaustive investigations which
have been recently carried out on the sugar beet the actual mechanism
of the sugar synthesis seems to be practically unknown. Two
alternatives seem possible, the simpler of which is that both glucose and
fructose are formed in the leaf region and are there subsequently
transformed into sucrose which passes by means of the leaf meristem
directly to the root: The alternative explanation, that the disaccharide
is a degradation product of starch, seems unlikely. Little or no starch
is found under normal conditions in the growing leaf and, moreover,
such a change would involve the partial transformation of maltose or
glucose into fructose. This would necessitate a fundamental change in
the sugar molecule, which seems improbable. Again, according to Grafe,
no definite sugars have ever been isolated in the degradation products of
cellulose, and thus the latter does not seem to be a likely source of the
sucrose,

The first alternative is supported by many arguments. Friedrick
Strohmer in liis valuable contribution to the Wiesner Fest-Schrift! shows
conclusively from the result of his own experiments and those of A.
Girard that the formation of sugars is restricted to the leaf region and,
moreover, that the roots of adult plants contain no reducing sugars save
in the early stages of growth.

It must be maintained that the evidence seems to be increasing that
formaldehyde is one of the initial substances formed during the natural
process of photo-synthesis of sugars, and it would thus appear that a close
parallel can, in this case, be drawn between natural and artificial

1. Uber Ausspeicherung und Wanderung des Rohrzuckers in der Zucker-Riibe.

SUCROCLASTIC ENZYMES IN BETA VULGARIS — 259

i synthesis. Fenton’s recent successful reduction of carbon-dioxide to
_ ____ formaldehyde' and the earlier work of Usher and Priestley,? who showed
that the same reaction proceeded in plant cells, has strengthened the
argument considerably. There seems litile doubt that the artificial
sugars obtained from formaldehyde contain large quantities of ketoses,
‘s and thus is can be readily understood that in the leaves of the beet the
_—s«swR@essary constituents for the formation of cane sugar may be formed.
The final stage of the process, the condensation of the glucose and
fructose, although theoretically a simple reaction, is one which the
organic chemist has been unable to duplicate.

Perhaps the most notable example of the comparative failure of
ordinary chemical methods to produce naturally-occuring compounds is
to be found in the meagre results which have attended attempts to
‘_ synthesis disaccharides in the laboratory. Moreover the methods followed
____ im the few successful cases must of necessity be widely different in their
ae nature from those of the natural process. In view of these facts Grabe’s
statement that the fundamental réle in the carrying out of these
| reactions must be ascribed to enzymes seems justifiable.*
| Assuming that cane-sugar is formed by enzyme action, two
alternative theories may be offered. The reaction may be occasioned
either by a special enzyme, termed the associating enzyme, capable of
} condensing glucose and fructose, or the hydrolytic action of invertase
* may be a reversible change which is capable under suitable conditions of
s producing sucrose from the constituent sugars. This latter view is
ee upheld by Gonnermann and Stoklasa, and brings the reaction into line

with the cases of reversible zymolysis studied by Croft Hill. This idea

has also recently received considerable support in the results obtained
by Kohl,* who subjected invert sugar solutions to the action of yeast
____ extract rich in invertase. A series of titrimetric estimations showed that
an -equilibrium point was reached, presumably owing to the partial
3 formation of sucrose. The result is interesting, but is not altogether in
agreement with the nature of reversible change, as apparently conditions
were realised in which both the fructose and glucose contained in the
invert sugar completely disappeared on continued action. Pantanelli,°®
in a paper on a similar topic, contributes the remarkable statement that

1. Trans. Chem. Soc., Vol. XCVIII, p. 687, 1907.
2. Proce. Royal Soc., Vol. LX XVIII, B, p. 318, 1906,

F “3. Macchiati, Comp. Rend., Vol. CXXXYV, p. 1128.

3 4. Beth, Bot, Centralblatt, Vol. XXTLU, 1, p. 64b-640, 1908.

5. Rendiconti Accademia Lincei, 5, Vol. XVI, pp. 419-428, 1907.

260 BIO-CHEMICAL JOURNAL

concentrated solutions of invert sugar undergo partial reversion when
preserved at room temperature, ‘ particularly when the solution is feebly
alkaline.” The reaction was accelerated by the addition of fungus
revertase. The first conclusion seems to have been arrived at by the
observation that the reducing power of the solution diminished. In view
of Lobry de Bruyn and van Ekenstein’s work’ on the interconversion of
hexoses in the presence of alkalies the result seems capable of a sae
interpretation.

Although opinion, in the meantime, seems undivided in attributing
sucrose formation in plants to enzyme action, it would appear that it is
still doubtful if the action is due to a definite specialised enzyme or is
merely the result of reversible zymolytie change.

The following research was undertaken in the hope that the
biochemical formation of sucrose by enzyme action could be detected
polarimetrically, and for evident reasons the sugar beet was selected for
experiment. The adoption of the polarimetric method seemed in this
case specially advisable as affording a more accurate index of alteration
in the composition of sugar mixtures than any method based on the
quantitative use of Fehling’s solution.

In view of the fact that the chemical activity of many enzymes is
increased by the presence of other enzymes, no attempt was made to
separate, even approximately, the individual enzymes from the various
mixtures obtained. The total product was tested in its action towards
suitable optically active substrata, and in view of the well-defined
selective action of enzymes, the method seems justifiable. As far as
possible, substrata were used which would readily react and would give
large polarimetric differences. The optical activities were determined
with an instrument displaying a tripartite field and sensitive to y}, of a
degree. Throughout the work standard two-decimetre tubes were used;
these were provided with a water-jacket and all the determinations were
made at 20°C., accurate to y°. As a rule the sodium-flame was used,
except in special cases where, on account of the unavoidable turbidity of
the solutions, the incandescent light was substituted.

Some difficulty was occasionally experienced in filtering the
solutions successfully, but this was overcome by shaking with finely-
divided ignited silica, a method which we found did not introduce any
experimental error.

For the purposes of the present investigation only the examination
of the enzymes capable of reacting with carbohydrates and glucosides

1. Ree. trav. chim. Pays-Bas, Vol. XTX, p. 1, 1900.

SUCROCLASTIC ENZYMES IN BETA VULGARIS — 261

was necessary, and accordingly in the first place the general nature of the
soluble enzymes present in the leaves of the adult plant was studied.
The plants used weighed on an average about 650 grams. The leaves were
detached at the junction of the stem, and after being finely divided, were
macerated with water in a sterilised mortar. The pulp thus obtained was
mixed with a large excess of water (two litres), and kept in a thermostat
at 30° ©. for five hours, during which time the mass was kept thoroughly
_ mixed by means of a powerful mechanical stirrer. The liquid was then
filtered under pressure and the bright-red filtrate rapidly diluted with
_ four times the volume of alcohol. The mixed enzymes were thus obtained
‘in the form of a grey precipitate, which was filtered off, washed with
queous alcohol and dried in a vacuum. The dry product gave very little
ganic residue on ignition, and was devoid of any action on Fehling’s
on even on prolonged boiling. The solubility in water was slight,
“gram of the substance requiring about 700 c.c, of water to effect
mmplete solution at 25°C. The aqueous solution, which became turbid
on the addition of traces of alkali, showed practically no activity when
examined in a two-decimetre tube. This result was unexpected, but
‘se ms to be due to compensation, as enzymes of opposite activity were
af er ards shown to be present.
In testing for the presence of probable enzymes, standard solutions
of the active substrata were mixed with excess of the solid enzymes, and
the mixture sterilised by the addition of a few drops of toluene. The
initial specific rotation was determined without delay, after which the
liquid was kept in a thermostat at 30°C., polarimetric readings being
taken every twelve hours. In each case a control experiment, in which
no enzyme was used, was conducted under parallel conditions, and
results were only accepted as positive when these control solutions
showed no alteration in rotatory power. ‘The following table shows the

“i st principal results obtained :

Enzyme Substrate (a) initial (a) final Diff.
Be IL. Invertese ...Sucrose * ... + 666° ... + 644°... 2-2°
a °2. Emulsin ...Amygdalin .. — 344° .. — 344°... nil
Mca? 3. Diastase =... Stareh ods - eee a EP se
4. Maltese _,..a-methyl-ghicoside +157°5° ... + 1284° .... 291°
5. Lactase — ... Lactose see + 63-1° ere 0-3°

*In view of the possibility that the enzyme action might be inhibited by the hydrocyanic
ade (ase from amygdalin, other substrata were afterwards used but a negative Welt was

In the case of Experiment 3, the dilute starch paste used was rapidly
liquefied and gave a dextro-rotatory filtrate. The concentration of

262 BIO-CHEMICAL JOURNAL

dissolved matter was estimated by evaporation of an aliquot part of the

solution and weighing the residue dried at 100°C. In this way it was
shown that the specific rotation of the solution gradually decreased
from +- 108°, when the first reading was made, to the constant
value + 52°. This ultimate complete conversion into glucose through
the intermediate formation of a more highly rotatory compound is in
agreement with the idea put forward by Payn! that the hydrolysis of
starch in the joint presence of diastase and maltase passes through the
following stages :—

Starch —> Dextrine —> Maltose —> Glucose

The chemical activity of the leaf enzymes used in the above
experiments was somewhat disappointing, few positive resulis being
obtained. This seems to be due to the method of preparation and
purification which would yield only the more soluble enzymes. A similar
result has already been obtained by Brown and Morris? in a study of the
enzymes present in foliage leaves. It seems in fact to be a general
experience that careful filtration of the original aqueous extract is not
desirable in the preparation of enzymes as the product obtained displays
very little reactive power. Better results are, as a rule, given by
straining the liquid extract of the macerated tissues through fine muslin,
and a similar method was therefore adopted in the case of the leaves of
the adult beet.

PREPARATION OF MIxEpD ENZYMES FROM THE LEAF

The leaves were macerated with water and extracted in the ~
thermostat as already described, the resulting liquid being strained
through several folds of fine muslin, On adding a large excess of
alcohol to the turbid filtrate, a brown sludge separated from which the
supernatant liquid was decanted away. After washing well with absolute
alcohol, the insoluble matter was filtered under pressure and washed with
50 per cent. alcohol until the washings were inactive and ceased to reduce
Fehling’s solution. The purified precipitate was then diluted with water
so as to give a mixture containing 1 per cent. of the dry enzymes, and
the liquid was rendered antiseptic by the addition of a little toluene. A
filtered sample was optically inactive and did not reduce Fehling’s
solution.

This method of obtaining the enzymes suspended in water neces-

1. Comp. Rend., Vol. LIII, p. 127.
2. Trans. Chem. Soc., Vol. LXII, p. 604, 1893.

SUCROCLASTIC ENZYMES IN BETA VULGARIS — 268

__ sitates a modification in the preparation of the test-solution. Where
possible, 20-per cent. aqueous solutions of the substrata were prepared,
and 25 c.c. introduced by means of a pipette into a standard 50 c.c. flask,
which was then diluted to the mark with the homogeneous enzyme
sludge. The control solutions were similarly prepared, the dilution to
half the original concentration being of course made with water. The
experimental solutions were filtered through several baryta filters and
the optical rotations determined without delay. All the solutions were
kept in the thermostat at 35°C., the rotations being determined at
regular intervals. The following positive results were obtained :—

Substrate used c (a) initial (a)”"” final difference
@methylgiucoside ... 9995 ... + 1570° ... + 1306° 2. 27-4°
ss: Buerose sa 10-006 dea + 66-2° ... + 314° ... °° 348°
Inulin 7 POS tn hs SB 88 4-2°
Starch ati — oa — = + 60° ... —

‘The results confirmed those already obtained with the purified enzymes,
but the actions were much more rapid. Although marked optical
= differences were observed in a few hours the experiments were in each
ease continued for twenty days. The joint presence of maltase and
diastase was confirmed, while inulase and lactase were again shown to
be absent. In this case, however, evidence was obtained that invertase
was present, as shown by the following figures :—

Experiment—Concentration of sucrose = 10-0060, initial (a)?” = + 66-2°
final (a)? = + 31-4°
Control— 7 ; = 10-0060, initial (a)? = + 66-6°
final (a) = + 63-2°

ie 3 The result was verified in a duplicate experiment, but in no case was
complete inversion obtained.

EXAMINATION oF THE ENzymMEs FROM THE Stem AND Roor ReGIons

After removal of the leaves, the roots were separated from the short
stem region and the enzymes isolated as described above in the form of
an aqueous sludge. Prolonged washing with dilute alcohol was, of
course, necessary to completely remove sugars and also the red colouring
matters. In addition, the bulky solid residue left in each case on the
muslin filter after extracting the macerated organs with water was
My utilised in the following manner:—The material, after being washed
a with water, was spread on sheets of filter paper and dried in a current of

a " -.. '
wer. : J ra

264 BIO-CHEMICAL JOURNAL

air at 30°C. After several days’ treatment, the brittle residue was
powdered finely in an agate mortar, again digested with a large excess
of water, filtered and dried. In this way we were able to avoid the
extensive decomposition which results when attempts are made to
desiccate the tissues without in the first place removing all soluble organic
matter. A similar method has been used by Brown. and Morris in
demonstrating the presence of diastase in foliage leaves’. We
thus obtained four preparations, viz.:—stem-sludge, solid stem, root-
sludge, and solid root, all of which were found to be capable of promoting
hydrolytic changes. The average yields obtained were :—

Weight of stem used .. 133 grams. Weight of solid stem obtained ... 6-5 grams.
Weight of root used ... 520 grams. Weight of solid root obtained ... 19 grams.

As usual each preparation was found to be inactive and to be deyoid
of any action upon Fehling’s solution. In preparing the test solutions
the method already described was adopted, that is, an aliquot part of the
solution was diluted to half the concentration with the stem or root
sludge. In the case of experiments with the solid stem or solid root, half
a gram of the powdered tissues was added to 25 c.c. of the test solution,
which was then diluted to 50 c.c. with water. The control solutions
were subjected to a similar dilution without the addition of any enzymes.
Sterilisation was effected by the addition of a little toluene or chloroform,
and heating was conducted in the thermostat at 30° to 35°C. The
optical changes were complete in a few hours, but the values given below
were those obtained after seven days’ treatment.

Test ror INVERTASE

Substrate = sucrose (‘ Kahlbaum’). ¢ (initial) = 5-000 c (final) = 2-500

Stem sludge ... initial (a) = + 66-10° ad final (a) = — 128°
Solid stem. » (a = + 66-80° wo (a a seer
Root sludge... » (ay = + 66-10° sii » (ay = + 618°
Solid root ss, » (a) = + 66-10° pes » (ay = + 620°

A well marked difference therefore exists between the results
obtained with the root and stem preparations. In the former the optical
changes observed were small and did not differ in any marked degree
from those shown by the contro] solutions.

It would thus appear that while invertase is present in the stem
region it is absent in the root. The result agrees with the general

1. Loe. cit.

SUCROCLASTIC ENZYMES IN BETA VULGARIS — 265

organs” during the period in which reserve material is being laid down.
Our observation is, however, at variance with those obtained by
~Gonnermann and Stoklasa.!

Test ror Emvtsin

ie ae Substrate = B-methylglucoside ¢ (initial) = 4-060 ¢ (final) = 2-030

a Stem sludge ... _ initial (a) = — 335° or final (ap = — 22-5°
= elites lw OM ae. » (@)y = — 221°
Root sludge. » (ae = — 335° a » (aj = — 22-7°

- Solid root oes » (ap? = — 335° “ul » (a) = — 25-1°

‘The above results were obtained twenty-four hours after commencing
i experiments ; meanwhile the control solutions suffered no alteration

E ‘rotatory power. As in each case the liquids reduced Fehling’s
28 actively after treatment with the enzymes, the results are
a | accepted as indicating the presence of emulsin in all the preparations. As
= Ee : a confirmatory test, salicin was substituted for S-methylglucoside as a
seach substrate. In each case the result was the same, the specific rotations
of the solutions diminishing about 14° in twenty-four hours, and the
presence of both glucose and saligenin was detected in the resulting
liquids.

Test ror MALTASE

Maltase was found to be present in both the root preparations, but
was absent in the stem as shown by the following typical results :—-

ele ‘Substrate = a-methylglucoside ¢ (initial) = 4-9992 ¢ (final) = 2-5005

—— Bolid root... initial [aR + 156-0" final [af + 1400° diff. = 160°
= Solid stem... Ps + 156-0° hes + 154°, 0-6°
= Substrate = Maltose (Kahlbaum) ¢ (initial) = 5-0010 —_¢ (final) = 2.5005

A Solid root ..._— initial fa” + 1321° final [a + 968° diff. = 353°
. Solid stem... - + 132-1° ” + ie", = «(3-7°
* In using maltose as a substrate we took the precaution of adding a

minute trace of caustic soda to the solution in order to promote
mutarotation. The optical changes observed are therefore not due to
stereochemical alterations in the sugar.

deat) ’

1, Czapek's Biochemie der Pflanzen, Bd. I, p. 375.

4

266 BIO-CHEMICAL JOURNAL

Test ror DrasTase

Substrate Starch solution c (initial) approx. 0-50

Stem sludge ... no initial a taken oes final [ap + 440°
Solid stem... ‘ " aes ” + 32-0°
Root sludge ... = ” asi a + 240°
Solid root... “ 9 at i + 28-0°

The final concentrations were determined by evaporation of a
measured volume of the solutions and weighing the residue dried at
100°C. All the products reduced Fehling’s solution actively even in the
cold. We are in the meantime unable to explain the fact that the end
values obtained are lower than those calculated for glucose; no
8-glucose was present as the rotations remained permanent on adding a
trace of alkali. The concentration would of course only be approximate,
but any experimental error thus introduced would be insufficient to
account for the discrepancy between the calculated value for glucose and
those experimentally found. Possibly inactive products may have been
formed, or a partial conversion into other hexoses may have taken place.
It will be noticed that in the case of enzymes extracted from the leaf the
hydrolysis of starch resulted in the formation of a reducing sugar which
displayed the correct rotatory power for glucose.

SuMMARY OF THE DistRIBUTION oF SucrocLAstic ENZYMES IN THE ADULT
BEET .

(1). Znvertase——Invertase was not detected in the carefully
purified mixture of enzymes obtained from the leaf. When, however,
the enzymes are obtained by filtering through muslin and are afterwards
washed free from reducing sugars, invertase is then found to be present.

Both the aqueous sludge containing the enzymes of the stem and
also the powdered stem contain invertase.

Similar preparations from the root contain no invertase.

(2). Diastase was found in all the preparations examined.

(3). Maltase is present in the leaf and root regions, but was not
detected in the stem preparations.

(4). Znulase seems entirely absent in the leaf region. Positive
results were obtained with all the preparations from the stem and root,
but the optical changes observed were irregular.

(5). Emulsin—The enzymes obtained from the leaf gave negative
results using amygdalin as a substrate; the stem and root preparations
on the other hand gave positive results with salicin and #-methyl-
glucoside.

(6). Lactase was absent in every case.

7

SUCROCLASTIC ENZYMES IN BETA VULGARIS 267
Arremptep Reverstste Zymonysis By THE Action or Beer EnzyMEs

The methods adopted in the attempted condensation of glucose and
fructose by enzyme action were similar to those successfully applied by
Croft-Hill' in the enzymatic synthesis of maltose from glucose.
As sucrose is incapable of forming an osazone, and as no suitable insoluble
derivative is known, the separation of which would serve to detect the
formation of the sugar in the presence of monosaccharides, recourse was
again had to a polarimetric method of following the reaction. The plan
adopted was to subject concentrated sterilised solutions of invert sugar
to the action of the various enzyme preparations, polarimetric readings

being taken at intervals. Change in rotation cannot, of course, in itself

at be considered evidence of sucrose formation. Other factors may play a

a « i including the possible self-condensation of both glucose and fructose.

experiments were therefore extended by hydrolysing the products
and ascertaining if the optical value for invert sugar was reproduced.
This joint evidence of a fall in laevo-rotatory power due to enzyme action,
and a subsequent increase to nearly the initial value of hydrolysis, would
justify the conclusion that condensation of the hexoses had taken place.
In the absence of positive results indicating self-condensation of glucose
or of fructose this double change in rotation would be significant of the
presence of sucrose, or at all events, of a glucosidic compound containing
both glucose and fructose. Before commencing our observations several
preliminary experiments were necessary. The preparation of a suitable
solution of invert sugar, displaying the maximum rotatory power,
presented some difficulty. In the first place pure crystallised glucose and
fructose (‘ Kahlbaum’) were dried until constant in weight, the former
at 110° C., and the latter in a vacuum. Equal weights of each sugar,
accurate to 1 milligram were then dissolved in water in a standard flask
and made up to the mark at 20°C. After allowing ample time for
mutarotation to take place, the rotations were determined in a two-
decimetre tube (t = 20°C.). The result was, however, unsatisfactory, as
the values obtained were not very uniform and were invariably too low.
This is shown in the following table : —
(ce. for glucose = 20-0010)

. 0° 9°
sa \¢. for fructose = 20-0013) (@), = — 21-2
(c. for glucose = 20-0015) . ;
2. efor fructose = 20-0006) (2), = — 11
(¢. for glucose = 20-0006) ai ig
s ic. for fructose = 20-0003) (2), = — 168

1. Loe. eit,

268 BIO-CHEMICAL JOURNAL

As the correct value for invert sugar is (a), = — 247° it would
appear that this method of directly weighing out the sugars is
inapplicable, probably owing to the difficulty in obtaining fructose in an
anhydrous condition. Although the solutions referred to above were used
in preliminary experiments, we prepared our invert sugar solutions by the
method recommended by Maumené.' Pure sucrose (Kahlbaum) in
quantities of 150 grams was dissolved in water (250 c.c.), and heated in
sealed flasks at 106° C. for fifty hours. The resulting liquid gave
(a) = — 241° calculating on the complete conversion into glucose
and fructose. The effect of hydrolytic agents on this solution was then
studied in order to ascertain if any sucrose remained unaltered. A test
portion was diluted to half the concentration with water containing
varying amounts of hydrochloric acid, and the optical changes on heating
observed. It is of course well known that hydrochloric acid, even in
dilute solution, readily decomposes fructose, and consequently suitable
conditions of acid, concentration, and temperature had to be determined
which would suffice to hydrolyse sucrose and yet be without action on
fructose. Using solutions containing 5°3, 2°6, and 13 per cent. of acid the
laevo-rotation of the invert sugar diminished steadily when the liquid
was maintained at 50°C. At 20°C., however, although a similar fall in
laevo-rotation was recorded in the case of the strongest acid solution, a
constant value was ultimately obtained. On the other hand, the solution
containing 2°6 per cent. of acid remained unaltered in rotatory power
when kept for six days at 20° C. This result proved the complete absence
of sucrose, and gave the desired conditions for carrying out a polari-
metric test for sucrose formation in the subsequent experiments.

In the first place the precipitated enzymes obtained from adult
leaves were used, but, as was expected from the feeble activity of the
preparation, no positive results were obtained. Even after an interval
of two months the optical activity of an invert sugar solution was found
to be quite unaltered by the action of the enzymes. Recourse was there-
fore had to the more active enzyme preparations previously referred to
as the ‘stem sludge’ and the ‘ root sludge.’ Only small optical changes
were, however, observed, but these were in the right direction, and were
supported by the evidence of hydrolysis. The results of one typical
experiment may be quoted :—

Initial concentration of invert sugar solution = 40-0100
Concentration when diluted with stem sludge = 20-0050
Initial specific rotation of the mixture = — 24-7°
After 350 hours’ action at 35° = — 21-2°
Decrease in laevo-rotation due to enzyme action = 3-5°
Increase in laevo-rotation due to subsequent hydrolysis = 2-1

1. Journal des fabricants de sucre, Vol. XX X1,p. 46.

ba EIS ee in.
et de

SUCROCLASTIC ENZYMES IN BETA VULGARIS 269

Control experiments showed that the enzymes were without action
on either glucose or fructose solution alone. It would thus appear that
the glycolytic enzymes described by Stoklasa are without action
on concentrated glucose solutions. The above data represents
about the average result obtained in five different experiments, and
_ eorresponds with the formation of approximately 4 per cent. of sucrose.
_ The addition of maltose did not effect any appreciable improvement in
_ the process and, moreover, prevented the hydrolysis being studied.
- It must be admitted, even if the above results can be correctly
_-_-—_—aseribed to sucrose formation, that the magnitude of the changes are
E i small. It appeared possible that this might be due to alteration in the
nature of the associating enzyme once the storage of sucrose had ceased,
we, i: and, consequently, in another series of experiments, young plants, which
had not begun storing, were used as the source of the enzyme preparations.
_ The isolation of the mixed enzymes was carried out as already described,
but no differentiation into leaf and root region was made, the entire plants
____ being macerated and extracted. The sludge finally obtained consisted
of a mixture of 445 grams of the mixed enzymes diluted to a litre with
_ The following experiment showed that the sludge possessed
associating powers : —

70 cc. of invert sugar solution giving (a) = — 23°7° were
introduced into a standard 100 c.c. flask and made up the mark with the
homogeneous sludge. A portion filtered at once gave in a one-decimetre
tube the rotation a, = — 686°. The solution was then kept at
35° C. in a thermostat for a week, when it was found that the rotation

had diminished to — 595°. The subsequent optical changes were only
small, and the constant value observed was a = — 570°. The total
change in a therefore amounted to + 116°. Expressed in specific
rotations the change becomes (a)®” = — 23°99 —> (a)®” = — 19°89,
and is, moreover, in the right direction. The subsequent hydrolysis was
carried out by diluting a test portion to half the original concentration
with water containing sufficient hydrochloric acid to give a 2°6 per cent.
acid solution. The initial rotation was (a)®* = — 19°3°, and on stand-
= ing at 20° this gradually altered to the constant value
a (a) = — 227°, the actual change in a being — 0°99°. This result
ae can only be explained on the assumption that a glucosidie product had
been formed possessing a less laevo- or more strongly dextro-rotation than
invert sugar, and which is, moreover, capable of undergoing hydrolysis
to give an equimolecular mixture of glucose and fructose. Assuming

=
ae
=

a te
is" A ee
a...

270 BIO-CHEMICAL JOURNAL

that sucrose is the only glucosidic product thus formed, the result indicates
the production of about 6 per cent. of the disaccharide. The above
observation was controlled by a duplicate experiment in which a sample
of the invert sugar solution was diluted with water in place of the enzyme
sludge and kept in the thermostat for an equal time. No appreciable —
alteration in rotatory power was, however, observed even after three
months. It was also shown that the sludge was without action on glucose
or fructose solutions alone; only in the joint presence of both sugars were
any optical alterations observable.

These results are summarised below :—

Nature of Experiment Length of tube Initial Final Behaviour on
in decimetres ae Ane A hydrolysis
Invert sugar and enzyme 1 — 6-86 — 575 16-6 — 555° —> — 654°
sludge
Control sol. of invert 1 —14-43 —14-37 O-4 No change
sugar
Glucose (20%) and 2 20-21 20-13 O-4 No change
enzyme sludge
Fructose (20%) and 2 —34-65 —34-80 O-4 No change

enzyme sludge

The column marked A contains the optical changes expressed in
percentages, and it will thus be seen that a marked difference exists
between the first experiment and the subsequent controls.

The result was verified in a duplicate series of experiments in which
the treatment with the enzymes was continued for three months: once
wore the control solutions gave perfectly negative results, but the invert
sugar solution underwent similar alterations to those quoted above :—

Initial (a) = —24-05° Final (a) = — 18-10°, Diff. = + 5-95°.
Change of rotation on hydrolysis (a) =—i81° -> —,27°2°,

A slight irregularity will be noticed in the permanent end value
obtained on hydrolysis. This we are in the meantime unable to explain,
but nevertheless it is evident that the rotation changes are of the same
nature as those discussed above, and are in agreement with the idea that
about 6°6 per cent. of sucrose had been produced in the reaction.

It is of course premature to claim the above results as a successful
bio-chemical synthesis of sucrose, but it is difficult to find any other
explanation of the optical changes other than that already suggested,
viz., that the aldose and ketose combine to form a glucosidic derivative,
and sucrose is the only compound of this type now known.

Se ae ee ee ee ee ee py a NP on Sees ~_-\i as
. = oe. (pM oue, ad ay eee ad P

SUCROCLASTIC ENZYMES IN BETA VULGARIS 271

Discussion OF THE ReEsvu.ts

vi earns soneiderations of interest to the plant physiologist accruing from
- this work group themselves around the following heads : —
(a) The topography and work of the enzymes.
as (6) The synthesis of a glucosidic compound, presumably sucrose, by
the action of beet sludge on invert sugar.
i (ec) The difference in magnitude of the results obtained in vivo as
fe coe geeg with that in vitro.

ex (a) 1. The topography of the enzymes here worked out may be

Leaf Stem Root
+ + 0
+ on + -
+ wi 0 -
0 7 +
0 + +

A’ significant tie in oe enzyme distribution is the absence
fi of invertase from the beet root. Kastle and Clark! found that
a x a inulin- and starch-producing plants such as the artichoke
.. and the potato, even in the tubers, when the inulin and starch were
undergoing storage, invertase was present in larger quantity than
either inulase or diastase. In the beet root, when sucrose is being
stored the appropriate enzyme for its formation and hydrolysis is absent.
Since in the artichoke and potato respectively the inulin and starch are
formed in loco, the conclusion to be drawn, on analogy, in regard to the
_ beet seems to favour the hypothesis that sucrose is not formed in loco in
_ the root, but is only stored there after undergoing translocation as sucrose
_ from the other organs of the plant.

% iN (a) 2. The varied associations of the enzymes in the different plant
= are is a striking point. To take the case of diastase, which occurs in
all paris of the beet, it is found that in the leaf it is associated with
invertase and maltase, in the stem with emulsin in addition, while in the
root a third variation in the environment of activation of diastase is
produced by its association with maltase, inulase and emulsin. The
questions which naturally suggest themselves are as to whether the
diastatic activity is affected by the different environment in each case,
--—s and as to whether a given combination of enzymes may not inhibit the
e appearance of some other enzyme which @ priori might be expected to be
present. This latter bears on the absence of invertase in the root in an

1. Amer. Chem. Journ., Vol. XXX, p. 422, 1003.

272 BLO-CHEMICAL JOURNAL

environment containing an association of diastase, maltase, inulase and
emulsin. That some interaction of the enzymes of an acceleratory,
retardatory or inhibitory nature occurs is quite in agreement with the
doctrine propounded on the result of experimental research by several
investigators?,

(a) 3. Besides the differences in the environmental spheres of activation
resulting from the varied association of the enzymes themselves in the
different plant organs, other factors enter to complicate the result. For
example, the associated enzymes of the leaf are subjected to different
light conditions from those in the root; in the leaf to alternation of light
and darkness, in the root to continuous darkness. That light affects the
results in the case of invertase action is shown from the recent researches
of Kohl,? who has found that in darkness inversion gives place to the
opposite process sooner than in light. Girard’s discovery® that the amount
of sucrose in the beet leaf is much increased towards evening may bear
directly on this point. Other factors affecting the result are, the presence
of products of hydrolysis, differences in the osmotic pressure in the cells
of various organs, and differences in the reactions of the cell contents.
In the last two cases the ‘intensity factor’! would be affected, on the
one hand by alteration in the concentration of the enzyme and on the
other from the acceleration or retardation of the enzyme action due to
increased or diminished acidity.

(b) 1. Of the experiments on reversible zymolysis the first series
made with pure enzyme extracts of beet leaves on invert-sugar substrates
gave no positive results. It is, however, a common experience that pure
enzyme extracts are less active than sludges. This was strikingly shown
in the negative results obtained in testing for invertase by the action of
the leaf extracts on sucrose, whereas, when the sludge was substituted,
invertase was proved to be present. The presumption is that by the action
of the leaf sludge on invert sugar, reversible zymolysis would have taken.
place. The second series of experiments with stem sludge on invert sugar
gave figures corresponding with the presence of 4 per cent. of sucrose—a_
result to be expected from the ascertained presence of invertase in the
stem. The third series with the sludge of seedling plants gave the higher
figure (6 per cent. sucrose), indicating the greater activity of the plant at
the beginning of the storage as compared with the diminished activity of

1. Bayliss, The Nature of Enzyme Action, p. 657, and references.
2. Abstr. Bot. Centr., Vol. CVITI, p. 137, 1908.

3. Compt. Rend., T. XCVIT, p. 1305, 1883.

4. Visser, Zeit. f. physik. Chem., Vol. LIT, p. 283, 1905.

BA “a a
: i “ pie. 3 Fa 4. 2

SUCROCLASTIC ENZYMES IN BETA VULGARIS 278

the older plant at the end of the storage period. The reasons for regarding
the products as sucrose have already been stated.

(b) 2. That the cane-sugar stored in the beet root is formed from
antecedent monosaccharides by reversible zymolysis in the organs
containing invertase (viz., in the leaf and stem), and thence translocated
as such seems highly probable.

The alternative view that the sugars travel downwards as
monosaccharides and are subsequently condensed into the disaccharide!
meets the diffusion difficulty. The absence of invertase from the root as
here shown, and the results of Strohmer’s researches proving that
practically no reducing sugars occur in the root, while, according to
Girard?, sucrose is present in all parts of the plant in the earliest stages

___ f development, militate against this view.

(6) 3. In relation to the translocatory difficulty in the first view,

reference may be made to the researches of Hanstein® and Puriewitsch*

on the diffusion of disaccharides in the maize endosperm, to Peklo’s
recent discovery® that the sieve tubes of the beet serve as_ the
sugar-conducting channels, and to Pfeffer’s dictum on the great
regulatory faculty of the cell cytoplasm in relation to the materials to
be diffused.

(6) 4. That the metabolism of the sucrose during the second spring to
supply the young shoots takes place in the stem® is supported by the fact
that invertase is found in that organ.

(c) 1. The apparently relatively unlimited quantity of disaccharides
capable of being formed in the living plant is in marked contrast to the
small results obtained experimentally in vitro. This is not surprising
when it is remembered that the reaction is presumably a reversible one.

_In vitro the equilibrium stage attained is permanent owing to the fact that

the reaction products are not removed, while in the plant the equilibrium
phase is only momentary owing to the continual removal of the products
as they are formed.

Maquenne, Compt, Rend., 1. CXXI, p. 834, 1805,
Compt. Rend., 'T. CLL, 1887,

Flora, p. 419, 1894.

Ber. d. Bot. Gea., p. 206, 1896.

Bot. Centr., Vol. CVIIL, p. 239, 1908.

Strohmer, loc. cit.

ere ep

274

THE OUTPUT OF ORGANIC PHOSPHORUS IN URINE

b

By G. C. MATHISON, M.B., B.S. (Mexn.), Sharpey Scholar.
From the Physiological Laboratory, University College, London
(Received April 22nd, 1909)

The existence of organic phosphorus compounds in normal urine has
often been asserted and as often denied. In a previous paper (1) I have
shown their undoubted existence. The present investigation deals with
the quantity of organic PO, excreted in the urine of healthy persons on
an ordinary diet. The results obtained by many previous workers are un-
reliable owing to the employment of unsuitable methods. Ehrstrom (2),
Gumlich (3), Keller (4), Le Clere and Cook (5), and older workers such as
Lépine (6) and Zuelzer(7) attempted to determine organic P,O, by
methods entailing titration of urine with uranium acetate. This method
gives a value greater than inorganic, indeed sometimes greater than total
P,O,, so that it is valueless for the determination of organic P,O,. —

Le Clere and Cook (5), whose dictum is quoted with approval by
F. G. Benedict (8), state that there is insufficient evidence of the existence
of organic phosphorus, despite the fact that in twenty-four hours’ urine
from a dog they obtained a difference of 0°093 gram P,O, between total
and inorganic, in rabbits a difference of 0°055 gram. Even though one
agree with these workers that the method they employ is not sufficiently
exact to afford evidence of the existence of organic phosphorus, one
cannot agree that it is sufficiently exact to afford evidence against such
existence. .

Oertel (9) was the first to use a sound method of estimating organic
phosphorus. He precipitated phosphates by means of calcium chloride,
and determined organic PO, in the filtrate. This was evaporated to
dryness, fused with KOH and KNO,, precipitated with ammonium
molybdate, dissolved in ammonia, re-precipitated with magnesia mixture,
incinerated and estimated as pyrophosphate. The process is thus very
lengthy and involves several manipulations.

Oertel obtained values for the output of organic P,O, in twenty-four
hours ranging from 0°12 gram (5 per cent. of total P,O,) to 0°03 gram,

(15 per cent, of total P,O,). He considers 0°05 gram to be the usual ~

ORGANIC PHOSPHORUS IN URINE +275

_ quantity. Mandel and Oertel (10), employing the same method, found in
the twenty-four hours’ urine of three individuals an average of 0°024
gram organic P,O,, equal to about 2 per cent. of the total.

Bornstein (11) made some observations on the output of inorganic and
organic PO, on an ordinary diet and on plasmon. He estimated organic
PO, in the filtrate after barium chloride, by a method somewhat similar
to that of Oertel. His results are here summarised :—

Percentage of
Nitrogen Total P,O, Organic P,O, P,O;
as Organic
Average of l4days ... one 14-0 2-09 0-058 2-8
Highest values sins — 14-2 1-82 0-16 8-8

Bornstein himself considers that there is some mistake in the highest
gee, and rejects them in his average.
Bock (12) used methods entailing uranium acetate titration, but also
fin some cases estimated total P,O, by Neumann’s method, and inorganic
P 2U, with calcium chloride or barium chloride. In rabbits he found as
much as 0°29 gram of organic P,O,, equal to 11 per cent. of total, in
_ twenty-four hours’ urine, in cats from 0°04 gram, 26 5 per cent., up to
0-20 gram, 11 per cent.

The present observations were carried out on the urines of healthy
3 individuals on an every day diet during January, February and March.
. All these persons followed sedentary occupations. The estimations were
-——s garried out by the methods described in a previous paper (1).
4 Samples of urine from different individuals gave the following

values : —
Tanne I—InorGanic ann OrGanic Puosruorvs

Grams P,O, in 100 c.c. urine

” Tnorganic P.O Percentage of
Total P,O, PO, pee cee PO, as
By difference In filtrate Organic °
8. 0-092 0-086 0-006 0-004 55
K (0-124 O117 0-005 0-005 40
re : (0-121
. p (0-148 ( 0-145 (0-003 5-0
| ? (0-145 , 0138 (0-007 0-007
ps: M | 0-008 0-090 0-008 O-O10 9-0
a : > . “ee “ee 0-009

Since these results were considerably higher than those usually
cited, further observations were made on the daily output of different
individuals, usually over a period of several consecutive days.

276 BIO-CHEMICAL JOURNAL
Taste [I—Ovrrevr or Organic PO, ry 24 Hours
Grams P,O,

: : wf ’
Subject Day Quantity a Po, OF. FOr One Po,"

M. act.25 = I 1320 — 3-00 0-303 10-1 _
Ir 1030 — 2-30 0-155 6-7 _
i Il 1000 — 2-43 0-180 75 —
a IV 1240 — 2-75 0-148 5-4 ~
ie V 1070 _ 2-45 0-225 9-0 —
Average — — 2-58 0-202 77 _—
M. aet.25 I 1200 16-44 2-41 0-168 7-0 6-8
II 1140 12-77 1-90 0-114 6-0 68
Il 1200 15-28 2-45 0-120 4-9 6-2
ve IV 1050 14-59 2-05 0-121 6-2 Tl
: V 1500 15-90 2-05 0-180 78 6-9
i VI 1050 13-46 2-31 0-140 6-0 65
Average — 14-74 2-19 0-140 6-3 6-7
L.aet.27. I 1100 10-18 2-10 0-104 5-0 4-9
: I 1230 9-76 1-90 0-041 21 5

; I 1400 11-02 2-41 0-063 2-5 4-6

Average — 10-32 2-13 0-068 3-2 49

D. aet.40 = 1850. 16-63 2-84 0-27 9-5 5-9
a II 1150 14-78 2-61 0-01 0-4 5-6

uj Iu 2050 16-43 2-77 0-15 55 5-9
Average oe 15-94 2-59 0-14 51 58
P.aet.30 =I 1700 13-71 2-32 0-223 10-0 59

Organic P,O, averaged 0°15 gram per diem, equal to 62 per cent.

of total P,O,.

The highest output was 0°35 gram, the lowest 0:04 gram.
It will be noticed that the N : P,O, ratio was fairly constant in any

one individual, but that it varied greatly in different individuals.

The values obtained for organic phosphorus are considerably higher
than those cited by most other workers, even by those who employed
accurate methods. This difference must be ascribed in the latter cases to
individual variations in output. :

Tue Errecr or INGEesrion or GiycerorpHosPHoRIC ACID

Although the subject was not on a rigid diet, it was thought worth
while to try the effect of adding a large amount of organic phosphorus,
in the form of glycerophosphoric acid, to the diet. For several days an
approximately similar diet was adhered to, except that on one day
glycerophosphoric acid was added. ‘Two series of observations were made
on the same subject.

ORGANIC PHOSPHORUS IN URINE 277

Tasie Ll]—Krrecr or Incestion or GrycerorHospHoric Acip

(i) Glycerophosphoric acid (Merck) containing 1-44 grams Organic P,O,, 0-075 grams
Inorganic P,O, added to diet early on sixth day.

. Subject Day Nitrogen Total P,O, Organic P,O, 20s = ma

-~‘Maet.25 LIV 14-99 2-24 0-146 6-4 6-5
fat (average)

» Vv 13-46 2-07 0-140 6-0 65

” Vis 13-6 2-76 0-165 6-0 438

ae vil 13-77 2-29 0-162 7-0 6-0

Si (ii) Sodium Glycerophosphate, containing 2-4 grams Organic P,O, and 0°125 grams Inorganic
____ PO, added to diet on second day.

oo I 14-2 2-47 0-168 6-8 5-7

bs Ii* 10-44 3-73 0-113 3-0 2-8

ae a Il 10-55 0-16 O-174 8-0 4-9
i IV 14-63 2-74 0-150 5-3 5-3

‘The increase of organic P,O, is well within normal variations—no
significance attaches to it. The same might be said of the increase of
total P,O,, but for the marked alteration in the N : P,O, ratio. It is
obvious that a great part of the ingested SAvcarcullinaphite has been
_exereted as inorganic phosphate; it is probable that a considerable
portion was not absorbed and would be found in the faeces.

The experiments of Bergmann (13) are of some interest in this
connection. He injected into a dog subcutaneously several grams of organic
P.O, in the form of glycerophosphoric acid. He found a marked increase
in the inorganic P,O,, none in the organic. He used titration methods
whieh would only show large changes. The increase in inorganic P,O,
was so great, however, as to leave no doubt that the glycerophosphoric
acid had been decomposed here without intervention of alimentary
ae processes. It has been asserted that many organic phosphorus compounds
are absorbed as such. To test the probability of this assertion, I have
subjected sodium glycerophosphate solutions to the action of active
preparations of pepsin, of trypsin, and of fresh pancreatic juice, both with
and without enterokinase.' The solutions were incubated for weeks at
39°C. Inorganic phosphates were estimated at the beginning and at
intervals during the experiment. In no case was any increase in the
inorganic phosphates found; the glycerophosphate remained unchanged.

. 1, The juice was obtained from dogs after injection of secretin. It was used without
enterokinase because some authors have asserted that enterokinase destroys the lipase
present.

278 BIO-CHEMICAL JOURNAL

It is probable, therefore, that the ingested glycerophosphate in the
experiments detailed above was absorbed unchanged. As the glycero-
phosphate used was synthetic it does not follow that natural glycero-
phosphoric acid is unaffected by digestive processes.

Tue Errecr or Exercise

This was investigated on two occasions. The urine was collected
over four or five days, an approximately regular diet being taken during
this period. On the second day a sharp twenty mile walk was taken, on
the other days no exercise beyond leisurely walking a couple of miles.
The walk was followed on both occasions by slight stiffness, but beyond
this no fatigue was felt.

Taste LV —Errecr or EXercisé

Subject Day Quantity Nitrogen Total P,O, Organic P,O, Percentage P,O; _N
as Organic

PO;

M.aet.25 I 1440 15-10 2-769 0-181 6-5 5-5
e: * 1550 15-23 2-81 0-168 5-9 54

Fy Itt 1200 13-96 2-58 0-114 4-4 61

a IV 1320 16-01 2-93 0-146 5-0 5-4

V 1510 14-30 2-71 0-110 41 53

5 I 1360 13-09 2-18 0-163 1-4 6-0

= II* 1710 14-01 2-44 0-171 7-0 57

Me Il 1450 15-48 2-70 0-120 47 57

. IV 1250 13-94 2-67 0-134 5-0 5-2

*Twenty mile walk during first half of this day.

These figures do not show any increase of organic P,O, after exercise.
The diet was not sufficiently rigid to enable any deductions to be drawn
from the nitrogen and total P.O, figures.

No statement can as yet be made as to the origin of the organic
phosphorus of urine. As far as can be gathered from the present results
and from a long series of observations, on which Dr. Aders Plimmer is
at present engaged, the quantity of organic P.O, is not affected by food.
It is thought that some indication of its origin may be given by
investigation of pathological conditions in which gross changes in
lymphoid or nervous tissues are present.

ORGANIC PHOSPHORUS IN URINE 279

SuMMARY

" aT, Organic phosphorus compounds are normally present in the
urine. Contrary statements are due to the employment of incorrect
methods.

2. In young adults on an sanlikiog diet the organic P,O, was
usually more than 01 gram per day. Occasionally it fell titer this,
and in one case it reached 0°3 gram. f

,, 3. The percentage of the total PO, present in organic combination
anes considerably from day to day. In the cases examined it averaged
6 per cent. of the total.

ae 4. The addition of a large quantity of organic phosphorus in the
form of glycerophosphoric acid to the diet had no distinct effect on the
m output of organic P,O,, while it increased the total P,O; output.
_ Glycerophosphoric acid was not broken down by gastric or pancreatic
digestion in vitro, so it was probably absorbed unchanged.

| 5. In the observations made, vigorous exercise was not followed
by increased output of organic P,O,.

6. The N_ : P,O, ratio was fairly constant in any one

individual on a fairly regular diet. It differed greatly in different

individuals, and also in the same individual when the diet was irregular.

REFERENCES

Mathison, This Volume p. 233.

Ehrstrom, Skand. Archiv., p. 83, 1903.

Gumlich, Zeitech. /. physiol. Chem., Vol. XVITI, p. 508, 1894.
Keller, Zeitsch. /. physiol. Chem., Vol. XXIX, p. 146, 1900.

Le Clere and Cook, Journ. Biol. Chem., Vol. II, p. 203, 1906-7.
Lépine, Comp. Rend. Acad. des Sciences, Vol. XCVIII, p. 238, 1884.
Zuelzer, Semiologie des Harns quoted by Keller.

F. G. Benedict, Metabolism in Inanition, 1907, p. 410.

Ocrtel, Zeitech. {. physiol. Chem., Vol. XXVI, p. 123, 1898.
Mandel and Oertel, New York Univ. Bull. of Med. Sciences, Vol. I, p. 165, 1901.
Bornstein, Pfliger’s Archiv., Vol. CVI, p. 66, 1904-5.

Bock., Arch, /. Exp., Path. u. Pharm., Vol. LVIII, p. 236, 1907.
Bergmann, Arch. /. Exp. Path. u, Pharm., Vol. XLVII, p. 76, 1901.
Plimmer and Bayliss, Jour. of Physiol., Vol. XX XIII, p. 439, 1906.

PERE Seesueaeere

280

ON THE RELATIVE HAEMOGLOBIN-VALUE OF THE
RESISTANT ERYTHROCYTES DURING THE HAE-
MOLYSIS OF BLOOD WITH HYPOSMOTIC SODIUM
CHLORIDE SOLUTION, AND ON THE PERMEABILITY
OF THE ERYTHROCYTES TO WATER AS A FACTOR
IN THE PRODUCTION OF HAEMOLYSIS

By U. N. BRAHMACHARI, M.A., M.D., PH.D., Lecturer in Medicine
at the Campbell Medical School, Calcutta.

(Received May 10th, 1909)

In a previous paper! I have pointed out that the dark coloration
described by Wright, and obtained by mixing one part of blood with two
parts of a progressive dilution of saline does not represent the point of
complete haemolysis. This point is obtained in the observations of

McCay? and my own observations by mixing one part of blood with two

parts of on to aS saline solution. It may, for the sake of convenience,

40 50
be called Wright’s haemolytic point.

This point probably represents the stage at which a large number of
the erythrocytes undergo haemolysis as the result of osmosis and rupture.
The corpuscles that do not haemolyse at Wright’s haemolytic point will
be termed in this paper the resistant corpuscles.

By quantitatively estimating the amount of dissolved haemoglobin in
20 cb.mm. of the supernatant fluid obtained after centrifugalisation of a

mixture of blood and two volumes of 2 ‘saline solution, where 2 is any
. x
number from 20 upwards, I have made out the curve of haemolysis with

hyposmotie saline solutions (see fig. 1). |
From the curve below it will be seen that the very beginning of

haemolysis starts with an saline solution. Then the degree of haemolysis

i N N . N NV sees
suddenly increases from 50 to 30 saline. From 30 to a5 = it

is somewhat gradual, while from Po upwards it increases very slightly

with the higher dilutions.

1. Bio-Chemical Journal, Vol. TV, p. 59, 1909.
2. Ibid., Vol. III, p. 97, 1907.

P
a

81

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282 BIO-CHEMICAL JOURNAL

The fact that some of the erythrocytes haemolyse with higher
dilutions of saline than others leads to the conclusion that either they
are less permeable to water or they can bear the tension of distension
from osmosis better than others, and therefore do not rupture so readily.
I have, however, already pointed out that osmosis and rupture alone
cannot explain the whole phenomenon of haemolysis with hyposmotie
saline solutions, and that one has to take the question of mass action into
consideration in explaining it’. The presence of erythrocytes containing
partially discharged haemoglobin among the sediment corpuscles goes
against the theory of rupture.

The relation of the amount of haemoglobin in the resistant corpuscles
to the total amount in the sample of blood under examination appears to
me from observations in health and disease to have an important bearing,
and I would suggest that this be called the relative haemoglobin-value of
the resistant erythrocytes. It may be expressed as the quotient obtained by
dividing the amount of haemoglobin in the resistant corpuscles by that
of the total blood. |

The method by which I estimated the amount of haemoglobin in the
resistant corpuscles is described as follows:—In all cases the blood was

haemolysed with two parts of a saline solution, with which in the case

of healthy individuals Wright’s haemolytic point is with certainty
obtained. After thoroughly mixing 5 cb.mm. of the blood with 10 cb.mm.

of x saline, the mixture is centrifugalised as thoroughly as possible,

and then the sediment is washed several times with N saline till the

10

supernatant fluid at the top is perfectly colourless. The sediment is now
dissolved in a small quantity of distilled water with the addition of a
drop or two of chloroform, and then the amount of haemoglobin is
estimated by a Haldane’s haemoglobinometer. In those cases in which _
the amount of haemoglobin in the resistant corpuscles is less than 10 per
cent., 10 or 20 cb.mm. of blood is taken and then treated with 20 or 40

cb.mm. of x saline respectively, and the amount of haemoglobin in the

resistant corpuscles is then estimated. This number divided by two or
four, as the case may be, gives the amount of haemoglobin in the resistant
corpuscles of 5 cb.mm. of blood.

The accompanying table gives the relative haemoglobin-value of the
resistant corpuscles in the blood of some of my students as well as in some

cases of anaemia in my wards :—
1. Loe, cit.

—_—
eeseeees

HAEMOLYSIS OF BLOOD

TasLe I1.—Heatra

Haemoglobin in
ie tedanent

Corpuscles in
5 cb.mm. of
blood

ERESESER

ANAEMIA

_ Tasre I. 3

Haemoglobin in
the Resistant
Co cles in
5 cb:mm. of

blood

Taste III

Haemoglobin in |

the Resistant
Corpuscles in
5 cb.mm. of
blood

Relative Haemo-
globin-value of
the Resistant
Corpuscles

0-336
0-416
0-416
0-391

Relative Haemo-
globin-value of
the Resistant
Corpuscles

0-200
0-133
0-285
0-277

Relative Haemo-
globin-value of
the Resistant

erythrocytes

0-350
0-413
0-467
0-417

Relative Haemo-
globin-value of
the Resistant
erythrocytes

0-571
0-547

283

It will be seen from the above tables that while in health the relative
haemoglobin-value of the erythrocytes varies within small limits; in

anaemia it varies within much wider limits.

Thus, in some cases, it is

284 BIO-CHEMICAL JOURNAL

much below the normal, in others it is almost the same as normal, while
in others again it is above the normal. In kala-azar it is generally below
the normal, while in ankylostomiasis it is above the normal. The forms
of anaemia in which this value is increased or diminished and its clinical
significance can only be determined by further investigation.

PERMEABILITY OF THE ERYTHROCYTES TO WATER AS A FACTOR IN THE
Propvuction oF HAEMOLYSIS

An explanation may here be offered as to the cause of the differences
of the haemoglobin-value of the resistant corpuscles in health and disease.
It is possible that the resistant corpuscles are less permeable to water or
can bear the tension of distension better than those that haemolyse. This
permeability, or the power of resisting rupture, is probably altered in
anaemia, being increased in some and diminished in others, while in
others again it remains normal.

The entrance of water into the erythrocytes may, therefore, to some
extent, be dependent upon their specific permeability, and this may be
independent of the force of osmosis. So, also, their power of resisting
rupture from distension after the entrance of water into their substance
may vary in the different erythrocytes. That these are important factors
in the phenomenon of haemolysis is borne out by the following facts :—

If one part of human blood is mixed with one part of ee saline solution

and then treated with two parts of = saline, we find that the amount

of haemolysis is much greater than when the pil saline contains 1 per —

10
cent. formol. The presence of formol cannot in any way change the
concentration of the salts in the corpuscles, and its action must result
either in increasing the resistance of the erythrocytes to rupture from
osmotic distension or diminishing the permeability of water. Similarly,
again, when blood is allowed to crenate between the slides for twenty-four

hours or more, and then treated with = saline soiution, we find that
some of them still remain crenated. Now, if crenation were simply due
to osmosis, then the corpuscles would swell up and lose their crenation

by re-absorption of water when ‘treated with * saline solution. The

fact that some of them do not lose their crenation shows that they have
become less permeable to water. In other words, along with crenation
the outer portion of the erythrocytes undergo some changes, as a result

ir.

HAEMOLYSIS OF BLOOD
wel do not allow the free passage of water into their structure
| osmosis. The same is also borne out by the fact that

) gipaiolaer aaa -eceur in the blood in some forms of
This cannot be due to any stronger concentration of saline in

1, as in anaemia the organ _ Benes much rt egies increased
: m (see Tables IV and V). ee

Ryyokeoniina YRAmeaiow

2 Panes se 7 8

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PAM brati <

286

THE ISOLATION OF THE CONIUM ALKALOIDS FROM
ANIMAL TISSUES, AND THE ACTION OF LIVING
CELLS AND DECOMPOSING ORGANS ON THESE
ALKALOIDS

By WALTER J. DILLING, M.B., Cu.B. (Abdn.), Carnegie Scholar in
Pharmacology.

From the Laboratory of the Institute of Pharmacology, University of
Rostock, Germany

(Received May 10th, 1909)

The literature on the isolation of the conium alkaloids is very limited,
and the only treatises requiring mention are those of Harley! and
Zalewsky.? Zalewsky’s method consisted in extracting the organs with
acidified water, followed by alcohol, and in shaking out the final residue
with petroleum ether after making it alkaline with ammonia. This author —
has given no quantitative results, and from the fact that he frequently
describes scaffold-like crystals as occurring in the final residue, one is
inclined to believe that the residue in many cases consisted of ammonium
chloride, which gives dense precipitates with phospho-molybdie acid, as
he describes.

PRELIMINARY OBSERVATIONS

On account of the volatility of pure coniine, it is impossible to obtain
a satisfactory result unless the base is converted into one of its salts which
are not volatile at 100°C. The hydrochlorate of coniine is very suitable
for the purpose, as it crystallises in long, double-refracting, silky needles,
which are easily recognisable. Coming to the estimation of the amount
of coniine present, there is a choice of three methods, namely :—(1) The
weight of the residue; (2) estimation of pure coniine by titration with
normal acid using suitable indicators, such as iodeosin, haematoxylin,
cochineal, lacmoid, or congo red; (3) estimation of the alkaloid or its salts
by titration with some precipitating reagent, such as Mayer’s solution.
In the following experiments I have used a combination of the first and
third methods, and, in applying the latter, I have estimated the amount of
the alkaloid by the number of drops from a capillary tube required to

1. Old Vegetable Neurotics, pp. 19 and 80, 1869.
2. Untersuchungen tiber das Coniin. Dissert., Dorpat, p. 17, 1869.

CONIUM ALKALOIDS FROM ANIMAL TISSUES 287

titrate the residue which was acidulated with a definite quantity of dilute
hydrochloric acid. This method gave in test cases results which were
correct to the fourth decimal place in grams. It will be noted that in
many of the experiments the weight of the residue and the amount of
alkaloid present, as estimated by Mayer’s reagent, do not agree; this is
due to the fact that one cannot get a pure alkaloidal residue without
going through processes which would entail serious loss of the alkaloid.
In such cases the amounts estimated by Mayer’s reagent may be taken as
correct.

I. Isonarron or ContineE By DISTILLATION WITH AN ALKALI

_. The distillation method I have used was that ordinarily employed
for such purposes; I have not adopted the method of distilling in hydrogen
gas, as I found this unnecessary, also it is not readily applicable to the
distillation of animal organs. When coniine hydrochlorate is distilled
with sodium hydrate, I have found that a certain amount of ammonia is
present in the distillate, and also that the crystals of coniine hydrochlorate
re-obtained from the distillate are not nearly so strongly double-refracting
as they usually are. If 10 mg. of coniine hydrochlorate is used, the whole
of the coniine distils over in the first 12 to 20 c.c. of fluid, and the alkaloid,
when estimated by Mayer's reagent, was present to the amount of 9°5 mg.
On the other hand, by distilling the coniine hydrochlorate with sodium
carbonate, almost no ammonia is produced, and the crystals obtained from
the distillate are strongly double-refracting, and, again, when using
10 mg. of the salt the whole of the coniine is contained in the first 20 c.c.
of fluid distilled, and this amounted in test cases to exactly 10 mg.
Experiments done with sodium bicarbonate showed a more marked
production of ammonia but no effect on the refraction of the crystals.

When, however, coniine has to be isolated from organic matters there
is always found in the distillate a large amount of ammonia, and, on
evaporating the distillate with hydrochloric acid, ammonium chloride
forms the greatest proportion of the residue. This salt I have removed
by treating the residue with a mixture of two parts of absolute alcohol
and one part of ether, or with pure chloroform, which is much more
satisfactory. The organic matters were distilled immediately. after the
addition of the alkaloid.

288 BIO-CHEMICAL JOURNAL

(1) Jsolation from Urine

The residue, left after evaporation of the neutralised distillate, is
usually so large that it requires to be treated two or three times with the

above solvents.
200 c.c. human urine f 200 c.c. human urine

with 10 mg. coniine . with 10 mg. coniine
hydrochlorate hydrochlorate
Residue from chloroform ... Double-refracting, needle- ~_ ly double-refracting,
like crystals P -like espace and
iregular crystal, not
double-refracting*
Weight of residue iss 0-007 gms. a 0-013 gms. ies
Estimated alkaloid by Mayer ... 0-0054 gms. ose 0-0083 gms.
On boiling Mayer’s ppt. with
sodium-hydrate one ... Distinct smell of coniine ... Strong smell of coniine

From the above experiments it will be seen that 83 per cent. of 10 mg.
of coniine hydrochlorate can be regained from urine.

(2) Isolation from Blood

Forty c.c. of calf’s blood with 5 mg. of coniine hydrochlorate was
distilled with 100 c.c. of water and excess of sodium carbonate. The
crystals of the residue being not quite typical, they were made alkaline
by sodium hydrate and the freed base shaken out with ether and re-
converted into the hydrochlorate. The crystals obtained were long,
transparent, double-refracting needles. A solution of these in acidified
water gave dense precipitates with Dragendorff’s and Rohrbach’s reagents,
and also with phospho-wolframic and phospho-molybdic acids, and, on
boiling with lime water, a strong smell of coniine was. given off. This
shows that a sufficient amount of 5 mg. can be regained from 40 c.c. of
blood for identification purposes.

(3) Isolation from Liver
100 horse’s liver 100 gms. horse’s liver 100 horse’s liver

th 10 mg. coniine with 10 mg. coniine with 10 mg. coniine
hydrochlorate hydrochlorate hydroe ’
Residue from chloro- Long, needle-shaped = Long, needle-shaped § Pure, long, needle-
form crystals, strongly crystals, strongly shaped Be ba ve
double-refracting double-refracting strongly ble-
refracting
Weight of residue... 0-0045 gms. 0-005 gms. 0-0085 gms.
Estimated alkaloid by
Mayer ee oes 0-00416 gms. 0-005 gms. 0-0083 gms.
On_ boili pt. with
sodium hydrate ... Strong smell of coniine Strong smell of coniine Strong smell of coniine

These results demonstrate that one can obtain back from liver not
less than about half and not more than 83 per cent. of the coniine salt.

1. As to what the other crystals present were, one cannot give any opinion. Certainly
they did not seem to affect the titration by Mayer’s reagent.

at a
BEY as
ne
re
a
a
ae
.
>

CONIUM ALKALOIDS FROM ANIMAL TISSUES 289

(4) Isolation from Spleen}
Seen ’s spleen wi
= Ae horse’s sp with

. coniine hydro-
chlorate
Residue from chloroform 0 oat ave , transparent, double-
eae needles
Weight of residue Wee ood eee si 0-0085 gms.
On boiling Mayer’s ppt. with sodium hydrate ... Strong smell of coniine

Thus 83 per cent. of alkaloid can also be regained from spleen.

Il. Exrracrion or Contrne By TREATMENT WITH ALCOHOL AND
SvuBsEQUENTLY SHAKING OvT witH ETHER

The finely minced organs containing the alkaloidal hydrochlorate

_ were extracted three times with fresh portions of absolute alcohol. After

filtering, the alcohol was evaporated off and the residue again extracted
with fresh alcohol; this was filtered, evaporated, and the residue treated
with water, any insoluble matter being removed. The watery solution

was then made alkaline with sodium hydrate, and the freed base removed

by shaking out with ether. In earlier experiments, the ether was
evaporated at a low temperature, but in later cases it was acidified with
hydrochloric acid before evaporation, in order to obtain the crystalline
hydrochlorate. Urine was treated by the same process except that it was
evaporated to syrupy consistence with excess of hydrochloric acid, before
adding alcohol. When dealing with fatty organs, the ether frequently
separated as a muddy layer. This trouble has been avoided either by
previous shaking of the still acid water with ether or by evaporating the
muddy ether which has separated from the alkaline water with
hydrochloric acid, and extracting the residue with water, filtering, and
evaporating the watery solution.

(1) Eztraction from Urine

100 c.c. human urine 100 c.c, human urine
with 10 mg. coniine with 10 mg. coniine
hydrochlorate h orate
Residue from ether sav .. Double-refracting, needle- Double-refracting, needle-
like crystals and some like crystals and some
Weight of pe rte oe matter
Estimated alkaloids by Ma jun 0-0046 gms. ége 0-0054 gms.
On boili Mayer's ppt. with
sodium hydrate Ome .« Nosmell of coniine* .-» Distinct smell of coniine

The minced spleen containing the alkaloid was first treated with tannic acid in presence
Cl to precipitate with albumin as much of the fatty matter as possible. The filtrate was
then rather easier to distil. Tannic acid tot Guabealie of EICL does not precipitate coniine salts.

2. The probable explanation of this is that since I had been on with pure coniine
just before and as the nose becomes rapidly insensible to this smell, I had failed to detect the
slight odour which must have been present.

2
=-

290 BIO-CHEMICAL JOURNAL

(2) Extraction from Blood

Fifty c.c. of calf’s blood with 5 mg. coniine hydrochlorate. The blood
was first acidified and coagulated by heat, the coagulum was filtered off,
and the filtrate evaporated to a syrup and treated as with urine.

Residue from ether.—Long, double-refracting, needle-like crystals,
which, when dissolved in acidulated water, gave a dense precipitate with
Dragendorft’s reagent, but only slight precipitates with phospho-molybdic
and phospho-wolframic acids, and Rohrbach’s reagent; on boiling the
solution with excess of lime water a distinct smell of coniine was evolved.

(3) Extraction from Liver

The following table will give some idea of the results which are
obtained when the coniine is isolated as a free base. It also shows that
by the method of extraction one may isolate substances from liver which
show alkaloidal reactions. =e

ie eeaged ners prt fhe
Pig’s liver 80 gms. without alkaloid Slight yellow ppt. Faint white ppt. Faint yellow ppt. -
Wb je + 10 mg.coniine HCl Good yellow ppt. Slight white ppt. Slight yellow ppt.

- + 10 mg.coniine HCl Good yellow ppt. Faint white ppt. Faint yellow ppt.

In the following results the ether was acidified before evaporation.

= horse’s liver with go horse’s po
10 mg. coniine
Tichyadcoblacete
Residue from ether A ... Transparent, needle-like ... gpoemeine 5 double- |
crystals, slightly double- gp Arr ner arranged
refracti along with in sheaves
some resinous matter
Weight of residue eos ee 0-003 gms. PP —
Estimated alkaloids by Mayer... 0-0021 gms. ob 0-00125 gms.
On boiling Mayer’s Ppt. with
sodium hydrate .»» Faint smell of coniine .«-. Slight smell of coniine

(4) Eatraction from Spleen

~ One hundred grams horse’s spleen with 10 mg. coniine hydrochlorate.

Residue.—Long, double-refracting, needle-like crystals with some
resinous matter.

Weight of residue.—0°009 grams.
Estimated alkaloid by Mayer.—0°0029 grams.

On boiling Mayer’s precipitate with sodium hydrate.—Distinct smell
of coniine.

——————

—— es le

CONIUM ALKALOIDS FROM ANIMAL TISSUES 291

(5) Eatraction from Kidney
_ Fifteen grams rabbit’s kidney with 10 mg. coniine hydrochlorate.
Residue.—Long, double-refracting needles, which, when dissolved in
acid water, gave dense precipitates with alkaloidal reagents and gave off
a strong smell of coniine when boiled with lime water.

III. Precrerratioy or THE ALKALOID BY MEANs oF PuosrHo-WotrraMic
Acip

As phospho-wolframic acid gives precipitates with coniine hydro-
chlorate in presence of hydrochloric acid up to dilutions of 1 : 10000, the
following method was adopted for the isolation of the alkaloids by this
means:—The organs containing the alkaloid were coagulated by heat,
the coagulum removed, and the filtrate treated with phospho-wolframic
acid and some dilute hydrochloric acid till complete precipitation had
occurred. The precipitate was filtered off, washed with water containing

some phospho-wolframic acid, and then drained free of excess of fluid.

The partially dried precipitate was rubbed up in a mortar with excess of
barium hydrate and the freed alkaloid extracted with absolute alcohol.
After being acidified with hydrochloric acid, the alcohol was evaporated
off and the residue extracted with chloroform, filtered, and evaporated,
when one ought to obtain the hydrochlorate of coniine pure. Any barium
hydrate which dissolves in the aleohol may be removed by means of carbon
dioxide or by extracting the residue of chlorides as above with chloroform.
Urine was treated directly with the acids, but it is safer to precipitate it
twice, since the first precipitate is very copious.

(1) Precipitation from Urine

100 c.c. human urine 100 c.c. human urine
with 10 mg. coniine with 10 mg. coniine
hydrochlorate hydrochlorate
Residue from chloroform --- Double-refracting needles ... Double-refracting needles
Weight of residue te ove 0-008 gms. 7 0-006 gms.
alkaloid by Mayer ... 0-0042 gms. ai 0-0046 gms.
On boiling Mayer's ppt. wi
sodium hydrate... «» Faint smell of coniine ... Distinct smell of coniine

(2) Precipitation from Blood
Fifty ¢.c. calf’s blood with 5 mg. coniine hydrochlorate.
Residue.—Long, needle-like crystals, strongly double-refracting.
Weight of residue.—0°004 grams.
Estimated alkaloid by Mayer.—0'0017 grams.
On boiling Mayer's precipitate with sodium hydrate.—Faint smell
of coniine.

292° BIO-CHEMICAL JOURNAL

(3) Precipitation from Liver

100 horse’s liver 100 gms. horse’sliver 100 horse’s liver
with 10 mg. coniine — with 10 mg. coniine wi nydrcodlonenie

hydrochlorate hydroc lorate
Residue from chloro- Long, needle-shaped Double-refracting, orga double-
form crystals in sheaves ; needle-like fs crystals
strongly double- crystals er on fatty
refracting matter
Weight of residue... — 0-008 gms. 0-005 gms.
Estimated alkaloid by am
Mayer 0-0037 gms. 00025 gms. 0-0005 gms, '
On boiling Mayer's ppt.
with sodium hydrate Distinct smell of coniine Smell of coniine Faint, primer: recognisable
smell

(4) Precipitation from Spleen
One hundred grams horse’s spleen with 10 mg. coniine hydrochlorate.
Residue from chloroform.—Long, double-refracting, needle-like
crystals, |
Weight of residue.—0'008 grams.
Estimated alkaloid by Mayer.—0'0021 grams.
On boiling Mayer’s precipitate with sodium hydrate.—Distinct oni
of coniine.
(5) Precipitation from Kidney
Fifteen grams rabbit’s kidney with 10 mg. coniine hydrochlorate.
Residue from chloroform.—Double-refracting, needle-like crystals
which, dissolved in acid water, gave dense precipitates with alkaloidal
reagents and gave off a strong smell of coniine when boiled with lime
water.

ITV. Precrprration spy Kravut’s REAGENT

This process proved in my hands quite useless for coniine, as the
precipitate was of such a nature that it passed readily through a filter,
and, again, it was found impossible to get rid of the iodine completely.

CONCLUSIONS ON THE ISOLATION OF CONIINE

It will be observed that, in the case of the distillation, the best results
are in three cases 83 per cent., while with the alcohol and ether process
and precipitation method the results vary considerably, and in most
instances the results are considerably below 50 per cent. If one makes
an average of all the figures one finds that the average return by
distillation is 65°7 per cent., while the other two methods only show half —

1. This liver proved very difficult to filter after boiling.

CONIUM ALKALOIDS FROM ANIMAL TISSUES 293

this amount. Again, the distillation method can be carried through in
at most three hours, while the other two occupy eight hours at least, and
the alkaloidal salt obtained in the end is much purer in the case of

“distillation, as may be surmised by the small differences between the
weights of the residues and the amounts of alkaloid found by Mayer's

e ‘ reagent. Taking everything into account, only one conclusion is possible,
og that, for practical purposes and satisfactory results, distillation is the
7 . most valuable method for isolating coniine from tissues.

Action or Livine CEetts on ContTINE

Im order to ascertain whether living cells had any power of
decomposing coniine or in any way interfering with its recognition in the
animal organism after death, finely minced liver containing coniine
d lorate was mixed with 100 c.c. normal saline solution, to which
had been added 1 c.c. of chloroform and 1 c.c. of toluol to prevent

fe
= a8" C. for a definite period. The alkaloid was re-isolated by distillation.

gms. rabbit's 60gms. rabbit's 30 gms. rabbit's 190 gms. horse’s 100 gms. horse’s
bade with 20 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg.
coniine hydro- coniine hydro- coniine hydro- coniine hydro- coniine hydro-

chlorate chlorate chlorate chlorate
_ Time on water bath ... 16 hrs. 18 hrs. 24 hrs. 12 hrs. 18 hrs.
Residue from ane
pee double- Long needles, Some long, A few needle- A few long,
*releacting slightly double- like crystals, double-
double- refracting not double- refracting
a crystals refracting needles refracting crystals
= Weight of residue .- 00065 gms. 0-003 gms. 0-010 gms. 0-002 gms. 0-002 gms.
Estimated alkaloid ‘i
_ Mayer... «... 00065 gms. 00021 gms. 0-0037gms. No ppt. 0-0004 gms.
“7 with
oh cop Sg lem ... Distinct smell Faint smell of Distinct smell No smell Faint smell of
es of coniine coniine of coniine coniine

; _ All these livers were re-distilled with dilute sulphuric acid, the
s distillates neutralised with sodium carbonate and evaporated to dryness.
| To the residue absolute alcohol and excess of concentrated sulphuric acid
by. were added, and in all the above cases a very strong smell of butyric ether
Ye was evolved, mixed in some cases with the smell of acetic ether. A test
experiment, done with liver alone, gave a distinct smell of butyric acid,
zs but not so powerful as that of the above cases.

Conelusions.—If one takes the average amount of alkaloid which may
be regained from liver by distillation as 58 per cent.—a low estimate—
it will be seen from the table that there is a remarkable loss of alkaloid

294 BIO-CHEMICAL JOURNAL

after the cells of the liver have been allowed to act on it even for a short
time. ‘The powerful smell of butyric acid would seem to suggest that the
coniine may probably be broken up into this substance. Whether this
suggestion is correct or not, I am unable as yet to say, but I hope to follow
this up shortly. ¥

Action oF Decompostne Trssurs on CoNIINE

To ascertain whether decomposing tissues had any influence on
coniine, minced liver with the alkaloidal hydrochlorate was mixed with
100 c.c. of normal saline solution and left in a water bath at 38° C. for a
definite period. The alkaloid was isolated by distillation.

60 gms. rabbit’s 160 gms. horse’s 100 gms.horse’s 100 gms. horse’s 100 gms. horse’s

liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg.
coniine hydro- coniine hydro- coniine hydro- coniine hydro- coniine

chlorate chlorate chlorate chlorate
Time on water bath ... 18 hrs. 24 hrs. 24 hrs. 24 hrs. 24 hrs.
Residue from chloro- :
form +» «se Some trans- Dae. Detiasemnent Needle a lle
nt refracting needles, crystals, not
ouble- needles, not some double- needles, not
refracting typical of double- refracting typical of
crystals, coniine refracting, and not coniine
which were hydro- others not deliquescent jac
very deli- chlorate, c
quescent and very and very
deliquescent deliquescent
Weight of residue - 0-009 gms. 0-004 gms. 0-01 gms. 0-003 gms. 0-009 gms.
Estimated alkaloid by c
Mayer aoe - 0-005 gms. 00021 gms. 00-0025 gms. 90-0021 gms. 0-0008 gms.
Boiling Ppt. with
sodium hydrate ... —? Peculiar smell, —) Peculiar smell, Strong and
resembling resembling peculiar
ammonia ammonia

sm
; ammonia —
Conclusions.—It will be apparent that the maximum amount of
alkaloid found after twenty-four hours was 2°5 mg., but the crystals of
the residue were only partially double-refracting and, at the same time,
very deliquescent, while the smells evolved in all cases were ammonia-like.
In Experiment III it should have been easily possible to obtain the ;
uranium nitrate reaction for coniine, but this failed; in Experiment I,
however, a positive reaction was got, probably since the quantity of
liver was small and the time for decomposition short. I am, therefore,
of the opinion that no coniine was present in the distillate from the latter
four experiments, but that the substance obtained was a body possessing —

1. On treating the Mayer’s precipitate in this case with sodium carbonate, carbon
eee = uranium nitrate, and shaking up with toluol—no red colour was obtained
in the liqui

2. The same reaction in this case, however, gave a red colour in the toluol which would
indicate that coniine was present.

re

CONIUM ALKALOIDS FROM ANIMAL TISSUES 295

some of the characters of coniine hydrochlorate, while lacking others.
_ The suggestion is possible that many of these crystals were cholin bydro-
chlorate, which is double-refracting and very deliquescent. This salt also

erystallises in needles, and when boiled with sodium hydrate a smell of
trimethylamine is given off which would correspond closely to the smell
perceived above. The cholin hydrochlorate must have passed through the
__ filter in a deliquesced state along with the chloroform extract.

IsoLATION OF CONHYDRINE

, The isolation of conhydrine proved more difficult, on account of the
fact that it cannot be distilled from watery solutions, even when these are
saturated with calcium chloride or under pressures as low as 10 mm. of
_ mercury. Conhydrine is also less soluble in ether than in water, and
_ chloroform, in which it is readily soluble, gave unsatisfactory results, so
_ that it was discarded in favour of repeated shakings with fresh ether.
3 = conhydrine forms a very deliquescent hydrochlorate, and was
_ therefore isolated as the free base, care being taken to evaporate solutions
____ of this at low temperatures. With reference to the process of precipitation,
___ phospho-wolframic acid is again the only suitable method, but its delicacy
only reaches to dilutions of 1 : 1000. The extracts of the organs were
‘therefore evaporated to small bulk before adding the reagent, and urine
was first precipitated with lead acetate in presence of hydrochloric acid,
in order to avoid bulky precipitates with the reagent. For the estimation
of the amount of alkaloid recovered, phospho-wolframic acid was
_ substituted for Mayer’s reagent, as the latter is not sufficiently delicate.

(1) Extraction from Urine by Alcohol and Ether

5O0c.c. human 100c.c. human 100c.c. human 100c.c. human 200 c.c. human

: v wi urine with urine with urine with urine with
— 10 mg.conhy- 1l0mg.conhy- 1l0mg.conhy- 10 is ate conhy- 10 mg. conhy-
= drine rine rine rine + drine
ether - Double- Needle-like, Double- Double- Double-
4 i double- refracting, refracting refracting,
needles and refracting needle-like needles and needle and
a the erystals crystals flat, oblong plate -like
ae: having crystals, crystals, the
one angle cut having an latter having
vps out angle cut an angle cut
ea? es out out
ss Weight of residue - 0-008 gms. 0-005 gms. 0-012 gms. 0-015 gms. 0-014 gms.
Maid by phosp.-
7 wolf. acid ... 0-0012 gms. 0-0017 gms. 0-0045 gms. 0-0075 gms. 0-0037 gms.
hydrate ... Distinct smell of Distinct smell Strong smell of Very strong Strong smell of
eonhydrine of conhy- conhydrine smell of ydrine

rine

296 BIO-CHEMICAL JOURNAL
(2) Extraction from Liver ih: Alcohol and Ether .

s. horse’s liver "4 horse’s liver 100 horse’s liver
sas 10 mg. conhy- Ome. conhy- with 10 mg. Mus 1
drine
Residue from ether ... No shies! Neill fuadiale Double-
pe or double- crystals of ob bad
form with one .
. the angles cut out
Weight of residue --- 0-002 gms. 0-003 gms. 0-009 gms, 3
Estimated alkaloid by ; i
phosp.-wolf. acid . 0-0005 gms. 0°00075 gms. 0-0037 gms.
On si sang, Be ppt. with
ydrate - Faint smell of Distinct smell of Strong smell of
conhydrine conhydrine conhydrine

(1) Precipitation from Urine by Phospho-wolframic Acid

100 c.c, dog’s urine 100¢.c. humanurine 100 ¢.c. human urine —
with 10 mg. conhy- with10mg.conhy- with 10 a conhy-
drine drine

Residue from chloroform Colourless, imperfect, Imperfect, double- double-
double-refracting refracting crystals Ima, dub

crystals
Weight of residue --- 0-004 gms. 0-006 gms. ~ 0-009 gms.
Estimated alkaloid by
are -wolf, acid =... 0-0015 gms. 0-0025 gms. 0-006 gms.

ppt. with :
pariirey i al -.. Faint smell of conhy- Distinct smell of Strong smell of conhy-
drine . eonhydrine = — drine

(2) penises from Liver by Phospho-wolframie Acid

oo horse’s liver 100 gms. horse’s liver 100 gms. cow’s liver —
10 sheng conhy- with gin conhy- with 10 mg. conhy-
drine

Residue from chloroform lar, mee ive No crystals Tra’ t needles,
racting crystals double-refeaaia
and very deli-
quescent
Weight of residue --» 0-004 gms. 0-003 gms. 0-005 gms.
Estimated alkaloid by
phosp.-wolf. acid... 0-0012 gms. 0-0025 gms. _ 0-0025 gms.
On boili pt. with
sodium hydrate ... Distinct smell of Distinct smell of oe smell of -
conhydrine conhydrine ydrine

CONCLUSIONS ON THE ISOLATION OF CONHYDRINE

The figures given above show that, by neither of these two methods
are results got which are in any way constant. The higher figures are
those obtained from later experiments and show probably about the limit —
attainable by these processes. One can, however, say that conhydrine can |
be isolated from both urine and liver in quite appreciable amounts, even
when the quantity added consists of only 10 mg. in 100 c.c. or grams of
substance.

0 ees eS eee et

CONIUM ALKALOIDS FROM ANIMAL TISSUES 297

Action or Livinc Ceits on ConHYDRINE

ee “The method used was the same as that described for coniine. The
alkaloid was extracted by alcohol and ether.

100 gms. cow’s liver srs horse’s liver a . horse’s liver

with 10 conhy- 10 conhy- 10 mg. conhy-
dino dtine drine
Time on water bath... 18 hrs. 38 12 hrs.
Residue from ether ... Thin gummy layer, No crystals A few double-
no refracting
a like crystals
a Weight of residue ... 0-011 gms. 0-003 gms. 0-012 gms.
alkaloid by
_ phosp.-wolf. acid ... 0-00076 gms. 0-001 gms. : 0-0037 gms.
On bol
: i . Doubtful smell of Faint smell of conhy- Distinct smell of
eonhydrine drine conhydrine

Action or Decomposinc Tissues on CoNHYDRINE

_ The alkaloid was treated in the way described for coniine and isolated
by means of the alcohol and ether process.

100 gms. horse’s liver with 100 gms, horse’s liver with

10 mg. conhydrine 10 mg. conhydrine

Time on water bath oe 18 hrs. 18 hrs.

Residue from ether --- Gummy skin, no tals ... Resinous residue and some

= double-refracting, oblong
crystals

Weight of residue _ 0-008 gms. hs 0-008 gms.

Estimated alkaloid by

’ phosp.-wolf. acid i. 60-0015 gms. eee 0-002 gms.

sodium 5 sega ..» Faint smell of conhydrine ... Distinct smell of conhydrine

Conclusions.—The above results show that, on account of the fact that
the method of isolation does not give constant returns, it is impossible
to draw any definite conclusion as to whether or not conhydrine is affected
by the action of living cells or decomposed tissues.

IsoLaTION oF PssuD0-CONHYDRINE

On account of the small amount of this alkaloid which I possess, I
have limited this research to a very few experiments, which, however,
show fairly satisfactory results. The difficulties encountered in the
isolation of pseudo-conhydrine are the same as those detailed under
conhydrine, and they were avoided by the same methods. Pseudo-
conhydrine cannot be distilled from watery solutions.

ar

298 BLO-CHEMICAL JOURNAL
(1) Extraction from Urine by Aleohol and Ether

60 ‘p human oe 100c.c. human urine 200 ¢.c. human urine
wit mg. o- with 10mg. pseudo- with 10
* sanlericles yes

conhydrine conh
Residue from ether _... Double-refracting, Double-refracting, alae
needle-like small, needle-like small, needle-
crystals crystals
Weight of residue _... 0-014 gms. 0-006 gms. 0-007 gms.
Estimated alkaloid by
phosp.-wolf. acid ave 0-0062 gms. 0-0035 gms. 0-0017 gms.
On boiling ppt. with
sodium hydrate --. Strong smell of Distinct smell of Faint smell of

pseudo-conhydrine —_ pseudo-conhydrine —_ pseudo-conhydrine
(2) Extraction from Liver by Alcohol and Ether

100 horse’s liver 100 gms. horse’s liver —
with 10 mg. pseudo- with 10 pseudo-
conh ae conhytenas
Residue from ether ... eA No distinct crystals --- Small hair or needle-like
crystals, double-refracting
Weight of residue... ave 0-002 gms. -. 0-007 gms.
Estimated alkaloid by phos.-
wolf. acid ... bed ue 0-0005 gms.* «» 0-004 gms.
On boiling ppt. with sodium
hydrate... soe ‘ No smell of alkaloid .-- Distinct smell of pseudo-
, conhydrine

(1) Precipitation from Liver by Phospho-wolframie Acid
100 grams horse’s liver with 10 mg. pseudo-conhydrine.
Residue from chloroform.—No crystals.
Weight of residue.—0°002 grams.
Estimated alkaloid by phospho-wolframic acid.—0°0015 grams..
On boiling precipitate with sodium hydrate.—A faint smell of pseudo-

conhydrine.
Conclusions.—The results indicate that, with urine, it is possible to
isolate 35 per cent. of the alkaloid added, while, with liver, about 40 per
cent. can be regained. The single experiment done with phospho-wolframic
acid shows a fairly good return for that method, namely, 15 per cent.

Action or Decomposinc TissvuEs oN PsEUDO-CONHYDRINE

One experiment was done with this alkaloid by leaving 10 mg. in
100 grams of horse’s liver in a water bath for twelve hours, as described
for coniine. The alkaloid was extracted by alcohol and ether.

Residue from ether.—A few, irregular, double-refracting crystals in
a gummy matrix.

1. Liver difficult to filter.

CONIUM ALKALOIDS FROM ANIMAL TISSUES 299

2 Weight of residue.—0°003 grams.

Estimated alkaloid by phospho-wolframie acid.—00007 grams.

On boiling precipitate with sodium hydrate.—No smell which
resembled pseudo-conhydrine.

No definite conclusion can be drawn from this, since, in one case, as

small a quantity was regained from fresh liver, although that was due to

difficulties in carrying out the process.

ScuMMARY

1. The most satisfactory method for the isolation of coniine from

wall animal tissues is that of distillation.

é. ifiy 2. Coniine appears to be decomposed both by the action of living

i a cells and by decomposing tissues.

» a: Conhydrine and pseudo-conhydrine can be isolated from animal
tissues by extraction with alcohol and by precipitation with phospho-
wolframic acid, but these methods do not give sufficiently constant results
to allow of any definite conclusions being drawn as to the action of living

cells or decomposing tissues on these poisons.

I have, finally, to thank Professor Kobert of Rostock for procuring
for me the materials used in this research and for his kind assistance and
advice. I wish also to acknowledge my indebtedness to Professor
MacWilliam and Professor Cash for their courtesy in advising me with
regard to the arrangement of the results.

800

SOME OBSERVATIONS UPON THE ERROR IN THE
OPSONIC TECHNIQUE’

By ERNEST E. GLYNN, M.A., M.D. (Canras.), M.R.C.P., Lecturer in
Morbid Anatomy and Clinical Pathology, University of Liverpool,
Pathologist, Royal Infirmary, Liverpool, anv G. LISSANT COX,
M.A., M.B., B.C. (Canran.), Holt Fellow in Pathology, University of
Liverpool.

From the Department of Pathology, University of Liverpool
(Received May 14th, 1909)
INTRODUCTION

Anyone who has compared the figures obtained for the opsonic index
of the same serum as estimated quite independently by two observers, will
be aware that the difference between their results is often considerable
even after extensive experience of opsonic technique, and when the
precaution has been taken of enumerating many leucocytes.

We have recently calculated a large number of indices three times,
employing tubercle bacilli and staphylococci, and consider that a detailed
account of the experimental errors in our own work, together with a
résumé of the errors obtained by other workers, may be of some interest
to those engaged in this line of research. There also arises the larger
question: is the Wright technique, even including all its most recent
modifications, so hopelessly inaccurate that no deductions whatever can be
drawn from it? |

In a paper at present in the press? we have detailed the results of
calculating eighty consecutive indices, three times, ie., 240, with
staphylococcus, and forty consecutive indices, three times, i.e., 120 with
tubercle.

The staphylococcus indices were calculated on seventeen different
days, and the tubercle indices on ten different days, almost invariably
twelve indices, or four sets of three, on each day.

The technique of Wright and Douglas was adopted.

One half of the indices calculated were the result of comparing the
degree of phagocytosis obtained with different sera, but the same
leucocytes, i.e., they were opsonic indices; the other half the result of
comparing the degree of phagocytosis obtained with different strains of
leucocytes, but the same sera; these we have called ‘ Cytophagic Indices.’

1. The greater part of this paper formed a portion of a Thesis for the degree of M.D. Cantab.
2. Journal of Pathology and Bacteriology, Vol. XIV, No. 1.

ERRORS IN THE OPSONIC TECHNIQUE 301

_ According to the Wright School, the latter indices should always be
unity, because the “phagocytic power of corpuscles from different sources ’
_ is‘thesame."' We have demonstrated that the inherent phagocytic power
of corpuscles is not always the same, and the cytophagic indices in our
series of observations vary from about 1°2 to about 17.

This fact, however, does not affect the present question, viz., the
_——s aeeuracy of the Wright technique for measuring phagocytosis, and we
have included all our figures, both of opsonic and of cytophagic indices,
__—s im order to increase the number of observations available for statistical

; ‘The Method by which the indices were calculated is briefly as follows :—
A sample of serum and of corpuscles were drawn from three normal
i “men, *G,’ ‘L,’ and ‘ A,’ i.e., three samples of each were prepared. Twelve
_ separate phagocytic mixtures were put up from these, and the counts
of D ‘ined enabled us to calculate twelve indices, three opsonic indices for
__ *A’ and for ‘L’ respectively, and three cytophagic indices for ‘A’ and
: for ‘L’ respectively; the washed leucocytes and serum of ‘G’ furnishing
the control.
ae The method by which these indices were calculated from the various
_ combinations of sera and leucocytes is tabulated below.

Tasce I, Grvine THE various ComBINATIONS OF WasHED Levucocytes oF ‘G,’ ‘ L,’

ee ap ‘A,’ UsED IN THE Puacocytic Mrxrures, AND THE MEeTHODs oF CALCULATING -
i tHE Inpices, ‘G's’ Serum or LevucocyTes BEING USED As ‘ ConTROL’
Source of Serum in Source of Leucocytes Number of
phagocytic mixture in phagocytic mixture phagocytic mixture
A. A.) :
A. A. ) “** .
A. G. 2.
A. L. 3.
ia G. A. 4.
. Ga. G.) 5
: G. G.) :
4 G. L. 6.
i .
7 i + :
7 L. G. 8.
- L. L.)
L. Li 9.

Fractions used for the caleulation of ‘ A’s’ three opsonic and three
cytophagic indices :—
A's opsonic indices ane te ; ;
A's cytophagic indices eid ; : ;

| 1. Practitioner, May, 1908,

302 BIO-CHEMICAL JOURNAL

The same principle was adopted in the calculations of ‘ L’s’ indices.
It is clear that ‘ A’s’ three opsonic indices were obtained from six distinct
phagocytic mixtures; therefore, any difference between the three indices
is due not only to errors from counting, but also to errors in ‘putting up”
the phagocytic mixture, preparing and staining the films. This is a more
thorough and practical way of ascertaining the degree of error inherent
in Wright’s technique than that of recounting the same slide—a method
adopted by some observers.

It will be noticed that the counts obtained from phagocytic mixtures
1,5, and 9 are really the mean of two separate estimations. This tends to
increase the accuracy of our indices somewhat, and gives us a slight
advantage in comparing our errors with those of other writers. The same
holds good for the other sets of triple indices. |The reasons for this
somewhat complicated method of calculating the indices, together with
an example fully worked out with figures, are given elsewhere.

The bacterial emulsion was prepared as recently recommended by
Fleming.!

It is important to note that the washed erythrocytes of these three
individuals were not agglutinated by any of the combinations of sera
employed.

Fleming’ who recently contributed a valuable paper on the accuracy
of the opsonie technique from Wright’s* laboratory, states that ‘a
diminution in the number of washed corpuscles in the opsonic mixture
causes an increased amount of phagocytosis.’ In order, therefore, to
eliminate errors from this source and ensure that the relative amounts of
normal salt solution and washed corpuscles taken up in the opsonic pipettes
were the same in all the triple estimations, the tubes containing them
were placed vertically between the palms of the hand and vigorously
rolled to and fro immediately before a quantum was removed; thus the
corpuscles and salt solution were always well mixed.

Ineubation—The phagocytic mixtures, consisting of equal parts of
corpuscles, serum and bacterial emulsion in salt solution, were placed for
fifteen or twenty minutes in a patent incubator at 37°C. This apparatus
consists essentially of a metal box filled with water, into one side of which
two horizontal and parallel rows of narrow metal tubes are inserted.
Each tube is open to the air in front, but is surrounded by water which
is maintained at a constant temperature by a single flame of gas.

1. Practitioner, May, 1908.
2. Fleming, Practitioner, May, 1908, p. 618.
3. Wright, Lancet, 1907.

ERRORS IN THE OPSONIC TECHNIQUE 303

It was found, even when the apparatus was most carefully regulated,
that the temperature of the tubes in the centre of the upper row was liable
to be half a degree C. or more higher than in the periphery of the lower
a row, on account of deficient circulation of the water.

This defect was completely remedied by constantly agitating the
water in the incubator with a rotating paddle. We do not know whether
a difference of }°C. between the individual tubes will exert any
appreciable effect upon the amount of phagocytosis, but it is advisable
that all scientific apparatus should be as perfect as possible, especially in
the technique so full of pitfalls as the opsonic technique. The readings
of temperature were taken with a microscope from a thermometer
graduated in tenths of a degree C. After incubation the contents of the
a pipettes were remixed and three films prepared from each one.
| The smears were made in the usual way by placing the drop at one
, end of the slide and drawing it out with another narrower slide held at an
i? angle, great care being taken to make the termination of the smear aa
; rectangular as possible. (Vide diagram.)

DIAGRAM OF SLIDE wiTH BLOOD SMEAR.

Staining.—The staphylococcus films were stained by Leishman’s
method, using accurately measured quantities of stain and distilled water,
and rocking the slides at intervals to ensure uniform staining—a very
important point. The tubercle films were fixed in saturated corrosive
sublimate solution, and stained by pouring upon them boiling carbol
fuschin, decolourised in 2 per cent. sulphuric acid, washed with 5 per

* cent acetic acid, and counter-stained in methylene blue.

Counting.—The number of bacteria phagocytosed by sixty polymor-
phonuclear leucocytes were enumerated in two out of the three films made
from each phagocytic mixture. Thirty cells were taken from the two
borders beginning at the corners B 2 and passing backwards to B1. Thus
the same part of every film was examined. All the eosinophiles and all ”
polymorphs with indistinct edges owing to damaged cytoplasm were

304 BIO-CHEMICAL JOURNAL

eliminated from the counts. Clumps of more than three leucocytes were
also omitted.

When the batch of films was finished, the figures were added up, and
if there was a deviation from the arithmetic mean of the counts of any two
duplicate films of more than 5 per cent., another sixty leucocytes were
taken from the third uncounted film and an average struck from the three
sets of figures. Half the total number of leucocytes counted (120) were
selected from one film and half from another, in order to diminish the
errors due to improper smearing, unequal staining, or inaccurate
counting, and, lastly, because it is obviously advantageous, when there
is a marked difference between the figures obtained from the two films,
to count an additional sixty from the third film.

If the figures when added up were contrary to expectation, recounts
were not made, nor were an extra sixty cells counted in the hope that
‘all might be well.’ In fact, no recounts were made except in the case
of some half dozen slides, because the counter was interrupted or found
his attention wandering. No counts were discarded on the grounds that
the films were improperly stained. The usual number of polymorpho-
nuclear leucocytes counted was sixty, but occasionally eighty or fifty,
according to the strength of the emulsion. The number once fixed by an
examination of the first film was invariably the same for all the batch.
As duplicate films were used, it is clear that the total number of cells
counted from each phagocytic mixture was usually 120, sometimes 180
or 100.

Elimination of Auto-suggestion—In order to eliminate any auto-
suggestion in counting, all the stained films, usually twenty-four in all,
were placed in numbered compartments, the figures upon them being
rubbed off. An attendant handed these unnumbered films indiscriminately
to the observer, who thus remained ignorant of which films he was
counting.

Every film was counted by the same individual, E. E. G.

STATEMENTS OF THE Wricut Scuoot Recarpinc Errors or TecuniQue

Three definite statements have been made by the Wright School
regarding the accuracy of the method.

1. In 1907 Wright! said the error ‘in the case of normal bloods in
the hands of a good worker ’ is ‘ rarely greater than plus or minus 5 per cent.’

1. Lancet, p. 427, August 17, 1907.

ERRORS IN THE OPSONIC TECHNIQUE 805

2. Next year Fleming,+ who had carefully investigated the
matter, concluded that ‘duplicate estimations of the tuberculo-opsonic
index of tuberculous patients can be performed, the results differing from
each other by less than 20 per cent., except in rare instances (two in
fifty-two)’; that is to say, he admits that the limits of error are at least
twice as wide as those laid down by Wright.

We have examined the figures given on Table VIII, in which thirty-
eight duplicate indices are calculated by two observers putting up separate
opsonic mixtures; fourteen pairs were from normal and twenty-four pairs
(not twenty-six as stated by Fleming) from tuberculous persons. We
find that the average difference between the fourteen pairs of normal

indices is 0'076, the maximum being 0°29, and between the twenty-four

pairs of tuberculous indices 0°068, the maxima being 0°21 and 0°20. The

ee average difference between the whole thirty-eight pairs is 0'071; nine

ee =
ee = a hal

out of these, or 25 per cent., differed by more than 10 per cent., and two,
or 5 per cent., by more than 20 per cent.

_ 3. Fleming stated in 1908 that ‘two practised observers can count
same slides and obtain results in almost all cases within 10 per cent.’

a _ Analysing the figures in Fleming’s table, VII, we find that the average

difference in forty-one indices obtained by two observers A and B counting
the same slides works out at 0°064 per cent. Seven of these differed by

- more than 10 per cent., the maximum difference being 20 per cent.

The comparison of counts from the same slides is, however, a very
unsatisfactory way of testing the accuracy of the method, for all errors
associated with putting up the phagocytic mixtures are excluded.

Meruops or CatcuLatinc Errors

_ The errors in this paper have been estimated in three ways.
Method A.—We have calculated the percentage deviation from the
arithmetic mean of the two counts of duplicate slides made from the same
phagocytic mixture. It has already been mentioned that usually sixty
cells were counted on each slide, and that if there was a deviation from
the arithmetic mean of more than 5 per cent. another set of sixty cells was
counted from the third slide.

Method B.—The simplest and most practical way of estimating the
degree of error in opsonic work is to compare the difference between
indices calculated from the duplicate phagocytic mixtures. We have
already alluded to Fleming’s paper, where the average difference between

1. Practitioner, p. 634, May, 1908.

806 BIO-CHEMICAL JOURNAL

thirty-eight pairs of indices calculated by two observers from duplicate
phagocytic mixtures is 0°07.

In our paper three sets of indices have been caleulated from
triplicate phagocytic mixtures; in order, therefore, to compare our results
with those of Fleming we have taken the mean of the difference between
each of the three arranged in pairs. For example, on July 20th the three
indices of A were :—(1) 0°778, (2) 0889, (3) 0°825. The difference between
1 and 2 is 0'11, between 1 and 3 is 0°05, and between 2 and 3 is 0°06. Now,
011 + 0:05 + 0°06 = 0°22. The mean of the differences of the three pairs
is, therefore, 0°07.

Our error so calculated is 0°17 for all duplicate staphylococcus indices
and 0°15 for all duplicate tubercle indices, which compares rather
unfavourably with Fleming’s figures of 0°07 for tubercle, though it is
within his 20 per cent. limit.

As previously mentioned, half our indices were opsonic, the other
half we have called cytophagic; that is to say, they were a comparison of
the phagocytic power of different strains of leucocytes put up with the
same serum. For some reason, probably accidental, the error in the
cytophagic indices, calculated by Method B, is slightly less than in the
opsonic. The figures being—staphylococcus, 0°14 and 0°20, and for
tubercle, 0°14 and 0°15 respectively.

Method C.—Krrors have been estimated in another way also, by
taking the maximum deviation from the mean of the triple estimation.
For example, the mean of the three indices 0°778, 0°889, and 0°825 is 0°831.
The maximum deviation from the mean is, therefore, in this case 0'058.

The application of this method to Fleming’s figures would give 0°035
for the average maximum deviation for the mean.

These methods of estimating the error of technique may be rather
elementary for a statistician, but they have the merit of being easily
understood by the non-mathematical mind. Method B is the most
valuable; it deals with indices, not counts, gives the most consistent
results, and can most easily be compared with duplicate estimations of
other workers.

| LS eee ee a ee eee
a “4s TT Wn. oe ‘ a al

ERRORS IN THE OPSONIC TECHNIQUE 307

TABLE ‘IT, SHowrne Averace DirreRENCE BETWEEN DupticaTe AND TRIPLICATE
Inpices or THe Same Serum BuT From Serarate Puacocytic MIXTURES A8
CALCULATED FROM THE Resutts oF DirFERENT OBSERVERS

No. of | Approximate Approximate
Observer Average indices number number of Organism

figures counted per cell

.. Two 007... 16 100 2 Tubercle
‘Ais op O10. 6 100 2,3 rf
art O12... 6 100 2-0 ;
c es O10. 6 100° 2-3 fs
Sie ah O15 .... 120 120 825 peri Aa
a... 2G, O17 ... 20 120 35 ... Staphylococeus
- Strangewayst F.G. & W 036,045 36 ... 50 1-2... Tubercle
ey We Cee we he ss
al F. G. 051,055 18 ace Eee

* Nore.— exact strength of emulsion employed by s workers (in Table VIII)
be ascertained, but we assume it is similar to that in Table VII, viz, approximately 2.
. pp- 630, 633, May, 1908

+ See page 313. } See page 309, Table IIT.

Various UnravourasLe Opinions UPON THE ACCURACY OF THE
Orsontc TECHNIQUE
" Many writers, especially in America, have recently impugned the
- aecuracy of the opsonic technique.
After working at the tuberculo-opsonic index for several months
and eliminating clumping and fragmentation of the bacilli in the emulsion
by exposing their culture to direct sunlight, Jeans and Sellards! conclude
that ‘the limits of error in our technique, at least, are so great as to
_ render the method inapplicable for clinical work.’ It may be added that
_ they followed the method of Wright as closely as possible.
-____ Moss,? who superintended very extensive comparative tests carried
out by three observers simultaneously, comparing the percentage and
_ greatest percentage variations from different counts, concludes that ‘ none
__ of the present methods of estimating the opsonic content of the blood
seem sufficiently accurate to be of practical value.’
_ ~—s«éDr. Bolduan,’ of the Department of Health of the City of New York,
‘a found that, ‘for reasons not yet understood, duplicate and triplicate tests
made on the same serum at the same time and under apparently identical
conditions often yield widely divergent results.’
Simon‘ speaks of the ‘ phantastic curves’ and ‘absolutely absurd’
results which Wright’s index sometimes gives.
Thomas® remarks ‘ that, aside from technical difficulties, the question

Bulletin of the Johns Hopkins Hospital, p. 234, June and July, 1907.
Bulletin of the Johns Hopkins Hospital, p. 234, June and July, 1907.
Long Island Medical Journal, Vol. 1, No. 10, p. 6.

Journal of Experimental Medicine, Vol. 1X, No. 5, pp. 488, 489.
Journal of American Medical Association, p. 1249, October 12, 1907.

Peer

308 BIO-CHEMICAL JOURNAL

of personal equation evolved in opsonic determinations is so serious as to
practically nullify the value of the method in most instances.’

Potter’ concludes that Wright’s method of estimating the opsonie
indices in bacterial infection is hardly accurate enough to compensate for
the amount of time involved in its application.

Jiigens,* discussing the well-known difficulty of phagocytosis of
clumped bacilli, quotes an example where an index was raised from 0°95
to 113 by the inclusion of two out of one hundred leucocytes which had
ingested twenty-seven and twenty-nine cocci respectively.

W ork of Fitzgerald, Whiteman and Strangeways.—A valuable enquiry
into the accuracy of the opsonic index was undertaken by Fitzgerald,
Whiteman and Strangeways,® in 1907, on account of ‘the very
unsatisfactory and discordant results’ obtained in the pathological
laboratories of Oxford and Cambridge Universities. One of the
investigators (Whiteman) had received instruction in the technique at
St. Mary’s Hospital, London, and had been engaged for a year in the
estimation of indices, and was under the impression that: the error in
his work was seldom greater than 10 per cent.’

In the first part of their research they made counts and estimated
indices from phagocytic mixtures and smears prepared by themselves,
and obtained very unfavourable results. The most important of all the
statistics are those in Table IV, page 124, which give various opsonic
indices obtained by two observers, who put up the opsonic mixture and
calculated the indices of the same sera absolutely independently.

Two capsules of serum were taken daily from a tuberculous patient,
viz., R.1 and R.2, and from two normal persons used as controls, viz.,
W.1 and W2 and F.G.1 and F.G.2.. They were numbered by a
disinterested observer S. to eliminate the unconscious influence on the
results of knowing which blood was being dealt with. As a rule these
observers F.G. and W. put up the blood within a short time of each other;
the washed corpuscles were usually taken from the same individual F.G.
For the sake of simplicity we have modified the headings in Table IV and
have not quoted all the indices calculated from the various combinations
of sera, including those by using S.’s control, and have added columns
showing (1) the variations between the duphicats indices of different
observers, and (2) the variations between the duplicate indices of the same

1. Journal of American Medical Association, p. 1815, November 30, 1907.
2. Berliner Klinischer Wochenschrijt, p. 641, May 30, 1908.
3. Bulletin jor the Study of Special Diseases, Cambridge, Vol. I, No. 8.

ERRORS IN THE OPSONIC TECHNIQUE 809

ers. The sera from R.W. and F.G. were divided into two capsules
purposes of convenience, so all four indices on February 4th, for

example, ‘in which W.’s serum was taken as control, should be theoretically

the same.
a TABLE ILI, SHowrne VaRIATION IN THE Opsonic INDEX OBTAINED IN CoMPARISON
ia or THE Same Sera BY DirFERENT OBSERVERS
e Wik
cam LGpecais ie mixtures put u Opsonic mixture put u
ao coe & akiedioes anlosinded - |:Vacia- Variations in | Vtia- and indices calculated.
be : by F.G tions in indices of tions in by W
Capsules of at rors 2 bp “or Capsules of
ier moe same same
4 | ~~ serum from observer . observer serum from
which indices | Index | F. G Fr G&W Index | which indices
| __ ealculated. -G. & W. parr He
ay t Control Control Patient
CRi CW. | 156 | 959 | 070 | 045 | oo, | O86 | CW. CRI
jc C.W.2 1-06 0-20 | 0-95 061 | CW2 CR2
P/ CRI CFG1| 147 | 455 || 006 | 002 | ogg | 141 | CFRGI CRI
| GR2 CF.G.2 | 0-95 0-46 | 0-54 0-93 | CFG2 CR2
2 {CRI CW.l | 075 | ogy || O10 | 050 | goog | 085 | CW. CRI
Sp oms CW2 | 1-35 0-38 | 0-22 113 | CW2 CR2
2 (CRI CFG1| 109 | 4,, || 062 | 009 | og | 171 | CFGI CRI
Re CR2 C.F.G.2 | 1-60 O-1l | 0-42 18 | CF.G2 CR2
. CR. C.W.1 146 | os 0-47 | O72 | 905 0-99 | CW.l CRI
a CR2 C.W.2 | 0-89 0-10 | O15 074 | CW2 CR2
| |GR1 CFG1| 090 | gig || 060 | 047 | os | 153 | CFGI CRI
| GR2 CF.G2) 1-06 0-17 | 0-33 073 | CFG2 CR2
HOR CW. | O49 | oye | O33 | O13 | oo, | O82 | CWI CRI
CR2 CW.2 | 0-95 0-38 | 0-08 087 | CW2 CR2
22 {CRI CW. | 072 | p99 0-25 | 016 | 9.99 047 | CW1l GRI
toms CW.s | 1-61 1-14 | 105 056 | CW2 CR2
>/GRI CFG1/ O83 | 44, | O21 | O31 | ogs 104 | GF.G1 CR!
GF.G.2 | 1-21 0-17 | 0-69 552 | OFG2 CR2
Total 4:59 12-83 3-25
SUMMARY

Average difference between nine duplicate estimations by the same
observer F.G.=0'51, by the same observer W.=0'36, and between 36
_ duplicate estimations by the two different observers =0°36.

__ These writers also give an exactly similar series of indices, calculated
from capsules C.W.1 and C.R.2 and C.W.2 and C.R.1, instead of from
capsules C.W.1 and ©.R.1 and C.W.2 and C.R.2.

310 BIO-CHEMICAL JOURNAL

We find in this series the figures are, nine duplicate estimations by
observer F.G.=0°55, by observer W.=0°40, and 36 duplicate estimations
by the two observers =0'45.

This table has been analysed at length for three reasons :—

1. It gives the figures which can be most satisfactorily compared Bs,

with ours.

2. The figures compared are the indices, i.e., the results of the
method, which is a more practical test than statistics of the percentage
differences between the highest and lowest counts obtained from various
slides, ete.

3. As the indices compared are calculated from duplicate phagocytic —

mixtures the errors due to ‘ putting up,’ as well as counting, are included,
which is again a more practical test of the technique than a comparison
of indices obtained from two observers counting the same slides.

The table shows that the average difference between duplicate indices

calculated by F.W. and S. is more than twice as great as our own and

more than four times as great as that of the workers quoted by Fleming.

This inaccuracy may be partly ascribed to inexperience in technique, the -
enumeration of only fifty cells and employing too weak an emulsion,

points which will be referred to later.

Other tables, II, III, XII, give the phagocytic counts obtained for
two different capsules of the same blood, and their percentage differences,
and also the percentage difference between the highest and the lowest
phagocytic counts obtained each day with successive sets of fifty or one
hundred cells. Thus on February 4th, 1907, F.G. found two counts from
duplicate phagocytic mixtures were 65 and 36 respectively; the difference
thus being 29. Now there are three possible ways of calculating the
percentage difference of these figures :—

I. By placing the lower figure 36 in the seaman:
29 x 100

II. By placing the higher figure 65 in the denominator.
29 x 100
III. Placing the mean of the two figures (50°) in the denominator.
29 x 100
505 = B7-4

It is clear that in I the error is largest and in IT smallest.

er

ERRORS IN THE OPSONIC TECHNIQUE $11

a Fitzgerald, Whiteman and Strangeways frequently calculate the

percentage differences between the figures by placing the lower figure in
____ the denominator, thereby making the error as great as possible. But they
have no right whatever to assume that the lower figure is more correct
than the higher. As it is impossible to determine which figure is more
correct, the only reasonable method would have been to place the mean of
the two figures in the denominator (Vide III). Fitzgerald, Whiteman
and Strangeways by adopting Method I have made their errors appear

a as high as possible.

In Tables IT and III, Fitzgerald, Whiteman and Strangeways show
“the phagocytic counts obtained for two different capsules of the same
blood and their percentage difference.’ For example, on 4th February,
1907, the counts obtained from two different capsules of the same serum
eS (fifty cells counted) were :- -
a We Capsule I.—36.
tak Capsule II.—65.
‘oad Now these writers estimate the percentage difference between these
_ two counts as 80°6, in the manner we have described on page 310, but the
percentage difference from the arithmetic mean would be, however, 28°7.
As all our percentages have been calculated by the latter method, we
have re-calculated the figures given by Fitzgerald, Whiteman and
Strangeways in their Tables II and III, and can therefore compare them
directly with our own.

Taste IV, SHowrmnc Percentage DevIATION FROM THE ARITHMETIC MEAN OF
Two Puacocytic Counts Opratnep From Two Different CapsULES OF THE
_ Same Serum

Observer No. of duplicate Organism No. of cells A percent-
; ettatlons counted on each age deviation
slide from the arithme-
tic mean
F. G. ea 22 -» Tubercle ae 50 “ 14:3
w. hee 51 ase fe sai 50 <3. 14-9
E. G. née 30 td e dda 120 et 5-3
E. G. wie 79 ie Staphylococcus ... 120 nv 7:3

On account of the ‘inconsistent results’ obtained by Fitzgerald,
Whiteman and Strangeways in their own experiments, a new series of
observations was made by them upon eight slides, one from a normal and
; seven from tuberculous persons, prepared in another laboratory, the
i: reputation of which should guarantee the opsanic technique being

312 BIO-CHEMICAL JOURNAL

‘unquestionable.’ From these slides several thousand leucocytes were
counted in sets of twenty-five by one observer, F.G, The normal slide was_
unknown. In Table XII they give the percentage difference between the
highest and lowest phagocytic count obtained on each slide in consecutive —
sets of 25, 50, 100 and 500 cells, and. found if averaged for single cells to
be 128°77, 70°82, 34°6 424, respectively, the maximum difference in the
figures for 500 cells being 9°9 and the minimum 0'1. ‘They state that this
table proves clearly that ‘the percentage difference on the results
decreases enormously the greater the number of the cells that are counted.’

If the observers had adopted the usual method of calculating per-
centage differences these figures would have been much more favourable.
However, taking them as they stand, it is clear that if 500 cells are
counted their technique will give fairly accurate results, though,
of course, no account is taken here of errors due to putting up the
phagocytic mixtures.

Analysing further the counts of these eight slides, the observers show
that the more cells counted the more the figures for the normal and
tubercular sera tend to approximate. When the index is based upon
1,000 cells for the normal and control sera, the seven indices are 0°78,
1°04, 0°88, 0°86, 0°109, 0°98, 0°95 (Table XI, p. 136). They point out,
further, that ‘if only fifty cells are counted this might in most cases be
sufficient to account for the differences recorded between normal and
tubercular blood.’ The suggestion obviously is that if sufficient cells are
counted Wright’s positive and negative phase would cease to exist.
There is considerable force in this objection. But two facts must be
remembered: firstly, that the normal limit for tubercular indices is
probably, as Fleming has pointed out, 09 and 11, not 0°8 and 12. ~
Second, it is quite possible that either the control serum was not normal or
that the tubercular sera were drawn ata time when the indices happened
to be nearly within the normal limits.

The occurrence, however, of a positive or negative phase has been
noted by numerous independent observers again and again, and cannot
be disproved by the examination of eight slides, even if they came from
a laboratory above suspicion, and a thousand cells were counted from each.

Greenwood! has recently published a paper entitled ‘A Statistical
View of the Opsonic Index.’ His conclusions, which are based upon
Strangeway’s counts of these eight slides and six slides counted in
Wright’s Laboratory, are the following :— |

1. Proceedings Royal Soc. Medicine, Vol. II, No. 5, p. 154.

ERRORS IN THE OPSONIC TECHNIQUE 313

(1) Phagocytic distributions are markedly asymmetrical.

(2) This asymmetry, although reduced, is not removed by
emulsions of (from the experimental standpoint) maximal thickness.

(3) The mode of a phagocytic distribution is a more reliable
constant than the mean.

(4) A corollary of (1)—Positive and negative deviations will
not occur in random sampling equally often.

Another set of figures which may compare with the results of

Fleming and ours are those of Lloyd Smith, Radcliffe and Crossley.

(1) Here thirty-two duplicate indices for tubercle were estimated by two

observers counting the same slides. Analysing the figures obtained from

their charts I and II, we find the average difference between the duplicate
indices is 0°26, the maxima being 1-1, 06 and 0°4. To the table we have
also added some figures by French.

Taste V, SHowine AVERAGE DIFFERENCE IN INDICES OBTAINED BY DIFFERENT
OBSERVERS RECOUNTING THE SAME SLIDES

Paper by Observer Average Numberof Approximate Approximate Organism
difference pairs of number of number of

indices cells counted _ bacteria
compared per slide per cell

Fleming ne 21s 9066.0 0 Sa 100'** ‘Ja 2 =... Tubercle
Smith* eka 6 xa 0-26 ami inert ? eas bB1...3 5
French+ re nce 0-09 ane ee OO. isa | Spe =

le 3 . 103 pe + ohlsyin® | a Pe ae a

*. Lancet, July 18, 1908.
+. Practitioner, 1906, p. 70.
t. This is from an example, purposely given, of a very bad result.

Dr. Hort? sent capsules of the same blood drawn from tubercular or
non-tubercular persons to ‘experts’ in ‘well-known laboratories’;
5 2a frequently duplicate capsules of a particular blood were sent to the same
expert ‘unknown to him.’ Hort gives the result of three duplicate
observations on three sera by the same observer T., the average difference
being 0°10; and a similar number by the same observer O. and the same
observer B., with an average difference of (12 and 0°10 respectively.

The figures obtained from examining the same serum in different
laboratories were much more divergent, especially in the case of expert A.,
whose indices were usually far too high. For example, Test 3, observer

=
aan
en

1. Lancet, July 18, 1908,
2. Practitioner, p. 70, 1906,
3. B. M. J., February 13, 1909.

314 BIO-CHEMICAL JOURNAL

¥e tuberculo-opsonic index, 1°34; observer T., 0°67; observer T., 0-56. 2
Test 6, tuberculo-opsonic index, observer A., 2°20; observer O., 0°96;
observer O., 0°82; observer B., 0°82.

A. made no duplicate observations. The average error of T., B., and
O. is slightly less than our own, though their figures are based on six —
indices instead of 120. It is noteworthy, however, that their emulsion
yielded about 2°3 bacilli per cell, but that of expert A. only 11.
Some Sources or Error vi
Assuming that the technique is carefully performed and none of the
fallacies recently described by Fleming, such as agglutination of the
erythrocytes, are introduced, two sources of error require special
attention—the emulsion and the counting.

The Emulsion

Quality of the Emulsion.—We were’ surprised to find that the counts
of tubercle bacilli were rather more accurate than those of staphylococci,
in spite of the fragmentation, beading, and comparatively large and
frequent clumps obtaining in the former organism and the small size of
the clumps, rarely more than three cocci, in the latter. ,

Quantity of Bacilli in the Emulsion—The quantity of bacilli in the
emulsion is perhaps even more important. The figures published by
Fleming show that the Wright School employed in 1907 a tubercle
emulsion yielding about two, and a staphylococcus emulsion about three
bacteria per cell in normal serum.

In Tables VI and VII we summarise the average errors in the four
sets of triple indices estimated on the different days with different bacterial
emulsions. The errors are grouped according to the strength of the
emulsion employed each day. Thus, twelve indices with staphylococeus
were estimated on July 23rd, May 28th, and August Ist, respectively,
and on each of these three days the average number of staphylococci per
leucocyte was less than 19. The average, however, for the three sets
combined is 1°4 staphylococci per cell. The average error for this strength

of emulsion, as calculated by Methods A, B, and C is 6°5, 0°25, ete. (See
first line of table.)

1. Reyn and Kjer-Petersen of Copenhagen (Lancet, 1908, March 28, p. 919) in a most
valuable paper criticise Wright’s theory and technique rather severely, but we are unable to
compare any of their results with ours.

ERRORS IN THE OPSONIC TECHNIQUE 315

‘Tasie VI, Ixiusrrarine Trae Retationsare Berween Error anp STRENGTH OF
Se EmuLsIon

I—SrapnHyLococeus ALBus
INDICES
SLIDES F : =
Error—Method A Error—Method B Error—Method C
A per- Average No. of indices
EmvuLsion par Sager Average difference maximum upon which
_ No. of bacteria per cell tion the between triple indices | deviation from
~ ari taken in pairs arithmetic are
‘Limits of Average mean of mean of triple . based

counts cell counts counts
19 6-5 0-25 0-32 0-13 36
n 2 and 2-9 5-1 0-17 0-19 0-13 60
m3and3-9 3-4 3-7 0-17 0-20 0-14 60
4and49 3-7 4-5 0-14 0-22 0-13 24
Sand5-9 5-1 3-5 0-13 0-16 0-11 12
B 6-3 40 0-11 0-15 0-10 48
rag: 3-5 48 0-17 0-21 0-12 240
“f ; Il—Tusercie
19 1-7 2-5 0-22 0-22 0-16 12
n2and2-9 2-5 6-7 0-16 0-24 0-14 48
a3and 3-9 3-3 6-2 0-16 0-15 0-13 24
é 4-6 5-7 0-09 0-13 0-08
verage 3-2 5-9 0-15 0-19 0-12 120
Vil—Summary or Precepine TABLE
I—Srarny.Lococcus ALBus
: INDICES
SLIDES ‘ ~
Exror— ¥rror— . Error—
Method A Method B eit ont c
Average tage Average
tage Average difference diminution maximum No. of
Seviation between triple indices in error deviation indices
__—_—s Evuision from the taken in pairs on 120 from upon which
No. of bacteria per cell —_ arithmetic cell —_ arithmetic _precedi
Ne 5 = — of counts mean of figures
ofgroup Average du te p= ee triple are based
Pu et count (i) on 120 (ii) on 60 all
percell (60 cell cell counts cell counts (on 120 cell

; counts) counts)

Between land2-9 1-9 5-6 0-20 0-24 16-6 0-13 96
Between 3and49 3-9 41 0-16 0-21 23-8 0-13 S4
Between Sand 6-9 60 3-9 0-12 O15 20-0 0-10 60
Average 3-5 48 0-17 0-21 0-19 0-12 240
. Il—Tusercie
Between land 2-9 23 5-9 0-18 6-23 2-7 O15 60
Between 3and49 41 59 0-12 0-14 14:3 0-10 60
Average 3-0 59 O-15 0-19 210 0-12 120

~

316 BIO-CHEMICAL JOURNAL

These tables demonstrate that there is a most definite connection
between the error and the strength of the bacterial emulsion. This is
especially obvious when large numbers of indices are grouped together,
and when the error is calculated by Method B, which is the most practical
and useful of the three methods. Naturally there are discrepancies,
particularly in Table VI, but when it is remembered that we are
dealing with two separate organisms, that the homogeneity of the
emulsion, the quality of the film, and the accuracy with which they were
counted by the observer (E. E. G.) necessarily varied somewhat from day
to day, and, lastly, that the methods of calculating errors are somewhat
rough, the fact that so few discrepancies occur is all the more striking.

The following deductions may be drawn from Table VII, in which
the indices are arranged in the largest groups.

1. The error steadily diminishes as the strength of the
bacterial emulsion increases as shown especially by Method B.

2. An emulsion of tubercle bacilli yielding about two or four
bacilli respectively per cell, gives approximately as accurate results,
as an emulsion of staphylococci yielding four or six cocei per cell.
That is to say, the emulsions of staphylococcus should be somewhat
stronger than those of tubercle. This also has been recommended
by the Wright School.

We give below the percentage diminution in the error by increasing
the strength of the emulsion.

Tubercle emulsion increased from 2-3 to 4-1 per cell, diminishes error from 0-18 to 0°12 = 33%
Staphylococcus _,, » 1939 ,, rm 0-20 to 0-16 = 20%
. * » 89to6 ss 0-16 to 0-12 = 25 %

These figures indicate, as far as our work is concerned, that increasing
the concentration of a tubercle or staphylococcus emulsion from two to
four bacteria per cell diminishes the error by about 25 per cent.

The inaccuracy of Fitzgerald, Whiteman and Strangeways’ work
must partly be ascribed to the employment of an emulsion averaging only,
as a rule, one to two bacilli per cell.

Lloyd Smith and his co-workers also employed a very weak emulsion,
averaging, if one may judge from page 148, about one tubercle bacillus
per cell in the control.

Allusion has already been made to the fact that in Hort’s figures
observer A., who obtained the most divergent results, employed the
weakest emulsion.

The importance of a strong emulsion is confirmed by an analysis of

ERRORS IN THE OPSONIC TECHNIQUE 317

_ Fleming’s figures, in which two observers, A and B, calculated forty-one

duplicate indices by counting the same slides. Separating the indices

into two groups, viz., those calculated from counts giving more than two
bacilli per cell and less than two bacilli per cell, we find that in twenty-
seven pairs of indices the average error was 0°07, and the strength of
emulsion 1°6 bacilli per cell, and in fourteen pairs the average error was
0°05, but the strength of emulsion 2°4 bacilli per cell.

In our thirty-six observations (Table VI) with emulsions averaging
4°6 tubercle bacilli per cell, i.e., about twice the strength used in Wright's
laboratory, gave an average error of 0°09, i.e., almost equal to Fleming’s
error of 007 for indices from duplicate phagocytic mixtures. This fact

indicates that one of the main factors in the superiority of Fleming’s

workers lies in tlie greater uniformity of their emulsion, prepared, no
doubt, from a strain of tubercle bacilli specially selected after continuous
Probably the less homogeneous the emulsion the stronger it must be.
There must be, however, an optimum strength for every emulsion above or
below which the accuracy of the counts diminish. This optimum strength
will depend not only on the nature of the organism and the tendency to
clump, but possibly on the personal equation of the observer.

All the facts, then, favour strong emulsion and support the
conclusions of Greenwood,’ who, arguing from statistical considerations
alone, stated ‘ that it is better to work with tolerably thick emulsion giving
an average for normal serum of not less than three bacilli per cell.’

Owing to the laborious nature of the research from which the figures
are obtained, most of the counting was done after the completion of the
technique, so that, unfortunately, we did not realise the great importance
of employing strong emulsion till too late. It must be remembered,
however, that a high phagocytic count per cell demands considerably more
time than a low one.

Counting
Method of Counting

Fleming holds that it is a ‘ great mistake’ to count an ‘arbitrary ’
number of leueocytes, for the observer should display some ‘ intelligence,’
and the number counted should depend on the regularity of the count.
This system is not a fair test of the accuracy of the opsonic technique
unless the observer, having decided upon the number of leucocytes which
give a true estimation of the phagocytosis of each slide, resolutely adheres

1. Practitioner, May, 1908, p. 645.

818 BIO-CHEMICAL JOURNAL

to his decision, even though he find the indices eventually calculated are
contrary to expectation, or differ considerably from those of another
observer. If such should happen, an enthusiastic and optimistic worker
is exposed to the insidious temptation of either recounting the suspected
slide or counting an additional number of cells in the hope that all may
come right.

Perhaps our error might have been less had we counted in the manner
suggested by Fleming, but then we might have unconsciously imprerrs
our results.!

Fitzgerald, Whiteman and Strangeways appear to have counted any
polymorpho-nuclear cells indiscriminately on any part of the slide. Moss
noted that the highest counts occur towards the end of the slide, and
particularly is this evident if larger groups are considered. He attributed
this result to a sorting out process when making the smear, whereby the
largest cells tend to be left to the end.

As previously stated, we made it a rule to count thirty cells on pa
vf the two borders of a film from the end of the smear, where the
leucocytes are most abundant, passing backwards towards the beginning.
Analysing the results of this method, it appears in a consecutive series of
311 slides that the number of bacteria in the first ten of the thirty cells
averages 2'7 per cent. less than in the last ten cells counted.

In another series of 100 slides the figures were 2°6 per cent. less for
tubercle and 1°9 per cent. less for staphylococcus.

These results contradict Moss’s statement, but he divided his slides
into zones 1 em. wide and counted 150 cells in each zone. Our figures,
however, suggest the advisability of counting as far as possible
corresponding portions of every slide. We believe that leucocytes should
never be selected from the middle zone of the smear (AA in the diagram),
for here, the film being thicker, the leucocytes tend to be contracted, so
that their cytoplasm stains more intensely, and the contained bacteria
are more difficult to count.

Of course all damaged leucocytes in which the outline is indistinct,
owing to damaged cytoplasm, must be rigorously excluded from the
counts.

Number of Cells Counted

We have already drawn attention to the fact that the technique of
Fitzgerald, Whiteman and Strangeways, as tested by their own figures in
Table II, is much less accurate than our own, but they enumerated the
bacteria in only fifty cells, selected apparently indiscriminately, and

1. See footnote, p. 321. :

~~

4

ERRORS IN THE OPSONIC TECHNIQUE 319

employed an emulsion yielding about two bacteria per cell, while we
counted 120 cells, selected with method, and the emulsion averaged 3°2

bacteria per cell.

In order to equalise the conditions as far as possible, we have re-
calculated our indices from figures obtained by counting the first sixty
cells only, instead of the whole 120. (See Tables VI, VII.)

These tables demonstrate that even with a weak emulsion yielding on
the average 2°3 bacteria per cell, our average error is 0°23 for tubercle as
compared with their figures of 0°51 and 0°55, 0°36 and 0°40, 0°36 and 0°45.
Even after making due allowances for the slight advantage to us of
enumerating sixty instead of fifty cells, of using some phagocytic mixtures

in duplicate (see p. 302), and from the somewhat stronger emulsion, it is

clear that our work is decidedly more accurate than theirs, as it is less

accurate than that of Fleming.

Ii will be noticed that on one occasion the error appears less on the

Bae a first sixty than on the total 120 for tubercle. This is an accident, and

would be more than neutralised by the counts of the second sixty cells;
thus, on July 20th, the average error for staphylococcus was 0-09 with

0 cells, 0-05 with the first sixty, but 0°135 with the second sixty.

The following conclusions may be drawn from perusal of Table
VII :—

1. The error diminishes by doubling the number of cells counted in
the case of both tubercle and staphylococcus by some 20 per cent., but not
by a half. This agrees with Greenwood’s comment— the belief that a
sample of fifty cells is twice as good as one of twenty-five indicates a
somewhat primitive state of knowledge.’

2. Sixty cells with an emulsion of four bacteria per cell gives
approximately as accurate results as 120 cells with an emulsion yielding

_ about two bacteria per cell, in the case of both tubercle and staphylococcus.

(Sse Chart, p. 320.)

Fitzgerald, Whiteman and Strangeways speak of the absolute
necessity of ‘enumerating at least 1,000 cells.’ But ‘even’ by so doing
a percentage difference of 25 might occur. If this statement were
invariably true, then Wright’s technique is practically useless, not only
for the estimation of opsonins, but also for any experimental work in
which degrees of phagocytosis are compared.

The figures given below, however, should convince the most hostile
critics that Wright’s technique measures something! They represent
four sets of indices, each estimated nine consecutive times on the same
day, August 10th, 1908. These indices are calculated from twenty-seven

320 BIO-CHEMICAL JOURNAL

separate phagocytic mixtures, nine being used as controls in series I and
II, and a second nine in Series III and IV. 120 cells were counted, and
there were no duplicate estimations.

‘A’ ‘L’
I Itt I IV
Opsonic indices Cytophagic indices Opsonie indices Cytophagic indices
1-34 ies 0-90 ee 1-36 sch 0-98
1-08 Ps 0-79 be 1-20 a 0-86
1-05 er 0-78 — 1-13 7 0-85
1-04 pie 0-76 sia 1-08 Pp 0-85
1-02 os 0-73 “— 1-05 ae 0-84
1-00 oye 0-73 a 1-03 ah 0-83
1-00 abe 0-73 iby 1-03 “Sh 0-77
0-96 cas 0-62 ws 0-93 it 0-73
0-86 sat 0-58 sae 0-88 va 0-63
Average 1-04 0-73 1-08 oe 0-81

If we have exhibited some of the work of Fitzgerald, Whiteman and
Strangeways in an unfavourable light, it is because we believe that
we have discovered the main reasons for their imperfect technique. They
deserve the best thanks of everyone interested in opsonic work, for the
courageous publication of their results.

V2 g Bact frer Kel.

1572 25 3 $5 4 4G 5 5S 6

Soa
Toons 20 Ds Siu
, eed
hlinenvrdius' “i SS eodatts

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ERRORS IN THE OPSONIC TECHNIQUE 821

SUMMARY AND CONCLUSIONS

1. The accuracy of the opsonic technique of Wright and Douglas has
been vigorously impugned by many writers, notably Simon, Bolduan,
Strangeways and Greenwood,

2. In 1907 Wright stated that the error in estimating the tubercular
opsonic indices for normal bloods is rarely more than plus or minus 5 per
cent.

3. Next year Fleming pointed out that duplicate estimations of
tuberculo-opsonic indices usually differ from each other by less than
20 per cent., provided the observer counts ‘ intelligently.’

4. We have estimated a large number of consecutive indices with
tubercle and staphylococcus three times, and find that the average
difference between each set of triple indices taken in pairs is 0°15 with
tubercle and 0°17 with staphylococcus. These figures may be compared
with 0-07 obtained by Fleming’ and 0°51 and 0°55, 0°36 and 0°40, 0°36 and
0°45 obtained by Fitzgerald, Whiteman and Strangeways in a series of
duplicate estimations with tubercle.

5. We adopted all the precautions recently advised by Fleming in

7 our work. The possibility of auto-suggestion influencing our counts was
eliminated.
; §. The accuracy of our technique was closely dependent upon two

variable factors:—{a) The number of cells counted; (6) the strength of
the bacterial emulsion.

7. The error steadily diminishes as the strength of the bacterial
emulsion increases. By increasing the concentration of a tubercle or
staphylococcus emulsion from two to four bacteria per cell the error
diminishes about 25 per cent.

8. An emulsion of tubercle bacilli yielding about two or four bacilli

respectively per cell gives approximately as accurate results as an
emulsion of staphylococcus yielding four or six cocci per cell.

9. The error diminishes by doubling the number of cells counted
from 60 to 120 in the case of tubercle and staphylococcus by some 20 per
cent.

1. Greenwood concludes that ‘Dr. Fleming's counts show signs of not being random
sam but selections.’ We are not clear whether this criticism applies to these figures from
the itioner. (Greenwood, Proc. Roy. Soc. Medicine, Vol. I, No. 5, p. 151.)

322 BIO-CHEMICAL JOURNAL

10. Sixty cells with an emulsion of four bacteria per cell give
approximately as accurate results as 120 cells with an emulsion of
about two bacteria per cell, in the case of both tubercle and staphy-
lococeus. |

11. Assuming the truth of Wright’s dictum that the opsonic index

is an index of the ‘ power of phagocytic response’ (although we claim to __

have demonstrated elsewhere that this dictum is not true), the inaccuracy
of the opsonic technique is such that at present we attach no importance
to an index between 0°8 or 1°2 (even estimated by an expert) in the
diagnosis and treatment of disease, unless the observation had been
repeatedly confirmed.

12. We entirely disagree with those crites who appear to maintain
that the technique of Wright and Douglas is practically useless as a
means of comparing degrees of phagocytosis. :

323

THE RELATIONSHIP OF DOSAGE OF A DRUG TO THE

, SIZE OF THE ANIMAL TREATED, ESPECIALLY IN
REGARD TO THE CAUSE OF THE FAILURES TO
CURE TRYPANOSOMIASIS, AND OTHER PROTOZOAN
DISEASES IN MAN AND IN LARGE ANIMALS

By BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio-
Chemistry, University of Liverpool.

From the Department of Bio-Chemistry, University of Liverpool
(Recewved June 11th, 1909)

It is an almost universal custom at the present time in describing
scientific work of a pharmacological or therapeutic nature intended either
to establish a lethal or a curative dose, to state the dose as so much per
kilogram of body-weight of animal or man employed as the subject of
_ experiment or treatment.

‘Many of the observers who use this method of expressing results are
aware and state that it is only roughly accurate, and that experiments
must be made from species of animal to species of animal on account of
idiosyncrasies.

The object of this note is to point out, that, quite apart from
idiosyncrasies, and alteration in the species of animal, this method of
stating dosage in reference to body-weight is not only inaccurate, but rests
entirely on a wrong principle for many kinds of drugs, which, even in
the same animal species, act upon two individuals of different size, not
proportionately to their weights, but proportionately instead to their
body-surfaces or, in other words, proportionately to the two-thirds powers
of their weights, which leads to quite different doses.

For example, the dose of many drugs which can be given to children
or infants to preduce a given therapeutic result, is often many times larger
than the proportionate dose for an adult, on the basis of body-weights.
There are a few cases such as the preparations of morphia where the child
has a marked idiosynerasy or sensitiveness to the drug, but in the majority
_ of cases the balance is entirely in the other direction, and an adult of say
150 pounds weight cannot be given 15 times-the dose of an infant of
10 pounds, but much more nearly a dose of 6 times as much, which is the
two-thirds power of the ratio in the two weights.

Not only is this relationship of importance in regard to the method
of expressing dosage and determining the approximate dose in man or
large animals, from experiments made upon smaller animals. It is also

§24 BIO-CHEMICAL JOURNAL

of the utmost importance in that it naturally sets a limit to our power of |
applying therapeutic agents against disease in larger animals, and allows
a cure with ease in smaller animals, which is difficult or impossible in man
or large animals, simply because they are large and not because of any
particular sensitiveness to the drug.

For example, it is perfectly easy to cure trypanosomiases by atoxyl or
other organic arsenical preparations, or better still by a proper combination
of arsenic and mercury, in small animals such as the mouse or rat. The
difficulty increases with rabbits, but in a large number may still be
surmounted, as also in small monkeys. But with donkeys, cattle, horses
and men, the difficulty is enormously increased, the trypanosomes can
scarcely be driven out from the circulation by such sub-lethal doses as it
is possible to give, recurrence svoner or later takes place and the animal
or man succumbs. This is the common experience of all workers in the
fight against the trypanosome group of diseases, and is particularly well
seen in the work of Moore, Nierenstein and Todd upon treatment by atoxyl
followed by mercury. Like other workers we were in all cases able at
once to drive the parasites out of the peripheral circulation in rats with
atoxyl and by then giving mercury were able to keep them permanently
from recurring in a high percentage of cases (about 60 per cent.).

But when we came to test this greatly improved result as seen in rats
to the larger mammalia, our results took on quite another complexion,
and of 15 donkeys we were unable ultimately to save one. In the first
case, on account of the limitations in the relative dose we could not
satisfactorily and at once with a single dose drive the parasites out from
the peripheral circulation. In some of our experiments for considerable
intervals no parasites could be found even by most careful examination,
but since in other donkeys similarly treated, a small number of parasites
kept persistently present and finally became inured or ‘Fest’ to our drugs,
we could not be free from suspicion, that the parasites in very small number
so as to escape actual observation, were always present, and that our
practically complete failure with the large animals, using a method of
treatment almost perfect for the rat, was due chiefly to this cause. That
is to say, in the large animal the dose cannot be given proportionately to
the body-weight and so the trypanosomes cannot all be killed.

Exactly the same results follow for the admirable treatment by
antimony, introduced first by Plimmer and Thomson, good results follow
and often a high percentage of absence of recurrence in rats and small
animals,’ but in large animals and man the percentage is much lower,
although some cases of cure have been noted.

1. I can completely confirm this result from a large number of experiments made in
collaboration with Dr. T. T. Bark in this laboratory. We found that even smaller doses than
recommended by these authors drove the parasites out. Also a careful search of all the heavy
metals, elements of the phosphorus group, and rare elements, gave negative results, except in
the cases of antimony and arsenic only.

RELATIONSHIP OF DOSAGE TO BODY-SURFACE 325

A like result has been obtained with other organo-arsenical

4 _ compounds.

In all these cases it is to be observed (with the possible exception of the
dog') that the difficulty is not one of idiosynerasy either to parasite or drug
in a particular animal species, but that definitely in each case, and from
each group of observers, we have the uniform report that small animals
are easily treated and large animals including man are difficult of
treatment.

It is hence of peculiar interest to review the situation from the general
point of view, and try to find whether there is any physico-chemical
basis which may supply an explanation. This is particularly so, since a
side-light is cast upon the general bio-chemical question of what regulates
the maximum therapeutic dose possible with such drugs in these diseases

due to protozoa or other micro-organisms.

The establishment of the minimum lethal dose of atoxyl proves at

once that this dose is not proportional to the body-weight, but relatively falls

off very rapidly as the body-weight is increased.

Thus, a large sized rat of say 140 grams weight can safely be given
abont 0-4 c.c. of a 5 per cent. solution of atoxyl, that is about 0°02 gram.
Now, if the dose could be given proportionately to body-weight, a man
of 70 kilograms weight, since he weighs 70,000 grams or 500 times as much
as the rat, ought to be able to safely stand 500 times as much atoxyl,
that is 0°02 x 500=10 grams or in 5 per cent. solution =200 c.c.

As a matter of fact, he not only cannot stand approximately this
quantity, he can only stand a small fraction of it, the highest dose which
can be given being one gram or one-tenth of the above amount calculated
from relat've body-weight, and this is not an idiosyncrasy of man, but is
true for all large animals.

If now instead of hody-weights, the two-third power of the body-
weights be taken for the purpose of estimating the relative doses. We have
that the ratio of weights is 1:500 and the two-thirds powers of this ratio
gives the ratio approximately of 1:63, and if 0°02 grams is the dose for the
rat, on this basis the dose for the man will be 0°02 x 63=1°26 grams.

Now this very closely represents the amount found by actual
experimentation, as 1 gram of atoxyl has frequently been given as a
therapeutic dose in man.

When the cause of this relationship is further investigated, it is found

1. It may also be noted that the dog has a very small area of intestine relatively to its
body-weight.

326 BIO-CHEMICAL JOURNAL

to lie in the selective action of arsenical and antimonial compounds upon
cells lying upon certain surfaces of the body. .
These substances are selectively taken up by epithelial surfaces.

It is well known that arsenic and antimony and indeed most of the
heavy metals such as zinc, silver and copper, when taken by the mouth

act as surface irritants upon stomach and intestine. It is less well known
and only for certain heavy metals, but is none the less a general law, as
experiments have shown me, that all the heavy metals are selectively taken
up by the cells lining the intestine, and kill by extensive inflammation of
the intestinal mucosa. Thus I have found that after subcutaneous injection,
of soluble salts of Jead, tin, bismuth, iron, copper, nickel, cobalt, or
antimony, there is extensive inflammation of the gastro-intestinal tract,
often accompanied by multiple hemorrhage and ulceration.

The same inflammatory processes in the intestine are found after all
forms of arsenic including atoxyl.

This shows that the intestinal mucous membrane acts as the
excretory organ for these foreign substances, and hence takes them up
selectively from tne circulation.

Accordingly the minimal lethal dose or the full therapeutic dose will
be limited by the concentration to which that dose gives rise in the
epithelium cells of the intestine.

Now, placing on one side variations in length of intestine, and
variations in length of intestinal villi, this means that the maximum dose
is proportional to the area of intestine which the animal possesses.

Hence, if for simplicity we suppose that the animal from small size
grows symmetrically bigger in all proportions so as to form a bigger
animal which is a fae simile of the smaller, then we must inevitably
have from purely geometrical considerations of the dimensions of solid
figures, that when the length, or linear dimensions are say doubled, the
surfaces external and internal of the body increase four-fold, and the
cubic dimensions, whch are of course proportional to the body-weight,
increase eight-fold.

Put otherwise this means that the dimensions of surfaces such as
area of skin, lung area and intestinal area increase as the two-thirds power
of the body-weights.

Now a great number of drugs including, as above mentioned, atoxyl
and all salts of heavy metals attack cells spread out on a surface and
hence the bigger animals cannot stand proportionately large doses, and,
for example, an animal eight times as heavy as another cannot be given

RELATIONSHIP OF DOSAGE TO BODY-SURFACE — 327

eight times the dose, but only 84, that is approximately four times as
mich. .

a But the result of this is that the concentration of the drug in the

tissue fluids and blood of the heavier animal will only be approximately

_ one-half of that in the smaller animal.

Suppose now that both animals have been affected by trypanosomes
and we have been attempting to kill them in both, then it is obvious that
there is far more margin to come and go on in the smaller animal than
in the larger one

The trypanosome is swimming about in the blood plasma of the
ee. animal, it may be a rat in one case and a horse in the other, the factor
sit ‘has to deal with is the concentration to which the drug can be raised
in that plasma, it is indifferent what the host may be. But as regards the

host, the drug is taken up by the intestinal cells selectively, and the
< ‘amount of drug which can be given is not determined directly by the
- concentration in the plasma, but by the number of columnar intestinal
cells, that is by the area of intestine which is proportionately greater in
the smaller animal. ) .
; This fact that the surfaces are relatively larger in smaller animals
than in larger animals, has been thoroughly appreciated in regard to body-
temperature and its variations.

A small animal is more susceptible to changes of temperature than
a large one, it requires more food proportionately to its weight than
a larger animal, and for the same reason its temperature goes up and
down more readily because the fly-wheel of weight is smaller relatively
_ to the amount of surface through which heat exchanges are going on, that
is the working part of the engine. It is also obvious since the surfaces
are the working parts of the body, and these are relatively greater in
_ proportion to the amount of material re-acting (¢.e. body-weight) that
all the processes are quickened up in the small animal, so that there is
an exceedingly rapid heart-beat and respiration in all small mammalia.

In cold-blooded animals and invertebrates, a different chemical
constitution in the protoplasm with a different reactivity and speed of
oxidation sets a new limit.

Returning to the pharmacological aspect, we find that this same
peer Gs factor of relative surfaces and volumes is in operation, but has not hitherto
§ been sufficiently recognised, and that it lies fundamentally at the bottom
: of the difficulty of treating man or large animals.

A careful consideration of the experimental facts known regarding
the action of atoxyl and similar therapeutic agents upon trypanosomes has

328 BIO-CHEMICAL JOURNAL

led me to the belief, that the intestine not only acts as above pointed out
in limiting the possible therapeutic dose, but also that the cells of the
intestinal mucosa, are essential intermediaries in preparing or elaborating
in a bio-chemical fashion from the atoxy] or other substance administered,

a substance which is peculiarly deadly for the trypanosome. Wri,
This is shown by the fact so well known to all workers upon &

trypanosomiasis, that substances can be found out by the score which
even in minimal concentration are deadly to trypanosomes in saline in
a watch-glass, yet these substances can be subcutaneously injected into
animals infected with trypanosomiasis without producing the slightest
obvious effect wpon the parasites. .
The explanation here probably is that the said substances are thrown
out of solution by the plasma proteins, which are not present to protect
the parasites in the experiment with the trypanosomes in saline in vitro.

A still more remarkable and instructive experiment, well known also
to workers on the subject, is that trypanosomes may be treated in vitro
in saline, with concentrations of atoxyl, and other trypanocidal drugs,
‘many times higher than the lethal concentration required to destroy them
after subcutaneous injection in the animal, without producing the
slightest apparent effect upon the parasites, which go on moving about
vigorously for hours.

The inference is obvious, viz.: that there must be formed intra vita
by some bio-chemical process, an organic body especially lethal to
trypanosomes.

Now it has been shown that the seat of bio-chemical activity after
administration of arsenic or antimony is the intestinal mucosa, and it
would seem probable that this is also the situation of manufacture of the
trypanocidal virus.

If this be the case, it is clear that the larger animal is doubly
handicapped, for in the first place the therapeutic dose is lowered because
of the relatively smaller number of intestinal cells to take up the drug,
and their consequent rapid poisoning and death of the animal if the
attempt be made to give the relative dose; and secondly, that this
relatively smaller number of cells or laboratories must first act upon the
drug before it can be turned out as a trypanocide upon the parasites.

These reflections suggest certain lines of experimentation and
treatment, which, since I am not myself in a position to experiment upon
larger animals, I here outline for others who may desire to test them.

In the first place, an attempt might be made to prepare the
trypanocidal material by giving gradually increasing doses of atoxy! in

RELATIONSHIP OF DOSAGE TO BODY-SURFACE 329

healthy animals, and then using an extract of the intestinal mucosa in
full doses as a therapeutic agent in man if it were found to be active in
small animals. Such an extract of the intestinal mucosa ought to be
active against trypanosomes in vitro.

Secondly, it appears to me that an attempt might be made to increase
the production of anti-trypanocidal virus by giving atoxyl by mouth
simultaneously with subcutaneous injection.

Lastly, as a kind of opposed treatment, the attempt might be made to
increase the possible subcutaneous dose by giving an antidote by the
mouth to the arsenic or antimony just before injection, so as to protect
_ the intestinal cell at the first pressure of the drug in the circulation due
to the subcutaneous injection.

SUMMARY

1. In the case of substances which act by stimulation or
Seifsmmation of surfaces, such as the intestinal tract, the maximum dose
is proportional not to the body-weight, but to the two-thirds power of
the body-weight.
2. This leads to important differences in dosage in man and large
animals.
© 3. It also shows that the possibilities of treatment are diminished by
: a natural means in man and large animals. These animals have naturally
___ less intestinal, and other, surfaces per unit of weight; accordingly they
ean only take up proportionately less drug, and if any remedial substance
is manufactured by the surface cells, they can only manufacture
relatively less of this than the smaller animal.
4. Also in general terms, uptake and output of poison or infection
_ are relatively more rapid in the small animal. The small animal and
child are hence at the same time more susceptible to onset of infection, and
have more power of recuperation when infected.

Nore.—The practical suggestion may be made that for the great
majority of drugs the method of stating dosage as so much per kilogram
should be abandoned.

The following method is suggested :—

The dose for an animal of an observed weight should be determined,
then by taking the two-thirds powers of the two weights, the dose for an
animal of, say, 1 kilogram, can readily be calculated. This should be

"-BIO-CHEMICAL

stated se tiecliaie Mend doe kilogram
From this dose, the dose for an a

kilogram animal, ‘not multiplied by 4, oe t -th
which i is 16. Here it is 3 obvious ae the usual: baal

therapeutics, the dose for low weight and nips vel
fall and rise in direct proportionality to the w

lessened ratio. The heavier person requires a little
proportionately to increased weight, and vice versa.

"PROPOSALS FOR THE NOMENCLATURE OF THE LIPOIDS*

‘By OTTO ROSENHEIM.
From the Physiological Laboratory, King’s College, London
(Received June 16th, 1909)

_ The term ‘lipoid’ was used fifty years ago by Kletzinski, but has
only obtained importance since Overton (1901) re-introduced it in
connection with H. Meyer's and his own theory of narcosis.1 It is now
generally accepted as a generic name for all those ‘ fat-like ’ constituents
of animal‘or vegetable cells which can be extracted by means of ether or

_ similar solvents. If we accept this definition which from its origin is

mainly a biological one, we are forced to include amongst the lipoids

substances which have very little in common from a chemical point of

‘The fundamental importance of these substances in biological processes

is being more and more demonstrated by exact experimental investiga-

___ tions, especially in relation to problems of immunity. These investigations

have drawn attention to the incompleteness of our chemical knowledge

__ of these substances, and stimulated research on their chemical constitution,
a thorough knowledge of which seems to be the first essential condition for
the elucidation of the biological questions.

Li Unfortunately there still exists a great confusion in the nomenclature

. 3 of these substances. This confusion is mainly due to three reasons:

| (1) Substances which are evidently identical have received different names

from different observers; (2) the same name has been applied to different
substances; (3) several names which have no chemical meaning are used

‘in a general physiological and histological sense.

ah r In order to arrive at a uniform nomenclature, it seems desirable (1)

to omit, for the present, all those names which have been given to

7 ‘substances which are either insufficiently characterised, or the existence

: of which has not been verified by later researches; (2) to dismiss altogether

7 the names given to substances which do not represent definite chemical

compounds, and (3) to adopt, in the case of different names for the same

+ ‘4 substance, those names which were proposed by the original discoverer.
Ms, The following noménclature is based on the classification introduced
a by Thudichum, the value of whose pioneer work in this field is now

iam: recognised, after it has been neglected for nearly thirty years.

A paper read before the Physiological-Chemical Section of the Seventh International
ES of Applied Chemistry, May, 1909.

1, Overton, Studien tiber die Narkose, Tena; 1901.

332 BIO-CHEMICAL JOURNAL

This classification offers the advantage of easily allowing for extension
and of having been already adopted in its main outlines by the majority of

modern workers (Schulze, Winterstein, Hammarsten, Erlandsen, Bang,

Thierfelder, Frankel, etc.).
We may distinguish three large groups of lipoids:

(I) The Cholesterin group (free from both chjecaed and ss

nitrogen).

(II) The Cerebro-Galactosides (free from phosphorus but con-
taining nitrogen).

(III) The Phosphatides (containing both phosphorus and
nitrogen).

(1) The Cholesterin group. The main representative of this group is
cholesterin. The name cholesterin has the advantage of long and inter-
national usage to recommend it in preference to ‘ cholesterol,’ which latter
name only represents one characteristic of the latter substance, namely, its
alcohol nature. This group also includes the vegetable cholesterins,
usually called phytosterins. Pigments such as /ipochromes, as well as
odoriferous substances which have not yet been chemically characterised

may also be provisionally included in this group, for at any rate they
resemble cholesterin in being free from both phosphorus and nitrogen.

(II) The Cerebro-Galactosides represent a group of nitrogenous
phosphorus-free substances which are characterised by the fact that its
members furnish galactose on hydrolysis. The general name of this group
which was first used by Thudichum, is preferable to the shorter name
‘Cerebrosides’ since ‘ cerebrose’ is identical with galactose. Two sub-

stances belong to this group, namely, Phrenosin and Kerasin, the latter of

which has not yet been so well studied as the former.

In adopting the name Phrenosin a number of names must be discarded,
which have given rise to a great deal of confusion. Phrenosin was
recognised by Thudichum as the main phosphorus-free constituent of the
mixture ‘ Protagon ’ (=Cérébrote of Couerbe, 1854; Cerebric acid, Fremy,
1841), and he proposed this name in preference to ‘ Cerebrin,’ as the latter
term had been ee to widely different substances.

The name ‘cerebrin’ was originally given by Kiihn (1828) to a

mixture of phosphatides and cholesterin; it was used by Gobley (1850) —

for a substance which, according to its preparation (boiling with sulphuric
or hydrochloric acid), must have been a partially hydrolysed product,
still containing phosphorus. Miiller (1858) for the first time applied this

1. The general name, ‘ Sterin.” has been proposed for this group by Abderhalden. Apart —

from other objeetions, the possible confusion with ‘Stearin,’ from which the name is derived,
will probably stand in the way of its general acceptation.

NOMENCLATURE OF THE LIPOIDS 333

name to a phosphorus-free substance, obtained by a process in which brain
had been coagulated by means of baryta or lead acetate. Miiller’s
_ ‘cerebrin’ probably represented also a partially hydrolysed product and
has not since been obtained. Some time after Thudichum described
phrenosin, Gamgee obtained the same substance and called it provisionally
*Pseudo-cerebrin.” Parkus (1881) and Kossel and Freytag (1893) again
used the name ‘cerebrin’ for a phosphorus-free substance similar to
phrenosin, in the preparation of which, however, boiling baryta had been
used. Finally Wérner and Thierfelder (1900) gave the name ‘ Cerebron ’
to a phosphorus-free substance, which they prepared mainly by fractional
__ erystallisation from so-called ‘ protagon,’ and which was found to be
_ identical with Gamgee’s ‘ pseudo-cerebrin.’
It seems certain that ‘cerebron ’ and ‘ pseudo-cerebrin’ are identical
with phrenosin, and it is therefore desirable to retain the original name
4 scorn, and to dismiss the others.
__ Kerasin was first obtained by Thudichum. Parkus described a
similar substance, obtained by his baryta process, which, however, he
called ‘ Homocerebrin.’ The name kerasin was again used by Kossel and
Freytag for their preparation, which resembled that of Thudichum’s. The
substances called by Bethe ‘ amino-cerebric-acid glucoside ’ is also probably
identical with kerasin.
ie (III) The Phosphatides. This name was proposed by Thudichum
e for a group of substances which contain both phosphorus and nitrogen.
| Tt was re-introduced by Schulze and by Ilammarsten, and is now
generally accepted, the name ‘lecithans’ proposed by Koch not being
- general enough. Thudichum demonstrated the existence of a whole series
of these substances, where formerly the presence of only one, namely
‘Lecithin,’ has been assumed. He introduced the principle which guides
me ~ modern workers in the classification of these substances. The
_ phosphatides, according to this, may be divided into several sub-groups,
according to the ratio of nitrogen to phosphorus contained in them. For
the present the constancy of this ratio (after repeated re-crystallisations
and fractionations) offers the only index for the chemical entity of these
substances.
We may distinguish the following sub-groups : —

(1) Monoamino-monophosphatides ..........N: P = 1: 1
(2) Diamino-monophosphatides —............ B:P = 23.1
(3) Triamino-monophosphatides ............. N: P=8:1
(4) Triamino-diphosphatides .................. Ni: P= 3:2
(5) Monoamino-diphosphatides .......,....... BLP «= ): 3

834 BIO-CHEMICAL JOURNAL

It is obvious that this list may be easily extended as ——
possessing a different N : P ratio are isolated.
(1) Monoamino-monophosphatides. This sub-group comprises Lecithin

and Kephalin. he term ‘lecithin,’ which was formerly given toa i
mixture of phosphatides, should be restricted to those monoamino-mono-

phosphatides which are soluble in ether, and not precipitated from their
solution by alcohol. The nature of the fatty acids contained in them is
still uncertain, but oleic acid seems to be the characteristic one. Kephalin
(or kephalins) first described by Thudichum, is soluble in ether, and
precipitated from this solution by absolute alcohol. It probably contains
as its characteristic fatty acid a still more unsaturated acid than oleic acid.
(Zuelzer has given the name Myeline to a substance obtained from egg
yolk, which is evidently similar to or identical with Thudichum’s
Kephalin).

Another substance belonging to this group has recently been isolated
from ox pancreas by Frankel and Pari. This monoamino-monophosphatide
which has been called Vesalthin, furnishes on hydrolysis myristie acid,
besides an unknown unsaturated fatty acid and a base different from
choline.

Thudichum includes in this group another substance called by him
Myeline. In view of the fact, however, that this substance has been very
little studied, and in order to avoid confusion with the general term
‘myelination ’ as used in the physiological sense, it seems desirable for the
present to dismiss the name altogether.

(2) Diamino-monophosphatides. | The main representative of this
group is Sphingomyelin. This phosphatide, which, in distinction from
the waxy lecithin and kephalin, is a crystalline white substance, was also
first obtained by Thudichum. It represents the main phosphatide of the
so-called ‘ protagon’ mixture. Its oceurrence in brain (and probably in
the cortex of the adrenals) has been confirmed by Rosenheim and Tebb,
who prepared it by a new method. A similar diamino-monophosphatide
was obtained as a cadmium salt by Erlandsen from the heart muscle, and
by Thierfelder and Stern from egg yolk.

(3) Triamino-monophosphatides. A phosphatide belonging to this
group has recently been isolated by Frinkel and Bolaffia from egg yolk.
They gave the name Neottin to this triamino-monophosphatide.

(4) Triamino-diphosphatides. A substance belonging to this class
is described by Frankel and Nogueira, who obtained it from ox kidney.

(5) Monoamino-diphosphatides. The first phosphatide of this group
was discovered by Erlandsen in heart muscle, and called by him Cuorin.

NOMENCLATURE OF THE LIPOIDS 835

Recently MacLean demonstrated the presence of another member of this
group inegg- yolk. Both substances are highly unsaturated and yield on
hydrolysis a base different from choline.

In this classification the lipoids which contain sulphur, although
possibly of considerable importance, have not yet been considered, as they
have so far not been isolated in a sufficiently pure state to warrant their

group as Cerebro-sulpho-galactosides, or if found to contain phosphorus
as well, in the third group as amino-sulpho-phosphatides.

Itis proposed to discard the following names : —

Cérébrote

Cerebric acid Mixtures of various lipoids.
Protagon

Cerebrin

Pseudo-cerebrin Identical with phrenosin.
Cerebron

Hlomocerebrin— identical with kerasin.
Myelin—for reasons explained above.

| ‘The following table contains a list of the substances which are

included by the proposed classification.
I. The Cholesterin group.
1. Cholesterin.
2. Phytosterins,

3. Lipochromes, etc.
Il. The Cerebro-Galactosides.
1. Phrenosin.
: 2. Kerasin.
: IL. The Phosphatides.
am 1. Monoamino-monophosphatides N: P = 1: 1.
a. Lecithin (or Lecithins).
6. Kephalin (or Kephalins).

ec. Vesalthin.
2. Diamino-monophosphatides N: P = 2: 1
Sphingomyelin.
8. Triamino-monophosphatides N: P = 3: 1.
Neottin.
4. Triamino-diphosphatides N: P=8: 32
=1: 2.

5. Monoamino-diphosphatides N : P
Cuorin.

es

In the sisal’ state of our Land desi
to defer the introduction of new names until a com)
hydrolytic cleavage products makes it evident that the sv
is fundamentally different from those previously describe
of the lipoids (except cholesterin) in a chemically pure, 3
impossible at present as that of the proteins and we
just as in the latter case, mainly on the results of c
further information about these substances. =

-

vi hutersee

avr t oe inees

e. Par a a ys
i WAY Bet eee oe Fe

837

A COMPARISON OF THE METHODS FOR THE ESTIMA-
TION OF TOTAL SULPHUR IN URINE

By STANLEY RITSON, A.K.C.
From the Physiological Laboratory, King’s College, London
Communicated by Prof. W. D. Halliburton, F RS.
(Received July 16th, 1909)

It is well known that urine contains sulphur, not only in the form
_ of sulphates, both inorganic and ethereal, but also in the form of less
highly oxidised organic compounds, generally spoken of (following
__E. Salkowski’s! suggestion) as ‘neutral sulphur. The organic
{ compounds in question are very diverse and include thiocyanic acid and
he ; its salts,? eystine and closely related bodies,* taurine and tauro-carbamic
acid, methyl merecaptan,5 ethyl sulphide,® thiosulphuric acid,’
sulphurous acid,’ urochrome,® oxyproteic acid,!° uroproteic acid,!
and uroferric acid.!?

The quantitative determination of the total amount of sulphur
present in the urine depends upon the fact that this neutral or unoxidised
sulphur can be converted by the aid of suitable oxidising agents into
sulphuric acid. The various methods in use differ mainly in the different
oxidising agents chosen. They may-be arranged in three groups :—

. 1. Virchow’s Archiv., Bd. LVIII, 8. 472, 1873.
_—__— 3. Leared, Proc. Roy. Soc., Lond., Vol. XVI, p. 18, 1870; R. Gschleiden, Tageblatt d. 47,
: prune gy deut. Natur, 7 u. Aertzte in Breslau, | 4 (quoted from E. Abderhalden’s Lehrbuch
"= d. physiol. Chem., 11 Aufl., 8. 343, 1909); I. Munk, Virchow’s Archiv., Bd. LXIX, 8. 354.
Sp 3. Stadth Zeitsch. }. physiol. Chem., Bd. TX, 8. 125, 1885; E. Goldmann u. E.
ibid., Bd. XII, 8. 254, 1888.
4. E. Salkowski, loc. cit.
5. M. Nencki, Arch. /. exper. Path. u. Pharmak., Bd. XXVIII, 8. 206, 1891.
| se cco gem neliee etiaresdtett
J. J.

. J. Abel, loc. cit., for d Presch found in case of typhus that a compound which

isolated from the urine in small. uantities =v rg acid on distillation with acids.

the case IT can find in w * Faerie / uric acid has been mopens 3 in urine of man.

Quoted from A. Heffter, Ergebnisse d. Asher and Spiro), Jg. I, Abt. I, 8. 458, 1902.
Striim

in case of fever, bane rs Heilk., 1876. (Quoted from Dixon Mann, Physiol.

8.

iy and Path, of Urine, 2nd Edit., p. 18, 1908.)

Ps 9. St. Dombrowski, Zeitsch. /. physiol. Chem., Bd. LIV, 8. 204, 1907-8.

or Aller peng pe tt ghee ge Soups ote ie ie Wap sage No. 33, 8. 577, 1897;
K. Panck, Ber. ¢. Dest. chem. Gesslioh. Jg. XXXV, 8. 2059, 1902; St.

go i, Bt. Dombrowski u. K. Panek, Zeitach. }. physiol. Chem., Bd. XLV, 8. 83, 1905 ;
F. Pregl, Pfliiger’s Archiv., Bd. LXXV, 8. 87, 1899.

bf 11. Max Cloetta, Archiv. /. exper. Path. u. Pharmak., Bd. XL, 8. 29, 1897.
4% 12. 0. Thiele, Zeitsch. /. physiol. Chem., Bd. XXXVII, 8. 251, 1903.

338 BIO-CHEMICAL JOURNAL

1. Methods in which the residue left upon evaporation of a certain
definite volume of urine is effected by fusing with a mixture of sodium

carbonate and saltpetre. These are merely applications to the urine of
Liebig’s original method of sulphur estimation. Full directions as to —

the procedure are given in Savelieft’s' and Moreigne’s? papers.
9

oxidising agent. Schulz* and Mohr* have described methods utilising
this reagent. Schulz originally carried out the process in a special closed
apparatus, believing that volatile sulphur compounds might be formed,
but in his last communication on the subject he states that this is not
the case, and now carries out the oxidation in an open Kjeldahl flask.
Konschegg,° who also uses fuming nitric acid, adds a small quantity of
potassium nitrate, probably in order to effect more complete oxidation.
3. Methods in which the oxidation is brought about by sodium
peroxide. The introduction of this reagent, for the purpose of sulphur
estimations, we owe to Hempel.® It was subsequently used by Hoehnel,?
Glaser® and by Asboth,® for the estimation of sulphur in organic
materials. Ina later paper Asbéth,!° who subsequently employed sodium
peroxide for the estimation of sulphur in organic compounds, suggested
that the method might also be useful for the determination of the total
sulphur in urine.!! Modrakowski!* simplified the method by omitting
the addition of sodium carbonate to the peroxide, which formed a part
of the original process. More receptly Folin! has modified the Asbéth-

1. Savelieff, Virchow’s Archiv., Bd. CX XXVI, 8. 197, 1894.
2. Moreigne, Bull. de la Soc. Chim. (3), XI, 975, 1894.
3. H. Schulz, Pfiger’s Archiv., Bd. LVII, 8. 57, 1894; also ibid., Bd. CXXI, 8. 114,

4. P. Mohr, Zeitsch. /. physiol. Chem., Bd. XX, 8. 556, 1895.
5. A. Konschegg, Pfliger’s Archiv., Bd. CX XIII, 8. 274, 1908.

6. W. Hempel, Zeitsch. /. anorg. Chem., Bd. TI1, 8. 193, 1893.
7. M. Hoehnel, Archiv. d. Pharm., Bd. CCXXXII, 8. 225, 1894.
8. ©. Glaser, Chemiker Zeitung, Jg. XVIII, 8. 1448, 1894.

: A. von Asbéth, Chemiker Zeitung, Jg. XTX, 8. 599, 1895.

A. von Asboth, ibid., Jg. XTX, 8. 2040, 1895; A. Edinger recommended sodium

vxide for the estimation of o anically combined sul hur before ory xiv Ronen
Fis uesd an aqueous solution of the peroxide. Zeitsch. f. analyt. Chem.,
1895; Ber. d. Deut. chem. Gesellech., Jg. XXVIII, 8. 427, 1896.

11. §. Lang used the Asboth method to estimate total sulphur in urine, but gives no control
analyses. Zeitsch. /. physiol. Chem., Bd. XXIX, 8. 305, soto.

12. G. Modrakowski, Zeitsch. /. physiol. Chem., Bd. XX XVIII, 8. 561, 1903. F. Clark,
Journ. Chem. Soc., 1893, I, p. 1093, had previously suggested that sodium carbonate was
unnecessary. Almost immediately after Modrakowski’s paper, Neumann and Meinertz (Zeitsch.
}. physiol. Chem., Bd. XLITI, 8. 38, 1904) advised the admixture of potassium carbonate.

13. O. Folin, Journ. of Biolog. Chem., Vol. I, p. 155, 1906. See also T. B. Osborne, Journ.

of Am. Chem. Soc., Vol. XXIV, p. 142, 1902; J. A. Le Clerq and Dubois, ibid., Vol. XXVI,
p. 1108, 1904; W. L. Dubois, sid. Vol. XXVIL, p. 729, 1908.

2. Methods employing concentrated fuming nitric acid as the '

TOTAL SULPHUR IN URINE 339

- _Modrakowski method in several particulars (especially with regard to
_ the addition of a little water before the final fusion in order to obtain
- complete fusion with the aid of comparatively little heat, and to protect
the crucible against corrosion). In all these methods the fusion is
accomplished by heat applied to the exterior of the crucible, preferably
by means of a methylated spirit burner.

Pringsheim, however, utilising an observation of Parr’s,! showed
that the oxidation may be most easily brought about by the introduction
of a red-hot iron nail into the mixture. He first employed this method
for the estimation of the halogens, phosphorus and arsenic,? and later,

_ following Konek? and other observers,‘ to the estimation of sulphur® in
‘organic combination. Recently, Abderhalden and Funk® have applied
Pringsheim’s method to the estimation of total sulphur in the urine.
__It is obvious that in any trustworthy method, the results obtained
‘with the same urine should agree infer se; and in the comparison of
different methods, the method which gives the highest results will in the
absence of other indications be the most correct. On both these points
there are differences of opinion with regard to the different methods:
thus Osterberg and Wolf? state that while the method of Asboth-
_ Modrakowski, carried out according to Folin’s directions, invariably
gives higher figures than the Schulz method, the results obtained by the

latter method do not agree well inter se; Konschegg states that his
modification of Schulz’s procedure gives considerably higher figures than
the latter; Abderhalden and Funk claim that the Pringsheim
modification in their hands also gives higher figures than Schulz’s
method; and Gill and Grindley® say that Konschegg’s figures are
invariably higher than those obtained by both Osborne’s and Folin’s
modifications.
‘Taking all these statements into consideration, and bearing in mind
4. that no corroborative observations have yet appeared on the Pringsheim
bi method, it seemed advisable to investigate the degree of accuracy of the
= various methods in a series of urines.

1. 8. W. Parr, Journ. Am. Chem. Soe., Vol. XXII, p, 646, 1900.

- ee 2 Ae oo ok Ber. d. Deut. chem. Gesellach,, Ig. XXXVI, 8. 4244, 1903: Amer.
Chem. Journ., Vol. XX XI, p. 386, 1904; Zeitech. |. angewand, Chem., Bd. XVII, 8. 1454, L904,

3. F. von Konek, Zeitsch. /. angewand. Chem., Bd. XVI, 8. 516, 1903.

4. C. Sundstrom, Journ. o are ees Vol. XXV, p. 184, 1903; J. D. Pennock
sage ae eegieap der peer XV, p. 1265, 1

5. H. H. Pringsheim, pagel! ad et OO Jg. XLI, 8. 4267, 1908.

6. E. Alderhalden and C. Funk, Zeitsch. /. physiol. Chem., Bd. LVIII, 8. 331, 1908.

7. E. Osterberg and C. G. L. Wolf, Biochem. Zeitech., Bd. IX, 8. 307, 1908.

8. F. W. Gill and H. 8. Grindley, Proceed. of Soc. of Biolog. Chem., Vol. VI, p. 11, May, 1909.

ois ene

340 BIO-CHEMICAL JOURNAL

In comparing the different methods, I have taken as my standard of
agreement the one adopted by Abderhalden and Funk, namely,
variations of 0:1 milligr. on either side of the mean value, when the
sulphur is estimated in 10 c.c. of urine. re

As there seemed to be no necessity of confirming the correctness of
the old Liebig method,! I limited myself to the following, viz.:—those a
of Schulz, Konschegg, Asbéth-Modrakowski, and Pringsheim
(Abderhalden-Funk modification).

Schulz’s Method. The following are the analytical results I have
obtained with four different urines:

Urine A
5 c.c. urine gave 0-0358jgr. BaSO, = 0-0098 gr. sulphur in 10 c.c. urine
me i: » 0-0362 gr. » = 0-0100 gr. ‘i Ac ie
Urine B
5 ¢.c, urine gave 0-0348 gr. BaSO, = 0-0096-gr. sulphur in 10 c.c. urine
” ” 0-0353 gr. ” => 0-0097 gr. ” oo ”
Urine ©
5c.c. urine gave 0-0510 gr. BaSO, = 0-0140 gr. sulphur in 10 ¢.c. urine
” ” ” 0-0501 gr. ” = 0-0138 gr. ”? ” ”
Urine D
5je.c. urine gave 0-0154 gr. BaSO, = 0-0042 gr. sulphur in 10 ¢.c. urine
ios as » 00150gr. ,, =00041 gr. ,, oe res

it will be seen that (contrary to Osterberg and Wolf’s statement)
the figures obtained agree very well inter se, the maximum variation being
only 0-2 milligr. Mohr, who used a method almost identical with that =
of Schulz, also obtained concordant results. My results by the Schula
method are, however, lower than those obtained by other methods, as "
will be immediately seen.? Rr?
Konschegg’s Method. 'The following are my analytical figures :—

Urine A
5c.c. urine gave 0-0378 gr. BaSO, = 0-0104 gr. sulphur in 10 c.c. urine
” ” ” 00375 gr.» = 0-0103 gr. ” ” ”
Urine B *
5c.c. urine gave 0-0369 gr. BaSO, = 0-0101 gr. sulphur in 10 c.c. urine
” ” ” 0-0384 gr. ” = 00106, ° ” ” bh
Urine C
5.0, urine gave 0-0528 gr. BaSO, = 0-0145 gr. sulphur in 10 c.c. urine
” ” ” 0-0524 gr. ss = 0-0144 gr. ” ” ”
Urine D
5c.c. urine gave 0-0165 gr. BaSO, = 0-0045 gr. sulphur in 10 c.c. urine
” ” ” 0-0163 gr. ” = 0-0045 gr. ” ” ”

1. See however O. Folin, Journ. of Biolog. Chem., Vol. I, p. 156, 1906.

2. This confirms the statement of Konschegg, Osterberg and Wolf, Abderhalden and Funk
(loc. cit.) and of Sherman who used the nitric acid method for the estimation of sulphur in organic
compounds. (Journ. of the Amer. Chem. Soc. Vol. XXIV, p. 1100, 1902).

TOTAL SULPHUR IN URINE 341

| Pringsheim’s Method (Abderhalden and Funk's modification). The
_ following are my analytical figures : —

Urine A
10 c.c. urine gave 0-0740 gr. BaSO, = 0-0102 gr. sulphur in 10 c.c urine
” ” ni 0-0744 gr. ow ; = 0-0102 gr. ow ” ”
Urine B
10 c.c. urine gave 0-0798 gr BaSO, = 0-0110 gr. sulphur in 10 c.c. urine
” ” ” 0-0800 gr. ” = 0-01 10 gr. ” ” ”
Urine C
10. c.c. urine gave 0-1032 gr. BaSO, = 0-0142 gr. sulphur in 10 c.c. urine
” ” ” 0-1043 gr. ” = 0-0143 gr. ” ” ”
Urine D
10 c.c. urine gave 0-0322 gr. BaSO, = 0-0044 gr. sulphur in 10 .c. urine
” ” » 00318 gr os = 0-0044 gr. ows ” ”
Urine E
10 c.c. urine gave 0-0756 gr. BaSO, = 0-0104 gr. sulphur in 10 c.c. urine
5 c.c, ” ” 0-0383 gr. ” = 0-0106 gr. ” ” ”

It will be seen, in confirmation of what Abderhalden and Funk
state, that the figures are very concordant.
If the figures obtained by the Konschegg method are compared with
_ the above obtained by the Pringsheim method, it will be observed that
the Konschegg figures are a little higher (1 to 2-1 per cent.) than those
obtained by this method, and 5°2 per cent.! on the average higher than
those obtained by Schulz’s method.

Asbéth-M odrakowski Method. Were different results were obtained,
according to differences in the details of the modus operandi. In most
of my estimations I carried out the process in the following manner :—
I added the urine gradually to the sodium peroxide to avoid loss by
spitting,” evaporated down on the water bath to complete dryness, and
subjected the residue to complete fusion over a spirit burner, maintaining

it in the fused condition for ten to fifteen minutes; cooled; added
1-2c.c. of distilled water, and more peroxide and again subjected the
mixture to complete fusion, prolonging the fusion for twenty to thirty
minutes. I used between three and four grams of sodium peroxide for
the preliminary fusion and eight grams for the final; the quantity of
urine used was 25 c.c. Using this method I obtained, in all the
estimations I performed, agreement between the individual determinations
made on the same urine, and also higher results than by any of the other
methods.* On the average the figures were 8 per cent. higher than those

- actin ard ale eee wach ing Aone by Konschegg himself.

2. Recommended by Modrakowski, loc. cit.

3. M. A. Deamouliére, Journ. de Pharm. et de Chim, (6th series}, Tome XXIV, p. 294, 1906,
states that Moreigne’s and Modrakowski’s methods give accurate results in estimation of total
sulphur in urine; the vremerinme serthdapmelland Mee! By Ret deen joomla
~~ sd ieee aoe h cen figu (really sight! ee ae

onstration, the same Tes wer) as
Na,CO,—KNO, method, accords with my results. . ‘

342 BIO-CHEMICAL JOURNAL

given by the Konschegg method. I was unable to detect any evolution
of sulphuretted hydrogen, either during the evaporation or the subsequent
acidification of the fused mass, such as Gill and Grindley cc

The following are my analytical figures :—

Urine B

25 c.c. urine gave 0-2036 gr. BaSO, = 0-0112 gr. sulphur in 10 ¢,c. urine ‘ .
” we 0-2053 gr. ” = 0-0113 gr. ” ” ” - Rated .
Urine © a
25 c.c. urine gave 0-2724 gr. BaSO, = 0-0150 gr. sulphur in 10 ¢.c. urine
” ” 0: 2798 gr. ” = 0-01 54 gr. ” ” ”
Urine D
25 c.c, urine gave 0-0966 gr. BaSO, = 0-0053 gr. sulphur in 10 ¢.c. urine
es » 0-0970gr. , =00053¢gr._,, pe 2

This aaaa’ as I have already described it, is in the main identical
with that described by Folin, except that he does not mention the
preliminary fusion, and maintains that ten minutes’ final fusion is
sufficient. The number of analyses I have carried out according to his
directions is not great; they agree well inter se, but they are never higher
than those obtained by Pringsheim’s method, and in the one case where ©
I can make the comparison they are lower than those obtained by
Konschegg’s method.' The following are my analytical figures : —

Urine A
25 c.c. urine gave 0- Ay BaSO, = 0-0102 gr. sulphur in 10 c.c. urine
” ” ” 01858 gr. os = 0-0102 gr. ” ” ”
Urine E “a
25 ¢.c. urine gave 01858 gr. BaSO, = 0-0102 gr. sulphur in 10 ¢.c. urine eee
” » 01880 gr. 5, = 0-0103 gr. ” ” ”

Putting all the results together, the following table shows the mean
figures expressed in grams of sulphur per 10 c.c. urine.

Method Urine A Urine B Urine C Urine D Urine E
GIEER © cobcn sdoavecccnkudvac coensiaee 0-0099 0-0097 0-0139 0-0042 ~
Konschegg sheesh eigdmagiiia na odteed 0-0104 0-0104 0-0145 0-0045 —
i RIE Yana vectcabasvudeobadenta 0-0102 0-O11L0 0-0143 0-0044 0-0105 ’
Asboth-Modrakowski ............ — 0-0113 0-0152 0-0053 — ’
Asboth-Modrakowski (Folin) .... 0-0102 _ —- = 0-0103

From the above table it will be seen that Schulz’s method gives the
lowest, whilst the Asbéth-Modrakowski method gives the highest figures;
the Pringsheim, Konschegg and the Folin’s modification of the Asbéth-
Modrakowski method all give intermediate figures. _

Conelusion. The sodium peroxide method carried out according to
Asbéth-Modrakowski (as described above) gives the highest figures in
the estimation of the total sulphur in the urine, and must therefore be
considered to be the most trustworthy of the methods at present in use. —

1. Gill and Grindley, loc. cit., state that the difference is even greater than I have obtained.

343

ber THE USE OF BARIUM PEROXIDE IN THE ESTIMATION
_ OF TOTAL SULPHUR IN URINE

By STANLEY RITSON, A.K.C.

From the Physiological Laboratory, King’s College, London
Communicated by Prof. W. D. Halliburton, F.R.S.

(Received July 16th, 1909)

——— Although the Asbéth-Modrakowski method gives the best results
for the estimation of total sulphur in urine (see preceding paper), it has
the disadvantage of being somewhat lengthy. In metabolic experiments,
where it is necessary to make a large number of estimations, it is essential
that a process should be adopted which can be carried out rapidly.
From this point of view the Pringsheim method seemed to be the best,
_ provided it could be modified so as to give figures equal to those obtained
by the Asbéth-Modrakowski method. With this end in view it occurred
to me that the use of barium peroxide, in the fusion of which a higher
_ temperature is obtained than with sodium peroxide, might lead to a more

complete oxidation. The introduction of a barium salt would have the
additional advantage of shortening the method, as the barium sulphate

is formed during the actual process of the oxidation.' I therefore tried
fusing with barium peroxide instead of sodium peroxide, but found that
fusion did not take place easily.

This difficulty was overcome by the addition of sodium peroxide to
____ the barium peroxide in the proportion of seven to one. The process was
__ then carried out exactly as in the Pringsheim method, the details of
____ this new method being as follows :—

Applying this idea to the Konschegg method, I found that the addition of barium
slices’ does not give any better results than when it is absent. The following are my mean
ee co ee oop pee 10 oe. urine :—

* Method Urine A Urine B Urine C Urine D
Konschegg ... Fs .. O-0104 0-0104 0-0145 0-0045
Barium-nitrate-nitric acid . 0-0102 0-0109 0-0144 0-0042

The addition of barium Tati in the Asbéth-Modrakowski method did not yield con-
cordant results, and as the method is afin ig 2 I did not pursue it further.

In the literature the only references I find with regard to the utilisation of a barium
salt in the estimation of sulphur were :—

(2) H. Weidenbusch, who, at Liebig’s suggestion, estimated sulphur in albuminous
materials by fusing » paste formed of the substance, barium nitrate and nitric acid. He gives
ae a ber « Ann., Bd. LXT, 8. 370, 1847. pate

um peroxide to estimate sulphur in cit S308, sub
. — finally fusing with sodium peroxide, Zeitech. /. analyt. Chem. Ba. Ba, XXXI

EM Ot a a ae a CMT ety AY,
ee ee ey a a

B44 BIO-CHEMICAL JOURNAL

Ten c.c. of urine were measured into a nickelled steel crucible, as
recommended by Pringsheim,! and made alkaline with sodium carbonate.
After the addition of 0:4 grm. lactose, the mixture was evaporated down,
on the water-bath, to a syrupy residue. Without further drying, the
residue was carefully mixed with 8 grms. of the oxidising agent, consisting —
of 7 grms. of sodium peroxide and 1 grm. of barium peroxide.?

The crucible is next immersed up to three-quarters of its height in
distilled water contained in a larger porcelain crucible or basin. A
red-hot iron nail is introduced through the hole in the lid, and in a few
seconds the reaction is completed. When the crucible has cooled down
sufficiently it is overturned into the water, the basin being covered by a
clock glass. The contents of the basin are then transferred quantitatively
into a 500 c.c. Erlenmeyer flask and raised to boiling point. Concentrated
hydrochloric acid is added gradually to the boiling fluid until the ferric
oxide (derived from the iron nail) has gone into solution. A small excess
of hydrochloric acid and a few c.c. of alcohol are then added, and the
boiling continued for a short time. This serves to drive off the chlorine,
which is always formed by the action of the excess of sodium peroxide
on acidifying the solution with hydrochloric acid. It was noticed that
under the above conditions the oxidation (as judged by the absence of
carbonaceous particles) is complete, whilst without the use of barium
peroxide filtration is very frequently essential in order to remove particles
of carbon. The barium sulphate which is now present in the form of a
granular precipitate is then collected on a weighed Gooch crucible, dried,
ignited, and weighed as usual. It was found as a further advantage of
the barium peroxide addition that the barium sulphate precipitate settles
and filters very easily, probably owing to the physical conditions under
which it is formed.

Using this method my analytical figures were the following :—

Urine B

10 ¢.c. urine gave 0-0857 gr. BaSO, = 0-0118 gr. sulphur in 10 c.c. urine

” ” ” 0-0837 ” 0-0115 ” ” ” 2)
Urine C
10 c.c. urine gave 0-1112 gr. BaSO, = 0-0153 gr. sulphur in 10 ¢.c. urine
” ” ” 0-1122 ” = 0-0154 ” ” ”

1. The crucible, together with the perforated lid, is obtainable from Messrs. Kohler,
Leipzig.

2. A certain amount of care must be exercised in the addition of the begining. Aten
I found it advisable to add about 0-1 to 0-2 gramme at a time at the
addition of about half the required amount, the remainder may be added in a Paste Fs
The barium peroxide used was tested for sulphur with negative results.

TOTAL SULPHUR “IN URINE 345,

= Urine D
“10 c.c. urine gave 0-0472 gr. BaSO, = 0-0065 gr. sulphur in 10 ¢.c. urine
” ” ” 0-0488 ” = 00067 ” ” ”

_ From this it will be seen that the results agree well inter se. If we
pare the mean figures obtained by this method with those obtained

out the use of barium peroxide (Pringsheim method) it will be seen
ve considerably higher results. Compared with the Asbéth-

\ditions the oxidation is even more complete than in the Asboth-

cowski method.

ere Urine Bs Urine ~——dUrrine D
Pringsheim method =... —«.. O00 0-0143 0-0044
Asbéth-Modrakowski method... 0-0113 0-0152 0-0053

New method 3 we OOLIT 0-0154 0-0066

346

A CONTRIBUTION TO THE BIO-CHEMISTRY OF
HAEMOLYSIS :—

(a) CHANGES IN SOLUBILITY OF THE LIPOIDS IN

PRESENCE OF ONE ANOTHER, AND OF CERTAIN >

UNSATURATED ORGANIC SUBSTANCES.

(6) THE BALANCING ACTION OF CERTAIN PAIRS OF
HAEMOLYSERS IN PREVENTING HAEMOLYSIS.

(c) THE PROTECTIVE ACTION OF SERUM PROTEINS
AGAINST HAEMOLYSERS.

(d) THE EFFECTS OF OXYDISING AND REDUCING
AGENTS UPON HAEMOLYSIS.

By BENJAMIN MOORE, M.A., D.Se. (R.U.1.), Johnston Professor of
Bio-chemistry, University of Liverpool; FREDERICK P. WILSON,
M.D. (Liverpool), saxn LANCELOT HUTCHINSON, M.D.
(Liverpool).

From the Department of Bio-chemistry, University of Liverpool
(Received July 22nd, 1909)

The subject of haemolysis, and the relationship of lipoid substances
to this process of laking of the blood corpuscles, is one which is at the
present time exciting very general attention from physical chemists,
biological chemists, and clinicians alike because of its important relation-
ships to the chemistry of colloidal solutions on the one hand, and of its
valuable applications to the diagnosis of disease on the other.

In earlier papers more directly concerned with the subject of the
digestion and absorption of fats, it was shown in 1897 by Moore and
Rockwood! and in 1901 by Moore and Parker? that the salts of the bile,
and the products of fatty cleavage of a lipoidal nature, possessed when in
common solution some kind of an affinity, apparently of a physico-
chemical nature, which had the effect of increasing the solubilities of the
fatty acids and soaps.

This remarkable change in solubilities was shown by Moore and
Parker to extend to other lipoids, such as lecithin. It was further
demonstrated that the unsaturated oleic acid and its sodium soap had a

1. Proc. Rey. Soe., Vol. LX, p. 438, 1897; Journ. of Physiology, Vol. X XI, p. 58, 1897.
2. Proc. Roy. Soc., Vol. LXVIII, p. 64, 1901.

BIO-CHEMISTRY OF HAEMOLYSIS B47

most peculiar effect in increasing many times the solubilities of the fully
saturated palmitic and stearic acids and their sodium soaps. These when
in a state of purity were found to have practically a zero solubility in
either water or bile salt solution.
| Moore and Parker showed in the case of lecithin, which they prepared
_ from egg yolk, that the lecithin, when it was added to a solution of bile
salts or to bile at body temperature, did not form an emulsion or fine
suspension as in the case of treatment with water, but gave instead a
Fae water clear solution. This solution was then more effective than the
se! “veda in dissolving other lipoids.
oe tes - They also found, probably on account of this mutual effect upon

: _ effective solvent than a considerably stronger solution of the separated
i cand re-dissolved bile salts.
PD _ These earlier results on mutual solubility appear to us to possess a
‘bearing, which will be pointed out later, upon the process of haemolysis
of the lecithin-containing corpuscles by other lipoids, such as sodium
- oleate, the bile salts, and saponin-like bodies, and for this reason we quote
here certain of the figures given by Moore and Parker which definitely
show the mutual effects.
+The solubility of the fatty acids and soaps was found to be as
follows :—

‘Oleic Acid : solubility in distilled water less than 0-1 per cent. ; solubility in 5 per cent. bile salt
solution, about 0-5 per cent. ; solubility in 5 per cent. bile salts plus one per cent. lecithin, 4-0 per

cent.’
pe _ * Palmitie Acid : in distilled water less than 0-1 per cent. ; in 5 per cent. bile salts, about
___ O1 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 0-6 per cent.’

____ * Stearic Acid : in distilled water less than 0-1 per cent. ; in 5 per cent. bile salts less than
’ O-1 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 0-2 per cent.’

Sodium Oleate : in distilled water, 5-0 per cent. ; in 5 per cent. bile salts, 7-6 per cent. ; in
SPUN UeaA hile. salts glue 1 for cent. lecithin, 11-6 per cent

* Sodium Palmitate : in distilled water, 0-2 per cent. ; in 5 per cent. bile salts, 1-0 per cent. ;
in 5 per cent. bile salts plus 1 per cent. lecithin, 2-4 per cent.’
* Sodium Stearate : in distilled_water, 0-1 per cent. ; in 5 per cent. bile salts, 0-2 per cent. ; in
5 per cent. bile salts plus 1 per cent. lecithin, 0-7 per cent.’

* Lecithin. “Pure” lecithin is practically insoluble in water, the addition of as little as
0-1 per cent. causes an opalescence and further additions give rise, as is well known, to a kind of
emulsion. But when lecithin is added to a 5 per cent. solution of bile salts, or to bile, the appear-
ances observed are quite different.’

*The lecithin dissolves to a clear brown-coloured solution and the amount taken up is
surprising ; thus a 5 pot cent. solution of bile salts takes up no less than 7 per cent. of lecithin

848 BIO-CHEMICAL JOURNAL — | is

at a temperature of 37°C. On cooling, part of the lecithin is thrown out of solution as a finely
suspended precipitate or emulsion which glistens with a silky lustre when the test-tube containing
it is shaken so as to set the fluid in motion. At ordinary room temperatures of 15° to 20°

a considerable amount of lecithin, 4 to 5 per cent., is, however, still retained in solution.” “AS
SP be, a

‘The power of lecithin in increasing the solubilities of the fatty acids and soaps,

in great part why lower solubilities are obtained in experimenting with pure bile salt solutions, in

than with bile. The lecithin naturally occurring in bile thus increases the solvent power of that
fluid in the intestine for fatty acids and soaps.’

We have quoted at length these earlier experiments upon the mutual
effects of different lipoids in common solution upon one another, because

they appear to us to have some bearing upon haemolytic phenomena. For

example, sodium oleate or sodium linoleate have a strong laking effect
upon the red blood corpuscles. Now the red blood corpuscles contain
lecithin, but the above experiments ‘show that the presence of lecithin in
solution increases the solubility of oleates. In haemolysis of this type it
is hence obvious that the converse result is being obtained and that the
oleates or linoleates are laking the corpuscles, because lecithin is more
soluble in presence of the oleates or linoleates.

We shall also see that the bile salts and the members of the saponin-
digitalin group of glucosides are all unsaturated compounds like the oleates
and linoleates, and that they increase by their presence lipoid solubilities,
and hence are powerful laking agents.

These results upon solubility were confirmed and extended in several
papers by Pfliiger! and others, and Pfliiger laid particular stress upon the

effect of the presence of sodium carbonate and of oleic acid and oleates in :

raising the solubilities of the other constituents.

The above experiments upon solubility of lipoid materials and their
derivatives may now be considered in relationship to haemolysis. ° |

A very considerable portion of the stroma of the red corpusele is
lipoidal in character, that is to say, is soluble in ether or similar solvents.
The amount is placed at one-third of the dry weight by Pascucci,? and of
this a large amount consists of mixed lecithides, containing unsaturated
fatty acids in the molecule.

Accordingly, any constituent in a serum or suspending saline which
possesses the property of increasing the solubility of these lecithides must
tend to lake the corpuscles by dissolving up the stroma. Such an action,
as shown by Moore and Parker, is possessed by the bile salts, and they
accordingly act as powerful haemolysers.

1. Arch. f. d. ges. Physiol. Bd. LXXXII, 1900, 8. 303, 381; LXXXYV, 1901, 8. 1;
LXXXVIITI, 1902, g 299, 431; XC, 1902, 8. 1.

2. Hofmeister Beitriige, 1905, Vol. VI, p. 543; Iscovesco (* Les Lipoides,’ p. 13, 1908) places —

the amount of lipoids in the dried corpuscle at a lower value than one-third.

BIO-CHEMISTRY OF HAEMOLYSIS 349

This haemolytic power of the bile has long been known qualitatively ;
it has just now been followed out quantitatively in this laboratory by
MacLean and Hutchinson with the most interesting results, recorded in
the paper immediately succeeding this one.!

In the same fashion, we have seen that oleic acid and oleates were
found experimentally to raise the solubilities of the practically insoluble

i palmitates and stearates in the presence of bile salts. Also, even in the
__ absence of all bile derivatives, the solubilities in water obtained by Moore
and Parker for the separate sodium salts of the acids, oleic, palmitic, and
___ stearic on the one hand, and for the mixed sodium soaps of naturally
--—s geeurring fats of pig, ox and sheep on the other, clearly show that the
_ presence of sodium oleate increases the solubility of the other soaps.
These experimental results must be the basis of the results obtained
_by many observers that sodium oleate is a powerful haemolyser, while, as
ae demonstrated by Noguchi,? the sodium palmitate and sodium stearate

aS aes inert.

A chemical point of great importance is that both the oleic acid and
the bile acids are unsaturated bodies containing in each case doubly-linked

__ earbon atoms in an open chain, and this suggests the general law, first
enunciated by St. Faust and Tallqvist,? that the haemolytic property is
_ associated with this absence of saturation.

At the outset of our work, we were unfortunately unaware of the
a existence of St. Faust and Tallqvist’s paper, and we must express our
Px regret that for this reason we were unable in a preliminary communication
to do justice to their most interesting work upon the subject.

These authors, in following out in a highly interesting fashion the
causes of a pernicious anaemia due to the intestinal parasite,
___—C Botriocephalus latus, were able to separate from the dried bodies of the
___— parasites a material consisting to a large extent of an unstable compound
_ £ cholesterin and oleic acid. This substance was shown to be a

cholesterin ester of oleic acid of the type first separated from blood serum
by Hirthle.® This cholesterin-oleic ester had a most powerful haemolytic
effect even in small quantities, and on further testing the matter, St. Faust
and Tallqvist discovered that the haemolytic action was due to the oleic
acid, and that sodium oleate gave a like result, while saturated soaps or
their esters gave no effect upon the blood corpuscles.

1. See page 369.

2. Noguchi, Journ. of exper. Medicine, Vol. VIII, p. 92, 1906.

3. Arch. f. exper. Path. u. Pharm,, Vol. LVIS, p. 370, 1907.

4.

5.

Journ. of Physiol,, Proo. Physiological Society, March, 1909.
. Zeitach, |. Physiol. Chem., Bd, XX1, 1895-6, 8. 331.

850 BIO-CHEMICAL JOURNAL

This result led the authors to the generalisation that such haemolytic
action was associated with the want of saturation of the oleic acid. This
was tested by employing other unsaturated acids such as acrylic, tiglie,
cinnamic and erucic acids, and it was found in each case with the free —

acids there was marked haemolysis, although in the case of the sodium __

salts of tiglie and cinnamic acids there was no haemolytic activity.
St. Faust and Tallqvist further demonstrated in support of their view, that
when acrylic acid is hydroxylated into hydracrylic acid the latter is almost
without action upon blood corpuscles.

Now one of these acids has a double bond which has been split up in
the other, as shown by the formulae given below, and this is probably the
cause of the difference in activity.

Acrylic acid. Hydracrylic acid
II
if 2
Coon COOH

Strongly haemolytic Not haemolytic

We have ourselves been able in the present series of experiments to
demonstrate that an unsaturated glucoside with strongly haemolytic
properties, isolated by Moore from the seeds of Bassia longifolia (Mowrah -
seeds), called ‘Mowrin,’ loses its haemolytic properties when it
becomes saturated by bromination.

It is difficult to explain why the sodium salts of tiglic and cinnamie
acids do not haemolyse, for they are unsaturated compounds. It may be
that a certain conformation of molecule in addition to the double linkage
is necessary in order to confer upon the haemolytic molecule certain
physico-chemical properties which we shall subsequently see all these :
haemolysers possess, and that the want of saturation really confers laking
power because of the physical properties of solution, ete., which attach
themselves to it, and not because the double bond is broken to allow a firm
combination between the two compounds.

St. Faust and Tallqvist do not appear to have tested other acids and
salts than those mentioned above, nor to have proceeded to the further
generalisation that a similar lack of saturation characterises other laking
agents such as the bile salts and saponins.

Believing that the view is one of somewhat far-reaching importance,
we have in the present experiments tested it with a number of haemolytic
substances, and have always found that substances of this nature which
were haemolytic were also unsaturated or possessed of a good deal of
residual chemical affinity.

BIO-CHEMISTRY OF tlAEMOLYSIS 351

Connected with the above two points of haemolytic power and want

of chemical saturation, these bodies—often of widely different origin and

chemical constitution—possess always a well-marked group of common
properties, physical and physiological, which are so striking when placed
in juxtaposition as to indicate that they and the laking process are all
closely connected together and have a common cause.

Further, these common properties, which will presently be stated,
are such that in spite of the fact of chemical unsaturation running through
the whole group, it is difficult, or indeed impossible, to draw any definite
conclusion as to whether these bodies act by forming a feeble labile
chemical union, or by physically altering the properties of the solvent so

a, that it now dissolves the lipoids.

The energy phenomenon at play is evidently one due to interaction

between dissimilar chemical molecules, but whether it consists wholly, or
as an initial stage, in a lowering of the surface tension at the interface
bes between lipoid and solvent, or whether there is first a labile chemical union
between lipoid and unsaturated acid causing an accumulation on the inter-
ae face, so leading to a negative surface tension and hence to solution of the
___ lipoid and to haemolysis, it is impossible to say in the present state of our

knowledge.

It may be remarked, however, that the combating physical and
chemical hypotheses are not so very widely apart as the two camps of
adherents suppose, for in either case, the interaction is between dissimilar
molecules or aggregates at an interface, and this does not differ widely
from chemical action. We do not know what are the initial ‘ physical ’
stages of ‘ chemical ’ combination.

It might be asked, what is the nature of the energy change which

eatises accumulation of a dissolved substance on an interface and lowering
_ of surface tension, if it be not chemical attraction and the preliminary

stage of a chemical reaction? Leaving these more abstract considerations
of chemical combination versus physical adsorption, we may now return to
the characteristic chemical and physiological properties of the group of
haemolytic agents which we are discussing.

Prorertizs or HAEMOLYSERS

Physical Properties—All these substances are colloidal in aqueous
solution, although some of them diffuse very slowly through parchment
paper; they do not crystallise out of aqueous solution, and they give rise
to thick syrups as they are evaporated down to more and more concentrated

352 BIO-CHEMICAL JOURNAL

solutions. Even in dilute solution they all froth easily, showing petenly

that the surface-tension is lowered,!

Chemical Properties —All show a great tendency to form —
compounds, which are very easily hydrolysed by dilute acids. For
example, the bile salts with amino-acids, such as glycocoll or taurin; ;
oleic acid with cholesterin to form cholesterin esters, and with glycerol to
form fats; saponin, digitalin, mowrin, and other haemolysers of that
type are glucosides; the lecithides are not only conjugated compounds
themselves, but unite in feeble union with a vast number of substances of
biological origin, such as snake venoms and tox-albumens.

This property of conjugating chemically is, as we shall see, of the
utmost importance in connection with haemolysis, where it also occurs,
and may cause active haemolysis or an anti-phase, according to how it is
directed. .

Physiological Properties—Vhe physiological properties of the whole
group are closely related, and are, no doubt, dependent upon the above
physical and chemical properties. Thus the soaps, the bile salts, and the
whole saponin-digitalin group, are characterised by a very intensely bitter
taste. Introduced directly into the circulation, they are all poisonous, and
all affect the heart, causing slowing and stopping. ‘This is in all proba-
bility due to a common cause, viz., combination with the heart lecithides.

That same physico-chemical property which attacks the red blood
corpuscles by means of the attraction for its lecithin and causes haemolysis,
causes attack, always of a common type with minor variations, upon the
heart, due here also to chemical attraction between soap (sodium oleate),
bile salt, saponin, mowrin, digitalin, or what not, of this large group of
unsaturated bodies on the one hand, and the heart lecithide on the other.
So variations in reactivity are caused within the heart cell, and accom-
panying modifications in heart beat. Here it is to be remarked that it is
an integral change inside the cell of which the lecithide is a vital part that
occurs, and is not a mere question of altered permeability of a lipoidal
membrane. .

These peculiar properties are shown in varying degree by different
members of the group, but taken together they form a good set of
characteristics for a very widely distributed’ group of substances all
possessing haemolytic properties.

1, This is known to be so with the soaps, experiments with other haemolysers are in progress,

as

BIO-CHEMISTRY OF HAEMOLYSIS 353

Tue Batanctnc Action or HAEMOLYSERS

~-Oné of the most interesting experiments in haemolysis is that of Sachs
and Altmann,! demonstrating that two bodies, each of which is strongly
haemolytic in itself, can be so admixed in common solution that no
haemolysis whatever results, the two haemolysers balancing each other.
Thus, it was found that when sodium oleate was added in just the

proper quantity to a strongly active haemolytic serum no haemolysis

resulted, and that as the amount of oleate was gradually increased above
this balancing amount, the mixture gradually became haemolytic again.
This result has been stated to be due to the neutralising of comple-

“a ment, the sodium oleate acting as an anti-complement. We think,
however, that there is clear evidence against this view. In the first place,
as we shall see later, an ordinary serum which is not haemolytic to the
corpuscles being used, it may, in fact, be their own serum, is strongly

ee. _ protective against the haemolytic action of sodium oleate.

=_

We have followed this question up in detail, as shown by the protocols

ae 3 of our experiments, and have successively removed or destroyed
(a) immune body, (4) complement, and (c¢) the lipoids from the active
- serum. In all cases we have found that no one alone of these substances

is responsible for the neutralizing of the haemolytic activity of the sodium
oleate.

The further fact that not only is the laking power of the sodium
oleate destroyed, but also the natural activity of the pig’s serum, or the
invoked activity of a specially sensitized serum, appears to us to clearly
demonstrate that the soaps of the unsaturated fatty acids, oleic and
linoleic, possess a selective affinity for the immune body, or actively
laking, substance, of these haemolytic sera. That is to say, in the active

_ Serum the immune body and the sodium oleate or linoleate combine and
_ mutually destroy each the other's laking power, so that the mixture in due

proportion is quite inert upon the blood corpuscles.

But in case the immune body has been inactivated by heating,
then the sodium oleate or linoleate is still captured and held by the serum
proteins, so that no laking oceurs until much more of the oleate or
linoleate has been added than would have sufticed to cause complete and
rapid laking in a saline suspension, where there is no protein to present a
counter-attraction and binding agent, so that the first trace of oleate or
linoleate at once attacks the lecithides and other lipoids of the corpuscles,
When the serum proteins are present, although inactive themselves, they

1. Berl. klin. Wochensch., pp. 494, 609, 1908.

354 BIO-CHEMICAL JOURNAL

form binding material for unsaturated lipoids, such as the oleates and

linoleates. Yao
The action of lecithin and cholesterin of a similar type can be.

explained on similar lines rather than on the view that these substances

wy
aig

behave as active anti-complements, lee

On the other hand, as our experiments also prove, two lipoids of
nearly allied nature which do not therefore combine with each other, or
mutually adsorb each other, such as oleate and linoleate of sodium, show
no balancing action whatever, but produce a distinctly additive effect. So
that whether shown by the smaller amounts which will produce complete
laking in a given time in presence of each other, or better by observing the
laking times of two minimal amounts of oleate and linoleate separately,
as compared with the time spent for laking with the halves of these
amounts acting in consort, the result always comes out that the haemolytic
effect consists of the two added factors of oleate action and linoleate action;
there being no reduction whatever due to action between the -two
haemolysers, such as is seen between either of them and the active
haemolytic body of a sensitized serum or a serum naturally haemolytic.

We may hence enunciate the law that if two given haemolysers are
capable of combining or adsorbing with each other, they will tend to
balance each other, and the effect on corpuscles will be less than either
acting alone; but if no adsorption is possible between the two, the effect
in common solution upon the corpuscles will be the sum of the effects of
the two.

Errects or Ox1pizinc and Repucinec AGENTS uron HaArMoLysis

These experiments were suggested by analogies between the mode of
action of the peroxidases and haemolytic serum, in that heating to
56° C. destroys the tissue peroxides and so stops the action of the

_ peroxidases in a somewhat similar way to that in which heating to 56° C.
inactivates a haemolytic serum by destroying complement.

The results of experiment showed that it was not possible to replace
the destroyed complement of an inactivated serum by means of hydrogen
peroxide or other form of peroxide, so that the haemolytic agent or
immune body can hardly be regarded as a peroxidase ferment.

Yet the experiments yielded the very interesting information that
addition of an alkaline reducing agent, sueh as ammonium sulphide, even
in very small amount, entirely inactivated an active serum, and
contrariwise an oxidizing agent, such as hydrogen peroxide in alkaline

. — ae

BIO-CHEMISTRY OF HAEMOLYSIS 355

solution, very much increased the haemolytic power. ‘The peroxide alone,
_ or lrydrogen sulphide alone, in absence of alkali, had very little action;

but the addition of a trace of ammonia at once produced the inhibiting

action in the ease of the sulphide, or favouring action in the case of the

peroxide. .

In view of the fact that ammonia and other alkalies by themselves
possess a laking effect, it may be emphasized that the amounts being used
lay below the laking amounts when used alone, as shown by control

experiments.

EXPERIMENTAL Mretruops AND REsULTS

The sodium salts of five fatty acids were taken, namely, the sodium
salts of stearic, palmitic, erucic, oleic and linoleic acids.
In this list the first two are sodium soaps of saturated fatty acids,

a 3 : belonging to the acetic acid series; sodium erucate and oleate belonging
; _ to the acrylic series, are unsaturated sodium soaps, each having one doubly-
_ linked carbon atom in their formula; sodium linoleate belonging to the

linolie acid series is still more unsaturated, having three doubly-linked

carbon atoms in its constitution.

To test the haemolytic power of these soaps, solutions varying in
strength from 0°01 M to 0°001 M were made up by the simple procedure of
weighing out the requisite amount of pure free acid and neutralising with
the calculated amount of decinormal alkali. Thus, taking C,,H,,0,
as the formula for oleic acid, this gives a molecular weight of 282, which

is equivalent to 0°282 grams in 100 c.c. for a centimolecular solution.

This weight of oleie acid was therefore weighed out in a beaker and

__— neutralised with 10 c.c. of 011M NaOH, and the volume made up to
—___—«* 100 e.c. by adding normal isotonic saline solution. The weaker molecular

strengths were made up by adding proportionally more normal saline.
The other sodium soaps were made up in a similar manner.

For the experiment a series of test-tubes were taken, and in each was
placed a certain known quantity of the sodium soap, whose haemolytic
properties it was desired to test, and 1 c.c. of a 5 per cent. emulsion of
sheep's red blood corpuscles; the volume of each tube was then made up to
5°5 c.c. by adding normal isotonic saline solution. When the contents of
the tube were completed, they were placed in a thermostat at a temperature
of 37° C, and observations made.

It should be mentioned that in this and the subsequent experiments
the emulsion of sheep’s red blood corpuscles was made by defibrinating

356

BIO-CHEMICAL JOURNAL

fresh blood and then washing and centrifuging the corpuscles three me
in ree saline, and finally the washed red blood corpuscles were made up

into a 5

The results of several experiments with the soaps above mentioned —

gave the following results : —

In the tubes containing:

0°2 c.c. = sodium stearate.
M : ;

02 o.c. Too. sodium palmitate.
M .

0°2 c.c. Too sodium erucate.
M_

0°8 c.c. 1000 sodium sruaite,
M 3

0°2 c.c. 00 sodium oleate.
M .

0°4 c.c. 1000 sodium oleate.
M .

0°2 c.c. 1000 sodium oleate.
tee

0-4 c.c. 4000 sodium oleate.
M . .

02 c.c. 00. sodium linoleate.
M Tore

08 c.c. 7000 sodium linoleate.
M a

0:4 c.c. 1000 sodium linoleate.
M / :

0-2 c.c. 1000 sodium linoleate.
M , :

0°4 c.c. 4000 sodium linoleate.

It will be seen that in the case of the saturated sodium soaps of stearic
and palmitic acid no haemolysis was observed with the above strengths, ‘fia
but with very much weaker strengths of the unsaturated soaps complete r
haemolysis was obtained, and that sodium linoleate, which has the greater
number of doubly-linked carbon atoms, possesses also the strongest laking

per cent. emulsion in normal isotonic saline. (eae

« met

No haemolysis in 24 hours.

No haemolysis in 24 hours.
Complete haemolysis in 24 hours.
No haemolysis in 94 hours.
Complete haemolysis in 1 hour.
Complete haemolysis in 8 hours.
Slight amount of laking after 20 hours.
Merest trace of laking after 20 hours.
Complete haemolysis in 16 minutes.
Complete haemolysis in 50 minutes.
Complete haemolysis in 153 minutes,
Complete haemolysis in 20 hours,

Slight amonnt of laking in 24 hours

ae oe ee

action, completely haemolysing the sheep’s red blood corpuscles within

twenty hours, even in a concentration equivalent to 0:000004 M.
oleate, which has one doubly-linked carbon atom, is also a powerful
haemolytic agent, but not so active as sodium linoleate, though more

Sodium

powerful than sodium erucate, which is an equally unsaturated soap, but mad

with a different molecular constitution, and with corresponding physical

ce eee IE OS ney
eerste 2 Le Oa eo

BIO-CHEMISTRY OF HAEMOLYSIS 357

_ properties showing less typically the common character of the class of
i On the same grounds, some observations were also made with a
_ glucoside mowrin and the sodium salt of mowrie acid, one of the products
prepared from the glucoside by hydrolysis. These preparations, which are
a unsaturated bodies, were prepared by Moore from the seeds of Bassia
____ dongifolia, commonly known as Mowrah seeds.
ms The following are the haemolytic results obtained with these
substances, using 1 c.c. of 5 per cent. emulsion of sheep's red
blood corpuscles and a total volume of 5°5 ¢.c., and following the same

M
1 c.e. "00 Mowrin. Complete haemolysis within 1 minute.
¥ ; M i
oe 22'S 0.0. 10000 ” Complete haemolysis within 4 hours.
= M Ue
$x 05 cc. Too00 ” No haemolysis in 24 hours.
at M
1 cc. Too Sodium Mowrate. . Complete haemolysis in 15 minutes.
M .
1 cc, 1000 PP ts Trace of haemolysis in 20 hours.
ae : ue No haemolysis
- “hod O75 c.c, 1000 ” ” ysis.

It will thus be seen again that these unsaturated bodies, especially
the glucoside mowrin, are also powerful haemolytic bodies.
. If, however, the sodium salt of mowric acid is brominated, the haemo-
lytie action is markedly weakened, for instance, 1 c.c. of 0°01 M sodium
-__- mowrate haemolyses in fifteen minutes, but of an exactly similar quantity
if the brominated sodium mowrate be used, the time required in this case
____ for complete haemolysis is five hours.
_ ————s*Tn view of the fact that there are present in the normal organism
_-—s many ~=unsaturated haemolytic lipoids, it is of interest to note the
. protective action that the animal's serum is able to exert on behalf of its
own red blood corpuscles.

In order to study this action as regards the three unsaturated
haemolytic soaps used in the previous experiments, a series of test-tubes
were taken in which various haemolytic quantities of these soaps were
placed, and 3 c.c. of fresh sheep’s serum added to each tube; the serum
and soap were then incubated together for half an hour at 57°C., after
, which the sheep's corpuscles were added and the tubes replaced in the
. thermostat; no haemolysis occurred in any of the tubes, even though

"

358 BIO-CHEMICAL JOURNAL

1 c.c. O'01 M of each soap was used, which amount alone would in the
case of sodium oleate and linoleate have laked an equal quantity of sheep’s

red blood corpuscles almost instantaneously, and eight minutes would haye _
sufficed for sodium erucate. whe

Further observations showed that 0°5 c.c. sheep’s serum will exactly a

protect 1 c.c. 5 per cent. emulsion of sheep’s red blood corpuscles against
0°35 c.c. of 0°01 M sodium linoleate. Sheep’s serum will also protect its
own corpuscles against the natural haemolytic action of pig’s serum; for
example, we found that le.c. of 5 per cent. emulsion of sheep's red
blood corpuscles is completely haemolysed by 0°5 c.c. fresh pig’s serum
within an hour, the addition, however, of 2 e.c. of sheep’s serum will
completely inhibit this action.

Cholesterin also has an anti-haemolytic action, though not very
marked, 1 c.c. 0°002 M cholesterin emulsion being able to inhibit the
action of 0°7 c.c. 0001 M sodium oleate. Difficulty was experienced in
obtaining the cholesterin in a suitable medium to work with, as the solvents
of this compound, such as acetone, etc., are mostly haemolytic. In these
experiments, therefore, an emulsion of finely suspended cholesterin in
normal saline was used, its strength being approximately 0°01 M.

This protective action of serum and cholesterin will be again referred
to later on, when we shall have pointed out an action which Sachs and
Altmann first described in the case of sodium oleate, and which they
termed the behaviour of sodium oleate as anti-complement. __

Experiments are described below showing not only that this action
can be extended to other unsaturated-soaps, but also that it is, to a certain
extent, independent of the presence of either complement or amboceptor,
and therefore the term ‘ anti-complement’ has been omitted and the word
‘balancing’ used in its place, as more accurately. describing the action.
For not only is the haemolytic property of the pig's serum on sheep's red
blood corpuscles gradualiy inhibited as the amount of soap increases, but
also after this action has been completely balanced, the serum on its own
part further inhibits the haemolytic action of the soap.

Pig's serum is naturally haemolytic for sheep’s red blood corpuscles,
but if to pig’s serum is added a certain quantity of sodium oleate there is
an inhibition of haemolysis, and a point can be found where, owing to

interaction between these two substances, no haemolysis occurs, although”

the quantities used of each haemolytic agent are such that if either was

usel separately complete laking of the sheep’s red blood corpuscles would
ensue,

1.
4
q
‘

BIO-CHEMISTRY OF HAEMOLYSIS 359

For instance, tubes containing :—
___ @. -1-¢.e. sheep's red blood corpuscles—0°5 c.c. pig's serum—4 c.c, normal saline gives
complete haemolysis within half an hour.
- M
b. 1 c.c. sheep’s red blood corpuscles—0°7 c.c. 100 sodium oleate—3°8 c.c. normal
saline results in complete haemolysis in 3 minutes.

But :—
M
e. 1c.c. sheep's red blood corpuscles—0°7 c.c. 100 sodium oleate—0°5 c.c. pig’s serum
—8-3 c.c. normal saline results in almost complete inhibition of haemolysis.

The same holds good for sodium linoleate, and sodium erucate, _— the quantities
_ vary in each case.

| The results of an experiment are shown graphically in fig. 1. Many
similar experiments were carried out giving parallel results : —
Tm all cases the sodium salt of the acid, the serum and saline were
incubated together for three-quarters of an hour at 37° C. prior to adding
the sheep’s red blood corpuscles. When the contents of the tubes were
completed they were again placed in the thermostat at 37°C., and
observations made from time to time.
f The slight variations in these results are probably due to variations in
the ‘titer’ of the different supplies of pig’s serum that were used, as the
solutions of oleate and linoleate of soda were the same in each experiment,
and, moreover, it may be mentioned that the difference between the
balancing points for the two unsaturated soaps is constant in all experi-
ments, in each the amount of 0°01 M sodium linoleate required to balance
05 c.c. pig’s serum being 13 ¢.c. in excess of the quantity of sodium oleate
required for the same result.
It is interesting to note that although according to the previous experi-
____- ments sodium linoleate alone isa stronger haemolytic agent than sodium
oleate, yet it does not seem to be as powerful as the oleate in balancing the
action of pig’s serum.

These experiments, while showing the balancing action of sodium
oleate and linoleate, seemed also to point to the existence of another.
inhibitory action which might be independent of the existence of comple-
ment in the pig’s serum.

Some pig’s serum was therefore inactivated by heating it at
56°C. for half an hour, and after making sure that the serum was
completely inactivated, exactly similar experiments were earried out as
before. It was then found that the haemolytic action of the sodium

by ; linoleate was inhibited up to the same point when using the inactivated as

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BIO-CHEMISTRY OF HAEMOLYSIS 861

when using the fresh serum; that is to say, whereas 1 c.c. 0°01 M sodium

_ linoleate will alone haemolyse 1 c.c. of 5 per cent. emulsion of sheep’s red

blood corpuscles almost instantaneously, yet when used with either (5 c.c.
of the fresh or the inactivated pig’s serum its action was almost completely
inhibited. As a further step, some pig’s serum was taken in which both
the complement and the amboceptor had been removed. This was
accomplished by heating some pig’s serum to 55° ©. for half an hour. to
destroy the complement, and then adding excess of sheep’s red blood

corpuscles (fresh pig’s serum being powerfully haemolytic for sheep’s red

blood corpuscles), incubating the mixture together for an hour at 37°C.
and then centrifuging; the clear supernatant serum being again treated
with sheep’s red blood corpuscles until by tests it was evident that all the
ambocepior had combined with the corpuscles, and the clear serum

contained neither of its two haemolytic factors.

The effect of this serum on the haemolytic properties of sodium

“4 - lineleate was then tested in an exactly similar way to that used in the
___ immediately preceding experiments, and it was found that even thus

depleted it maintained its protective power intact, and, moreover, that this
factor was not in any way diminished. With these results before us, it
will be at once apparent that although in the fresh state pig’s serum is a
powerful haemolyser of sheep’s corpuscles, yet if we remove from this
serum one or both of its haemolytic factors it then exerts a powerful
protective action on the red blood corpuscles, an action which in the case
of the sheep’s red blood corpuscles is more potent than that exercised by
the animal’s own serum, for it will be observed that 0°5 c.c. of sheep's
serum was able to protect 1 c.c. of a 5 per cent. emulsion of sheep’s red
blood corpuscles against the haemolytic action of 0°35 c.c. 0°01 M sodium
lir.cleate, while under similar conditions 0°5 c.c. of inactivated pig’s serum
was able to completely inhibit the haemolytic action of 0°6 c.c, 0°01 M
sodium linoleate on a similar quantity of sheep’s red blood corpuscles.

We have already mentioned that cholesterin has the power of
inhibiting, up to a’certain point, the haemolytic action of various soaps.

The question arises, is the above protective action due, as Iscovesco
thinks, to cholesterin 7 |

An attempt to investigate this by trying to extract this inhibitory body
from pig’s serum by means of ether gave a negative reply to this question.

To carry out this experiment 100 c.c. of fresh pig’s serum was poured
‘into a separating funnel and 150 c.c. of ether added, the contents being
then shaken up together for fifteen minutes, allowed to stand, and then the

362 BIO-CHEMICAL JOURNAL

serum which collected in the lower part of the funnel was withdrawn.

This process was repeated three times, using fresh ether each time. The

_ three portions of ether were then collected into one flask and the ether
slowly distilled off at a temperature of 37° C., the last traces being removed _

by means of asuction pump. The residue containing ether extractives wis 4
then shaken up with 30 c.c. of warm normal saline, forming ins a
white opalescent soapy emulsion.

The action of this emulsion was tried on some sheep’s red blood
corpuscles, but it was found neither to have any haemolytic action alone
nor any inhibitory action against other laking agents.

The serum, which had been carefully separated from the ether, was
then placed in a flask and all trace of ether removed by bubbling air
throug), it was then tested with sheep’s red blood corpuscles, and it was
found that although now it had no haemolytie power, yet its protective
action was intact. |

The result would appear to be evidence indicating that although z
cholesterin undoubtedly has considerable inhibitory power, yet it does not >.
account for the protective action of the serum, as the extraction with ether

:
would probably have removed the greater portion. :
_ F

Appitive Errect on Harmorysts or Two Cioseiy ALLIED HAEMOLYSERS
WHICH CANNOT THEREFORE CoMBINE witH Eacu OTHER B
re
The preceding experiments show that two dissimilar haemolysers, 4

such as the haemolytic substance of pig’s serum for sheep’s corpuscles and oe
sodium oleate or linoleate, so far from supplementing, balance each other. — i
The present experiment demonstrates that sodium oleate and sodium
lincleate uscd in common solution aid each other, the effect being
approximately additive.
Thus, using the same technique as previously described :—
I. Sodium oleate, 1:25 ¢.c. of 0-001 M + sheep’s r. b. c. 1 ¢.c, of 5 per cent. emulsion dye
+ normal saline to 5¢.c. Result—complete laking in 26 minutes. ae
II. Sodium linoleate, 1 c.c. of 0°001 M + sheep’s r. b. c. 1 c.c. of 5 per cent. emulsion -
+ normal saline to 5c.c. Result—complete laking in 19 minutes.
III. The same quantities of the two together, viz., sodium oleate 1-25 c.c. of 0001 M
+ sodium linoleate 1 c.c. of 0-001 M + sheep’s r. b. c. 1 c.c. of 5 per cent. emulsion + saline
to5c.c. Result—complete laking in 11 minutes.
IV. Half the initial amounts of the two sodium salts gave the following results, viz, :—
Sodium oleate, 0°62 c.c. of 0-001 M + sodium linoleate 0:5 c.c. of 0-001 M + sheep’sr. b. c.
1 c.c. of 5 per cent. emulsion + saline to5c.c. Result—laking in 28 minutes.

|

There is accordingly no balancing here, and the effects of the two
used in common solution are practically a purely additive function.

PIO-CHEMISTRY OF HAEMOLYSIS 363

When sodium linoleate and fresh lecithin are used as a pair of
haemolysers,-a very distinct balancing action is obtained. Thus, 0°2 c.c.
_ of 0°01 M sodium linoleate laked 1 ¢.c. of 5 per cent. emulsion of sheep’s
red blood corpuscles in four minutes; but when exactly the same amount
_ of the sodium linoleate is first treated with 0°5 c.c. of an emulsion
- -—s ¢orresponding to 001M lecithin, the haemolysis is delayed for about
-__ two and three-quarter hours.

This illustrates very clearly the constituent in the red blood corpuscle
which is attacked in haemolysis, and also shows that the haemolyser and
=" the lecithin of the corpuscles enter into at least a quasi-combination,
so limiting the amount of corpuscle haemolysable by a given amount of

_ haemolyser.
wl he
_ ee omaars or Oxipizinc AND Repvucine AGENTs To HAEMOLYSIS

_ Hydrogen Peroxide and Haemolysis.—At the outset of this investi-
an attempt was made to test whether complement could be replaced
by hydrogen peroxide.

_ Pig's serum, which, as is well known, has a haemolytic action on
sheep's red blood corpuscles, was inactivated, and to different dilutions of
is ‘Serum vary ing strengths of hydrogen peroxide were added. Repeated
experiments yielded discordant results; a strength of hydrogen peroxide
- which on one occasion caused haemolysis, failed to do so a second time.
Investigation showed that the hydrogen peroxide had an acid reaction,
and this was probably the cause of the uncertain results.

On using dilutions of Merck’s perhydrol, much more uniform results
es were obtained. Except in very strong solutions—-up to 1 in 50 in normal
_ saline—perhydrol had no haemolytic action on fresh sheep’s red
corpuscles. Added to inactivated pig’s serum it had no complementary
_ action, and, indeed, the haemolytic actioa of the stronger solutions seemed

; aa be inhibited by the serum.
Another oxidizing agent, quinone, was tried in varying strengths in a
similar way. No haemolytic or complementary action was obtained.

Added to fresh pig's serum, neither perhydrol nor quinone interfered
with its haemolytic power.

: : Similarly, oxidase containing solutions from fresh vegetable juices
ee: could not be used as substitutes for complement.

~ Thus, we have been unable to trace the nature and action of comple-
ment in haemolysis, but our experiments led us incidentally to certain
observations upon the effects of reducing and oxidizing substances on
haemolysis which are here recorded.

a bas. we ss ee ial ms; at “ee
pe ae | :

364 BIO-CHEMICAL JOURNAL

Errecr or Repuctnc AGENTS oN COMPLEMENT

We next tried the effect of various dilutions of ammonium sulphide on
the haemolytic action of pig’s and other sera. The stock ammonium

sulphide used for dilutions was of a strength 0'4 M. Table I gives the

details of the experiments. The results show that in dilution up to 1 in
1,000 the ammonium sulphide inhibits the haemolytic action of the sera

used.

Equivalent solutions of hydrogen sulphide and ammonia in normal

saline have not such inhibitory action.

In all experiments the tubes were all made up to 5 c.c. with normal

saline.

The sheep’s red corpuscles were used in the form of a 5 per cent.

suspension in normal saline.
Fresh guinea-pig’s serum was used as complement.

The anti-sheep haemolytic rabbit’s serum was of such a titre that
le.c. of a 1 in 1,500 dilution dissolved 1 c.c. of a 5 per cent. suspension of

sheep’s red blood corpuscles in half an hour.

Taste I
1 c.c. of fresh pig’s serum + 1 ¢.c. ammonium sulphide (dilution lin 50) + 1 e.c. r. b. e.—No haemolysis
% asi (dilution 1 in 100) Ms af
Pa ¥ (dilution 1 in 200) e- e,
9 ” (dilution 1 in 300) ~ Partial haemolysis

1c.c. of inactivated serum) +1 ¢.c. ammonium sulphide (dilution lin 50) + 1 ¢.c. r. b. ¢. —No heelys

+ O'Le.c. complement |

pepe 2 il eo

= a (dilution 1 in 100) Cs 3
ks (dilution 1 in 200) = Haemolysis F
” ” (dilution 1 in 300) its ae f
1 c.c. inactivated rabbit's serum (dilution 1 in 20)) +1 ¢.c. ammonium sulphide + 1 ¢.c. r. b. ¢.—No pm
+ 0°1¢.c. complement j (dilution lin 50)
” ” (dilution 1 in 100) ~ a aa
- a (dilution 1 in 200) ~ Slight haemolysis —
* a (dilution 1 in 300) He Complete haemolysis
1 c.c. fresh pig’s serum + 1 c.c. r. b. ¢. Haemolysis
1 c.c. inactivated pig’s serum + 0-1 ¢.c. plclinteehdain 45 ice tate a
1 c.c. inactivated rabbit’s serum + 0-1 c.c. complement + 1 ¢.c. r. b. c. ht be
1 c.c. inactivated pig’s serum + 1 ¢.c. r. b. ¢. No haemolysis

1 c.c. inactivated rabbit's serum + 1 c.c. r. b. c. :
1 c.c. ammonium sulphide (dilution 1 in 50) + 1 ¢.c. r. b. ¢.

All tubes were made up to 5 c.c. with normal saline.

The sera and ammonium sulphide were incubated together at 37° C.
for half an hour, r.b.c. emulsion then added, and whole incubated for one

hour.

BIO-CHEMISTRY OF HAEMOLYSIS 365

A comparison of the effects of equivalent strengths of sodium hydro-
oxide, ammonium hydrate, and ammonium sulphide on sheep’s r.b.c. are
_ given by Table IT..

Tasre IT

w NaOH + 1c.c. r. b. c, — Haemolysis in half an hour

2. O65 c.c. =e NH,OH + lc.c. r. b. c. — Slight haemolysis next day

l.. 0506.0. =~

3. OD e.c. X. (XH,)8 + Lee. r. b. c. —- No haemolysis
All tubes made up to 5 ¢.c. with normal saline. |
In Tables III, IV and V are shown the ‘ balancing ’ action of sodium
hydrate with fresh and inactivated serum; also the influence of perhydrol

on this action. All tubes were made up to 5 c.c. with normal saline,
incubated at 37° C. for two hours, and results noted.

Tasre III
le.c. fresh pig’s serum + 1 c.c. r. b. ¢ Haemolysis
N
» * + 0-5¢.c. 0 NaOH ... ane nae . | No haemolysis
N
a + 0-5 c.c. Te NaOH __.... =e a aa Bs
" ” ” + 0-5 ¢.c, Sy NaOH eee oes see see Haemolysis
” ” + 0-5 c.0. naon ese eee eee oss ”
N
. ” ” + 0-5 c.c. 30 NaOH ... dee ‘a ue fe
i. N
fo 1% 05 c.c. 75” NaOH + loo.r. boo. ... a mt aa te ike ii ‘
N
a4 0-5 c.c. 5 NeOH + leer. boc. ... te & te oan ti we ”
: = ;
05 c.0, “gp NaOH + Loc. rb. c. . cee ce tee nee nee nes «

Pata). :
05 cc, “g5 NaOH OS eo oe

ll. O5e.c. x Naot + locr. bo, ... as nae eae “on See ae 1.
Tasix IV
1. 1... fresh pig’s serum + 1o¢.c.r, boc... eae exe ee yes Haemolysis
a 2. O5c.c. x NaOH + 1o.c. r. b. c. Be ST eke.” | es ie
. 3. Le.c, fresh pig’s serum + 0-5 0.0. ~*~ noon + loo. r. bo. sie No haemolysis
4. ” ” ” + Loe. web = aes Haemolysis
ql te Sd aileeion)
5. ” ” ” + Leo, perh A
a Rag Deri tm
6. 1.0. perhydrol (1 in 30 dilution) + 1.0. r. b. ©. ioe, am oe No haemolysis
¥; ” (1 in 60 dilution) + 1 ¢.c. r. b. o. iad: |. a oe a

‘ oa a ii cd Tee « en pe

366 BIO-CHEMICAL JOURNAL a

TABLE V

N
1 c.0, inactivated pig’s serum + 0-5 ¢.c. 7 NaOH + le.c.r.b.c. Slight ies ns 4 hours

” se ”

" . i ae ¢.c, = oe Haemolysis in 9 lig
(i in 60 dilution) . &
+a + leo. rb. ce. + Lec. perhydrol No haemolysis ye
(1 in 30 dilution) a.
is ss » + 1¢.c, perhydrol No haemolysis

(1 in 60 dilution)

The haemolytic action of ammonia in various strengths on sheep's
r.b.c. is shown in Table VI.

Taste VI

N 4
l. B3e.c. 70 ammonia + le¢.c.r. b,c. ... ar Haemolysis in 2 or 3 minutes
2. 2c.c. es ro bie sie ”
3. 1-5c.c. -: » tee bes Partial haemolysis in 2 hours
4. leo.e. pa " a aoe Slight haemolysis in 2 hours
5. OF5c.c. ,, » sos oa No haemolysis in 2 hours
6. 0-5 ¢.ce. ” ” eee "eee ” ”

All tubes were made up to 5c.c. with normal saline.
Table VII gives the effects of adding ammonia to fresh and.
inactivated pig’s serum, and of variations in the technique. é

Taste VII

ae
l. lee. aT Ye ammonia + 1 c.c. fresh pig’s serum ... —... ... Slight haemolysis next day
2. 0°75 c.c. o = pes is ... Very slight haemolysis next day
3. 0-5 c.c. s cc} ‘e a aaa 99 * i
4. lose. * ammonia + 1 c.c. inactivated pig’s serum ... ... Pale brown colour next day
5. 0-75 c.c. ” ” oo ” ”
6. 0-5c.c. ” ” ” ”

Tubes made up to 4 e.c. with normal satiaes incubated for one hour at
37° C., then 1 c.c. r.b.c. added to each tube and incubated for two hours, —

N Act
1 c.c. 70 ammonia + 1 ¢.c, fresh pig’s serum + 1¢.c.r.b.c. Slight haemolysis next day =~

8. 0-75 ¢.c. Pa ‘9 ... Very slight haemolysis next day
9. 0-5 c.c, ” ” eco ” ”

10. Ic.e. 77 ammonia + 1 c.c. inactivated pig’s serum + 1 c.c. r. b. ¢. Pale brown colour .

ll. 0°75 ¢.c. : ” ” ” ” ”

12. 0-5c.c. ” ” ” ” ”

Tubes made up to 5 c.c. with normal saline and r.b.c. at onée, and
incubated for two hours. -

BLO-CHEMISTRY OF HAEMOLYSIS 367

Having found that quinone alone did not much influence haemolysis,
“we next tried the effect of quinone with ammonia added. The alkali
promptly turns the quinone black, or brown in weak dilutions, probably
turning it into hydroquinone, which then undergoes some further change.
Quinone added to a dilution of ammonia, which by itself had no haemo-
lytic action in two hours, promptly turned black and produced instant
haemolysis. Inactivated pig’s serum inhibits this action to some extent.
- Hydroquinone alone has no haemolytic effect, but with ammonia produces
the same results as quinone. The results obtained are set forth in

Table VIII.
Peery! |, Taste VIII
Jo *mmonia +loor. bo. ... ae See ee or sal een «» No haemolysis
wy om ” + 1 c.c. 0-1 % quinone ee eee — nee ties Dark brown color
Haemolysis in 2
” ” va ° eas se ans Si ... Brown colour
No haemolysis in
” ” ” » + 1¢.c. inactivated pig’s serum... Black colour
; No haemolysis
| ¢.¢. 0-1 %, quinone + 1 c.c. r. b. c. ve os Be apd ae ae a an ph =
0-1 % hydroquinone + 1 c.c. r. b, ¢. be Fe fo ade ae he *
oa 4 N ” ” ” re + 1 c.c. inactivated pig’s serum Fe Ea
OS cc. 0 ammonia + | ¢.c. r. b. c. + 1 ¢.c, 0-1 % hydroquinone ot pie ... Black colour
Haemolysis
i, ” ” * Pa + 1c.c. inactivated pig’s serum Brown colour
. No haemolysis
Ole. Jo *mmonia + loc. r. b,c, + 1e.c, 01% hydroquinone ... 4. «ss» Dark brown colo
om No haemolysis
ah ” ” ” ” + lc.c. inactivated pig’s serum Brown colour
ag No haemolysis

All tubes made up to 5 c.c. with normal saline.

SuMMARY OF REsULYTS

1. The substances concerned in haemolysis, including thereby both
the haemolytic agent outside and the substance attacked within, have a
powerful mutual effect upon one another's solubilities.

2. Instances are given of such increased solubilities, and the
favouring effects upon haemolysis noted. As a result of such increased
solubility lecithides are dissolved out from the mass of the corpusele, so

setting free the haemoglobin also, and laking is the result.

3. It is noted that all the haemolytic class of unsaturated soaps of

368 BIO-CHEMICAL JOURNAL

fatty acids, saponin, mowrin, digitalin, the various bile salts, possess
common physical, chemical and physiological properties, and are all
unsaturated biddise capable of bromination, ete.

4. The similar action upon the heart of the haemolytic bodies i is si
probably due to combination between these and the heart lipoids. — ilies

5. Although want of saturation exists, it is probable that the first

fundamental step is a ‘ physical’ one of lowering of surface tension with
accompanying tendency to solution. But no hard and fast line can be
drawn between so-called physical and chemical action.

6. The balancing action of haemolysers is discussed, and it is shown
that this is not obtained with closely similar haemolysers, where instead an
additive action is seen. This suggests that balancing is due to a
combination or interlocking of the two haemolysers, whereby nothing is
left free to touch the corpuscles.

7. Where sera and such haemolysers as sodium oleate balance, the
first call is between the active body of the serum and the oleate; next, in
absence of the active body or of complement, the serum proteins alone,
although not active in themselves as haemolysers, possess a superior binding
power over the corpuscles for the oleate, and hence act as protectors, so
that much more oleate in excess must be added before the corpuscles are
attacked. Accordingly, as is well known, a mere trace of oleate suffices
to break down corpuscles in saline suspension, but in serum suspension
many times more oleate must be added before any result is obtained.

8. It follows from this that oleates, etc., do not act as anti-comple- peep
ments, and ought not to be described as such; it is most.probable that
they possess no specific relationship whatever to complement.

9. Sodium oleate can also be balanced by lecithin for similar reasons.
10. Under conditions specified, and in alkaline solution, oxidizing
agents favour haemolysis, and reducing agents restrain it ; but an oxidizing

agent alone cannot replace complement in an active haemolytic serum, —
and it is not probable that complement has the nature of a peroxide body.

369

‘OBSERVATIONS ON THE HAEMOLYTIC ACTION OF
+ CERTAIN BILE DERIVATIVES

By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of
Aberdeen, anv LANCELOT HUTCHINSON, M.D. (Liverpool).

From the Bio-chemical Laboratory, University of Liverpool
(Received July 22nd, 1909)
Our knowledge of the processes involved in the laking of red blood

_ corpuscles is at present in an obscure and unsatisfactory condition, and
_ despite the amount of research carried out on haemolysis and the number
__ of theories advanced to account for different results, many cases still
remain unexplained.

___ In connection with this subject, one very interesting point has lately
__ been advanced by St. Faust and Tallqvist, and afterwards by Moore; these
____ observers found that haemolytic action, when brought about by such bodies
as the fatty acids, occurred only in cases where the acid employed was one
_ of an unsaturated variety containing in its molecule one or more double
bonds, while the corresponding saturated bodies produced no effect. In
view of this observation, it is likely that the state of combination of the
carbon atom constitutes an important factor in many cases of haemolysis

produced by chemical agents.

The strong probability, on chemical grounds, of certain bile products
being constituted by bodies of an unsaturated nature suggested the present
‘investigation; the curious ‘results obtained do not seem capable of
explanation by any of the theories put forward at present.

SUBSTANCES USED

The present paper deals with the results of haemolysis by three
= bile derivatives—the sodium salt of glycocholic acid, cholalic acid and
____ tholeic acid. These were prepared as follows : —

Soprum GLycocnoLaTe

Fresh ox bile was freed from pseudo-mucin by treatment with
"4 alcohol; after the evaporation of the aleohol, neutral acetate of lead was
aa added, the resulting precipitate separated off and decomposed by heat in

ee "a. a a et ee ae ee ee ae ee ee ey
eer ee eee
Be ee = a Mange , : ;

870 BIO-CHEMICAL JOURNAL

the presence of a sodium carbonate solution. The mixture was evaporated
to dryness and the residue extracted with alcohol; the alcoholic extract was
filtered, the filtrate evaporated to dryness and the residue dissolved in
water. This watery solution of sodium glycocholate was now decolorised

by animal charcoal, and the free glycocholic acid thrown out by means of

a dilute solution of hydrochloric acid. The acid was thoroughly washed
with water, then dried and again dissolved in alcohol. To this alcoholic
solution sodium carbonate was added, and the whole again evaporated to
dryness. Residue was dissolved in a little cold alcohol, and the mixture
filtered. This process of purification by dissolving in alcohol was
repeated; the alcoholic solution was then evaporated to dryness and the
residue dissolved in a little water, filtered, evaporated to dryness over the
water bath, and ultimately dissolved in normal saline solution, and in this
solution utilised for the experiments.

CHoLanic Acip

Ox bile was boiled for thirty hours with one-fifth of its volume of
30 per cent. caustic soda, the total volume of the mixture being kept
constant by the addition of water from time to time. The solution was
then saturated with carbon dioxide and evaporated almost to dryness.
Residue was extracted with 96 per cent. aleohol, and the extract diluted
with water so as to contain not more than about 20 per cent. of alcohol;

it was then treated with a solution of barium chloride, the precipitate

filtered off, the filtrate treated with weak hydrochloric acid, and the
precipitated cholalic acid separated off and thoroughly washed with water.
This acid was now changed into the sodium salt, and the above process of
treatment with barium chloride, etc., repeated twice. The free acid
ultimately obtained was dissolved in alcohol, and by the addition of sodium
carbonate again changed into the sodium salt; the alcoholic solution was
filtered, evaporated to dryness, the residue dissolved in a little cold aleohol
and filtered; filtrate was evaporated to dryness, residue dissolved in water,
filtered, again evaporated to dryness and final residue utilised for
experiments. oe

CuoLteic Acip

Choleic acid was prepared from the precipitate obtained by barium
chloride in the preparation of cholalic acid: this precipitate, which
consisted of an impure salt of barium choleate, was purified on the lines
described above; it was also utilised in the form of the sodium salt.

HAEMOLYTIC ACTION OF BILE DERIVATIVES = 3871

EXPERIMENTS

_ The first set of experiments was carried out with the sodium salt of
- cholalic acid. In all cases red blood corpuscles from the sheep were used ;
these were carefully washed four times in the ordinary way with normal
galt solution, and then made into a 5 per cent. emulsion with isotonic saline.
: + + a For convenience in preparing the somewhat varied strengths of
-__ gholalic acid utilised in the experiments, two solutions were made—one a
* + deci-molecular and the other a centi-molecular; in each case the molecular
: t of the salt was taken as corresponding to the formula C,,H,,0;Na,
= all solutions being made in normal saline.
Sie! For each experiment a series of tubes, each containing 1 ¢.c. of the
above emulsion of sheep’s red blood corpuscles, was taken, and to this was
added a measured amount of sodium cholalate. By the addition of the
- necessary amount of normal saline, the final volume of each tube was
a tained constant, and in all our experiments amounted to 5 c.c. The
+ ie were then placed in the incubator at 37°C., and observations
. ‘constantly taken at short intervals.
As the result of repeated experiments, it was found that this salt
_ displayed well-marked haemolytic properties, but at the same time
* ¥ exhibited some peculiarities so striking as to amount to what may be
die peageectively termed a haemolytic paradox.

In tubes containing a moderately strong dose of sodium cholalate,
the corpuscles were completely laked in intervals of from one to two hours;
___ when the substance was present in somewhat smaller quantities, haemolysis

was delayed for three or four hours; on still further reduction of the
a amount of substance, however, the peculiar fact was observed that
haemolysis took place in a shorter and shorter space of time, until a point
__-was reached at which a minimum dose gave a maximum effect. With
_ such an amount this minimal-optimum dose often caused complete
laking of the corpuscles in six minutes, or even less; while on the other
E.: hand, a dose several times as great might take hours to produce a similar
2H effect.
When exhibited in amounts smaller than this minimal-optimum dose,
the effect became gradually less, until ultimately a strength was used
beyond which no further haemolysis could be obtained.

The accompanying chart gives a good idea of the results obtained;

in a long series of experiments the laking always took place as indicated.

'%

=}
7

a.
a
i :
-

872 BIO-CHEMICAL JOURNAL |

Cuart No. [

Soptum CHOLALATE

al - eT ee Shs TO Rie TR we The eae - can —- : ~~
io)
Time during which sodium cholalate had been acting 2/2 )8 8 ¢ 8 a
-—— + aus Ban ae | _
. Masti, 1
I co. gpien's 8. Bi that oh secsbecvsiees} cavetnqos tense +4 ¢.c. sodium cholalate — >> > >>}
¥ at oy Fy ¥ OS saline...... +3 ss Re ~~ eS q
‘ eT 2 + 8.0.1 sodium cholalate | —|—|-|—|—/ | -\4 ,
reece se j pat
” ” +2 4, ‘ ” +2 Fe oe —|-|— | ae es 5 4
ae ei t
” + 3 ” ” + 1 os ” -—l—| ei rie 7 J ++
PPT aia : +09 Tost tee -|-|-}-|-|-|-|=|=}
” ” + 3-2 ” ? + 0-8 ” %9 gm Bek fiom. = = int = = +
” ” + 3-4 ” ” + 0-6 ” 7 ‘be: i "ex -| -|-|- +44
+ ae
ed ” 7 35 ” ” + O05 ” ” A ms fy. eS ‘Lee uy a +++
”? ” + 36 9 "9 = 0-4 ” ” | Tiel abe —+> ‘ ;
»” ” + 3:7 oh . ote O-3 . ” >) | = +> 4
” ” + 2 a ary + 0-2 99 ” - $444
J if
” ” + 3 ” ? 2 x 0-1 *9 ” $+44444 ¥.
+ » +35,, 4 +005 ,, s |_| —| —| —jedledled .
7” ” + 36,, ” + 0-04 ” ” | -|-|-|-|-}- ’ Bs ere ;
” ” oa 37 ” ” + 0-03 ” ” \— ons aed Gees loa Dee ‘ as
” ” + 38,, ” + 0-02 ” ” bi Si —} =} a) eae ss Fe
-- - i

” ” + 3-9 ” ” + 0-01 os + — -|-|-/='- = ERLE _—

a,
” ” + 4 ” ” (control) > - -| -| a — _- ae én _— —

fe Eee i x

Norr.—The dct of red seein i was rayidly added last of all, the difference in time
between the first and last tube being barely 4 minutes. Immediately after the corpuscles were
added, the tubes were placed in a thermostat [37° C.] where they were observed.

The times were calculated from the moment when the tubes were — in the thermostat. “2 ss
In measuring amounts of sodium cholalate less than 0-2 ¢.c., an 100 solution was used. * es

%

+ Denotes point at which laking became complete. wn
The results are also shown graphically plotted in fig. 1, in which the
ordinates represent time of complete haemolysis in minutes, and the
abscissae, the concentrations of the sodium cholalate, in ¢c., as indicated, a) i
of 0°1M solution added to a total volume of 5 c.c. when completely — 5
made up.

HAEMOLYTIC TION OF BILE

sodium cholalate

M
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b

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lume of 5 c.c.

ted abov

ica

CHOLALATE

to maintain a constant

emulsion) + amounts, as ind

ts

A
)
+ sufficient normal saline

Each test tube contained 1 c.c. sheep's r. b.

3 es & 8

—) ssApouteuy oyopdutoo soy poaynbea seynuyut uy eur,

| a an _ ———
a

874 BLO-CHEMICAL JOURNAL

The following reproduction also shows these results; the tubes were
left to stand for one hour and then centrifuged; by this means a more
suitable condition for photographic purposes was obtained. Haemolysis
is distinctly seen in tubes 1, 6, 7 and 8, the remainder being quite free
from laking, with the exception of Nos. 2 and 9, where a slight action has
taken place. }

From these results, it is obvious that the haemolytic power of the
so-called minimal-optimum dose is much more marked than that obtained
when much larger amounts are used, and that between these two points
there lies a sort of neutral ground where the haemolytic power is very
much reduced. In the experiment recorded in the chart it is seen that

hhh O OR aig

PHOTOGRAPH SHOWING HagEMotytTic PAarRApox
Tubes with black appearance are those in which laking has occurred.

The following table indicates the amount of sodium cholalate in each tube in the photograph :—

No. 1. 8c.c. = Sodium cholalate | No. 6. 08 c.c. a Sodium cholalate
9 M | a hs ee Oe ae rv 99
9 ire. “i 7 . M
Me. ae Pe Pe ea Ee Se TE RC. Se mts "
» & O8c.c. ,, eS i SL OR gles Sh 7 r
» 8 O5cec ; | sie. OS: 8. Fr:

0'1 c.c. of O'1 M. sodium cholalate lakes completely in five minutes, whereas
it takes thirteen minutes for 4 c.c. of 0°2 M solution to produce the same
effect; in other words, a certain minimum dose gives in five minutes an
effect equivalent to that produced by a dose about eighty times as great in
thirteen minutes.

HAEMOLYTIC ACTION OF BILE DERIVATIVES — 375

In another series of experiments performed under the same conditions,
but using the sodium salt of choleic acid, similar results were obtained.

When a strength of 4 c.c. 0°1 M sodium choleate was used, haemolysis
was complete in about siz minutes; in this strength laking was quickly
followed by an action on the liberated haemoglobin, as indicated by a

_ dark brown coloration of the liquid. With 0°8 c.c. 0'1 M solution the full

effect was in evidence in about eleven minutes; when, however, only 1 c.c.
~ 001 M was used, haemolysis was almost instantaneous, the corpuscles being
- completely laked within one minute.

This salt is a much more powerful agent than the corresponding

~ compound with cholalic acid. Whereas with sodium cholalate a solution
sof 03. c.c. 0°01 M required about eighty minutes for complete laking, and a
or oun strength corresponding to 02 c.c. 0°01 M gave only the merest indication of
-_ a positive reaction after twenty hours’ observation, with sodium choleate

_ in strength of 0°7 c.c. 0°001 M complete haemolysis was obtained in one
hundred and sixty minutes, and with 0°6 c.c. 0°001 M in twenty hours; even
~ with 0°5 c.c. 0°001 M, traces of haemolysis were evident after this time.

The full results of one experiment are indicated in Chart 2.

Similar experiments, giving in the main the same kind of result,
were performed with sodium glycocholate. Here, however, there was not
the same marked difference between the haemolytic action of large and
small amounts as was seen in the case of the above described salts; this is
due to the fact that the action of the minimal-optimum dose is in this case
not nearly sorapid. In other respects, as indicated by the chart, the same
general result is in evidence. The results are shown also in fig. 3.

On the other hand, haemolysis was ultimately obtained with smaller

amounts than in the case of sodium cholalate, though, even in this respect,

it did not appear to be so powerful as the choleic salt.

A few observations were also made with free cholalic acid; consider-
able diffieulty was experienced in procuring a suitable method of
application owing to its lack of solubility in inert substances. We
succeeded, however, in emulsifying a small amount with normal saline,
and with this emulsion similar results to those ébtained with the sodium
compound were noticed; that is to say, with a certain small amount of
emulsion a much more marked haemolysis was produced in a given period
than was in evidence after the exhibition of perhaps nine or ten times as
much in a much longer space of time.

876 BIO-CHEMICAL JOURNAL
Cuart No. Il
Soprum CHOLEATE Minutes
Tine panes which sodium choleate had been acting vised bac’ hick Ga 7 |e S18 |
1 c.c. sheep's r. b. ©. 4+ .ceeeeeeereeeeeeee + 40.0. F sodium choleate | — —| — ++ t++4444 .
el of lee ee, : ~~ $$444444444 =
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ey sy. SBeetial = ws sgt! —|—|—|—| “so > 9 >
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” oe +. o » (control) -|-|-'-l-}-1- aed ford ee tee bow os ae be

+ Indicates complete ‘haemolysis.

Norr.—The same conditions existed during this experiment as in the preceding Chart a
except that the tubes were not placed in the thermostat for 8 minutes after adding the corpuscles.
The corpuscles were added during 2} minutes, and the times are calculated from the time when
the last tube received its corpuscle.

O Trace of haemolysis.
These results are shown graphically in Fig. 2, where ordinates show as
in Fig. 1.

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Each test tube contained 1 c.c. sheep's r. b. c. (5% emulsion) + amounts in cubic centimetr

HAEMOLYTIC ACTION OF BILE DERIVATIVES

t normal saline to maintain a constant volume of 5 c.c.

cien

+ suffi

377

than a larger amount.

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The curve shows well the paradoxical result that a small amount of the haemo!

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878

HAEMOLYTIC ACTION OF BILE DERIVATIVES — 879

Cuart No. Ill

Soprom GLYCOCHOLATE

Times in minutes 25,3085 4044 80! 20 hours
1 o.e, sheep's r. b. c. + 1.0. saline + 3 cc. 3 sodium glycocholate 444444444
Pa ae PS ne cw HT ” : —|-|-|- o> +
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a 5. eee als OE si ¥ Oe Se 2 ed
a » +4 ww (Control) 0 | A
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+ = Complete laking.
_ Times calculated from time at which tubes were placed in thermostat.

380 BIO-CHEMICAL JOURNAL

Of late years, it has been repeatedly demonstrated that the serum of
an animal is able to exert a well-marked protective action against

haemolytic agents on behalf of its own red blood corpuséles. We
therefore tried the effect of substituting 1 ¢.c. of fresh sheep’s serum in

place of 1 c.c. normal saline in a series of tubes in which sodium cholalate
in various strengths was used as a haemolytic agent; here it was found
that the serum exercised a very marked inhibitory action on the haemolytic
power of this substance. The results of such an experiment are seen in
Chart 4.

It was found, for instance, that 1 c.c. of 0°1 M sodium cholalate, which
when used alone was able to completely haemolyse the 1 c.c. of sheep’s red
corpuscles within five minutes, was, when used in conjunction with 1 c.c.
of sheep’s serum, absolutely devoid of haemolytic action. Such a result
indicates very marked power indeed in the inhibition and prevention of
haemolysis by the protective action of normal serum.

In examining the chart, one apparent contradiction will be noticed ;
in the case of the tube containing 3 c.c. 0°1 M solution of sodium cholalate,
haemolysis was complete in twenty-two minutes. Such an amount,
however, when used alone, is capable of completing haemolysis only after
eighty minutes, so that in this particular case the serum has increased
instead of diminished the haemolytic action.

This result is easily explained in the light of our former observations; —

if we assume that the amount of sheep’s serum used was insufficient to

neutralise the effect of the total amount of cholalic acid salt present, but —

had neutralised its equivalent amount, it is obvious that in this case a
reduced amount of sodium cholalate would how be present in an active
form, and since a weak dose (within certain limits) is more powerful than
a stronger one, an increased action might naturally be expected.

Some sheep’s serum was then inactivated by heating it at 56°C. for
one hour; it was then tested to make sure that the process was complete;
this was done by means of several controls against inactivated pig’s serum
which normally lakes sheep’s red corpuscles. Ina series of tubes similar
to those used in the preceding experiment, 1 c.c. of the inactivated serum
was substituted for the fresh material; it was found that the protective
action was in no way diminished as the result of inactivation; indeed, if
anything, it seemed more marked. It may, therefore, be assumed that in
these cases the presence or absence of complement is immaterial in so
far as the protective action of the serum is concerned.

A similar series of experiments were carried out using the sodium salt
of choleic acid, and similar results obtained. Chart 4 shows these results
epitomized.

> >

.

a

‘

7

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881

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OF BILE DERIVATIVES

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HAEMOLYTIC ACTION

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NOILIGIHN] NIUALSAIOHO—'G “ON LUVHO “Ff DLT

382

HAEMOLYTIC ACTION OF BILE DERIVATIVES 383

According to Liebermann, this inhibitory power of the blood serum
is dependent on the serum-albumin. Iscovesco,! on the other hand,
_ attributes it to the cholesterin normally present in blood serum. Gerard
and Lemoine? have studied the anti-toxic action of cholesterin as applied
to tubercular poisons. Ranson’ has also demonstrated the anti-haemolytic
power of cholesterin on certain chemical substances.

; In view of these results, we have tried the effect of cholesterin on the
% haemolytic action of sodium cholalate. Using an 0°002M emulsion of
cholesterin, it was found that even in strengths of 0°0008 M solution it
ce exercised a considerable anti-haemolytic action, and in the presence of
weak doses of sodium cholalate was sufficient to entirely prevent a laking

These results are demonstrated in Fig. 4, Chart 5. When using
04 cc. 0-1 M sodium cholalate, the amount of cholesterin used (2 c.c.
002 M) was sufficient to delay the usual laking effect for about thirty
minutes; with 0-5 c.c. 0°01 M there was a delay of seventy minutes, and in
the case of 0°4 c.c. 0-01 M haemolytic action was totally abolished.

- In all these experiments, the cholesterin emulsion and the sodium
___ cholalate were incubated together at 37°C. for one hour previous to the
addition of the red blood corpuscles. —

The above are some of the results obtained with the substances
mentioned ; it is hoped to extend them at a later period. No theoretical
considerations with regard to them have been advanced, since much more
work must be done before any satisfactory explanation can be forth-
coming; they are merely given as facts obtained as the result of repeated

experiments.

ConcLuUsIONS

Such bile derivatives as the sodium salts of cholalic, choleic, and
at glycocholie acids are capable of producing haemolysis in the ordinary
- way when strong doses are used, but exhibit marked peculiarities when

‘ present in considerably weaker amounts :—

1. It was found that under similar conditions the same haemolytic
effect can be produced in a given time by widely divergent amounts of the
salts. Between these two points lies what may be termed a more or less

- 1. ‘Les Lipoides,’ 1908.
2. Congris de Médecine, 1907. Soc, Médic. des Hospitaux, Paris, 1907.
3. Deutsche med. Wochenachr., 1901.

a

384 BIO-CHEMICAL JOURNAL

neutral zone, in which haemolysis is very considerably delayed depending
on the relative amounts of haemolytic agent employed. The minimum
dose giving the maximum effect in the shortest time we have designated
the ‘ minimal-optimum haemolytic dose.’ For instance, it was found that
this minimal-optimum dose in the case of sodium cholalate was 0°1 c.c.
01 M solution; this quantity, mixed with 1 ¢.c. of 5 per cent. sheep’s red
corpuscles (the total volume being made up to 5 e.c.), gave complete
haemolysis within five minutes. Stronger solutions gave a much less
marked effect, a solution eight times as strong requiring one hundred and
fifty-five minutes for a similar result, while a dose eighty times as large
as the minimal-optimum dose required thirteen minutes to complete
haemolysis.

2. In all these cases the addition of cholesterin produced a marked
anti-haemolytic effect.

3. Ina similar manner, the addition of fresh sheep’s serum exercises
a markedly anti-haemolytic action; in some cases, however, an apparent
augmentation is in evidence. This is obviously due to the fact that the
amount of serum used was unable to completely neutralise the laking
action of the bile salts; this would result in a smaller relative amount of
active haemolytic agent being present, and hence a more powerful effect
would be seen (within certain limits), in accordance with the above
observations, .

4. This inhibitory action of serum does not depend on the presence of
complement; an inactivated serum acts equally well. |

We wish to thank Professor Benjamin Moore for advice and assistance
in the above investigation.

| THE PHARMACOLOGY OF APOCYNUM CANNABINUM*

By J. C. W. GRAHAM, M.A., M.D., B.C. (Cantab.).
Communicated by Prov. W. E. Dixon.
From the Pharmacological Laboratory, Cambridge
(Received August 25th, 1909)

) L INTRODUCTION.

Action on 1. Vascular System,
(a) Heart.
(i) Frog.
(ii) Mammal.
(iti) Relative Toxicity of Apocynum.
(b) Blood Vessels.
(c) Blood Pressure.

2. Toxic effects.
3. Muscular System.

. Comparison or APocyNUM CANNABINUM WITH OTHER MEMBERS OF THE CARDIAC
Group or Drugs.

I
Apocynum is a member of the group of cardiac tonics, and is official in
the United States. It has been credited with being the most powerful
indirect diuretic known. Descriptions of the drug are given by Paine!
and two Russians, Gliski? and Gvozdinsky.* In this country records* of

we patients suffering from pleuritic effusion have been given, under the

influence of Apocynum it was thought that the fluid subsided ‘ rather

quickly.” Hauseman® first suggested that Apocynum might contain a

cardiac poison allied to the digitalin group, and in 188% Schmeideberg
isolated two so-called active principles, apocynin and apocynéin. Rose
Bradford showed that its principal action was upon the heart. Sokoloft®
showed that the drug caused slowing of the heart’s action, * enlargement
of the pulse’ wave and marked rise of blood pressure. Petteruti and
Somma’ found different results were obtained according as to whether they
used the decoction or the tincture, the decoction acting chiefly on the
stomach and intestines causing catharsis and emesis, when this action was

*A t was made towards the expenses of the research by the Scientific Grants Committee
of the “British Medical Association.

386 BIO-CHEMICAL JOURNAL

absent there was diuresis and acceleration of the heart beat. The tincture
was stated to be unirritating to the gastro-intestinal tract even in large

doses. The emetic and cathartic action of the decoction was attributed to

the admixture of the bitter fibre of the wood with the bark of
the root.’ Further experimental work was carried out by Dortschewski,®
Klopotovitch! and Lapshin. Since my experiments have been completed,
I find that Laidlaw and Dale have been working with a crystalline active
principle isolated by H. Finnemore.'! Some apology is, therefore,
necessary for the late publication of this paper. :

The preparations used in my experiments were (a) Tinctura Apocyni
Cannabini. One part of the root to ten parts of 60 per cent. alcohol.
(6) Apocynin.

The animals employed were frogs, rabbits, dogs and cats. Frogs were
used for simple injections into the dorsal lymph sac, and for demonstrating
the action of the drug on striated and unstriated muscle, the gastrocnemius
muscles and a sectional ring of the stomach being used respectively.
Perfusion experiments were also performed by tying a cannula into the
hepatic vein. The effect of the drug on the heart in situ, and also in
connection with recording apparatus, was also observed. The frogs were
always pithed, except those used for simple injections.

Observations were made with rabbits on the effects of injecting both
large and small quantities of the drug; the action on the vascular system
was also determined.

Dogs and cats were employed for obtaining cardiometer and oncometer
records; experiments on the urinary flow were performed on dogs.

Chloroform, A.C.E. mixture and urethane were used as anaesthetics.
In a few cases no anaesthetic was used, the animals being pithed.

II. Action

Fe Vascular System.

(a) Heart. (i) Frog. The most important action of Apocynum is
seen in its effects on this organ. In a general way Apocynum acts on
cardiac muscle in the same manner as it acts on both striated and
unstriated muscle. The tonus is always increased, the systole and
diastole are greater in amplitude. Cardiac slowing is a marked feature.

On placing on the heart a few drops of a crude extract of the root of
Apocynum a marked effect is immediately apparent. The beat is slowed
and a clear rise. of tonus occurs. 3

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 387

Such experiments are of little value, since the effect of a drug applied
to the outside of the heart is not necessarily the same as when the drug is
given by the circulation, but they suffice to indicate the probable effect.

Fig. | shows the movements of a frog’s heart recorded by the suspension _method,'so! that
the systole is represented by the upstroke. A saturated alcoholic solution of Apocynin was
used for injection into the subcutaneous tissues. Section I shows the normal beat. In IT
is seen an increase of systole and diastole with some slight slowing after a subcutaneous injection
of 10 minims of the Apocynin solution. An increased effect is noticed in III and IV after applying
the solution directly to the heart. Section V gives the results of placing crystals of Apocynin on
the heart. The greatest amplitude and most marked slowing of the beat is seen in the first part
of VI, after this, the beat gradually quickens until it becomes very quick and irregular (delirium
cordis) and the heart dies in systole.

The experiment illustrated in fig. 1 is particularly interesting, as it
shows that the substance Apocynin is active, and that it causes an effect
like the tincture. It is also of some interest to observe the delirium cordis,

as this is a condition particularly difficult to obtain in the frog.

After a subcutaneous injection of 14 minims of tincture of Apocynum
in the frog, a similar series of events occurred, and death ultimately

- occurred in systole.

These simple experiments show that the crude extract of Apocynum,
the glucoside Apocynin, and the ordinary standard tincture, all give rise

to the same series of effects in the frog. The heart beat is slowed and

increased in amplitude, later in the action the beat becomes quicker and
irregular and death occurs in systole. The development of these effects
was also seen in tracings of perfused frog’s hearts.
(ii) Mammalian heart. Experiments were undertaken to demonstrate
the effect of small doses of Apocynum on the isolated mammalian heart.
Fig. 2 represents the records of an experiment in which the drug was introduced (5 c.c.
Tinct. Apocyni, | in 6,000) into the side tube of the perfusion apparatus so that after considerable
dilution with the Ringer’s solution in the apparatus all the drug would rapidly pass through
the isolated heart. In other words, five minutes after the injection, no Apocynum would be
in the ciroulating fluid. Section I shows the heart beating normally. II shows the effect five
minutes after injection; the rate of the beat is the same, but the amplitude of the systole is
increasing. Tracings LII and IV show still later effects, 20 and 25 minutes after injection the
rate of beat still remains about the same, 42 to the minute. In V, a record taken 30 minutes
after injection, the heart is beating much more slowly and ventricular beats are being lost,
so that two auricular beats are shown to each ventricular beat, and in the later portion of the
tracing the ventricular beats entirely cease for a time. This stage is rapidly succeeded by that
of delirium cordis in which the heart beats very rapidly and the tonus of the cardiac muscle rises
(VI, VIL, and VIII). The heart ultimately stops in systole (IX and X) fifty minutes after the

injection.

It may be noted here that the death of this heart, which is typical of
many experiments, results from a very small dose of the drug, in spite of

BIO-CHEMICAL JOURNAL

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390 BIO-CHEMICAL JOURNAL

the facts that the administration was of short duration, that the small
quantity of fluid containing the drug was quickly washed away by the
constant flow of fresh saline solution during the whole of the experiment,
and that only one dose was given. Evidently not all the drug administered
is removed by the constantly flowing saline solution, but it would seem
that some part of the dose remains behind in a kind of fixed combination
with the cardiac muscle, and that it is this portion of the drug which is
responsible for the production of the ultimate toxic effects. This may be
taken as an example of cumulative action. I would like to compare this
drug with strychnine or atropine which, when administered, induce a
certain definite action but are rapidly excreted. Apocynum, on the
contrary, is fixed by the heart, and continues to produce an effect on the
heart when no drug is present in the coronary circulation.

In other experiments the heart was perfused with the Ringer’s
solution, but after a short time this was replaced by a saline solution of
the drug, so that the heart was bathed during the remainder of the experi-
ment with a diluted tincture of Apocynum (1 in 6,000): this is a condition
which would approximate in a remote degree to that prevailing during
life while any drug was continuously and regularly administered, and
which was absorbed gradually into the circulation. Such experiments”
showed what may be regarded as the therapeutical action of the drug,
systole was augmented, diastole prolonged, and the tonus was unaltered;
later the poisoning action was shown by the acceleration present. The
acceleration was further increased ; so rapid was the heart that diastole was
incomplete, the heart beats followed one another too rapidly for complete
diastole to occur. Marked arhythmia was present and tonus was increased,
a condition which only occurs when the heart is beginning to die. In
some tracings, groups of two following beats were seen, so that there is a
greater interval between each group of two following beats than between
the individual beats of each group. This is a condition which appears in
that form of irregular pulse known by the names of coupled beats, allor-
rhythmia, pulsus bigeminus (twin pulse), and pulsus biferiens. It is well
known clinically that this type of pulse may at any time appear during
the administration of Digitalis, especially when the effect is becoming
cumulative, and it seems also that this is one of the toxie effects of
Apocynum. Experimentally, this form of heart beat is only seen when
the heart muscle has been rendered ‘ hyperirritable’ by a drug of the
digitalis group. One heart died in a state of extreme contraction, having
been at work for three hours,

:
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oO ee Se ee

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 391

Some further experiments were performed with the cardiometer.
Cats anaesthetised with urethane were used for this purpose, and the drug
was given directly through the circulation. Apocynum in small doses
increases the output of blood from the heart; the effect comes on slowly,
but is very prolonged. This effect is, of course, the true criterion of a
cardiac tonic, an increased cardiac output being the true sign of
stimulation.

The action on the rate of the heart beat varies according to the stage
of action of the drug. In the therapeutic stage the heart is slowed, and is
quickened in delirium cordis, which constitutes the poisoning stage of the
drug.

The slowing effect of Apocynum on the heart, as indicated by the pulse
and also the influence on the respiration, was shown generally by the

% following simple experiment, for which I am indebted to Professor
Dixon. A small-sized dog was given 5 minims of Tincture of Apocynum
subcutaneously. The data given in the following table indicate the
aad pranees i in the pulse rate and respiration :—

Time Pulse Respiration
4.30 ‘is 114 wid 20
4.45 sae 120 wad 16
4.55 - 76 on 14
5.0 me 96 wt 16
5.6 Ai 108 eek 20
5.15 hee 108 ay 16

Similar results were obtained on man.

(iii) Relative toxicity of Apocynum on the heart. For this purpose
solutions of various strengths of the substance Apocynin and of the
Tincture of Apocynum were injected into the dorsal lymph sac of the
frog; Dixon and Haynes!? having previously shown that this forms a
suitable means for determining the relative activity of the cardiac tonics.

Effects of injections of Apocynin : —

Exp. I. 5.30 p.m. le.c. of an 0-5 iien. of Apocynin was injected into the dorsal lymph
sac of a frog weighing 19 grammes.
eae less brisk, respiration quickened, animal crawls about but does
up.

5.55 p.m. ota ps of two or three respirations occur in
Soll conmendan’ Sencar Responds ert to forcible pricking stimulation.
6.20 p.m. By this time the frog had completely recovered.

-Exr, I. O45 Rt Reale Ue BE seiation fen talented tite the doom! lymph one of a A9p

12 grammes.
4.45 p.m. this time the had fully recovered, having been only sh for the
pm. By Bee edaaer tian eden od ng y sluggish

892 BIO-CHEMICAL JOURNAL

Exr. III. 3.60 p.m. A frog weighing 14 grammes was injected with 3 c.0. of 0-5 solution.

3.56 p.m. Frog motionless in dorsal position, pupils widely dilated, rere, to
pinching toe.

4.10 p.m. Breathing very feeble, struggles at times.
4.25 p.m. Turned on to its feet, pupils contracted.
5.0 p.m. Recovered but slightly, slow in its movements.
Exr. IV. 5.0 p.m. A frog weighing 16-5 grammes was injected with 3 0.0. of a 1:25 per cent.
solution of Apocynin.
5.5 p.m. Heart beats 60 per minute.
5.13 p.m. Heart beats 48 per minute.

5.15 p.m. Animal very feeble, does not respond to stintulation. The heart was now
on , the ventricle was feebly contracting and ceased in diastole at
p- m. The auricles and sinus esiehll still contracted in groups of rio
pee at a time.

5.35 p.m. Dead.

Effects of injections of the Tincture of Apocynum :—

Expr. I. 4.45 p.m. Frog weighed 20 grammes. 15 minims of the tincture injeoted.
4.50 p.m. Animal helpless in dorsal position.
5.6 p.m. Slight corneal reflex.
5.20 p.m. Dead.

Exp. II. 4.40 p.m. Frog weighed 12-5 grammes. 7 minims of the tincture injected.
5.5 p.m. Crawls with difficulty.
5.15 p.m. Slight corneal reflex.
5.20 p.m. Dead.

Exr. III. 4.38 p.m. Frog weighed 15 grammes. 3 minims injected.
5.5 p.m. Recovers from the dorsal position with difficulty.
5.30 p.m. Dead; ventricle firmly contracted.

Expr. IV. 3.53 p.m. Frog weighed 17 grammes. 2 minims injected.
4.30 p.m. Dead.

Expr. V. 3.55 p.m. Frog weighed 12 grammes. 1 minim injected.
5.25 p.m. Leaps and falls on its back, recovery difficult, tumbles over.
5.30 p.m. Crawls with difficulty.

5.35 p.m. No recovery from dorsal posture, moves limbs. Hind limbe —_—-
before upper ones.

5.45 p.m. Dead.

These simple injection experiments serve to demonstrate the
comparative inactivity of Apocynin, and the toxicity of the tincture of
Apocynum upon the heart. Smaller amounts than one minim of the
tincture had no fatal effect. One minim may therefore be regarded as the
minimum lethal dose for the standard tincture of Apocynum.

From the results of two of the experiments in each of which the frogs
died in 35 minutes, one from the effects of Apocynin and the other from
the tincture of Apocynum, it appears that 3 c.c. of a 1°25 per cent. solution
of Apocynin is about equivalent to 15 minims of the tincture of Apocynum
in toxicity. Approximately the 1°25 per cent. solution of Apocynin is

TSR ee Re ee el
oe

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 393

_ 8} times less toxic than the tincture of Apocynum. For the sake of
comparison, the following table of the minimum lethal doses of the most
commonly used cardiac tonics is appended : —

Drug M.L.D. (in minims) per 100
grammes of
Tinet. Digitalis (13)... ond fos oe 12-5
Tinet. Strophanth. (13) ee 1-47
Tinct. Scillae (13) iy sae has no 12-5
Tinct. Apocyni ... on Re wa os 8-33
Apocynin (1°25 per cent. sol.) at if ~ 20-15

From this it seems that the tincture of Apocynum is about one and a half

times more toxic than the tinctures of Digitalis and Squill, and nearly
_ six times less toxic than the tincture of Strophanthus. The substance
_____ Apoeynin possesses a fair degree of activity, although it is the least toxic
‘* _of the series in the above table.

All the experiments show that Apocynum acts on the heart somewhat
similarly to Digitalis in causing slowing of the heart beat. The principal
____ action is to raise the tonus or condition of continued contraction of the
ee muscle of the heart, especially in toxic doses the ventricle may be so
firmly contracted that it appears quite pale. This is well seen in the

frog’s heart and also in the mammalian heart when mounted on the
3 perfusion apparatus. If a sufficiently small dose of the drug is
f administered the first effect usually noticed is a gradual increase in the

extent of the heart beat, the increase in the excursions of the recording

lever increasing until the vertical height of the tracing becomes two or

three times greater than it was before the drug was administered. This

increase in amplitude of beat is accompanied by a decreased rate of beat,

which is more marked in Batrachian than in Mammalian hearts. The

amount of slowing produced varies from rather less than half the original
rate up to not more than three times as slow. The heart at this stage is
quite regular, relaxation is more complete and so is contraction; under
favourable cireumstances the contraction of the auricles may equal the
contraction of the ventricles in extent. This condition of affairs should
represent the most perfect therapeutical effect of the drug.

When this is being most completely manifested a change in the
character of the beat more or less suddenly takes place, some quickening
and irregularity occur. The ventricular portion of the beat may be
dropped, and each chamber of the heart in mammals may contract or not
independently of the rest without any order or sequence. This condition
of irregularity, combined with extreme rapidity of beat, constitutes the

34 BIO-CHEMICAL JOURNAL

condition of delirium cordis, and is a toxic effect of the drug. In
mammalian hearts the delirium ends in fibrillary twitching. Most of the

slowing of the heart beat is due to the action of the drug on the nerve

terminals of the vagus in the heart muscle; for if these terminals be
paralysed by atropine the slowing produced by the Apoeynum is much
less decided, although there is a very slight amount seen, which must be
due to the direct action of the drug on the muscular tissue of the heart.
The slowing effect gives place to a stage of quickening; during this
quickening the vagus nerve appears to be paralysed, since no slowing of
the beat can be obtained either by electrical stimulation of the nerves or
the injection of a drug such as pilocarpin or muscarin, which are known
to excite vagal endings. Now, Apocynum is not a paralytic drug; so far
as we know, it depresses no nerve endings, and its effect in preventing
vagus inhibition has a more ready explanation than the supposition that
it paralyses vagal endings like atropin. Apocynum acts essentially on
heart muscle by increasing its irritability, and in the later stages of its
action, this irritability shows itself by marked acceleration. The vagus
nerve is intact, but such is the irritability of the cardiac muscle that it has
no longer the power to hold it in check. The acceleration of beat,
accompanied in its later stages by irregularity, stimulates that part of
the heart known as the excito-motor area, and so the rapidity of beat is
increased continually until fibrillary twitchings occur. The amount of
work done by a heart under therapeutic doses of Apocynum is increased,
or in other words, the output of blood is increased. The inerease in the

amount of work is due to the combined action of the Apocynum on the —

nervous and muscular structures of the heart. The increased force of the
beat is due to the stimulant effect of the drug on the muscle alone, the
stimulant effect on the vagus endings produces some inhibition, so that
the heart becomes slower, the diastole is prolonged, and the heart is more
completely filled with blood. The systole is also increased, not only in
strength, but also in length, so that there resvlts a more complete
emptying of the heart in systole.

(6) Blood Vessels. The general effect of Apocynum on the blood
vessels was shown by the perfusion of a pithed frog. The hepatic vein of
a fair-sized frog was displayed, and a small cannula tied into it. This
cannula was then connected with a simple perfusion apparatus. One leg
of the frog was cut off below the knee, so that the perfusing fluid as it
circulated through the body of the frog dripped from the cut end of the
limb, The drops were counted at short intervals for periods of one

Oe

—-

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 395

minute; the frog was first perfused with normal saline solution, and then
with a solution of 1 in 60 Tinet. Apocyni. This experiment distinctly
showed that Apocynum is a powerful vaso-constrictor, as it diminished the
flow from 31 to 6 drops per minute. This effect is a peripheral one,
as the brain and spinal cord of the frog were destroyed, so that no central
yvaso-motor influence was at work. It may be noted that this diminution
of flow was not due to any decrease of the action of the heart, as at the
end of the experiment this organ was still beating vigorously. The vaso-
* constriction must be due to the direct action of the drug on the nerve
endings or muscular coats of the blood vessels.
_ The vaso-motor effects of Apocynum were further demonstrated by
ae smother experiment.
ieee he record in Fig. 3 was taken from a pithed cat and the blood pressure recorded in the usual
with a mereury manometer, the changes in the intestinal vessels were recorded by an
eter. Two c.c. of a | in 4 solution of the tincture were injected by the side tube into
ugular vein, and at once a very marked rise of blood pressure is seen. This was accompanied
y a lowering of the oncometric curve, which indicated a diminution of the intestinal volume
owing to the powerful contraction of the intestinal vessels, after this the volume increased
_-—s owing to the dilation of the organ as a whole, no doubt brought about by the great increase in
the cardiac output. The volume of the intestine at about the maximum blood-pressure is
the resultant of two factors, namely increase of size from the distension caused by the greater
blood-pressure, and decrease of size owing to the great vaso-constriction as shown by the volume
pulse. During the supervention of delirium cordis the intestinal volume again underwent
decrease ; owing to the marked fall in blood-pressure the intestine was less distended and the vaso-
constriction factor became evident. At the end of the experiment the blood-pressure fell
rapidly and suddenly to zero, and intestinal pallor was noticeable. The administration of
Atropin had no effect on the final result as the vagal influence had already been annulled by
the Apocynum : this is due to the increased irritability of the heart muscle which can no longer
2 be controlled by the vagus.
a __ In pithed dogs similar experiments were performed with like results.
) Further experiments were undertaken on the renal vessels, and the same
type of constriction as in the splanchnic vessels occurred. In every case,
then, in which this drug was perfused through the vessels or given
internally, vaso-constriction resulted. To determine whether the drug
acts on muscle or nerve endings, the pulmonary vessels of a mammal were
perfused. The first effect was a short dilatation, followed by a profound
and prolonged constriction; adrenalin, which was first injected, having
no influence on the blood flow. Therefore, since we know that the
pulmonary vessels are not innervated, the action of Apocynum must be on
the muscle fibre. The constriction of the blood vessels produced by
Apocynum is well marked, not only during its therapeutic action, but also
during the development of its toxic effects. Finally, all the experiments,

ed i
ne

896 BIO-CHEMICAL JOURNAL

such as perfusing a frog’s body or a mammal’s lung, or recording changes
in the volume of kidneys, loops of bowel, and forelimbs, show very
conclusively that this drug is a powerful vaso-constrictor. And it is to be
noticed that this effect is a peripheral one, due to the direct action of the
drug on the muscular tissues of the blood vessels, since the constriction
still takes place when the vaso-motor centre in the medulla is destroyed

and also when the drug is perfused through blood vessels which have no

Fic. 3.

Cat—pithed. Artificial respiration. Upper tracing taken from intestine enclosed in oncometer ;
lower tracing records blood pressure, Delirium cordis—death.
2 c.c. ¢ strength Tinct. Apoc. introduced per jugular vein at lst arrow. Atropin at 2nd arrow.

Time 20 seconds.
Scale 3 linear
vaso-motor nerve endings; the integrity, or otherwise, of the nervous
system makes no difference to this action of Apocynum on the vascular
tissues.
¢) Blood Pressure. In every experiment where the blood pressure
has been taken, certain changes in the tracings of the blood pressure have

always been recorded. In a pithed dog an immediate rise of blood pressure

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 3897

is recorded after the injection of 15 c.c. of Tinct. Apocynum. The

slowing of the heart beat was reflected in the tracing. This is the
therapeutical effect of the drug, but in this case it quickly gave place to
rapidity and irregularity of the heart beat and a further rise of blood

pressure; this condition is entirely due to the enormous dose of the drug

given; it is, of course, never seen when the dose is moderate. The delirium

cordis is increased by the further injection of 5 c.c. of the tincture, and
the blood pressure ultimately falls to zero.

: This experiment was typical, and the same results with similar effects
are obtained in cats and rabbits after massive doses of the drug.

The following experiment shows the effect of the drug on the blood
with the heart atropinised. A rabbit was pithed and connected
3 the artificial respiration pump; the blood pressure was recorded in
fy e usual way. After two injections of 1 c.c. of an 05 per cent. solution
io _Atropin, the cardiac terminations of the vagus were paralysed, and,
consequently, the heart rate increased and the blood pressure rose to some
se “extent; some slowing of the heart beat and a rise of blood pressure occurred
after a hypodermic injection of 20 minims of Tinct. Apocynum; delirium
cordis eventually prevailed. In this experiment the slowing produced was
: “entirely due to the influence of the drug on the heart muscle alone.

3 The action of Apocynum on the blood pressure is due to the
combined effect of two or more factors. In all the experiments the general
effect of Apocynum has been to produce a rise of blood pressure, and this

occurs in spite of the slowing of the heart due to the stimulation of the
vagal endings. ‘The rise in blood pressure depends on two factors, (1) the
increased output from the heart per minute, (2) vaso-constriction. It

_ is when these factors, namely, the cardiac slowing, with the increased
strength of beat resulting in augmented output, and vaso-constriction are
most in evidence that the drug produces its therapeutical effect. The rise
of blood pressure is thus the mean result of the above changes. Some-
times before the typical rise of blood pressure occurs there is a fall in the
normal blood pressure; this is due to the sudden stimulation of the vagus
overshadowing for the time being the other effects of Apocynum. I have
more than once in this way seen the heart die straightway from vagal
inhibition. When delirium cordis supervenes the blood pressure undergoes
the most extreme variations, or it may at once fall to zero.
Fig. 4. A cat was anaesthetised with urethane and the blood-pressure recorded in the usual
manner. The tracing shows a very perfect delirium cordis, in which the vagal influence is

entirely eliminated as no slowing effect is produced by the injection of 5 ¢.c. of an 0-5 per cent.
solution of Pilocarpin. Three oc, of Tinet. Apocynwn had previously been injected.

898 BIO-CHEMICAL JOURNAL

With large doses the irritability of cardiac musele rapidly inereases,
ind the heart beat quickens; and hence in this stage the blood pressure

ereatly, both on aecount of imereased cardiac output and

rises Vaso-

constriction. Still later, as the irregularity of auricles and ventricles

furthe Increases, they take on their own rhythm ; sometimes the auricles

Fia 4.

— 1 A —_—S—S ee eeeeeaeqF Oe

Pilocarpin | 5 c.c. 0°5 per cent. solution.
Shows perfect delirium cordis in which vagi have no control
of Tinct: Apocyni Ni

Cat— Urethane after 3 c.c,
slowing effect after pilocarpin

Full Seal

may force blood into the ventricles during diastole of the latter, in which

case when the ventricles contract the blood pressure rises. Sometimes

there is no sequence between auricles and ventricles, and then ventricular
contractions fail to keep up blood pressure. At this stage records of blood
pressure show great variations, sometimes bounding up to 50 mm. of

mercury for half a minute, and sometimes falling almost to zero.

Besides the action on the heart and blood vessels, Apocynum has
-infiuence on the kidneys. The kind of action the drug has on the
excretory function of the kidney is indicated by the following experi-
g Speen Atonthaln and morphia were injected into a dog, anaesthesia being

_ maintained in the usual way by the A.C.E. mixture. The blood pressure
| recorded by a mercury manometer and the artificial respiration pump
nnected with the trachea. The abdominal cavity was opened, and one
ter was dissected out and a cannula inserted. The urine was collected
ing glass every two minutes Sixteen and eighteen drops of
odin ua periods of time, witha rise of blood pressure ; twenty-

ion of 1 .c. of a ‘i in 10 solution of Tinct. Apocynum.
Amount of Urine flowing in two minutes
2+3.c.c.
ba 2:2 ¢.c
eee 2-1 c.c.
Bt des . 1-7 e.c.
1-8 e.c.
1:3 e.c.
1-0 c.c.
pe 1-2 e.c.
on > 08 ¢.c.
0-5 c.c. (1 in 2 sol.)
. Tinct. Apocynum injected
ode) 0-6 e.c.
gee 0-4 e.c.
: 0-9 o.¢.
ll e.c.
13 c.c.
1-2 ¢.c.
1-3 cc.
0-8 c.c.
0-8 c.c. urine blood-stained

‘y ¥ “5 S

* eu sun 88s gas

ane ! 2 ec,
“ee 3-2 e.c.
3-2 c.c.

43 cc.
55 o.0,
1-5 c.c.

l-2c.c. Delirium cordis
2 ec.

= 4 . : oe 0 o.c,
my :
Be

ya bk ie

3 THE PHARMACOLOGY OF APOCYNUM CANNABINUM 399.

ie

400 BIO-CHEMICAL JOURNAL

It is to be noticed that the increase of urinary flow is coincident with
the rise of blood pressure. Apocynum is sufficiently irritant in character
to cause the appearance of blood in the urine. The administration of a

saturated solution of Sodium Sulphate by the jugular vein causes a very
much greater diuresis than Tinct. Apocynum; the latter is very feeble as

a direct diuretic.

From this it appears that Apocynum causes a feeble inerease of
urinary flow in the healthy animal; if the action is continued long enough
and the dose is sufficiently large, haematuria may result. The slight
diuresis is not due to any direct action on the kidney, but is due to the
increase of blood pressure and passage of a greater quantity of blood

through the kidney; because the flow of urine in normal animals always

bears a direct relationship to the condition of the renal vessels and blood
pressure. If the renal vessels are much contracted the flow of urine
diminishes, but if they are only slightly contracted and the blood pressure
is considerably raised, the flow of urine is slightly increased. In patients
with cardiac disease and a failing heart, when there is back pressure and
venous congestion and oedema of the kidneys as well as in other organs,
little urine is secreted. The kidney is particularly sensitive to venous
blood, and ceases to excrete as soon as the oxygen reaches a certain point.
Now it is just in these cases that Apocynum produces so great a diuretic
effect. It acts, then, not by any direct influence on the kidney, but by
improving the condition of the circulation and so sending arterial blood
once more to the kidneys. The kidney vessels are at the time of diuresis

actually constricted, but this constriction must not be very complete,

otherwise the diuresis will lessen. As in the case of Digitalis, the imerease
in urinary flow is due neither to the ris? of blood pressure nor to any
action on the excreting arrangements of the kidney, but to the increased

quantity and quality of the blood which is driven through the kidney —

owing to the greater force of the heart beat. The diuretic effect of
Apocynum is a purely dynamical one, and is quite different to the action
of such a diuretic drug as Caffeine, which produces dilation of the kidney
vessels as well as a rise of blood pressure; the very marked flow of urine
which follows an injection of a solution of Sodium Sulphate (see table) is
an example of a third method of diuretic action, and occurs probably
because the salt solution has some kind of direct action on the renal
epithelium.

aa - cen
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a
4
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THE PHARMACOLOGY OF APOCYNUM CANNABINUM 401

2 Toxie effects.

- ‘The drug bes a penetrating bitter Gist; which gradually increases
until a feeling of nausea supervenes. Large doses have a very toxic effect
on the mucous membrane of the alimentary canal.

Experiment. A rabbit weighing 1,350 grammes was taken, and for

eight consecutive days 5 minims of Tinct. Apocynum with 10 minims of

normal saline solution were injected into the marginal vein of the left ear.

“3 On the sixth and seventh days the animal was noticeably ill and

exhausted ; it was constantly vomiting and had lost some weight. Death
occurred suddenly on the eighth day.

On examination the most obvious changes had occurred in the
_ alimentary canal. The whole of the stomach and intestines were closely
- ~ marked with haemorrhagic spots and areas, chiefly situated on the side of
‘the gut distal to the attachment of the mesentery. On opening the
| fomach and portions of the intestines these haemorrhagic areas were found
: 2 “te correspond with ulcers of various shapes and sizes in the mucous
membrane. The ulceration was general throughout the whole length of
aa the alimentary canal, from the lower end of the oesophagus to the rectum.

The ulcers varied in size from 2 cm. square, with complete denudation of

‘epithelium, to mere punctures. These small punctate ulcers easily
perforated on washing portions of the gut through from a pressure tap,
when the water sprayed out in many small streams.

The main blood vessels appeared to be normal, but some small white

__. selerotic patches were noticed in the aorta a short distance above the semi-

__Iwnar valves. The heart was larger than would be expected. Another

_ small sclerotic patch was noticed in the wall of the left ventricle just below
ee ‘the semi-lunar valves. The remainder of the organs appeared healthy.

ih Experiment. A second rabbit was used weighing 1,200 grammes.
“Two minims of Tinct. Apocynum were injected into the ear veins at
intervals of one, two or three days over a period of seventy days altogether.
At the end of this time the animal was killed. The left ear was
oedematous and swollen to three or four times its ordinary size. The left
eye was very prominent, and the conjunctiva extremely swollen and
oedematous.

On opening the thorax, some slight amount of fluid was found in the
pleural sacs, both lungs were oedematous. The pericardial sac was
distended with fluid. There was no increase of fluid in the abdominal
cavity.

402 BIO-CHEMICAL JOURNAL

One other effect of Apocynum deserves mention here. The mucous
membrane of the alimentary canal, like all other plain muscle in the body,

is stimulated; peristalsis is increased, and may give rise to nausea,
vomiting, and diarrhoea. This effect is, no doubt, partly local; —

stimulation of the mucous membrane causing local reflexes through
Auerbach’s plexus, with resulting peristalsis. But this is not the whole
explanation, since Apocynum still causes some increased movements of
the alimentary canal even after absorption, or the same effect can be
induced by injecting the drug directly into the circulation of an animal.

The results of the experiments serve to illustrate the extremely
irritant action of Apocynum on the tissues of the body. In the first
rabbit to which the larger dose was given, the intestinal ulceration may be
regarded as a secondary lesion occurring at the points of elimination of the
drug into the intestine. As far as the naked eye appearances go, the
frequent haemorrhagic areas point to profound changes in the blood
vessels of the intestine, and these changes are certainly correlated with
the appearance of the patches of sclerosis seen in the aorta.

Further evidence of the influence of Apocynum on the vascular
system is afforded by the lesions found in the case of the second rabbit;
these were localised oedema, pulmonary oedema and slight hydrothorax,
and distinct hydropericardium.

3. Muscular System. ;

(a) Striated muscle. The action of Apocynum on muscle is
comparable to that of Veratrine and Barium. The most characteristic ~
action of Apocynum on striped muscle is the delayed relaxation and
constantly increasing rise of tonus; in this respect Apocynum exactly
resembles Digitalis and Squill. It can be shown that this drug has the
same type of action on all forms of muscle, but that the effect on the more
delicate cardiac muscle overshadows the others. |

(6) Unstriated muscle. This is thrown into a state of prolonged
contraction, and the tonus is also raised so that in isolated preparations
the death of the muscle occurs in contraction. Plain muscle throughout
the body is affected in this way. The typical effect on this tissue may be
simply observed in ring preparations of the frog’s stomach, in which the
movements are recorded by the suspension method, so that the down stroke
of the tracing indicates the contraction (systole) of the preparation. .

Apocynum applied directly to the preparation causes a decided
contraction, and the tonus of the muscle is permanently raised; and this

THE PHARMACOLOGY OF APOCYNUM CANNABINUM 403

effect is obtained in spite of the fact that the tincture which was here
employed-contains alcohol, the action of which is to reduce tonus.

This effect on plain muscle may be regarded as representing the type
of action occurring in all plain muscle throughout the body. I would
observe here, however, that the alimentary canal shows irregular colicky
contractions, the spleen and bronchioles are induced to contract, there is
marked vaso-constriction and the tonus of the bladder and uterus is
increased. The effect of Apocynum on the uterus was determined on the
isolated uterus of a cat.

Experiment. Fig. 5. The uterus was suspended with its lower end fixed in an oxygenated
saline solution and the movements recorded by suitable levers. The introduction into the
saline solution of a trace of tincture of Apocynum causes a great increase in the muscular tonus.
The peristaltic movements become less and less and ultimately cease, but the muscle remains
in a state of tonic contraction. The uterus in this case was a pregnant one, and 0-2 c.c. of the
tincture was administered at the arrow mark.

The type of action shown in the above experiment is very similar to
that of digitalis, except that the effect of Apocynum on the uterine muscle
is of a much more intense character.

Pregnant uterus of Cat. 0°2 c.c. Tincture
gp bor given at arrow produces rise
of tonus and decrease in size of con-
tractions. Death in systole.

Full Scale.

II]. Comparison or Apocynum CANNABINUM WITH OTHER MEMBERS
or THe Carpiac Group or Drves

Apocynum will be compared only with Digitalis, Strophanthus and
Squill.

The experiments show that, of the four drugs, Apocynum stands
sécond in relative toxicity on frogs’ hearts. Strophanthus has the greatest
toxic action, Digitalis and Squill, which are about equal in toxicity, have
a less toxic action than Apocynum.

of the other three drugs; in several experiments delirium cordis set he a
almost immediately after the administration of the drug, and in others no- ie
—o

so-called therapeutical effects were obtained, also no tracings show

obtained from Apocynum.

ae
ey

generally such wide variations of blood pressure at ae stage as ‘those’ .

This drug is the most irritant of the four towards mucous metal Be

as shown by the intense ulceration produced in the alimentary canal after iy
the intravenous injection of moderate doses of the drug. Strophanthus
has been shown to be the least irritating to mucous membranes, and is i ine
consequence the most readily absorbed. Apocynum is the most powerful
vaso-constrictor of this group of drugs, and its property of constricting mee

the blood vessels stands in direct relationship to what is really the specific
action of the drug, that is its ability to increase the tonus of muscular
tissue of every kind. In connection with this action, a greater rise of
blood pressure is produced by Apocynum than by either Digitalis,
Strophanthus or Squill; it is known that Strophanthus produces a
comparatively small effect on the blood pressure, Apocynum causes a more
immediate and sudden rise of blood pressure than Digitalis or Squill.

There is no special difference in the action of these drugs on the kidney,
which in each case is an indirect one, but Apocynum appears more likely —

to produce haematuria owing to its pronounced irritant properties.

REFERENCES

1. Gould, Year Book of Med. and Surg., p. 472, 1904.

2. Lancet, 1894, Vol. I, 841.

3. B.M.J. Epitome I, paragraph 447, 1896.

4. Murray, Physiolog. Act. and Therapeut. Val. Apoc. bined. M.B. Thesis, Therapeutic
Gazette, 1890.

5. Hale White, Txt. Bk. Pharmacol. and Therapeut.

6. Dabney, Therapeutic Gazelte, Nov. 15, 1898. .
7. Il Policlinico, Nos. 10-14, May-July, 1894. ,
8
9.

.S

B.M.J., Vol. I, p. 1714, 1897.

Lancet, 1896, Vol. I, paragraph 447.
10. B.M.J., Epitome 1, paragraph 447, 1896.
11. Journ, Physiol., March 27, 1909.
12. Brit. Pharm. Conf., p. 387, 1905,

the Heart, Bio-Chem. Journ., Vol. I, No. 2, 1905.

a Ss “* ? *
on | ee

< 4 a
eat Li

13. Haynes,»G. §., The Pharmacological Action of Digitalis, Strophanthus and Squill on 4

THE PHYSIOLOGICAL EFFECTS OF SELENIUM COM-
- POUNDS WITH RELATION TO THEIR ACTION ON
GLYCOGEN AND SUGAR DERIVATIVES IN THE
TISSUES

By CHARLES 0. JONES, M.D. (Liverpool).
“3 Communicated by Prof. Benjamin Moore

% ‘ From the Bio-Chemical and Physiological Departments, University of
Liverpool

(Received September 6th, 1909)

“ . Historicat

ae Dibditis were ins by id in 1985. He found that on adding traces of
_ selenium and tellurium salts to the water in which plants grew, that
although no influence on the growth of the plants took place, yet selenium
ey : was absorbed. The same was found true of algae and infusoria by
__ Bokorny in 1893. Scheurlen, in 1900, seeking a substance which
-__ contained loosely-bound oxygen, to grow bacteria in absence of atmospheric
oxygen, tried sodium selenite, and found that though the bacteria were
unaffected, yet they were coloured with the reduced selenium. This
selenium was found entirely in the cell, none being found in the media.
’ A careful study of these effects was conducted by Klett, who found that
_ bacteria and moulds were not as a rule hindered in their development by
traces of selenite of sodium, but a few, such as the bacillus of malignant
i oedema and symptomatic anthrax, were arrested in growth. He also
found the bacteria coloured with the reduced selenium, the surrounding
media being colourless, and that as the amount of selenite was increased
growth was inhibited. He concluded that the reduction of selenite to
selenium took place in the protoplasm of the bacterial cell, and not outside

the cell by secondary action of metabolic products.

Action on animals.—Gmelin appears to be the first who investigated
the effects of selenium and tellurium salts on animals. He found that
they were poisonous, that they produced a deposit of the reduced element
on the intestinal walls, and that the animal gave off a garlic-like odour.
This odour was also noticed in the animal’s breath by Hansen, who
attributed it to ethyl selenide; this observer found also that after a large

—_
f

406 BIO-CHEMICAL JOURNAL

dose the animal vomited, and that the vomit contained selenium. On —

making sections of the animal’s organs he noticed that they all contained
selenium deposited in granules. The work was continued by Rabuteau,

who observed that after a large dose vomiting, profound dyspnoea,
anasthesia, opesthotonos, and death from asphyxia took place. The —

post-mortem findings were intense congestion and ecchymosis of the whole
intestinal tract, also of the liver, spleen, lungs, and kidney. The right
side of the heart and large blood vessels held a multitude of small
prismatic crystals of unknown chemical composition. Rabuteau concluded
that these crystals acted as a mechanical obstruction and caused death.
These results were not confirmed by Czapek and Weil, who could not find
any crystals or mechanical obstruction, and concluded that selenium was
very similar in its action to tellurium, arsenic and antimony, and that
death was due to paralysis of the so-called excito-motor ganglia. They
further noticed marked distension of the abdominal capillaries. The
blood was normal, but it gave off a marked garlic-like odour. This odour
was noticed by Wohler to be similar to methyl] selenide, which he was
then preparing. Hofmeister confirmed this, proving by analysis that they
were the same, and further showed that all the organs gave off the odour,
but that it was most pronounced in the testes and lungs, and marked in
the blood, liver and kidney. If the organs were placed in an ineubator
the smell was intensified, but blood loses the smell. Hofmeister concluded
that all the organs could absorb selenium and form methyl selenide from
it; lastly he discovered that the reduction to selenium and the formation

om methyl selenide were independent of one another. On heating an .

organ to 55° C. the formation of methyl selenide ceases, but the organ will
still reduce selenite to selenium. The explanation suggested was that the
reduced selenium was slowly built up into a soluble compound in the
alkaline blood and was changed in the lungs to methyl selenide. The
meth] groups he supposed to be derived from cholin, creatinin, and other

methyl-bearing substances. he effects of selenium and tellurium salts

on metabolism were investigated by Mead and Gies, and Woodruff and
Gies, who found that selenium salts had little or no effect on metabolism,
but that the ether-soluble substance in the faeces was increased. This
they attributed to diminished absorption. They also examined the vomit
resulting from selenium salts, and found that there was complete absence
of free hydrochloric acid, the pepsin was unaltered, and on addition of
hydrochloric acid digestion proceeded at a normal rate. Ptyalin, on the
other hand, was markedly affected by selenium salts,

PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 407
r

The compounds of selenium are thus extremely toxic; but if the
_ amount present is very small, the cells are able to reduce it, forming an
inert substance, and can then continue their metabolic changes.

This research was undertaken to endeavour to find out how this
reduction is accomplished and also to find out more exactly the cause of
death in selenium poisoning.
Two compounds of selenium were used in this reasearch, viz., sodium
_ selenite and sodium selenate, both obtained from Kahlbaum.
_ Sodium selenate is a comparatively stable salt, easily soluble in water
and neutral in reaction.
~~ he lethal dose of selenate for a moderate sized rat (about 80
—— was found to be 0°6 c.c. of 0°125 per cent. solution.
Selenite of sodium is an extremely unstable salt. It is stated by
“Mead and Gies to be reduced by all protoplasm, and they have seen
_ reduction take place in contact with fresh meat. It is even said that
reduction takes place in contact with all organic matter. If this is so,
__ it is difficult to see how any can be absorbed if given by the mouth, so
_ that in this research all doses were given hypodermically.
i. The preparation used was found to be acid in reaction, the acidity
. of 1 gramme being equal to 2°75 c.c. normal sulphuric acid. It is
distinguished from selenate most easily by the insolubility of the selenites
of copper, cobalt and nickel. Cobalt salts give a mauve precipitate visible
1 in 800 of water; copper salts give an apple-green precipitate visible
1 in 1,200 water; nickel salts give a green precipitate visible 1 in 1,600
_-__— water. Owing to its instability the selenite solution was always made
fresh as required.
_--_—__._- The lethal dose for a moderate sized rat was 04 c.c. of 0-125 per cent.
solution. The lethal dose for a moderate sized rabbit was 0:5 c.c. of 2 per
cent. solution. The lethal dose for a moderate sized cat was lc.c. of
2 per cent. solution.

From these results it appears that selenate of sodium is only two-
thirds as toxic as selenite.

After a small dose no symptoms were observed, and even the
appetite was unaffected. As the dose was increased and was just sub-
lethal, it was observed that, after about ten minutes, the animal became
‘restless; this was followed by movements of the mouth, tongue and nose.
As there were now present a peculiar garlic-like odour in the breath,
it is probable that these movements are due to stimulation of the nerves

Pree a ee ee Seer

fae

408 BIO-CHEMICAL JOURNAL

of taste and smell. Very shortly afterwards retching and vouitiiaaia :
commenced. If the dose has not been too great, recovery soon takes
place, no after effects being noticeable. If the dose be too great, the
vomiting and retching continue and somnolence passes on to unconscious
ness and death. ee .
It may be mentioned, as death has been aseribed by previous vocal bane
to dyspnoea, that laboured breathing was seen in one case only. It was
in this case due to excessive reduction of the salt to selenium and
consequent embolism of the pulmonary vessels. The paralysis, con- —
vulsions and other symptoms noticed by former investigators were —
probably due to the same cause, and are in no way connected with death —
from chemical poisoning with selenium salts.

Macroscopic AND Microscorrc CHANGES IN THE TISSUES

The macroscopic post-mortem changes were very few. The liver was
usually soft and friable. All the organs gave off the garlic-like odour
noticed in the breath. The right side of the heart was distended and
full of clot. The splanchnic vessels were enormously dilated.

The microscopic changes were more pronounced, and were investi-
_ gated as follows:—The tissues were immediately placed in formol,
dehydrated with acetone, embedded in paraffin and stained with eosin
and haematoxylin. 7

The most noticeable feature of all the sections was a golden-brown er
amorphous deposit found in almost every organ. It is chiefly found =
around the blood vesséls and between the cells, but some is also to be SS
seen inside the cells. It was suggested that this substance might be iron,
but it gave none of the staining reactions for iron. On grinding up the
organs with sand this substance could be extracted, and formed a brick-
red deposit. This was found to be identical in every way with the -
amorphous form of selenium produced on reducing sodium selenite in the
test-tube. It volatilized with heat, burning with a blue flame, and gave _ |
off the well-known horseradish smell of selenium. a a

This golden-brown deposit was found, if the dose was laripesit in almost | 7 q
every organ, the whole of the tissues being flooded with it, but this is han
not the cause of death, for if a just lethal dose is given there is no such
flooding and death still takes place. Physiologically this deposit is inert,
for if a small dose of a selenium salt is given and the animal killed some
time afterwards, this deposit will be seen in the cells, which are evidently —
still capable in its presence of performing their metabolic changes
without noticeable change.

PILYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 409

E The action of selenium salts on different isolated physiological
____ systems prepared by the usual methods was next investigated, and they
were found to be without action on (1) a muscle-nerve preparation,
(2) isolated heart muscle, (8) nervous mechanism of heart, (4) higher
nerve centres, (5) blood pressure, (6) intestinal movements. The urine
was also normal, traces of selenium salts were found present, but never
any solution that reduced Fehling’s solution. The blood was occasionally
_ found altered; the most frequent change was a slight lymphocytosis
- followed by a more marked increase in the polymorphonuclear leucocytes.
The red blood cells were fpund normal both in their number and in
_ their haemoglobin contents, and spectroscopically the blood was found
normal. In small amounts, selenate had no effect on gastric digestion,
aor had it on pancreatic, while selenite had an inhibiting effect on
“pancreatic action, this being in part at least due to its acid reaction.
The reduction which takes place in the tissues was next investigated.
_ After heating an organ to 60°C., as Hofmeister showed, it will still
yeduce selenite to selenium. This was repeated, and it was further seen
that higher temperatures do not stop this reduction. . It therefore seemed
Sale probable that this effect was not due to an enzyme, but was some direct
_ chemical effect. A fresh solution of sodium selenite was therefore made,
and 5e.c. added to 0-5 gramme of each of the following carbohydrates
with aseptic precautions. The mixtures were then placed in an
incubator at 35°C. for twenty-four hours and again examined.
Reduction was shown by the solution becoming pale brown, while
if the reduction were intense a fine brick-red powder became deposited.

Glycogen io No action
Tnulin hs Profuse reduction
POLYSACCHARIDES ... ; ea
Starch rye No’action
Dextrin Fe No action
H é ‘ Mannite ry No action
ee eee oe | Duleite he No action
Arabinose Ae Reduction
PeNTOSES . 4 Rhamnose sx No action
a Xylose on No action
4 Maltose ie Faint reduction
Glucose al Reduction
HEXOSES ... cee cee +e" Galactone vse Slight reduction
Lactose es Slight reduction
Levulose can Profuse reduction
Ammo Hexose ...._—_... 1 Glucosamine is Profuse reduction
4 ; 8 | Saccharose ose No action
4 ComPouND BOGARS... ++ | pom ce ae

410 BIO-CHEMICAL JOURNAL

The reduction of inulin is due to the formation of levulose.

Tt will be seen that reduction takes place with arabinose, levulose,
glucose, and the sugars yielding glucose. The reduction by glucose and
levulose was then tried at a low temperature, viz., 30°C., and it was’
found that glucose caused no reduction at this temperature even after
several weeks, while levulose caused a profuse reduction. When the two
sugars were mixed it was found that no reduction occurred at 30° C., and
both came down at 38°C. It was thought that perhaps this reaction
with more rapid heating might serve to differentiate the sugars, but on
heating a solution of the sugars with sodiym selenite solution in a test-
tube, it was found that levulose commenced to reduce at 58°C., while
glucose, lactose, galactose and maltose all reduced at about the same
temperature, viz., 72°C.

The derivatives of glucose and its compound sugars were next tested
as reducing agents for selenite, with the following results :—

Glucose ica Reduction
Gluconic Acid oa No action
Glucuronic acid ins No“action
Saccharic acid aey Reduction
Mucie acid oT No action
Furfurol Ele No action.

The above reductions at first sight would appear due to the aldehyde
or ketone groups in the sugars, but here we are met with the fact that
while rhamnose and xylose, which contain aldehyde groups, have no
action, yet saccharic acid, which has neither aldehyde nor ketone group,
acts very strongly. We tried the action of benzaldehyde, formaldehyde
and acetone, but found they all gave negative results.

Other possible degradation products of the sugars were tried, for
example, lactic acid and acetic acid were without action, but formic acid
had a powerful reducing action.

It is interesting to note here that selenite is reduced by arabinose,
levulose, glucose, maltose and lactose, all of which are found in, or are
excreted from, the human body. The only exception is xylose, which is a
necessary constituent of the nucleoproteids, but in such a position an
energetic sugar would be a source of danger to the animal. These results
cannot be explained on the structural formulae at present ascribed to the
different sugars, so that the reduction rests more on a physiological basis
than a purely chemical one. It was next ascertained whether proteins,
fats, or other substances of animal origin, would perform the same

PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 411

reduction. Although treated in the same way as the carbohydrate or
heated together directly in the test-tube, no action was found associated
with any one of them. The following substances were all tried, viz. :—
Olein, oleic acid, palmitin, palmitic acid, potassium palmitate, erucic
acid, lecithin, cholesterin, glycerine, uric acid, hippuric acid, urea,
gelatin, glycocoll, tyrosin, casein and creatinin.
From this it appears probable that this reduction can be performed by
; ae glucose and a few allied sugars, and cannot be produced by organic matter
from which carbohydrates are absent.

To ascertain whether glucose is the agent which accomplishes this
_ reduction in animal organs, the following method was adopted. Ten
- Sma of finely minced liver were taken with aseptic precautions, and
to this were added 5 grammes of yeast, 50c.c. of distilled water and a
. - few drops of toluol. A control was prepared in the same way, but
without any yeast. Both vessels were placed in an incubator for twenty-
four hours, they were then heated to 70°C. to destroy the yeast and
_ glycolytic enzyme, and 1 gramme of sodium selenite added to each. After
___ being in an incubator at 38° C. for a few hours, both were examined. The
control which contained glucose from the glycogen was of a deep red
—__ eolour, showing that quantities of selenium were present. The flask
which had its sugar destroyed by the yeast showed no red colour,
so that no selenite had been reduced. This experiment makes it probable
that in the absence of glucose selenite is not reduced, and, accordingly,
that reduction of selenite to selenium in a cell indicates the presence of
glucose.
a _ Since selenite of sodium is reduced in the cells by glucose, it appeared
as necessary to ascertain where, especially in the body, this reduction takes
place. As has been already seen, if a large dose is given the whole
organism is flooded with the selenium. A rat was therefore given several
small doses, and was then killed, and the organs quickly removed and
examined histologically.

The spleen was found to contain abundance of selenium, as well as
an excessive number of leucocytes.

The portal vein was examined and found to also contain selenium
and leucocytes, and the same was found true of the liver; while the
“vessels leaving the liver, the lungs, kidney and intestine, were found to
be quite free from selenium.

‘: In some cases the liver cells show destructive changes. The nucleus

412 BIO-CHEMICAL JOURNAL

stains less deeply while in others the nucleus has disappeared, the cells
being mere shadows.

It therefore seems probable that reduction takes place firstly in the

spleen. The reduced selenium is brought by the blood stream, only a
small amount by leucocytes to the liver. The liver also reduces any
selenite that has escaped the spleen. If the dose is not excessive no
selenium is allowed to pass the liver.

DiIsAPPEARANCE oF GLYCOGEN FROM THE LivER

Finding that selenite is reduced by glucose in the liver and spleen
suggests that the glucose must be derived from the liver glycogen, and
this was found to be the case. ‘Two well-fed rats were taken, and to one
was given an injection of sodium selenite just sublethal. As soon as it
began to recover, which happened in a few hours, another injection was
given. ‘Treated in this way, in a time varying from three to seven days,
the animal dies. The control rat was then killed, and the livers from
the two animals contrasted as to their glycogen content by the following
method: —The livers from both rats were quickly removed, cut in pieces
in each case, and placed separately in boiling water acidified with acetic
acid. The pieces were then ground up in each case in a mortar with hot
distilled water.

In the rat which had been injected with selenite, there resulted a
perfectly clear pale orange coloured solution, which gave no coloration
with iodine and no precipitate with alcohol or basic lead acetate.

The fluid from the normal rat’s liver gave an opalescent solution,
which on addition of iodine gave a dense brown coloration.

This experiment, on account of its importance, was repeated several
times, with the same result. It may be stated that the dose given must
be carefully regulated so as to be just sublethal. If an over-dose is given,
the animal will die, due probably to not sufficient glucose being available
to reduce the selenite; glycogen will then not have disappeared entirely.
This disappearance of glycogen was also found to occur in both frogs and
rabbits.

This gradual using up of glycogen and glucose made it interesting
to find out if any other metabolic changes took place at the same time.
A well-nourished cat was used for the experiment. The normal

excretions were examined and estimated daily for a week, during which

period the urine was invariably found to be acid, A small injection was

4
a

PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 413

then made of ('5 e.c. of 0°25 per cent. solution of selenite; the only change
following this small dose that took place was that the urine next day had
become alkaline; this continued so throughout the experiment. The dose
was gradually increased, and it was noticed that the amount of urea
excreted on the day following the injection had fallen considerably, but
had returned to normal on the following day. As the dase increased, the urea
decrement became greater, and an increased number of days were required
_ for the return to the normal. When the dose became excessive the animal
yomited. No urine was excreted the following day. The next day the
excretion of urea was exceedingly small, and continued small for some
days, only very slowly returning to the normal. The total amount of
nitrogen excreted showed no relation to the urea; it kept steady all
through the periods when the urea fell. When an excessive dose was
reached the total nitrogen fell to about one-third the normal amount and
~ gradually returned to normal.
~ It would thus appear that while the wrea was excreted in less amount
the nitrogen was got rid of in some other form. That it was not excreted
as uric acid nor as ammonia seems probable as these showed little or no
change throughout the experiment. Sulphates and phosphates showed
no change.
| The most striking effect was noticed in the excretion of chlorides.
Until the dose had become excessive the amount of chlorides thrown out
had continued steady. After the animal vomited the excretion of chlorides
suddenly dropped, so that the daily amount excreted became half or less
pe than half the normal amount; thus, in the first cat the average daily
a output was 1°28 grammes; this fell to 0°061 grammes.
4 In the seeond cat, as will be seen in the following table, the average
daily excretion of chlorides for fifteen days during which it was having
- increasing amounts of selenite was 0°0749 grammes. After 0°75 c.c. of
2 per cent. solution had been injected the animal vomited. No urine was
passed the following day. The next day the amount was only 00305
grammes, that is, less than half the normal amount. The following three
days there was very little increase. The following day, as will be seen
in the table, the whole of the retained chlorides were thrown out, the
amount excreted afterwards returning to normal again.

Vomited 00805 0°0835 00881 OO 0° 1820 o-os1li
No urine

Injection

414 BIO-CHEMICAL JOURNAL

The vomiting took place about ten minutes after injection. The if 2 ae
vomit consisted of the stomach contents in a state which showed that there =
had been no interference with digestion until the injection was given, =

THe animal during this period was under the influence of the drug, rr:

and it is only when it reaches an excessive amount that vomiting occurs. = |

This vomit was found, as already pointed out by Mead and Gies, to be
free from any trace of free hydrochloric acid. The reaction was acid,
and quantities of organie acid were present.

It therefore seems probable that the hydrochloric acid is suddenly
withdrawn from the stomach to serve some other necessary purpose, and
if 02 per cent. hydrochloric acid be added to the stomach om
digestion will proceed normally.

This withdrawal of hydrochloric acid is accompanied by a iieiilly
diminished excretion of chlorides in the urine. This observation coincides
with the withdrawal of hydrochloric acid from the stomach. While the
chlorides are retained, appetite and digestion are in abeyance; after a
certain period, varying from one day to five days, during which time the
excretion of chlorides is only about half the normal amount, the whole
of the chlorides retained are thrown out, their purpose having been
fulfilled. Then the animal regains its appetite and the exeretion of
chlorides becomes normal.

Still another interesting fact bearing on the subject was noted: when
the animal had lost its appetite, although it refused fresh meat it would
still eat salt meat. Possibly the excess of chloride helped the return of
hydrochloric acid. :

Lastly, with these changes there was also a remarkable loss of weight.
One cat lost 38 per cent. of its total weight, the average daily loss being
26 grammes. A second cat lost 335 grammes during the first week, or

16 per cent. of its weight, and in one day it even lost as much as 65

grammes. This loss of weight is too great to be accounted for by

diminished consumption of food alone, for until the dosage became large
the animal still retained its appetite, and even after a large dose it only |

refused its food for a day or two, the appetite gradually. returning.

It was observed by Mead and Gies that after a dose of a selenium
compound the amount of ether-soluble substance in the faeces was
increased. This we found true for small doses, but on investigating the
effect of large doses we found that the amount of ether-soluble matter in
the faeces was very much diminished, and that the relative amounts of
neutral fat, fatty acid and soap (reckoned as oleic acid) were altered.

=
a Te ee

eS

id pit
a |) OU! * = a

hy ent
ie,

om?

—
a
=

ae sia hlY aeaiala a ae

PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 415

_ The faeces were dried and extracted with ether in a Soxhlet apparatus,
the acidity of the fat being titrated with 01 N alcoholic potash, using
phenol phthalein as indicator. After an injection of selenite of sodium
the total amount of fat in the faeces became very much reduced and was
at its minimum the second day after injection. The excretion of fatty
z acid was less affected or increased in amount, so that the normal proportion
_ of neutral fat to fatty acid being 2 to 1 on the second day, the fatty acid
~ became equal to, and sometimes greater than, the amount of neutral fat.
This may be seen from the following table, where the amount of fat has
been worked out to a constant: —-

Fat Free fatty acid
0-5 0-284
0-5 0-292
0-5 sie 0-290
Injection of selenite
0-5 a 0-350
0-5 0-530
0-5 0-170
0-5 0-280

- from then onward.

- These observations seem to show that glucose, or a derivative, is the
means by which the body protects itself from the toxic effects of selenium
salts, and death may even in certain cases be due indirectly to the using
up of the glucose; but this cannot be the cause of death after a single
large dose, therefore the effect of selenite on the living cells of the liver
was next investigated. A fresh liver was finely minced, using aseptic pre-
-__-vautions, and 10 grammes were weighed out in every case for the purposes
of the experiment. To this were added 50 c.c. of sterile distilled water
and a few drops of toluol. The substance to be tested was added to one,
and the control and the one containing selenite were placed in an incubator
at 38° C. for varying periods of time. They were then boiled and filtered.
The filtrate and solid matter were each separately estimated by Kjeldahl’s
method.

SeLenate or Soprem
Soluble Nitrogen Insoluble Nitrogen

Normal cas nde 0-0883 ce 02125
Selenate 0-5 gramme ue 0-1076 < 0-1704
Normal soe ae 0-1674 set 1618
Selenate °° wed ais 01268 = 02047
Selenate 1%) an v3 01570 0-1751

We see that there is no definite effect on autolysis caused by selenate
of sodium.

oo :
¥ ¢
i te

416 BIO-CHEMICAL JOURNAL
Seiexttr or Soprum oft.
Third day ; inte a
Soluble Nitrogen Insoluble Nitrogen —
Normal boiled ee ii 0-0192 Sis OS7LL |. nite
Selenite 0-5 gramme ae 0-0211 sing 0-2708
Normal liver ont ous 0-0883 = 02123
Seventh day , pie ta
Soluble Nitrogen _ Insoluble Nitrogen mr
Normal liver sae “a 0-1911 Pie 0-1497 © ‘
Selenite 0-5 grammes aire 0-0762 nee 0-2636—
Selenite 1 gramme ... Be 0-0566 de 0-2763
Tenth day Roy +.
Soluble Nitrogen Insoluble Nitrogen
Normal liver das oe 0-1674 ons 0-1616 i i
Selenite 0-5 grammes Sc 0-0636 a OTL ©
Selenite 2 grammes ... 0-0745 oat 0- 1047

It is evident here that selenite of sodium has a very marked inhibitory
effect on autolysis. The presence of selenite in a cell in sufficient amount —
would seem to inhibit all metabolic changes and destroy the cell. As the
action of selenate of sodium in the body is similar to selenite in its ulterior —
effects, and as selenate is not poisonous to the cells, it seems evident as
its toxic effects are present only when it has been reduced in the body ated
to selenite. ate

The ease with which the glucose molecule is broken up by soit a
selenite and selenate in the body suggested that if diabetes mellitus were —
due to the inability of the animal cells-to break up the glucose molecule,
as the believers in the oxidation theory hold, then the presence of selenite
or selenate which effects this splitting up should lessen the amount of 3
sugar in the urine, for it is well known that the degradation products of
glucose can be easily dealt with by the diabetic. Selenate of sodium was
the salt used, being less easily reduced in the intestine than the selenite. —
The patient was under the care of Dr. J. Hill Abram, whom I have to —
thank for trying the drug, and also Dr. A. F. Jackson for the care with — er
which he performed the sugar estimations. The patient was a case of ae
severe diabetes with twelve months’ history. an :

Per diem
~~ ots eT aay
Average sugar, Urea,
in grains ‘in grains
Common diet as yi 5232 so 538
Special diet ba as 2212 3 307

Selenate ee Gen 3087 Ra 455

PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 417

The dose of selenate given was 5 minims of 1 per cent. solution,
gs gradually increased until 25 minims was given.

As will be seen, there was an actual increase in excreted sugar while
taking selenate, which seems to show that diabetes is not due to any lack
of oxidation power nor to any difficulty in breaking up the glucose
molecule.

ConcLUSIONS

These observations make it probable that selenate is reduced in the
body to selenite, so that the action of selenite only need be considered.

When an injection of selenite is given, it is quickly taken up by the
blood stream; only a small quantity is excreted by the kidney, the
oa ‘remainder is carried to the spleen and liver, where it is reduced by glucose
to selenium. According to Zsigmondy, this reduction with glucose can,
_ with the aid of the ultra microscope, be seen to take place fairly easily
outside the body, particles of the reduced selenium being visible in about
two minutes, so that the living cells would have no difficulty in effecting

This reduction does not appear due to the aldehyde group of the
‘sugar, but more probably is due to some special configuration of the
glucose molecule, in which it is closely resembled by arabinose and
levolose. This glucose, as required, is furnished from the glycogen of
the liver, but when this is becoming exhausted fat is called upon.
Whether the fat is used up as such or 1s transferred into sugar, it is
___, diffieult to say. The excretion of excess of fatty acid after an injection
ay when sugar is urgently required, would point to the glycerine being
___ possibly required for transference to sugar, but it is clear that there is no
effort on the part of the organism to form sugar from proteins. If it were
possible an effort would be made by the cells to manufacture glucose from
protein to save themselves from destruction, but that no such action
occurs is shown by the excretion of nitrogen remaining low, even until
death. One must conclude that the organism cannot under such conditions
transfer protein into sugar.

The evidence as to the transference or at least equivalence of fat and
sugar is better. There is complete disappearance of fat as well as
glycogen and sugar, which points to their transference or utilization.
The loss of weight also suggests the same. In a moderately fat cat the
loss of weight was about 38 per cent. This would just about represent
the weight of fat, glycogen and glucose. Méchel’s estimate of fat in a

418 BIO-CHEMICAL JOURNAL —

moderately fat dog being 26 per cent., the remaining 12 per cont: would ‘

account for the glycogen and glucose. bees

It is necessary to consider at this stage the suddeu disepppaha |

of hydrochloric acid, the holding back of the chlorides by the organism,
and the extraordinary relish for sodium chloride. These factors are all

present when selenite has just been given, and when there is a sudden
demand for glucose on the part of the cells. The most likely explanation —
is to be found in an observation by Eckhard that a one per cent. solution _

of common salt introduced into the blood caused glycosuria. Fisher
showed that other sodium salts had the same effect, and that the stronger
the salt solution the more glycosuria resulted, even up to 73 per cent. of
sugar was found. volts

Excess of sugar in the urine represents excess of sugar in the blood,
and therefore it would be possible for an animal holding back its chlorides
by using its hydrochloric acid to fix some sodium salt, and taking sodium
chloride in its food to raise its blood sugar content. If this sugar is picked
out by the spleen and liver, and also possibly by leucocytes, it must result
in a wonderfully increased power of reducing selenite to selenium, and so
saving the cells from the poisoning effect.

If the organism is unable to neutralize the selenite, either because
the selenite is in too great excess or because the available stores of glucose
are used up, then selenite will act on and destroy the cells. The action is
on the ferments of the cells, causing all metabolic changes to cease.. It is

curious to note here what a slight effect it has on bacteria and a powerful —
effect on ferments, quite a contrary effect to toluol, chloroform and similar _

antiseptics. It seems to show that the defences in single cells are much
more highly developed than is the case in cells which rely on others for
protection.

It is interesting to consider here whether glucose may not poise ,

be the means by which all reduction processes take place in the body.
The well-known reductions taking place in the organism, such as methy-
lene blue and Prussian blue, can be accomplished with ease by glucose in
faintly alkaline solution. Considering the universality of occurrence of
glucose in the body cells, and its well-known power of reduction outside
the body, which is immensely multiplied within by cell activity, it would
hardly seem necessary for the cells to require any other means of reduction.

I take this opportunity of expressing my indebtedness to Punta

Benjamin Moore and Professor C. §. Sherrington for their kind
assistance and advice.

LITERATURE

, Verauche tiber die Wirhetigen des liaryib es: Miubicas che. au} den thierischen Organismus
1824, p. 43.

Ann, de Chemie wnd Pharm., UXXXVI, p. 208.

Gazette hebd. de Med. et de Chem., XVL, pp. 194-241.

Botanisches Centralblatt, XXII, p. 35.

k and Weil, Arch, fiir exp, Path. und Pharm., XXXII, p. 438,

, Chem. Zeit., XVII, 2, p. 1098. Tbid., XVIII, 2, p. 1739.

r, Arch. fiir exp. Path. und Pharm., XXXII, p. 198.

Arch. f. Physiol, 1895, p. 225.

Jen, Zeit. j. Hyg. u. Inj. Krank., XXXII, 135.

420

THE EFFECT OF WORK ON THE CREATINE CONTENT
OF MUSCLE

By T. GRAHAM BROWN anp E. P. CATHCART.
From the Physiological Laboratory of the University of Glasgow
(Received September 30th, 1909)

Previous Work

The question—Does work influence the amount of creatine in muscle,
and lead to a change in the output of creatinine in the urine, has long
been a debated one. Much of the older work on this point is of little
value owing to the defective methods then available for the estimation
of creatine and creatinine. The papers of Weber (1) and van
Hoogenhuyze and Verploegh (2) give good accounts of these earlier
investigations. ;

Of the older workers, Liebig (3) found an increase of creatine in
muscle after work; as did also Sarokow (4), Sczelkow (5), and Monari (6).
Nawrocki (7), and Voit (8) thought that there was a slight decrease. Of
modern workers Weber (loc. cit.), using Folin’s method, found that
work caused a slight decrease in the creatine content of muscle. He
also found that traces of creatine and creatinine could be demonstrated
in the Ringer’s fluid with which, using the method of Langendorff, he had
perfused an isolated heart; while Mellanby (9), also using Folin’s method,
obtained results from which he concluded that work had no
influence on the creatine content of muscle.

As regards the effect of work on the excretion of creatinine in the
urine there is also a difference of opinion. Meissner (10), employing
very defective methods, found an increase of creatinine in the urine on
the day of exercise, followed by a decrease on the succeeding day.
Grocco (11), and Moitessier (12) also found an increased output on the
day of exercise. Gregor (13) concluded from personal experiment, using
a creatinine free diet, that exercise increased the output of creatinine
in the urine. Voit (loc. cit.), Hoffmann (14), Oddi and Tarulli (15), using
the older methods found no increase. Van Hoogenhuyze and Verploegh
(loc. cit.), using Folin’s method, found that, with an ample diet—creatinine
free, work left the excretion of creatinine unaffected, but that when the
diet was defective, as during the complete fast of their subject, La Tosca,
exercise resulted in a slight increase of creatinine in the urine. Weber

_ EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 421

(loc. cit.) also came to the conclusion that, if work were performed during
a fast, a rise in the output of the creatinine followed. Shaffer (16)
agreed with the results of van Hoogenhuyze and Verploegh. One of us
(E. P. C.), although not investigating this particular point, working in
conjunction with Drs. Kennaway and Leathes (17), was unable to detect
any definite rise in the output of creatinine even after severe work
under very different conditions, in all of which, however, the diet was
ample. We may therefore accept that, provided the supply of food is
sufficient, work does not bring about any increase of creatinine in the
urine.

PRESENT INVESTIGATION

As regards the results on stimulating frog muscle we have already
made a communication (18), in which we showed that there is an increase
in the amount of total creatinine in stimulated isolated frog muscle
(ordinary nerve muscle preparation), whereas, when the circulation is
left intact as in the decerebrated frog, there is always a slight decrease
in the amount of the total creatinine present.

The following table gives the results with frog muscle :—

Taste I
Serres A. (Isolated nerve muscle preparation.)
Experiment Per cent. of total Per cent. of total Difference
creatinine in creatinine in
normal (controls) stimulated

A 0-32 0-36 12 % increase
B 0-30 0-32 T% ss
c 0-32 0-36 13% on Be
D 0-36 0-39 oe '

Serres B. (Muscle in situ, circulation intact.)
A 0-37 0-26 lb-6 % decrease
B 0-35 0-29 62% ,, 33°G6
Cc 0-30 0-23 9-1 % OT
D 0-32 tly 0-24 77%

We have now carried out a series of experiments on rabbit muscle
under different conditions, and have obtained constant results.

The methods we employed were as follows:—The rabbit was deeply
anaesthetised with ether and was kept under the influence of the
anaesthetic till the end of the experiment, when it was destroyed. The
extensor muscles of the right knee were exposed by means of a skin incision
and removed, the amount taken being usually 10 to 15 grammes. The

422 BIO-CHEMICAL JOURNAL

bleeding points were secured and care was taken not to injure the great
vessels of the thigh. All visible fatty and fibrous tissue, as well as blood,
was rapidly removed from the excised mass, which was then weighed,
transferred to a small mortar, minced fine by means of a pair of sharp
scissors, then rubbed up with finely powdered glass, water being gradually
added until a fine suspension was obtained. This mixture was next
carefully transferred to an Erlenmeyer flask, filled up to 150 e.c. with
distilled water, some chloroform and thymol solution added, thoroughly
shaken up, and then placed in a hot water oven at 50°C., where it was
left, being repeatedly shaken, for eight to ten hours. At the end of this
period the flask, after faintly acidifying its contents with acetie acid.
was put on the steam bath, or boiling water bath, for thirty minutes and
then filtered. The solid residue was extracted with boiling water (as a
rule by boiling the residue with water in a porcelain basin) five to seven
times and filtered through the original filter. The united filtrates were
then concentrated to 40 c.c., and the content of creatine + creatinine
estimated by Folin’s colorimetric method. The same procedure was
carried out in the case of the stimulated extensor muscles. of the left
thigh, care being taken (i) that the corresponding muscles were taken
for examination, and (ii) that the amount taken differed by several
grammes from that taken from the right (unstimulated) side—sometimes
more being taken, sometimes Jess—-so that the readings on the colorimeter
were widely apart, thus obviating to a considerable extent any possible
personal bias. As an additional precaution one of the observers frequently
manipulated the solutions in such a manner that the observer who took
the readings was unaware which of the two solutions he was examining.
The reason why definite corresponding muscles were chosen was that as
the result of several experiments we carried out with different kinds of
muscle (red and white) we are inclined to believe that there is some
difference in their creatine content. That such might be the case is
suggested by such work as that of Bonhéffer (19) and of Paukul (20), —
who showed that in muscle structural differences might be associated
with functional. So far we have not performed a sufficient number of
experiments on this point to be able to draw definite conclusions. —__
As regards our method of stimulation, immediately after the removal
of the extensor muscles of the right side those of the left were transfixed
by a pair of electrodes, one of which passed through the muscles close —
to the patella and the other near their origin. Through these electrodes

the muscles were stimulated with rapid alternating faradic shocks, A a

_ EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 428

Berne coil was used, the primary current being about 5 volts and the
secondary coil was placed between the 2,000 and 4,000 unit marks. The
muscles were stimulated over a period of thirty to forty-five minutes,
but the secondary currents were not allowed to pass continuously through
them, being short-circuited for five seconds in every ten seconds.

We carried out control experiments to determine the accuracy of
our chemical methods, and found that, for small amounts of muscle,
the average difference between the extensors of the two sides, neither
having been stimulated, was a little over 1 per cent. of the total creatinine,
and for larger amounts (10 to 15 grammes) was about 2°3 per cent.

The general result of the whole series of experiments has been
that, with the circulation intact, stimulation of the muscles brings about
a constant, although small, decrease in the amount of total creatinine
(i.e., creatine + creatinine) extracted from the stimulated muscle.

_ Bxperiment II.—Rabbit, weight 1,550 grams. 12 grams muscle removed before stimulation
(stimulation 30 minutes) and 11 grams after. Total creatinine before stimulation, 43-42 milli-
grams = 0-361 %, and after, 30-84 milligrams = 0-280 %, i.e. a decrease of 22-4 % as a result
of stimulation.

Experiment I1V.—Rabbit, weight 1,850 grams. 7-5 grems muscle removed before and 17
grams after 40 minutes’ stimulation. During the period of anaesthesia a free supply of pure
oxygen was given. Total creatinine before stimulation 28-8 milligrams = 0-334 %, after stimu-
lation 54-6 milligrams = 0-321 %, i.e. a decrease of 4-3 % as a result of stimulation.

Experiment V.—Rabbit, weight 1,450 grams. 9-2 grams muscle removed before and 16
grams after 30 minutes’ stimulation. Total creatinine obtained before, 37-84 milligrams
= 0-411 %, and 52-4 milligrams after = 0-327 %, i.e. a decrease of 20-4 %.

Experiment VI.—Rabbit, weight 2,200 grams. 10 grams muscle removed before and 12-5

grams after 30 minutes’ stimulation. A free supply of pure oxygen was allowed during the
period of anaesthesia. Total creatinine before stimulation 30-60 milligrams = 0-306 %, and
37-68 milligrams after = 0-301 %, i.e. a decrease of 1-6 %.

Experiment IX.—Rabbit, weight 2,250 grams. 9-1 grams muscle removed before and 14-2
grams after 35 minutes’ stimu.ation. Total creatinine in muscle, before stimulation 39-04
milligrams = 0-429 %, and 51-42 milligrams after = 0-361 %, i.e. a decrease of 15-8 %.

Experiment X.—Rabbit, weight 1,900 grams. 11-5 grams muscle removed before, and
14-8 grams after 35 minutes’ stimulation. Total creatinine in muscle, before 43-24 milligrams
= 0-375 %, and 50 milligrams after = 0-337 %, i.e. a decrease of 10-1 %.

oO
As an example of two control experiments done, the following may
be given, No. 7 with our maximum error, and No. 12 with our minimum.
Experiment VII.—Rabbit. * 15 grams of muscle removed from right, and 13-1 grams from
left thigh. Total creatinine, in muscle from right side 53-08 milligrams = 0-354 %, and from
left side 48-0 milligrams = 0-366 %, i.e. a difference of 3-3 %.
Experiment XII.—Rabbit. 8 grams of muscle removed from right thigh and 11 grams from

left. Total creatinine, in muscle of right side 36-93 milligrams = 0-461 %, and from left side
50-31 milligrams = 0-457 %, ie. a difference of 0-86 %,. ey

424 BIO-CHEMICAL JOURNAL he.

If Mellanby’s figures be examined, although he draws the conclusion.
from them that work is without influence on the creatine content of
muscle, it will be noted (/oc. cit. pp. 459-460) that his frog’s musele
stimulated when isolated always show a slight increase in amount of total
creatinine present, perhaps somewhat smaller than ours, and, in the two
experiments on rabbits, which he did with their circulation intact, there —
is in both cases a slight decrease.

As already mentioned, van Hoogenhuyze and Verploegh have come
to the conclusion, in which they are supported by other investigators, |
that work does not increase the output of creatinine in the urine when
the diet is sufficient.

It was thought that the state of the nutrition of the animal, or,
perhaps, variations in the carbohydrate reserve, might influence the
total creatinine content of worked muscle. The experiments given above
may be divided into two series, one in which the animals were kept on
a very low cabbage diet for four or five days preceding the experiment,
and the other in which the animals were allowed to feed freely on bran,
turnip, and carrot for the same length of time. In every case there was
a decrease in total creatinine, but the decrease was somewhat greta: in
the badly fed than in the well fed animals.

Taste II.

Serres A. Badly fed
Experiment Totalcreatinine Percent. present Decrease Mean decrease

in milligrams in muscle per cent. " per cent.
a) ee ee
5 {8 mk BARB ang
FRE» here BAB 0 0 BE Promise
52-0 17-63
Serres B. Well fed cs)
4 {h 546 0-321 “3
ot SA et
a(R a0 Hae
21-7 7-23
» b “ r
“ 60 ‘UY L

EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 425

Other experiments with frogs were carried out along these same lines,
previous to the rabbit experiments. In these the frogs were given large
quantities of glucose for two days before stimulation, but the results
which we obtained by this method were not very concordant.

Another point which appeared in the course of our work was the
constant gain in fluid which occurred in the muscle as a result of
stimulation. We found that, in the muscles of both frogs and rabbits,
the average gain after thirty to forty minutes’ stimulation was about
2 per cent. We discovered subsequently that this figure was almost
identical with that given by Ranke (21). In our experiments the fresh
muscle was weighed as soon as possible after removal, then dried at
100° C. till constant weight was obtained.

In every experiment, although our method is not calculated to
elucidate the question, we examined the muscle extract for preformed(?)
creatinine, but found that the amounts present varied very largely in
quantity, the variation being also found in the control experiments.
We are inclined to agree with Mellanby that muscle contains
no preformed creatinine, or at most mere traces of this substance. The
variation in the amounts found in our experiments were due in all
probability to a partial conversion of creatine to creatinine during the
process of extraction. There was, however, one interesting fact which
may be mentioned in this connection, and that was that the percentage
amount of preformed (?) creatinine present in the above experiments with
rabbit muscle showed, with the exception of one experiment, a decrease
when there was a decrease of total creatinine.

The expenses of this research have been defrayed by a grant from
the Carnegie Trust.

426:

ee ee ee OS ee ae ee Pe , ae oo
Se a ee ee
wogl sah > a edt Oe ee ee

“Weber, Arch. f. exp. Path... Pharm., Vol. LVIIL, p. 93, 1907. 123 tage
~ Liebig, Ann. d. Chem. u. Pharm., Vol. LXII, p. 257, 1847 (cit. in 2). sivas at.

. Sezelkow, Centralbl. j. d. med. Wiss., p. 481, 1866 (cit. in 2).

~ Voit, Zeit: f. Biol., Vol. IV, p. 77, 1868.

Gregor, Zeit.’ f. physiol. Chem., Vol. XXXI, p. 98, 1900.°° . . .
» Hoffmann, Arch. f. path. Anat., Vol. XLVIIL p 908 lade Siete oii ee
% Oddi and Tarulli, BoWl. dell. Acad. med. di Roma, Vol. XIX, 1893 (cit. Maly., Vol.

_ Shaffer, Amer. Jour. of Physiol., Vol. XXII, p. 445, 1908.

’° BIO-CHEMICAL JOURNAL § 9 =

REFERENCES

van Hoogenhuyze and Verploegh, Zeitech. f. physiol. Ohem., Vol. XLVI, p. 416, 1905,
Sarokow, Arch. f. path. Anat., Vol. XXVIII, p. 544, 1868. sits tig ee

Monari, Arch. ital. de Biol., Vol. XIII, p. 1, 1890.
Nawrocki, Centralbl. j. d. med. Wiss., p. 416, 1865 (cit. in 2).

Mellanby, Jour. of Physiol., Vol. XXXVI, p. 446, 1908.

Meissner, Zeit. f. rat. Med., XXXIV, p. 297, 1868.

Groeco, Maly. Jahresbericht, XVI, p. 199, 1886. :)

Moitessier, Compt. Rend. Soc. Biol., Vol. XLII, p. 573, _ (cit. Maly; aL vie
- p. 182, 1891). thot vei

p. 522, 1894).

Cathcart, Kennaway and Leathes, Quart. Jour. of Medicine, Vol. 1,’p. 416, 1908
Graham Brown and Cathcart, Jour. of Phasiel. (Fyos. Phyotal. See.) Yo). XE YT iam
Bonhaffer, Arch. f. d. ges. Physiol. Vol. XLVII, p. 125, 1890. |
Paukul, Arch. {. Physiol., p. 100, 1904.

Ranke, Tetanus, 1866, p. 69.

427

THE ACTION OF EXTRACTS OF THE PITUITARY BODY

By H. H. DALE, M.A., M.D.

From the Welleome Physiological Research Laboratories,
Herne Hill, London, S.E.

(Received October Ist, 1909)

I. Iwrropvuctory

Though the activity of pituitary extracts was discovered by Oliver
and Schafer (1) almost simultaneously with that of suprarenal extracts,
the conceptions of the nature of the action of the former are as yet far less
precise. A comparison of the two was inevitable, and it has more than once
heen suggested that their action, at least as regards vaso-constriction, is
of the same kind and produced by stimulation of the same structures.
Herring (2) advanced this view as regards the arteries: a more recent
Observation by Cramer (3), of the action of pituitary extract on the pupil
of the frog’s eye (enucleated), lends support to the same idea: still more
recently an account given by Bell and Hick (4) of the action on the uterus
emphasised the similarity between the action of extracts from the two
organs. I thought it worth while, therefore, to bring together a number
of observations, made at different times and in different connexions,
which appear to me to indicate that such correspondence as exists is wholly
superficial and illusory. In the first place it must be admitted that the
actions of pituitary and suprarenal extracts have superficially several
points of suggestive similarity. Both raise the blood-pressure, peripheral
yaso-constriction being a principal factor in the effect (Oliver and Schiifer) :
in both cases the active principle is limited to a small, morphologically
independent portion of the gland, developmentally related to the central
nervous system in the one case, as to the sympathetic system in the other.
Attention is drawn to these points of similarity by Schafer and
Herring (5), who state that ‘here the parallelism ends’: but the
divergence of which they make specific mention is that the pituitary
extract has an additional effect on the kidney. Since they attribute this
to a separate active principle, no true divergence is indicated between
the pressor principles of the two organs. It has been shown (Langley (6),
Brodie and Dixon (7), Elliott (8) ) that the action of adrenaline reproduces
with striking accuracy the effects of stimulating nerves of the true

428 BIO-CHEMICAL JONNTRNAL

sympathetic or thoracico-lumbar division of the autonomic system. An
examination of the action of pituitary extract on various organs and
systems containing plain muscle and gland-cells will indieate whether its
action has more than a superficial resemblance to that of adrenaline by
showing whether its effects, or any group of them, can be similarly
summarised by relating them to a particular element of the visceral
nervous system. Incidentally evidence will be discussed which throws
light on the contention of Schafer and Herring that two active principles
exist in the extract, one acting on the circulatory system, the other
specifically on the kidney.

The extract used in my experiments, except where otherwise stated,
was a 5 per cent. decoction of the fresh posterior lobes of ox pituitaries.
The posterior lobes were dissected clean from the rest of the gland and
from dura water, weighed in the moist condition, pounded with sand, and
boiled with water faintly acidulated with acetic acid to produce
coagulation. The exfract, filtered from coagulum, is a clear colourless
fluid giving a faint biuret reaction. For experiments on isolated organs
the extract was prepared with Ringer’s solution and carefully neutralised
before use.

Il. Tae Errect on THE Crrcv.atory System

It has been mentioned that pituitary extract causes a striking rise of
blood-pressure, chiefly due to arterial constriction. If the action had any
relation to innervation by the sympathetic system we should expect to
find that the effect on the arteries was accompanied by an increased
frequency and force of the heart-beat, corresponding to the effect of the
cardio-accelerator nerves. It was pointed out by Schafer and Oliver that
this was not the case: the beat of the heart usually becomes slower, even
after exclusion of vagus action, though it may be somewhat augmented.
Reference will be made later to the action of the extract on the isolated
heart, which enables the effect to be studied in its least complicated form.

We should further expect to find, if the action were like that produced
by sympathetic nerve-impulses, that the action on the arteries showed
irregularities of distribution corresponding to that of sympathetic nerves.
It was of special interest, therefore, to examine the action on those arteries
which have been shown to be exceptional in their innervation and in their
reaction to adrenaline.

The pulmonary arteries. Brodie and Dixon showed that the
peripheral branches of the pulmonary artery are exceptional in that their

THE ACTION OF EXTRACTS OF THE PITUITARY BODY 429

muscular coats are not under the control of sympathetic nerves, and made
the interesting parallel observation that adrenaline, perfused through the
pulmonary vessels, produces no vaso-constrictor but a small vaso-dilator
effect. With segments of the main branches of the pulmonary artery,
treated as isolated organs, others have obtained definite constrictor effects
with adrenaline (Meyer (9), Langendorff (10) ). It is clear that there is
no real discrepancy between the two sets of observations: the only
conclusion justified by the evidence is that the sympathetic nerves send
motor fibres to the muscular walls of the pulmonary artery and its main
branches, but that the innervation stops short of the peripheral arterioles,
the calibre of which is alone concerned in determining the rate of
' perfusion under constant pressure, as measured by Brodie and Dixon.

In a few experiments with isolated rings of large branches of the
pulmonary arteries of large dogs and goats, I observed contraction on
adding small quantities of the pituitary extract to the Ringer’s solution
in which the rings were suspended. Since these experiments were made
similar observations have been published by de Bonis and Susanna (11).
Since, however, I obtained even more pronounced constriction of the strips
of pulmonary artery on adding adrenaline, these results only add another
to the cases already known in which adrenaline and pituitary extract
both cause constriction of an artery, and are of no significance for our
present enquiry. I owe to Professor Dixon the opportunity of making
with him observations on the effect of pituitary extract on the peripheral
pulmonary arterioles. The observations were made in connéction with
experiments concerning action on these arterioles of certain organic bases.
The lungs were perfused with Ringer's solution, or defibrinated blood
diluted therewith, according to the method described by Brodie and Dixon.
After it had been shown that either adrenaline or p. hydroxyphenyl-ethy-
lamine caused only a slight acceleration of the rate of perfusion, 1 ¢.c. of
the pituitary extract was introduced into the circulating fluid. As soon
as the extract reached the lungs there was a pronounced retardation of
the outflow. The observation was repeated several times, in different
experiments, with uniform result. Here, then, is a clear case of vaso-
constriction produced by pituitary extract on a system in which no such
constriction is produced by adrenaline or substances of similar action.

The coronary arteries. ‘The innervation of the coronary arteries
‘cannot be regarded yet as definitely settled, even the more recent
observations being by no means concordant. Maas (12) found that the
vagus supplies vaso-constrictor fibres to this system: Dogiel and

430 BIO-CHEMICAL JOURNAL

Archangelsky (13) found that vaso-constrictor fibres are contained in the
accelerator nerves: on the other hand Schafer (14) could not find any
evidence for vaso-motor nerves to these arteries, and observed no
constriction of them under the influence of adrenaline. The last
observation was confirmed by Elliott (8), who found the outflow from a
perfused segment of ventricle increased by adrenaline. Langendorff
observed that adrenaline caused relaxation of an isolated ring of coronary
artery, and this has been confirmed by de Bonis and Susanna. Still more
recently Wiggers (15) has found evidence of vaso-constriction when
adrenaline is added to a fluid perfusing the coronary arteries. From all
this conflicting evidence emerge the facts that the coronary arteries are
slightly, if at all, controlled by vaso-motor nerves, and that the
constrictor effect of adrenaline on the peripheral branches, if it exist at
all, is very weak compared with the effect of that principle on other
arteries.

In this instance | made no experiments with isolated rings of artery,
but such have recently been published by Pal (16) and by de Bonis and
Susanna. ‘These observers agree in finding that pituitary extract causes a
marked constriction of a ring cut from a large coronary artery. De Bonis
and Susanna also confirmed Langendorff’s observation that adrenaline
causes relaxation of such a ring, so that in this case the action of the two
principles is again contrasted.

My own experiments were made with the isolated heart of the rabbit,
perfused with oxygenated Locke-Ringer solution, by Langendorff’s method
as modified by Locke. There are several errors involved in the
measurement of the coronary outflow from such a preparation. These
have recently been discussed by Wiggers. The outflowing Ringer’s fluid
always accumulates to a certain extent in the right auricle and ventricle,
and, as Schafer pointed out, a certain amount may pass the semi-lunar
valves and so reach the left ventricle. With small hearts I have not
found that these defects seriously disturb the average rate of outflow: the
principal drawback is that the dripping of the fluid from the heart is
rendered irregular by the accumulation of fluid in the right side of the
heart during diastole, and its ejection by the systole. With a small,
rapidly-beating heart the quick and irregular succession of small drops
which results can be averaged and converted into a regular series of large
drops by a simple device. I used a large glass funnel, placed immediately
beneath the recording lever. A skein of threads, hanging loosely from
the heart and lever into the mouth of the funnel, ensured the delivery

= eget - _-

THE ACTION OF EXTRACTS OF THE PITUITARY BODY 431

into it of all the fluid leaving the heart, without at all interfering with
the record of the contractions. The funnel was fixed in an inclined
position and over the lower opening of the stem was drawn a short length
of rubber tubing, the diameter of which could be reduced by a clip. This
device converts an irregular series of drips and splashes into a regular
series of large drops, which fall at a constant rate so long as the average
rate of the drippings from the heart remains constant. These large drops
were recorded on the smoked drum by the ordinary arrangement of
receiving and recording tambours. When the beat of the heart and the
rate of the coronary outflow, as shown by the drop recorder, had become
constant, a small quantity of the filtered and warmed pituitary extract was
introduced into the bulb of the heart-cannula by means of a hypodermic
syringe, the needle being thrust through the wall of the rubber tube
leading to the cannula. Fig. 1 shows a typical effect. It will be seen
that the outflow from the coronary sinus becomes very much slower as
soon as the extract reaches the heart. The effect shown in the figure is
quite typical, and I know of no other drug which, in doses not immediately
fatal to the heart-muscle itself, will produce so pronounced a constriction
of the coronary arteries. That the effect is genuinely due to constriction,
and not to viscosity or mechanical accident, can easily be ascertained
from the fact that a second dose, introduced when the effect of the first
has subsided, produces a very small change in the rate of outflow. This
is quite in accordance with the observation, first made by Howell (25),
that a second dose of the extract, given intravenously when the effect
of a first large dose has passed off, produces hardly any rise of arterial
blood-pressure.'

One other point needs mention. It is clear from what has been said
above that a weakening or stoppage of systole might lead to an apparent
temporary retardation of the coronary outflow by allowing accumulation
in the right side of the heart. The phenomenon illustrated is not of that
kind. It is a prolonged effect, which persists to some degree for upwards
of half an hour after the injection, and its maximum coincides with a
phase of increased ventricular activity. There is no room for doubt,
therefore, that the coronary arterioles afford another example of an
arterial area slightly, if at all affected by adrenaline, stimulated to intense
constriction by pituitary extract.

The effect on the ventricular beat of the isolated heart

1. It is of interest to note that Dr. W. H. Harvey, to whom I communicated my observa.
tion of the constricting effect of pituitary extract on the eoromary arterivs, has produced
sclerotic changes in these arteries by repeated injections of the extract.

JOURNAL

BIO-CHEMICAL

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THE ACTION OF EXTRACTS OF THE PITUITARY BODY 488

can also be studied in fig. 1. It will be seen that, immediately
after the injection, it becomes slightly slower and considerably
more vigorous: later, with persistent retardation, it becomes
weaker than before the injection. Similar effects, in the same order, have
been previously described by Hedbom (17) and by Cleghorn ( 18). It is
difficult, however, to decide how far these changes in ventricular activity
are due to primary action on the cardiac muscle, how far to reduction of
the oxygen supply by coronary constriction. Neither effect is modified
by previous atropinisation, so that there can be no question of the
peripheral vagus-mechanism being concerned. There is further, in the
ease of the effect on the heart-beat, as in that of the coronary constriction,
no resemblance whatever to the effect of accelerator nerves or of adrenaline.
The safest conclusion is to regard the action on the coronary arteries as
certainly a primary effect of the extract, that on the heart-beat as probably
in part due to direct effect on the heart-muscle, and in part secondary to
the altered rate of coronary perfusion. It should be noted, in this
connection, that under conditions of natural circulation, in which the
effect of coronary constriction would be antagonised by the great rise of
systemic pressure, the secondary weakening of the beat is not usually
observed. .

The renal arteries. Schafer with Magnus (19), and later with
Herring (5), found that the kidney expanded when pituitary extract was
injected intravenously. It was of interest, therefore, to examine the
effect of pituitary extract on the rate of perfusion through the renal
vessels. The perfusion was made with oxygenated Ringer’s solution
under constant pressure, as for the isolated heart, the outflow from the
renal veins being measured by the drop-counter. The kidneys used were
those of cats and dogs. Both kidneys of the cat were perfused, the
cannulae being inserted into segments of aorta and vena cava. From the
dog one kidney was used, with cannulae in the renal artery and vein.
The pituitary extract was added by injection into the circulating
Ringer’s fluid. The following results were obtained :—

Rate or Ovtriow ty Drops rer

Ingection or Prrurrany Extnacr 20 seconps
Before injection After injection
Bxperiment 1.—Cat. 5 minims 34 20
Experiment i.—Cat. ist. 5 minims ay 27
2nd. 10 minims 20 31
Kaperiment [11.——Dog. Lat. 5 minims a4 20
2nd. 10 minima 20 22

434 BIO-CHEMICAL JOURNAL

It will be seen that the first injection causes in each case a decided
though small constriction. The genuineness of the phenomenon is again
shown by the failure of second injections, which even slightly reduce the
resistance of the constricted arteries. Similar results were obtained by
Houghton and Merrill (24), in the course of experiments made to determine
whether the extract locally excites the renal epithelium to secretion. On
the other hand Pal states that isolated rings of the proximal portion of
the renal artery were constricted, while rings from more peripheral
portions were relaxed by the extract. On the whole the evidence obtained
with isolated organs suggests that the marked swelling of the kidney in
its natural relations must be chiefly due to a relative insensitiveness of
the renal arteries towards the vaso-constrictor effect of the extract. It
might seem, at first sight, that even this implied, as Pal concludes, an
action of the vaso-constrictor principle on some nervous structure, and
not on the muscular coats of the arteries themselves. This, however, is
by no means the only instance of an exceptional reaction of the renal
arteries towards general stimulants of plain-muscle contraction. The
various drugs of the digitalis series, for example, injected in small doses,
cause expansion of the kidney and diuresis, especially in the rabbit; but
the result of most experiments on the artificial perfusion of these drugs
through the vessels of the excised kidney, especially of the dog and the
cat, has been to demonstrate a marked constrictor action even on the
vessels of that organ. There is no reason at all for supposing that these
drugs act on nervous structures, and there is as little in the case of the
pituitary extract. The anomalous reaction of the kidney vessels in their
natural relations is clearly a similar phenomenon to their reaction to the
digitalis series; but since the pituitary extract acts more powerfully on
the arterioles and less on the heart than digitalis and its allies, the
phenomenon is presented by the former in an exaggerated form.

The Spleen. The spleen may be regarded, in so far as its contractile
activity is concerned, as belonging to the circulatory system. Schafer
and Magnus showed that pituitary extract caused contraction of the
muscular capsule. I have repeated this observation with a like result. A
plethyomographic record of the effect is shown in fig. 2.

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THE ACTION OF EXTRACTS OF THE PITUITARY BODY

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436 BIO-CHEMICAL JOURNAL

Ill. Tue Uterus

In a paper on another subject (22) I mentioned incidentally the
powerful uterine contraction produced by pituitary extract. I have
since extended the observation, finding, as expected, that the action,
like that on the arteries, is possessed by extracts of the posterior lobe
only.

Bell and Hick, working with the extract which I myself used,
appear to have obtained a comparatively small effect on the rabbit's
uterus in the resting (i.e., non-pregnant and non-oestrous) condition. This
is quite contrary to my own experience. They worked exclusively with
the rabbit. This animal is not really suitable, however, for our present
enquiry, since its uterus responds, under all conditions, to the stimulus
of sympathetic nerves or adrenaline, by contraction. In the cat, on the
other hand, as was shown independently and almost simultaneously by
Cushny (20), by Kehrer (21), and by myself (22), the uterine tone and
contractions are inhibited in the non-pregnant, stimulated in the pregnant
animal, by sympathetic nerves or supra-renal preparations. I regard
it, then, as of great significance that in the uterus of the cat, as well as in
that of the dog, the guinea-pig, the rat, and the rabbit, I have always
observed, in all functional conditions, powerful tonic contraction as the
effect of applying pituitary extract. The results were obtained by
intravenous injection into the anaesthetised or brainless animal, and also
by Kehrer’s method of adding the extract to a bath of warm oxygenated
Ringer’s solution, in which the isolated horn of the uterus was s0
suspended as to pull on a recording lever. The effect, under these
conditions of adding a few drops of pituitary extract to the 200 c.c. of
Ringer’s solution in the bath, is illustrated in figs. 3 and 4. So
little, in my experience, is the effect dependent on the condition
of the uterus as regards oestrum or pregnancy, that the uterus of a virgin,
half-grown cat responded to the pituitary extract by as marked a tonic
contraction as was given by any of the numerous pregnant or multiparous
organs examined.

The effect of pituitary extract on the uterus, then, shows again the
absence of parallelism to the effects of sympathetic nerves, the effect of
the extract being always tonic contraction, even when stimulation of the
hypogastric nerves produces pure inhibition of tone and rhythm.

THE ACTION OF EXTRACTS OF THE PITUITARY BODY 487

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488 BIO-CHEMICAL JOURNAL
IV. Ovner Orcans Contarxinc Praty Musee

The intestines and the urinary bladder give no such marked response
to the pituitary extract as the organs hitherto mentioned. In a dog
anaesthetised with A.C.E. mixture I observed, indeed, a distinct inhibition
of intestinal movements when the extract was given intravenously, even
when the splanchnic nerves were cut. This might be regarded as
indicating a similarity of action to sympathetic nerves. An isolated
loop of intestine, however, the rhythm and tone of which are
immediately inhibited by adrenaline, contracts, though but feebly, when
pituitary extract is added to the bath. It is probable, therefore, that
the inhibition, seen under normal conditions of circulation, is due to the
intense anaemia which the vaso-constrictor action of the extract produces.

The bladder of the cat, when the extract is injected intravenously,
usually exhibits a temporary weakening, followed by more prolonged
increase of tone. Neither is of any great extent. A guinea-pig’s bladder,
suspended in the Ringer-bath, contracted feebly when pituitary extract
was added.

The plain muscular coats of the intestines and the bladder contract,
then, like other plain muscle, in response to pituitary extract, but their
sensitiveness thereto is small in comparison to that of some organs. The
retractor penis of the dog, a convenient sheet of plain muscle for
examination in the Ringer bath, contracts, as might be expected, when
the extract is added (fig. 5).

No effect could be detected on pilo-motor muscles or on the
mammalian pupil.

‘VY. Guanp CELLS

Schafer and Herring found that the extract caused secretion neither
of saliva nor pancreatic juice, which observations I have confirmed. In
its failure to evoke salivary secretion the extract is again contrasted to
adrenaline. The profuse flow of urine which the extract causes, as first
shown by Schafer, in conjunction with Magnus (13) and with Herring
(3), can hardly be regarded as a true glandular secretion.

VI. Tue Action arrer Ercoroxtne

I have shown (23) that the specific ergot alkaloid ergotoxine, when
injected intravenously in certain doses, annuls all motor effects of
sympathetic nerves and adrenaline, so that the latter produces, in the cat,

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THE ACTION OF EXTRACTS OF THE PITUITARY BODY 439

a fall of blood-pressure and relaxation of the pregnant uterus in place
of the customary rise and contraction. Ergotoxine may be given,
however, in any quantity without affecting the contraction of arterial and
uterine muscle produced by a subsequent injection of pituitary extract

(fig. 6).

AcTION OF ENZYMES, ETC., ON THE EXTRACT

Schafer and Herring (5) state that peptic digestion reduces the action
of the extract on the blood-pressure without affecting the action on the
kidney, but that neither action is affected by tryptic digestion. They also
obtained results which they regarded as indicating that oxidation by
H,O, destroys the pressor action more quickly than the diuretic action.
Certain obvious precautions seem to have been omitted: there is no

_ indication that they controlled the activity of their enzymes or the

response of their animal. A negative result should obviously not be
accepted as indicating destruction of the agent unless a positive effect
could subsequently be obtained with the untreated extract. Adopting
these precautions I have failed to confirm them on all points. Digestion
for twenty-four hours with a peptic extract of proved activity and 0:2 per
cent. HCl failed to alter in any perceptible degree the pressor or
diuretic action of my extract. I can only conclude that the peptic
extract used by Schafer and Herring contained some antagonistic
depressor substance, or that their animal was for some reason
unresponsive to the pressor effect. On the other hand every active
preparation of trypsin which I have tried has reduced the action on the
blood-pressure and on the urinary flow practically to ni after a few hours’
digestion. Commercial trypsin, ‘liquor pancreaticus,’ pure pancreatic
juice obtained by secretin and activated by enterokinase—all gave the
same result. In all cases a subsequent injection of the original extract
produced the usual rise of blood-pressure and acceleration of the flow
of urine (figs. 7 and 8). It may be suggested, in the absence of evidence
for control on that point, that Schafer and Herring were using an inactive
preparation of trypsin: at least it is clear that the tryptic
preparations used by me contained something which was not present in
theirs. In my experience oxidation with H,O, failed likewise to
discriminate between the pressor and diuretic activities. Both effects
‘ were smaller after oxidation than those produced by a subsequent

injection of the original extract; but that either had suffered greater
change than the other was not apparent.

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THE ACTION OF EXTRACTS OF THE PITUITARY BODY 448
Excretion. Arremper to Propuce Iuuunitry

The fact, discovered by Howell, that second doses are relatively
ineffective, suggests that the active principle is not readily destroyed or
rendered inactive in the body. I found that the urine of a cat, excreted
in response to an injection of the extract, had a pressor action, like a
dilution of the extract, when tested on another cat (fig. 9). Probably the
active principle, therefore, is at least to some extent excreted unchanged.

The refractory state to further injections has nothing to do with a
true ‘immune’ reaction. In the serum of a rabbit, treated for a month
with increasing injections of the extract, I could distinguish no trace
of a body neutralising the physiological activities of the extract.

. Discussion OF THE RESULTS
It is clear from the foregoing that the characteristic action of
extracts of the posterior lobe of the pituitary body is stimulation of plain
muscle fibres. Different organs containing plain muscle show a varying
sensitiveness of response to the extract, the arteries, the uterus and the
spleen being conspicuously affected. This unequal distribution of effect
cannot, however, in any way be related to inequalities of innervation by
nerves of the true sympathetic or of the autonomic system as a whole.
Ergotoxine, which excludes motor effects of true sympathetic nerves,
and of drugs acting through those nerves or like them, leaves the action
of pituitary extract intact. Neither atropine nor curare affects its direct
action in any degree. The muscle of the mammalian heart is possibly
affected to some extent by the extract, apart from effects secondary to
constriction of the coronary arterioles: Herring’s observations on the
frog’s heart render this most probable. No effect could be detected on
the response of voluntary muscles, either to direct or indirect stimulation.
The active principle is then essentially a stimulant of involuntary, and
especially of plain muscle.

The question of the diuretic effect needs some further discussion.
Houghton and Merrill (24) have recently taken the somewhat extreme
view that this is entirely secondary to the rise of blood-pressure. They
state that the rise of blood-pressure produced by adrenaline is
accompanied by a similar diuresis, This latter observation is directly
‘opposed to the experience of others, and I have never myself been able
to confirm it. Further it was shown quite clearly by Schafer and
Herring that a second injection of pituitary extract may cause distinct

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THE ACTION OF EXTRACTS OF THE PITUITARY BODY 445

diuresis without any perceptible rise of blood-pressure. While such an
observation, which I have been able repeatedly to confirm, sufficiently
disproves the statement that the diuresis is secondary to and runs
parallel to the actual rise of systemic pressure, it does not remove the
possibility of the dependence of the diuresis on vascular effects. A
redistribution of the blood in the system, caused by the comparative
irresponsiveness of the renal arterioles, is conceivable without actual rise
of general systemic pressure, especially if the arterial constriction is
accompanied by weakening of the heart’s action, due to the depressor
constituent which the extract always contains, the action of which,
moreover, is much more evident in the case of a second injection.

The differential action of enzymes and oxidation on the supposed

: pressor and diuretic principles, alleged by Schafer and Herring, has not
_ been confirmed in my experiments. On the contrary I have found that
whatever destroyed one action destroyed both. Their other evidence for

the existence of two principles seems to me also inadequate. They lay
stress on the difference in the time relations between the two effects and
the relatively greater effect of second injections on diuresis. The
difference in time-relations of a diuretic and pressor effect is, however,
a familiar phenomenon in cases where there can be no question of the
presence of more than one active principle. If strophanthin, for example,
be injected intravenously into a dog or cat, the immediate effect on the
diuresis is usually a distinct retardation: later, as the rise of arterial
blood-pressure passes off, there is generally a secondary acceleration which
often persists after the blood-pressure has regained its original level. A

similar sequence of events was recently observed by P. P. Laidlaw and

myself in experiments, in course of publication, on the action of a pure,
erystalline active principle from Apocynum. Such a difference in
time-relations cannot, therefore, be accepted as necessitating the presence
of two principles. The relatively greater efficacy of a second injection
in causing diuresis as compared with its pressor effect can also be
interpreted in another way, as indicated above. The blood-pressure
tracing is complicated by the presence of the heart-depressing principle :
it is not a fair index of the degree of vaso-constriction in this instance.
An apparently greater relative efficacy of second injections can also be
observed in the case of the uterus, when the effect on that organ is

‘compared with that on the arterial pressure. I have frequently seen,

as the result of a second injection, marked contraction of the
uterus accompanying a very slight or no rise of blood-pressure.

1 tam il ai all | alll 2 ae oat oe eae
ae ee a See, Be
‘ 7 sila ' Khe

446 BIO-CHEMICAL JOURNAL |

It does not seem justifiable, however, to draw from this catia the —
conclusion that the principle acting on the plain muscle of the te rus is
different from that which acts on the plain muscle of the arteries. It is, —
of course, true that nothing short of the isolation of a single pr
principle, producing both pressor and diuretic effects, would 1 ke the
view that two principles exist untenable. While Tae fu i
evidence, however, the conception of both effects as due to one one | :
seems to me adequate and simpler.

CoNncLUSIONS

1. The action of extracts of the posterior lobe of ne
is a direct stimulation of involuntary muscle, without any
innervation. The action is most nearly allied to that of the ¢
series, but the effect on the heart is in this case bl that
muscle intense.

ig

2. The active principle is excreted in the urine.

3. No true immune reaction is produced by —
the extrect.

4. The evidence advanced in proof of the exiatinee of
pressor and diuretic principles is inadequate. .

SSRPFSERE Serene apeywe .

-—- « Re ae hw ee ee ee
—_— vr eS eo a ee Sie a
: .

_ THE ACTION OF EXTRACTS OF THE PITUITARY BODY

REFERENCES

Oliver and Schiifer, Journ. of Physiol., XVIII, p. 277, 1895.
Herring, Journ. of Physiol., XX XI, p. 429, 1904.
Cramer, Quart. Journ. of Exper. Physiol., 1, p. 189, 1908.
Bell and Hick, B.M.J., 1909 (1), p. 777.
Schafer and Herring, Phil. Trans., 1906.
Langley, Journal of Physiol., XXVIT, p. 237, 1901.
Brodie and Dixon, Jbid., XXX, p. 476, 1904.
Elliott, Jbid., XXXII, p. 401, 1905.
age mags f. Biol., XLVIIT, 1906.
vendorff, Zentralbl. f. Physiol., XX, _p. 551, 1907.
s and Susanna, Zentralbl. j. Physiol.. X XI, p. 169, 1909.
Pfliiger’s Arch., LXXIV, p. 281, 1899.
el and Archangelsky, ibid., CXVL., p. 482, 1906.
x, Arch. de Sci. biol. de St. Petersbourg (Pawlow Festschrift), p. 251, 1904.
rs, Amer. Journ. of Physiol., XX1V, p. 391, 1909.

n, Skand. Arch. {. Physiol., VUIL., 1898.
n, Amer. Journ. of Physiol., U1, p. 273, 1899.
and Magnus, Journ. of Physiol., XXVIL, p. ix (Proc. Phys. Soe.).

Barger and Dale, Bio-Chem. Journal, U1, p. 240, 1907.
Houghton and Merrill, Journ. of the Amer. Med. Assoc., LI, p. 1849, 1908.
Howell, Journ. of Exper. Med., Lil, p. 2, 1898.

447

¢

448

A METHOD FOR THE ESTIMATION OF THE UREA,

ALLANTOIN, AND AMINO ACIDS IN THE URINE © &
By DOROTHY E. LINDSAY, B.Sc., Cannzom Scuoran. \
Sz
Communicated by Prof. D. Néel Paton »
From the Physiological Laboratory, University of Glasgow 3 aay

(Received October 2nd, 1909)

In investigations involving the determination of the distribution of
nitrogen in the urine, the separation of the various nitrogenous
constituents is a matter of no little difficulty, and involves so much time
that it is almost impossible to carry through any prolonged series of
observations.

The object of the present investigation is to determine how far the
distribution of nitrogen can be more rapidly determined indirectly by
taking advantage of the differences in solubility and stability to reagents
of the various substances.

The chief substances in which nitrogen occurs in the urine are: —

Urea, purin bodies including uric acid, creatinin (with or without creatin),
ammonia, allantoin, amino acids (including hippuric acid), sulphur-
containing bodies and bodies of unknown composition. a

The present investigation is confined to the determination of urea, ial
allantoin and amino acid nitrogen.

For the determination of Urea Bohland (1) recommended the
precipitation of the other nitrogen-containing substances by
phosphotungstic and hydrochloric acids. The urea was then estimated

in the filtrate. He claimed that the results got by this method gave only — E as
urea nitrogen and ammonia nitrogen, all other nitrogen-containing bodies __ et
being precipitated. sg

Later Schéndorff (2) showed that the amino acids (glycocoll, leucin, =
&c.) are not precipitated by phosphotungstic and hydrochloric acids. He
also showed that while creatinin is precipitated, as Bohland had already
found, creatin is not. Allantoin also is nof precipitated; a statement later
confirmed by Mérner (3).

Pfaundler (4) experimented with various samples of phosphotungstic |
acid and found that, if Merck’s preparation is used, ammonia is is Ce |
precipitated. ig

_ ee be ae lt 7 i
eas 7

ESTIMATION OF UREA, ETC., IN THE URINE 449

The nitrogen obtained by this method therefore includes urea, amino

acids (with hippuric acid), creatin and allantoin nitrogen.

Morner and Sjéqvist (5) recommended a method for the estimation of
urea in which the urine is precipitated with a saturated solution of barium
chloride in which five per cent. of barium hydrate is dissolved, and an
aleohol ether mixture containing two parts aleohol to one of ether. After

twenty-four hours it is filtered, the filtrate evaporated at 50° to small
bulk, after the addition of a pinch of magnesium oxide to drive off the
ammonia, and the nitrogen in it estimated by Kjeldahl’s method.
Creatinin, hippuric acid, and also some amino acids (leucin, bile acids)
are not precipitated, and their nitrogen is thus included in the amount
obtained by this method.

Folin (6) proposed a method of heating the urine with crystallised
aa. ium chloride and hydrochloric acid. At the temperature thus
__ employed—150° C.—urea and allantoin alone are decomposed to give off
ammonia. Thus the nitrogen obtained by this method includes allantoin
nitrogen in additionsto*urea nitrogen and ammonia nitrogen.

______- Mérner, in a moré recent paper (7), develops what may be called the

__-Mérner-Folin method by which urea alone yields its nitrogen. The bulk

a of the nitrogen-containing substances are precipitated as described above,
____ with barium chloride, barium hydrate and alcohol ether solution, but the

evaporated filtrate is heated with magnesium chloride and hydrochloric
acid as in Folin’s method. Allantoin is by this procedure almost entirely
removed before heating with magnesium chloride, thus avoiding Folin’s
_. error. The creatinin, &., which are not precipitated in the Mérner

_ method are not decomposed in the Folin method. Thus the Mérner-Folin
___ method gives only the nitrogen which is contained as urea.

ee ‘In the present investigation a slight modification of this method
_ was adopted, a modification first employed by Underhill and Kleiner (8),
viz., the use of an alcohol ether solution containing equal parts of

__ aleohol and ether, since as Haskins (9) had previously observed,
oa allantoin is more insoluble in such a solution than in the one employed
iby Mérner. In addition a few drops of hydrochloric acid were at once
_ added before the evaporation of the alcohol ether filtrate, thus preventing
a the escape of ammonia, the nitrogen in which is thus included in the

results. This addition of acid was also recommended by Folin, instead of
i driving off the ammonia by means of magnesium oxide as practised by
1 Méruer,

450 BIO-CHEMICAL JOURNAL

The method in detail as employed by me is, 5c.c. of the urine are
mixed with 5c.c. barium chloride, barium hydrate solution, and 100 ¢.c.
alcohol-ether (50 c.c. absolute alcohol, 50 ¢.c. ether). After twenty-four
hours it is filtered, an aliquot portion of the filtrate is taken, and a few
drops of hydrochloric acid immediately added. This is then evaporated
almost to dryness at a temperature of 50°C. in an apparatus described
by Haskin; 20 grs. magnesium chloride are then added and a small piece
of paraffin wax to prevent frothing, and the whole is heated for three
hours on an electric heater described by Catheart (10).

The direct determination of Allantoin has always proved difficult and
unsatisfactory. The similarity between its properties and those of urea
renders complete separation extremely difficult. Poduschka’s method
(11), in which allantoin is precipitated by silver nitrate in alkaline
solution, is comparatively simple, though somewhat lengthy, but, as
Salkowski says, the precipitation of the silver compound with ammonia
is one of the most difficult of chemical procedures.

Wiechowski (12) proposed using mercuric acetate, which precipitates
allantoin but not urea, instead of the usual mercuric nitrate, by which
urea too is precipitated. Allantoin when thus precipitated shows a strong
tendency to crystallise. The details of the method are, however, long and
troublesome, though giving accurate results, and it is impracticable for
a long series of daily observations.

For the estimation of the Amino acids various methods have been
suggested.

Fischer and Bergell (13) proposed a direct method which depends on
the power of napthol-sulphonie acid to combine with amino acids. The
urine was shaken with an ethereal solution of 8 napthalene sulpho

chloride and the amino acid compound subsequently precipitated with

hydrochloric acid. This method was later modified by other workers.

Glaessner (14), in criticising the method, states that he, as others also, ©

did not recover on an average more than 60 per cent. of the amino acids.

Neuberg and Manasse’s direct method (15) depends on the fact that —

on shaking a urine with a strongly alkaline solution of a-napthol
isocyanate, hydantoic acid is formed, which is precipitated after
acidification. Glaessner found this method not at all constant. Some
good results were obtained, but in general they were quite unreliable.
Pfaundler (16) endeavoured to devise an indirect method for the

estimation of amino acids. His method was to precipitate the urine first

with phosphotungstic acid, The precipitate and filtrate were then each

.
=

ESTIMATION OF UREA, ETC., IN THE URINE 451

heated with phosphoric acid. The urine nitrogen was thus divided into
four fractions.
I. Substances precipitated by phosphotungstic acid.
(a) Nitrogen easily removed by heating with phosphoric acid.
(b) Nitrogen not thus easily liberated.
II. Substances not precipitated by phosphotungstie acid.
(a) Nitrogen easily removed by heating with phosphoric acid.
(6) Nitrogen not thus easily liberated.
Of these four fractions, II (a) consists mainly, if not- entirely, of
amino acids.
Kruger and Schmid’s method (17), also an indirect one, was based on
_ the fact that amino acids do not give off ammonia when heated with
concentrated sulphuric acid at 160° to 180°C. They estimated the
_ nitrogen in the filtrate from phosphotungstic acid and also the nitrogen
_ which was obtained after heating with half the volume of concentrated
} sulphuric acid. The difference between these two should correspond to
the amino acid nitrogen.

e, Glaessner (18) proposed a somewhat similar method for the estimation

of amino acids. He precipitated with phosphotungstic acid and

_ evaporated the filtrate to dryness at a low temperature. The residue was

freed from water and then extracted with alcohol-amyl aleohol for six

hours. It was then filtered and a Kjeldahl nitrogen estimation done on

the residue. This gave the amino acid nitrogen. The difficulty of this
method is to ensure that the urea is completely dissolved.

Present Meruops

_____ It appeared possible that the combination of certain of these methods
_ —-Bohland, Folin, Mérner-Folin—might yield definite results as to the
distribution of nitrogen, and the method which I employ is as follows :—-

Kstimations of the nitrogen present are made by these three methods,
and by the differences between them the nitrogen in urea, allantoin and
amino acids is determined.

(a2) Bohland nitrogen, using Merck’s phosphotungstic acid, which
leaves unprecipitated amino acids, hippuric acid, creatin, allantoin and
urea.

'(6) Folin nitrogen, which includes urea, allantoin and ammonia
nitrogen.

452 BIO-CHEMICAL JOURNAL

(ce) Mérner-Folin nitrogen, which includes urea and ammonia . os]
nitrogen.

The ammonia nitrogen was separately estimated by Folin’ 8 method
and subtracted from (6) and (ce). i

~The difference then between the nitrogen of (a) and (6) ideal give
the amount of amino acid nitrogen and creatin nitrogen present, hippuric
acid being included as an amino acid. The creatin can be determined
separately by Folin’s method.

The difference between the nitrogen of (4) and (c) should hee the
amount of allantoin nitrogen present.

To verify the method estimations were made on solutions ovata 3
varying amounts of urea, allantoin and amino acids. The nitrogen in
each solution was determined by Kjeldahl’s method.

I. A solution was made consisting of 25 c.c. of a 2 per cent. solution
of urea, 25c.c. of a 0°5 per cent. solution of allantoin, and 10c.c. of a
(5 per cent. solution of alanin. 5c.c. of this solution contained 00016 |
grs. nitrogen as allantoin and 0°0022 grs. nitrogen as alanin. |

Bohland nitrogen required 9 c.c. ws acid = 0-0126 grs. nitrogen

Folin d » Ih 0.0. o = 060-0104 »

Mérner-Folin __,, » S5Occ. ,, = 00088 ,,
B. — F. = 0-0022 grs. nitrogen = 100 p.c. alanin nitrogen *
F -M-F.= 00016 _,, = 100pe.allanton , ¢

II. Solution consisted of 25c.c. of a 05 per cent. solution of ©
allantoin, 25¢.c. of a 1 per cent. solution of glycocoll, 25 c.c. of a 2 per
cent. solution of urea, and contained 0°042 gre. nitrogen as allantoin and
0044 grs. nitrogen as glycocoll.

Folin nitrogen required 13-1 ¢.c. jy acid = = 0-275 grs. nitrogen

Mérner-Folin _,, a 8-9 o.c. ms = 0234 i
F.— M.-F. = 0-041 grs. nitrogen = 97-6 p.c. allantoin nitrogen

IIT. Solution consisted of a mixture of a 2 per cent. solution of urea
and a 0°5 per cent. solution of allantoin, 5 c.c. of which contained 0°0034
grs. nitrogen as allantoin.

Folin nitrogen required 15-260.0. -\-acid = 0-02136 gre. nitrogen
Mérner-Folin ,, rm 12-9 0.0. sa > = OORT oF —
F, — M.-F, = 0-00825 gra. nitrogen = 95-5 p.c. allantoin nitrogen = 9 |

ESTIMATION OF UREA, ETC., IN THE URINE 458

2] IV. Solution consisted of a mixture of a 2 per cent. solution of urea
wnd a t per cent. solution of glycocoll, 5 e.c. of which contained 0°0035 grs.
nitrogen as glycocoll.
| Boland nitrogen = 15-2 ¢.c. —\ acid = 00212 grs. nitrogen
Folin - « = 8760. , = 00178 7
fe B. — F. = 00034 grs. nitrogen = 97 p.c. glycocoll nitrogen
_ V. Solution consisted of a mixture of a 0°25 per cent. solution of
alanin and a 1 per cent. solution of urea, containing 0°45 grs. nitrogen as
urea, and 0'037 grs. nitrogen as alanin.
eg Bohland nitrogen = 34°15 cc. acid = 0-478 grs. nitrogen
Folin oe = 31-6 ec. 7 = 0-442 ”
B. — F. = 0-036 grs. nitrogen = 97 p.c. alanin nitrogen

: These results are summarised in Tables I and II.

TABLE I
No, of solution Grs. allantoin F.—M.-F. Pc. allantoin —_Solution contained
nitrogen
= A 0-0016 0-0016 100 Urea, alanin, allantoin
IL. 0-042 0-041 97-6 Urea, allantoin, glycocoll
LL. 0-0034 0-00325 95-5 Urea, allantoin
No. of solution Grs. aminoacid B. — F. P.c. amino Solution contained

nitrogen acid t

L 0-0022 0-0022 100 Urea, allantoin, alanin—

IV. 0-0035 00034 97 Urea, glycocoll

v. 0-037 0-036 97 Urea, alanin

_ In order to ascertain whether these methods held when applied to
nes, estimations were made on urines to portions of which known
1ounts of allantoin and amino acids had been added.

Folin ni = O16 gra. ni
trogen grs. nitrogen

os oka re ”
F. — M.-F.
ae aed vols nitrogen was added
Folin masta i 8 Be
Mamner-Fotin » 0/1036 ,,
F.— M.-F. = = 00168
Increase of difference = 0-0168 grs. nitrogen = 105 p.c. allantoin nitrogen

P. — M.-F. = 00005 =" 98-0 p.c. allantoin nitrogen

as : i. ,
454 BIOCHEMICAL JOURNAL
I Goose win tlio

10 ¢.c. urine + 10 ¢.c, water. le tacit
Folin nitrogen = 0-00582 gre. nitrogen ty al ea
10 ¢.c, urine + 5 ¢.c. water + 5 ¢.0. allantoin solution ontainin
0-0096 grs. nitrogen —— h bani
Folin nitrogen = 0-01456 grs, ows 8 .
Difeence — 00824 gr. nitrogen. 8548p. al Hante
1V. Goose's urine. ite
15 c.c. oo + We. is water. 4. - =e
0-00798 :

Fh ting + Malar yl sation 0
clined: sliveeek a Naiiaeeanes nitrogen |
Folin = 0-00798

B. - F, = 0-00546 gra
Increase of difference = =
enemas RE a sors

The results are summarised in Table Til.

TABLE IL
No. — Grs. ni added) Grs. ni P.c. ni
i en arc wel aaowere Se ian
L : 0-016 0-0168 105 ye ee
IL. 0-0096 . 0-0095 98-9
UL. 0-0096 0-00924 95°6
IV. 0-0091 0-00924 WL
* age : ‘
REFERENCES oes
. rh 4) Bet
Arch. }. d. g. Physiol., XLILL., p. 30, 1888. /

1 Seed
2.. Areh. f. d. g. Physiol., LX, p. 15, 1896. — Ly op, vara. ihe aes
3. Skan, Arch. fur Physiol. XIV, p. 2981908, Hike
4. Ztech. j. physiol. Ohem., XXX, p. 75, 1900. ,

5. Skand. Arch. }. Physiol., U1, p. 448, 1891.

6. Ztach. j. physiol. Chem., XX X11, p. 504, 1901.

7. Skand. Arch. }. Physiol., XIV, p. 297, 1903.

8. Journ, of Biol. Chem., IV, p. 166, 1908,

9. Journ. of Biol. Chem., Ul, p. 243, 1906.

10. Proc. Physiol. Soc.. XXXV, 1906.

ll. Archiv. j. exp. Pathol. u. Pharmak., XLIV, p. 60, 1900. .
12. Beitr. z. chem. Physiol. u. Pathol., X1, 1907.
13. Ber. d. d. Chem, Gesellsch., XXXV, p. 3779, 1902.

14. Ztach. j. exper. Path. u. Therap., IV, 1907.

15. Chem. Berichte, XXXVIU, p. 2,359. :

16. Ztach. j. physiol. Chem., XXX, p. 75, 1900.

17. Ztsch. f. physiol. Chem., XXX, p. 556, 1900.

18. Ztech, j. éxper. Path. u. Therap., TV, p. 338, 1907.

Fon THE NATURE OF THE SO-CALLED FAT OF TISSUES
AND ORGANS

By HUGH MacLEAN, M.D., Carnegie Fellow, University of Aberdeen,
_ axypj OWEN T. WILLIAMS, M.D., B.Sc. (Lonp.), M.R,C.P.,
_ Hon. Asst. Phys. Hosp. for Consumption; Lecturer in Pharmacology,

University of Liverpool.

a From the Bio-Chemical Laboratory, University of Liverpool

(Received November 5th, 1909)

The question of the nature of the fatty substances present in animal

research in this direction has elicited some noteworthy facts. On the
other hand, the general problem of fat metabolism in almost all its details
till awaits solution, and though the processes undergone during fat
orption are now fairly well understood, we have absolutely no
knowledge of the methods utilised in the body in connection with the
_katabolism of these substances. The recent interesting observations of

_ Leathes and of Hartley point to the liver being an active agent in the

ss preparation of fat prior to its final oxidation in the tissue cells; it is,

however, not improbable that other organs may also be capable of
_ participating in this preliminary desaturation of the fatty acid radicles.

NaTuRe OF so-caLLep Tissur Far

S For many years it has been recognised that fatty substances may be

_ present in, or at least derived from, an organ which, on ordinary
ac pical or microscopical examination, appears to be absolutely fat-
4 and gives no trace of reaction to the specific fat stains. This
‘masked * fat can, however, be rendered visible under particular
circumstances, and the question of its origin has given rise to one of the
_ greatest controversies in the annals of pathological chemistry; it is now
generally accepted as arising from a combination of fat and protein

Ms a _ normally present in the tissue, the fat becoming evident only as the result
of certain post- or ante-mortem changes, by which the compound is
broken up and the fat liberated.

The recognition of the presence of this combined fat explained the
fact that ordinary solvents, such as ether, are ineapable of extracting all
the fat from an organ, and it was found that better yields were obtained
by the addition of auxiliary substanees, such as aleohol and chloroform,

456 . BIO-CHEMICAL JOURNAL

Various modifications have been suggested, but in most cases the general
principle of fat extraction adopted and recommended by the different
investigators depends on thorough extraction of the tissue by combinations

of the above solvents with the aid of heat. That an organ can be freed

from fat-like substances by the above means may be granted, but it is
exceedingly doubtful whether the actual substances present in the extract
represent with any degree of exactitude the fatty substances as they
actually existed in the tissue prior to extraction. Of late, attention has
been chiefly focussed on the fatty acids obtained, but the nature of the
compounds in which these fatty acids are actually present in the tissues
has been to a great extent lost sight of. It seems obvious, however, that
a correct knowledge of the nature and disposition of the ‘fat’ in the
animal organs is the only way by which a key to the difficult problem of
fat metabolism is likely to be found.

Experiment shows that in many cases the greater part of the fat
obtained is really not fat in the ordinary sense of the word, but to a great
extent complex combinations of fatty acids with glycero-phosphorie acid
and a nitrogen-containing compound—the so-called phosphatides.

Though it has been long known that tissues and organs contain
‘lecithin,’ it is hardly recognised that very much of the ‘fat’ they do
contain may be present in this or in an allied form. What ordinary
neutral fat can be extracted from an organ seems to be generally
interstitial, and is present as stored fat, just as glycogen represents stored
carbohydrate. On the other hand, fat which is actually being made use
of by the living cells seems to be represented to a very greet extent—if,
indeed, not altogether—by phosphatides.

These substances, as pointed out by Heffter,! are Pen fe labile,
and undergo partial decomposition when heated to a temperature of over
50° C.; even an acid reaction has a similar tendency. Under these
circumstances it might be expected that the nature of the material
obtained by the different extraction methods would vary in its com-

position, and investigation proves that this is actually the case. This

fact in itself indicates that the general extraction methods do not suffice
for the determination of what appears, after all, to be one of the
fundamentally important points in such investigations—the general
nature of the fatty substances originally present in the tissue. It is
obvious that such methods as that recommended by Dormeyer,? in which

1. Arch. j. exp. Pathologie, XXVIII, p. 97, 1891.
2. Arch. f. d. ges. Physiol., Vol. LXV, p. 90, 1907.

NATURE OF FAT OF TISSUES AND ORGANS 457

___ digestion with pepsin and hydrochloric acid is utilised to separate off the
___ masked- fat, must also result in more or less marked disintegration of the
more labile fatty substances. The varying saponification values obtained
in the following experiments show that there must be marked differences
in the different extracts prepared by various methods.

EXPERIMENTS WITH DIFFERENT Extraction METHODS

Dog’s liver was taken, and an equal amount used for each experiment.
The final extracts obtained were then saponified in the usual way with
| alcoholic potash, and the saponification values compared.

Bale (1) Noel Paton’s Method.—Liver was cut up into small pieces and placed in methylated
“a spirit for a week ; the spirit was then poured off into an evaporating basin and the liver pounded
____ im @ mortar and thrown again into the spirit. The whole was then dried on a water bath, the
= temperature being always kept below 80° C. The contents of the basin were then put into a
= ‘Soxhlets apparatus and extracted with ether for about 12 hours. The syrupy extract obtained
was then dried and saponified.

Saponification value = 215.

(2) Liver was cut up and extracted for a week with cold alcohol ; it was then treated with
~ hot alcohol for several hours, and with ether as described on page 458. Ether extract was
separated and the ether distilled off at a low temperature.

Saponification value = 248-2.
(3) Liver was thoroughly ground up with calcium sulphate until a fine dry powder was

obtained. This was repeatedly extracted with ether in the cold. Ether was then distilled
off at a temperature below 50°.

Saponification value = 234-84.
The essential features of these experiments, given in tabular form,
are as follows, and show that both the nature of the extracting substances
and the order in which they are used, play a part in determining the
nature of the final extract obtained. While the saponification values are
given as one instance indicating a difference in the extracts, there are
other variations with regard to the amounts of free fatty acids, etc.,

present :—
Saponification
Organ used Nature of extraction value of extract
Dog's liver Placed in methylated t for 7 days; dried 215
in all at 80°. Total we prt vith ether
experiments for 12 hours,
‘ First cold alcohol ; then hot alcohol ; then ether. 248-2
Dried with CaSO, Extracted with cold ether , 23484

458 BIO-CHEMICAL JOURNAL

An extension of such experiments showed that not only are different
results obtained by different methods of extraction, but that the same
method may give variations in the nature of the substances obtained: the —

chief explanation seems to be the one that naturally presents itself, se,
that during the ordinary process of extraction, in which heat is

invariably employed, there is, together with the abstraction of the fat-
like substances from the tissues, a more or less well-marked disintegration

of the extracted products themselves; the extent to which this destructive —
change becomes manifest depends no doubt to a great extent on the —

amount of heat utilised during the process. 1 nl

Again, the above methods are absolutely useless for determining
another most important point-—the relative amounts of free and combined
fatty substances in the tissues. It may well be that the extremely labile
nature of these substances causes their extraction to be inevitably
associated with a certain amount of change in their constitution, but it is
obvious that this tendency is exaggerated by the use of hot solvents, and
the only rational method would appear to be extraction at as low a

temperature as possible. By a careful combination of solvents used in

the cold, a fair idea can be obtained of the nature and disposition of these

fatty substances in the tissues, and it is hoped to publish shortly a set of

experiments giving information with regard to these important details.

In all our experiments it was noticed that the saponification values
obtained were very high, and the following samples indicate the nature of
the figures found in the case of different organs from the dog. oa a

The animals were anaesthetised with chloroform, quickly bled, and —
transfused with saline. The organs were then removed, transfused
separately, and then minced and weighed in the moist condition.

Meruop or Extraction

Tissues were extracted for several days with cold alcohol, the
being changed several times. The residue was then treated for one to two
hours with bo‘ling alcohol, a reflux condenser being used. After removal
of the hot alcohol, the residue was extracted several times with ether.
Both alcoholic extracts were mixed, the alcohol evaporated, and the
residue taken up in ether. This was mixed with the ether extract of the

tissue, and after the chief part of the ether was driven off, residue was _
dried in vacuo over H,SO,. Saponification was carried out with

alcoholic potash.

+

NATURE OF FAT OF TISSUES AND ORGANS 459
Saponification figures of ‘fat’ from organs of dog

are Dog A Dog B
Blood = bes a. 153-89 a5 _
Connective tissue... tear? 223-9 Fa 195-2
Liver : 231-6 ‘ne 230-5
Kidney 264-6 ait _
Muscle piss nS me 254-5 =# 264-5

3 a. 190 and 200; Gaicebiedis ae substances as cholesterol are
sg to a varying extent, but these and other similar substances would

4 be obtained. The alternative niplapaton seemed to point to the
ence of phosphatides as the cause of the high values, and
igation showed that this was the case. It occurred to B. Moore

sited fat or free fatty acids, and observations made by us at his request
- indicates that the same holds good for certain other organs; it is very
probable that the free fatty acids found in extracts obtained by the
Be _ ordinary methods are to a considerable extent disintegration products of
____ tissue phosphatides. The high saponification values were found to be
eaused by combination of part of the sodium with phosphoric acid and
. glycero-phosphoric acid liberated during the process of saponification.
__ Every organ and tissue naturally contains more or less neutral fat in
the interstices of its substance; but though it would seem that the
preponderating portion of the ‘ fat’ combined with protein in the bioplasm
as masked fat is present as phosphatide, in addition some of the
_phosphatide is present in free form, perhaps as a phase in its passage to
_ combination; free phosphatides would, on this view, constitute
___ preliminary substances which subsequently pass on to actual combination
in the tissues. In a normal organ, therefore, the less microscopical
___ evidence there is of fat, the less neutral fat is actually present; while the
-__- eombined fat—a phosphatide—seems to represent one of those steps in that
__ gynthetical elaboration of fats which appears to be a necessary prelude to
actual assimilation. It is not improbable that phosphatides represent a
necessary step in the elaboration of fatty substances destined ultimately to
undergo actual assimilation into living matter. That such substances

460 BIO-CHEMICAL JOURNAL

are essential for the vital processes seems indicated by their presence in all
living cells hitherto investigated; it cannot be doubted that one of the
steps which ordinary fat undergoes in the cell is a transformation into
phosphatide, and probably in these bodies the desaturation of the fatty
acid radicle is brought about. Whether this elaboration is necessary for —
the ultimate oxidation of all fats, or whether we have here wholly or in
part a process somewhat analagous to the endogenous metabolism of
protein, can as yet be but conjecture. The fact, however, that
phosphatides contain practically all the constituents (even iron, according
to Glikin) of nucleo-proteins, is not without significance, and it is not
unlikely, as partly suggested by Hammersten, that they may be the
source of the cell nucleo-proteins.

The marked amount of phosphatide obtained in two experiments in
which pig’s liver was used is shown by the following figures. The
phosphatides were separated by means of acetone. As full details of
similar experiments will be published later on, the general outlines alone
are given here. In the first experiment cold alcohol was followed by hot
alcohol, and then by ether. In the second case the more rational method
of cold extraction, first with ether and then with alcohol, was carried out:
this method, which seems to give the best results, has the advantage of
furnishing a good idea of the amount of ‘ free’ and ‘ masked ’ phosphatide
and other substances; at the same time the necessary manipulation does
not tend to cause a disintegration of the extracted substances.

Experiment I Pig
(1) Cold alcohol extract = 18-2 grm. { Fhospaatide =17-0084) nized = 2623
(2) Hot alcohol extract = 11-3692 grm. veren eee cane
(3) Cold ether extract = 2-9608 grm. \veetae m4 AS Tae mixed = 220

Total extract = 32-5300 grm. | FasPen t= *7 abe, orm.

Phosphatide = 84 _— per cent. of total extract.

Fate, &. = 16 per cent. of total extract.

Experiment II
Liver 20 grm. dried substance
Ether extract {Phvsshatide = OS416 cox | Proportion of Fat to Phosphatide = 2: 1
Alcoholic extract | Phraphotide ve seen ane Proportion of Fat to Phosphatide = 1:20

NATURE OF FAT OF TISSUES AND ORGANS 461

= This liver showed fat distinctly on examination with the naked eye,
__ and the total amount of neutral fat was somewhat large. On the other
hand, it will be noticed that the alcoholic extract contained about 95 per
cent. of phosphatide; the amount of ether-soluble fat other than
_ phosphatide in the liver is probably intimately connected with the
digestive processes, and is a much more variable constituent of the liver
_ than the more complex phosphatides. This acetone-soluble ‘ fat’ would
likely be reduced in amount as the result of a period of starvation.
rae Tn short, it would seem that the essential fat of the liver, and probably of
—s gertain ~=other organs, is really phosphatide, and under certain
circumstances, if care be taken to avoid disintegration during the process
___ of extraction, it may be practically the only one found in any appreciable
amount in the combined part of the ‘ fat.’

“dl _ i = ee ee a a —— Se ree Re Pe
Re yy a ee ei ie whe te ahs
oo ie ee *

ee

462

THE OSMOTIC PRESSURE OF LIQUID FOODS
By JUDAH L. JONA, B.Sc. (Adel.) bags

From the Physiological Laboratory, Melbourne University

Communicated by Proressorn W. A. OsBoRNE
(Received November 7th, 1909)

One of the admitted functions of the stomach is the osmotic
equilibration that takes place between the blood and the fluid food
swallowed. Hypertonic solutions are diluted and hypotonic solutions
have salts, ete., added until isotonicity is attained, though it may be a
matter of debate whether the diluting (or concentrating) fluid is
physiologically secreted or is due to physical diffusion. That the lining
cells of the mucous membrane of stomach and gut would be injured by
prolonged contact with a hypertonic fluid may be stated a priori. Even
the mucous membrane of the mouth is open to injury in this way—-witness
the ‘ roughness’ produced when a piece of confectionery is retained for a
few minutes between the teeth and cheek. Also the discomfort which
follows the intake of such substances as strong salt solutions, very strong
soups, or peptone solutions which ‘irritate’ the stomach, is thus easily
explainable. 7 A

The object of the present research was to determine the osmotic
pressure of the fluids ordinarily admitted to the stomach, and at the same
time to discover whether the sense of taste afforded us any guidance in the
choice of fluids with reference to their osmotic pressure, more
particularly as regards the rejection of the hypertonic. Of the food-
stuffs ordinarily eaten, the vast majority are in solid or gelatinous or
colloidal form. To such substances the consideration of osmotic pressure
cannot apply. The actual fluid foods admitted to the stomach are milk,
the ordinary beverages, fruit juices, beef-tea, meat extracts and soups.

In tea, coffee, or cocoa there is usually a sugar addition which varies with

personal taste, whilst in beef-tea, soups, ete., common salt is invariably = R
an added ingredient. The osmotie pressure of milk has been determined |
so frequently that I have not thought it necessary to make any
confirmatory experiments. In the case of soups the ‘salting to taste’ els
was carried out by a laboratory attendant, who was not aware of the sit

THE OSMOTIC PRESSURE OF LIQUID FOODS 463

purpose of the research. ‘The Beckmann freezing point method was
employed throughout. A mixture of ice and salt water was used to
produce the requisite cold, but care was taken to prevent excessive
_ supercooling. In none of the recorded readings was the degree of super-
cooling more than about 15° C. — Crystallisation was started by
i inoculation with a fragment of frozen distiJled water. The stirring was
“carried out by a simple elock-work mechanism. Centigrade scale

employed throughout.

REsvULtTs.

__ Coffee.—(2 spoonfuls of sugar in ordinary breakfast cupful.) (1) 4 0-341°C.; (2) A 0-343°C.
" Pea Infusion.—(2 teaspoonfuls, about 12 c.c., of tea in 200 ¢.c. boiling water.) Allowed to infuse
5 minutes. (1) A 0-052°C.; (2) A 0-049°C.; (3) A 0-050°C.
TT pear 10. 0 Infusion, 50 c.c, water, 25 c.c. milk, 10 grms. of sugar.) Tasted ‘ just nice.’
| (1) A 0-457° C. ; (2) A 0-458°C,; (3) A 0-456° C.
nd infusion, made with 200 c.c. more water added to leaves from infusion and allowed
: to stand 35 minutes. (1) A 0-026°C.; (2) A 0-025°C.
Lemon Juice.—(As used in Melbourne Hospital.) Strained juice of lemon (one lemon) 33 c.c. in
250 c.c. distilled water. (1) A 0-126°C.; (2) A 0-125°C.; (3) A 0-122°C.
100 ¢.c. of diluted juice + 1 teaspoonful (5 grms.) cane sugar added. Tasted ‘ just gight.’
aa (1) A 0-487°C.; (2) A 0-485°C.
as “Beer.—{Carlton draught XXX Beer.) (1) A 2-407°C.; (2) A 2-409°C.
a : aif sne.—{Cheap Australian claret.) Cooled to —5°C., but ice would not separate out.
_ Wine.—Kept between 77°C. and 80°C. for 35 minutes, and then boiled briskly for about
: 7 minutes to get rid of alcohol, at end of which time boiling point rose to about
103°C. 75 o.c. of wine subjected to this treatment yielded 45 0.0.
(1) A 3-240°O.; (2) A 3-238°C.; (3) A 3-241°C.
Foodstuffs
‘Treacle. —Diluted with water to 1 in 7 and this solution gave A 1-730° C.
Peptonised Milk.—Benger’s peptonised milk as used at the Melbourne Children’s Hospital,
it are Carlton, Melbourne, Victoria.
Pat e's Milk ‘ A.’—(Milk 3, water 1, peptonised 20 minutes. Boiled. Sweetened with cane
sugar about 1 oz. to 1 pint milk.) One drop gave pink biuret reaction.
(1) 4 0-652° 0. ; (2) A 0-656° C.
Benger’s Milk ‘ B.—(Milk 2, water 1, peptonised 20 minutes. Sweetened.) (1) 4 0-630° C.
(2) A 0-628° C.; (3) 4 0-626°C.
Peplonis ed Milk.—(Milk 4, water 1, peptonised 20 minutes.) (1) A 0-548°C.; (2) A 0-546 C.
_-—- Sowps.—An ordinary soup which had been served up but rejected as unpalatable on account
a of salt taste. A 1-984° C.
A Vegetable Soup was made of the following ingredients ;—Carrot, 100 grms. ; parsnip, 110
‘ grms.; turnip, 55 grms.; spring onion, 47 grms.; celery, 25 grms.; parsley,
: 12 grms.; water (distilled), 1500 c.c, Brought to boil and kept simmering
for 2} hours. Strained. Salted to different degrees.
Vegetable Soup Plain.—(1) & 0-374°C.; (2) 4 0-873" ©. ; (3) A 0-372" C.

464 BIO-CHEMICAL JOURNAL
Vegetable Soup Salted.—

Soup + salt to 4 per cent. A 2-°757°C.

Soup + salt to 2 per cent. A 1-536°C. ee
Soup + salt to $ per cent. (1) A 0-851°C.; (2) A 0-856°C. dat

Pe opp

Soup + salt to } per cent. (1) A 0-586°C.; (2) A 0-584°C.; (3) A 0-582°C.

Soup + salt to } per cent. A 0-780°C. ot ae i

The verdict of the taster was :—The unsalted vegetable soup possessed a 7A

very flat and unsatisfactory taste. A, B, C, and D distinctly too salty. A and B
distinctly unpleasant taste. E was about right.
Beef Tea.—Made with about 6 c.c. meat extract (Fitzroy Brand, Queensland manufacture) in
1000 c.c. distilled water.
Beef Tea Plain.—{1) A 0-141° C.; (2) A 0-139° C.; (3) A 0-140°C.
Beef Tea Salted.—
Beef tea + salt to 2-5 per cent. (1) A 1-626°C.; (2) A 1-626°C.; (3) A 1-625°C.
Beef tea + salt to 1-25 per cent. (1) A 0-882°C.; (2) A 0-887°C.; (3) A 0-886°C.
Beef tea + salt to 0-625 per cent. (1) A 0-546°C.; (2) A 0-544°C.; (3) A 0-643°C.
Beef tea + salt to 0-416 per cent. (1) A 0-419°€.; (2) A 0-416°C.
Sample C was much the tastiest—just salted to taste. A and B having
too much salt, and sample D and the original not enough salt.
Bee} Tea made with about 6 c.c. meat extract in 1000 c.c. boiling distilled water. A 0-160° C.
Beef Tca Salted.—Salt added till the flat and unsatisfying taste of the beef tea was abolished,
but still no salty taste perceptible.
Taster A. (1) A 0-330°C.; (2) A 0-331°C.
Taster B. (1) A 0-329°C.; (2) A 0-330°C.
Beej Tea Over-salted.—A 1-922° C.

Sap Pr

Sugar Solutions

Dextrose Solutions. —100 c.c. taken into mouth in sips of 25 c.c., each sip kept in mouth 4 minute,
spat out ; in } minute another sip taken, kept in } minute, spat out; and so on
for four sips. .
10 per cent. Dextrose Solution. A 1-156°C.
10 per cent. Dextrose Solution Salivated. (1) A 1-068°C.; (2) Al 066° C.
5 per cent. Dextrose Solution. A 0-566°C.
5 per cent. Dextrose Solution Salivated. (1) A 0-536° CG. ; (2) A0-532°C.; (3) 40-534°C,
Cane Sugar Solution, 20 grms. in 150 c.c. (13-3 per cent). 100 c.c. treated in similar manner as
dextrose solution above, 7 c.c. Saliva were added by this process to the 100 0.0.
sugar solution, and there was an after secretion for several minutes.
Cane Sugar Solution (13-3 per cent.) A 0-868°C.
Cane Sugar Solution Salivated. (1) A 0-788°C.; (2) A 0°792°C. ;

Fruit Jwices

Lemon.—Weight 140 grms. Peel and connective structure 90 grms. Yielded 40 o.c. strained
juice. (1) A 0-937°C.; (2) A 0-940°C.; (3) A 0-939°C.

Orange.—Orange 135 grms. Yielded 50 0.0. strained juice. (1) A 1-100°C.; (2) A 1-101°C. ;
(3) A 1-100°C.

Pineapple Jwice.—From fresh Queensland pineapple. (1) A 1-462°C.; (2) A 1-464° ©. ;
(3) 4 1-460° C.

Cocoanut * Milk.’—{ About 150 0.0. were yielded by the nut.) (1) A 0-521°C.; (2) 4 0-518°C, ;
(3) A 0-518° C. ar

THE OSMOTIC PRESSURE OF LIQUID FOODS 465

Saline Aperients
Magnesium Sulphate Solution.—(15 grms. in 100 ¢.0.) A 1-136°C.
Balanced Saline Aperient,—(The stronger one) as recommended by Professor W. A. Osborne
in a paper in the Intercolonial Medical Journal of Australasia, July 20th, 1909.
(1) 4 0-864° C. ; (2) A 0-862°C.; (3) 4 0-861°C:
Saliva produced from Sucking Confectionery.—Barley-sugar stick (about 12 grms.) sucked for
15 minutes led to prodution of 72 c.c. Saliva. (1) 4 1-008°C.; (2) A L-006°C.

Saliva from about 30 grms. boiled cane sugar. Sweetmeat — 100 c.c. (1) A 1-488°C.;
(2) A 1-484° 0.

GENERAL CoNCLUSIONS

It will be seen from the above experiments that of all the fluid foods
which are admitted to the stomach, alcoholic beverages and fruit juices
alone are hypertonic. Further, it may be safely stated that in no case is
___ a fluid admitted in which hypertonicity is due to the mineral ingredients
alone. When, therefore, we find the kidney elaborating a fluid (urine)
with sufficient piline ingredients to render it hypertonic, we must regard
the high concentration of this fluid as so much external work done and of
sufficient moment to be taken into consideration in calorimetric

experiments on an animal or on the human subject. These experiments

also demonstrate that we must ascribe to the sense of taste a distinct

osmotactic character. Not only is this sense potent in testing the food

qualitatively, but also from the quantitative standpoint of molecular

concentration. Even those hypertonic fruit juices which are admitted to
Z __ the stomach are passed, so to say, under protest, for their taste is

- recognised as astringent or highly acid, and are apt to be followed by a
sense of thirst.

The mechanism is faulty, however, when dealing with alcoholic
beverages, a fact which we may ascribe to the artificiality of fermented
liquors, and their manufacture and consumption being restricted to man
only. The great majority of fluid foods are, however, hypotonic, and
thus a margin is left for the addition of hydrochloric acid and other
constituents of the gastric juice. With regard to alcoholic beverages, it
may be stated that a solution of alcohol in pure water, isotonic with the
blood, would only be about 15 per cent. As this percentage is almost
invariably exceeded in fermented (and, of course, distilled) liquors, and as
other substances are present in addition, the high osmotic pressure of the
beer and wine tested is not surprising.

The association of the raising of osmotic pressure of beverages with

466 - BIO-CHEMICAL JOUR

the induction of thirst is made use of in some depar
the excessive salting of wines and the over-sugaring 0:
In the case of cane sugar, a solution isosmotie with the t
about 11 per cent., whereas the fluid which reaches the s
of even the slow methods of ingestion of sweetmeats, |
process of sucking confectionery, is much higher tha
for the disagreeable after-results bet often ex
indulgence in such delicacies.

lke’ in this work.

4 THE RELATIONSHIP OF DIASTATIC EFFICIENCY TO
AVERAGE GLYCOGEN CONTENT IN THE DIFFERENT
TISSUES AND ORGANS

By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of
Aberdeen.

From the Bio-chemical Laboratory, University of Liverpool
(Received November 17th, 1909)

+ The first demonstration of an enzyme capable of hydrolysing glycogen
is associated with the names of von Wittich (1) and of Claude Bernard (2).
5 The brilliant researches of the latter observer, which ultimately led up
to the discovery of glycogen in 1855 (3), formed the basis on which the
probability of the presence of such an enzyme rested, but the actual
proof of its existence was first furnished by von Wittich; a short time
. ipietwards independent evidence was advanced by Bernard, both observers
____ having found the substance in the liver.
atti _ These discoveries, however, were not allowed to pass unchallenged,
J and to Pavy (4) belongs the credit of having settled beyond dispute the
fact that the liver really contains a substance of the nature of an enzyme
which is capable of converting glycogen and starch into sugar, and acts
quite independently of the vitality of the tissue cells from which it is
i derived.
2 ‘yy - __-‘The presence of such an enzyme both in the animal and vegetable
a organism is now universally admitted, but its exact function is still in
certain quarters a matter of controversy; the fact that glycogen occurs
in the body tissues, and that the diastatic enzyme! possesses the power
be é transforming this substance into dextrose and intermediate products,
suggests that the normal function is directly associated with the conversion
of glycogen into the less complex substance—sugar.

Pavy (5), however, is unable to accept this view, and suggests that
the enzyme is really a product of the dead or dying cell which is generated
from an inactive pre-existent zymogen. On the other hand, analogy with
other intracellular enzymes points to the probability that this diastatic
enzyme actually exerts its influence during life in the same manner as

1 * diastatic enzyme ’ is used in this paper to indicate the substance or substances

vilok ah co i transfrmed into red maltose, dextrose, eto. ; it is
hy which lycogen si ¥ SSISnOnT Gauiidaedaeti thes farmaiton il detvoee dossnceaeaile

or glycogen.

468 BIO-CHEMICAL JOURNAL

its activity is evidenced in vitro—as a hydrolyser of glycogen—the
difference being solely one of degree, and its activity being called forth

in response to some condition not yet understood. That the conversion

-f

of glycogen is not regulated by the condition of the animal is suggested —
by the experiments of Kisch (6) carried out on adult muscle; his results _

seem to indicate that regulation of sugar formation from glycogen is
apparently not brought about by the needs of the tissues for sugar.

If, then, the function of this ferment is to act on the glycogen
present in the organs and change it into sugar in response to some
unknown body stimulus, it might be expected that the amount of glycogen
normally present in any particular organ would bear some general
relationship to the richness of that organ in diastase. It has been known
for many years that the chief seat of glycogen in the animal body is the
liver; the muscles may also show a very considerable amount, while the
other organs contain but traces. Consequently the literature of the
subject abounds with data concerning the diastatic activity of the liver
and muscles, but comparatively few observations dealing with the other
organs are available; it has generally been recognised that many or all
the tissues of the body contain traces of a glycogen hydrolysing ferment,
but until quite recently no attempt has been made to investigate the
relationship of average glycogen content to amylolytic efficiency.

A short time ago, however, an interesting series of observations have
been undertaken by Mendel and Saiki (7); for their experiments muscle
and liver of pig embryos of various sizes was utilised. In the case
of the liver it was found that the diastatie activity increased markedly
with the growth of the embryos, while in the case of muscle, which
showed in general a well-marked initial activity, the increase was not
nearly so pronounced. Hence it would seem that the diastatic activity
of embryonic liver and muscle varies directly with the normal glycogen
content, for it has been shown by Lockhead and Cramer (8) that the
liver of foetal rabbits is at first very poor in glycogen, but that after
twenty-five days a considerable amount is present; again the estimations
of Mendel and Leavenworth (9) indicate that in the very young pig
embryos no glycogen at all may be found. Thus, the foetal liver is at
first very poor in glycogen, and apparently also in glycogen hydrolysing
substances, while on the other hand the embryonic muscles may contain
a fair amount of glycogen even at very early age, but tend to show only
slightly increased amounts as age advances; in correlation with this
slight rise in glycogen content the increase in diastatic power is
correspondingly slight.

. —
-*

DIASTATIC EFFICIENCY 469

e So far, therefore, as the subject has been investigated there is, in
_ the case of embryonic tissues, undoubted evidence pointing to a
_ relationship between diastatic efficiency and glycogen content, but whether
varying diastatic activity is directly dependent on the relative glycogen
content, or whether the increase of diastatic power constitutes a factor
which directly influences the amount of glycogen, is still obscure. An
answer to this question might help us in attributing its proper function
to the diastatic enzyme, for it is not impossible that its chief sphere of
action during life is synthetical rather than destructive. If the view
entertained by several authorities with regard to glycogen is correct—
that under normal circumstances it does not pass on to any marked extent
to form dextrose, but is utilised in other ways—then it might perhaps be
expected that the measurement of post-mortem amylolytic activity
___ im an organ would afford some clue to its glycogenetie power during life.
It will be shown, however, that the amount of diastatic enzyme actually
_ found in different adult tissues bears no apparent relationship to the
glycogen content of the same tissues; it is thus obvious that amylolytic
efficiency and glycogen storage do not necessarily go together. It might
be argued that the comparative absence of glycogen in an organ did not
of itself imply the inability of that organ to form glycogen, for the latter
substance might be formed and carried away in the blood; or again it
might be immediately utilised at the seat of formation by the tissue
cells. That the blood does not carry the glycogen as such from an organ
is suggested by the very low glycogen content of this fluid, and the
- conception of a simultaneous process of formation and destruction of
glycogen by which this substance might be kept at a minimum, can
hardly be entertained if we accept the theory that it is as dextrose that
_ earbohydrate is utilised by the tissues; such a procedure would be quite
out of harmony with the ordinary methods of nature, for in that case
formation of glycogen would be but a useless step; only on the theory
that glycogen is utilised as such by the tissue cells could the latter
possibility be entertained.
ee This apparent lack of correlation between glycogen content and
glycogen hydrolysing capacity makes it much more difficult to understand
what the amylolytic activity as evidenced post-mortem really means in
connection with the vital processes. It may well be that these enzymes
exert their destructive action chiefly as the result of some interference
which threatens the life of the organ.
It is well known that the kidney, for instance, contains normally

470 BIO-CHEMICAL “JOURNAL

but a very small amount of glycogen, and yet it is often found re bem sh

richer in diastatie enzyme, per unit weight, than any organ in he

body. On the other hand, muscle which generally contains a_ a
amount of glycogen is often very weak in diastatie effect. The wil 3
figures, taken from an experiment recorded by Piek (10), are ——
with regard to this point; they indicate the results of some conten
estimations of amylolytic activity of different adult tissues. + 19a

It was found that a a head ee *

100 grms. kidney digested 2°37 grms. glycogen in 3 hours. ROA
100 grms. liver digested 0°69 grms. glycogen in 3 hours. — iat has es
ia ee
100 grms. blood digested 0°31 grms. glycogen in 3 hours. Pee re
Here it is seen that kidney possessed between three and four times the
diastatic power of liver and about eight times that of blood. Experiments .
made by the writer gave in general very similar results, and showed that —
almost invariably the kidney is the organ which possesses, weight for
weight, the greatest amylolytic efficiency. Often other organs were also
found to have quite a marked effect. It-is interesting to note that in __
the case of another endoenzyme—erepsin—it has been shown pie 2 ia
that the kidney possesses far and away a greater ereptic action than any
other tissue, with the exception of small intestine. The richness of the
kidney in these and perhaps other enzymes is not easily explained in
the light of our present knowledge of vital processes. war s(t neers
In order to demonstrate the comparative efficiency of the different sate
organs the following procedure was adopted. - Shue:

‘et a Ady i"
Estimation oF AmyLonytic AcTIVITY oa ay}

The majority of observers conducting research on amylolytic wet
have chosen as an index of activity the amount of reducing substances
formed in a given time. Hither the dried tissue or the expressed His ue
juice was incubated with a starch or glycogen solution of known stret sth, 3 2
and subsequently the extent to which reducing substances had 1 ‘beeitt
formed estimated by Fehling’s or some similar alkaline copper solution. 7%

This method supplies information as to the activity of the enzyme
in terms of its power of forming products which reduce copper solutions,
but affords no information as to the relative amounts of these substances”
actually present—dextrose, maltose, etc. For general comparative work, —
however, such detailed information is not necessary, and this method is: ae
quite suitable. i? =

i oS ee eee se

DIASTATIC EFFICIENCY 471

In other cases the amount of glycogen present before and after
=a incubation was—calculated, and the difference taken as an index of
amylolytic action. This again merely indicates the power of the enzyme
to transform glycogen into substances soluble in alcohol, but gives no
-__ indieation as to the extent to which this change has been carried on

towards the formation of the final products. As exact estimation of
glycogen, especially where many experiments have to be undertaken, is a
tedious process, a good indication can be obtained by substituting starch
for glycogen and testing with iodine in order to ascertain when the starch
ceases to give its characteristic iodine reaction. This method has been
used and recommended by Wohlgemuth (11), but in my experiments on
dried tissues it was found occasionally to give somewhat indefinite results ;
_ only a few experiments, however, were carried out, and so far as they
: a went the results were in general similar to those obtained by the reduction
ace method. On the whole the most satisfactory method was found to be the

estimation of the reducing power, and dried tissue was mostly employed ;
in a few experiments moist fresh tissue was also used.

Merruop

The method utilised is in general the same as that made use of by
many observers. The various organs were obtained as soon as
possible after death, freed from adherent fat, and quickly cut up into
small pieces; these were washed in normal saline solution in order to free
them from blood, dried by being pressed gently in a cloth, and finally
passed through a mincing machine. The finely divided substance was

| _ placed in « large volume of alcohol and left there for 48 hours. The
: aleohol was then filtered off and the tissue dried in vacuo over H,SO,,
and ground to a fine powder by a coffee mill. To 50 c.c. of a 1-2%
solution of soluble starch or glycogen, 1 gm. of the dried powder was
added, and the mixture placed in the thermostat at 37° and left there for
18 to 20 hours. Small flasks of about 100 c.c. capacity were found to be
most convenient, and the utmost precautions were adopted in order to
ensure the sterility of all vessels used; toluol, alone or with sodium
fluoride, was always added to the fluid. The flasks were thoroughly
shaken up from time to time, and next day the contents were boiled in
order to coagulate the albumin; after filtration a measured volume
(10-30 ¢.c.) was taken and gradually added to excess of boiling Fehling’s

solution. After boiling for about 5 minutes, the cuprous oxide was

472 BIO-CHEMICAL JOURNAL

collected on a weighed Gooch crucible containing asbestos; after thorough
washing of the precipitate with distilled water, the crucible was
heated in order to oxidise the lower oxide, and the precipitate | hen
weighed as cupric oxide. Parallel experiments with boiled controls: were

also made. In all cases the ordinary Fehling’s solution was employed, x,

diluted with an equal volume of water, and in every comparative
experiment the same amount of Fehling’s solution was used; this
precaution was necessary in order to obtain accurate results, and likely
depends on the power of caustic alkali to dissolve small amounts of
cuprous oxide.

At first some difficulty was occasionally experienced in filtering a too

finely divided cuprous oxide precipitate; it was found, however, that this —

could be overcome in all cases by boiling both the sugar-containing fluid
and the alkali before bringing them together, or by adding the sugar
solution very slowly to the boiling Fehling’s fluid; by adopting this small
precaution a well defined red precipitate was easily obtained in every
case, and no difficulty arose in filtering.

Some preliminary experiments showed that this method, when
carried out carefully as above described, gave practically identical
results in parallel experiments with the same tissue. Wohlgemuth,
however, raises the objection to results based on reduction methods, that
they do not necessarily indicate the diastatic activity, since there may be
present glycolytic enzymes which destroy part of the sugar as it is formed.
The evidence for the presence to any appreciable extent of active
glycolytic enzymes in the tissues is by no means very convincing, as may
be seen by consulting the literature of the Conheim controversy relating
to the alleged glycolytic action of muscle juice combined with pancreatic
extract. In my experiments in which it is sought to establish the
different diastatic powers of the various tissues, it -is obvious that

glycolytic effect is of most importance from a comparative point of view —

in cases where low results were obtained. Some experiments made on
this point showed that mixtures of sugar and tissue, treated exactly as in

the diastatic experiments, did not tend to decrease in strength to any :

appreciable amount; the results obtained indicate that this factor is of
little importance in determining the results of diastatic efficiency
caleulated in terms of reducing substance. In one experiment the
following figures were obtained :— ; ‘i

10 e.c. sugar sol. = 01126 grm. CuO.

DIASTATIC|ANNIchincy 178

50 c.c. of Bare sugar séletion digested with 1 grm. of following
gans for 20 hours; other 50 ¢.c. + tissue boiled and used as control :—-

(10 e.c. used) (10 c.c. used) solution ad doe
Liver 0-1130 grm. ek 0-1109 grm. ~ + 0-0004 grm.
= Bent. Olli 5: ellie eNO 5 id ~ 00005...
‘% ; z Lung 00-1046 - ae 0-1090 - aes we 0-0080 -

Muscle 06-1102 ,, | isa Os ual ~ 00024 .,

a at any rate, any glycolytic. action that may exist is much too
all and indefinite to be of any appreciable account in determining
r static activity, especially from a general comparative point of view.

|Rerative Drastatic Errvictency or Dirrerenr Orcans Nx
Terms or CuO

given below. In almost all cases where sufficient material was available,
two sets of parallel experiments were carried out; by this means the
is possibility of accidental results due to contamination by micro-organisms
was excluded. In a it cases in which the was too small to vield

: Tae Amount of digest used = 20 c.c. Result in grms. CuO

Kidney = 0-3708
Langs = 0-3008
Liver = 0-2720
Heart a 02566
Stomach = 2210
Bladder na 01984
Masele = 1448

Here it is seen that kidney shows the greatest diastatic action, while
slung comes second and liver third; muscle is apparently weaker than any

of the tissues examined. Almost identical comparative results were

found in another cat examined, except that the lung was less active.

474 BIO-CHEMICAL JOURNAL

Experiment II.—Rabbit

1 germ. dried tissue + 50 c.c, ie cent, starch solution + toluol. ted 19 hours, —
20 c.c. digest u Result in grms.
Kidney = 02694
Liver me 0-2028
Stomach = 0-1572
L = 01546
Bladder = 0-1348
Muscle ad 0-0621

In the case of another rabbit, experiments were made with the fresh
tissue in order to test the relative effects immediately after death. The
animal was killed by bleeding, and the organs removed as quickly as
possible and cut up into small pieces. They were then thoroughly minced
up and 1 grm. of each quickly weighed and then digested with the above
starch solution + toluol. Only kidney, liver and muscle were used for
this experiment.

- ) Amount of Result in
Kidney 5 minutes after deeth; ts: ee | Wen: are

Liver 10 7 “ 7 0-0516
Muscle 12 = o 0-0294

In order to ascertain whether there was any difference if the organ
was not used quite fresh, it was tested again three hours after death,
exactly as mentioned above.

” ?

Result—Kidney = 0-1016 grms, CuO
Liver = 0-0584 ”
Muscle = 0-0318 9

In another rabbit the following figures were obtained; same starch
solution as above + 1 grm. tissue were used, and digestion went on for
20 hours 30 minutes :—-

20 c.c. digest used.

Kidney 10 minutes after death 0-1872 CuO

4 hours ” => 0-2000 ”
Liver 5 minutes after death = 0-0640 _,,
4 hours ” = 0-0599 ”
Lung 14 minutes after death = 00576 _,,
4 hours hm = 00521 .,,

From these results it appears that tissue does not lose any of its:
activity on standing, at least for a few hours; other experiments show that
no change is apparent after standing for a very much longer time.

Here, again, it is seen that kidney possesses the most marked amylo-
lytic efficiency, while liver has a very much less marked effect and musele
comparatively little. In one rabbit, however, it will be noticed that liver
tissue was twice as active as in the next case, while muscle was about equal
in both. In the last case, lung is as effective as liver, though lung at
most contains only traces of glycogen under normal circumstances. In

another case it was found that muscle was somewhat richer in diastatic
ferment than liver.

eee Reg: ih a ae i os

DIASTATIC EFFICIENCY ees...

Here, when tzeated a as other dried tissues for 18 hours, it was

Kidney = 0-2264 grms. CuO
Liver = 01136 pm
Muscle = 0-1200 ”
Lung = 0-0716

These results indicate sufficiently well the marked fluctuations that
2 “may exist in the corresponding tissues of the same species of animal.

Experiment ITT
_ Ptc
In this case only one experiment was made, and the lung gave higher
results than any of the other tissues examined. Liver was also somewhat
more active than kidney, while skeletal muscle was lowest of all. In this

In many experiments carried out on animals of other species, it was
often found that lung had a relatively high value.

‘Eaperiment IV
Doe
; The figures obtained from two dogs examined showed that kidney
_ was again most effective, but the difference was not so marked as in some
other animals, It is likely that an increase in the number of experiments
would have shown greater variations than are here indicated.
1 grm. dried tissue + 50 ¢.c. 2 per cent. starch solution + toluol. Digested 20 hours.

10 ¢.c. digest used. Result in grms. CuO

= 0-1612

es $1

roe = 0-1051

Bladder - O1214

— = 1333

uscle - 01051
Small intestine > 01850!

1. Be Gee Meare etter of tathatey yuanooaaie face 4 is
ory comaga Pe ee eee ee, juice, this tissue has

ees ee oe

476 BIO-CHEMICAL JOURNAL

Experiment v

Sueep ‘vt a

were phone: both in the solpele of corresponding organs of Z arn
animals and in the relative efficiency of the different tissues d

the same animal. Sometimes kidney was found to be very aia
active than liver, or any other organ; in other cases liver and k
displayed practically an equal effect. In a few cases the heart
exerted but very slight diastatie action. Some of the ° \
obtained are indicated by the following figures from two
animals :—-

1 grm. dried tissue + 50c.c. 14 per cent. starch solution + toluol + NaF. Digested
20 ¢.c. digest used. Result in grms. CuO
(1) ae:

Liver = 0-1876
Kidney = 0-1796
Muscle = O44
Heart = O- 1384

Lungs i 01204

Kidney ¥ ieetikes 0-2576

Liver = ~ 01846
Lungs = 0-1642
Stomach = 0-1401

Muscle = ~ 00920
f. 0-074) i

Se

A few investigations were carried out with sheep's antiaar in orde
ascertain whether there was any change in diastatic activity after t
fresh moist tissue had stood for a considerable time at room a e
in a cold chamber.

2 grms. liver were taken at different times atter death ona dig
with starch solution in the usual way, under exactly similar co:
The results indicate that liver does not lone. its full diastatic sti
very considerable time: :

Result in CuO given by

10 ¢.c. digest
} hour after death = 0-0852
24 hours ie ' = 0-0754

¢ 86 ” ” = 0-0835

og

DIASTATIC. EPFICIENCY 477

E In another case liver was left for several days in aleohol, part being
takenout-at different times and dried in the usual way. No difference
could be detected between liver treated for 24 hours with alcohol and the
same liver treated for four days; the figures given are merely comparative,

_the same amount of dried tissue being used in each case :—

Result in grms, CuO

=: 0-0876

48.» ” = 0-0851

, Sa = 0-0836
Samples of powdered teens kept in a dry state did not seem to have
changed in the slightest degree after several months. No indication of
any difference in the action of the liver when used as soon as possible after
death, and a very considerable time afterwards, was observed, though it is
possible that this may not obtain for the glycogen actually deposited in

__ the liver itself when acted upon by the cells immediately after the death of

cs the animal.

‘That diastatic enzymes in general are exceedingly stable bodies,
_ especially when protected from moisture, is proved by many researches.
Thus Sehrt found active diastatie ferments in the tissue of mummies
several thousand years old, and lately it has been shown by White (12)
that the seeds of such cereals as wheat, maize and barley contain diastatic
enzymes, which, if stored dry, retain their activity for twenty years or
more; that these substances are necessarily comparatively stable bodies is
obvious from the observation that barley diastase remains unaffected in
efficiency after being subjected to temperatures varying from — 200° C. to

+ 138° C.

These facts indicate that observations on human tissues which can
generally be obtained not sooner than 18 to 24 hours after death, probably
give quite a fair picture of what would be produced by fresh tissues. In
the cases of normal organs examined, the results were generally of the
same order as those obtained for other animal tissues. Kidney was
generally much more active than any other part, liver being second and
lung third. Muscle was again about the bottom of the list; great
variations were, however, in evidence, and only a few cases were
investigated.

Tissues were also obtained from two patients who had died from
diabetes; it has been shown by Bainbridge and Beddard and by others
that diastatic activity is present in diabetes, but the relative power of the
‘different organs has not been investigated. In these two cases, however,
no appreciable variation from the normal was observed; in No. 1 the
marked effect of the kidney over all the other organs is well brought out,

24 hours in alcohol

2 a= Sa rere, oa ee rt i a a a er me

478 BIO-CHEMICAL JOURNAL

Experiment VI
Drapetic Tissues

1 erm. tissue + 50 e.c. 1 per cent. starch + toluol + NaF. Digested 20 hours.

30 c.c. digest used. No. 1
Result in grms, CuO
Liver = 0-1191
Kidney = 0-2642
Muscle = 0-0959
Heart = 0-0820
Lungs = 02214
No. 2 ’
Kidney = 0-1498
Liver | 0-1089
Lungs a= 0-1186
Muscle oa 0-0707

In these two eases, at any rate, it would seem as if the diabetic
condition had at least no tendency to cause diminished diastatic activity ;
obviously many more cases would be required before any definite statement
could be made.

GENERAL SUMMARY

Just as the present investigation was almost finished, several papers —
by Wohlgemuth (15) on tissue diastases appeared. One contribution
dealing with the relative diastatie power of certain organs of the rabbit—
liver, kidney, muscle—states that kidney was most powerful, followed by
muscle and liver in the order mentioned. The figures given show fairly
marked variations, and in some cases liver and muscle appeared to be about
equal; sometimes muscle was more powerful than liver.

From these investigations, and those given in this paper, it is quite
obvious that there are considerable variations in the relative diastatic
activity displayed by different animals; at the same time there is no very
definite relationship between the results obtained for the different tissues
of two animals of the same species. The experiments given above are
intended to indicate chiefly the comparative results obtained with animals
of the same species, for sometimes different samples of soluble starch were
made use of. At first it was intended to make comparative observations
on many different species, but the extent to which variations manifested
themselves in animals of the same species indicated that such an

investigation would lead to no definite results unless a very great number
of animals were used,

DIASTATIC. EFFICIENCY 479

= The results obtained, however, give sufficient data for an answer to
ep question of the existence of correlation of diastatie efficiency to
recogen content. —

; Tust as the liver normally contains the most glycogen, and the
scles a considerable quantity, while such organs as the kidney, bladder
id lungs contain but traces, it might be expected that these same organs
a 1 show diastatic power in the order of their glycogen storing power.
s, however, i is not the case, and sometimes an organ containing at most
mut vis mere trace of glycogen-—e.g. lung—shows more marked amylolytic
ower than liver; again, muscle may contain less ferment than any other

Tt is obvious from these results that there exists no definite correlation
ven glycogen content and diastatie efficiency in the case of adult tissues.

REFERENCES

Pfliiger’s Archiv., VU, p. 28, 1873.

Comples Rendus, LXXXV, p. 519, 1877.

Bernard, Legons (Cours d’Hiver, 1854-55).

Journ. of Physiology, XX, p. 391.

The Physiology of the Carbohydrates, 1895.

Beitrage z. chem. Physiol., VIII, p. 210, 1906.

. American Journ, of Physiology, XXI, p. 64, 1908.

8. Journ. of Physiol., XXV, p. 11, 1906; and Pror. Roy. Soc., B. LXXX, p. 263, 1908.

9. Amer. Journ. of Physiol., XX, p. 117, 1907.

10. Hofmeister’s Beitr., U1, p. 174 (1903).

1. See Noel Paton, Journ. of Physiol., XXII, p. 423, 1898,
- Proc. Roy. Soc., B. LXXXI, p. 417 (October, 1909).

Biochem. Zeits., Ba, XX1, 8. 380, 484 (October, 1909).

480

THE OSMOTIC PRESSURE OF THE EGG OF THE COMMON

FOWL AND ITS CHANGES DURING INCUBATION

veri
-

By W. R. G. ATKINS, M.A. (Trinity College, Dublin). -—
(Received November 17th, 1909) 2

Having shown in a previous paper’ that the blood and eggs of birds
ure not isotonic, it seemed of interest to study the changes, if any, taking
place during incubation, thus tracing the pressure variations from germ
cell to chick. This difference in osmotic pressure between the blood and —
the egg was quite unexpected; its magnitude may be seen from the
following table, in which A denotes the depression of freezing point of
the fluid below that of pure water and P stands for the osmotie pressure,
which was caleulated from the formula P = 12:06 A — 0°021 A®

Number of

Bird experiments A P in atmospheres
Common fowl (Gallus bankiva) 15 0-607° ©. 7-31 Blood
12 0-454" C. 547 Egg
Duck (Anas) 8 0-574° C. 6-92 Blood
9 0-452° C. 5-45 Egg
Goose (Anser) 4 0:552° C. 6-65 Blood
1 0-420° ©, 5-06 Egg

The above difference in osmotie pressure is accounted for by the
diminution in the inorganic salts of the egg as compared with the blood
serum. This was ascertained by estimating the chlorides in egg-white %
and plasma by Volhard’s method, after incineration, but as only .
0-03 to 0°02 grm. of chlorine was found in the ash owing to the small
quantity of material available, the following table is not of great

aceuracy :
Duck

Plasma, per cent. chlorine Egg white, per cent. chlorine
0-278 oom 0-080
0-276 ms 0-088
0-312 ay 0-088
vi 0-104
Mean 0°287 °,, Mean 0-090 °,,
As NaCl 0-473 °,, As NaCl 0-148 %

To examine the changes during incubation, eggs were placed in a
Hearson egg-incubator and maintained at 40° to 41°C. Samples were __ 4
taken out at intervals, and their freezing point determined with a r
Beckmann thermometer, care being taken to avoid freezing out of the
' Proe. Royal Dublin Soe,, Vol, X11 (N.S.), May 1909,

THE OSMOTIC PRESSURE OF THE EGG 481

nt, water, thus leaving a too concentrated solution. The zero of
@ ruometer was re-determined frequently, both in the apparatus
sds suai in powdered ice and water. Vigorous and continued stirring is
necessary in dealing with viscous liquids such as eggs. It is to be
regretted that, possibly owing to the lateness of the season—August and
ptember-—most of the embryos had died before the shell was broken.
is is not a very serious drawback, however, as owing to the variations
the freezing points of fresh eggs of Gallus, from 0-427° C. to 0-480° ©.
in a dozen determinations, quantitative results proportional to the time
of incubation are not to be expected. The experiments are tabulated

maize present Noembryo Musty eggs
0-480° C. - i
0-458°C. -- -—~
0-605° C. 0-532° C. 0-620° C.
0-565" C. — 0-656° C. embryo present
0-605° C. 0-538° C =
0-565° C. pas as
0-563" C _ —
590°C. 535° C. _
— 0-550" C. _
0570 C. 05388" C =
0-590" C, — =
OGIEC. -_- 0-633°C. embryo present
0-598°C, * 533°C. co

ras — 0-658°C. embryo present
= ; -- 0-680°C. embryo present

Control egg. in room at 15° to 20° C. for twenty days A = 0-480° (,

In these determinations the whole of the liquid contents of the egg

tube containing the thermometer. It may be seen from the table that
_ there is a rise in the numerical value of the depression of freezing point
throughout the period of incubation, the final value, 0°590° C. to O-611° C.,
being about that given by the blood, 0°591° to 0-662° C., mean 0-607° C.
On the other hand, eggs containing no embryos show a similar rise, but
to a much less extent; apparently, as the figures range from 0:532° to
- 0550° C., there is an initial rise due possibly to evaporation, although the
egg chamber is nearly saturated with water vapour. Thus the rise in
osmotic pressure during incubation may be divided into two parts :—

(a) The rise, as shown by unfertile eggs, probably due to evaporation,

(b) The rise due to metabolism of the embryo.

Oa,

482 BIO-CHEMICAL JOURNAL

In connection with the latter it is to be noted that both yolk and
white become much less viscous during incubation if there is a developing

embryo, probably owing to the presence of an enzyme Maier cd

reserve materials. “3

Eggs with or without embryos, but which were musty, gave high -
values in every case, 0°680° C. being the maximum obtained, though itis

quite reasonable to expect that if bacterial action had been more
extensive a much higher figure might have been reached. More attention
was paid to this effect of bacteria in the second series of experiments than

in the first, so this, combined with the impossibility of detecting

putrefaction in its initial stages, may account for some of the high values
of A obtained for eggs with embryos in the early stages of incubation, —

The difference in osmotic pressure between the blood and eggs of
birds, together with the gradual rise in pressure of the egg to approximate
isotonicity with the blood, may, perhaps, be accounted for by the

following speculation. There is much evidence that the ontogeny of an—

organism is a more or less abbreviated repetition of its phylogeny; by
extending this view, based on morphological grounds, to physiology,
there is reason to believe that birds are descended from ancestors with a
lower osmotic pressure, about five or five and a half atmospheres. _ Fossil
remains point to the reptilia as the class from which birds developed. It
remains to examine the osmotic pressures of the blood of this group. The
following determinations are available :—

A P
Thalassochelys Caretta, 1. = — 0615 ... Bottazzi and Dueceschit
= — 0-602 -- Rodier ?
Emys europoea = — 0-463 to-- 0-485 ... Bott. and Duce.

(freshwater species of above)

Thus the osmotic pressure of the blood of the only freshwater
reptile which I have been able to see recorded, is not very different
from that of the eggs of birds, being, in fact, within the limits of
variation, It may seem fanciful to regard the osmotic pressure as a
hereditary character transmitted with great regularity, but sueh a
possibility seems well worth serious attention in view of the high
elaboration of the organs regulating this pressure in all the vertebrates,
a regulation not quantitative only, but qualitative. In this connection
Loeb’s researches (see ‘ Dynamies of Living Matter’) show how marked is
the effect of a qualitative difference in the salts present upon developing

1 Quoted from Rodier.
* Station Zoologique d’Arcachon, 1899,

a
5
4
;

THE OSMOTIC PRESSURE OF THE EGG 488

embryos. The whole question of the constancy of osmotic pressure is
diseussed-at tength by E. H. Starling (see ‘ Fluids of the Body’), where
_ Macallum’s interesting views on the origin of the blood plasma ‘salts from
the waters of a pre-Cambrian ocean are also considered. So apart
2 altogether from the, possibly misleading, agreement in osmotic pressure
___-with certain reptilia, it seems not unreasonable to suppose that birds are
_ descended from a stock which possessed a considerably lower osmotic
pressure.
Turning now to the egg membranes which are easily observed, the
existence of a fair degree of semi-permeability may be demonstrated by
the experiment figured by Bergin and Davis (‘ Principles of Botany’), in
which a chip having been taken out of the rounded end of an egg, over
_ the air space, without piercing the outer membrane, a glass tube of
narrow bore is inserted into the pointed end and cemented in position.
_ The contents of the egg will slowly rise in the tube to a height of over a
metre when the egg is placed upright in a few centimetres depth of
distilled water. If the yolk be pierced it will colour the column, which
will stay at its upper limit for several days and then slowly sink. That
_ this membrane is somewhat permeable to sodium chloride may be seen by
adding a few drops of silver nitrate solution to the water in which the egg
is standing, when a turbidity or precipitate is produced. If, however,
_--—s the outer membrane be pierced, the rise in the tube will only amount to a
: few centimetres, for the inner membrane is much more permeable than
RE the outer, as seen by the silver nitrate test. It is to be noted that, while
the two membranes are in contact throughout the greater part of the egg,
at the blunt end they are separated by the air-space.

Acetic acid also penetrates the two membranes, for if an egg be
placed in the dilute acid till the shell is dissolved, and then washed and
placed in distilled water, it will be found that the egg swells greatly—to
; nearly twice its former volume, in fact. The water surrounding it
a becomes acid, even after numerous changes. If now the egg be placed
first in a strong solution of sodium chloride, and then in water, it will
swell again, and in this condition may be freely handled without risk of

uncture. If the membrane be cut after the shell is dissolved, the egg
will be found to be coagulated by the acid, having the appearance of
having been boiled.

The yolk is also enclosed in a delicate membrane with some degree of

semi-permeability, for on carefully breaking a fresh egg into water, and
rinsing to remove the white, the yolk swells considerably and becomes a

184 BIO-CHEMICAL JOURNAL

paler yellow with a turbid appearance. The germinal dise soon disappears "

from sight, apparently sinking into the yolk, which by the dilution is now .
of a lower specifie gravity, in which the dise can no longer float. This is
evidence that the dise is surrounded by a membrane either impermeable, _

or more probably less permeable, to water than the yolk membrane. In _

this distended condition the yolk membrane is very delicate, being
ruptured by the weight of its own contents if the water be drawn oft from
around it. In spite of this slighter permeability of the germinal dise as
compared with the yolk membrane, there seems to be no doubt that the
dise is normally approximately isotonic with the egg as a whole.

In the above experiment I have been unable to observe whether the
dise really sinks into the yolk; it becomes lost to view, but it is just
possible that in the imbibed condition it may not be practicable to
distinguish it from its surroundings.

That the germinal dise is not necessarily absolutely isotonic with the
fluid surrounding it, but is rather in a state of osmotic equilibrium with
it, seems extremely likely from the researches of Moore and Roat
(Bio-Chemical Journ., p. 55, Jan. 1908) on the equilibrium between the
cell and its environment. These authors showed that there was normally a
difference of 0°02° to 0-03° C. between the freezing point of the serum and
red blood corpuscles of the pig, and that diluting the blood affected serum
and corpuscles unequally. ‘Che work of Dakin on the variations in the
osmotic pressure of marine vertebrates by change in the external medium
(Bio-Chemical Journ., p. 475, Dee. 1908), shows that in these cases also
the systems are in equilibrium rather than isotonic,

SUMMARY

The osmotic pressure of the egg of Gallus bankiva, as calculated from
freezing point depressions, rises during incubation from about 55

atmospheres to about 73 atmospheres, the latter value being approxi-_

mately that of the osmotic pressure of the blood of the same bird.
Bacterial action during incubation may cause the pressure to rise to
over eight atmospheres. The view is put forward that birds are

descended from organisms with an osmotic pressure of five atmospheres
or less.

I have much pleasure in thanking Professor A. F. Dixon for his
advice and the loan of apparatus, and also Professor H. H. Dixon for
permission to carry out the work in the School of Botany.

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