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http://www.archive.org/details/cu31924003195850

THE VENOM OF HELODERMA
BY

LEO LOEB

WITH THE COLLABORATION OF CARL L. ALSBERG, ELIZABETH
COOKE, ELLEN P. CORSON-WHITE, MOYER 8. FLEISHER,
HENRY FOX, T. 8. GITHENS, SAMUEL LEOPOLD,

M. k. MEYERS, M. E. REHFUSS, D. RIVAS,

AND LUCIUS TUTTLE

WASHINGTON, D. C.
Published by the Carnegie Institution of Washington
1918 Gt

N2,8 808%
CARNEGIE INSTITUTION OF WASHINGTON

PusBLicaTIoN No. 177

Copies of this Boot

were first issued

MAY 10 1943

PRESS OF GIBSON BROTHERS
WASHINGTON, D.C,

\ PREFACE.

Our investigations into the properties and toxic action of the venom of
Heloderma were undertaken at the request of Dr. S. Weir Mitchell, and carried
out with the aid of grants from the Carnegie Institution of Washington.

In order to be able to extend the researches in various directions, I asso-
ciated with myself a number of collaborators, who undertook the study of
various parts of the problem. If, notwithstanding this partition, the work
has, as I hope, preserved its uniform character, it is due to the fact that by
far the greater part of these investigations was carried out in the Laboratory
of Experimental Pathology of the University of Pennsylvania, of which I had
charge at that time. This made possible the constant correlation of the
results, and the data obtained in the study of one problem became available
for and were used in the investigation of other problems. The chemical
analysis of the venom which Doctor Alsberg undertook was done elsewhere.
The histological study of the changes produced by the venom in the central
nervous system was partly done in the Laboratory of Neuropathology of the
University of Pennsylvania, through permission of Dr. W. G. Spiller.

Dr. D. T. MacDougal, Director of the Department of Botanical Research
of the Carnegie Institution of Washington, on various occasions kindly obtained
for us living specimens of Heloderma suspectum.

To Dr. S. Weir Mitchell and to the Smithsonian Institution we are
indebted for a specimen of Helodermg horridum.

I am under obligation to Doctor Calmette, of the Pasteur Institute of
Lille, who very kindly put at my disposal some of his cobra antivenin.

I wish to thank all my collaborators for their faithful cooperation, and
especially to acknowledge the assistance given me in the preparation of this
work on various occasions by Dr. Moyer 8. Fleisher and Dr. Ellen P. Corson-
White.

Lro Logs.
BaRNARD FREE SKIN AND CANCER HospPITAL,
St. Louis, Missouri, March 1912.

Ill

CONTENTS.

A. Anatomy of the Poison Gland of Heloderma. By Henry Fox.....
B. Structural Changes produced in the Poison Gland by Injection of
Pilocarpine. By Henry Fox (with the collaboration of Leo Loeb).
C. Transplantation of the Venom Gland. By Henry Fox and Leo
DOOD) west s te adiah obs gS we O ASG ARE De EES CEASE Pee eR ES
D. Solubility of the Venom Granules. By Henry Fox and Leo Loeb

II. General Properties and Actions of the Venom of Heloderma, and Experi-
ments in Immunization. By Elizabeth Cooke and Leo Loeb (with some
additional experiments by Moyer S. Fleisher).................0.-020000-

Review of the Literature. 0.0.0... 00000 c ccc eee
Method of Collecting the Venom and Factors which influence the

Amount of Secretion... 4.00/05 ag aaalvers se aoes Peds AwALESE SEES
Influence of Pilocarpine on the Secretion of Venom................
Effect of Heat on Venom...............0 000 cece eects
Effect of Standing in Solution on Toxicity of Venom...............
Hiflechiol Dialy Zing oy pia oncaaien te yawn ne a kidehin hacia we oa ghgudad
Effectuot: Filtrationiss.s ies nc easce oo none nad den eevard Rees BEG Mea ha A

Effect of Roentgen Rays on Secretion of Venom Gland.............
Effects of Venom upon Animals... ......0...00 000002 c cee eee
Condition of Mouse’s Eye produced by Injection of Heloderma Venom.
Anatomical Lesions produced by Injection of Venom...............
Histological Changes produced by Injection of Venom..............
Influence of the Method of Administration on the Toxicity of the Venom
Effect of placing Collodion Capsules containing Venom in the Peritoneal
CA NAUY A. ssln cece secneu Gr rstenet chien eh ite asa Ete As Sete ce espace ce ceo UAT Boa SMES
Administering Fractional Doses of Venom..................0.-655
Susceptibility of Various Species of Animals to Heloderma Venom. . .
Toxicity of the Organs and Excretions of Heloderma...............
Influence of Venom on Coagulation Time of the Blood..............
Influence of Inoculation of Pieces of Venom Gland into Mice........
Immunization against Heloderma Venom..................--+0005
Protective Power of the Serum of Immunized Rabbits.............
Precipitins in the Serum of Rabbits immunized against Heloderma

III. Bacteriology of the Saliva of Heloderma Suspectum. By D. Rivas ........
IV. Influence of Heloderma Venom upon Blood Pressure, Diuresis, Peritoneal
Transudate, and Intestinal Fluid. By Moyer S. Fleisher...............

Y. Effect of the Venom of Heloderma Suspectum on the Isolated Heart. By
Thomas Stotesbury Githens.......... 20.0... c cece eect teen eee

VI. Experimental Production of Acute Gastric Ulcer in the Guinea-Pig. By M. E.
ReHMMSS soe pds aie certs haste Ss Gee ES Edo EEO RNR Se Fee tg eee sees

v

VI

. Hemolytic Properties of Heloderma Venom.

CONTENTS.

. Histological Changes of the Central Nervous System produced by Heloderma

: 5 Reopoldgewae ccdusia} eens enact ROO a ROE 2
ete ae By Elizabeth Cooke and Leo

LOOD tise Sed He sa 309. see ecient tale ap ateeaa a Ales demas Salup eat lene tna ako a nn oA

. Action of Heloderma Venom on the Cellular Elements of the Blood within the

Living Organism. By M. K. Meyers and Lucius Tuttle...............,

. The Influence of Heloderma Venom upon Phagocytosis. By ual Tuttle.
- Can the Presence of Antibodies to the Venom of Heloderma Suspectum be

Demonstrated in the Blood Serum of this Animal by the Method of Com-
plement Fixation? By Ellen P. Corson-White ..................

1. The Chemical Character of Venom.
2. The Enzymes of the Venom and of the Venom Gland.

PAGE,

137

143

171

187

193

199
205

211

THE VENOM OF HELODERMA.

INTRODUCTION.

While considerations of a practical as well as of a theoretical nature le«l
to the study of snake venoms, the interest in the venom of Heloderma is more
purely scientific and of comparatively slight practical importance. No death
of a human being has come to our knowledge that can be attributed to the bite
of a Gila monster. A bite from this animal is in man either followed by no
symptoms at all or by a local swelling, perhaps extending to the shoulder, if
the bite affected the upper extremity. The swelling disappears within a short
time, but it may be followed by a long-continued weakness of the hand, with
sensations of discomfort lasting over several years. The preparation of an
antivenin so desirable in the case of snake bites is therefore of little importance
in the case of Heloderma. From a theoretical point of view, on the other hand,
a study of the venom of Heloderma is of considerable interest. We have
learned much of the mode of action and of the constitution of snake venoms
through the extensive studies of S. Weir Mitchell and Reichert, Calmette,
Lamb, Flexner and Noguchi, Keyes, Fraser, von Dungern and Coca, Bang,
and especially Faust, with many others.

Relatively little of a definite character is known concerning the venom of
Heloderma. The investigations of Santesson and van Denburgh and Wight,
which followed the observations of S. Weir Mitchell and Reichert, were neces-
sarily limited in their scope, mainly in consequence of the small amount of
venom at their disposal. Data which permit a comparison between the mode
of action and constitution of snake venoms and the venom of the only other
poisonous reptile known, the Heloderma, are of great theoretical interest. An
intimate understanding of the different steps by means of which the venom
gains entrance into certain cells and exerts its destructive action on the con-
stituents of these cells is of fundamental pathological importance. The number
of unknown factors coming into play during these processes is, however, very
great, and the number of equations with which we can operate must be pro-
portionately increased. We undertook, therefore, to supply the data which
will render more accessible the venom of Heloderma for future detailed study of
certain problems. Not rarely our own investigations had to cease when the
point was reached where more far-reaching studies promised to vield results
of importance, and the further cultivation of this field had to be left to future
investigations. Under these circumstances it may not be without interest to
point out some of the results of our investigations, and especially those findings
which may serve as a starting-point for future research.

The poison glands of Heloderma and of snakes differ somewhat in position
and structure. The poison gland of snakes is considered to be homologous

to the parotid of mammals, while in Heloderma it corresponds to a sublabial
1

2 THE VENOM OF HELODERMA.

gland. Their microscopic structure also differs somewhat. In Heloderma the
intralobular duets alone contain the typical granules and the end acini do not
seem to be concerned in the production of the venom, while in snakes the
acini as a whole are believed to secrete the venom. Doctor Fox has not been
able to find mucus-gland cells in Heloderma, while in snakes they seem to
occur. The mechanism of the poison gland also differs in Heloderma and in
snakes. In the latter the contraction of 2 muscle-coat surrounding the gland
expresses the venom, while in Heloderma the contraction of certain muscles
makes tense a fibrous facia which then presses the venom from the gland.
Differences also exist in regard to the structure of the teeth of Heloderma and
poisonous snakes. In different though related species the same end, /. ¢., the
secretion of venom during the process of biting, is achieved through means
which are insome respects similar but not identical. Different glands can be
adapted to a similar purpose and variations can occur in the mechanism of
the expulsion and in the chemical constitution of the venom. If we consider,
furthermore, the differences in the constitution of the venom, we may conclude
that the production and secretion of venom are effected through different
means in various, even nearly related, species of animals.

No clrect proof has so far been given that the granules are the bearer of
the venom within the cell, or that the granules are concerned in the production
of the venom. Our observations on the action of pilocarpine upon the venom
gland suggest, however, very strongly that the granules do carry the venom
or its precursor. Pilocarpine increases the secretion of the venom and causes
at the same time a rapid disappearance of the grannles. After repeated in-
jections of the pilocarpine at intervals of 24 hours the new formation of the
granules becomes imperfect, and pilocarpine correspondingly fails to call forth
an increased flow of venom. After transplantation, the toxic character of
the gland is retained and we find granules present in a certain number of
lobules. If we consider the great lability of the granules, it becomes probable
that these granules have been newly formed in the transplanted glands and
are not merely the granules that existed before transplantation. On the whole,
the gland preserves its character very well after transplantation, especially
when we consider that the gland functionates under modified conditions where
an expulsion of the secreted material is no longer possible.

The significance of cell-granules has been the subject of much controversy.
Therefore the data which we give concerning the behavior of the granules in
the poison gland may not be without interest. The granules are, on the whole,
very labile; many dissolve spontaneously in the venom. Distilled water, weak
acid, and extremely weak alkali cause their dissolution. Addition of NaCl
solution, especially of hypertonic solutions, renders them more stable. In this
respect we notice a similarity between the granules of the poison gland and of
the blood-cells of Limudus, which latter I studied on previous oceasions.' Both
kinds of granules are labile and are preserved in a similar manner by salt solu-
tion. These and certain other observations seem to point to the conclusion

'Polia Haematologica, vol. 4, 1997; PAiixer’s Archiv, vol. 131, 1910.

INTRODUCTION. 3

that the granules consist of a proteid rather than a lipoid material, although a
mixture of both may be present.

After addition of slightly hypertonic salt solution, a relatively stable sus-
pension of granules (with some admixture of nuclei) can be obtained, and
through centrifugation it will perhaps be possible to separate the granules and
the liquid part of the venom. A comparison of the toxicity of these two con-
stituents of the venom could thus be made and direct proof obtained of the
significance of the granules in the production or fixation of the venom.

There exists much difference of opinion in regard to the real function of the
poison gland. Does it merely take up from the blood the poisonous material
produced by other cells, or does it actually manufacture the venom from non-
poisonous substances supplied through blood or lymph? Calmette and Faust
accepted the first view. In snakes the blood and, as Flexner and Noguchi
found, even the ova contain a venom very similar to that secreted by the poison
gland. Calmette and Faust believe, therefore, that the cells of the poison
gland have a selective affinity for the venom circulating in the blood. Inas-
much as Calmette noticed that the heat resistance of the poisonous substance
present in the blood differs somewhat from that of the venom secreted by the
poison gland, he assumes that a precursor which is poisonous and has been
produced by other cells is slightly modified in the poison gland and then
secreted. This difference in the heat resistance of the two poisons he regards
as a reason sufficient to exclude the view of Phisalix and Bertrand, who regard
the venom circulating in the blood as being manufactured in the venom gland
and eliminated in the blood through a mechanism related to internal secretion.
They found, accordingly, that after extirpation of the venom gland of snakes
the blood lost its toxic properties.

In Heloderma conditions are less complicated. Here the venom can not
be found anywhere in the body except in the poison gland. Neither does the
blood become poisonous after extirpation of the poison glands, as should be
expected if the poison glands served merely as a place of elimination for the
venom prepared elsewhere. We can therefore be certain that in the case of
Heloderma the poison gland is the place where the venom is produced out of
non-poisonous material carried to the gland. Such a conclusion is also in
accordance with the view that the granules of the gland are the carriers of the
venom.

In its action on animals the venom of Heloderma bears a close resemblance
to the venoms of some snakes, especially the Colubride. It affects mainly the
central nervous system. The marked local, especially the hemorrhagic, effects
that characterize the venom of some vipers are absent. It affects very early
the respiratory center and, as in the case of cobra venom, death is mainly due
to a failure in the action of this center. Accordingly, we may find, even
within the first hour after injection of the venom, microscopic changes in the
ganglia-cells. The changes here are therefore noticeable at least as early as in
the case of snake venoms that have been investigated from this point of view;
but the changes do not go so far as after injection of the venom of some Colu-

4 THE VENOM OF HELODERMA.

bride studied by Hunter and Lamb. In contradistinction to these venoms,
the venom of Heloderma, although principally a neurotoxin, does not entirely
destroy the ganglia-cells. Its effect on the heart is very slight in vitro as well
as in vivo. Furthermore, it is of interest that the action of the venom on the
heart is reversible and that removing the venom can restore the heart action
(Githens).

We also observe, in contradistinction to certain snake venoms with the
power to dissolve muscle-tissue, that the venom of Heloderma does not exert a
lytic effect upon the heart-muscle. The marked primary fall in blood-pressure
which takes place after injection of the venom must therefore be due to a
direct or indirect vasomotor action of the venom on the blood-vessels. In
this respect our results agree with those obtained by van Denburgh and
Wight. Fleisher, however, did not observe the strong antagonistic action of
adrenalin upon the venom noticed by the previous investigators. Further-
more, he could establish the fact that the diminution in the flow of urine during
a long-continued injection of venom dissolved in much 0.85 per cent NaCl
solution is not due to a direct injurious action upon the kidney on the part of
the venom, but corresponds to, and is merely the result of, the decrease in
blood-pressure. The interchange of fluid between the blood and body cavities
is not markedly influenced by the venom during an infusion with 0.85 per cent
NaCl solution. The elimination of fluid into the small intestines is, however,
increased under these conditions, and correspondingly we find that if death
follows an injection of venom, the small intestine contains much fluid; espe-
cially is this true of the guinea-pig.

Two sequels of poisoning with the venom of Gila monster deserve special
mention, inasmuch as I have found no reference to them in the literature on
snake venoms, so far as the latter was accessible to me: (1) In the mouse we
find very frequently a rupture of blood-vessels near the optic nerve and pro-
trusion of the eyeball with subsequent opacity of the cornea or lens. (2) In
guinea-pigs we found ulcers and hemorrhages in the wall of the stomach. The
investigations of M. E. Rehfuss have shown that these ulcers are due to a
digestive action of the gastric juice; that the hemorrhages are usually second-
ary; that this effect is not specific for venom, but is found after the administra-
tion of a great variety of poisonous substances in cases in which the animal is
markedly affected by the toxic substance. Thrombi are present in the neigh-
borhood of the lesions, but are not the cause of the lesions. The factor that
makes the mucosa accessible to the digestive action of the gastric juice has yet
to be determined. Comparative studies in different species of animals (her-
bivorous and carnivorous animals) ought to yield results of interest.

Other structural changes produced by the venom are slight and seem to
be especially noticeable after chronic poisoning. Even the slow action after
introduction into the peritoneal cavity of collodion capsules containing venom
caused no marked structural changes; they certainly formed a much less promi-
nent feature than the great loss of weight which we observed in such animals.
A study of metabolism under these conditions might be of interest and throw
light on the disturbances of metabolic equilibrium in states of chronic poisoning.

INTRODUCTION. o

On the whole, we may conclude that the structural changes (even in the
brain) which we find after the administration of venom are not the cause of the
primary pathologic effects, but either accompany or follow the latter. Later,
however, these functional disturbances may give rise to secondary structural
changes, a consideration which probably applies also to the changes which
Leopold found in the ganglia-cells of animals injected with poison of Gila
monster.

The effect of the venom of Heloderma upon the cellular elements, as well
as upon the coagulation of the blood, is less marked than those found by pre-
vious investigators in the case of the venoms of various snakes. If there exists
any effect at all upon the coagulation of the blood, it is certainly very slight.
After intravenous injections of extracts of the poison gland of Heloderma, we
observed an insignificant retardation in the coagulation of the blood, an effect
frequently observed after intravenous injection of various tissue extracts in a
quantity too small to cause intravascular clotting.

The venom of Heloderma possesses no direct hemolytic action. It becomes
hemolytic only in combination with some activators, especially lecithin and
certain blood sera, and even here the dose of lecithin necessary for activation
is relatively great. In the case of heloderma, as well as cobra venom, the
hemolytic is somewhat less heat-resistant than the neurotoxic substance,
while the neurotoxin of the venom of Heloderma is somewhat more heat-
resistant than that of cobra venom; the hemolysins seem to show approxi-
mately the same degree of heat-resistance in both cases. Even in combination
with activators the hemolytic property of heloderma venom is comparatively
weak as compared to the cobra venom. Moreover, it differs from cobra venom
in not being activated by that constituent of fresh serum which is apparently
identical with complement. Heloderma venom can be activated by certain
blood sera, but the latter then retain their activating power after having been
exposed to a temperature that destroys or somehow invalidates the comple-
ment. Our knowledge concerning the finer mechanism through which the
complement participates in various reactions, or is prevented from participat-
ing, is at present too indefinite to make advisable a discussion of the cause of
the difference in the behavior of the two venoms; but we may mention that it
may possibly depend merely on a quantitative difference in the hemolytic
strength of both venoms.

Blood-sera not having an activating influence upon hemolysis inhibited it
similarly as they inhibit hemolysis by soap or saponin. A certain specific
relationship seems to exist between the inhibiting serum and the blood-
corpuscles used; in certain cases the hemolysis of the blood-corpuscles of a cer-
tain species seems to be inhibited, especially by the serum of the same species.

A similar relationship has been apparently observed by Besredka in some
other connection (Annales de 1’Institut Pasteur, xv, 1901). Besredka found
that sheep serum, for instance, protects only sheep corpuscles against a hemo-
lytic rabbit serum and not the corpuscles of another species. This apparent
specific relationship between serum and blood-corpuscles deserves a further
study.

6 THE VENOM OF HELODERMA.

The discovery of Neuberg that a certain parallelism exists between the
hemolytic power of certain toxic substances and their contents in lipase sug-
gested the responsibility of the lipase for the hemolytic effect. It was therefore
of interest to compare the heat-resistance of the lipolytic and hemolytic sub-
stances in heloderma venom. After heating it to 60° C. for 30 minutes, its
hemolytic power is not impaired. No appreciable loss is noticeable after heat-
ing it 10 minutes to 100° C. After heating it to 100° C. for 30 minutes a great
part of its hemolytic power is lost, and after exposing it to a temperature of
120° C. for 15 minutes in the autoclave it has lost all of its hemolytic power.
According to Alsberg, lipase of heloderma venom is weakened after heating to
60° C. during 30 minutes; it is destroyed after an exposure to 100° C. lasting
10 minutes. The lipolytic power of venom is therefore distinctly less heat-
resistant than its hemolytic power; the venom is still hemolytic after its
lipase has been destroyed through heating. We may therefore conclude that
lipase and hemolytic principle are not identical in the venom of Heloderma and
probably not in snake venoms. Of course the possibility exists that after all
lipase and hemolytic principle have some components in common.

We are not in a position to state definitely through what mechanism the
venom of Heloderma causes hemolysis. Certain facts suggest, however, that
lecithin and venom may increase the permeability of the erythrocytes. In the
case of cobra venom von Dungern and Coca and also Manwaring believed that
the venom splits the lecithin and that a splitting of lecithin is the real hemo-
lytic agent. In the case of heloderma venom we saw that the amounts of
venom and of lecithin required for hemolysis stood in inverse ratio.

It seems to me that two facts indicate that the venom-lecithin mixture
increases the permeability of the erythrocytes. In the first place, I would thus
interpret the observations of Bang and Overton, who found that the action in
vitro of cobra or crotalus venom upon tadpoles is counteracted by addition of
CaCl. Now, according to Jacques Loeb and W. J. V. Osterhout (Science,
Dec. 15, 1911, Jan. 19, 1912), CaCle inhibits the entrance into the cells of vari-
ous salts of monovalent atoms. We may assume that it acts in a similar
manner in combination with venom, but in other cases CaCl, may produce its
inhibiting influence by inactivating soaps, as has been previously pointed out
by Noguchi. In asimilar manner I would interpret the observation of Goebel.
According to him, corpuscles suspended in 0.85 per cent NaCl solution, which
can not be hemolyzed by cobra venom alone, are hemolyzed if the latter are
suspended in isotonic solutions of saccharose. It seems to me that this fact is
similar to the results which I obtained in the course of my investigations into
the influence of external conditions upon the blood-cells of Limulus (Folia
Hemotologica, Bd. 4, 1907; Pfluger’s Archiv, 1910, Bd. 131). In this case I
have shown that solutions of non-electrolytes behave somewhat similarly to
H.0 and produce similar lytic changes. We should therefore expect that cor-
respondingly they are also more favorable to hemolysis than are salt solutions.

It would be of interest to study thoroughly the various factors in their
relation to hemolysis which I investigated previously in their significance for

INTRODUCTION. 7

the blood-cells of Lonulus. I found many analogies between the action of
these factors on colloids of a proteid character and on the living cells.

The venoms of snakes, as far as they have been investigated in this respect,
do not only possess the power to hemolyze erythrocytes, but also other kinds of
cells, especially leucocytes. In contradistinction to snake venom, venom of
Heloderma does not possess this destructive power. No dissolving or agglu-
tinating effect upon leucocytes is noticeable, which may even show great
phagocytic power, notwithstanding the presence of venom (Tuttle). After
addition of heloderma venom phagocytosis takes place in vivo as well as in
vitro and apparently in undiminished strength.

After a subcutaneous injection of heloderma venom a marked increase in
the number of polynuclear leucocytes takes place in the peripheral circulation.
This reaction was especially marked in animals during the process of immu-
nization, where relatively large quantities of venom could be administered.
How far this reaction depends merely upon differences in distribution of leu-
cocytes, or on an actual increase in the output of polynuclear leucocytes into
the blood-vessels from the bone-marrow, has to be decided by further investi-
gation (M. K. Meyers and Lucius Tuttle).

As previously stated, the heloderma venom has nodirect hemolytic effect;
it does not digest muscle, it does not destroy leucocytes, and, we may add, it
has no noticeable effect upon eggs of Echinoderms.

Cobra venom and other snake venoms have a very decided lytic effect
upon various kinds of cells; they even are able to destroy bacteria. In this
respect heloderma and cobra venom differ very markedly, while in the manner
in which both venoms affect the living animal organism there exists much
similarity; both are principally neurotoxic, causing lesions in nerve-cells and
exerting their lethal effect by paralysis of the respiratory center.

The similarity between certain snake venoms and the venom of Heloderma
is also apparent in the parallelism that exists between the doses lethal for vari-
ous species. Against the venom of Heloderma as well as of Cobra, Bungarus
ceruleus, Enhydrina valakadien it is found that the white rat is relatively more
resistant than most of the other mammals tested, but this increase in resist-
ance does not extend to all the snake venoms. Thus, according to Fraser
and Gunn (Phil. Transactions Roy. Soc., t. 202), the white rat is even more
susceptible to the venom of Echis carinatus, which resembles in its action
crotalus venom, than the rabbit or the guinea-pig. A further similarity is
the following: cold-blooded vertebrates are more resistant toward cobra and
certain other snake venoms, as well as against heloderma venom, than are
the warm-blooded animals. Certain differences, however, exist among cold-
blooded animals in regard to their susceptibility toward heloderma venom.
Especially is this true of toads, which show a marked degree of resistance.
During different stages of development, the susceptibility toward heloderma
venom may vary. Tadpoles are undoubtedly more susceptible to heloderma
venom than adult frogs. All the invertebrates which we tested were immune
against the action of venom, while Fundulus, a salt-water fish, was susceptible.

8 THE VENOM OF HELODERMA.

Correspondingly, invertebrates also show no or only very slight susceptibility
to cobra venom. As we mentioned above, echinoderm eggs, although not
affected by heloderma venom, are affected by cobra venom. The similarity in
the lethal dose of heloderma venom pro kilo animal which we found in the case
of most mammalians tested is surprising indeed if we consider that a number
of variable factors determining the lethal dose comes into play, and that these
factors might be expected to vary in the case of different species. Further-
more, we have to consider the difference in size and number of nerve-cells in
various species. It appears doubtful what we really measure in determining
the lethal dose. Is it the amount of venom entering each ganglia-cell of the
respiratory center? The fact that notwithstanding these complications a great
similarity exists in the lethal dose pro kilo animal indicates that the amount of
venom absorbed and reaching the respective ganglia-cells must be very similar
in different species. In the case of the white rat, is the respiratory ganglia-cell
less sensitive, or does less venom get into contact with each ganglia-cell, or
are the ganglia-cells less permeable? These questions we can not answer at
present. Asin the case of other poisonous animals, we found Heloderma to be
immune against very large doses of its own venom when injected subcutane-
ously. Whether they are susceptible to an intracerebral injection of venom,
as (according to Phisalix) snakes are in the case of their own venoms, we have
not had an opportunity to investigate.

In this connection an observation of A. Fluhner (Archiv f. Exp. Path.,
1910, 63) is of special interest. This author found that the heart of toads is
not immune against the venom of Bufo, which is principally a heart-poison.
Natural immunity of a species against its own venom here, as well as in the
case of snakes, is therefore not dependent upon a lack of susceptibility to the
poison on the part of those cells upon which the toxic substance principally
acts. We might here also recall the fact that the erythrocytes of Heloderma
are not immune against the hemolytic action of the venom of this animal.
We may therefore conclude that the natural immunity is based on other fac-
tors. Secondary mechanisms evidently exist which protect the sensitive cells
against the action of the poison.

Phisalix believed that the natural immunity of poisonous animals against
their own venom depends upon the presence of antitoxin in the circulation.
In the case of Heloderma such antitoxic substances certainly do not exist, not
even in animals whose poison gland had been removed some time previously,
in order to obviate a neutralization of antitoxin which might be present by the
venom discharged into the blood through a process of internal secretion. There
exists, however, another mechanism through which a natural immunity could
be obtained. Should some provision in other organs prevent the venom from
reaching the brain and acting upon the nerve-cells, the latter would be pro-
tected against the injurious effect of the venom. Suchan explanation of natural
immunity was suggested by Wolff-Eisner several years ago. It. seemed, there-
fore, worth while to compare the absorptive action of the pulp of various organs
for the venom of Heloderma, and furthermore to make a comparative test of

INTRODUCTION. 9

the behavior of the organs of different species. Other considerations made
such an investigation advisable. It is necessary for a toxin to get into contact
with the surface of cells before it can exert its injurious influence. One of the
means through which such contact between cells and poison can be accom-
plished is adsorption.

Only a few of the results obtained in this study can here be referred to.
We found that organs like the liver and kidney adsorb at least as much venom
as the brain. In this respect heloderma venom seems to differ from certain
other poisons which have been examined, as, for instance, cobra venom. On
the whole, the brain closely resembles lecithin in its adsorbing power and it is
very likely that the adsorptive power of the brain is due to the lipoids and not
to the proteids of the brain, which latter seem, however, to adsorb some other
toxic substances, as, for instance, rabies virus. Emulsions of lecithin, how-
ever, adsorb more heloderma venom than emulsions of brain-matter. In both
cases, especially the small particles of the emulsified material, adsorb a great
part of the venom, and it needs therefore filtration through a Berkefeld filter
to determine accurately the quantity of venom adsorbed. We may conclude
that conditions comparable to those found in the case of tetanus toxin, in
which brain-substance has a special antitoxic power, do not exist in the case
of heloderma venom. Furthermore, the combination between lecithin and
venom, or brain and venom, is only a loose one, inasmuch as after an injection
of the residue obtained through centrifugation the animals die, perhaps after
a previous absorption and splitting of the lecithin, while after adsorption of
venom by charcoal the adsorbed venom is not at all or only very slowly given
off and has therefore become innocuous.

Our comparative studies of adsorption by the organs of various species
point to the conclusion that the organs of Heloderma and of species nearly
related to it adsorb venom in a larger quantity than the organs of species not as
nearly related to Heloderma. Our experiments seem, therefore, to indicate
that certain organs of Heloderma might be concerned in the natural immunity
of this animal against its own venom. We can, however, not yet regard as
conclusive this part of our investigations. Considering the large number of
variable factors present in such experiments, additional investigation, espe-
cially a comparative study of the adsorption of heloderma and certain other
venoms by the organs of different species, seem to be desirable in order to
place this theory of natural immunity on a solid basis. On the other hand, we
must fully realize that adsorption experiments with organ-pulp in vitro are
only a very crude imitation of conditions that exist in the body after injection
of the venom, and that normally functionating organs might take up and fix
to themselves a very much larger quantity of venom than the organ-pulp.

Furthermore, we have to consider the fact that according to our obser-
vations blood-serum markedly inhibits the adsorption of venom even by such
a strong adsorbent as charcoal. Assuming that circulating blood should act
in a similar manner, adsorption of the venom in the body would be greatly
interfered with.

10 THE VENOM OF HELODERMA.

An interesting consideration suggested by various investigators is that
adsorption of the hemolyzing substance represents the first step leading to
hemolysis. We furthermore know that blood-serum inhibits venom hemoly-
sis. Does serum exert this inhibiting action by preventing adsorption of the
hemolyzing agency by the corpuscles? From our results it is clear that the
chemical character of the adsorbent is not the determining factor in adsorption.
Carmine, kaolin, aluminium oxide, and especially charcoal are adsorbent
substances. The electric charge of the adsorbing material, therefore, does,
at least in this case, not seem to be the principal factor that determines
the strength of adsorption.

The marked tendency of the venom of Heloderma to be adsorbed became
apparent very early in our studies. After boiling cobra venom and thus pre-
cipitating its albuminous material, its toxic principle, which the poison of
heloderma venom resembles so closely in its action, is found in the liquid part,
while the venom of Heloderma is partly adsorbed by the coagulum produced
during the process of boiling, and its tendency to be so readily adsorbed by
many substances interfered markedly with its chemical analysis. Doctor
Alsberg found that the majority of the methods used so successfully by Faust
in his analysis of cobra and crotalus venom were not applicable in the case of
heloderma venom, on account of the readiness with which it is carried down
with various precipitates; but by a modification of one of the methods used by
Faust for isolating the crotalus toxin, he succeeded in obtaining the heloderma
venom in a state in which it no longer gave the biuret reaction, thus proving
that its poisonous principle is also a substance free from proteid or only sec-
ondarily combined with it. It shows also in this respect its close relation to
snake venoms.

A more extended chemical analysis of the venom of Heloderma was impos-
sible on account of the great difficulty we incurred in obtaining quantities of
venom sufficient for this purpose. The same difficulty prevented us from
carrying out studies in immunity as far as we had planned to do. In order to
produce an antivenin against heloderma venom through injection of venom
into animals, relatively enormous quantities of the venom would have to be
used—very much more than was at our disposal. We had therefore to con-
tent ourselves with demonstrating the possibility of active immunization of
rabbits against the venom of Heloderma.

Precipitins are much more readily found than antitoxin in the serum of
rabbits repeatedly injected with venom. If we consider the chemical char-
acter of the venom as established by Alsberg, and furthermore the fact that
this precipitin produced in rabbits reacts not only with the venom but also
with the blood of Heloderma, although less markedly, there ean be little doubt
that the precipitin was produced in response to the injection of certain proteid
material admixed with the poison of Heloderma proper, and not of the venom
alone. Inasmuch as this precipitin reaction is quantitatively stronger with
venom than with the blood of Heloderma, we may conclude that the proteids
of the venom gland and of the blood are not identical—a further confirmation
of the chemical specificity of the different organs of the same individual.

INTRODUCTION. 11

It may also be mentioned that the formation of precipitates, which takes
place when the serum of injected rabbits and venom are mixed, may possibly
account for a slight decrease in the toxicity of the venom, occasionally noticed
after addition of venom to the serum of rabbits immunized against venom.
We may assume that this precipitate included a certain portion of the venom
and thus retarded its absorption and exerted a very slight protective influence.
An antivenin which, according to Phisalix, is responsible for the natural im-
munity of some poisonous animals against their own venom, can not be found
in the blood of Heloderma, as we have already mentioned. Neither does the
blood-serum of Heloderma contain any substance which fixes complement in
combining with a constituent of the heloderma venom (E. P. Corson-White).
Venom itself, however, is capable of binding a considerable quantity of com-
plement in a similar manner, as has been found in the case of snake venoms.
It appears doubtful whether this effect is due to the toxic substance proper or
to admixed proteid material. After repeated injections of venom, symptoms
of general anaphylaxis could not be observed. The sterile abscesses which
appear in animals that have received frequent injections of heloderma venom
may perhaps represent a local anaphylactic reaction related to the Arthus phe-
nomenon, to which a similar reaction observed after injection of snake venom
had indeed been compared by previous investigators.

In this case also the reaction is perhaps not due to the toxin proper, but to
the proteid material admixed with the venom; and this reaction could therefore
be in part avoided, perhaps, if the filtered fluid only were injected without the
precipitate found on heating, a procedure which entails a certain loss of venom,
inasmuch as the precipitate includes some toxin which has been adsorbed.

The specificity of antivenin against snake venoms has been an object of
much controversy. We thought it therefore of sufficient interest to inves-
tigate the effect of Calmette’s cobra antivenin upon the venom of Heloderma.
Both the venom of Heloderma and of Cobra are essentially neurotoxic and
secondarily hemolytic. Both venoms are therefore very similar in their effect
upon animal tissue. On the other hand, Heloderma is systematically less
nearly related to the Cobra than any of the snakes. Inasmuch as it has been
maintained that even among snakes the various neurotoxins are specifically
different and that cobra antivenin neutralizes mainly the venom of cobra, it
was not probable that cobra antivenin would exert any influence upon helo-
derma venom; but our investigations leave little doubt that Calmette’s cobra
antivenin does exert antitoxic effect upon heloderma venom. This action is,
however, very slight and naturally very much less marked than in the case of
cobra venom. Our observations confirm, therefore, the belief that there exists
a graded specificity, and also prove that this specificity is less limited than has
been assumed by various investigators.

The difference in the heat-resistance between the neurotoxin of heloderma
and of cobra venom makes it very improbable that both substances are iden-
tical, although they may be related to each other. The results of Faust,.
which show a difference in the constitution of ophiotoxin and of crotalotoxin,
make this conclusion almost certain.

12 THE VENOM OF HELODERMA.

The question how far the different effects of one and the same venom
depend on one toxic principle, or on the presence of multiple poisonous bodies,
is a problem much more difficult to decide. There can be little doubt that not
all actions of one venom are due to one single substance. We now know that
some of the ferments of the venom of Heloderma are not at all produced in the
venom-gland, but are evidently admixtures derived from elsewhere. Fur-
thermore, there can be little doubt that the substances present in some snake
venoms, causing or preventing the coagulation of the blood, are not identical
with the neurotoxins or with the hemolysins of the same venom. Still more
difficult to answer is the question how far other constituents of various venoms,
the neurotoxin, hemolysin, and other cytolysins, and hemorrhagin are identical.
The evidence we have concerning this point appears conflicting. On the one
hand, Faust’s fundamental investigations into the chemical constitution of
venoms seemed to simplify these problems. According to Faust, ophiotoxin
exerts both a neurotoxic and hemolytic action, crotalotoxin in addition a hem-
orrhagic action comparable to arsenic, emetin, and sepsin. The preponder-
ance of the local effects in the case of crotalotoxin is said to be due to the greater
molecular weight of this venom as compared to ophiotoxin, which increases its
colloidal properties and causes it to be more slowly absorbed. On the other
hand, a good deal of evidence can apparently be adduced against this view.
We might especially mention the following facts:

The heat-resistance of the so-called neurotoxin, hemolysin, and hemor-
rhagin is not identical in the same venom. This applies to the heloderma
venom, where the neurotoxin is distinctly more heat-resistant than the
hemolysin. Their resistance to the effect of certain chemicals varies. The
hemolytic principle can be extracted through some substances in which the
neurotoxin remains undissolved. The different constituents of a venom showa
different tendency to be adsorbed by certain materials. Various venoms differ
in the number and quantities of the various toxic constituents which they con-
tain. The antivenin produced through immunization with a certain venom
does not neutralize al! the toxic constituents of another venom. Whether the
ophiotoxin and crotalotoxin of Faust are able to call forth the production of
antivenin, we do not yet know.

It appears improbable that these substances could give rise to the produc-
tion of antibodies, graded in accordance with the relationship of the animals
from which the venoms were derived. There exists, therefore, a certain dis-
crepancy between the results of the purely chemical analysis of the venoms
andl between the evidence obtained through the application of so-called
biochemical methods. In weighing the evidence we have to take into con-
sideration the fact that some of the conclusions based on the latter methods
are of such a remarkable character that we may find some difficulty in accept-
ing them. Thus studies of hemolysins and other cytolysins of snake venoms
by the method of selective adsorption lead to the conclusion that a single
venom contains an almost endless number of various cytolysins, and espe-
cially hemolysins. A venom would therefore constitute a mixture of poisons

INTRODUCTION. 13

of an almost incredible complexity. If, on the other hand, we should reject
such a conclusion we must be aware that much of the evidence as to the differ-
ence between hemolysins and neurotoxins of a single venom is of a similar char-
acter. At present we can only point out these apparent contradictions; it
remains for further studies to solve these problems.

Provisionally we may suggest that perhaps certain proteid constituents of
the venom are responsible for some of these apparent discrepancies; it may be
that these proteids are able to increase or diminish certain functions of the
poisonous principle and that the effects of heat and certain chemicals concern
rather these secondary substances than the poison proper. As far as immuni-
zation is concerned, these proteids may perhaps, to use the terminology of
Ehrlich, supply a multiplicity of haptophore groups, all linked to a uniform
toxophore group, which latter would represent the poisons isolated by Faust.
Future investigation will also have to analyze the mechanism through which
the neurotoxic effect is produced. Do we have to assume that the structure of
the various body-cells is in the main similar, as far as the conditions of perme-
ability of the outer protoplasmic layer are concerned? Are the neurotoxic and
hemolytic effects produced in a similar manner? A number of observations
point to the importance of a change in cell permeability as one of the factors
through which the poison exerts its injurious effect, as the significant action of
CaCl, and of sugar on the hemolytic action of venom already discussed indi-
cates. Furthermore, the observations of Noguchi, that the later stages of the
developing ova of Fundulus were more resistant to the action of venom than.
the earlier stages, are in agreement with my observation in regard to the action
of neutral red on Fundulus eggs. In this case I also found the later stages.
more resistant than the earlier ones, and I could furthermore show that this
difference in resistance depended upon a difference in the permeability of the
egg-membrane for neutral red. (Elizabeth Cooke and Leo Loeb, Ueber d.
Giftigkeit. einiger Farbstoffe fuer die Eier von Asterias und von Fundulus,
Biochem. Zeitschrift, Bd. 20, 1909.)

That a certain venom destroys various kinds of cells through a similar
mechanism is perhaps indicated in the observations of Flexner and Noguchi,
who found that cobra venom which exerts a strongly neurotoxic action has
also a very marked lytic effect upon other kinds of cells. This lytic action of
cobra venom surpasses that of certain Viperide in correspondence with the
stronger neurotoxic action which cobra venom exerts in the living body. On
the other hand, the dissolving action of certain venoms on muscle seems to be
due to a different mechanism, and accordingly we find in this case crotalus
venom exerting a more powerful influence than cobra venom.

It is to be hoped that future investigations will definitely answer these
questions and will thus deepen our understanding of the conditions which
determine the function and death of the various cells of the animal body.

I.

MORPHOLOGY OF THE POISON GLAND OF HELODERMA,

A. ANATOMY OF THE POISON GLAND OF HELODERMA. By
Henry Fox.

B. SrructuRAL CHANGES PRODUCED IN THE POISON GLAND BY
INJECTION OF PiLocaRPINE. By Henry Fox (wiry THE
COLLABORATION OF LEO LOEB).

C. TRANSPLANTATION OF THE VENOM GLAND. By Henry Fox
AND LEO LOEB.

D. SoLuBILITY OF THE VENOM GRANULES. By Henry Fox
AND LEo Logs.

A, ANATOMY OF TITLE POISON GLAND OF TELODERMA.’

By Henry Fox.

GROSS ANATOMY OF THE POISON-GLAND.

The gross anatomy of the poison gland was studied in both the common
species (Heloderma suspectuim) and the less familiar Mexican form (Heloderma
horridum);a considerable number of the former and one of the latter species
were available. So far as this material was concerned, it showed that the
general structure and relations of the gland were similar in both species—a
result which is at variance with the statement of Professor Stewart (Proc. Zool.
Soc. London, 1891), who asserts that in Heloderma horridwm there is only a
single opening to each gland, while in Heloderma suspectwm there are four or
five. In the single horridium examined I found several openings exactly as in
Heloderma suspectum.

Hic, 1,—Ventral view of 2 dissection of head of Helv-
derma suspectum, showing poison glands in
situ. Dissection on right is deeper than
on left. Natural size.

Fie. 2.—One-half of a dissection of the head seen from
ventral side. The poison gland has been
pulled outwards in order to show nerves
entering its mesial surface. Natural size.

Each poison gland is of large size and is situated on the outer side of the
anterior half of the lower jaw, immediately under the skin, from which it is
separated by thin sheets of connective tissue. On the surface its position is
indicated by a prominent swelling underlying the lower jaw.

The gland is closely invested by a capsule of fibrous tissue. From the
inner surface of this capsule septa extend into the body of the gland; the larger
of these septa divide the gland into three or four primary subdivisions or lobes,
while the smaller, which arise in part from the investing capsule and in part
from the primary septa, penetrate the lobes and there form a network dividing
them into numerous lobules (see figs. 7 and 8).

1A ll the illustrations accompanying this and the following chapters were made by Dr. Fox.

7

18 THE VENOM OF HELODERMA.

The lobes are the primary subdivisions of the gland. They vary from
three to four in number and are arranged in a longitudinal series. They in-
crease in size from front to back. Each lobe is in reality a structurally inde-
pendent organ, the different lobes being separated by complete fibrous parti-
tions and opening by separate apertures to the exterior; they are bound into a
compact whole by the investing fibrous capsule (¢f. figs. 1,3, and 4). In form
they are roughly club-shaped, their upper excretory portions are narrowed,
while their lower, glandular portions are rounded and swollen (figs. 3 and 4).
Each lobe is a sac, containing a relatively large central excretory cavity, which
narrows at its upper end to form a short excretory canal. This canal opens on
the outer side of the jaw close to the base of the tooth. The wall of the sac is
of considerable thickness and consists of lobules of glandular tissue. Numer-
ous fine tubules, representing the intralobular ducts, open from the lobules
into the central cavity.

The general position and structure of the gland is shown in figs. 1 and 3.
It consists of four lobes, directed from above downwards and backwards.

Fic, 3.—Lateral view of head, showing poison gland consisting of four independent sacs. Each sac is cut tangentially,
showing its central duct or cavity. Natural size.

Fig. 4.—Enlarged view of same poison gland shown in fig. 3. Slightly diagrammatic. Designed to give a clearer
view of external openings of poison ducts and their relation to the teeth. Portions of three bristles are
shown inserted in the last three openings.

Their upper ends converge close to the anterior border of the lower jaw. This
convergence would seem to indicate the formation of a common excretory duct,
but, as already mentioned, such a common duct is not formed, each duct open-
ing quite independently of the others, as was determined by the use of bristles
and the ejection of the secretion under water. In the latter case a thin stream
of fluid escaped from each opening.

As regards the course of the ducts, my observations are in accord with
those of Stewart (1891) and opposed to those of Fischer (1882) and Shufeldt
(1890). The last two authors described the ducts as originating from the
mesial surface of the gland, passing upward through the lower jaw and open-
ing within the mouth at the base of the teeth which they supplied. Stewart
maintained that “the ducts pass directly from the gland to their openings,
which are situated to the outer side of a fold of mucous membrane intervening
between the lip and the jaw.”” Stewart’s contention is undoubtedly correct.
The so-called ducts of Fischer and Shufeldt are, as Stewart asserts, the
branches of the inferior dental nerve which pass out through foramina in the
lower jaw and enter the mesial surface of the gland (see fig. 2).

The statement of Shufeldt that the ducts open at the base of the teeth is
not strictly correct. They lie, as Stewart observes, to the outer side of the

ANATOMY OF THE POISON GLAND OF HELODERMA. 19

teeth, from the base of which, I may add, they are separated by the outer wall
of the dental sacs. These are cup-shaped folds of mucous membrane com-
pletely surrounding the basal portions of the teeth. Immediately external to
them is a shallow groove which is limited at the outer side by the prominent
fold of mucous membrane mentioned by Stewart as intervening between the
lip and the lower jaw. This fold is connected to the jaw by a series of vertical,
obliquely transverse folds which lie in the regions between the teeth. They
interrupt the continuity of the groove already mentioned and subdivide it into
a series of shallow depressions, into the more anterior of which the ducts of the
poison gland open. It would seem probable that these depresssions serve as
temporary reservoirs for the secretion. The tips of the upper teeth project
into these depressions when the mouth is closed (see fig. 4).

The teeth of Heloderma have been fully described by Cope (1900). Both
upper and lower teeth are grooved, although no evidence of a poison gland has
been found in connection with the upper teeth, and even in the lower teeth the
poison ducts are associated only with the anterior three or four pairs. The
latter show no features distinctly differentiating them from the remaining
teeth. The grooves are formed by projecting folds of enamel which extend
along both the anterior and posterior edges of the tooth. The anterior fold is
the stronger and its groove correspondingly deeper. The secretion is doubt-
less carried along the groove by capillarity. The teeth are hollow within
when dry, but there is no sign of a perforation at the apex. In life the internal
cavity is filled by a fleshy papilla arising from the jaw margin. Most of the
teeth are firmly united to the bone, but the younger teeth are quite free and
easily detachable.

Shufeldt has described a tendinous expansion arising from the outer sur-
face of the superficial muscles close to the hinder margin of the mandible and
spreading out over the gland. Posteriorly it is rather narrow and strong, but
anteriorly, as its fibers diverge, it expands to form a thin sheet closely adherent
to the gland. Shufeldt is inclined to believe that, although it contains scarcely
any muscle fibers, by its contraction the venom of the gland can be ejected
through the ducts. As to the nature of this contraction he says nothing, but
I think it may be considered as purely passive, due to the tensions produced
in it by the movements of the jaw. Such movements produced artificially in
the dead animal do, as a matter of fact, cause ejection of the venom. I may
add to Shufeldt’s account by stating that the superficial muscles from which
the tendinous expanse arises are on the outer side of the posterior half of the
mandible. From this point the fibers pass forward and downward and then
bend inward under the lower jaw in precisely the position for the movements of
the jaw to produce the tensions mentioned. In fig. 1, on the right side of
the figure, this part of the tendinous expansion can be seen arising from the
muscles immediately behind the angle of the lower jaw. Anteriorly it has been
cut away along with the skin in order to show the poison glands.

Concerning the homology of the poison gland with the mouth glands of
other vertebrates, it is obvious from its relative position that it is not com-

20 THE VENOM OF HELODERMA.

parable with the mammalian submaxillary, as is so frequently asserted in text-
books. The Jatter gland opens by its duct on the floor of the oral cavity
mesial to the lower jaw, whereas the ducts of the venom gland of Heloderma
open to the external side of the jaw and in close association with the lips. From
the relative position and composite structure of the poison gland, I agree with
Professor Stewart in considering it as the hypertrophied representative of the
sublabial glands of other reptiles and mammals.

Fic. 5.—Ventral view of dissected head and neck,
showing the more superficial blood-vessels.
Drawing slightly diagrammatic, approxi-
mately natural size.

. Submental vein.

- Inferior labial vein.

. Lingual artery.

. Anterior jugular vein.

. Hyoid cartilage.

. Facial vein.

. Pharynx.

. Internal jugular vein.

. Gsophagus.

. Common carotid artery.

. Left aortic arch.

_
SrPBNOMPwiWo

=

The nerve-supply of the gland is furnished by branches from the inferior
dental nerve. Four or five of these branches leave the inferior dental eanal
through a series of minute foramina perforating the outer side of the dentary
bone; one branch, however, passes through a slightly larger opening in the
articulare. The nerves enter the gland by its mesial surface, 7. ¢., the surface
applied to the bone (fig. 2).

The vascular supply of the gland is derived from the inferior dental artery,
branches of which pass outward through the foramina, already mentioned as
transmitting the nerves, and with the latter enter the gland on its mesial side
(fig. 6). Here they pass in along the interlobular septa, the ramifications of

ANATOMY OF THE POISON GLAND OF HELODERMA. 21

which they follow to all parts of the organ. Most of the blood leaves the gland
by a vessel which extends along its entire ventral edge. The relations of this
vessel are essentially those of the submental vein of mammals. At its hinder
end it is joined by the inferior labial vein. The common trunk thus formed is
the facial vein. This is continued backward along the sides of the face, finally
joining the internal jugular. Near its anterior end the submental vein gives
off a smaller vein which curves mesially under the lower jaw and joins a vein,
evidently the anterior jugular, which lies on either side of the trachea (fig. 5).
Some of the veinlets in the upper portion of the gland appear to communicate
with the inferior labial vein.

a
WIA

ar
W My I,
On ay

Fic. 6.—Ventral view of approximately half of head, showing arte-
rial supply of poison gland. Drawing partly diagram-
matic; approximately natural size.

. Poison gland, turned outwards.

. Mandible

Inferior dental foramen with inferior dental artery and

nerve.

Pterygoid bone, outer half removed.

Quadrate.

Internal carotid artery.

. External carotid artery.

Lingual artery.

Posterior auricular (?) artery.

Common carotid artery.

. Left aortic arch.

=
SEEN R wR

—
=

HISTOLOGIC STRUCTURE.

For the minute study of the gland, pieces were fixed in various fluids, but
the most satisfactory fixation was obtained by the use of Bensley’s modification
of Kopsch fluid. (See ‘The finer structure of the glandula submaxillaris”
by B. A. Cohoe, in Amer. Journ. of Anatomy, vol. v1, No. 2, 1907, p. 171.)
This fluid was especially favorable for the preservation of the granules in the
secreting cells, the other fluids used being rather defective in this respect.
The sections were stained mostly in Benda’s iron hematoxylin, followed by
eosin, erythrosin, or Bordeaux red. Some sections were doubly stained in
safranin and gentian violet.

22 THE VENOM OF HELODERMA.

As already mentioned in the section on the gross anatomy, the entire
poison gland is invested by a continuous capsule of fibrous tissue. From this
several relatively thick septa extend inward, completely dividing the gland
into three or four entirely independent parts or lobes. Each lobe is a pear-
shaped sac. The center of the lobe is occupied by a relatively capacious
lumen, which I shall term the central collecting duct. At its upper end this
narrows gradually to form the excretory duct, which opens at the apex of the
lobe. The walls of the sac are thick and formed chiefly of glandular tissue.
From these walls minute intralobular tubules open into the collecting duct.

Fic. 7.—Part of section of normal poison gland, showing typical histological structure (Zeiss oc. 4, obj. 8 mm.).

Fach lobe is subdivided into a large number of lobules by septa which are
given off from the investing capsule and the fibrous partitions separating the
lobes. These septa extend through the gland tissue as far as the central col-
lecting duct (figs. 7 and 8). Each lobule is further cut into several smaller
lobules by secondary septa which interpenetrate it in all directions, and this
subdivision continues until the terminal acini are reached, each of which is
separated from its neighhors hy a delicate septum (figs. 7, 8, and 9).

ANATOMY OF THE POISON GLAND OF HELODERMA. 23

In its general structure each lobe may be described as a compound tubular
gland. The axis of the gland is formed by the common lumen of the collecting
and excretory ducts. From this lumen at all levels are given off numerous
smaller tubules, the intralobular tubules, which, branching repeatedly, radiate
toward the periphery of the containing lobule to form the terminal acini.
Topographically there is no sharp distinction between the terminal acini and
the intralobular tubules; both are perfectly continuous, show approximately
the same diameter throughout, and contain a wide and clear lumen. This
lumen is always open; it is never obscured by the surrounding cells, even when
the latter are swollen with the secretions (figs. 7, 9, and 12).

Fic. 8.—Outline drawing of a section of poison gland, showing three lobules. Drawing was made to show arrange-
ment of inter- and intralobular septa and distribution of granule-secreting cells; the latter indicated by
dotted portions of the epithelium of the tubules. The epithelium is merely outlined, none of the constit-
uents, excepting the granules, being shown (Zeiss oc. 4, obj. 8 mm.).

In any lobule of considerable size there may be distinguished two regions:
(a) a peripheral zone formed of more or less clearly defined clusters of terminal
acini and (b) a central area composed of intralobular tubules. The latter con-
verge from the periphery toward the center of the lobule and there unite to form
a few central tubules which open into the large collecting duct (figs. 7 and 8).

We shall now consider in order the structure of the cells in (1) the central
collecting duct, (2) the intralobular tubules, and (3) the terminal acini. In
each of these parts, as previously noted by Holm, the cells are characteristic.

24 THE VENOM OF HELODERMA.

In the central duct the epithelium is one cell-layer thick and is thrown into a
series of low undulations. In form the cells are columnar and, as Holm
notices, are of slightly smaller dimensions than the intralobular tubule cells.
According to Holm, there is no granulation in their protoplasm, but this is not
strictly true, judging, at least, from some of my preparations—preparations
which in other respects are perfectly satisfactory, and therefore probably reli-
able in regard to this point also. These preparations show that in nearly all of
the collecting-duct cells the peripheral portion of the cytoplasm is crowded with
exceedingly minute granules, the crowding being so dense that under ordinary
magnification (7. ¢., é inch obj.) the whole appears as a hyaline mass. These
granules are totally unlike the large, clearly defined granules of the intra-
lobular tubules; they are much smaller than the latter, do not stain at all with

Fia. 9.—Section of portion of normal lobule, showing terminal acini, and several intralobular ducts. Note clear
alveolar character of protoplasm i in cells of peripheral acini and presence of granules in many cells of
intralobular ducts (Zeiss oc. 2, obj. DD.).

iron hematoxylin, and absorb cytoplasmic stains only slightly. The nuclei of
these cells are confined to their basal portion, where they are embedded in a
rather compact mass of ordinary cytoplasm (fig. 11).

According to Holm, the central collecting-duct cells have no secretory
functions. Iam inclined to concur in this view, although I have observed cells
which show faint traces of seercting. I have, for instance, frequently seen
small granular threads extending out from the cells into the lumen of the duct,
but it is possible that these may have been forced out of the cells by the pres-
sure incident to the excision of the gland.

The cells of the intralobular tubules are columnar, but are more capacious
than those of the central collecting duct (fig. 10). They are the typical
granule-secreting cells. The entire inner portion of the cells bordering the
lumen, including in average cases from one-half to four-fifths of their extent,

ANATOMY OF THE POISON GLAND OF HELODERMA. 25

shows what I interpret as an alveolar or foam structure, the clear spuces or
vacuoles being quite large and conspicuous, with only very thin strands of
cytoplasm separating them. In these vacuoles lie the fully formed granules,
typically a single granule to each vacuole. The granules, like those of serous
glands generally, are characterized by their large sizc, definite form, and the
avidity with which they absorb such stains as iron hematoxylin and gentian
violet. In some cases the cells are so numerous and so densely crowded within
the cell that they quite obscure the alveolar structure of its protoplasm. In
other cases they are much less numerous and may be entirely absent. A feat-

Fic. 10.—Section passing through point of union of two intralobular ducts, showing granule-secreting epithelium.
Normal gland (Zeiss oc. 2, obj. y's).

ure which has also been noted in other glands is that while all or nearly all of
the intralobular cells of a single lobule contain numerous granules, those of
neighboring lobules may show few or no granules.

The mode of formation of the secretory granules appears to me to conform
to the mode observed by E. Miller (Archiv f. Anat. u. Physiol., 1896, 8. 317 ff,
cited from Metzner in Nagel’s Lehrbuch der Physiologie, 1907). Minute,
dark-staining particles appear in the nodes of the cytoplasmic (=spongio-
plasmic) reticulum. These apparently increase in size and definiteness and

26 THE VENOM OF HELODERMA.

finally become the typical secretion granules. The latter, when fully formed,
become detached from the reticulum and come to lie in the vacuoles. Some of
the cells in fig. 10 show the early phases in the formation of the granules. In
no case have I seen any distinct trace of filar structures within the cells, such as
have been described by a number of authors in the gland-cells of the parotid,
pancreas, etc.

In the process of secretion the inner membrane, facing the tubule lumen,
bursts, and the secretion is extruded. Whether the granules are extruded
bodily or whether they first undergo liquefaction previous to extrusion are
questions which my material does not enable me to answer. J have frequently
observed large, swollen, vesicle-like bodies and irregular, solid particles in the

Tia. 11.—Portion of epithelium of central duct at a point where an intralobular duct opens into it. The sections show
sudden change in histological character in passing from one structure to the other. Note minutely
granulose character of central duct cells and clear alveolar character of intralobular duct cells. Compare
cag oo duct cells with typical secretion granules of intralobular ducts by comparing this figure
wit. g. 10.

gland lumen close to the ruptured inner wall of a cell, but whether these were
normal appearances or artifacts due to manipulation of the excised gland pre-
vious to fixation Iam unable to say. By whatever means the granules are
liquefied or dissolved, they doubtless disappear as distinct structures at the
time that the secretion is extruded.

A number of authors, who hold to the view that the granules are dissolved
within the cell, believe that the vacuoles represent the dissolved granules.
‘These authors maintain the reticular theory of cytoplasmic structures, accord-
ing to which the spongioplasmic reticulum is composed of anastomosing
threads, instead of the more viscid covering of the vacuoles postulated by the

ANATOMY OF THE POISON GLAND OF HELODERMA. 27

alveolar theory. The poison gland of Heloderma is not a suitable object for
testing the validity of the various protoplasmic structure theories, but I have
chosen to interpret the facts in conformity with the alveolar theory, since on
general grounds that appears to me to have most in its favor. The vacuoles
appear to me to be always present, although in the empty gland-cells they are
frequently quite minute, owing apparently to the shrinkage of the cells and the
consequent condensation of its cytoplasm. They are present and of full size
in many cells in which the early stages of granule formation, as evidenced by
the presence of the minute particles in the nodes of the reticulum, were alone
visible. I am inclined, therefore, to view the vacuoles as relatively constant

Fic. 12.—Section through part of single lobule of poison gland, showing terminal acini (Zeiss oc. 4, obj. 3mm, ap. 0.95).

constituents of the cell protoplasm. Whether at the moment of secretion the
granules dissolve in the liquid of the vacuoles, or are instead extruded into the
gland lumen, where they quickly dissolve, can not be settled by the material
examined.

The outer or basal portions of the granule-forming cells are filled by a
relatively dense, homogeneous cytoplasm, similar to that which nearly all
authors have described for that portion of the gland-cell. The protoplasm
appears to be alveolar, but the vacules are extremely minute and closely
crowded together. The nucleus is embedded in this cytoplasm.

The nucleus of the gland-cells is typically quite large and conspicuous, and,
as already mentioned, is embedded in and surrounded by a relatively dense

28 TILE VENOM OF HBLODERMA.

perinuclear cytoplasm occupying the basal portion of the cells. In form it is
spherical or elliptical, but I have observed no constant relation between the
form of the nucleus and the secretory phase of the cell. Shrunken nuclei were
observed in cells entirely filled with granules, but in all other cases the nuclei
were of approximately the same average size. I have not observed in Helo-
derma the changes in the nucleus described by Matthews in the pancreas and
by Launay in the poison gland of the viper, as associated with the changes in
activity of the cell. The nuclci of the active cells, as far as I have observed,
show no constant differences from those of the inactive cells.

The cells of the terminal acini (fig. 12) are slightly smaller than the intra-
lobular duct cells. In form they approximate to the cubical type. Their
cytoplasm is distinctly alveolar, this structure being clearest on the side facing
the lumen. At the base it is much denscr, resembling that in the correspond-
ing part of an intralobular tubule cell. The large secretory granules, so evi-
dent in the latter, are entirely lacking in these cells, though minute granular
particles are rather thickly scattered through the cytoplasm. The interspaces
of the reticulum are occupied by a clear ground-substance, which failed to stain
with the usual mucus stains. The nuclei showed no constant differences from
those of the intralobular duct cells.

B, STRUCTURAL CHANGES PRODUCED LN THE POTSON
GLAND BY INJECTION OF PILOCARPINE,

By Henry Fox.

(With the collaboration of Leo Loeb.)

In order to study the different stages of activity of the poison gland of
Heloderma under various conditions, the animals were injected subcutaneously
with pilocarpine. The gencral effects of the pilocarpine were strikingly similar
to those described in the case of salivary glands. (See Cohoe, Jour. of Anat.,
vol. VI.)

EXPERIMENT I.

An animal (No. 1) was for the first time injected with 0.1 grain of pilocar-
pine. Immediately before the injection a portion of the poison gland was
removed and fixed in Kopsch fluid to serve as a control. The animal was then
injected and three pieces of the gland were removed at intervals of 15, 35, and
50 minutes, respectively.

(1a) Guanp ReMmovepD Previous To THE INJECTION.

This was found upon microscopic examination to be entirely normal.
Numerous granules were observed crowding the cells of the intralobular tubule,
the undifferentiated cytoplasm being limited to an extremely thin layer at the
base of the cells. A copious granular secretion was found in the lumina of the
tubules, but it is doubtful if this condition is normal—it may have been forced
out of the cells into the tubules as a result of the handling consequent upon the
removal of the gland.

(18) Gianp Removep 15 MINuTES AFTER INJECTION OF PILOCARPINE.

Sections showed most of the intralobular duct cells devoid of granules,
though a moderate number retained a few. As a rule the cells presented the
usual character of stimulated gland-cells, being quite or almost free of granules
and with large, clear vacuoles inclosed in the meshes of the cytoplasmic retic-
ulum. Within the tubules the amount of secretion visible was extremely small,
in marked contrast to the quantity observed in the normal gland. If the latter
condition was normal, one evident effect of the stimulation with pilocarpine
was a rapid solution of the extruded granules or an expulsion of the secreted
material from the gland proper. One remarkable feature of pilocarpine stimu-
lation shown in this case is the extreme rapidity of its action as compared with
its action on salivary glands. According to Cohoe, the submaxillary gland
of a rabbit showed all the granule-cells loaded with granules after 3 hours’
stimulation with a dosage of 0.014 gm. per kilo of body-weight of the animal.

29

30 THE VENOM OF HELODERMA.

(1c) Guanp Removep 35 MINUTES AFTER INJECTION OF PILOCARPINE.

The appearance of this gland was very similar to the preceding, the chief
difference being that the number of intralobular duct cells containing granules
was considerably less and that the granules were fewer in each cell; but in one
place near the cut surface of the gland granules were observed in abundance.
In the lumina of the tubules a moderate quantity of the granular secretion was
present, the amount being slightly more abundant than in 1s.

(1p) Guanp Removep 50 MINUTES AFTER INJECTION OF PILOCARPINE.

Granules appeared to be almost entirely absent in the intralobular duct
cells, only a few cells in the more distal portions of the tubules showing any
clear trace of them. The greater number of these showed the granules in what
appears to be an incipient stage of formation, namely, as dark-staining nodular
thickenings of the cytoplasmic reticulum. The majority of the granules were
minute, but some were larger and closely resembled the typical granules. Fre-
quently the granule-containing cells were observed to be much swollen, the
adjoining cells, which had not yet begun to reform the granules, being com-
pressed into a narrow space.

A few cells were observed each containing two nuclei, usually in process of
disintegration; but such cells were excessively rare, and, owing to the difficulty
of finding and of clearly identifying them, it is doubtful if they are normal con-
stituents of the gland. They are probably not a result of the pilocarpine
stimulation.

In the lumen of the tubules a granular secretion was present, but less in
amount than in the unstimulated gland. At certain places in this secretion
were observed many apparently pycnotic cells and nuclei. Whether these
were desquamated into the lumen in the normal process of secretion or were
merely detached as a result of the pressure to which the gland was subjected in
the process of removal from the animal we are unable to solve through a study
of the sections.

ExpEniment II.

An animal (C) was injected with 0.1 grain of pilocarpine, a portion of the
unstimulated gland having been previously removed and fixed in Kopsch fluid
as acontrol. The animal was then injected and 10 minutes after the injection
another portion was removed and fixed.

(C1) Normat, UNSTIMULATED GLAND.

This gland appeared to be normal, though in many of the tubules the cells,
or a considerable number of them, were devoid of granules; but such a condi-
tion is not unusual for normal glands, as shown, for example, by the series of
sections on which the description of the normal gland was based and in which
many of the lobules were mentioned as showing only a few granule-containing
cells, while in others the cells were crowded with granules. A similar condi-
tion is shown by this gland. In many lobules numerous cells containing the
granules were observed, while in others the cells were empty.

STRUCTURAL CHANGES IN THE POISON GLAND. 31

(C*) Guanp 10 MINUTES AFTER INJECTION OF PILOCARPINE.

Sections showed the granules still present in a considerable number of
cells, but the latter were less numerous than in the unstimulated gland. The
number of granules in each cell was also less. The lumen of the tubules con-
tained an abundant granular mass.

EXPERIMENT III.

An animal (D) was injected with 0.1 grain of pilocarpine and, after an in-
terval of 24 hours, pieces of the gland were removed and fixed in Kopsch fluid.
Examination of the sections indicated that the gland had almost regained its
normal activity. Some of the lobules were apparently perfectly normal, the

Fic. 13.—Two thin sections of intralobular tubules of a gland 24 hours after injection of pilocarpine. Some cells show
initial steps in granule formation (Zeiss oc. 4, obj. 3 mm., ap. 0.95).

number of granule-containing cells being nearly if not quite as numerous as
in similar lobules of the normal gland. In other lobules the number of such
cells was much less and in many instances they contained only a few granules.
In many cells the granules were apparently in the process of formation, appear-
ing as minute particles at the intersections of the cytoplasmic network.

Fig. 13 represents a section passing through two intralobular tubules. The
greater number of cells, it will be observed, are filled with a relatively dense
cytoplasm similar to that which in normal granule-containing cells forms a
basal layer (cf. fig. 10). In other cases the cytoplasm is more clearly alveolar.
In these cells granules are usually present, in some cases in early stages of
formation, in others almost fully formed.

32 THE VENOM OF HELODERMA.

Compared with the eclls of the normal tubules, the cells of the gland, after
injection of pilocarpine, appear much shrunken. The free surface of the cells
also appears to be less regular, though in neither case have we been able to
observe a limiting membrane, the naked protoplasm appearing to be in direct
contact with the lumen (cf. fig. 13).

A small amount of secretion was present in the tubules. This was similar
in character to that observed in other glands, both normal and stimulated.
As shown in fig. 13, it consisted of a finely granular matrix, in which were em-
bedded a number of relatively large vesicular masses. These bodies are cells
apparently detached from the epithelial lining. They contain a finely granular
cytoplasm which shows in some cases a clear alveolar and in others an irregular
structure. Occasionally, as shown in fig. 13, a few small secretion granules
may be found in the vesicles. In the figure nuclei are not shown in these bodies,
but in other sections the nuclei, usually much shrunken, have been seen.
Sometimes free nuclei occur in the secretion.

In the same figure some of the cells are scen giving off a clear secretion
which, as it leaves the cell, assumes the form of a vesicle. In the lumen, how-
ever, as indicated in one part of the figure, these vesicles soon lose their regular
outline and disintegrate.

EXPERIMENT IY.

An animal (B) was injected with the usual dosage of pilocarpine and on the
following day the injection was repeated. Two days after the first injection
the animal was again injected, making altogether three injections at intervals
of 24 hours. Immediately before the last injection a portion of the gland was
removed and fixed in Kopsch fluid. A second portion was removed and fixed
15 minutes after the injection.

(B!) A GLAND STIMULATED ON Eacu or THE Two Precepine Days.

This gland was removed immediately before the third injection. After
having been twice stimulated at intervals of 24 hours, it had been given for 24
hours opportunity to recover from the effects of the stimulation. Examina-
tion of the sections showed that it had done this very imperfectly. The gran-
ules were found to be rather numerous in the cells of a few lobules, but they were
either entirely lacking or exceedingly scarce in most of them. The limen of
the tubules was found to contain a considerable amount of secretion.

(B?) Guano 15 Minutes Fottowine Tuirp Insection or PILocaRPINE.

Careful examination of the sections failed to reveal granules in any part of
the gland. The ducts contained considerable granular material in which dis-
integrating cells and vacuolar vesicles were scattered.

EXPERIMENT V

Simultaneously with the preceeding animal, another individual (C) was
injected on three successive days. The third injection was made with 0.125
grain of pilocarpine. One part of the gland was removed previous to this

STRUCTURAL CHANGES IN THE POISON GLAND. 33

injection and 17 minutes after the last injection of pilocarpine another part
was removed. This experiment gave results similar to Experiment IV, the
chief difference being the amount of secretion present in the lumen of the
tubules. In the present instance this secretion was scanty, whereas in B it
was quite abundant. In that part of the gland removed previous to the third
injection, the cells containing granules were very few and were found in widely
separated parts. In the part removed 17 minutes after the third injection, no
granules were seen in any part.

CONCLUSIONS.

The experiments with pilocarpine warrant us in drawing the following
conclusions:

(1) Injection of pilocarpine in doses of 0.1 grain begin to act almost imme-
diately upon the poison gland, causing within a period of 15 minutes an almost
complete disappearance of the secretion granules from the intralobular duct
cells.

(2) Recovery of the gland from the effects of a single injection of pilo-
carpine is noticeable as early as one hour after the injection, and is indicated by
a swelling of the intralobular duct cells and a formation within them of minute
thickenings of the cytoplasmic reticulum.

(3) Full recovery from the effects of a single injection would appear to
require at least an interval of 24 hours.

(4) Two injections of the usual dosage of pilocarpine, the second injection
following the first after an interval of 24 hours, appear to exert a stronger
action upon the gland, as after 24 hours’ rest a gland so stimulated shows only a
partial recovery from the effects of the injection.

(5) Two injections of pilocarpine, carried out as mentioned in the preced-
ing paragraph, appear to have no effect upon the ability of the gland to respond
to the action of pilocarpine. A gland injected twice with the usual dose of
pilocarpine responded just as readily as a normal gland to the action of the
drug after 24 hours’ rest, although the gland had only imperfectly recovered
from the first two injections of pilocarpine.

C, TRANSPLANTATION OF THE VENOM GLAND.
By Henry Fox anp Lro Lors.

With one exception all the experiments mentioned under this heading
were carried on in the early summer of 1909. Two series of transplantations
were planned. In the first series a portion of the gland was cut out and trans-
planted to the right side of the thorax of the same animal, where it was inserted
within the muscles just underlying the skin. In the second series another por-
tion of the same gland was transplanted to a similar place on the left side of a
different individual. The transplanted glands were then removed after inter-
vals of one, two, three, and four weeks, respectively, fixed, and sectioned. In
every case where a transplanted gland was removed, a piece of the normally
situated injured gland and one of the uninjured gland were removed and
studied as controls.

Serizs I.
ExpreriMentT A.—GuaND EXAMINED ONE WEEK AFTER TRANSPLANTATION.

The animal H was operated upon on June 4. A portion of the right gland
was cut out and divided into two pieces. One piece, designated H!, was trans-
planted to the right thorax of the same individual; the other, designated K’*,
was transplanted to the left thorax of another animal, K.

One week later (June 11) the piece H! was removed and fixed in Kopsch
fluid. Previous to fixation a small section was cut off and transplanted into
an albino mouse, which died a day or two later. Examination of the gland
showed the central area completely necrotic, the entire mass being composed
of an irregular aggregation of opaque, more or less hyaline particles, among
which were scattered numerous, small, densely-staining granules representing
necrotic nuclei. Surrounding the necrotic area more or less completely was a
middle zone of pyenotic elements. In this zone the individual cells were recog-
nizable, but much shrunken; their nuclei were shriveled and closely pressed
against the basement-membrane.

The peripheral portion consisted in part of an extensive zone of nearly
normal gland-tissue. In this zone the terminal acini were recognizable, but
their typical structure was obscured by a process of desquamation whereby the
central lumen had become largely filled with necrotic cells. The living cells of
the acini were quite large and apparently normal, except that the cytoplasm
was less clearly alveolar in structure and tended to collect in minute droplets,
thereby giving the cells a decidedly granular appearance. As a rule, the
nuclei were also more shrunken than those of the normal gland-cells. In many
cases the chromatin was densely aggregated throughout the entire nucleus; in

35

36 THE VENOM OF HELODERMA.

other cases it formed discrete particles or knots, the remainder of the nucleus
being nearly clear. None of the cells on this gland were observed to contain
typical secretion granules.

Experiment B.—G.Lanp ExaMINeD Two WEEKS AFTER TRANSPLANTATION.

On the same day that animal H was operated upon, another individual,
K, was also operated upon. Itsright gland was exposed and a portion removed.
This was then divided into two pieces, one piece, K', being transplanted to the
right thorax, while the other, H’, was transplanted to the left thorax of H.

Two weeks later, June 18, the former piece, K', was removed and cut into
two pieces. The larger piece was fixed in Kopsch fluid, the smaller trans-
planted to an albino mouse, which died shortly afterwards. In this gland the
same zones were observed as in the preceding, 7. e., a superficial zone of nearly
normal gland-tissue, a middle zone of pyenotic elements, and a central area
of necrotic material.

The areaoccupied by the apparently normal gland-tissue was considerably
larger in this than in the preceding, H', and it had a much more healthy aspect.
In H' this area formed a very thin zone, while in K? it formed nearly a third of
the entire transplanted piece. In some of the tubules and acini a large and
distinct lumen was present, quite normal in every respect, and in some of the
cells of the tubules the typical secretion granules were observed, whereas in
the transplanted piece, removed one week after transplantation, no trace of
these was evident. In many of the tubules and acini the lumen was more or
less occluded by dead and desquamating cells in a manner similar to that
found in H'. The nuclei of these cells in the peripheral tubules were appar-
ently quite normal, in strong contrast to those of H!, which were nearly all
shrunken. Some of the nuclei of the gland Kt! presented the same shrunken
aspect, but they were the exception and not the rule.

The middle pycnotic zone was variable. Those cells lying nearest the
normal layer were rather indefinitely outlined, but had nuclei of nearly normal
form and size, with a clear ground-substance and somewhat scattered chromatin
particles. The deeper cells were more degenerated, with small shrunken nuclei
in which the chromatin was massed in lumps.

The necrotic portions had no special features to warrant description. It
was similar in every respect to the corresponding portion in H!.

ExpEerIMeNtT C.—G.Lanp Removed THREE WEEKS AFTER TRANSPLANTATION.

An animal (M) was operated upon on May 31. As in the other casesthe
right gland was exposed and a portion of it removed. One half, M’, of this
was then transplanted to the right thorax, while the other half, N*, was trans-
planted to the left thorax of another individual, N. Three weeks later the
transplanted gland M! was removed. Part was fixed in Kopsch fluid and the
remainder transplanted to a mouse, which died within an hour after the
operation.

As shown in typical sections, this gland appeared to consist of equal parts
of normal and necrotic elements. Toward one side all the lobules were normal;

TRANSPLANTATION OF THE VENOM GLAND. 37

toward the other they were necrotic. A few lobules near the center were inter-
mediate, being in part necrotic, in part consisting of normal gland-tissuc.

In the living lobules the cells were in general large and quite irregular,
differing in no visible respect from normal gland-cells. In most of the living
lobules the cells appeared in good condition; in some, especially those near
the center, a narrow zone of pycnotic cells intervened between the living cells
and the necrotic mass. In many of the lobules the tubules and acini showed
a large and distinct lumen which contained a mass of more or less translucent
particles and numerous desquamating cells or cell-groups.

The cytoplasm in the living cells was moderately dense and finely reticu-
late with minute granules. But no typical secretion granules were found in
them.

In some acini indistinct mitotic figures were observed, but were very scarce.

In the intermediate zones there were numerous dying cells. Some of the
cells were apparently living, but showed no distinct outlines or regular struc-
ture. Other cells were entirely vacuolar, owing to liquefaction of their con-
tents. Many of their nuclei were large and vesicular, with an almost colorless
ground-substance and scattered chromatin knots.

In the necrotic portion of the gland were observed numerous connective-
tissue cells apparently invading it from all directions. Their nuclei were rather
regular in outline, sometimes rounded, sometimes elongated, and occasionally
constricted on one side. Each showed a clear ground-substance through which
were scattered numerous fine chromatin particles, while part of the chromatin
was aggregated near the center into a conspicuous nucleolus-like mass.

EXPERIMENT D.—GLANnD REMOVED Four WEEKS AFTER TRANSPLANTATION.

A fourth animal (N) was operated upon on May 31. A portion of its
right gland was removed and divided into two pieces. One piece, N!, was
then transplanted to the right thorax of N; the other, M’, was transplanted
to the left thorax of M. The gland N! was removed June 28 and fixed in
Kopsch fluid. A part of it was transplanted to a mouse, which died after a
few days.

This piece of gland was more nearly like the normal gland than any of the
other transplanted pieces removed at earlier periods. The peripheral lobules
were all quite normal in appearance. Thus in one lobule selected for study
the terminal acini were to all appearances quite normal and constituted the
bulk of the lobule. Each acinus was separated from adjacent acini by clear
fibrous septa, similar to those separating normal acini. The cells were typical
in form and contained an alveolar cytoplasm in which coarse granulations were
present at the nodes of the reticulum. The nuclei were situated close to the
basement-membrane, and in many cases were rather more shrunken than normal
nuclei. This shrunken condition was not, however, pronounced, while in other
cases the nuclei were apparently quite normal. Each nucleus contained a
clear nuclear sap in which were scattered grains of chromatin and a central
chromatin knot, the entire chromatin arrangement being essentially like that

38 THE VENOM OF HELODERMA.

observed in normal nuclei. In this lobule, the lumina of the smaller acini
were either empty or contained only an insignificant amount of a coarsely gran-
ular mass. In the larger acini or tubules this mass was much more copious.
In some cases, especially in the largest tubules, it also contained free nuciei,
which in one instance were observed aggregated in a dense cluster in the center
of the tubule.

In the lobule just below the one described in the preceding paragraph,
but in a deeper part of the gland, the conditions deviated somewhat from the
normal. In this lobule the intralobular ducts were numerous and in most
cases of relatively large diameter. They contained a granular mass with
occasional desquamated cells or nuclei. They were lined by a single layer of
epithelium which, in those tubules or those parts of a tubule nearest the
surface of the gland, consisted of either columnar or cubical cells, while in the
deeper tubules or deeper parts of a larger tubule the cells were much flattened.
In that portion of the lobule nearest the surface of the transplanted piece, the
various tubules and acini were separated by clearly defined fibrous partitions,
but in the deeper portion of the lobule these were less distinct and were largely
replaced by solid aggregations of rounded or spindle-shaped cells. Usually
these cells were clustered about the tubules and were not always clearly separ-
able from the epithelial lining of the ducts. A mitotic figure was observed
in one of these cells. The origin of these cells we have not been able clearly
to determine. They may possibly be derived from the proliferating epithelium
of the tubules, their usually close association with the latter favoring this
view; on the other hand, they may be derived from proliferating connective-
tissue cells.

Since these cell-aggregations largely grouped themselves about the vari-
ous tubules, there were left between them narrow, clear spaces with indications
of a very loose connective tissue through which were scattered minute granules
of apparently necrotic material.

In another lobule lying to one side of the one last described, the conditions
were still less normal. The superficial part of the lobule alone showed tubules
with a distinct lumen; in the remainder of the section the outlines of the orig-
inal tubules were rather indistinctly indicated by clusters of rounded cells, the
cells of each cluster being grouped more or less concentrically around the
center, which in one case at least showed signs of a lumen. These clusters
were observed to be separated by an anastomosing network of loose connective-
tissue septa in which necrotic particles were abundant and in which numerous
elongated or spindle-shaped cells were scattered.

In the deeper parts of this lobule most of the solid cell-clusters were very
small—considerably smaller than any tubules observed in normal glands. A
number of these appeared to be grouped in a radial manner about a common
center and invested by an indistinct layer of necrotic connective tissue. These
masses appeared to correspond to single tubules, the solid cell-mass of the
latter having degenerated into a number of cell-clusters between which con-
nective-tissue cells then penetrated.

TRANSPLANTATION OF THE VENOM GLAND. 39

One lobule, which was situated beneath the superficial zone, showed three
well-defined areas in which the tubules were in different conditions. In the
more superficial area there was a fairly broad cap of clearly defined tubules and
acini, in each of which a large lumen was present. These tubules were sepa-
rated by thin, fibrous septa, which, however, were much more nearly normal
in the superficial part of the zone than in the deeper part, where the fibrous
elements were largely necrotic and partly replaced by thin layers of elongated
cells. The epithelial lining of the tubules was not entirely normal, the cells
showing considerable shrinkage, with loss of clearly defined outlines, in many
instances. Each tubule in this zone contained a considerable amount of gran-
ular material through which, in some cases, were scattered a few nuclei.

Below this superficial area came a middle zone of the lobule which was
almost entirely occupied by four exceptionally large vesicle-like tubules.
These tubules were considerably larger than any of the normal intralobular
tubules. They may perhaps have been formed by the fusion of two or several
adjoining tubules as the result of the degeneration of the intervening septa and
of the desquamation of some of the epithelial elements. Each was lined by a
single layer of greatly flattened cells, resembling very much the epithelium of
the lung alveoli. These large tubules contained considerable granular matter,
in some cases with, in others without, desquamated cells and nuclei. In the
largest tubules these desquamated cells were collected into two compact
masses, one situated in the center of the lumen, the other to one side, close to a
point where the main body of the tubule opened widely into a smaller vesicle.
In the case of the latter the origin of such masses by the aggregation of desqua-
mated cells was clearly shown, one cell of the mass with its nucleus being
observed detaching itself from the epithelial wall.

The deepest zone of this lobule consisted of several circular or ovoid solid
masses of round cells, each mass being separated from its neighbors by a sep-
tum formed of rather loose connective tissue.

In this gland mitotic figures were observed in fair numbers, indicating a
rapid proliferation of the cellular elements of the gland.

Regular, clearly defined secretion granules were not abundant in any of the
lobules, even in the almost normal ones near the surface, but they occurred in
one or two of the latter, and in the cells of some of the tubules were almost as
abundant as in normal cases.

The lobules in the interior of the gland were largely necrotic, but fre-
quently one or several tubules were preserved or were represented by solid
cellular masses, similar to those already described. The necrotic material,
however, was much less dense than in the transplanted glands already de-
scribed, and interspersed amongst it were numerous apparently normal cells,
some clearly rounded, others ovoid or spindle-shaped, of connective-tissue
origin. We see, therefore, proliferating connective tissue substituting part of
the necrotic material.

40 THE VENOM OF HELODERMA.

EXPERIMENT E.—Guanp Examinep Two Monrus arrer TRANSPLANTATION.

It was considered advisable to observe the changes produced in the gland
after a longer period of transplantation. Accordingly, on January 18, 1910,
we removed a piece of the right poison gland of a Heloderma and inserted it
into the same side of the thorax of the same animal. On March 18 the trans-
planted gland was removed and fixed in Kopsch fluid. Typical sections
showed almost the entire gland to be composed of fibrous connective-tissue
through which were distributed numerous connective-tissue cells, mostly
spindle-shaped. In most cases the connective tissue of the different lobules
had so completely fused as to form a nearly homogeneous mass, in which it was
almost impossible to recognize the outlines of the original lobules, tubules, or
acini. Remnants of the gland were, however, present in the form of large

Fia. 14.—Section of peripheral part of a transplanted gland removed from the thoracic region of a Heloderma one week
after transplantation. The nearly normal living elements of the peripheral portion and the pycnotic and
necrotic mass of the deeper portion are shown (Zeiss oc. 4, obj. AA).

vesicles, similar in all essential respects to the large vesicle-like tubules de-

scribed under Experiment D. Some of the vesicles were close together, but

others were separated by broad partitions of connective tissue. The cells of
the vesicles were mostly rather flat, but some of them were cubical or columnar.

Closely associated with one group of these vesicles were several tubules
of normal size and approximately normal appearance. Other tubules or fol-
licles of small size were observed scattered through the general fibrous tissue,
but were few in number. A considerable part of the transplanted gland-tissue
at some distance from the vesicles just described was entirely necrotic. Along
its edge next to the connective tissue the necrotic material was being replaced
by ingrowing masses of connective-tissue cells.

TRANSPLANTATION OF THE VENOM GLAND. 41

Series IT.

In this series a portion of the poison gland of one individual was excised
and transplanted to the left side of the thorax of a different individual.

EXPERIMENT A.—GLAND REMOVED ONE WEEK AFTER TRANSPLANTATION.

On June 4 a part of the right poison gland of individual K was trans-
planted to the left thoracic wall of H. On June 11 this was removed and
fixed in Kopsch fluid. A portion of this gland is shown in fig. 14. In its
general features sections of this gland resembled those of H', as described under
Series I. The chief difference was in the size of the intermediate pycnotic
zone, which was either very narrow or absent in this gland. The periphery
of the gland consisted largely of a relatively broad zone of apparently normal
gland-tissue, aggregated into tubules and acini, all of which were provided with
clear and distinct lumina.

The inner necrotic portion formed fully three-fourths of the entire mass
of the transplanted piece. Here the various lobules and their subdivisions
were distinctly visible, and even the lumina of the tubules were recognizable,
but the cellular elements were fully necrotic. No typical secretion granules
were observed in any of the cells of this gland.

ExpPERIMENT B.—Guanp RemMoveD Two WEEKS AFTER TRANSPLANTATION.

This piece of gland was obtained from individual H on June 4 and was
transplanted to K, from which it was removed on June 18. This piece,
designated K’, was nearly normal throughout. Only in the very center of the
section was a relatively small necrotic area. The cells of the approximately
normal portion had the usual size, but their cytoplasm appeared rather more
granular than normal. The typical secretion granules were also present, but
were confined to the intralobular ducts of a few lobules. In this specimen the
nuclei, even of otherwise apparently normal cells, appeared to be much shrunken.

As mentioned above, the necrotic area was confined to the central part
of the transplanted piece. In places the pycnotic and necrotic elements were
inclosed by an investing sheath of fibrous tissue. Usually the tubules of these
areas were distinct, but in most cases they lacked a distinct epithelial lining
and their lumina were greatly occluded by large quantities of desquamated
cells and necrotic matter.

In all tubules, both normal and pycnotic, there were large accumulations
of desquamated cells.

EXPERIMENT C.—GLAND REMOVED THREE WEEKS AFTER TRANSPLANTATION.

This piece of gland, M’, was obtained from individual N on May 31 and
transplanted to M, where it was allowed to remain until June 21, when it was
removed and fixed in Kopsch fluid; part of it was transplanted to a mouse
just after the second removal, the mouse dying within a few days. M? was
rather less normal than piece K’, removed two weeks after transplantation,
and in many respects closely resembled the piece H?, removed after one week.
Most of the peripheral portion, forming about one-third of the total mass,

42 THE VENOM OF HELODERMA.

consisted of apparently normal tissue aggregated into acini and tubules, each
with a clear and distinct lumen, the latter usually containing more or less
granular and desquamated material. The cells in general were regular in size,
structure, and contents. The typical secretion granules were mostly absent,
but in one lobule they were observed in profusion, the cells in which they
were found having the appearance of normal intralobular duct-cells. In the
deeper portions of the peripheral zone the cells were not so regular and the
lumina of the tubules were less distinct. The interior of the piece consisted
entirely of necrotic material.

EXPERIMENT D.—Guanp REeMovED Four WEEKS AFTER TRANSPLANTATION.

The gland used in this experiment was obtained from individual M on
May 31 and was transplanted to the left thorax of individual N. There it
remained until June 28, when it was removed and fixed in Kopsch fluid. A
small piece of it was at the same time used to inject a mouse, which died
shortly afterwards. The most prominent feature shown by this gland was
an extensive proliferation of small round cells, either of connective-tissue
origin, or leucocytes emigrated from the blood-vessels. These cells were
exceedingly abundant about the periphery of the gland, where they formed a
dense layer of considerable average thickness. Beneath this layer was a zone,
not very thick, of gland-tissue, which, however, was in most lobules not
entirely normal. In one or two lobules some of the acini seemed to be entirely
normal, each with a clear lumen, but usually the cells were more or less
shrunken, while the lumina were either indistinct or filled with desquamated
cells or disintegrated material. Here and there in these same lobules some
of the larger tubules showed a distinct lumen, but the cells lining it were much
more flattened than in normal cases.

The small round cells mentioned above extended into the lobules along
the septa. The latter showed only slight traces of fibrous tissue, and were
almost entirely composed of strands of round cells. In the deeper layers of
the transplanted piece, where the acini were mostly pycnotic, the round cells
spread out beneath the lobule, frequently forming dense masses aggregated
about the dying gland-cells or congested blood-vessels. From these points the
small cells were found to radiate into the subjacent necrotic areas.

The necrotic region of the transplanted piece was very extensive. Com-
pared with the same region in the transplanted pieces removed at an earlier
period, it was distinguished by a great reduction in the amount of actual
necrotic material. In the earlier stages the latter was so abundant as to make
the necrotic regions more or less strongly opaque. In the present gland, on
the other hand, the actual quantity of purely necrotic material was small,
the entire region consisting almost exclusively of a loose fibrous tissue, with
large, clear interspaces and numerous widely scattered round cells.

In a few places the round cells were observed to form dense strands
extending from the periphery into the necrotic portions. Usually, however,
the infiltration appeared to be irregular, the cells dispersing in all directions
from the aggregations at the base of the lobules.

None of the typical secretion granules were observed in this gland.

TRANSPLANTATION OF THE VENOM GLAND. 43

Serres III.

In Series III portions of those glands from which the pieces transplanted
in the preceding experiments had been cut were examined. ‘They were used
partly as control and partly to determine whether regeneration of the excised
portions took place. For the latter purpose the sections were made at right
angles to the superficial blood-clot which filled the cavity produced by the
excision. Examination of the sections showed the glands to be normal in every
respect. No sign of regeneration of the missing parts of the gland was observed.
Measurements of the cells showed that no perceptible shrinkage or hypertrophy
had taken place.

Series IV.

In Series IV pieces taken from the normal, uninjured gland of the animals
in which the gland of the other side had been partly excised were examined.
They were used as controls. These glands were perfectly normal in every
respect. Typical cells were measured and were found to have the average
size of cells of glands taken from animals that had never been operated upon.

SERIES V.

Only one experiment was made in Series V. A part of a normal gland,
taken from Gila monster N, was transplanted to a turtle. The latter died 3
days later. The transplanted piece was then removed and fixed in Kopsch
fluid. Microscopic examination of this piece showed the superficial portions
apparently quite normal. The cells were of the average size and their con-
tents, although somewhat granular, were not much more so than may be seen
frequently in preparations of the normal gland. Inthe intralobular duct-cells
the large, typical secretion granules were often present, sometimes quite filling
up the entire space within the cell. There was very little desquamation in this
region. The deeper lobules were much disorganized, although the form of the
individual tubules and acini had been fairly well preserved. The elements
were pycnotic and there was much desquamation, the lumina containing large
quantities of both disintegrated material and necrotic cells and nuclei.

SUMMARY.

(1) Pieces of the poison gland, transplanted to the muscles and subcu-
taneous regions of the thorax, continued living in their peripheral portions for a
period of at least a month, while in one case a few tubules were observed alive
after an interval of 2months. The central portions of the transplanted glands
always undergo necrosis. In the course of from three to four weeks the
necrotic material is largely absorbed and its place taken by connective tissue.

(2) There appears to be no marked difference between_the pieces trans-
planted into the same animal from which the piece of the gland had been
excised and those transplanted into other individuals of the same species.

(3) The transplanted glands show little, if any, power of regeneration.
The cells of individual tubules may, however, divide by mitosis and replace the
necrotic cells of the same tubules, but any further regeneration apparently does.

44 THE VENOM OF HELODERMA.

not take place, although an extensive desquamation of cells takes place in the
tubules and acini of the transplanted glands.

(4) In the living portions of the transplanted pieces, the regular secretion
granules were observed in those pieces which had been removed two, three, and
four weeks after transplantation, none being seen in those removed a week
after the operation. The transplanted glands retain, however, their toxic prop-
erties at all times, as shown by the fact that when pieces of them are used to
inoculate mice the latter quickly die; but whether this is due to the venom
already present in the gland at the time of transplantation, or to venom newly
formed and secreted after transplantation, is not determinable from these
experiments.

(5) Because of the abnormal conditions under which the gland-tissue lives
after transplantation, various abnormalities in its structure are found. The
number of the typical granules is in every case diminished. Some changes
which we observed are probably due to the absence of an effective excretory
duct. Thus the collection of desquamated cells or cell detritus in the ducts,
the flattening of certain tubule cells, and the dilation of some ducts may be due
to this factor. The frequent shrinking of cells and nuclei and the substitution
of the typical tubules by clusters of cells markedly differing in their shape from
the normal gland cells are results of the abnormal conditions under which cells
live after transplantation.

(6) In the glands from which pieces had been cut. no regenerative process
could be observed, nor had a noticeable compensatory hypertrophy taken place
in the gland of the other side within 4 weeks after the operation.

REFERENCES.

Couor, B. A. The finer structure of the glandula submaxillaris of the rabbit. (Amer.
Journ. of Anat., vol. 6, No. 2, pp. 167-190, Jan. 1907.)

Corr, E. D. The crocodilians, lizards, and snakes of North America. (Rept. U.S. Nat.
Mus., 1898, Washington, 1900.)

Duasé, A. Venin de l’heloderma horridum. (Comptes rendus de la soc. de biologie, pp.
134-137, 1899.)

Fiscupr, J.G. Anatomische Notizen itiber Heloderma horridum. (Verhandl. des Vereins
fiir naturw. Unterhaltung zu Hamburg, v, pp. 2-16, 1882.)

GEGENBAUR, C. Vergleichende Anatomie der Wirbelthiere. (Leipzig, 1901.)

Harvey, B.C. H. The study of the structure of the gastric glands of the dog and of the
changes which they undergo after gastro-enterostomy and occlusion of the py-
lorus. (Amer. Journ. Anat., vol. 6, No. 2, pp. 207-248, 1907.)

Hom, J. F. Some notes on the histology of the poison glands of Heloderma suspectum.
(Anat. Anz., x11, pp. 80-85.)

Matuews, Atpert P. The changes in structure of the pancreas cell. A consideration of
Tas of cell metabolism. (Journ. Morph., xv, Supplement, 1899, pp.
171-216.

OppeL, A. Lehrbuch der vergleichende mikroskopische Anatomie der Wirbelthiere, Th.
1, Jena, 1900.)

Metzner, R. Die histologische Veranderungen der Driisen bei ihrer Tatigkeit. (Hand-
buch der Physiologie des Menschen, edited by Nagel, p. 899, seq.)

Launay, M. L. Sur quelques phénoménes nucléaires de la secretion. (Note de M. L.
Launay, présentée par M. Joannes Chaten, Comptes Rendus, 136, 1903.)

‘Suureipt, R.W. Contributions to the study of Heloderma suspectum. (Proc. Zool. Soc.,
London, 1890, p. 148.)

Suureipt, R. W. A note acknowledging the correctness of Stewart’s views. (Nature,
XL, p. 514, 1891.)

Srewart, C. On some points in the anatomy of Heloderma. (Proc. Zool. Soc., London,
1891, p. 119.)

). SOLUBILITY OF THE VENOM GRANULES,

By Henry Fox anp Leo Lors.

Whenever a piece of the fresh poison gland is cut into small particles and
the contents of the latter squeezed out into 0.85 per cent (NaCl) salt solution,
there results a cloudy emulsion. Microscopic examination shows this to con-
tain innumerable granules of two kinds—spheres and rods. The rods were
abundant and were at first supposed to be distinct from the spheres. Nothing
like them had been observed in sections of the fixed and stained gland, though
in some cases, especially in the formative stages, the intracellular granules
were frequently seen to be more or less elongated, but they never showed the
clear outlines of the rods as seen in the emulsion. It occurred to us that they
might be composite granules; that is, composed of two or several spherical gran-
ules arranged in a row. To determine this point they were examined under
the oil-immersion objective. In some cases, indeed, the composite nature of
the rods appeared fairly evident; in such instances the rod could be seen to be
cut across by what appeared to be one or more transverse walls, appearing
much like the intercellular partitions in the chain of a rod-shaped bacterium.
In other cases the rods showed no clear indications of a composite structure.

The following tests were made to determine the solubilities of the granules.
We also wished to examine in a comparative manner the behavior of the rods
and spheres. We found that both behaved on the whole alike. This fact.
renders still more probable the conclusion that the rods and spheres represent
the same chemical substance and differ only morphologically.

RECORDS OF TESTS.
A. SoLutions or Hyprocuioric Ac.

(A}) 1 c.c. of N/10 HCl added to 1 ¢.c. of the suspension: Granules dis-
solved in 13 minutes. Microscopic examination, 10 minutes later, showed no
clear indications of granules. Some minute objects of doubtful nature were
observed.

(A’) 0.1 e.c. N/10 HCl added to 0.5 ¢.c. suspension: Granules partly dis-
solved in 14 minutes. Microscopic examination after 7 minutes showed no
certain traces of granules, but free nuclei of blood-corpuscles were present.

(A3) 0.1 ¢.c. N/10 HCl added to 0.9 ¢.c. of the suspension: Liquid re-
mained cloudy, and after an interval of 30 minutes was still slightly opalescent.
Microscopic examination, 7 minutes after addition of reagent, showed both
granules and rods present, though not in nearly as large a quantity as in a con-
trol. A second examination, after 31 minutes, showed no distinct rods or
granules. Free nuclei were moderately abundant.

45

46 THE VENOM OF HELODERMA.

In more dilute solutions of HCl (N/100 to N/250) the granules and rods
seem also to become to a great extent dissolved, but here a finely granular pre-
cipitate forms in the mixture of solution and acid, and this precipitate makes it
difficult to distinguish the preformed from the granules of the precipitate.

B. Tests wits Sopium HypRatTe.

(B') 1 cc. N/10 NaOH added to 1 ¢.c. suspension: Granules dissolved
after interval of 14 minutes. Microscopic examination after 10 minutes showed
the liquid clear, except for traces of an occasional granule.

(B’) 0.1 cc. N/10 NaOH added to 0.5 ¢.c. suspension: Granules dis-
solved in 40 seconds. Microscopic examination after 5 minutes showed the
liquid clear, with a few nuclei of blood-corpuscles or perhaps also of other cells.

(B*) 0.1 ¢.c. N/10 NaOH added to 0.9 c.c. suspension: Granules dissolved
in 5 seconds. Microscopic examination, after 9 minutes, showed the liquid
clear, with no signs of granules.

(B*) 0.5 c.c. N/100 NaOH added to 0.5 ¢.c. suspension: Granules dis-
solved in 5 seconds. After 27 minutes the liquid was very clear. Microscopic
examination after 21 minutes showed the liquid almost clear, with no distinct
trace of granules. In alkaline mixtures the fluid becomes clearer than in the
acid solutions of corresponding strength. If the alkaline solutions through
addition of NaCl are made isotonic with a 0.85 per cent NaCl solution, some of
the granules remained somewhat longer preserved than with hypotonic alka-
line solutions made up with distilled water.

C. Tests witH So.tutions or NaCu.

(C!) 1 ec. 0.85 per cent NaCl added to 1 ¢.c¢. suspension: Granules not
dissolved after 13 minutes. Microscopic examination after 3 minutes showed
granules and rods in abundance.

(C*) 0.1 ¢.c. 0.85 per cent NaCl added to 0.5 ¢.c. suspension: Granules
not dissolved after 1 minute. Microscopic examination after 8 minutes gave
same result as preceding observation. Other similar experiments gave the
same results. After 3 hours the fluid had become less cloudy. Microscopic
examination at the same time showed that the granules were extremely scarce,
only a few distinct ones being seen.

(C®) 0.5 ¢.c. 1.5 per cent NaCl added to 0.5 c.c. suspension: Granules
not dissolved after 30 seconds. Microscopic examination after 2} minutes
showed the rods and granules well preserved. The same mixture examined
after 3 hours showed microscopically only small numbers of granules preserved.

In 0.85 per cent NaCl solution a slow but continuous solution of the gran-
ules takes place. They remain better, but not definitely preserved, in a
hypertonic NaCl solution, made up by mixing equal quantities of the emulsion
of granules and of a 1.7 per cent NaCl solution.

D. Tests wirn DistinLep WATER.

(D') 1 ¢.c. H,O added to 1 c¢.c. suspension: Granules partly dissolved
after 14 minutes. Microscopic examination after 5 minutes showed number of

SOLUBILITY OF THE VENOM GRANULES. 47

granules to have decreased much more than in control. In other experiments
with distilled water a large number of the granules disappeared within the first
few minutes.

In mixtures of the emulsion and distilled H,O the granules are not as well
preserved as in 0.85 per cent NaCl solution; they are, however, not as rapidly
dissolved as in a diluted solution of alkali. Glycerin also causes a disappear-
ance of many granules; at least the granules become less visible a short time
after the mixture with the glycerin.

In one experiment (87), 1 c.c. absolute alcohol was added to 0.5 ¢.c. emul-
sion. The liquid cleared almost immediately, but showed a slight cloudiness.
Microscopic examination, after 3 minutes, showed no granules, only an albu-
minous precipitate in which a few indistinct granules were intermixed and a
few apparent nuclei.

E. Tests with ForMALIN.

In a mixture of equal amounts of emulsion and of 10 per cent formalin dis-
solved in 0.85 per cent NaCl solution instead of water, the granules and rods
remain distinctly better preserved than in a 10 per cent solution of formalin in
distilled H,O. The limited number of animals which could be used for these
experiments, and the fact that each series of experiments meant the sacrifice of
an animal, necessitated the limitation in the number of our experiments; yet
we believe our results of sufficient interest to warrant publication. We hope
that our experiments may be repeated, and, if necessary, in some details cor-
rected in future investigations, but we believe that the following summary will
on the whole be found to be correct.

SUMMARY.

(1) The granules are readily soluble in weak solutions of HCl. They dis-
solve quickly in N/20 and N/50 solutions, but only more slowly dissolve in a
N ‘100 or N/200 solution.

(2) The granules dissolve very quickly in weak solutions of NaOH, even
such minute strengths as N/100 and N/200 dissolving them nearly as quickly
as the stronger solutions.

(3) The granules are dissolved only very slowly in normal salt solutions.
Hypertonic solutions of NaCl preserve the granules better than isotonic
solutions.

(4) The granules disappear more rapidly when the normal salt solutions
containing them are strongly diluted with distilled water or with glycerin, but
solution is not quite as rapid as in the case of HCl and NaOH solutions.

(5) Solutions of formalin dissolved in normal salt solution preserve the
granules better than 10 per cent formalin prepared with distilled water.

(6) The rods and spheres react in a similar manner toward the various
reagents.

Under normal conditions the granules probably are dissolved before the
venom enters the gland-ducts. At least we do not find granules in the contents

48 THE VENOM OF HELODERMA.

of the venom gland-ducts. If we collect venom in the manner usually em-
ployed in our work, we find the fluid collected from the mouth of the animal to
contain cells of an epithelial character and leucocytes. Many of these cells
contain granules, and through disintegration of cells nuclei get into the sur-
rounding fluid. In one case we saw only leucocytes in a drop which we col-
lected, and the fluid was here free from granules. The large majority of gran-
ules found in the venom collected from the mouth are probably not derived
from the venom gland, but from epithelial cells of the mouth and perhaps of
some neighboring glands.

I.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM OF
HELODERMA. AND EXPERIMENTS IN IMMUNIZATION,

By ExizaBetH Cooke anp Lro Logs.

(Wire Some ADDITIONAL EXPERIMENTS By Moyer 8. FLEIsHEer.)

GENERAL PROPERTIES AND ACTIONS OF THE VENOM OF
ITELODERMA. AND EXPERIMENTS TX IMMUNIZATION.

By ExizaBpetu Cooke anp Leo Logs.

(WirH some ADDITIONAL ExprRIMEeNTS BY Moyer 8. I'LEeIsHEr.)

REVIEW OF THE LITERATURE.

The Gila monster is a reptile found in the southwestern portion of the
United States and also in Mexico. The Heloderma suspectwm is usually found
in the United States; in Mexico the Heloderma horridum is more common.
The bite of either species is supposed to have a distinctly poisonous effect;
however, the general evidence in the literature regarding the toxic effects of
the venom of these animals is distinctly contradictory. The physiological
action of the venom has been studied by several observers: Sumichrast (Note
on the habits of some Mexican reptiles, Ann. and Mag. Nat. Hist., vol. 13 (3),
p. 497, 1864); Boulenger (Proc. Zool. Soc., London, 1882, p. 631); A. Dugés
(Cinquantenaire de la Soc. de Biologie, Volume jubélaire, publié par la Société,
p. 134, Paris, 1899); Gorman (Bull. Essex Institute, Salem, Mass., vol. 22,
p. 60, 1890); and Boncourt (Compt. rend. de l’Acad. des Sciences, vol. 80, p.
676, 1875). Boncourt caused animals of different species to be bitten by helo-
dermas and noted that the bite was fatal to smaller animals, such as frogs,
chickens, rabbits, and guinea-pigs, while dogs and cats developed serious sym-
toms, from which, however, they usually recovered. Besides these observa-
tions, there have been reported instances of more or less serious disturbances
arising from accidental injuries to human beings, but in regard to these the
evidence is very conflicting and appears to be scarcely trustworthy.

Mitchell and Reichert (Medical News, vol. 42, p. 209, 1883; Science,
vol. 1, p. 872, 1883; Amer. Nat., vol. 17, 800, 1883) were the first to collect the
venom and to study in detail the symptoms produced by injecting it into ani-
mals in known quantities. They caused the helodermas to bite upon a saucer
edge and with the secretion thus obtained they made experiments upon frogs,
rabbits, and pigeons. Since that time several other investigators have col-
lected the venom and have investigated its properties.

Santesson (C. G. Santesson, Ueber das Gift von Heloderma suspectum
Cope, einer giftigen Eidechse, Nordiskt Medicinskt Arkiv. Testband tillegnadt
Axel Key, Nr. 5, 1897) obtained the poisonous secretion by causing animals to
chew upon small sponges, which he afterwards washed out with physiological
salt solution. The venom thus obtained he studied with reference to its chem-
ical constitution and with it also made a number of experiments on frogs,
rabbits, and mice.

51

52 THE VENOM OF HELODERMA.

Van Denburgh (Trans. Am. Phil. Soc., vol. 19, 1898, p. 199) and Van
Denburgh and Wight (Am. Jour. Phys., vol. 4, 1900, p. 209) collected the
secretion either by causing the helodermas to chew filter paper or to bite on
rubber. They made observations on the physiological effects of the venom
upon dogs, cats, and frogs, and also a few experiments to test its effect upon the
red blood-corpuscles.

We have studied the venom of both species in regard to its physiological
action, in its influence in causing hemolysis of red blood-cells, and in several
other ways.

METHOD OF COLLECTING THE VENOM AND FACTORS WHICH
INFLUENCE THE AMOUNT OF SECRETION.

A number of small ducts lead from the poison gland into a groove between
the lower lip and the teeth of the lower jaw. When the animal bites the secre-
tion gushes into this groove. The ducts open near the bases of the small
pointed teeth; consequently the venom is ejected in the neighborhood of the
several wounds made by the teeth, the grooves present on the outer side of the
teeth carrying the venom into the wound; there is, however, no means by which
the Heloderma can inject venom directly into an enemy, as does the Crotalus.

In order to collect the venom we caused the animals to bite upon a piece of
soft rubber and with a capillary pipette drew off the secretion from the lower
side of the rubber. This method of collecting has the double advantage of
yielding a maximum of the secretion and also secretion from the lower jaw
only. An animal in good condition will usually chew the rubber for several
minutes and each time the jaws close there is a gush of venom into the groove,
but the length of time which the chewing continues, as well as the amount of
secretion which flows out at each bite, is subject to great variations. Chief
among the factors which influence these variations are:

(1) The condition of the teeth. The teeth are easily and frequently in-
jured, and animals without teeth spit out the rubber as soon as it is placed in
the mouth. Probably the reason that animals with good teeth do not spit out
the rubber is that the teeth become caught in the rubber, which thus can not
be pushed out by the tongue.

(2) The time which has elapsed since the previous collection. If the
secretion is taken every day the amount falls off rapidly, and even if the ani-
mals chew well the flow is quickly exhausted and may at times be absent
altogether.

(3) The nutritive condition of the animal. We found that animals which
had not eaten food for some time yielded little or no secretion, but that soon
after they began to take food again the secretion became abundant.

Besides these factors it seems that the temperature and the length of time
since the last feeding also play a réle, but the evidence on these heads is not
sufficient to enable us to draw definite conclusions. It is also probable that
when animals are kept for a considerable length of time in captivity they grad-
ually lose the power of secreting venom.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 53

INFLUENCE OF PILOCARPINE ON THE SECRETION OF VENOM.

In order to obtain greater quantities of venom we injected subcutaneously
0.1 grain of pilocarpine into a heloderma from which all secretion obtainable by
the usual method had been collected. As a rule, in from 1 to 3 minutes after
the injection the flow began again and lasted now 20 to 30 minutes, reaching
its maximum at 10 to 15 minutes after injection of pilocarpine. The flow
induced by pilocarpine not only lasts longer, but is more abundant than the
ordinary flow and frequently reaches three or four times the amount obtained
without pilocarpine. However, the influence of pilocarpine can not be main-
tained permanently. By its use venom may be obtained from an animal
that has just previously yielded none, but if such an animal be injected with
pilocarpine on the succeeding day no secretion will be obtained in consequence.
We found that in animals yielding venom freely the first injection of pilocar-
pine caused increased secretion; the second injection (on the succeeding day)
caused a secretion where none was previously to be obtained; the third injec-
tion was without result. In those animals which secreted venom freely the
first injection of pilocarpine caused an increase in the secretion of venom; when
the second injection of pilocarpine was given on the day following the first
injection, the venom glands were again stimulated to secretion of venom,
although before the injection of pilocarpine it had been impossible at this time
to collect any venom; when a third injection of pilocarpine was given to such
an animal on the day following the second injection, no venom was secreted.*
This failure of the venom glands to respond to the third injection of pilocarpine
is probably due to exhaustion of the secreting-cells, an exhaustion from which
the cells are unable to recover in so short a period of time as 24 hours.— Asa
rule, the first injection causes also discharge of urine and feces, whereas suc-
ceeding injections do not have this effect. The variations in the reaction of
the Heloderma to succeeding injections of venom are perhaps partly to be
explained by the fact that the animals were fed but once or twice a week.
After the first two doses of pilocarpine have set up a genuine secretion of the
venom, a new formation of the venom takes place only very insufficiently, and
a third dose of pilocarpine is therefore without much effect. After a third
injection the gland-cells still secrete the very small amount of venom stored up
within the cells. Whether the pilocarpine, besides causing a real secretion of
the venom, also causes a discharge of venom already secreted, we can not state.

Twenty-one helodermas in all were investigated with reference to the
influence of pilocarpine upon the amount of secretion. In every case the first
dose brought about increase of secretion. The second dose also produced
increased flow when the animal had been previously yielding well, and only at
the third dose was there no increase. Three animals were subjected to more
than three successive injections and all of these died. Sixty animals—most

*In one case an animal which had responded to a first injection of venom did not give an increased amount of
venom under the influence of a second injection of pilocarpine given 40 hours later.

tT hese observations on the functional effect of pilocarpine are in good agreement with the observations con-
cerning the effect of pilocarpine on the morphology of the venom gland recorded in the paper by Fox and Loeb.

54 THE VENOM OF HELODERMA.

of them rats and guinea-pigs—were used in testing the comparative strength of
venom obtained with and without pilocarpine. The venom collected after
injection of pilocarpine was at least as active as the venom obtained without
pilocarpine.

Influence of injection of Pilocarpine on secretion of venom in Heloderma.

Experiment 1 (May 14):

10515" Inserted rubber in mouth; rapid flow of saliva.

10 25 Diminishing flow.

10 30 Flow ceased; 2 c.c. collected.

10 30 Injected 0.1 grain pilocarpine in 1 c.c. physiological salt solution; animal shows

immediate weakness; ceases to bite; no flow of saliva.

10 40 =‘ Flow of saliva beginning.

11 00 Good flow; mucus increasing; animal again bites well.

11 15 Flow finished; about 2 c.c. collected.

Experiment 2 (May 20):

Collected nearly 1 ¢.c. of saliva from large heloderma during 20 minutes; injected 0.1
grain pilocarpine in 1 ¢.c. physiological salt solution; collected nearly 1 c.c.
during next 20 minutes; urine and feces voided; no marked signs of weakness.

Experiment 3 (May 20):

Collected nearly 1 ¢.c. saliva from large heloderma in 30 minutes; injected 0.1 grain
pilocarpine; flow began in about 5 minutes; collected in 15 minutes about 2c.c.;
became viscid and flow diminished; collected in next 15 minutes about 1 c.c.

Experiment 4 (May 31):

Collected during 30 minutes about 0.7 c.c. saliva from one large heloderma; secretion
ended, injected 0.1 grain pilocarpine; 5 minutes later secretion began again;
during 30 minutes collected 1.5 ¢.c.

Experiment 6a (June 12):

Collected venom from 8 helodermas; got from 8 only about 0.4 c.c.; gave 2.1 grains each

of pilocarpine; collected about 1.4 c.c.
Experiment 6b (June 13):

Injected same helodermas with pilocarpine; one gave no venom before or after injection;
the other was not tried before, but gave after injection only about 0.2 c.c.; all
helodermas not tried on the 12th were tried and gave practically no saliva; to
be fed to-night.

Experiment 6c (June 14):

Two helodermas injected with pilocarpine on third successive day; no secretion.
Experiment 6, cont. (June 17):

One heloderma that received pilocarpine on three successive days (June 12-14) dead.
Experiment 7 (June 18):

Took venom from all helodermas (18); 4 that have been in the laboratory and have
eaten two successive nights gave most of it; 2 were injected with pilocarpine; 1
which had given none before gave scarcely any, the other (one that had been in
the laboratory) gave the usual increased secretion; collected altogether only
2.2 €.¢.

Comparative toxic effect of venom obtained with and without injection of pilocarpine.

Guinea-pig 29, 100 g.

May 20, 45 12™ injected 0.066 ¢.c. venom of heloderma No. 1 in 1 ¢.c. physiological
salt. solution; secretion obtained before injection of pilocarpine. 5 o'clock
weak; breathing slow, not difficult. 6 o'clock dead.

Guinea-pig 28, 500 g.

May 20, 45 10™ injected 0.066 c.c. venom from heloderma No. 1 obtained after
injection of pilocarpine. 5 o'clock paralyzed; breathing slow and quiet. At
6 o'clock dead.

Guinea-pig 31, 500 g.

May 20, 45 16™ injected 0.066 ¢c.c. venom from heloderma No. 2 obtained before
injection of pilocarpine. 5 o'clock animal affected, sits upright, able to right
itself when put on side. 6 o’clock symptoms graye; not likely to recover.
May 21 found dead.

Guinea-pig 32, 480 g.

May 20, 45 14™ injected 0.66 c.c. venom from heloderma No. 2 obtained after
injection of pilocarpine. First portion before secretion of mucus was abundant.
6 o'clock paralyzed; breathing quick, not difficult. 5» 20™ dead.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 55

Guinea-pig 33, 480 g.
May 20, 4» 17™ injected 0.066 ¢.c. venom from heloderma No. 2 obtained after
injection of pilocarpine; second portion after secretion had become very slimy.
5 o'clock paralyzed; breathing quick, not labored; occasional convulsive move-

ments of legs. 5h 20m breathing irregular, slow; jerking movements of head
and legs. 6 o'clock dead.
Rat 6, 120 g.

May 24, 6 p. m. injected 0.1 ¢.c. venom from heloderma No. 1 cenit before
injecting pilocarpine. 6" 25™ appears weak, unable to move easily. o'clock
somewhat better. May 25 found dead.

Rat 7, 120 g.

May 24, 55 66™ injected 0.1 c.c. venom from heloderma No. 1 obtained after inject-

ing pilocarpine. 6" 80™ very weak. 7 o'clock labored breathing; reflexes

absent. 75 10™ dead.
Venom before injection.
Rat 58, 100 g.
JUNE 7, 5" 35™ injected 0.1 ¢.c. venom. 6» 30™ dying. 8 o’clock dead.
Guinea-pig 48, 420 g.

May 31, 125 03™ injected 0.1 ¢.c. venom. 1» 30™ sits down; breathing rapid.
2 o’clock inclined to fall on one side; able to right itself. 2b 30™ breathing
difficult, jerky. 3h 25™ occasional violent jerks. 4 o'clock dead.

Rat 15, 61 g.
May 81, 12" 14™ injected 0.1 ¢.c. venom. 12» 30™ lying on side; breathing rapid.
12 40™ lying on side; breathing slow. 125 45™reflexesabsent. 12" 50™ dead.
Venom after injection.
Rat 59, 100 g
JUNE 7, 54 40™ injected 0.1 ¢.c. venom. 6% 30™ dead.
Guinea-pig 47, 600 g.

May 31, 12 o'clock injected 0.1 ¢.c. venom. 125 30™ lying down. 12> 40™
lying down; breathing rapid. 1» 30™ lies on side. 2 o'clock breathing very
forced; right legs paralyzed. 2'30™breathing difficult; slower. 25 45™ tetanic
convulsions when stimulated. 2» §0™ dying; convulsions when stimulated.
3h 10™ dead.

Rat 14, 98 g.

May 31, 125 10™ injected 0.1 ¢c.c. venom. 125 30™ lying on side; breathing

rapid. 12» 40™ lying on side; breathing slow. 12 45™ reflexes present.

125 50™ reflexes present. 1 o'clock reflexes absent. 15 10™ better; able to
move. 2 o'clock lies on side; apparently dying; reflexes present. 2b 20m
dead.

EFFECT OF HEAT ON VENOM.

Santesson (Nordickt Medicinskt Arkiv. Testband Ullegnadigt Axel Key,
Nov. 5, 1897) showed in a few experiments that boiling the venom for at least
10 minutes does not diminish its toxicity. We have found, however, that
boiled and unboiled portions of the same venom were equally toxic only when
the precipitate was not filtered off from the former. Some of the venom was
evidently carried down with the precipitate, but if precautions were taken so
that the precipitate was fine enough to be injected there was no appreciable
difference in the effect of the boiled and unboiled portions. In some cases
where the filtrate from the boiled venom showed a lower toxicity than the
original sample we found that the precipitate, mixed with physiological salt
solution and injected into an animal, was toxic. We found the same to be true
concerning the precipitate from unboiled venom, 2.e., the precipitate formed by
diluting venom with ten times its volume of physiological salt solution gives,
when injected into an animal, all the effects produced by injecting the precipi-
tate from boiled venom.

Since it was possible to heat the fresh venom to 100° without decreasing
its toxicity we were able to sterilize the venom before injecting it into animals.
This precaution was manifestly necessary, since it was found that the fresh

56 THE VENOM OF HELODERMA.

venom contained large numbers of bacteria, some of which proved to be ex-
tremely virulent for guinea-pigs.* In making use of sublethal doses and of
doses which kill only after a considerable interval, it was of course necessary to
eliminate the influence of micro-organisms. Almost all our experiments have
been made with venom sterilized either by boiling for 10 minutes or by being
kept for an hour at a temperature of 60°. The advantage of the latter method
is that the resulting precipitate is a much finer one and passes easily through
a fine hypodermic-syringe needle.

A large number of tests were made comparing the toxicity of heated and
unheated venom. The toxicity of the venom was tested in guinea-pigs, rats,
rabbits, mice, and tadpoles. The venom used in these experiments had been
heated either to 60°C. for an hour, 100°C. for 10 minutes or 30 minutes, or to
120°C. in the autoclave for 15 minutes. The injection of any of the heated
venoms led to the death of the animals almost as quickly as the injection of
unheated venom.

Thus it was apparent that heat of such degrees as we have used in these
experiments does not injure the toxicity of the fresh venom to any marked
degree. However, it was noted occasionally that when animals were injected
with small quantities of the heated venom (that is, quantities which were but
very little larger than the lethal dose), such animals lived longer after the injec-
tion than animals injected with corresponding quantities of the unheated
venom. Therefore it appears that the heating exerts some slight injurious
effect on the venom. This slight decrease in the toxicity after heating may
perhaps be due to an inclusion of some venom in the coagulum. It will pre-
sumably require some time for the included venom to be liberated; the action
of the venom is, therefore, distributed over a longer period of time and is con-
sequently less acute.

With dried venom we tested the influence of exposure to temperature of
80°C. for 1 hour, 100°C. for 1 hour, 120°C. (autoclave) for both 1 and 2 hours.
In these experiments only mice were used. It was found that the heating of
the dissolved dry venom did not diminish its toxicity, although in a few cases it
was observed that animals injected with the smaller doses of the heated venom
did not die quite as soon as animals injected with the same quantities of un-
heated venom. Santesson records the fact that the dried venom would not
endure heating to 110°C., while the fresh venom weuld. The reason for this
difference between Santesson’s observations and ours is not clear.

The action of heat upon various snake venoms differs; thus, the venom of
Colubridee (Naja, Bungarus, Holocephalus, and Pseudechis) and of Hydrophine
may be heated to 100°C. and even boiled for a short time without injury ; heat-
ing for a long time to 100°C. or heating to 120°C. diminishes or destroys the
toxicity. The venom of Viperidse (Lachesis, Crotalus, Vipera) is injured by
being heated to 70°C. and is destroyed at 80° to 85°C.

Unlike the heloderma venom, the venom of the Colubridz gives a pre-
cipitate which appears after heating to 72°C. and which is not tonic.

*Bacteria belonging to the group of the colon were especially virulent, as found by Dr, D. Rivas.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 57

The heloderma venom is rather more resistant to the destructive action of
heat than any of the above-named snake venoms, since even though heated to
120°C. the toxic action is weakened but not destroyed.

Below we give a few experiments showing the influence of heating on the
toxicity of the venom.

Boiled Venom (filtered).
Rat 12, 110 g.
May 28, 35 22™ injected 0.1 ¢.c. venom. 4. 0’clock very weak, appears dying.
45 30™ somewhat better. 6 o'clock a little better, able to slowly right itself if
placed on back. 6» 30™ still living, condition very bad. 6 25m dying.

Found dead.
Guinea-pig 41, 400 g.
May 27, 4 23™ injected 0.1 ¢.c. venom. 6 36" drowsy, occasional starts, able
to right itself when placed on side. 7 o'clock occasional convulsive jerks, posi-

tion drooping. 8 30™ occasional violent jerks, position abnormal. May 28,
9 a.m. much improved. May 29 recovered.
Unboiled Venom.
Rat 13, 110 g.
May 28, 35 35™ injected 0.1 c.c. venom. 4 o'clock slightly worse than rat 12.
4» 30™ improving, still worse than rat 12. 6 o'clock dying. 55 30™ dead.
Guinea-pig 41, 340 g.
May 27, 45 30™ injected 0.1 ¢.c. venom. 6» 30™ forced breathing; lies on side;
very weak. 7 o’clock dying. 75 05™ dead.
Precipitate from Boiled Venom used on May 28.
Rat 14, 100 g.
May 29, 3" 55™ injected. 6 o'clock rather sick. May 30, 9 a. m. found dead.
Boiled Venom (not filtered).
Guinea-pig 45, 800 g.
May 29, 3" 47™ injected 0.2 c.c. venom. 5% 30™ lying down, breathing labored.
55 45™ dying, occasional jerking movements. 6° §0™ convulsive jerking, ex-
tremely irritable. 6 o'clock dead.
Unboiled Venom.
Guinea-pig 46, 700 g.
May 29, 35 55™ injected 0.2 ¢.c. venom. 5% 30™ bad condition, slightly better
than guinea-pig 45. 6 o'clock dying. 65 05™ dead.
Venom Dried below 70°.
Mouse 41.
Nov. 13, 25 20™ injected 0.2 mg. intraperitoneally. 4» 25™ dead.
Same Dried Venom Heated 1 hour to 116°
Mouse 45.
Nov. 13, 3 15™ injected 0.2 mg. intraperitoneally. 5% 30™ dead.

EFFECT OF STANDING IN SOLUTION ON TOXICITY OF VENOM.

A 10 per cent solution of venom in physiological salt solution kept sterile
and allowed to stand for 9 days in the laboratory showed no decrease in viru-
lence; but venom sterilized and merely covered became, under the same con-
ditions, less toxic. This doubtless is due to the action of bacteria entering it
from the air. To guard against possible complications due to the presence of
bacteria in venom which had been allowed to stand, it was resterilized before
being used for inoculations.

June 8 a quantity of venom was sterilized and its strength tested on a
mouse and a rat. It was divided into two portions, one of which was kept
sterile and the other simply covered, so as to prevent the entrance of dust.
Both portions were left for 9 days in the laboratory at room temperature and
in the light. On June 17 they were heated to 60°C. for 45 minutes and used in
the following experiments:

58 THE VENOM OF HELODERMA.

Sterile Venom.
Rat 96, 120 g.

June 17, 12" 05™ injected intraperitoneally 0.1 ¢.c. venom. 1» 30™ found dead.
Mouse 17.
June 17, 12" 28™ injected intraperitoneally 0.01 ¢.c. venom. 1» 30™ found dead.

Not Sterile Venom.
Rat 97, 120 g.

June 17, 12> 08™ injected intraperitoneally 0.1 ¢.c. venom. 1» 30" living; sick.
2b 45™ dead.
Mouse 18.
JUNE 17, 125 31™ injected intraperitoneally 0.01 ¢.c. venom. 1» 30™ living; very

sick. 2 o'clock dying; killed.

Other experiments were also carried out with venom which had been
treated in a similar manner, that is, sterilized and kept at room temperature.
This venom was tested 2, 7, and 21 days after sterilization, and it was found
that while it had lost part of its toxicity it had lost far less than the unsterilized
venom which was kept on ice. Thus, on the day the venom was sterilized,
0.005 c.c. of unheated venom killed a mouse in from 25 minutes to 4 hours 30
minutes; 2 days later, 0.005 c.c. of sterilized venom killed a mouse in 8 hours;
7 days later, 0.01 c.c. of unsterilized venom killed a mouse in 24 hours, while a
similar quantity of sterilized venom killed in 6 hours; 21 days after the sterili-
zation, 0.03 c.c. of the sterilized venom killed a mouse in 40 minutes, while the
same quantity of the unsterilized venom killed in 24 hours.

In other experiments the venom, after being sterilized, was placed in the
thermostat and kept at a temperature of 37.5°C. instead of being kept at room
temperature. In these experiments also it was noted that the sterilized venom
kept better than the unsterilized venom. However, in all cases, the toxicity of
the venom when it had been dissolved diminished slightly after some time, even
though it had been sterilized.

Furthermore, we tested the power of thymol to prevent the deleterious
effect on venom of bacteria and of standing in solution. We found that at
periods 2, 7, and 21 or 23 days after mixing, unsterilized venom to which thymol
had been added had lost its toxicity in much the same degree as unsterilized
venom to which no thymol had been added. Sterilized venom, to which thy-
mol had been added, acted in much the same manner as the simple sterilized
venom; that is, became very slightly less toxic after several days.

Thus we note that unsterilized venom in solution loses its toxicity fairly
rapidly because of bacterial action. Sterilized venom in solution loses in toxic-
ity far more slowly, and here the loss of toxicity is probably due to the gradual
decomposition of the venom when kept in solution. The addition of thymol
does not appear to prevent the destruction of the venom, which, in spite of the
thymol, loses its toxicity as rapidly as unsterilized venom.

EFFECT OF DIALYZING.

The venom passes through parchment paper rather slowly. In our experi-
ments venom was diluted to ten times its original volume with physiological
salt solution, sterilized and put to dialyze against twice its volume of sterile
salt solution. The whole apparatus (made as sterile as possible) was kept
directly on ice. In 43 days—the longest period that we tried—dialysis of the

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 59

toxic substances was not complete. The results obtained by injecting into
animals, at different intervals, material from inside and outside the dialyzer
were as follows: At the end of 1 day the solution outside the dialyzer had
acquired scarcely any of the toxic substance and the solution inside showed
searcely any decrease in toxicity; at the end of 2 days the symptoms produced
by material from outside the dialyzer were marked but not grave, while death
followed the injection of a similar quantity of the solution inside the dialyzer;
at the end of 3 days the symptoms produced by the injection of fluid from out-
side the dialyzer were grave, but less serious than those produced by material
from inside the dialyzer; at the end of 4 days the symptoms produced by
material from outside the dialyzer were grave and with large doses sometimes
fatal, but they were still less severe than the symptoms produced by similar
quantities of the material within the dialyzer. Throughout there was noted a
gradual decrease of the toxicity of the solution inside the dialyzer. Where
large quantities of the solution were injected, control animals were injected
with equal volumes of sterile salt solution. All the solutions were sterilized
before injecting. In these experiments mice and rats were used to test the
toxicity of the solutions. In this respect the heloderma venom behaves in a
manner similar to the venom of the Colubride, which passes slowly through
vegetable membranes. The gradual decrease in toxicity of the solution within
the dialyzer and the corresponding increase in toxicity of the solution outside
the dialyzer is apparent in the following protocols:

Venom Dialyzing 24 hours.
Outside Dialyzer.
Rat 104, 100 g.
June 25, 4 o'clock injected subcutaneously 1 c¢.c. of solution. 4» 45™ slightly

affected. JuNE 26 recovered.
Mouse 26.
JUNE 25, 45 02™ injected subcutaneously 0.2 c.c. of solution. 4> 45™ respiration
rapid; otherwise well. JuNE 26 recovered.
Inside Dialyzer.
Rat 105, 110 g.
JUNE 25, 45 15™ injected subcutaneously 1 c.c. of solution. 4» 45™ very gravely
affected. 45 56™ dead.
Mouse 27.

JUNE 25, 45 16™ injected subcutaneously 0.2 c.c. of solution. 4» 45™ lying down;
breathing forced. 5 o'clock dead.
Venom Dialyzing 44 hours.
Outside Dialyzer.
Rat 24, 100 g.
June 5, 124 15™ injected intraperitoneally 4 c.c. of solution; appeared somewhat
drowsy all day, but recovered.
Rat 26, 100 g.
June 5, 125 17™ injected intraperitoneally 2 c.c. of solution; drowsy; recovered.
Rat 28, 120 g.
June 5, 125 18™ injected intraperitoneally 1 c.c. of solution; slightly drowsy;
recovered.
Inside Dialyzer.
Rat 25, 100 g.
June 5, 125 20™ injected intraperitoneally 4 c.c. of solution. 126 30™ lying
down; very much affected. 12» 40™ dead.
Rat 27, 100 g.
JUNE 5, 12 22™ injected intraperitoneally 2 c.c. of solution. 12 40™ breath-
ing difficult, lies on side. 125 45™ breathing very difficult, legs paralyzed.
125 47™ reflexes absent. 1 o'clock dead.
Rat 29, 120 g.
JUNE 5, 12 26™ injected intraperitoneally 1 c.c. of solution. 12% 45™ lies on
side. 1 o’clock dying. 1 30™ dead.

60 THE VENOM OF HELODERMA.

Venom Dialyzing 49 hours.
Outside Dialyzer.
Rat 109, 105 g. :
Jung 26, 65 10™ injected subcutaneously 1 c.c. of solution. 6 o'clock slightly
affected. JUNE 27 recovered.
Mouse 28.
Jung 26, 54 11™ injected subcutaneously 0.2 c.c. of solution. 6 o'clock affected,
but much less than mouse 29. JUNE 27 recovered.
Inside Dialyzer.
Rat 110, 110 g.

JuNE 26, 5% 12™ injected subcutaneously 1 c.c. of solution. 6 o'clock lying down,
very sick. JUNE 27 dead.
Mouse 29.
JUNE 26, 54 18™ injected subcutaneously 0.2 ¢c.c. of solution. 6 o'clock nearly

dead. JUNE 27 dead.
Venom Dialyzing 67 hours.
Rat 112, 100 g.
JUNE 27, 115 30™ injected subcutaneously 1 c.c. of solution. 12 o'clock lying
down. 12» 80™ lying down. 1 o'clock lying down. 2 o'clock improving.
JUNE 28 recovered.

Mouse 30.
JUNE 27, 114 30™ injected subcutaneously 0.2 c.c. of solution. 12 o'clock lying
down. 1 o'clock better. JUNE 22 recovered.
Rat 113, 110g.
JUNE 27, 115 32™ injected subcutaneously 1 c.c. of solution. 12 o'clock lying
down; about same as rat 112. 125 30™ lying down, not much worse than rat
112. 1 o’clock worse than rat 112. 2 o'clock dead.
Mouse 31.
JUNE 27, 114 33™ injected subcutaneously 0.2 ¢.c. of solution. 12 o'clock lying
down. 1 o'clock dead.
Venom Dialyzing 112 hours.
Rat 84, 85 g.
JUNE —, 10" 52™ injected subcutaneously 2 c.c. of solution. 2 o'clock sick, but
may recover. JUNE 14 well.
Rat 88, 120 g.
JUNE -, 115 20™ injected subcutaneously 1 c.c. of solution. 14 20™ seriously
affected. JUNE 14 recovered.
Rat 85, 85 g.
June —, 105 56™ injected subcutaneously 2 c.c. of solution. 2 o'clock dead.
Rat 89, 130 g.
June —, 115 22™ injected subcutaneously 1 c.c. of solution. 1» 20™ dead.
Mouse 13.
June —, 125 44™ injected subcutaneously 0.2 c.c. of solution. 1» 50™ seriously
affected. 2> 35™ dead.
Mouse 14.
June —, 125 4™ injected subcutaneously 0.2 c.c. of solution. 2» 20™ dead.

EFFECT OF FILTRATION.

The venom of Heloderma passes unchanged through a Chamberland filter.
Two similar portions of venom, one filtered, the other unfiltered, produce, when
injected into animals, identical results, that is to say, they bring about similar
symptoms and kill in approximately equal times. We found in ten rats which
we injected that exactly similar results were produced by equal doses of filtered
and unfiltered venom.

Again, it may be noted that the venom of Heloderma acts in a manner
similar to the venoms of the Colubride, which pass through a Chamberland
filter, while that of the Vipcridee does not.

Filtered Venom.
Rat 31, 140g.

Junge 5, 4524™ injected intraperitoneally 0.1 ¢.c. venom. 6 30™ drowsy.
JuNE 6, 9 a. m., found dead.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 61

Rat 33, 120 g.
June 5, 45 22™ injected 0.05 ¢.c. venom intraperitoneally. JUNE 6, well, no
symptoms.
Rat 54, 190 g.
June 7, 5" 27™ injected intraperitoneally 0.2 c.c. venom. 6» 30™ dying. 8
o’clock dead.
Rat 56, 120 g.
June —, 55 28™ injected subcutaneously 0.1 ¢.c. venom. 6» 30™ slightly sick.
8 o’clock dead.
Unfiltered Venom.
Rat 30, 140 g.
June 5, 4503™ injected intraperitoneally 0.1 ¢c.c. venom. 6» 30" drowsy.
June 6, 9 a. m., found dead.
Rat 32, 120g.
June 5, 4501™ injected 0.05 c.c. venom intraperitoneally. JUNE 6, well, no
symptoms.
Rat 55, 190 g.
Jung 7, 55 25™ injected intraperitoneally 0.2 c.c. venom. 6» 30™ dying. §
o’clock dead.
Rat 57, 130 g.
June -, 55 30™ injected subcutaneously 0.1 ¢.c. venom. 6» 30™ slightly sick.
8 o'clock dead.

INFLUENCE OF THE ADDITION OF ACID TO VENOM SOLUTION.

In connection with certain experiments which we performed it was of
interest to determine whether the addition of acid to a solution of venom in-
fluenced the toxicity of this substance. It has been shown that the venom, in
a solution of either fresh or previously dried venom, is neutral in reaction. We
added to 5 c.c. of a 10 per cent (fresh) venom solution 2 c.c. of a N/10 HCl
solution. No precipitate was produced. After allowing this mixture to stand
for some time we added 2 c.c. of N/10 NaOH solution in order to neutralize
the previously added acid.

Various quantities of this venom solution were injected into mice, and at
the same time other mice were injected with similar quantities of the pure,
equally diluted venom solution. In every case the mice injected with the neu-
tralized acid solution of venom died in approximately the same length of time
as the animals injected with the pure venom. The addition of acid to helo-
derma venom does not, therefore, change the toxicity of the venom.

Influence of addition of acid to venom solution.

Quantity | Time between injection and death.
of venom
injected. Controls. Acidified venom.
C.c. | hem. he m.
0.011 3 Bas
022 3 as Oe as
055 jane 1 20
055 2 15 Be ea
027 My ae 2

EFFECT OF ROENTGEN RAYS ON SECRETION OF VENOM GLAND.

In order to test the influence of exposure to Roentgen rays on the proper-
ties or secretion of the venom we exposed several of the helodermas to the Roent-
gen-ray illumination. Finding that single exposures produced no appreciable
result, we subjected two helodermas to a period of treatment extending over a
period of about 3 weeks. The lower jaws of the animals, with the venom

62 THE VENOM OF HELODERMA.

glands, were exposed every other day for 10 minutes. At the end of the treat-
ment we found that venom from these individuals, instead of having dimin-
ished in virulence, was somewhat more active than venom taken from other
animals which had not been exposed tothe Roentgenray, afact that we attrib-
uted to the influence of the long period of rest enjoyed by the glands of the
Roentgenized helodermas. Six rats injected with the first venom taken from
these animals showed that its toxicity had undergone no change that could be
attributed to the Roentgen-ray treatment.

EFFECTS OF VENOM UPON ANIMALS.

When a warm-blooded animal receives a lethal dose of heloderma venom
the first conspicuous effect is a disturbance of respiration. The breathing
becomes quickened and the respirations are forced. After a time the respira-
tory rate diminishes and the respirations grow shallow; finally they occur only
at long intervals, until after a period of respiratory spasm they cease alto-
gether. Meanwhile, other symptoms appear. The animal shows weakness
and falls down, and frequently, though not as a rule, has acute attacks of con-
vulsions. Reflex irritability becomes increased so that a slight sensory stim-
ulus is able to inaugurate an exaggerated response which resembles the re-
sponse obtained in an animal poisoned by strychnine. The hind legs hang as
if paralyzed and later the forelegs also appear paralyzed. If the skin of the
leg be pinched, however, a response is elicited, and this response may still be
obtained very shortly before the last respiration. The corneal reflex may be
obtained even later than the sensory skin-reflex.

It was noted that when mice were injected subcutaneously with lethal
quantities of venom, one hind leg became completely paralyzed and spastic at
a time when complete paralysis was not present in the other hind leg. This
unilateral spastic and paralytic effect was not due to the venom being injected
into or near this leg, since the fluid was usually injected under the skin of the
thorax. Not only was the one leg spastic and paralyzed, but sensation was
almost completely lost in this leg. Pinching the skin or pricking with a pin
produced no response; occasionally, however, pressure (pinching the entire leg)
would lead to a slight muscular response of the other leg. These symptoms
are general for all the mammals which we have used, viz, dog, cat, guinea-pig,
rabbit, rat, and mouse; but some of them appear more conspicuously in certain
species than in others. For instance, the apparent paralysis appeared most
marked in rats and mice and the strychnine-like response to stimulus was most
evident in guinea-pigs. The respiratory disturbances were substantially the
same in all animals, and it is to the interference with respiration that we must
attribute the immediate cause of death in the warm-blooded animal.

Besides the abnormal symptoms common to all, certain species of animals
show other disturbances. In dogs and cats injection of venom causes vomit-
ing and discharge of urine and feces. It also causes a very copious flow of
saliva and of tears. In cats a noticeable symptom is loss of voice. The ani-
mal makes continuous crying movements, but without the production of

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 63

sound. In rabbits and guinea-pigs there occurs occasionally a conspicuous
flow of tears, and guinea-pigs develop an extreme rigidity of the abdominal
walls. In order to see whether this symptom might be due to the operation,
we injected control guinea-pigs with salt solution and found that the abdominal
walls remained relaxed.

The heart continues to beat after the respiration has ceased, sometimes
for a considerable time—10 to 30 minutes in the warm-blooded animals and
many hours in frogs. The auricles beat after the ventricle has stopped. Occa-
sionally the cardiac rhythm is disturbed, the auricles beating oftener than the
ventricle. Asarule the heart stops in diastole, but may at times stop in systole.

Santesson records a curare-like effect of the poison, which was not mani-
fested in our experiments. We found that in frogs which had died several
hours previously as a result of the injection of venom, mechanical stimulation
of the sciatic nerve would cause twitching of the leg muscles, and the same
effect was observed in mice immediately after death.

Mitchell and Reichert believed the venom to be a heart poison. In the
few experiments that they made they used very large doses and brought about
death very suddenly. Probably by this means they caused a reflex stopping
of the heart, but that death is not due to a direct effect upon the heart we saw
in very many cases where mammalian hearts continued to beat for 30 minutes
and frogs’ hearts for several hours after death.

Denburgh and Wight (Am. Journ. Phys., 1900, 4, p. 209) had previously
reached a conclusion identical with ours; they also had decided that the imme-
diate cause of death was in respiratory failure. The observations of Denburgh
and Wight upon the physiological effects of heloderma venom are in substan-
tial agreement with our own. We have injected more than 360 warm-blooded
animals with heloderma venom, and although these have not all received con-
tinuous detailed inspection, nevertheless many have been kept under close
observation. From the history of these, we have selected the following typical
records:

Rabbit 2, 500 g.
May 30, 95 20™ injected intraperitoneally 0.15 c.c. sterile venom in 1 ¢.c. NaCl 0.85
per cent. 95 55™ signs of paralysis, unable to walk. 10° 10" drowsy.
10% 15™ unable to hold up head; breathing rapid. 105 20™ appears to be dying;
reflexes absent. 10+ 25™ breathing labored, slower. 10° 27™ dying; heart-
beat slow and weak. 10%35™ occasional gasps; heart-beat not felt. 10%40™
stopped breathing, heart still beating, very weak.
Rabbit 4, 1000 g.
May 30, 15 34™ injected 0.1 ¢.c. venom into ear-vein. 1+ 35™ pupils contracted.
1» 36™ lying down; heart-beat weak; breathing rapid. 15 37™ heart-beat weak,
fast; reflexes present, pupils contracted. 15 38™ heart-beat weak, slow; breath-
ing rapid, strained. 15 39™ pupils excessively contracted; ears bloodless.
1» 40™ convulsions; pupils dilate, reflexes present. 1 45™ occasional respi-
ratory gasps. 1» 46™ reflexes still present; convulsions. 1» 49™ reflexes
absent; heart action slow. 1 50™ dead; chest opened; heart still beating;
blood not clotted in heart; clots quickly after escape into chest cavity.
Guinea-pig 41, 600 g.
May 31, 125 00™ injected subcutaneously 0.1 ¢.c. venom in 1 c.c. NaCl 0.85 per cent.
12» 30™ lies down. 124 40™ breathing rapid. 14 30™ falls on side. 2 00m
breathing very strained, right legs paralyzed. 25 30" breathing difficult,
slower. 2 45™ lying on side; tetanic convulsions when stimulated. 2h 50m
dying. 3> 10" dead.

64 THE VENOM OF HELODERMA.

Rat 14, 98 g.

May $i, 12 10™ injected subcutaneously 0.1 ¢.c. venom in 1 ¢.c. NaCl 0.85 per cent.
12 30™ lying on side; breathing rapid. 12» 40™ lying on side; breathing slow.
12» 45™ reflexes present. 1500™ reflexes absent. 1» 10™ better; able to move
about. 2b 00" lies on side; apparently dying; reflexes present. 2» 50™ dead.

Dog 1, 10 kg. : .

May 31, 35 74™ given subcutancously 0.1 ¢.c. fresh venom. 35 26m vomited; urine
and feces voided. 3» 30™ lying down; very sick. 3% 50™ vomited. JUNE
1, died during forenoon.

Dog 2, 9700 g. ;

May 31, 35 17™ given subcutaneously 0.02 ¢.c. fresh venom. 3 30™ lying down;
trembles; occasionally much twitching. 3» 40™ voids urine and feces.
Recovered.

Dog 8, 11 kg.

May 31, 3" 30™ given 1.1. c.c. fresh venom subcutaneously. 3» 45™ vomited; urine

and feces voided. Very sick all afternoon. Died during night.
Cat 1, 3000 g.

June 1, 12"10™ give subcutaneously 0.4 ¢.c. fresh venom. 12420"™vomits. 12525™
pupils enormously dilated; vomits. 12» 30™ very nervous; cries if touched;
breathing difficult. 2» 0O™ breathing rapid, superficial, pupils still dilated.
Sick for some days, but recovered.

Cat 2, 3600 g.

JUNE 1, 12" 08™ given subcutaneously 0.09 c.c. of fresh venom. 12» 25™ pupils enor-
mously dilated. 12 40™ breathing difficult. 2>00™ breathing rapid, super-
ficial, pupils dilated. Recovered.

Cat 3, 3000 g.
June 1, 12 06™ given subcutaneously 0.045 c.c. of fresh venom. 12 25™ pupils enor-
mously dilated, vomits. 12 40™ pupils contracting. Recovered. JUNE
24 sick, killed; no lesions.
Cat 4, 3000 g.
JUNE 3, 25 12™ given 0.9 c.c. of fresh venom subcutaneously. 2» 20™ violent move-
ments of head from side to side, cries, pupils enormously dilated, eyes roll.
2» 35™ more quiet; eyes still roll, but head movements stopped. 25 §5™ lies

quiet, very nervous; makes crying movements, but has nearly lost voice; breath-
ing difficult, audible. 3 06™ no longer able to hold up head; voice gone.
June 4 living; takes no food. June 6 dead.

Cat 7, 1500 g.

Jung 8, 12555™ injected intraperitoneally 0.6 c.c. of fresh venom. 1» 05™ pupils
greatly dilated, losing voice. 1» 40 pupils contracted, voice gone; breathing
labored; tears secreted. Recovered.

Cat 9, 700 g.

JUNE 8, 25 25™ injected intraperitoneally 0.7 c.c. of fresh venom. 2b 85™ very much
affected; pupils dilated; tears and saliva flowing; breathing forced. 5> 00"
has remained in apparently dying condition all afternoon; breathing forced;
nearly paralyzed. 5» 30™ dead.

CONDITION OF MOUSE’S EYE PRODUCED BY INJECTION OF
HELODERMA VENOM.

We found that the injection of heloderma venom into mice caused fre-
quently a peculiar condition of the eye. This condition did not appear as a
rule when mice were injected with a large quantity of venom—a quantity
which was lethal within a comparatively short time—but appeared usually
when quantities of venom had been injected which caused death after many
hours or several days. At times the injection of a sublethal dose of venom was
sufficient to produce this lesion. However, its appearance was not constant,
even after the injection of a lethal dose.

The lesion was observed as early as from 1} to 6 hours after the injection
of the venom. At this time the eyeball appeared somewhat more prominent
than usual and the cornea showed a slight opacity. The protrusion of the eye
and opacity of the cornea progressed fairly rapidly (there was, however, con-

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 65

siderable variation in the rapidity of advance of these symptoms), until the
eye protruded about 2 or 3 mm. from its socket and the cornea assumed a
gray-white color. This appearance was observed as early as 3 hours after the
injection of venom.

If the condition continued to progress the gray-white of the cornea and
sclera now turned red and gradually assumed a dark red color. At times the
blood which had collected in the tissue about the eye-ball would break through
the conjunctiva anda more or less free oozing of blood about the eye would be
the result. Whether this breaking through of the blood was due to the pressure
exerted by the blood within the eye-socket, or whether it was due to external
trauma, we can not state. It is certain that when the cornea has become abso-
lutely opaque the animal is blind, and, in view of the blindness and the unusual
prominence of the eye, the mouse might easily rub the weakened eye against
some object and thus injure the conjunctiva in moving about the cage.

In those cases in which the condition progressed only as far as the stage
of corneal opacity, the animal seemed to be able to regain its vision after sev-
eral days; but when the condition progressed to the stage of subconjunctival
hemorrhage or actual oozing of blood, the eye-ball was lost, although the ani-
mal might otherwise recover from the injections. The stages of healing, either
the return to normal or the disappearance of the protrusion and hemorrhage,
progressed usually very rapidly, so that while the lesion usually reached its
maximum severity between 12 and 24 hours after the injection, no external
evidence of the lesion could be noted at a period 3 days after the injection.

It remained to be determined whether these macroscopic appearances
represented a pathological condition within the eye-ball or within the perioc-
ular tissue. We removed the eye-ball and periocular tissue of several mice
which had been injected with venom and which showed the various stages of
the conditions described above. These eyes were sectioned and studied micro-
scopically.

The microscopic examination of these tissues showed that in the earliest
stages of the condition there was a slight extravasation of blood in the loose
fatty tissue around the optic nerve, usually at some distance from where it
entered the eye-ball, but no evidences of any intraocular lesions were observed.
In later stages the hemorrhage in the periocular tissue became more evident
and spread forward in close proximity to the optic nerve and also laterally in
the fat tissue. Gradually the blood worked around the eye-bulb and appeared
under the conjunctiva. At no time was any blood found inside the eye-bulb.
The only abnormal condition within the eye consisted in the forward pressing
of the lens until it almost touched the posterior surface of the cornea. It is
possible that this condition of the lens was due to the manipulation of the eye
during its removal, or, perhaps, to the osmotic movements of the aqueous
humor during the fixation of the tissue, since it was noted in only a few of the
eyes examined.

These ocular lesions in mice that follow the injection of venom are, there-
fore, confined to the periocular tissue of the orbital space. There occurs first

66 THE VENOM OF HELODERMA.

a hemorrhage in the posterior portion of the orbital space which gradually
inereases. The eye-ball is thus pressed forward and exophthalmus develops.
In some unexplained manner the cornea becomes opaque. The blood works
its way forward in the periocular tissue until it appears under the conjunctiva
and this may at times, as a result of either the internal pressure or the trauma
of the conjunctiva, produce conjunctival oozing.

The cause for the primary hemorrhage in the posterior portion of the
orbital space is not clear. We have no evidence which would point to any
vascular lesions cwusing a greater permeability of the vessels for the red blood-
cells. The probable explanation must, therefore, be sought in an increase of
the intravascular pressure within the vessels of the brain and orbit sufficiently
great to causc a rupture of the thin and rather poorly supported vessels of the
orbit.

A somewhat similar condition has been noted by Emmert* in mice which
were injected with adrenalin. He found that when adrenalin was injected sub-
cutaneously into mice, the eye-ball protruded and the cornea became opaque,
but this author makes no mention of any hemorrhage. He also observed
that the lens was pressed forward.

ANATOMICAL LESIONS PRODUCED BY INJECTION OF VENOM.

The most conspicuous and common of the macroscopic changes to be seen
after death are found in the alimentary canal. These are particularly marked
if some hours have elapsed between the injection of venom and the animal’s
death. The serous layer of the intestines and stomach is usually markedly
congested. The intestines are much dilated with fluid and gas. In guinea-
pigs and rabbits—the only mammals investigated with reference to this point—
the mucous layer of the stomach was congested and showed small hemorrhages
and areas of self-digestion. To determine beyond question that these changes
were produced by the venom we made the following experiments: The abdom-
inal cavity of four guinea-pigs was opened and the condition of the intestines
noted. The abdominal wall was then sewed up and three animals were in-
jected subcutaneously with varying doses of venom and one with the same
volume of physiological salt solution. Immediately after death the intestines
of the animals injected with venom were found to contain much more fluid and
gas than they had contained before injection and also to show the other symp-
toms previously mentioned. The animal which had been injected with salt
solution was killed and found to have undergone no evident alteration of the
alimentary canal. ‘Two control guinea-pigs belonging to the same lot were
killed and likewise showed no abnormalities of stomach or intestine.

Inasmuch as certain of the snake venoms are perhaps excreted through
the gastric mucous membrane, it might be thought that the post-mortem
changes observed in this organ were due directly to a digestive action of the
heloderma venom. In order to see whether similar changes might not be pro-
duced by other methods of slow poisoning, we killed guinea-pigs by injecting
sublethal doses of potassium cyanide and of resorcin, repeating the injections

*Emmert, Virchow's Archiv, 1908, 194, 114.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 67

at intervals, so that the animals continued seriously affected until death finally
resulted. By means of both these poisons hemorrhages and self-digestion of
the stomach were produced. The self-digestion was not considerable in the
case of potassium-cyanide poisoning, but it was marked when death was caused
by resorcin. Particularly in one case, where the poisoning extended over more
than 24 hours, the mucous membrane was so changed that it came away
entire from the muscular layers.*

After death following the injection of venom the lungs always showed
more or less congestion and edema. These were the only invariable naked-
eye changes that we observed. At times liver, spleen, kidneys, and adrenals
showed congestion, but not very markedly and not invariably. Contrary to
the experience of Santesson, we never saw the slightest evidence of any local
disturbances. At the point of injection there was neither hemorrhage nor
swelling, or any other symptom of local effect.

Santesson records these local symptoms in frogs, and in order to observe
whether they might be specific in these animals we injected frogs with doses of
both fresh and dried venom, and of strengths sufficient to kill in long, short,
and medium intervals of time, but in no case did we observe any lesions at the
place of injection, either before or after death. We were also unable to con-
firm Santesson’s experience that the muscles at the site of injection become
softened and easily disintegrated, although we looked again and again for evi-
dence to this effect.

In all the reports concerning the local effect of the bite of the Heloderma
in man, mention is made of the rapid appearance of swelling and hemorrhagic
discoloration of the skin at the site of injury. We found that when fresh
venom was injected subcutaneously into an animal, no swelling or hemorrhage
appeared at the site of injection; and when venom was injected intramus-
cularly, no hemorrhage or swelling was noted. These negative results were
obtained with venom collected in the usual manner, namely, by allowing the
animal to bite on a piece of soft rubber and collecting only the venom which
appeared between the rubber and the lower lip of the animal. In some other
experiments we collected venom by allowing the animals to chew small pieces
of sponge; the venom was squeezed and washed out of these sponges. The
venom thus obtained was injected subcutaneously into the ear of a rabbit and
into the tissue-pad of a rat’s paw. Both of these animals developed marked
swellings at the site of inoculation, and later showed ecchymoses and hemor-
rhages in these areas.

It is possible that the local symptoms—swelling and hemorrhage—result
from some buccal secretion of the Heloderma. They certainly are not caused
by the secretion of the true venom glands. It is impossible, however, to abso-
lutely rule out mechanical injury as a factor in causing the appearance of these
local symptoms when an individual is bitten by a Heloderma. The animal has
very powerful jaws and its bite would easily bruise a large area of flesh and
skin. When the animal bites it clings tenaciously, and in endeavoring to
extricate a wounded part the injury might easily be increased.

*Cf. the paper by M. E. Rehfuss, which presents a more elaborate experimental study of these lesions.

68 THE VENOM OF HELODERMA.

The possibility that the local symptoms are due to the effects of micro-
organisms which enter the wound either at the time of biting or after the biting
can not be excluded, since in our experiments the venom obtained by allowing
the helodermas to chew the sponges was not sterilized before injections.

The following records of autopsies performed on animals which had been
injected with heloderma venom show some of the appearances which have
been mentioned above:

Rabbit 13.

JuNE 26, 2505™ received in peritoneal cavity 0.2 c.c. of venom. 5» 10™ dead.
Autopsy immediately; lungs, hemorrhagic and edematous; spleen. congestion
and hemorrhages; hemorrhagic points in omentum; blood in heart fluid; hemor-
rhagic points in stomach; much fluid in small intestine.

Rabbit 14, 2 kg.

JUNE 26, 3805™ received subcutaneously 0.4 c.c. of venom. 5> 40" dead. Au-
topsy immediately; lungs congested, hemorrhagic and edematous; ventricles not
contracted; small intestine contains much fluid; stomach shows self-digestion.

Rabbit 16, 2 kg.

JUNE 26, 35 29™ received subdurally 0.2 c.c. of venom. 5» 30™ dying; killed. Au-
topsy immediately; lungs edematous and emphysematous; spleen hemorrhages
and congestion; ventricle not contracted; much fluid in small intestine; stomach
shows hemorrhages and traces of self-digestion.

Rabbit 17, 1890 g.

JUNE 26, 3" 57™ received intravenously 0.2 ¢.c. of venom. 4e 45™ dead. 5» 10™
autopsy; auricles still beating; lungs hemorrhagic and edematous; ventricle not
contracted; small hemorrhages of the stomach.

Guinea-pig 105, 240 g.

JUNE 26, 25 40™ received intraperitoneally 0.1 ¢.c. of venom. 3% 20 dead. Au-

topsy immediately; fluid in intestine slightly increased; hemorrhages in stomach.
Guinea-pig 107, 220 g.

JUNE 26, 25 20™ received intraperitoneally 0.05 c.c. of venom. 3» 00™ dead. Au-

topsy immediately; fluid in intestine slightly increased; hemorrhages in stomach.
Guinea-pig 109, 220 g.

JUNE 26, 25 45™ received intraperitoneally 0.02 c.c. of venom. 5% 10™ dead. Au-
topsy immediately; much fluid in intestine; hemorrhages and self-digested areas
in stomach.

Guinea-pig 106, 240 g.
June 26, 24 42™ received subcutaneously 0.1 ¢.c. of venom. 34 20™ dead. Au-

topsy; fluid in intestine slightly increased; hemorrhages in stomach.
Guinea-pig 108, 220 g.

JUNE 26, 25 22™ received subcutaneously 0.5 ¢.c. of venom. 3> 25" dead. Au-
topsy immediately; fluid in intestine considerably increased; hemorrhages in
stomach.

Guinea-pig 110, 220 g.

JuNE 26, 25 48™ received subcutaneously 0.2 ¢.c. of venom. 6" 05™ killed; appeared

likely to die before morning. Autopsy immediately; fluid abundant in intes-

tine; hemorrhages and self-digested areas in stomach.
Guinea-pig 111, 260 g.
June 26, 45 10™ received subcutaneously 0.1 ¢.c. of venom. 5” 20™ dying; killed.
Autopsy; fluid slightly increased in intestine.
Guinea-pig 112, 260 g.

Juni 26, 4> 15” received intraperitoneally 0.1 ¢.c. of venom. 5” 10™ dead. Au-
topsy immediately; fluid slightly increased in intestine; small hemorrhages in
stomach.

Guinea-pigs 114 and 115 (controls).
JUNE 26, 65 15” from same lot; killed for controls; showed no abnormal fluid content of
intestine, no hemorrhages, and no self-digested areas of stomach.
Guinca-pig 99, 400 gy.
June 21, 2" 00" opened abdominal cavity and examined intestines; sewed up wall and
injected subcutaneously 0.05 c.c. of venom; no symptoms. 2h 35™ injected
0.1 ¢.c. of venom. 5” 00 dead. Autopsy; intestines contain much more
fluid and gas than at previous examination; hemorrhage in omentum; serous
layer of intestine slightly congested; mucous membrane of stomach hemorrhagic;
small self-digested areas.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 69

Guinea-pig 100, 420 g.

June 21, 2% 18™ opened abdominal cavity, examined intestines and sewed up wall;
injected 0.1 c.c. of venom subcutaneously. 5" 16™ dead; fluid and gas in-
creased in intestine; hemorrhages and self-digested areas in stomach.

Guinea-pig 102, 420 g.

JuNE 21, 2° 50™ opened abdominal cavity, examined intestine, sewed up wall, and
injected 0.15 c.c. of venom. 5% 30™ dead; fluid and gas increased in intestine;
hemorrhages and self-digested areas in stomach lining.

Guinea-pig 101, 440 g. (control).

Jung 21, 2» 40™ opened abdominal cavity, examined intestines, sewed up wall, and

injected subcutaneously 1 c.c. of sterile salt solution. 54 40™ killed; no changes,
Guinea-pigs 103 and 104 (controls).

JUNE 21, 6" 10™ killed two guinea-pigs from same lot for controls; intestines without

excess of fluid or gas; stomachs normal.

HISTOLOGICAL CHANGES PRODUCED BY INJECTION OF VENOM.*

We investigated the histological changes produced in the organs of rabbits,
guinea-pigs, and mice by the injection of heloderma venom. In some cases
the animals had died shortly after a single injection, in other cases the organs
examined were from animals which had received repeated injections of venom
over varying periods of time. In the animals which died of acute poisoning,
after a single administration of venom, no distinct histological changes were
noted in any of the organs. Some of these animals died but a few minutes
after the injection (rabbits injected intravenously with large doses), while
others lived as long as 48 hours after the injection (rabbits or guinea-pigs in
whose abdominal cavity a collodion capsule containing venom had been placed).

In the organs of animals dying a very short time after the administration
of venom—within 30 minutes—no changes whatsoever were noted. When the
animals died at periods between 1 hour and 48 hours after the administration
of the venom the only noticeable change was a general venous congestion in the
liver, lungs, and kidneys. In no cases were any degenerative changes noted in
the organs of rabbits, guinea-pigs, or mice.

In the animals which received repeated injectionst of venom over long
periods of time, changes were occasionally found in the various organs, but
these changes did not appear regularly, nor did they bear any relationship to
the length of time during which the animal had been injected with venom or to
the amount of venom injected.

The livers of none of the animals showed any degenerative changes. In
one liver, from a rabbit which had received injections for a period of more than
6 months and which during this time had received 758 mg. of venom (150 mg.
at the last injection), a slight increase of the connective tissue about the portal
vessels was noted and in places this connective tissue was pushing in between
the liver-cells. The liver of another rabbit which had received in all 0.96 c.c.
of venom during a period of almost 3 months showed a slight increase of the
connective tissue about the portal vessels. The livers of the other animals
injected showed no changes.

The kidney of one rabbit which died after having received 1,050 mg. of
venom during a period of almost 6 months showed an increase in the number of

*The microscopical examination of the various organs has been carried out by Dr. M. S. Fleisher.
{These were animals which we had attempted to immunize against heloderma venom by the administration
of gradually increasing dosesof venom. The results of these immunization experiments are reported later.

70 THE VENOM OF HELODERMA.

connective-tissue cells between the tubules and about the glomeruli. The
epithelium of the tubules was swollen and in many places obliterated their
lumen. The nuclei of some of these cells showed karyolysis and in some
tubules nuclei were seen lying free in the lumen. The glomeruli of this kid-
ney showed no distinct signs of involvement. One other kidney removed from
an animal which had received 67.5 mg. of venom during a period of 3 months
showed a slight increase of connective-tissue cells between the tubules. The
kidneys of the other animals examined appeared normal.

All the hearts examined were normal, except one which showed a slight
increase of connective tissue between the muscle-fibers and also a marked
degree of vacuolization in the muscle-fibers over a rather small area. Else-
where the muscle-fibers of this heart were normal. This animal, in which also
a connective-tissue overgrowth in the kidney was found, had received 67.5 mg.
of venom during a period of 3 months.

The lungs of several of the rabbits showed pneumonic areas. In these
areas the capillaries were congested and the alveoli contained desquamated
epithelium, polymorphonuclear leucocytes, and considerable granular detritus.
In the lungs showing this condition the large vessels were dilated with blood.
It is probable that this pneumonic condition represents simply a terminal
infection and can not be attributed directly to the influence of the venom.

The bone-marrow and spleen of animals that had received a number of
injections of venom showed more constant changes than did the other organs.
In bone-marrow, which showed no gross change, it was generally noted that the
giant cells were increased in number (in four or five cases) ; the polymorphonu-
clear leucocytes seemed to be increased in number, while the erythroblasts
showed a relative decrease. The histological picture suggests at least that
there was an effort to regenerate leucocytes or to produce a leucocytosis, and
this latter explanation is in agreement with the blood-picture noted in animals
which received repeated injections of heloderma venom.

The spleens of the rabbits which had received several injections of venom
showed several types of changes, one or more of which appeared in the spleen
of every animal examined. Most commonly we noted a change in the mal-
pighian corpuscles. These appeared smaller than normal and the cells forming
these bodies were less densely packed about the small vessels; in short, the
malpighian corpuscles were less prominent than normal. Almost as regular as
this change in the malpighian corpuscles was the proliferation of the endothelial
cells in the splenic pulp. This proliferation of the endothelial elements of the
splenic pulp was at no place very marked, but appeared diffusely throughout
the spleen. In afew cases we noted the splenic pulp to be less dense and cellu-
lar, and as a result of this thinning of the splenic pulp the connective-tissue
framework—especially the finer trabecule—became more prominent. The
cellular elements of the spleen pulp did not show any marked changes; no one
type of cell appeared to be unduly increased or diminished.

Thus we see that in the liver, kidney, and heart the venom does not pro-
duce any typical histological changes, but, when injected repeatedly over a

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 71

long period of time, it may lead to a slight degree of fibrosis in these organs.
In the lungs the venom itself does not produce any distinct changes and, as
stated above, the pneumonic condition which has been noted is most probably
due to a terminal infection. In the bone-marrow, however, changes are pro-
duced which probably represent an effort to produce leucocy tosis of the poly-
morphonuclear element.

INFLUENCE OF THE METHOD OF ADMINISTRATION ON THE TOXICITY
OF THE VENOM.

It was possible in certain cases to determine exactly the influence that a
certain mode of administration of the heloderma venom might have on the
toxic effect, and especially upon the lethal dose of the venom. We have tested
the influence of injecting the venom subcutaneously, intravenously, intraperi-
toneally, and subdurally, and also of injecting it into the stomach or intestines.

We found that the venom acts most quickly when injected intravenously,
and also that, when thus administered, the minimal lethal dose is smaller than
when administered in any other manner. When injected in this manner the
venom causes the death of the animal in a very short time, usually within 30
minutes.

The effect of the venom appears rather more slowly when it is injected
intraperitoneally, and the amount necessary to cause the death of the animal
is distinctly larger than when injected intravenously.

Although the subcutaneous injection of venom kills the animals rather
more slowly than does the intraperitoneal injection, it is probable that very
little difference exists between the smallest amount of venom necessary to kill
the animal when injected in these two different methods. As arule, the mini-
mal lethal dose appears to be the same, but occasionally an animal injected
subcutaneously would survive the injection, whereas an animal injected in-
traperitoneally with a similar quantity of venom died. The most important
difference between the effects of injecting venom subcutaneously and intra-
peritoneally probably arises from the fact that the absorption of venom from
the peritoneal cavity is more rapid than the absorption from the subcuta-
neous tissues.

When injecting venom subdurally, one difficulty to be met and overcome
is the probability that the injection of large quantities of fluid into the sub-
dural spaces may, of itself, have some injuriousinfluence. Although we injected
1e.c. of 0.85 per cent sodium-chloride solution subdurally in guinea-pigs with-
out producing any noticeable effects, it is possible that the injection of 1 c¢.c.
of the diluted venom solution may produce more harmful effect than the injec-
tion of 0.1 ¢.c. of pure venom solution. Indeed, our experiments would appear
to support this possibility. When guinea-pigs were injected subdurally with
venom which had been diluted (they received subdural injections of 1 ¢.c. of
fluid), they died in almost as short a time as the animals which were injected
intravenously, and the minimal lethal dose appeared to be approximately the
same whether administered intravenously or subdurally. On the other hand,

72 THE VENOM OF HELODERMA.

when very small quantities (between 2 and 8 drops) of the fresh undiluted
venom were administered subdurally to rabbits we found that animals injected
subcutaneously with similar quantities of venom died as soon or sooner than
those which were injected subdurally. Whether these contradictory results
represent a difference between the reactions of guinea-pigs and rabbits to the
heloderma venom, we can not state, but it appears most probable that the
differences are to be explained by the injection of the large quantity of fluid in
the experiments with the guinea-pigs. It therefore seems that when venom is
injected subdurally it is no more active than when injected subcutaneously.
Perhaps the dilution of the venom and the quantity of fluid injected determine
the rapidity with which the ganglia-cells are affected, and correspondingly the
time of death.

When venom was injected directly into the stomach of a guinea-pig, by
opening the abdominal wall and passing the needle of the syringe through the
wall of the stomach, no effects due to the injection were observed. The same
negative result was noted when venom was injected into the small intestines.
Whether no venom is absorbed from the stomach and intestines, or whether
the venom is so changed by the gastric or intestinal secretions that it has lost
its toxic properties, we must leave undecided.* Similarly, venom of Colu-
bride has very little or no effect when introduced into the intestinal tract,
while venom of Viperide causes intense irritation of the intestinal mucosa.

Yet another method of administrating the venom was tested by placing
collodion sacs containing venom in the peritoneal cavity of rabbits and guinea-
pigs. The results of these experiments will, however, be reported in another
place.

We used 124 animals in testing the influence of the method of administer-
ing the venom, and the following examples have been selected as typical cases:

Subcutaneous injection: Guinea-pig 58, 400 g. JUNE 12, 3% 55™ injected 0.1 c.c. of venom.
JuNE 13, died during night.

Intraperitoneal injection: Guinea-pig 57, 560 g. JUNE 12, 3 56™ injected 0.1 c.c. of
venom. Died during night.

Intravenous injection: Guinea-pig 64, 440 g. JuNE 12, 5% 05™ injected 0.1 ¢.c. of venom.
5» 30™ dead.

Subcutaneous injection: Guinea-pig 60, 420 g. JuNE 12, 3" 50™ injected 0.05 c.c. of
venom. No symptoms.

Intraperitoneal injection: Guinea-pig 59, 680 g. JUNE 12, 35 54™ injected 0.05 c.c. of
venom. No symptoms.

Intravenous injection: Guinea-pig 65, 680 g. JUNE 12, 4" 55™ injected 0.05 c.c. of venom.
5» 25™ dead.

Subcutaneous injection: Guinea-pig 62, 640 g. JUNE 12, 35 39™ injected 0.02 c.c. of
venom. No symptoms.

Intraperitoneal injection: Guinea-pig 61, 660 g. JUNE 12, 3 47™ injected 0.02 c.c. of
venom. No symptoms.

Intravenous injection: Guinea-pig 66, 680 g. JUNE 12, 4" 40™ injected 0.02 ¢.c. of venom.
5" 20™ dead.

Subcutaneous injection: Guinea-pig 51, 740 g. JUNE 12, 9» 30™ injected 0.2 ¢.c. of venom.
1» 40™ dead. ; :

Intraperitoneal injection: Guinea-pig 52, 620 g. JUNE 12, 9» 33™ injected 0.2 ec. of
venom. 1» 40™ only moderately affected. JUNE 13, recovered.

*Compare the chapter on the biochemistry of the venom, by Dr. Alsberg. It is not probable that the injurious
influence of peptic and tryptic digestion upon the venom is sufficient to account for the lack of any symptoms after
this mode of application of venom. Other factors, as adsorption by the contents of stomach or intestines, imperfect
penetration through the wall of the alimentary tract, probably cooperate.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 73

Intravenous injection: Guinea-pig 63, 600 g. JUNE 12, 9% 52™ injected 0.05 c.c. of venom.
10° 05™ dying. 10 09™ dead.

Intravenous injection: Guinea-pig 54,600 ¢g. Jung 12, 10 16™ injected 0.02 c.c. of venom.
Showed serious symptoms, but recovered.

Subcutaneous injection: Rat 71, 170 g. JuNnE 11, 115 45™ injected 0.2 ¢.c. of venom.
Showed serious symptoms for a while and continued drowsy during day. JUNE
12, recovered.

Intraperitoneal injection: Rat 75, 190 g. June 11, 115 46™ injected 0.2 c.c. of venom.

Serious symptoms during day. Worse than rat 71. JUNE 12, dead.

pecicube een Rat 79, 30g. June 11, 35 01™ injected 0.2 c.c. of venom. 3h {gm

ead.

Subcutaneous injection: Rat 72, 170 g. June 11, 115 48™ injected 0.1 c.c. of venom.
Symptoms same as Rat 71. June 12, recovered.

Intraperitoneal injection: Rat 76, 190 g. June 11, 11> 20™ injected 0.1 ¢.c. of venom.
Serious symptoms. JUNE 12, recovered.

seaabiae aaa Rat 80,120g. June 11,3" 20™ injected 0.1¢.c.of venom. 4520"

ead.

Subcutaneous injection: Rat 73, 170 g. June 11, 11° 52™ injected 0.05 ¢.c. of venom.
No symptoms. JUNE 12, living.

Intraperitoneal injection: Rat 77, 170 g. Jone 11, 115 55™ injected 0.05 c.c. of venom.
No symptoms. JUNE 12, living.

Intravenous injection: Rat 81, 130 g. June 11, 55 16™ injected 0.05 c.c. of venom.
6» 00™ dead.

Subcutaneous injection: Rat 74, 170 g. June 11, 114 58™ injected 0.02 ¢.c. of venom.
No symptoms. JUNE 12, living.

Intraperitoneal injection: Rat 78, 170 g. JuneE 11, 125 00" injected 0.02 c.c. of venom.
JUNE 12, living; no symptoms. 10 30™ second injection of 0.28 c.c. of venom.
1» 30™ dead.

Intravenous Peon Rat 82,125g. June 11,4» 20™ injected0.02¢.c.of venom. JUNE
12, living.

Subcutaneous injection: Guinea-pig 92, 400 g. June 21, 115 00™ injected 0.15 c.c. of
venom. 1> 15™ dead. ;

Intraperitoneal injection: Guinea-pig 95, 300 g. JUNE 21, 125 30™ injected 0.15 c.c. of
venom. 1» 15™ dead.

Subcutaneous injection: Rabbit A 1, 1500 g. Fes. 3, 10> 30™ injected 15 mg. venom.
Fes. 4, seems well. Fes. 6, dead.

Subdural injection: Rabbit A 2, 1500 g. Fes. 3, 25 30™ injected 8 mg. venom. Fes.
4, seems well. Fes. 7, dead.

Stomach injection: Guinea-pig 117, 340 g. JUNE 28, 35 15™ injected about 1 c.c. of venom
in 6 e.c. NaCl. 4" 10™ recovered from influence of ether; appears normal.

Juxy 1, living, normal.

Subcutaneous injection: Rabbit A 5, 2500 g. Fes. 3, 3 00™ injected 8 gtt. fresh venom.
35 30™ affected. 55 00™ improved.

Subdural injection: Rabbit A 6, 2100 g. Fes. 5, 45 30™ injected 8 gtt. fresh venom.

Fes. 6, dead. ae

Stomach injection: Rat 112, 140 g. JUNE 28, 35 35™ injected 1 c.c. undiluted venom.
Tied up stomach at point of puncture. 4 10™ appears well. Juty 1, living,
normal.

Subcutaneous injection: Rabbit A 9, 1080 g. Fes. 9, 12" 30™ injected 4 gtt. fresh
venom. 3» 00™ affected. 39 00™ dead.

Subdural injection: Rabbit A 10, 1100 g. Fes. 9, 12" 46™ injected 4 ett. fresh venom.
1» 30™ slightly affected. Fes 10, 25 00™ dying; killed.

Stomach injection: Rat 111, 120 g. JUNE 28, 125 39™ injected 1 c¢.c. pure venom.
12h 45™ respiration disturbed. 100" improved, but remains lying down.
2h 00™ somewhat better. 45 00™ somewhat better; may live. JUNE 27, dead.

Subcutaneous injection: Rabbit A 14, 1120 g. Fes. 13, 12> 00™ injected 2 gtt. fresh
venom. 2h 00™ affected. 2h 30™ dead.

Subdural injection: Rabbit A 13, 1120 g. Fes. 12, 12 00™ injected 2 gtt. fresh venom.
2 00™ much affected. 4> 45™ dead. The symptoms produced in this experi-
ment we attribute to the escape of venom from the wound into the peritoneal cavity.
With rat 117, where precautions were taken to ligature the stomach at the point
of injection, no symptoms appeared.

Subdural injection: Guinea-pig 93, 400 g. JUNE 21, 115 20™ injected 0.15 c.c. of venom.
115 28™ dead.

Subdural injection: Guinea-pig 94, 350 g. Jung 21, 125 05™ injected 0.1 c.c. of venom.
12 13™ dead.

Subdural injection: Guinea-pig 96, 350 g. Jung 21, 12" 05™ injected 0.1 ¢.c. of venom.
2» 45™ dies, having been almost dead since 24 07™,

74 THE VENOM OF HELODERMA.

Subdural injection: Guinea-pig 98, 350 g. JunE 21, 25 29™ injected 0.05 c.c. of venom.
2h 46™ dead; almost dead since 25 32™. ans ;
Subdural injection: Guinea-pig 97, 350 g. JUNE 21, 24 30™ injected 1 c.c. normal saline.

4» 46™ normal; killed. ’ tone
Injection into intestines: Rabbit B 3, 1700 g. JuNE 28, open abdominal cavity; inject
into intestines (small) 40 mg. venom. June 29, well and lively. JULY 7,
well and lively. ; ene:
Injection into intestines: Rabbit B 4, 1600 g. Jung 28, open abdominal cavity; inject
into intestine (small) 20 mg. venom. JuNE 29, well and lively. Joy 7, well
and lively.

EFFECT OF PLACING COLLODION CAPSULES CONTAINING VENOM IN
THE PERITONEAL CAVITY.

In several rabbits and guinea-pigs we tested the influence of venom placed
in the peritoneal cavity in collodion capsules. By this method we wished to
determine to what extent venom passed through the collodion capsule in the
animal body, and also the effect of venom being added continuously in very
small quantities.

Eight rabbits and two guinea-pigs were used in these experiments. The
collodion sacs were made by pouring a thin coating of collodion into a small
test-tube and removing this collodion coating after the ether and alcohol had
evaporated. The sacs were first tested with water and only capsules that
were perfect were used. The solution of either fresh or dry venom was placed
in the capsule, the neck of which was tied. The tied end of the capsule was
further sealed with collodion and the capsule containing the venom was placed
for an hour in a water-bath, the temperature of which was maintained at 80°C.
Thereupon the capsule was put into the peritoneal cavity under the usual
aseptic precautions.

We found the venom gradually passed out of the capsules and was
absorbed by the animals. The lethal effect was proportionate to the quantity
of venom put within the capsule. The number of capsules placed within the
peritoneal cavity appeared to influence the toxic effect. The greater the num-
ber of capsules, the greater was the aggregate surface of diffusion through
which the venom could pass out of the capsules.

Only two guinea-pigs were used in these experiments. One collodion cap-
sule was placed in the peritoneal cavity of each of these animals; in one case
the capsule contained 0.5 c.c. of fresh venom, in the other 24 mg. of dissolved
dry venom. The first animal died after 28 hours, while the second died in
24 hours.

Both animals showed marked weakness about 12 hours after the capsules
had been placed in the peritoneal cavity. The respiration was rapid and
usually shallow. They were very quiet and did not move about their cages
unless disturbed. From this stage they both passed on to a stage of distinct
paralysis, in which the respiration was deep and strained. Previous to this
latter stage, however, the guinea-pig which had received the capsule contain-
ing 24 mg. of venom developed marked convulsions which lasted for a short
time and were immediately followed by the paralytic stage. The animals did
not show any recovery from this dyspneic, paralytic condition; they died from
30 to 60 minutes after the appearance of this condition.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 75

The post-mortem appearances were essentially the same in both animals.
There was a slight amount of gelatinous exudate on the visceral peritoneum
where the collodion sacs had been in contact with the intestines. The peri-
toneum elsewhere was smooth and not injected. The intestines contained
considerable quantities of fluid and the stomach showed several small hemor-
rhagic and ulcerated areas.

The collodion sacs in both cases contained at the time of the autopsy con-
siderably less fluid than had been put into them at the beginning of the experi-
ment. It was, however, definitely shown that some venom still remained
inside the capsules. They were washed out with sodium-chloride solution and
various quantities of the solution were injected into mice. It is impossible to
state exactly how much venom was found in the sacs, but the quantity was
sufficient to kill some of the injected mice. In the case in which 24 mg. of
venom had been put into the capsule, approximately 3 mg. were found at the
end of the experiment; while in the other case only a very small quantity, pos-
sibly 0.02 c.c., of venom was recovered (about one-twentieth of the amount
originally introduced).

Of 8 rabbits, 2 received one capsule (1 of these later received three cap-
sules), 2 received two capsules, 3 received three capsules, and 1 received four
capsules.

The first of the animals, receiving in one capsule 30 mg. of venom, died 9
days after the operation. The other rabbit received in one capsule 50 mg. of
venom, but did not die; 25 days after the first capsule had been placed in its
peritoneal cavity, it received three more capsules, each containing 0.3 c.c. of
fresh venom. The animal died 2 hours after these last capsules had been
placed in the peritoneal cavity.

The three animals with two capsules received each 60 mg. of venom, 30
mg. being in each capsule. One of these animals died 5 days afterwards,
another in 2 weeks, while the third survived.

In the experiments in which three capsules were placed in the peritoneal
cavity each capsule contained 0.3 c.c. of fresh venom, each rabbit receiving 0.9:
c.c. of venom. One of these animals died in 40 hours, the others in 18 hours.

One rabbit, given four capsules each containing 0.3 c.c. of venom, died in
about 12 hours.

All of the animals which survived the administration of venom for several
days or weeks showed a marked loss of weight, which amounted in some cases
to as much as one-third of the original body-weight; otherwise, they showed
no symptoms of the poisoning, as they were usually active and their appetites
continued good. Death usually occurred suddenly. In a period of a few
hours the animal would develop weakness and prostration and death would
follow rapidly. Evidently a gradual accumulation of venom takes place in the
animal organism. The animals which died shortly after the capsules had been
placed in the peritoneal cavity showed signs of weakness soon after the opera-
tion. Prostration and dyspnea appeared rapidly and the animals appeared to.
die as a result of respiratory failure.

76 THE VENOM OF HELODERMA.

In none of the rabbits were any especial changes foundat theautopsy. In
one animal, however, which died 18 hours after receiving three capsules, each
containing 0.2 ¢.c. of venom, small hemorrhages and a few ulcers were noted in
the gastric mucosa. The capsules were always found to be surrounded by a
thick fibrinous exudate, which appeared to be partly organized in the cases in
which the animals survived.

In two cases we made hemoglobin, red blood-cell, and leucocyte counts
before and several days after the giving of the capsules. The results of these
are given below:

if
. No. of | Amount | - Hemo- | Red blood | Teuco-
Rabbit. capsules. | of venom ; Period. globin. | corpuscles. | cytes.
| {
mg. pct:
Ad 1 20 {Before..... 28 15.66 6,430,000 8,160
Sarees i 6 days after...... 16 6,430,000 | 18,040
A8 1 50 Beloreiie. o4.6 en es 10.90 5,420,000 9,120
fe aestite 24 days after...... 8.78 3,030,000 | 9,200

These experiments are too few in number and the results are too irregular
to allow of any definite conclusions. It appears, however, that this method of
administering venom has not a very marked effect on the elements of the
blood.

ADMINISTERING FRACTIONAL DOSES OF VENOM.

In pigeons and mice small sublethal doses of venom were repeatedly in-
jected in order to determine the rapidity with which the venom was destroyed
by or eliminated from the body. For this purpose we used six pigeons. The
following table shows the amounts injected, the time which elapsed between
the injections, and the ultimate result:

Influence of injection of fractional doses of venom into pigeons.

Time
. - between
First Time Second
injection. | elapsed. | injection. com Result.
and death.

mg. hre. mg. hrs. min.

0.3 3 0.3 hints eee Recovered.
4 3 4 “9 Do.
5 3 5 ie TBS Do.
6 3 6 2 00 Died.

6 Ps 6 1 00 Do.
6 1 6 40 Do.

From these experiments we learn that when the dose of venom usually
lethal for pigeons (0.8 mg.) is injected in fractional parts, with an interval of 3
hours between the injections, the animal survives the injection. If slightly
more than the minimal lethal dose be injected, with a similar interval of time
between the two injections, the pigeons still may survive. If, however, within
a period of 3 hours, as much as 12 mg. of venom be injected, thus half again as
much as the minimal dose, the pigeons die. When the interval between the
two injections of venom is shortened to an hour the animal dies more quickly
than when a longer time elapses between the two injections. It therefore

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 77

appears that in 3 hours the pigeon is able to eliminate or destroy a part, but
only a relatively small part, of the injected venom.

The results of similar experiments with mice were not as regular as those
with the pigeons.

Influence of administration of venom in fractional doses to mice.

First Time Second Time Third
injection. elapsed. injection. elapsed. injection. Death alter
mg. hrs. min. ma. hrs. mg.

0.04 1 00 0.04 is ee 3 hours.
04 1 00 04 E Shey 8 hours.
04 3 00 04 eh isin 12 hours.
04 3 00 04 ia aoa 12 hours.
.08 4 00 04 als ee Recovered.
04 1 00 04 2 0.04 Do.
04 2 00 04 1 04 8 hours.

04 2 00 04 2 04 Recovered.
04 1 00 04 2 04 0
03 .. 45 05 did i Do
03 45 05 Do
03 45 05 Do

It has been noted before that the reaction of the individual mouse to
the heloderma venom varies quite markedly and that frequently mice will die
as a result of the injection of a dose which usually would prove to be sublethal.
It seems that such has been the case in these experiments. While 0.15 to 0.12
mg. of venom may be considered as the lethal dose for mice, we find that some
mice which received only 0.08 mg. of venom died as a result of the injection,
even though 3 hours intervened between the injection of the two parts. On
the other hand, some mice which in two or three injections received a quantity
of venom equal to the minimal lethal dose survived. In view of these latter
experiments, it appears probable that the injection of a minimal lethal dose in
fractional parts does not necessarily cause the death of the mouse; we must,
however, consider that 0.12 mg. is not necessarily a lethal dose.

In the case of the pigeon, within a few hours a relatively very small part of
the venom is either eliminated or somehow transformed into such a condition
that it is no longer injurious to the sensitive parts of the central nervous sys-
tem of the injected animal.

These results agree with the effect we observed in animals in which the
venom had been introduced by means of collodion capsules. Here also, owing
to the slow diffusion of the venom, the lethal effect was much delayed and in
some cases the animals, which would have died if the same amount had been
injected directly, recovered.

SUSCEPTIBILITY OF VARIOUS SPECIES OF ANIMALS TO
HELODERMA VENOM.

We have tested the susceptibility of warm-blooded vertebrates, cold-
blooded vertebrates, and invertebrates to the venom of the Heloderma. In
comparing the resistance of the warm-blooded vertebrates to heloderma venom
we have used as a standard for comparison the minimal lethal dose per kilo-
gram of body-weight.*

*We have not compared the differences in functional disturbances, as these seemed to bear but little relation-
ship to the minimal lethal dose. In certain cases the animals were severely affected by the injection of venom, but
were relatively resistant to its lethal effect.

78 THE VENOM OF HELODERMA.

We have, except in one case, compared only the minimal lethal doses of
the dissolved dry venom. Although we have investigated the minimal lethal
doses of the fresh venom, we have not, as a rule, used the results of these experi-
ments in comparing the susceptibility of the various species, mainly on account
of the already mentioned marked daily variation in the strength of the fresh
venom. It has, however, been necessary in certain cases to compare also the
lethal doses of the fresh venom in order to supplement the results as shown by
the lethal doses of the dry venom, and in one case (cat) we have tested only the
lethal dose of fresh venom. We have noted considerable variations in resist-
ance among individuals of the same species, most especially when fresh venom
was injected. Dogs showed a considerable individual variation in regard to
sensitiveness, and in a lesser degree rabbits and mice showed similar variations.
The variability among animals of the same species showed itself always in the
direction of aresistance considerable belowthe average. We didnot find indi-
viduals showing more than the average resistance.

We note that among the warm-blooded animals tested pigeons are the
least resistant. The other animals all show relatively less susceptibility to the
lethal effect of venom in approximately the following order: dog, guinea-pig,
mouse, rabbit, cat, and rat. The dog, guinea-pig, mouse, and rabbit all seem
to have approximately the same degree of resistance to the venom, since the
lethal dose of the dry venom is very nearly the same for every kilogram of body-
weight of each of these animals. The rat, however, shows distinctly greater
resistance to the lethal effect of the venom than any of the other warm-blooded
animals.

Minimal lethal doses of venom for warm-blooded animals.

| Per kilogram of

Absolute quantities. body-weight,

. Average |
Subject. weight. j
Fresh | Dry Fresh Dry
venom. | venom. | venom. | venom.

gm. cc. mg. C.c. mg.
(1) Pigeon......... 250 0.02 0.8 0.08 3.2
(2) Dog.......00., 10,000 0.1 | 100 0.01 10+
(3) Guinea-pig..... 500 0.05 5 0.1 10
(4) M 15 0.005 | 0.15 | 0.25 10
(3) Rabbit. 1,500 0.05 | 18 0.04 12
(6) Cat 3/000 OO . Sea 0:3 ‘ae
(7) Rat 125 ae ie 0.8 40

It will be noted that the lethal dose of dried venom is approximately the
same for every kilogram of body-weight of either dog, guinea-pig, mouse, or
rabbit, the lethal dose being approximately 10 mg. for each of these animals.
We do not, however, find a similarity regarding the lethal dose of fresh venom
for these four classes of animals. The dog appears to be most susceptible to
fresh venom; the rabbit, pigeon, guinea-pig, mouse, and rat follow in the order
named. It is probable that this apparent variation in susceptibility to fresh
venom is due largely to the variation in the toxicity of the venom.*

*In order to obtain the dry venom, many specimens of fresh venom were mixed; thus the dry venom Tepresents
a substance of approximately average toxicity. On account of the great variability in the toxicity of fresh venom not
much importance can be attached to the minimal lethal quantities of fresh vonom in the case of dogs, in which the
number of experiments was necessarily small.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 79

The lethal dose of the fresh venom only was testedin cats. We found that
a cat weighing 3,000 em. was killed by the injection of 0.9 c.c. of venom; thus
the minimal lethal dose was 0.3 ¢.c. of venom pro kilogram of the body-weight.
This dose is considerably larger than the lethal dose of fresh venom for dogs,
guinea-pigs, or rabbits, and slightly larger than the lethal dose for mice.

Rats were, as stated above, distinctly more resistant to the injection of
venom than any of the other animals. A rat weighing 125 gm. was killed by
the injection of 5 mg. of dry venom or 0.1 ¢.c. of fresh venom. Thus the lethal
dose pro kilogram of body-weight for rats was 0.8 c.c. of fresh venom and 40
mg. of dry venom.

Cold-blooded verbebrates are distinctly less susceptible to the action of
venom than warm-blooded vertebrates. We have tested the lethal effect of
venom of frogs, toads, snakes, turtles, eels, heloderma, and fundulus.

The Heloderma shows a resistance toward its own venom which for pur-
poses of protection at least is complete. The injection of 2.25 c.c. of fresh
venom—a quantity sufficient to kill 45 guinea-pigs each weighing 500 grams—
produces no evident symptoms on the Heloderma, which weighs on the average
500 grams. A definite explanation accounting for this marked resistance of
the Heloderma to the toxic effect of its venom can not be given at present, but
in view of the results of experiments in which the adsorptive power of helo-
derma organs for heloderma venom was tested, it appears that the liver, and,
to a less degree, the kidney, of the Heloderma exert a specific adsorptive action
on the venom and this may explain, in part at least, the resistance of the Helo-
derma to its venom.* This resistance on the part of the Heloderma is not due
to antibodies in the serum of the Heloderma, since we have found that venom
which had been mixed with heloderma serum had not lost any of its toxic
power. The relative immunity of poisonous animals against their own venom
is evidently a general phenomenon.

We have found in a single experiment that the immunity of Heloderma
toward its own poison does not extend to the snake venoms. The heloderma
reacts very quickly toward rattlesnake venom, but, as we had for the deter-
mination of this point, only a small supply of snake venom of unknownstrength,
we can say no more concerning the lethal dose of this venom for helodermas
than that it appears not to greatly exceed the lethal dose for rats and guinea-
pigs.

The resistance of the snake to the toxic action of heloderma venom was
tested in only two animals, both water-snakes (Utania certalis). One animal
which weighed 80 grams died after the injection of 35 mg. of venom, while the
other survived the injection of 25 mg. and only died after the second injection
of a similar quantity. The lethal dose of venom for snakes appears to be
approximately 400 mg. pro kilogram of body-weight.

The toad (Bufo americanus) shows a marked resistance to the toxic effect
of venom. An animal weighing 50 grams was killed by the injection of 15 mg.
of dry venom; thus for toads the lethal dose of venom pro kilogram of body-
weight would be 300 mg., a quantity far greater than the lethal dose of venom

*C/f. the chapter on the adsorption of venom, by M. Fleisher and Leo Loeb.

80 THE VENOM OF HELODERMA.

pro kilogram for rats, which proved to be the most resistant of the warm-
blooded vertebrates tested. The toads showed very profound respiratory dis-
turbances and almost complete paralysis for some hours following the injection
of venom, but in spite of these alarming symptoms, unless the amount injected
was 15 mg. or more, the animals recovered from the injection.

As is well known, the toad is resistant to a number of poisons besides the
one contained in its own skin secretion. Certain of these poisons, notably anti-
arin and the poisons of the digitalis group, produce physiological disturbances
similar to those produced by toad venom, and it has been suggested that the
same mechanism by which the toad is able to protect itself against the latter
poison answers also as a protection against the former ones. However that
may be, the venom of Heloderma, toward which the toad shows also remarkable
resistance, has physiological effects quite different from those produced by
antiarin, digitalis, and the toad poisons; consequently, the same explanation
will not suffice here. We made no experiments to investigate the nature of
this resistance, as our supply of toads was limited.

Frogs (Rana climatans and Rana pipiens) proved tobe more susceptible to
the action of venom than toads. Five frogs weighing between 80 and 85
grams died after injection of 4, 5, and 6 mg. of venom; one frog weighing 84
grams received 5 mg. of venom and survived; two frogs weighing 80 and 75
grams, respectively, survived after injection of 3 mg. of venom; 4 mg. repre-
sents, therefore, approximately the lethal dose for a frog weighing 80 grams.

Tadpoles were found to be more susceptible than adult frogs. Tadpoles
weighing approximately 2 grams survived after the injection of 0.025 mg. and
died after injection of 0.05 mg. of venom. The lethal dose for a kilo of tad-
poles is, therefore, between 12 and 25 mg. of venom, while the lethal dose pro
kilogram of frogs is 50 mg.

Lethal doses of venom for cold-blooded vertebrates.

Average Tethal Lethal dose

Animal, . weight dose, in prokilo., in
in grams. | milligrams milligrams.

Largest dose
injected which
was not lethal,
in cubic centi-
| meters.
|
I

80 35 425

50 15 300

80 4 50

80 5 65
Heloderma........ 500 < 225
TEC is sna nantcamaiatn nee (?) ! oer wl
Fundulus,......... (?) O.1 1 | ied

Testing the susceptibility of various kinds of turtles to the heloderma
venom, we found that all reacted in relatively the same manner to the venom.
We used the spotted terrapin and painted terrapin in most experiments, but
also used the stink-turtle and mud-turtle. The injection of 5 mg. of venom
was found sufficient to cause the death of a turtle weighing 80 grams. The
lethal dose of venom for a turtle is, therefore, 65 mg. for every kilogram of
body-weight.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 81

We tested the effect of venom on only one eel; this animal, which weighed
100 grams, wasnot affected by the injection of 0.1¢.c.of freshvenom. Whether
this is nearly the minimal lethal dose we can not state.

The fundulus (F. majalis) was the only member of the fish family in which
the influence of venom was tested. We found that 0.1 ¢.c. of fresh venom was
the minimal lethal dose for these animals. Since the fundulus is a small fish
weighing probably 30 grams, it is evident that the lethal dose pro kilogram
would be very large, possibly 3 c.c. of venom.

Our investigations on invertebrate animals indicate that these animals are
immune to heloderma venom, or, if not immune, they at least possess a resist-
ance enormously greater than vertebrate animals. We injected individuals of
Sycotopus canaliculatus, Limulus, Nereis vivens, Phascolosoma gould, Asterias
forbesti, Mnemiopsis leidyi, and Gonionemus murbachii, without being able to
produce evident symptoms in any of these animals, although we injected them
with doses sufficient to kill more than 50 times the same weight of guinea-pig.
We also demonstrated in the case of Gontonemus that animals immersed in sea-
water containing 5 per cent of sterile venom continued their swimming move-
ments unchanged for many hours and died only after a length of time sufficient
for bacterial decomposition to render the sea-water unsuitable. Controls
placed in sea-water plus 5 per cent of sterile human saliva died somewhat later,
but as the sea-water plus venom contained far more decomposable material
than the sea-water plus saliva, no conclusion concerning the toxicity of venom
for Gonionemus could be drawn from this circumstance. We likewise found
that fertilized sea-urchin and starfish eggs placed in sea-water containing 3 to
7 per cent of venom continued to develop and formed a number of swimming
gastrule. That the number which reached this stage in the venom solution
was considerably less than in controls is not surprising, when we consider the
large amount of organic matter and the consequent putrefactive changes tak-
ing place in the sea-water containing venom.

Maximum quantities of venom injected* into invertebrates.

Animal, Maximum quantity.

Limulus. ...........0.
Sycotopus............
Phascolosoma........
Asterias.... cw
Mnemiopsis.
Nereis........

No toxic effects.

SrowwwnRads

COSSOSO,
oro

TOXICITY OF THE ORGANS AND EXCRETIONS OF HELODERMA.

Suspensions of the various organs of the Heloderma were injected in order
to determine whether any organ other than the venom gland secreted or held
the venom. The various organs tested were the liver, kidney, spleen, pancreas,
and lachrymal gland (?). (This latter was a gland which lay on the lower por-
tion of the eye-socket.) The bile, urine, and blood-serum were also tested.

“Where in our report the dose of venom is given in volume and not in weight, fresh venom was used; otherwise,
dried venom.

,
82 THE VENOM OF HELODERMA.

In making the suspensions, the various organs were first minced and
crushed and then added to a very small quantity of 0.85 per cent sodium-
chloride solution. This mixture was allowed to stand for about 2 hours before
being injected into the animals. In all cases the effects of the organ extracts
were tested on mice.

We found that the injection of 1 c.c. of the suspension of kidney, pancreas,
and spleen of Heloderma had no visible effect upon the animals. Two mice in-
jected with liver suspension, one with 1 ¢.c. the other with 0.5 ¢.c., died on the
third day after the injection, in all probability not as a result of the injection,
but due to starvation. Two other animals injected with 1 c.c. and 2 c.c. of
liver suspension, respectively (not at the same time as the other two), showed
noilleffects. Three animals were injected with heloderma egg which had been
mixed with an equal quantity of sodium-chloride solution; they received either
0.5 ¢.c., 1 ¢.c., or 2 ¢.c. of this mixture and none of them was affected by the
injection. Several animals which received variable doses up to 1 ¢.c. of the
suspension of the supposed lachrymal gland or in which picces of this organ
were placed in a subcutaneous pocket remained perfectly well. From the above
experiments it would appear that none of the organs of the Heloderma, includ-
ing the gland which is found in the eye-socket, have a toxic action.

Two mice were injected with urine of the Heloderma; one received 1 c.c.,
the other 2 c.c._ Neither of these animals showed any disturbances following
the injection.

Two mice were injected with serum of the Heloderma. Both received 2
c.c. of serum, but no toxic effect was evident.

One mouse injected with 0.25 c.c. of heloderma bile died after 2 hours,
while another mouse which received 0.1 ¢.c. of bile survived the injection. In
order to test the toxicity of bile, we injected a mouse with 0.5 e.c. of dog’s bile
and this animal died in 2 hours. It is evident that the toxic effect of the helo-
derma’s bile is due not to any content of venom, but to some substance con-
tained in bile, such as the bile-salts. In order to further show that the toxic
effect of the heloderma’s bile was not due to venom, we injected some mice
with various quantities of bile and venom and other mice with like quantities of
venom alone. The mice which received the injections of bile and venom did
not die sooner than those which received the venom alone. On the other hand,
the addition of bile to the injected venom did not diminish the toxicity of the
venom, since the mice which received like quantities of venom died approxi-
mately the same length of time after the injection, irrespective of whether bile
had or had not been added.

These results obtained with the organs and also with the eggs of Heloderma
differ markedly from those obtained with the blood of snakes and certain other
animals possessing venom-secreting glands. The blood of the latter has by
various investigators* been found to be very poisonous; while the blood of
Helodermaisinnocuous. Itis possible that the toxicity of certain organs of the

*Phisalix et Bertrand, Archiv. de Physiol. 1894; Calmette, Compt. Rend. de la Soc. Biol., 1894, x, ser. 1;
Wehrmann, Annales de l'Institut. Pasteur, x1, 810, 1897.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 83

Crotalus, observed by Flexner and Noguchi,* was entirely or partially due tothe
admixture of blood in the organs. These authors, as well as Phisalix,f estab-
lished the toxicity of the ova of venomous snakes, while we found the ova of
Heloderma to be devoid of any toxic effect. We may add that Fraser} de-
scribed an antivenomous action of the bile of serpents.

INFLUENCE OF VENOM ON COAGULATION TIME OF THE BLOOD.

The influence of the Heloderma venom on the coagulation time of the rab-
bit’s blood was studied in various ways. The first method consisted in inject-
ing large quantities of venom intravenously and shortly before the death of the
animal, collecting the blood through a glass canula into a small test-tube. We
injected intravenously doses of venom of 20 to 30 mg., an amount causing the
death of the animal in periods varying from 5to10 minutes. The coagulation
time for the blood in these cases varied from 4 to 11.5 minutes, apparently
independent of the amount of venom injected or the interval between the
injections and the taking of the blood. In one case, a large quantity of blood
collected in a flask coagulated in 12 minutes.

Since all the rabbits injected with venom died as a result of asphyxia
(death being due to respiratory failure), we considered it necessary to deter-
mine what influence asphyxia itself might have on the coagulation time of the
blood. We therefore compressed the trachea of a rabbit which was deeply
under the influence of ether and after 10.5 minutes of intermittent compres-
sion bled the animal. The blood collected in a small test-tube coagulated in
4 minutes; that collected in a small flask in 12 minutes.

The results of these experiments lead to the conclusion that the venom of
the Heloderma has no distinct influence on the coagulation time of the blood.

Influence of injection of venom on the coagulation of the rabbit's blood.

Hy wlio
Rabbit F Amount of venom | 2¢?Wween ee
Weight. — injection | Coagulation time.
No. injected. and taking
of blood.
gms. he om hem
613 1100 30 mg..........-. 10 00 4 00
616 1440 25 mg...........- 7 30 11 00
617 1280 20ME oe 665s euies 4 45 5 45
618 1700 15+5+5 mg...... 5 00 4 30
412 00 (in flask)
619 | 1200 | Asphysia......... 10 20 lf 43 Oo Gin ask)

aAfter last injection.

The second method of testing the influence of venom on the coagulation
time of the blood was to allow blood to flow through a canula into a tube con-
taining various quantities of venom solution. In these experiments we used
0.5 c.c., 1 ¢.c., 2 ¢.c., and 3 ¢.c. of a 1 per cent solution of the dry venom to 2
c.c. of blood. In every case the fluid in the test-tubes was made up to 5 c.c.
with 0.85 per cent sodium-chloride solution. Furthermore, two control experi-

*Jour. Path. and Bacteriol., vim, 1903. Compt. Rend. Soc. Biol., 1905, pv.
{British Med. Journ., i, 1897.

84 THE VENOM OF HELODERMA.

ments were made with the blood of the same animal, in which 2 ¢.c. of blood
were added to 3 c.c. of sodium-chloride solution and one in which 5 ¢.c. of undi-
luted blood were collected. The coagulation times of the various mixtures are
given below:

Influence of venom on the coagulation of the rabbit’s blood.

1 per cent
Blood. | venom NaCl
solution.

Coagulation
solution. time.

a

ANNNNNNS

From these experiments it is again evident that venom neither delays nor
accelerates noticeably the coagulation of the blood.

Inasmuch as in the preceding experiment the individual variations were
marked and the quantities of venom used were large, another similar experi-
ment was performed. Into a series of test-tubes each containing 2 c.c. of
venom dissolved in 0.85 per cent NaCl solution 2 c.c. of rabbits’ blood was
allowed to flow from a canula inserted into the carotid artery.

Surface of

blood coag-

Pate Beginning
Injection. ulated. coagulation.

coagulation.

he om. hk. m. h. m.

2 c.c. rabbit’s blood plus 2 c.c. 0.85 per cent NaCl... 6 20 es 10 40
2c.c. TaDeNt: 3 blood plus 2 c.c. 0.85 per cent NaCl contai 4 05 6 40 10 35

espace aa tet dette its he Pe ae eR NS tec spe Se Sac cn ak esi ahaa 4 25 8 15 9 50
2 c.c. rabbit! 83 blood plus 2 ¢.c. 0.85 per cent NaCl containing 2 mg. venom ; A 7 05 : fe
pag srabbibs blood pluie Mee. URS per seal NEOl oontailag Time vauoal 4 05 7 20 8 50
2,0. rabbit's biood plus 2.c.0. 0.85 per cont NaCl containing 12 mg.venom.| 3 00 coe 10 00
2 c.c. rabbit's blood plus 2 c.c. 0.85 per cent NaCl............2....0 000s 6 40 ll 55

The individual variations in this experiment, due to imperfections of tech-
nique, are relatively slight. There is little doubt that the admixture of venom
caused an insignificant acceleration of the coagulation that is not specific. Ad-
dition of a chemically inert foreign body in fine suspension would have had at
least an equally strong accelerating effect.

We also used a third method, which had been employed by Noc in his
studies of the influence of snake venoms on the coagulation of the blood. For
this we obtained 100 c.c. of rabbit plasma kept liquid through the addition of 1
gram of sodium citrate. This citrate plasma coagulates readily after addition
of CaCh.

So much 0.85 per cent NaCl solution was added that the total volume in
each tube was 2 c.c. It was found that 0.4 to 0.6 c.c. of 5 per cent CaCl,
caused a coagulation of this plasma in approximately 14 to 15 minutes. After

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 85

addition of 1, 2, 3, 4, 5, and 7 mg. of venom dissolved in 1 c.c. of 0.85 per cent
NaCl to 1 ¢.c. of citrate plasma, but without CaCl, the plasma remained
liquid for 24 hours, when the experiment was interrupted.

0.5 percent) posinni f
ginning off Complete
Plasma. a dd a coagulation. | coagulation.
C.c. €.c. he om. h. m
1 0.2 20 20 30 00
1 0.3 15 40 19 50
1 0.4 9 00 14 30
1 0.5 13 00 1 8
1 0.6 10 50 13 20
1 0.7 14 10 16 10

If we add venom to the citrate plasma and afterwards 0.4 c.c. 0. 5 per cent
CaCl, the various quantities of venom again do not exert any specific influence
on coagulation.

1 Bericent 0.5 Bet as Venom | Beginning of | Complete
plasma. added. added. coagulation. | coagulation.
C.c. C.c. mg. hem. ho om
al 0.4 1 8 15 13 45
1 0.4 2 8 5 14.5
1 0.4 3 7 55 13 55
1 0.4 5 6 55 14 50
1 0.4 7 9 45 14 35
1 0.4 10 10 55 14 35
1 04 6 55 11 35
hee 7 00 oe

In each case the venom had been dissolved in 0.85 per cent NaCl solution
and to each test-tube, so much 0.85 per cent NaCl was added that the total
volume of fluid in the test-tube was 3 c.c. In this case addition of venom
caused a quite insignificant delay in the coagulation of the citrate plasma which,
however, did not bear any direct relation to the amount of venom added.
From all these experiments we may conclude that venom does not exert any
distinct influence on the coagulation of the blood.

Our results differ from those obtained by van Denburgh and Wight, who
state that they found the venom of Heloderma exerting a distinct influence on
the coagulation of the blood. In order to clear up, if possible, the cause of this
divergence in the results obtained by ourselves and by van Denburgh and
Wight, who in some of their experiments had used fresh venom, while we
worked with solutions of dried venom, we made a few additional experiments
in which we injected into the ear-veins of rabbits an extract of the venom gland
of Heloderma after it had been filtered through filter paper. The extract was
prepared by rubbing up 0.87 gram of gland with 6 c.c. of 0.85 per cent NaCl
solution.

Fifty seconds after the injection of 0.5 ¢.c. of the extract into a rabbit
weighing 800 grams, the animal was in a dying conditon; 30 seconds later
blood was obtained by making an incision into the heart. Respiration had
ceased at that time, but the heart was still beating. Some of the dark-colored
blood was keptina porcelain dish; the rest was distributed into four test-tubes,

86 THE VENOM OF HELODERMA.

each receiving 1 to 2c.c. blood. After 1 minute 40 seconds there was the
beginning of coagulation in the tubes and 8 minutes after the withdrawal of
the blood the coagulation in the test-tubes was completed; 3 minutes later the
blood in the porcelain dish was a solid coagulum. In a control experiment
we withdrew in a similar manner, directly after death, the blood from a rabbit
whose neck had been broken. In this case the blood escaped more slowly from
the heart and consequently came into a prolonged contact with the tissues.
Coagulation here was completed in 1 minute 25 seconds.

In a third experiment the same venom-gland extract was used after it had
been standing on ice for several days; 30 seconds after the intravenous injection
of 1 c.c. of the extract, the rabbit, weighing about 2,200 grams, was weak and
its respiration forced; 3 minutes after the first injection a second injection of
1 c.c. of venom was given; 6 minutes after the second injection the animal died
and the blood was withdrawn through an incision into the heart; after 1 minute
10 seconds the blood was coagulated in the porcelain dish and almost coagu-
lated in the test-tubes. In the latter the blood had been shaken during the
process of coagulation and the clot somewhat retracted. In consequence of
this premature retraction some blood remained fluid in the test-tubes; this
blood became solid 3 minutes after the formation of the first clot.

We may conclude from these experiments that after injection of the gland
extract an exceedingly slight retardation in the coagulation of the blood may
take place. It is, however, very doubtful whether this retardation is to be
attributed to a specific effect of the venom; it is more probably due to the
influence of tissue extract, which produces a negative phase if injected in very
small quantity. However that may be, at the best the effect of the injection of
gland extract on the coagulation of the blood was very small in our experiments.

In contradistinction to the venom of Heloderma various snake venoms
have a pronounced action on the coagulation of the blood, some venoms accel-
erating, others inhibiting it.

INFLUENCE OF INOCULATION OF PIECES OF VENOM GLAND INTO MICE.

In the course of some work concerning the growth of pieces of the venom
gland transplanted into helodermas, the retention of the toxicity of the venom
in the transplanted pieces of gland was also studied. In order to determine
this, pieces of venom gland were inoculated into mice. The details of the
transplantation of these pieces of gland will be found in a previous paper deal-
ing with the morphology. It will be sufficient here to state that pieces of the
gland were placed under the skin of the same animal’s thorax and at the same
time under the skin of another animal’s thorax, so that each animal had two
grafts. The first mentioned piece will be spoken of as the auto-transplant, the
second as the iso-transplant. Four experiments were carried out in which the
pieces were removed 1, 2, 3, and 4 weeks respectively after the transplanta-
tion, and were then inoculated into mice. The pieces of venom gland were
placed under the skin of the mice by means of a trocar and canula in a manner
similar to that by which the routine mouse-tumor inoculations are made.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 87

Before giving the results of the expcriments with the transplanted glands
we will give the results of some experiments in which fresh venom glands were
inoculated into mice, as well as some in which the juice expressed from the
fresh gland was injected.

Two mice were inoculated with small pieces of heloderma venom gland
which had just been removed. Both of these mice died about 2 hours after
being inoculated.

A small quantity of the fluid expressed from the cut surface of a fresh
venom gland was measured and diluted with 0.85 per cent sodium-chloride
solution and various quantities of this mixture were injected into mice. The
results of the injection are given in the following table:

Injection of venom-gland fluid into mice.

pout aie

of venom- etween

gland fluid Result. injection

injected. and death.

C.c. he om.
0.01 15
0.005 1 00
0.002 40
0.0003 2 15
0.00006 2 15
0.00001 | Recovered.......) 9 .....

The fluid from the venom gland was in this case about 100 times as strong
as the venom obtained from the heloderma’s mouth (since the lethal dose of the
latter is 0.005 c.c. for a mouse). In another experiment 0.44 gram of a fresh
gland was cut in small pieces and in a mortar mixed with 3 c.c. of 0.85 per cent
NaCl. As much fluid as possible was pressed out of the gland and the mixture
was filtered through a paper filter. 100 c.c. of 0.85 per cent NaCl solution were
added to 1 c.c. of the filtrate. One mouse received 1 ¢.c. and another 0.5 c.c.
of this diluted extract subcutaneously. Both mice were soon affected and
died within less than an hour. Another mouse which received 0.1 c.c. of this
diluted filtrate was found dead 2 days after the injection; mice that received
still smaller doses remained alive. A mouse which received, therefore, the fil-
tered juice of about 0.7 mg. of the venom gland died in less than an hour.

The experiments in which the toxicity of the pieces of venom gland trans-
planted subcutaneously into helodermas were tested gave the following results:

Inoculation of transplanted pieces of venom gland into mice.

Period after Effect of auto- Effect of iso-
transplantation. transplant on mouse. transplant on mouse.
Died in 20 hours...... Died in 5 hours.
Died in 44 hours...... Died in 2 hours.
Died in 50 minutes....) Died in 9 hours.
Died in 30 minutes....) Died in 9 to 18 hours.

From these experiments it is evident that the fluid in the transplanted
pieces of gland retains its toxic effect even after a period of 4 weeks. Whether
there has been any diminution in the strength of this fluid we are unable to state.

88 THE VENOM OF HELODERMA.

In another experiment the piece of venom gland was placed under the skin
into the subcutaneous tissue of a turtle’s leg. Two days after the transplan-
tation, when the turtle seemed to be dying, the piece of gland was removed;
part was reserved for histological study and another part divided and inocu-
lated into two mice. Both of these mice died 45 minutes after the inoculation.
(The turtle died on the following day, that is, about 2.5 days after the gland had
been placed under the skin.) Even in the turtle the fluid of the venom gland
of the Heloderma retains its toxicity for a period of 2 days.

IMMUNIZATION AGAINST HELODERMA VENOM.

By giving repeated and constantly increasing doses of venom an attempt
was made to immunize animals against the venom of the Heloderma. For this
purpose rabbits, pigeons, and geese were used, the latter animals because they
were reputed to be resistant to various venoms, and especially to certain bac-
terial infections.

In immunizing the geese, we injected into the pectoral muscles of the ani-
mals very small doses (0.05 c.c.) of the fresh venom. The first or second
injection appeared to produce no effect, so that the quantity of venom was
increased to 0.07 ¢.c., and in every case the animals died after the third or
fourth injection. The attempt to immunize geese against venom was given
up after all four geese used in these experiments had died.*

Immunization of rabbits against heloderma venom.

. Date of first Amount Amount last Total amount No. of
Rabbit. injection. injected. Date of death. injection. injected. injections.
obsessed Dec. 5.1907 | 15 mg...... Apr. 20, 1908 1050 16
De Di wiesesecad Dec. 5, 1907 Apr. 4, 1908 152 9
x3 Dec. 10, 1907 e Jan. 8, 1908 33 5
xX4 Jan. 7, 1908 ‘ Mar. 7, 1908 67.5 cd
X 5... Dee. 21, 1907 3 Jan. 25, 1908 27.5 4
X 6. Jan. 20, 1908 : Jan. 31, 1908 34 3
YL: June 18, 1908 g June 23, 1908 Fi 1
Y3. June 24, 1908 3 Aug. 10, 1908 9
Y 4. Aug. 14, 1908 P Oct. 5, 1908 7
Y 2. ..{ June 18, 1908 3 Oct. 16, 1908 i
Y5.......] Aug. 20, 1908 : Nov. 6, 1908 : 10
Did 4 vse Dec. 29, 1908 5 MGs esazs Apr. 1, 1909 135 13
Vi eee Pree Do 6 TEs saree Mar. 3, 1909 88 10
Leis cuass dist |pisior see Do.. 3 Mes gence Feb. 11, 1909 51 7
Vig. Caeeennete| eeegiea Do.. 5 MG ies June 15, 1909 758 23
Vie Meeners eearertn Do 5 mg...... May 14, 1909 233 7

With the ten pigeons used in these experiments we were somewhat more
successful. 0.5 mg. of venom was given at the first injection, this dose produc-
ing no distinct effect. At periods varying from 1 to 33 days after this first in-
jection a second injection of a like quantity of venom was given. Four pigeons
died after the second injection of 0.5 mg. of venom. The six remaining pigeons
received, 5 to 7 days after this injection, 0.6 mg. of venom, and thereafter, every
5 or 10 days, slightly increased doses of venom. Four of the remaining pigeons
died; one after three, one after six, one after seven, and one after eight injec-

*In repeating these experiments, it will bo advisable to use dried instead of fresh venom, in order to eliminate
the variations in the strength of the venom asa complicating factor.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 89

tions. All of these pigeons, excepting the one dying after the third injection,
had received several injections of 1 mg. of venom, and it was following the in-
jection of this amount of venom that they died. The two remaining pigeons
received eight injections; the last injection being 1.1 mg.of venom. In view of
the poor results obtained with the majority of pigeons we discontinued the
experiments. It appeared that pigeons might survive one injection of a lethal
dose of venom and die from the effect of a second or third injection of a like
quantity.

In the experiments with rabbits we used in all 16 rabbits. The table
on page 88 gives the total amounts injected, the period of time over which
the injections extended, and the various doses of venom used.

It will be seen from the above protocols that the experiments in immu-
nizing rabbits were successful in so far as the establishment of an active immu-
nity against the venom is concerned. We were, however, unable to push this
immunity to as high a degree as it appeared desirable. The rabbits would at
times survive the injection of a large dose of venom and eventually die from
the injection of an only slightly increased dose. Both the fresh venom and
the dry venom were tried; but because of the variation in strength of the
fresh venom, the dry venom was used in most cases.

The injections were given every 5 or 7 days, provided the animal did not
show emaciation; if it had lost weight, no injection was given until the original
weight had been regained. As a further precaution in the first few injections
(in the experiments with rabbits Z1, Z2, Z4, and Z5), the venom was mixed
with a 1 per cent solution of calcium hypochlorite, a method of starting the
injections for purposes of immunization recommended by Calmette in his work
with cobra venom. At first equal quantities of venom solution and calcium-
hypochlorite solution were used, but gradually the quantity of hypochlorite
solution was diminished and the amount of venom increased. The quantity
of venom was increased very slowly until the amount injected was greater than
the lethal dose; then the quantities injected were increased somewhat more
rapidly.

Only four of the animals were injected with quantities of venom larger
than the usual lethal dose (Rabbits X1, X2, Z4, Z5), and only two of these
(X1 and Z4) received sufficient quantities to produce any distinct immunity;
these two withstood the injection of about eight times the dose lethal for ordi-
nary rabbits.

It is impossible to explain the death of the animals after the injection of an
only slightly increased dose of venom or after the injection of a minimal lethal
dose of venom. That we are not dealing with a typical anaphylactic reaction
is evident from the special experiments in which we tested the anaphylactic
power of venom. It appearsthatthe animals’ resistance to venom varied from
time to time and that the immunity produced was not a definite and stable
one. It not rarely happened that abscesses developed at the place of injection,
notwithstanding all the precautions taken to insure a sterile procedure. It
may be that after all conditions similar to the Arthus phenomenon of local

90 THE VENOM OF HELODERMA.

anaphylaxis were present. Other investigators have made similar observa-
tions in the immunization against snake venoms.*

Blood taken from two of the rabbits (X.1 and Z4) was tested for the pres-
ence of precipitins. Furthermore, the protective action of the serum obtained
from these animals was tested against venom and mixtures of venom and of
serum obtained from the immunized rabbits were injected into mice.

PROTECTIVE POWER OF THE SERUM OF IMMUNIZED RABBITS.

Mice were used in testing the protective power of the serum of immunized
rabbits.

Some blood was drawn from rabbit Z4 after it had withstood the injection
of 95 mg. of venom in one dose and after it had been under treatment for four
and a half months. This blood was allowed to clot and the serum was drawn
off after the blood had stood in the ice-box over night.

Mice were injected with venom, venom and immune serum, and venom
and normal-rabbit serum. Injection of 0.02 c.c. of fresh venom killed a mouse
in 35 minutes, 1 ¢.c. of normal-rabbit serum added tothe venom killed asecond
mouse in 20 minutes, while a mouse injected with a similar quantity of venom
which had stood for an hour mixed with 1 ¢.c. of immune-rabbit serum died
after 3 hours.

The results were practically the same when we first injected the serum and
then the venom as when they were injected together. Thus control mice in-
jected with either 0.015 or 0.01 c.c. of fresh venom died somewhat sooner than
mice which received similar quantities of venom but which had, in addition,
received 1 c.c. of either normal or immune-rabbit serum; the animals which
received the immune serum lived a little longer than those which received the
normal-rabbit serum in addition to the venom. When we injected 2 c.c. of
serum, followed, in 40 minutes, by the injection of 0.01 ¢.c. of fresh venom, we
found that the animal which received immunc-rabbit serum and venom lived
longer than a mouse which received venom alone, or a mouse which received
2 c.c. of normal-rabbit serum followed by 0.01 ¢.c. of fresh venom solution.
This mouse died as soon as the control animal.

The same rabbit whose serum was tested in the above experiments (Z 4)
was bled again a few days later before any further injections of venom had been
made. At this time only a very small quantity of blood was drawn. In all
the experiments with this serum the mice received 0.01 c.c. of fresh venom.
Two quantities of serum were used, namely, 1 ¢.c. and 1.5 ¢.c. of either the
immune-rabbit serum or the normal-rabbit serum. All the six mice used in
these experiments, those injected with venom alone as well as those injected
with venom plus serum, died in approximately the same length of time.

The other rabbit (X 1) was bled 4 months after the injections were begun,
when it had developed sufficient resistance to withstand the injection of a dose
of 100 mg. of venom. In these experiments mice were injected with venom
alone, with venom and normal-rabbit serum, and with venom and immune-

*Immunization with heloderma venom might perhaps be more easily accomplished if it were possible to sepa-
rate venom and proteid matcrial through heating, as can be dono in the case of cobra venom. This would obviate the
necessity of injecting accessory proteids that may add to the local anaphylactic condition.

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 91

rabbit serum. All the mice which received 0.2 mg. of dry venom died approxi-
mately at the same time after the injection, even when the injected venom
stood for an hour after having been mixed with 0.5 c.c.ofimmuneserum. Sim-
ilar results were obtained when 1.4 mg. of venom were injected, even though
1 c.c. and 2 ¢c.c. of immune serum had been added.

The injection of 0.4 or 0.6 mg. of venom plus 1c.c. of immuneserum was
not lethal when the venom and serum of the immunized rabbit were allowed to
stand until a precipitate had formed, whereas the injection of the same quan-
tities of venom without serum caused death.

We conclude from these experiments that although repeated injections of
venom into rabbits enable them to resist the toxic effect of large doses of venom,
no distinct antitoxin had been produced. It may be noted that in some cases
the previous injection of immune-rabbit serum or the mixing of the venom
with immune-rabbit serum before the injection of venom into mice delayed
slightly the lethal effect of the venom, so that we may perhaps conclude that
a very small amount of antitoxin had been produced. Had it been possible to
carry the immunization of the rabbits further, an antitoxin might have been
produced. It is, however, quite apparent that relatively enormous quantities
of venom—more than we could devote to this problem—would have been
required in order to accomplish the production of an antitoxin, and it was
doubtful whether, even in the case of perfect success, the results would have
been of sufficient theoretical interest to justify such an expenditure of venom
and of effort.

Antitoxic power of the serum of rabbits immunized against heloderma venom.

Amount of | Amount of
Serum from | Amount of . .
rabbit. enOT: ments ina Mouse died after:
Ag teen 0.02 c.c..... A 2 hours.
-02 c.c..... A ..| 20 minutes.
.02 c.c ai ..| 30 minutes.
VAs eee .015 c.c 12 hours.
015 c.¢ os ..| 12 hours.
.015 cc ..| 30 minutes.
DE teaceie' 01 .| 72 hours.
01 2 hours 30 minutes.
01 .| 2 hours.
Vie aera 01 6 hours.
-O1 3 hours
01 4 hours.
ZAP sssincs's 01 1 hour.
-O1 1 hour 15 minutes.
-01 1 hour 15 minutes.
Dy WF. wezcoasecs 01 2 hours.
01 2 hours,
01 2 hours.
P. al Ueereer tan o 6 hours
2 6 hours.
2 6 hours
pe eer . 08 Recovered
.08 Do.
08 Do.
>.) Ree .04 Do.
04 Do.
04 Do.
bo Beene 1.4 30 minutes.
D.@ eee 1.4 30 minutes.
1.4 30 minutes.
1.4 30 minutes.
>. Oe arene 0.4 Recovered.
0.4 1 hour.
D>. oh Ree ey 0.6 Recovered.
0.6 1 hour.

*Rabbit bled May 23,1909. Rabbit bled May 27,1909. {Standing 4 hours showed precipitate.

92 THE VENOM OF HELODERMA.

PRECIPITINS IN THE SERUM OF RABBITS IMMUNIZED AGAINST
HELODERMA VENOM.

We mixed the blood of the rabbits which had been immunized against
venom with various quantities of fresh venom, dried venom, or dried heloderma
blood, and noted the appearance of precipitates in these mixtures. We used
in these experiments the serum of two of the immune rabbits, X 1 and Z4, ata
time when they were able to resist an injection of 100 mg. and 95 mg. of venom,
respectively. In the following table the results of these experiments are given:

Precipitins in serum of rabbits immunized against heloderma venom.

Wits Fresa AND Drip Venom .

Amount of serum. 0.2 mg. 0.4 mg. 0.6 mg. Remarks.

1c.c. immune‘...

1c.c. normal... trace trace trace

0.1 ¢.c. immune’. + Precipitate appears sooner than with
0.1¢.c. normal..... trace trace trace 1 c.c. immune serum.

0.05 ce. normal........ trace trace trace Precipitate appears sooner than with
0.1 c.c. immune serum.

2 mg. 2 mg. Ol mg.
0.25 c.c. immunef...... + + se
0.25 c.c. normal........ 0 0 0

O.lec. | 0.025 c.c. | 0.0025 c.c.

0.25 c.c. immunef...... + + + Somewhat heavier precipitate than
0.25 c.c. normal........ 0 0 0 with dry venom.
2 mg. 0.2 mg. 0.01 mg.
0.025 c.c. immunef..... 0 trace 0
0.025 c.c. normal....... 0 0 0

0.1e.c. 0.025 c.c. | 0.0025 c.c.

0.025 c.c. immunef..... + + trace
0.025 c.c. normal....... 0 0 0

2 mg. 0.2 mg. 0.01 mg.
0.005 c.c. immune}..... 0 0 0
0.005 c.c. normal....... 0 0 0

0.1c.c. 0.025 c.c. | 0.0025 c.c.

0.005 c.c. immunef..... + + 0
0.005 c.c. normal....... trace 0 0

Wita DissoLvep HeLopeRMA BLoop WHICH HAD PREVIOUSLY BEEN Driep.

Amount of serum. 2 mg. 0.2 mg. 0.01 mg. Remarks.

0.25 c.c. immuneft...... +
0.25 c.c. normal........
0.025 c.c. immunef.
0.025 c.c. normal...
0.005 c.c. immunet. J
0.005 c.c. normal.......

cootot
eoccooo

eoococoo

*Scrum of rabbit X 1. {Serum of rabbit Z 4.

The serum of the immunized rabbits contains venom precipitins. How-
ever, the serum of normal rabbits may also contain precipitins for the venom,
but not in such large quantities as the immune serum. The serum of rabbit
X1 contained more precipitin than the serum of rabbit Z4.

In view of the fact that the smaller quantities of serum reacted more
rapidly with the venom than the larger quantities of serum (experiments with
serum of rabbit X1), we may conclude that there is an optimum proportion
for mixtures of the venom and immune serum. However, the degree of pre-

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 93

cipitation in the different tubes eventually becomes the same in all cases.
Whether the precipitins owe their origin to and react with the venom proper or
proteid material admixed to the venom, can not be decided without a deter-
mination of the chemical character of the venom.**

The serum of the immunized rabbits contains also a precipitin which re-
acts with the blood of the Heloderma. This reaction was, however, quite weak
and only a small amount of precipitate was found in serum-blood mixtures.
Whether this reaction is due to the same precipitin which reacts with the
venom or to a specific precipitin for the heloderma blood is doubtful. There
is a strong possibility that it is a specific heloderma-blood precipitin, since,
as we have stated earlier, the venom frequently contained small quantities of
blood which were dried and again dissolved simultaneously with the venom.

DOES BLOOD SERUM OF HELODERMA CONTAIN A VENOM ANTITOXIN ?

The antitoxic properties of the serum of both normal helodermas and the
helodermas from which the venom gland had been removed were tested. Be-
fore injection, the serum was allowed to stand mixed with the venom for periods.
of from 30 minutes to an hour at room temperature. These mixtures were
then injected into mice and at the same time control mice were injected with
the same quantities of venom which had not been mixed with serum.

Injection.
Experi- Result
ment No. Quantit ae
Venom. of aaa Kind of serum.

NO: ‘Logg (ON2) “Migaias|s oa ey os veal eacaieeais sy oa ds agecsaee wea Se Died in 4 hours.
Dies 2 .| Glandless heloderma*....... Died in 12 hours.
3.. iil sc2, 3. acelin \ocvis lu roa Bc cpaaeeuavesbats uid Died in 4 hours.
4, Glandless heloderma* ...| Died in 24 hours.
Discs OROT TMB sssare |asssaiaa: des snacarecellesensiapsonpaievalara: art aye ites atGveyite .| Died in 48 hours.
6 Glandless heloderma* .| Recovered.
qT Normal heloderma... .| Died in 3 hours.
8. Glandless helodermat .| Died in 3 hours.
Ds aveicllf O) OOD Ci aicinel vers e's si air traaywissiohaviuntie. atic bcbcdce eal .| Died in 3 hours.

10. Normal heloderma. .| Died in 2 hours.
11. Glandless helodermat .| Died in 2 hours.
125554] OL0L. C:0biseclaness scacona|teackins sears ts ta saan .| Died in 2 hours.
13. Normal heloderma... .| Died in 2 hours.
14, .| Glandless helodermat Died in 2 hours.
15.. al aiaagancroiatenctacareee ee eee weindrcraVitreraia va Died in 2 hours.
16.. .| Normal heloderma.......... Died in 2 hours.
I 5 .| Glandless helodermat....... Died in 2 hours.
WB ies rs QUO. Crea sissies eracerasa.aes wis 3 2) Pato narstets inves T0-05 ee HEME R Died in 2 hours,
19.. .| Normal heloderma.......... Recovered.

20.. .| Glandless helodermat....... Recovered.

21.. -| $e Saaudietne it ohals Be ses cae Recovered.

22... .| Normal heloderma.......... Died in 4 hours.
23.. .| Glandless helodermat....... Recovered.

24.. | ees See aig eataacained wines wlasoechatsrates ns aa lee Died in 2 hours.
25... Normal heloderma.......... Died in 45 minutes.
26.. ..-| Normal heloderma.......... Recovered.

2h ae ...{ Normal heloderma..........] Recovered.

28). 2x ...| Glandless helodermaf....... Recovered.

29.. ...{ Guinea-pig§..............0008 Recovered.

30.. ...| Guinea-pig§... Recovered.
31... seal Sheep] wisi sccceaanemewas ¢ Died in 6 hours.
32....] 0. eben sCrwecaza| MCC Y crass sacsausdadensas Died in 18 hours.
33....] 0. Apel RK ceoeee OK A ccicdtare dH ohalSaidiciadteioiaiesss Recovered.

* Poison glands had been removed 66 days previously (serum 1).
¢ Poison glands had been removed 30 days previously (serum 2).
t Poison glands had been removed 7 days previously (serum 3).
§ Injected at same time as mice injected with serum 3.
{| Injected at same time as mice injected with serum 1.
**T he results obtained by Dr. Alsberg make it almost certain that the precipitins formed owe their origin not
to the injection of the venom proper but to admixed proteid material.

94 THE VENOM OF HELODERMA.

We may conclude from these experiments that the addition of normal-
heloderma serum or of the serum of a Heloderma from which the venom gland
had been removed does not neutralize the venom. In a few cases the animals
which were injected with venom mixed with serum of a glandless heloderma
lived somewhat longer than controls injected with a similar quantity of venom;
and two animals recovered, while the controls died. In other cases, no effect
of the heloderma serum was noticeable. Inasmuch as guinea-pig serum and
ox serum had in a few cases a similar effect, we can not attribute to the serum
of Heloderma, when mixed with heloderma venom, any specific antitoxic effect.

We tested the serum of glandless helodermas, having in mind the possi-
bility that the venom gland might discharge some venom into the blood and
that the venom present in the blood might obscure a possible antitoxic property
of the serum.

We may, therefore, conclude that the resistance of the Heloderma to the
injection of large doses of venom is not due to any substance contained in the
circulating blood.

DOES THE INJECTION OF VENOM PRODUCE A STATE OF ANAPHYLAXIS?

We injected pigeons, mice, and rabbits with heloderma venom, and several
days or weeks later injected these animals a second time in order to determine
whether any phenomena of anaphylaxis could be found.

Twelve pigeons were injected with doses of venom varying from 0.2 to 0.6
mg.; at various times from 4 to 33 days after the first, a second injection was
given. Three animals died after the second injection, but in all these cases
death was due to the administration of an overdose of venom. The nine
remaining animals showed no ill effects whatever from the second injection.
The results of these experiments are given in the following table:

Experiments in anaphylaxis (pigeons).

First Time Second First Time Second
injection. | elapsed. injection. Result. injection. elapsed. injection. Result,
mg days. mg. mg. days. mg.
0.6 4 0.8 Died. 0.6 11 0.6 No effect.
0.6 5 0.8 Do. 0.5 12 0.5 Do.
0.6 5 1.0 Do. 0.6 13 0.7 Do
0.6 4 0.7 No effect. 0.5 17 0.5 Do.
0.6 5 0.6 Do. 0.5 33 0.6 Do.
0.5 5 0.5 Do. 0.2 33 0.6 Do.

Eleven mice were injected with venom on two separate occasions. One
lot of six mice was given the second injection 14 days after the first; another lot
of five was injected a second time on the twentieth day after the first injection.
None of the mice showed any symptoms after the second injection. In the
table following the amount of venom injected and the time interval between
the two injections are given:

GENERAL PROPERTIES AND ACTIONS OF THE VENOM. 95

Experiments in anaphylaxis (mice).

First Time Second First Time Second. 7
injection. elapsed. injection. | Result. injection, elapsed. injection. Result.
| am : = iat Loewe ene
|
mg. days. | mg. | \ mg. days. mg.
0.05 14 0.03 | No effect. 0.05 20 0.05 No effect.
0.05 | 14 0.03 Oo. 0.05 20 0.05 Do.
0.05 ' 14 i 0.03 i Do. 0.05 20 0.05 Do.
0.05 | 14 | 0.05 | Do. 0.05 20 0.05 Do.
00 | ww =! 005 | Do. 0.05 20 0.05 Do.
0.05 | 4 | 0.05 | Do.
|

Two rabbits were used in these experiments, the time interval between the
first and second injection being 36 days and 44 days, respectively. Although
both animals received quite large doses of venom at the second injection,
neither of them showed any symptoms as a result of the injection.

Experiments in anaphylaxis (rabbits).

; | |
; First Time |: Second
Weight injection. elapsed. injection. Result. |
gms. | mg. days mq. |
1200. cnisce | 6 44 12 | No offect.
1900.......] 15 36 12 Do.

In all the experiments (with pigeons, mice, and rabbits), both the first and
the second injections were given subcutaneously. It is quite evident that the
injection of heloderma venom into an animal does not noticeably increase the
susceptibility of the animal to a second dose of venom.

The venom of Heloderma, therefore, does not cause typical general ana-

phylaxis.

Ii.

BACTERIOLOGY OF THE SALIVA OF HELODERMA
SUSPECTUM,

By D. Rrvas.

BACTERIOLOGY OF THE SALIVA OF HELODERMA
SUSPECTUM.

By D. Rivas.

In the course of investigations into the toxic action of the poison of Helo-
derma suspectum, it was necessary to compare the effect of fresh and heated
saliva of these reptiles. It had been found* that if tested on guinea-pigs the
fresh venom caused, besides the typical symptoms common to both the heated
and fresh venom, certain inflammatory reactions which were absent in those
guinea-pigs which had been injected with heated venom. It was of interest,
therefore, to determine which organism might be responsible for the inflam-
matory lesions found in guinea-pigs after injection of fresh venom, and with
this point in view experiments were carried out to isolate the organism respon-
sible and to determine whether the organism was present frequently in the
saliva of these reptiles.

A guinea-pig which had received an intraperitoneal injection of fresh
venom and had been dead for about 3 hours was brought to me for bacterio-
logical examination.

Autopsy.—Point of inoculation slightly swollen and edematous; slight peritoneal exudate,
but no congestion. Spleen enlarged; other organs normal.

Microscopical examination.—Smears made from point of inoculation, peritoneal exudate,
blood, and organs showed, among other bacteria, a thin Gram negative bacillus which in
pure cultures corresponded to the morphological and biological features of Bacillus coli com-
munis; it fermented dextrose, produced indol, coagulated milk, did not liquefy gelatine, was
negative to test No. 1 and test No 3, and positive to test No. 2 (D. Rivas, Journal of Infec-
tious Diseases, vol. 4, November 1907, p. 641, and Journal of Medical Research, March 1908.)

After obtaining similar results with a number of other guinea-pigs which
directly after death presented the same post-mortem appearance as the first
animal, experiments were made to determine the virulence of the cultures iso-
lated. A number of guinea-pigs were used for the experiment; some were
injected subcutaneously and others intraperitoneally with a small amount of
the scraping of a 24-hour agar culture dissolved in sterile salt solution. All
injected guinea-pigs died, some a few days after the inoculation and others
on the day of the injection, and in one instance the culture was found to be
so virulent that the guinea-pig died 3 hours after the intraperitoneal injection.
Bacteriological examination of the point of inoculation, blood, and organs of
the dead guinea-pigs revealed in each case the presence of the bacillus used
for injection.

*Cf. the communication by E. Cooke and Leo Loeb.
99

100 BACTERIOLOGY OF THE SALIVA.

These results suggested a direct bacteriological examination of the saliva
of the Heloderma suspectum from which the venom had been obtained. The
saliva collected in a sterile pipette from the mouth of the animal was plated,
and the isolated colonies studied. Besides many other micro-organisms, B. colz
communis was found to be abundantly present in the saliva.

The fact that such a virulent strain of B. coli communis, able to produce
death in so short a time as3 hours after the injection, was found in the saliva,
seems to me to be worthy of special consideration, not merely as a contribution
to the virulency of B. coli communis, but more especially as indicating a possible
source of error in the study of the fresh venom of poisonous reptiles.

In our investigations, besides B. coli, B. pyocyaneus, and other pathogenic
and pyogenic bacteria were found, but attention was given to B. coli only,
because in this case this organism was found to be more virulent than the
others with which it was associated in the saliva.

In a further series, the frequency of the presence of B. coli in the saliva of
this reptile was investigated. The saliva of ten individuals of this species was
examined, in five of which B. coli was isolated after a single examination, and it
is probable that on repeating the examination of the negative cases, this organ-
ism (B. coli communis) would have been found in most, if not in all, of the ten
helodermas.

IV.

INFLUENCE OF HELODERMA VENOM UPON BLOOD-PRESSURE, DIURESIS,
PERITONEAL TRANSUDATE, AND INTESTINAL FLUID.

By Moyer 8S. FLEIsHER.

INFLUENCE OF HELODERMA VENOM UPON BLOOD-PRESSURE, DIURESIS,
PERITONEAL TRANSUDATE, AND INTESTINAL FLUID.

By Mover 8S. FLeisuer.

Van Denburgh and Wight,* studying some of the physiological actions of
the venom of Heloderma suspectum, came to the conclusion that the lethal effect
of the venom was due to its influence on respiration. The animals injected
with venom died as a result of respiratory failure; at the same time the blood-
pressure was lowered. In order to investigate further the influence of the
venom on blood-pressure we injected into rabbits large quantities of 0.85 per
cent sodium-chloride solution in which venom had been dissolved. We were
thus also able to study the effect of venom on the secretion of urine and the
transudation of fluid into the peritoneal cavity or into the intestines, as well as
the relation existing between the blood-pressure and the secretion of urine.

The 0.85 per cent sodium-chloride solution was allowed to flow into the
jugular vein of the rabbit at the rate of 4 c.c. of fluid per minute. By means of
a hot-water bath the infused fluid was maintained at a temperature of 39° C.
Quantities of fluid varying between 600 and 700 c.c. were infused into the
rabbits.

EXPERIMENTS WITH NON-NEPHRECTOMIZED RABBITS.

In a first series of experiments, rabbits with intact kidneys were infused
with the sodium-chloride solution, to which venom had been added in the
proportion of 20 or 30 mg. to every 800 c.c. of the sodium-chloride solution.
Quantities of urine secreted during the inflow of every 50 or 100 c.c. of the
NaCl solution were noted. At the end of the experiment, after the inflow of
fluid had been stopped, the animal was killed and the quantity of fluid in the
peritoneal cavity and small intestines was measured. By means of a mercury
manometer connected with the carotid artery of the rabbit, the arterial blood-
pressure was measured during the time of the infusion.

Immediately after the infusion of the venom-sodium-chloride solution
was started the blood-pressure of the rabbit became rapidly lower; after 20 to
50 c.c. had been infused, a fall of 10 to 40 mm. of mercury could be observed; in
our 5 cases the average fall of blood-pressure was 24mm. After the first rapid
fall the blood-pressure remained either approximately stationary or gradually
decreased slightly. After 600 or 700 c.c. of the venom-sodium-chloride mixture
had been infused the arterial blood-pressure was usually between 80 and 60
mm. of mercury (figs. 15 and 16). In only one of these experiments, toward
the end of the infusion, the blood-pressure fell below 50 mm. In this experi-
ment, the only one in which 30 mg. of venom was used, the animal died as a

*Van Denburgh and Wight, Trans. Amer. Phil. Soc., 1898, xrx, 199.
103

104 THE VENOM OF HELODERMA.

result of the fall of blood-pressure (see fig. 17). In all other experiments the
animals were alive at the end of the experiment.

During the time of infusion a large quantity of urine, an average of 590 c.c.
for 1,000 c.c. of infused fluid, was secreted.

In no case was any ascitic fluid found at the expiration of the experiments,
notwithstanding the fact that an average of approximately 300 c.c. of fluid
was retained within the bodies of the infused rabbits. Transudation of fluid
into the intestinal canal, however, took place. On the average 51 c.c. of intes-
tinal fluid were recovered for every 1,000 c.c. of fluid infused.

TasBLe I.—WNon-nephrectomized rabbits infused with venom-sodium-chloride solution.

: ; ;
| Quantity of venom. es | Urine. | Ascites. nee anal
|
ake C.c. c.c.
7 | 345 0 55
700 | 360 0 (b)
700 | 365 0 50
700 460 0 30
700 | 505 0 45
Average for every 1,000 c.c. infused .........).....2.... | 590. 0 51
Average for every 1,000 c.c. retained@ ......).......... Hake egrstee.tealla sear mcbeds ee 120
aNot measured. oRetained fluid equals fluid infused minus amount of fluid eliminated as urine.

Although the quantities of urine eliminated hy the animals infused with
venom-sodium-chloride solution were relatively very large, vet in a series of
control experiments with animals infused with pure sodium-chloride solution
even larger quantities of urine were eliminated, namely, on the average 860 c.c.
of urine for every 1,000 c.c. of fluid infused. The addition of venom therefore
diminished somewhat the secretion of urine.

During the early period of the infusion the quantity of urine secreted is
large in spite of the fact that the blood-pressure has fallen quite markedly. It
is only after 300 or 400 c.c. have been infused, at a time when the blood-pressure
has reached a level which, while fairly constant, is nevertheless below the nor-
mal, that the secretion of urine begins gradually to grow less. These experi-
ments do not yet permit us to decide whether the diminished secretion of urine
is due to the action of venom in lowering the general arterial pressure or to a
local action of venom on the renal vessels or renal cells.

The addition of venom to the infused sodium-chloride solution seems to
diminish the transudation of fluid into the peritoneal cavity; in previously pub-
lished control experiments* on non-nephrectomized rabbits infused with 0.85
per cent sodium-chloride solution, an average of 68 c.c. of fluid for every 1,000
c.c. of fluid which had heen retained was found in the peritoneal cavity.

On the other hand, the addition of venom has increased the transudation
of fluid into the intestinal cavity. In rabbits infused with pure sodium-chloride
solution we found* an average of 53 c.c. in the intestines for every 1,000 ¢.c. of
fluid retained, while an average of 120 ¢.c. was recovered if venom had been
added. Venom causes, therefore, an increase in the elimination of fluid into
the intestines.

*Fleisher, Hoyt and Loeb., Jour. Exper. Med., 1909, x1, 201.

INFLUENCE OF HELODERMA VENOM. 105

According to Porges and Pribram,* and Fleisher and Loeb,f the addition
of calcium chloride to an infused sodium-chloride solution causes a lowering of
the blood-pressure and a diminution in the secretion of urine. These observa-
tions suggested the possibility that the lowered blood-pressure and the lessened
elimination of urine in animals injected with venom-sodium-chloride solution
were due to some inorganic portion of the dry venom, acting in a manner simi-
lar to calcium chloride. Calcium chloride, however, as we have shown pre-
viously, diminishes the elimination of urine independently of its action on the
general arterial system. We incinerated, therefore, 20 mg. of the dry venom
and dissolved the ash in sodium-chloride solution; 20 mg. of venom contain on
the average approximately 13 per cent of ash; approximately 2.5 mg. of inor-
ganic material were therefore added to 800 c.c. of salt solution.

When this mixture of venom-ash and sodium-chloride solution was infused
into rabbits no fall, but even a slight rise, of blood-pressure was usually seen
after the first 50 or 100 c.c. of fluid had been infused. After this rise the pres-
sure either remained at the same level during the course of the infusion or sank
very gradually to normal. At no time did the pressure fall as low as it did
after the infusion of the dry venom. Thus the infusion of the venom-ash
sodium-chloride solution acts in very much the same manner on blood-pressure
as the infusion of pure sodium-chloride solution,{ and the fall of blood-pressure
resulting from the infusion of venom-sodium-chloride solution must conse-
quently be due to the organic portion of the venom (see fig. 18).

The addition of the venom-ash to the sodium-chloride solution does not
interfere with the elimination of fluid through the kidneys; for every 1,000 c.c.
of fluid infused 780 c.c. of urine were secreted. In these experiments the quan-
tity of fluid present in the intestines at the end of the infusion was so small that
it appeared useless to measure it; neither was any ascitic fluid found in these
experiments.

TaBLe 2.—Non-nephrectomized rabbits infused with venom-ash sodium-chloride solution.

Infused. | Urine. Ascites. cee
Average for every 1,000 c.c. infused:

Cc.c.
c.c. C.C. Urine .cecastise ay isiesnse ts taaeweane 780
600 425 0 Intestinal fluid.................24 5
700 625 0 5 Average for every 1,000 c.c. retained:
700 540 0 2 Intestinal fluid .................. 19
700 605 0 3
700 485 0 4

The fact that such a small amount of intestinal fluid and no ascitic fluid
were found in these experiments is due to the very small quantities of fluid (an
average of 144 c.c.) which were retained within the bodies of the animals. As
we have shown previously,{ when small quantities of fluid are retained within
the body, the quantities of peritoneal transudate and intestinal fluid produced

*Porges and Pribram, Arch. fiir exper. Path. u. Pharm., 1908, tx, 30.
{Fleisher and Loeb, Jour. Exper. Med., 1909, x1, 641.
tFleisher, Hoyt and Loeb, Jour. Exper. Med., 1909, x1, 291.

106 THE VENOM OF HELODERMA.

are very small as compared with the amounts produced when larger quantities
of fluid are retained.

The diuresis corresponded to the blood-pressure curve in these experi-
ments. During the infusion of the first 100 or 200 c.c. of venom-ash sodium-
chloride solution the secretion of urine increased quite rapidly ; and it continued
at about the same rate during the whole of the remainder of the period of infu-
sion. In this respect the venom-ash sodium-chloride solution produces approx-
imately the same effect as the pure sodium-chloride solution. The quantity of
urine secreted by the animals infused with venom-ash sodium-chloride solution
is only slightly less than the amount secreted by the animals infused with
sodium-chloride solution, but is distinctly larger than the amount secreted by
animals infused with venom-sodium-chloride solution.

The venom-ash exerts no distinct influence on cither blood-pressure or
secretion of urine, and the changes observed must be due to organic substances
of the venom.

In order to determine whether the lessened secretion of urine in the experi-
ments in which venom was added to the infused sodium-chloride solution was
due to the lowering of the arterial pressure, we carried out some experiments in
which we added adrenalin to the infused fluid. Adrenalin, when infused intra-
venously into animals, causes a rise of blood-pressure, and this rise 1s main-
tained during the whole period of the infusion; at the same time, adrenalin
increases the diuresis.*

In one experiment we added 1.5 ¢.c. of adrenalin to the 800 c.c. of sodium-
chloride solution which already contained 20 mg. of venom. In spite of the
presence of the adrenalin the infusion of the solution containing venom caused
in this case a very marked fall of the blood-pressure during the infusion of the
first 100 ¢.c. of fluid. Also in the later stages, after 400 or 500 ¢.c. had been
infused, the usual gradual fall of pressure was observed. In this experiment
adrenalin had little or no effect in preventing the action of venom on the blood-
pressure; neither had the diuresis curve been affected by the addition of adre-
nalin, for we noted the early rise in the curve and the later fall coincident with
the fall in blood-pressure (see fig. 19).

Three other experiments were made in a somewhat different manner.
The infusion was started with the usual venom-sodium-chloride solution (20
mg. of venom to 850 c.c. of sodium-chloride solution), and after either 150 or
200 ¢.c. of this mixture had been infused the adrenalin was added. In each of
two cases 1 c.c. of adrenalin (making thus approximately a 1 to 600,000 solu-
tion), and in the third experiment 6 c.c. of adrenalin (making a 1 to 100,000
solution) were added.

In all these experiments the usual marked fall of blood-pressure was
observed when the first 50 or 100 ¢.c. of venom-sodium-chloride solution were
infused. After the addition of the adrenalin (1 to 600,000) the blood-pressure
rose slightly, and it continued at approximately the same level until the con-

*Pleisher and Locb, Jour. Exper. Med., 1909, x1, 480.

INFLUENCE OF HELODERMA VENOM. 107

clusion of the experiment. The addition of small quantities of adrenalin
causes, therefore, a slight rise in the blood-pressure, and prevents, or at least
delays, to some extent, the usual fall after 500 or 600 ¢.c. of venom-sodium-
chloride solution has been infused (see figs. 20 and 21).

In the experiments in which the 1 to 600,000 adrenalin solution was used
the diuresis increased quite markedly after the infusion of the adrenalin, and
the secretion of urine reaches a relatively high level, when compared with the
secretion at a similar period, during the infusion of the venom-sodium-chloride
solution. This was true in two of the experiments at least. In one experiment
no decrease either of diuresis or blood-pressure was observed in the late stages
of the infusion, whereas in the other experiment a slight decrease in the secre-
tion of urine is noted simultaneously with a slight fall in the blood-pressure.

In a third experiment,* in which we infused a 1 to 100,000 adrenalin solu-
tion during the second period of the experiment, the rise of blood-pressure fol-
lowing the infusion of the adrenalin solution was more pronounced than in the
other two, and at the same time the secretion of urine was very markedly
increased after addition of the adrenalin (see fig. 24).

From these experiments we may conclude that adrenalin counteracts to a
small extent the blood-pressure lowering action of the venom. In view of the
fact that in one experiment the blood-pressure began again to fall even after
the addition of adrenalin, it is probable that this influence of adrenalin would
eventually be overcome by the depressing action of the venom. We further
note that simultaneously with the rise of blood-pressure following the addition
of adrenalin, the diuresis is also increased; it is therefore probable that the
diminution of diuresis resulting from the addition of venom to the infused fluid
is due not to a specific action of the venom on the kidneys, but to the lowering
of the arterial pressure.

The average amount of urine secreted per 1,000 c.c. infused was greater in
the experiments in which adrenalin was added to the venom-sodium-chloride
than in the experiments in which venom-sodium-chloride solutions alone were
infused. In these experiments, as well as in the former experiments with
venom but without the addition of adrenalin, the quantity of intestinal fluid
was increased, while the ascitic fluid was absent.

TaBLe 3.—Non-nephrectomized rabbits infused with venom-sodium-chloride solutions to which.
adrenalin was added.

. . Intestinal
Infused. | Urine. | Ascites. fluid.
| Average for every 1,000 c.c. infused:
Cc.
C.c. cc. cc .
: AD ING cs, ctuivijovs sya sr neagedae dak wanes 700
oe He 40 Intestinal fluid. ................008 91
700 490 0 26
700 550 0 25

“In this experiment a large quantity of venom was injected intravenously some time alter the infusion of adre-
nalin was started, but at a time when the influence of the addition of adrenalin had been clearly demonstrated.

108 THE VENOM OF HELODERMA.

Furthermore, we have in a few experiments injected in one dose a large
quantity of venom intravenously, while the sodium-chloride solutions were
being infused at the usual rate. The rabbit was infused in the usual manner
witha 0. 85 per cent sodium-chloride solution; after several hundred cubic centi-
meters of this solution had been infused, when the blood-pressure was high and
the diuresis markedly increased, the infusion was stopped for a few moments
while the venom solution was injected directly into the jugular vein. The in-
fusion was then continued as before, with 0.85 per cent sodium-chloride solu-
tion. In one experiment the rabbit was infused with venom-adrenalin-sodium-
chloride solution instead of sodium-chloride solution, both before and after the
injection of venom was given.

As stated above, the injection was given at a time when the blood-pressure
was either higher than usual or nearly normal and when the diuresis was
marked. Immediately following the injection we observed a marked fall of
blood-pressure, which was 24 mm., 62 mm., and 89 mm. of mercury in the three
cases respectively. Following this very marked lowering of the blood-pressure
it rose again 10 to 18 mm. during the inflow of the next 100.c.c. of fluid. When
a pure sodium-chloride solution was infused both before and after the injection
of the venom, the blood-pressure continued from there on at about the same
level (still quite markedly below the normal) until the end of the experiment
(see figs. 22 and 23). When adrenalin-venom-sodium-chloride solution was
infused both before and after the sudden injection of the venom, the pressure
fell again after the first rebound but later rose very gradually (see fig. 24).
In this latter case it is probable that exhaustion prevented the blood-pressure
from rising as much as it did in cases in which pure sodium-chloride solution
was infused.

The diuresis curve shows a very marked fall simultaneously with the fall of
blood-pressure. In experiments in which a pure sodium-chloride solution was
used in the period directly following the rapid injection of the venom, only 6 to
10 c.c. of urine were secreted during the infusion of the first 100 c.c. of fluid.
As the blood-pressure now became gradually higher, the secretion of urine also
increased, 20 to 60 c.c. of urine being eliminated, while 100 c.c. of fluid were
being infused. In the experiment in which venom-adrenalin-sodium-chloride
solution was infused the decrease in the diuresis, as well as in the blood-pressure,
was not as marked as in the experiments in which pure sodium-chloride solu-
tion was infused. The presence of the adrenalin in the infused fluid diminished
probably to some extent the fall of blood-pressure and decrease in diuresis.

The rapid injection of venom produces a fall of blood-pressure much more
marked than after the continuous infusion of the dilute solution of venom.
Accompanying this fall of blood-pressure, the secretion of urine is very markedly
diminished. As the pressure gradually rises the diuresis becomes again more
pronounced.

It is of interest to note that the quantity of venom injected in two cases,
namely, 12 mg., would be a fatal dose if injected intravenously into a rabbit
under normal conditions, while the 36 mg. which were injected in the other

INFLUENCE OF HELODERMA VENOM. 109

experiment is considerably larger than the lethal dose. Yet none of the ani-
mals succumbed to the injection! It appears, therefore, that the venom is
either very rapidly destroyed within the bodies of the animals or prevented
from combining with the central nervous system, either as a result of being very
rapidly eliminated from the body or in some other way. It seems most prob-
able that the failure of the venom to cause death under these conditions is not
due to the destruction but perhaps to the climination of the venom, since in the
normal uninfused animals the same possibilities for the breaking-down of the
venom exist, whereas the possibilities for the elimination of the venom are not
so good as in the infused rabbits.

These experiments make it highly probable that the venom acts only indi-
rectly on the diuresis by influencing the general arterial blood-pressure and
that it does not exert a direct influence on renal cells or vessels. With the fall
of blood-pressure the secretion of urine diminishes, while with the increasing
pressure the secretion of urine also increases.

EXPERIMENTS WITH NEPHRECTOMIZED RABBITS.

In further experiments we infused venom-sodium-chloride solution into
rabbits whose kidneys had previously been removed. Under such conditions
all the infused fluid was retained within the body of the animal, and we were
able to determine more exactly the influence of the addition of venom to the
sodium-chloride solution on the production of peritoneal transudate and intes-
tinal fluid. We carried out seven experiments. Inone 600, in four 700, and in
two 800 c.c. were infused. In all these experiments 20 mg. of venom had heen
added to 800 c.c. of sodium-chloride solution. An average of 104 c.c. of peri-
toneal transudate was produced for every 1,000 c.c. of fluid infused. In con-
trol experiments, carried out some time ago,* and in which pure sodium-chloride
solution was infused into rabbits, 109 c.c. of peritoneal transudate were pro-
duced for every 1,000 c.c. of fluid infused. The addition of venom to the
sodium-chloride solution has, therefore, little or no effect on the production of
peritoneal transudate.

Before the infusion was started, and at the time the kidneys were removed,
the small intestines were clamped at both the upper and lower ends, and at the
conclusion of the experiment the fluid contained in them was measured in each
case. An average of 130 c.c. of fluid was found in the intestines for every
1,000 ¢.c. of fluid infused. In previous experiments in which sodium-chloride
solution alone was infused,* we had found an average of 94 c.c. of fluid was col-
lected from the intestines for every 1,000 c.c. infused. Thus the addition of
venom to the infused fluid had increased the transudation of fluid into the
intestines, a finding in agreement with the results of similar experiments in
non-nephrectomized animals and also with the autopsy findings of animals that
had died after a single injection of venom.

*Fleisher, Hoyt and Loeb, Jour. Exper. Med., 1909, x1, 291.

110 THE VENOM OF HELODERMA.

Tasie 4.—Nephrectomized rabbils infused with venom-sodium-chloride solution.

. Intestinal
Infused. | Ascites. | quig,
G00 45 ee For every 1,000 ¢.c. infused:
700 68 84
ABCILES 5 34 savesieainacees 104
gt a 6 Intestinal fluid 777.2"! 130 c.c
700 82 106
800 120 135
800 70 90

Thus, in spite of the fact that the addition of venom causes a lowering of
the blood-pressure, the transudation of fluid into the peritoneal cavity is not
diminished and the transudation of fluid into the intestines is increased, if the
venom is added to the NaCl solution hefore the infusion.

CONCLUSIONS.

(1) The addition of venom to sodium-chloride solution which is infused
intravenously into rabbits causes a marked and rapid fall of blood-pressure.
If the infusion is extended over some time, the pressure continues to fall
gradually.

(2) This fall of blood-pressure is due to the organic constituents of venom.

(3) The addition of adrenalin to the venom-sodium-chloride solution coun-
teracts only to a slight extent the blood-pressure-lowering effect of the venom,
and it is probable that eventually if the infusion were continued over a suf-
ficiently long period of time, the influence of the venom would overcome
the action of the adrenalin.

(4) The addition of venom to the infused sodium-chloride solution dimin-
ishes the secretion of urine. Since the diuresis curve varies with the blood-
pressure curve it is probable that venom does not cause a decrease in diuresis
by a direct action on the kidney, but through its influence on the general arte-
rial blood-pressure.

(5) The rapid intravenous injection of a large dose of venom into a rabbit
which is being infused with sodium-chloride solution causes a marked and very
rapid fall of blood-pressure which is accompanied by a very marked decrease in
the secretion of urine. When the infusion is continued the blood-pressure rises
again and the diuresis increases.

(6) Animals which are being infused with sodium-chloride solution will
resist the intravenous injection of a quantity of venom which under normal
conditions would be lethal. This resistance is probably due to the rapid elim-
ination of the venom.

(7) The addition of venom to the sodium-chloride solution appears to
have no influence on the production of ascitic fluid, while it causes an increase
in the elimination of fluid into the intestinal cavity, notwithstanding the
owered blood-pressure.

INFLUENCE OF HELODERMA VENOM,

Experiment I.

(Rabbit, weight 2,200 gms.

In-

fused with sodium-chloride
solution plus venom (20 mg.

PROTOCOLS.
Experiment If.
[Rabbit, weight 1,900 gms, In-

fused with sodium-chloride
solution plus venom (20 mg.

IexrERIMENT III.

(Rabbit, weight 1,760 gms In-
fused with sodium-chloride
solution plus venom (30 mg.

to 800 c.c.).] to 800 c.c.).] to 800 ¢.c.).]}
Amount | Blood a Amount Blood ae : Amount | Blood- we
infused. | pressure. Urine. infused, pressure. Urine. | ; infused. ' pressure. Urine
| : | ‘
coi ay mm. Conary £0. mm, Ce 1 i Cy | mm. €.c
0 120 cece O° 190 7 / 0 128 ¢
50 90 fins 200 | Ok ee | 10 | 102
100 S6 50 300 5G 5 | 50 | 106
200 80 80 200. By) 20 | 100: 92 55
300 80 70 300 60 65 | | 200 82 | 60
400 2 60 400 fm) 80; | 800 BS
500 72 70 500 | 56 sO | 400 | 84 65
600 72 20 600 56 55 500 70 50
700 R 35 700 1 BA | 53, | 00 | 68 | 50
— — fof ' 700 50. | 30
Total bicndasscvne|ansadS? Potall tsa, chee 360 ie —-
‘ Totalirscss sia axe 365
# t

Experiment IV.

(Rabbit, weight 1,700 gms

In-

fused with sodium-chloride

IEXPERIMENT Y.

(Rabbit, weight 1,700 gms. IJn-
fused with sodium-chloride

EXPERIMENT VI.

(Rabbit. weight 1,700 gms. In-
fused with sodium-chloride

solution plus incinerated solution and __ incinerated solution and _ incinerated
venom (20 mg. to 800 c.c.).] venom (20 mg to 800 c.c.).} venom (30 mg. to $00 c.c.).]
Amount | Blood- : Amount | Blood- an Amount | Blood- .
infused. | pressure. Urine. infused. | pressure. Urine. infused. | pressure. Urine.
c.c. mm, C.c. ee: mm. c.c. C.c. mm. C6
0 110 aac 0 100 0 80 ae
100 110 K3 20. 4 120 20 86
200 84 100 100 110 63 50 100
300 90 75 200 100 8&2 100 90 41
400 88 90 300 110 50 200 96 104
500 86 95 400 14 75 300 84 95
600 86 90 500 114 70 400 96 85
700 86 92 600 116 85 500 100 85
— a= 700 116 85 600 104 90
Total |.......... 625 | —_ _ 700 106 105
Motalsh senese svais 540 — —
Total lissascas cass 605

EXPERIMENT VII.

[Rabbit, weight 1,700 gms.

In-

fused with sodium-cbloride

solution

plus

incinerated

venom (25 mg. to 800 c.c.).]

EXPERIMENT VIII.

[Rabbit, weight 1,700 gms. In-
fused with sodium-chloride
solution plus incinerated
venom (25 gm. to 800 c.c.).J

EXPERIMENT IX.

[Rabbit, weight 2,400 gms. In-
fused with sodium-chloride
solution plus venom (20 mz.
to 800c.c.); at 200 c.c. added
1e.c. adrenalin.]

Amount | Blood- . Amount | Blood- .
infused. | pressure. Urine. infused. | pressure. Urine.
c.c. mim. C.c. Cc. mm. c.c.
0 120 eect, 0 110
20 148 aig 20 130 |
100 150 20 100 126 45
200 150 45 200 120 60
300 154 65 300 122 75
400 150 80 400 124 95
500 150 90 500 106 90
600 150 100 600 110 90
700 150 85 —_— —
_ = Total fsarsisce satya 425
Total |.......... 435

Amount | Blood- -
infused. Blood. | Urine. |
c.c mm. C.c.
0 96
10 112
20 84
50 80 |
100 70 40 |
*200 72 50
300 76 80
400 76 95
500 80 95
600 82 90
700 82 100
Teta sccssiess ciate ais 550
“Added adrenalin.

111

112

[Rabbit, weight 1,800 gms.

THE VENOM OF HELODERMA.

EXPERIMENT X,

In-
fused with sodium-chloride
solution plus adrenalin (1.5
c.c. to 1,000 c.c.) plus venom

EXPERIMENT XI.

[Rabbit, weigbt 1.900 gms. In-
fused with sodium-chloride
eolution plus venom (20 me.
to 800 c.c.); at 150 ¢.c. added
1 c.e. adrenalin.]

20 mg. to 800 e.c.).]

EXPERIMENT XII.

[Rabbit, weight 1.700 gms. In-
fused with sodium-chloride
solution plus venom (20 mg.
to 800 ¢.c.); at 150 c.c. added
6 c.c. adrenalin; at 500 c.c.
injected J2 mg. venom intra-

venously.]

Amount | Blood- , ie
infused. | pressure. | Urine.
C0. mm. oC

0 100 nee
10 84 oe
20 72 ee
50 76 yeas

100 68 52
150 70 23
260 74 30
300 82 88
400 82 112
500 82 102
Injected 12 mg. venom and
started infusion again.
C.C. mm, C.c.
510 52 sat
520 60 15
540 64 sites
550 58 sight
600 56 36
650 50 man
700 60 20
58 30
Total |.......... 508

Amount | Blood- a Amount | Blood- .

infused. | pressure. | UTine- infused. | pressure, | Urine.

C.c. mm. c.c. eB. mm. c.c.

0 140 can 0 &4 ar

10 112 a 30 96 Nee

30 100 ee 50 76 ee

100 92 25 100 70 15

200 8&6 75 130 66 aa

300 90 100 *150 72 15

400 90 85 200 72 25

500 82 85 300 70 50

600 70 65 400, 74 75

700 64 50 500 72 80

—_ —_— 600 66 65

‘Total |esase ce ve { 485 700 62 69

Total |.........| 385

*Added adrenalin.

EXPERIMENT XIII.

{Rabbit, weight 1,900 gms. In-
fused with sodium-chloride
solution; at 300c.c. injected 12
mg. venom intravenously.]

{Rabbit, weight 1,800 gms.

EXPERIMENT XIV.

In-
fused with sodium-chloride
solution; at 300 c.c. injected 36
mg. venom intravenously.]

*Urine begins.

Amount | Blood- . Amount | Blood- ae
infused. | pressure, | Urine- infused. | pressure, | Uvine.
c.c. mm. CE. cc. mm, €ey

0 124 0 100 saute
80 128 en 20 120 wast
100 122 53 100 126 35
200 102 120 200 114 105
300 120 110 300 114 110
Injected 12 mg. venom and ‘
continued infusion again. ie ane
coc. mm, C.c.

20 = a C.c. mn c.c.
40 50 Bs 5 a re
90 54 ve 20 36 ee
100 58 (*) 30 4 ia
200 60 15 80 BH ig
300 62 42 100 52 "6
Oe a 200 52 10

Total |.........., 400 ri i Be
Total jo... 301

INFLUENCE OF HELODERMA VENOM. 113

In the following charts the straight line equals blood-pressure, the dotted
line equals serum.

i. B
120 mm 120 ce

20 mgm Venom + 800 cc NaCl;

80 mm 80 cc

adimm) f 40 cc

100 6¢ 200 cc 300 cc 400 cc 500 cc 600 cc 700 ce
Via. 15.
B U
120 mm 120 ce
20 mgm Venom + 800 cc NaCl;
80 mm 80 cc
f
i ia ies \
=a
ae
40 mm 40 ec
100 ce 200 cc 300 cc 400 ce 500 ce 600 cc 700 ce

Fia. 16.

114 THE VENOM OF ITBLODERMA.

B U
120 mm 120 ce
30 mgm Venom + 800 cc NaCl;
80 mm NM 80 ce
ran i a
cs: Se <=
40 mm 40 cc
'
100 cc 200 cc 300 cc 400 cc 500 ce 600 ce 700 cc
Fie. 17.
B U
120.mm 120 ce
— aaa
80 mm 80 ce
30 mgm Venom incinerated
+ 800 cc NaCl;
40 mm 40 cc

100 cc 200 cc 300 cc 400 cc 500 cc 600 cc 700 ce

Fre, ts,

INFLUENCE OF HELODERMA VENOM.

115

B a
20 mgm Venom + 800 cc NaCl;
+1.5 cc Adr. pro tL.
120 mm 120 ce
J
80 mm 7 RS 80 ce
40 mm 40 cc,
100 cc 200 cc 300 cc 400 cc 500 cc 600 ce 700 cc
Fia. 19.
U
120 mm 120 ce
80 mm 7 80 cc
rs
/ 20 mgm Venom + 800 cc NaCl;
at 200 cc added 1 cc Adr.
40 mm 40 ce
100 cc 200 cc 300 cc 400 cc 500 cc 600 cc 700 ce

Tig. 20.

116 THE VENOM OF HELODERMA.

B U
120 mm 120 ce
20 mgm Venom + 800 cc NaCl;
+ at 200 cc added | cc Adr,
80 mm i 80 ce
i
a
% |
40 mm 2 40 cc
100 ce 200 cc 300 cc 400 cc $00 cc 600 ce 700 ce
Fia, 21.
B U
120 mm 120 ce
Infused NaCl solution,
X at 300 cc injected —w
12 mgm Venom intravenously
and continued intusion
80 mm 80 ce

40 mm Ss t 40 ce

v

100 cc 200 ce 300 ce 400 cc 500 cc 600 cc

Tren, 22,

INFLUENCE OF HELODERMA VENOM. 117

120 mm =e, 120 cc

Infused NaCl solution,

x at 300 cc injected

30mgm Venom intravenously
and continued infusion

\

80 mm

40 cc

40 mm f = V

Lae

100 ce 200 cc 300 ce 400 ce 500 cc ~ 600 ¢c 700 cc.
Fie. 25
B U
80 mm A 80 cc
|
o <<
40 mm 40 ce
20 mgm Venom +800 cc NaCl;
x at 150 cc added 6 cc adr. sol to 600 cc NaCl Venom;
oat 500 cc inject 12 mgm.Venom intravenously.
100 cc 200 ce 300 cc 400 cc 500 ce 600 cc 700 cc

Fic, 24.

EFFECT OF THE VENOM OF HELODERMA SUSPECTUM ON
THE ISOLATED HEART.

By Tuomas StroresBury GITHENS.

EFFECT OF THE VENOM OF HELODERMA SUSPECTUM ON
THE ISOLATED HEART.

By Tuyomas Stotresspury GITHENS.

In order to determine whether or not the influence of the venom of the
Gila monster on blood-pressure was dependent upon a paralyzing action on the
cardiac muscle, and also to determine the effects of this venom on muscle-tissue
in vitro, two series of experiments were carried out, one on strips of the cardiac
muscle of the turtle, the other on the isolated heart of the frog. The results
seem to show that the direct action of the venom on the heart-muscle plays
only a small part in the toxic action exhibited when it is injected into the
living animal. The venom seems to exert a slight toxic action on the isolated
heart, but does not exert a noticeable cytolytic influence upon heart-muscle
tissue in vitro. These results agree with physiological tests which show that
the venom of the Gila monster, like some snake venoms, acts mainly on the
respiratory center, and that the heart continues to beat after respiration has
ceased. The strips of muscle which were exposed to the action of the venom
usually ceased to beat somewhat sooner than the controls, but not enough to
indicate a very marked toxic action on the heart. The isolated heart, trans-
fused with solutions of venom, was markedly affected only by very high
concentrations.

ACTION ON STRIPS OF TURTLE HEART.

The technique of this series was as follows: The heart was removed and
the ventricle cut into four longitudinal strips, extending from the base to the
apex, the auricle being used as a fifth strip. No difference was found in the
behavior of the auricle, except that the latent period was somewhat shorter. If
such strips are placed in 0.7 per cent NaCl solution, they will begin to contract
rhythmically in from 30 minutes to 2 hours, these rhythmic contractions con-
tinuing about the same length of time and then gradually ceasing. If small
amounts of calcium and potassium are now added to the NaCl solution, or if
the strips are transferred to Ringer’s solution, rhythmic contractions will again
begin. If the contractions cease they may often be induced to recommence by
transferring back to 0.7 per cent NaCl solution for a time and returning once
more to the Ringer’s solution. The length of time during which contractions
continue is more or less irregular, even under the best conditions. In our
experiments the controls lived from 9 to 36 hours.

In some experiments the venom was dissolved in 0.7 per cent NaCl solu-
tion and the strips placed in this on their removal from the body, and when the

121

122 THE VENOM OF HELODERMA.

contractions had almost ceased calcium and potassium chlorides were added.
In others, the venom was dissolved in Ringer’s solution and the strips trans-
ferred to this at the same period. In some of the experiments the strips were
allowed to remain in the venom solution until they ceased to contract; in
others they were left in the venom only part of the time. These differences
may be seen by referring to the protocols. The amount of venom varied in
the different experiments between 0.1 and 0.01 per cent.

In order to determine the effect of possible inorganic substances, some
tests were made with a solution of the ash from incinerated venom. This was
found to have no influence on the heart. Certain lots of venom were heated to
80° C. for an hour and then kept at 37° C. for 3 days. This heating did not
seem to influence the activity. The “heated venom’ of the protocols was
prepared in this way.

The extreme toxicity shown by sample B (see protocols 7 to 10) was evi-
dently due to some artificial contamination, possibly from the saliva or mouth,
and not to the venom itself, as the effect of this venom when injected was not
different from that of the others.

PROTOCOLS.

(1) Venom A.—Heart of small painted turtle (Chrysemys picta) removed
and the ventricle cut into five pieces, equal in size. Each strip placed, at 3
p. m., in normal salt solution containing venom as follows: No. 1, 0.07 per cent;
No. 2, 0.035 per cent; No. 3, 0.015 per cent; No. 4, 0.005 per cent; No. 5, con-
trol. All began irregular contractions which soon ceased. At 5 p.m. all were
beating; at 5" 30™ Nos. 1 and 2 ceased; at 7 p. m. No.4 ceased; at 9 p.m. Nos. 3
and 5 stopped.

(2) Venom A.—Heart of slightly larger painted turtle, treated as before.
The auricle in one piece was used as an additional control. Amounts of venom
asin1l. Heart removed at3 p.m. At6p.m. Nos. 1, 2, and 3 had ceased to
beat. Added 1 to 10,000 calcium chloride to each, which immediately started
contractions. At 6" 30™ No.1 contracted every 28 seconds; No.2 every 120
seconds; No.3 every 12 seconds; No.4 every 15 seconds; No.5 every 25 seconds;
No. 5aevery 6seconds. At 8" 30™ No. 1 and No. 2 have ceased entirely; No. 3
almost entirely; Nos.4,5, and 5a still beat strongly. At 9" 30™ No. 5a stopped;
at 6 a.m. No. 4 stopped, and soon after No. 5 stopped.

(3) Venom A.—Heart of painted turtle. Ventricle cut into four strips;
auricle used as No. 2. Placed at 3 p. m. in salt solution containing venom as
follows: No. 1, 0.1 per cent; No. 2, 0.05 per cent; No. 3, 0.025 per cent; No. 4,
ash of 0.05 per cent; No. 5, control. At 5 p.m. all beating feebly; add to each
0.03 per cent CaCl, and 0.01 per cent KCl. All begin contractions. At 6
p.m. all still beating, but more feebly. Add 0.01 per cent CaCl, contractions
increase. At 10> 30™ all have ceased. Transfer to plain 0.85 per cent. NaCl
solution; only No. 5 beats. At 11> 30™ transfer to Ringer’s solution without
venom for the night. The following day at 10 a. m. transfer to plain 0.85 per
cent NaCl solution and 5 minutes later to venom; Nos. 1 and 3 contract, No.
1 ceasing soonest.

(4) Venom A.—Heart of painted turtle. Ventricle cut in four strips,
auricle set as No. 2. Removed at 3 p. m. and placed in salt solution contain-
ing venom as follows: No. 1, 0.08 per cent; No. 2, 0.04 per cent; No. 3, 0.02 per

EFFECT OF VENOM ON ISOLATED HEART. 123

cent; No. 4, 0.01 per cent; No. 5, control. At 4" 30™ all beating feebly and
irregularly. Add to each 1 to 10,000 CaCl, all beats strengthened. At 6
p.m. all beating feebly. Make all solutions up to 0.03 per cent CaCl, and 0.02
per cent KCl. At7p.m.all have ceased. Transfer to normal NaCl solution
for 30 minutes and then back to the old mixture. All begin to beat and con-
tinue for several hours. At 8 a.m. transfer all to plain NaCl solution. All
but No. 3 begin to beat. At 3 p.m. Nos. 2, 4, and 5 are still beating. At 4
p.m. Nos. 2 and 4 stopped and No. 5 stopped soon after.

(5) Venom A.—Venom dissolved in Ringer’s solution containing venom as
follows: No. 1, 0.05 per cent; No. 2, 0.025 per cent; No. 3, 0.05 per cent, heated;
No. 4, 0.025 per cent, heated; No. 5, control. Heart removed at 12 noon and
placed in 0.85 per cent NaCl solution. 3 p.m. transfer to venom, all beat, No.
2 very feebly. 4" 45™ p. m. all still beating; transfer to 0.85 per cent NaCl
solution, all beat. 6 p.m. transfer to venom, all beat feebly. 8 20™ p. m. all
still.

(6) Venom A.—To determine whether the slight effect of this venom was
due to inorganic constituents, 0.0106 gm. of the venom was incinerated, the ash
weighing 0.0012 gm. The heart was removed in the usual manner and placed
in normal salt solution containing venom as follows: No. 1, 0.05 per cent
venom; No. 2, 0.025 per cent venom; No. 3, ash from 0.05 venom; No. 4, ash
from 0.025 per cent venom; No. 5, control. Heart removed at3 p.m. At8
p.m. only No. 1 beating, add 0.02 per cent CaCl, toeach. 12 p.m. all beating.
125 30™ a. m. No. 2 still, No. 5 very feeble. 7 a.m. all have ceased.

(7) Venom B.—Heart removed at 3" 30™ p. m. and at 65 45™ p. m., after
latent period, placed in Ringer’s solution containing venom as follows: No. 1,
0.04 per cent; No. 2, 0.04 per cent; No. 3, 0.04 per cent, heated; No.4, 0.04 per
cent, heated; No. 5, control. At 8 p.m. all had stopped and on transfer to
plain 0.85 per cent NaCl solution only No. 5 beats. This continued several
hours; the others never beat again.

(8) Venom B.—Heart removed at 1 p. m. and placed in plain 0.85 per cent
NaCl solution. At 5 p.m. placed in Ringer’s solution containing venom as
follows: No. 1, 0.08 per cent; No. 2, 0.04 per cent; No. 3, 0.02 per cent; No. 4,
0.01 per cent; No. 5, control. All stopped in about 3 hours, except control,
which continued for over 36 hours.

(9) Venom B.—Heart removed at 3 p. m. and placed in normal 0.85 per
cent NaCl solution containing venom as follows: No. 1, 0.016 per cent; No. 2,
0.008 per cent; No. 3, 0.004 per cent; Nos. 4 and 5, control. Only the controls
started to beat. The strips in the venom were immediately paralyzed and
none showed any tendency to recovery on being placed in normal 0.85 per cent
NaCl solution or Ringer’s solution. They became so soft that they rolled up
into a ball by their own weight when the hook on which they were hung was
turned over.

(10) Venom B.—Heart removed at 3 p. m. and placed in normal NaCl
solution containing venom as follows: No. 1, 0.004 per cent; No. 2, 0.002 per
cent; No. 3, 0.001 per cent; No. 4, ash from 0.004 per cent; No. 5, control.
Only Nos. 4 and 5 started to beat, the others passing into the relaxed condition
described in the last experiment.

(11) Venom C.—Heart removed at 4 p. m. and the strips placed in NaCl
solution containing venom as follows: No. 1, 0.002 per cent; No. 2, 0.001 per
cent; No. 3, 0.0005 per cent; No. 4, 0.00025 per cent; No. 5, control. At 7» 30™
p- m. all were beating very feebly; added to each CaCl, 3 to 10,000 and KCI 1
to 10,000. All start to beat. At 11> 30™ p.m. all still beating. At 12 p.m.
all have ceased except No. 5, which is still beating, but very slowly and feebly.

124 THE VENOM OF HELODERMA.

ACTION ON THE ISOLATED HEART OF THE FROG.

In this series hearts of leopard frogs (Rana pipiens) weighing from 60 to 70
grams were used. They were immersed in Ringer’s solution of the composition
NaCl 0.6 per cent, NaHCO; 0.03 per cent, CaCl, 0.035 per cent, KCl 0.03 per
cent, and were transfused by a similar solution in which the venom was
dissolved.

The venom solutions varied in strength from 0.01 to 2 per cent. Solu-
tions of less than 0.1 per cent had little if any effect. Solutions of 0.5 and 2 per
cent had respectively a moderate and a marked action in reducing the strength
of the contractions. Even after the heart had been exposed to the action of
such solutions for 15 to 30 minutes, washing out with fresh Ringer’s solution
restored the strip at once to its former strength, showing that no marked toxic
action had been exerted and that no permanent structural change due to
cytolysis had been produced.

PROTOCOLS.

Heart No. 1.—Ringer’s solution for 6 minutes until normal rhythm was
established. Venom 0.01 per cent, renewed every 5 minutes for 24 minutes.
Heart stops beating, but on single touch gives a long series of beats of same
rate and strength as before the action of the venom. 32 minutes, Ringer; 47
minutes, venom 0.02 per cent; 60 minutes, beats as strong as ever, becoming
slower; on touch, series of rapid beats; 70 minutes, Ringer, beats continue slow;
78 minutes, venom 0.1 per cent, beats continue to become slower but show no
change in force; 102 minutes, venom 0.5 per cent, beats continue 5 minutes,
with no loss in strength.

Heart No. 2.—Ringer’s solution to establish rhythm; 13 minutes, venom
0.2 per cent, beats grow weaker, but on renewing venom return to original
strength, 22 minutes, no loss of strength: 33 minutes, beats much reduced:
Ringer, beats immediately restored to full strength.

Heart No 3.—Ringer’s solution for 9 minutes; venom 0.5 per cent; 24
minutes, heart beating in groups but strongly ; 26 minutes, heart stops, stimula-
tion causes single beats or short groups; 39 minutes, Ringer, heart begins again
in widely separated groups, beats of full strength but apparently some loss of
spontaneous impulse.

Heart No. 4.—Ringer, heart beating strongly in groups; 20 minutes, venom
2 per cent, beats immediately, much weaker but no change in rhythm; 32 min-
utes, Ringer, beats immediately restored to full strength; 42 minutes, venom 2
per cent, beats weaker and grow weaker until 52 minutes when put into Ringer
solution, beats immediately restored to full strength, that is, about 1.25 to 1.5
times as high as those under the influence of venom 2 per cent.

VI.

EXPERIMENTAL PRODUCTION OF ACUTE GASTRIC ULCER
IN THE GUINEA-PIG.

By M. E. Reuruss.

EXPERIMENTAL PRODUCTION OF ACUTE GASTRIC ULCER
IN THE GUINEA-PIG."

By M. E. Reuruss.

It is here proposed to describe somewhat more in detail the results of our
experiments and to give a very short résumé of the contributions by some au-
thors bearing on the formation of hemorrhagic erosions and acute gastric ulcer
of the stomach. This was the most typical lesion found in guinea-pigs which
succumbed to the venom of Heloderma. The fact that little is known as to the
pathogenesis of hemorrhagic erosions and the possibility of their development
into typical gastric ulcer increases the importance of this work. The researches
of Gay and Southard} on serum anaphylaxis, of Boltont on gastrotoxic serum,
and of Rosenau and Anderson§ on production of gastric ulcer and hemorrhage
by the subcutaneous injection of diphtheria toxin into guinea-pigs, together with
the exhaustive anatomical study by Beneke of hemorrhagic erosions occurring
in the human stomach, represent the most important contributions relating to
this problem.

At a recent meeting of the German Association of Pathologists in Kiel,
Beneke{ called attention to the frequency of hemorrhagic erosions and cited
400 cases which he had collected. He pointed out the possibility of their de-
velopment into typical gastric ulcer. He says surgeons have frequently, within
the last ten years, noticed the occurrence of hemorrhage in the stomach as
evinced by the so-called “black vomiting” and even occasionally death. Ana-
tomically, hemorrhagic erosions of Cruveilhier are found in such cases, and
frequently associated with them are punctate ulcers. 27 per cent of Beneke’s
collected cases were post-operative, the great majority having been observed
after intraperitoneal operations. He claims the danger in their formation is
the greater the more the field of operation approaches the ductus choledochus
and ceeliac ganglion. The remainder of cases were found in a great variety of
diseases. From a morphologic point of view, he classifies his cases as follows:
(1) Typical hemorrhagic erosions. (2) Typical ecchymoses without ulcer.
(3) Simple ulcer, origin doubtful. (4) Ulcer plus ecchymoses. (5) Parenchy-
matous ecchymoses and apparent anatomical changes of the stomach.

Beneke believes that the lesion is a primary digestion; that hemorrhage is
secondary, and that the origin of but a small proportion is in ecchymoses. In
order that digestion may take place, some special injury to the mucosa must
occur, and this usually is brought about through a lack of oxygen. For in-

*A preliminary report of this work was published in the University of Pennsylvania Medical Bulletin. 1909.
tJourn. Med. Research, July 1908., vol. xrx,

tProceed. Roy. Soc., 1905-1906, series B, 77, p. 426; series B, 79, 1909.

§Journ. Infect. Diseases, 1907. WVerhandl. d, deutsch. Path. Gesellsch., Kiel, 1908.

127

128 THE VENOM OF HELODERMA.

stance, in the new-born dying of asphyxia, he found many small necrotic areas
inthe stomach. He believes that a local ischemia is reflexly produced through
some irritation of the central nervous system. This condition, combined with
the collapse of the circulation and increased gastric secretion, results in hemor-
rhagic erosion or ulceration. Beneke’s views in the main, however, are purely
hypothetical. His experimental evidence, consisting in (1) ligation of the
ceeliac artery, and (2) injection of adrenalin into the muscularis of the stomach,
is inconclusive; nor is his reasoning on the basis of anatomical observations
quite convincing. Beneke, therefore, merely suggested the primary digestive
nature of these phenomena without any experimental proof. Furthermore,
his theory as to the formation of these lesions is probably, in part at least,
incorrect, as will be demonstrated later.

Bolton* reports some very interesting results in his work on the action of
gastro-toxic serum. This serum, which is produced by the injection of the
mucous membrane of the stomach of the guinea-pig into the rabbit, is not abso-
lutely specific in the true sense of the word. It produces necrosis and ulcera-
tion of the mucous membrane of the stomach when injected subcutaneously
into the guinea-pig, but Bolton was unable to demonstrate any effect upon
the gastric glands in vitro. The gastric cytotoxin formed in response to the
injection of the gastric-cells seems to be a very complex body—increasing the
hemolytic power which is normally present in the blood—containing many
precipitins, most of which are absolutely specific, and containing no specific
agglutinin for the gastric granules. By means of this serum it seems impos-
sible to establish a chronic lesion, unless there is a perpetuation of the acutely
produced lesion either by (1) a secondary bacterial infection, or (2) hyperacidity
of the gastric juice. The actual necrosis and ulceration is produced by the
action of the gastric Juice on a cell which is functionally damaged. Hyper-
acidity increases the tendency toward ulceration.

Gay and Southard,f in their studies on anaphylaxis, also report anatomical
changes in the gastric mucosa. In studying the causes of death in serum in-
toxication, they found hemorrhages in a great many organs. The most mas-
sive and most frequently observed occurred in the stomach-wall. Thus 32 out
of 41 guinea-pigs dying within 24 hours of the second or toxic injection showed
gastric hemorrhage, while 59 out of the total of 86 showed the same phenome-
non. They do not consider the lesions as specific for serum intoxication in an
anatomical sense, but claim that focal cytolyses of wide distribution occur and
that this especially characterizes the action of the toxic phase. Certain dif-
fuse fatty changes take place in the gastric epithelia where the local action of
the gastric juice can come into play.

Rosenau and Anderson also describe an acute lesion of the gastric mucosa,
which they are inclined to regard as more or less specific because it is confined
to the pyloric end of the stomach. In their studies on diphtheria toxin they
found that 66 per cent of the 1,879 animals injected with this toxin and dying

*Proceed. Roy. Soc., 1905-1906, series B, 77, p. 425; series B, 79, 1909.
tJourn. Med. Research, July 1908, vol. xrx.

EXPERIMENTAL PRODUCTION OF GASTRIC ULCER. 129

acutely from the injection showed lesions of the stomach. Turck failed to pro-
duce gastric ulcer by injection of the diphtheria toxin into the stomach-wall;
only minute hemorrhagic foci were found in the duodenum. He also failed to
produce gastric ulcer by the introduction of the toxin into the mesenteric ves-
sels. He obtained, however, in ten weeks, necrosis in the duodenum and near
the pylorus. Rosenau and Anderson produce these changes by direct subcu-
taneous injection of the diphtheria toxin in a dose sufficient to cause the acute
death of the animal. When the toxin is completely neutralized by antitoxin
the stomach shows no lesions. Lesions may occur in infections with Bacillus
diphtherie as well as through the action of the toxins. These pathologic
changes—ulceration and hemorrhage—always occur at the pylorus and are
found in 50 per cent of the animals dying within 24 hours and in 75 per cent of
those dying between the third and fourth day after the injection. They enum-
erate the steps in the formation of the ulcer: (1) congestion, (2) hemorrhage,
(3) digestion. ‘

These observations represent more or less acute conditions in which toxic
agents are at work. Indirectly they have, as will be shown, some bearing on
our work.

METHOD OF PRODUCING ACUTE TOXIC ULCER OF THE STOMACH.

The beginning of our investigation was the observation that the subcu-
taneous injection of the venom of the Gila monster (Heloderma suspectum) into
guinea-pigs frequently produced ulceration and hemorrhage of the stomach.
The dose of venom necessary to produce this change depended on the toxicity
of the venom, and as this constantly fluctuates, the dose must vary with the
toxicity. In the beginning of our experiments we found that 1 ¢.c. of fresh
venom, diluted in the proportion of 1 part of venom to 19 parts of normal salt
solution, was sufficient to cause death in from 2 to 4 hours after the animal had
reached a state of marked intoxication. Later on, however, as much as 1.5 to
3 c.c. of diluted fresh venom was occasionally required for the purpose. The
dose of dried venom which exhibited approximately the same toxicity was from
12 to 15 mg., and a much greater dose was frequently given. The average
weight of the animals employed in our experiments was from 350 to 400 grams.
In larger animals the dose must be increased in proportion to the increase in
weight.

It is very important that the animal should be in a state of marked intoxi-
cation and remain in such a condition for a period of at least an hour. Under
such circumstances the animal is in a stuporous condition or has tremors, con-
vulsions, or later actual paralysis. This condition of intoxication is the most
important factor in the production of gastric ulcer; the dose of venom must be
chosen accordingly and the injections repeated with sufficient frequency to
insure the continuance of such an effect for a certain time. Following these
rules, we were able to produce gastric ulceration in 35 out of 41 animals injected
with venom alone—a little over 85 per cent. Of the 15 per cent in which no
change or only a doubtful change was visible, several had received insufficient
doses, and in the case of several others the toxicity of the venom had been

130 THE VENOM OF HELODERMA.

below normal. This ulceration was independent of the state of dying, inas-
much as animals killed at a much earlier period showed the same change, but
the changes were usually more marked in direct proportion to the degree of
intoxication manifested by the animal.

Tue TypicaL ULcrr.

The typical ulcer, usually accompanied by hemorrhage, was, on exposure
of the peritoneal surface of the stomach, generally seen as a purplish-red cir-
cumscribed patch. If ulcers were unaccompanied by hemorrhage—and this
was by no means rare—they appeared as rounded grayish points. On opening
the stomach, hemorrhage was usually seen; after removing the blood the char-
acteristic ulcer appeared, varying from1to 8 mm. in diameter. Occasionally,
by confluence, they would involve as much as 0.5 square inch of the gastric
mucosa. They were always found in the cardia and fundus, and were never
observed in the pyloric fifth of the stomach. They are found more frequently
at the greater curvature than at the lesser, but were by no means excluded from
the latter. Of interest is their occurrence in discrete clusters—most frequently
on the anterior cardiofundal region—or near the center of the stomach around
the greater curvature. This arrangement might be explained on the ground
that each territory involved was nourished by a certain blood-vessel and its
branches. We endeavored, therefore, to ascertain, by holding the entire mu-
cosa up to the light, what relation these ulcers held to the distribution of the
vessels, and found a certain proportion of the clusters bearing some relation to
the vessels.

The ulcers, when typical, are round, sharply circumscribed, with sloping
edges resembling a peptic ulcer in the stomach of man, with or without a hem-
orrhagic base. Frequently the ulceration reaches down to the muscularis, and
the ulcer then has a translucent appearance when viewed from the peritoneal
surface and has usually a number of hemorrhagic erosions combined with it.
These erosions occur so frequently and are seen so constantly, especially when
an animal is killed soon after injection, before a definite ulcer has had a chance
to develop, that in all probability the hemorrhagic erosion represents an early
stage of these ulcers. Indeed, all transitions can be found, from hemorrhagic
erosion to distinct, large, and well-defined ulcers. These ulcers bear a striking
resemblance to the ulcers described by Bolton,* and, to judge from his descrip-
tion, the appearance of both kinds is identical. The fact that Bolton’s lesions
were acute also favors this view of the identity of both varieties; but we do not
know whether the condition of marked intoxication, so important in the pro-
duction of venom ulcers, was equally present in his animals, nor whether the
anatomical position of the ulcers was the same in both cases. Both of these
ulcers differed from those produced by Rosenau and Anderson in that the latter
were confined to the pyloric region. Furthermore, as will be shown later,
hemorrhage was not the primary factor in the ulcers produced by the injec-
tion of venom.

*Proc, Roy. Soc., 1905-1906, series B, 77, pp. 425, 428; series B, 79, 1909.

EXPERIMENTAL PRODUCTION OF GASTRIC ULCER. 131

Microscopically a section through a well-formed ulcer shows a sharply cut
crater resembling a peptic erosion. The walls of the mucosa dip in on either
side, disclosing the muscularis or submucosa as the floor of the ulcer. In the
ulcer proper we find granular débris consisting of digested mucosa, food par-
ticles, and occasional hemorrhages. Frequently, however, the walls, instead of
being sharply cut, sloped gradually, giving the ulcer the appearance of an exca-
vation. Another form is seen where three-fourths of the mucosa takes a more
or less basic stain and sloughs off, leaving a few rows of intact cells below. Fre-
quently a number of polynuclear leucocytes are seen in the base of the ulcer,
together with a little fibrin; but connective-tissue proliferation does not take
place, which is in accordance with the acute character of the lesion. Usually
hemorrhagic areas are seen between the sloughed-off part of the mucosa and the
intact mucosa below. This might suggest hemorrhage as the primary cause of
the ulcer, but far more frequently areas of mucosa are seen which take a more
or less basic stain. Under the high power various stages of nuclear degenera-
tion are seen in these areas. The large majority of the nuclei are shrunken and
do not take the stain. The protoplasm of the gland cells is granular and fre-
quently has entirely disappeared in the upper layers, leaving nothing but poorly
stained, shrunken nuclei and connective-tissue fibrils. This area contrasts
strongly with the gastric tubules below, in which the cells are intact.

Interesting is the fact that in these superficial areas, when all other ele-
ments are necrotic, the acid cells stain well and seem able to resist digestion
much better than the parietal cells. This was observed in a number of in-
stances, and is in accord with Bolton’s observations on the effect of the gastro-
toxic sera on the gastric cells in vitro.

Every intermediate stage has been observed from slight change with no
sloughing of the mucosa to actual ulcer, and frequently without hemorrhage. In
this very early stage not even capillary hemorrhages can be seen. Ontheother
hand, small submucous hemorrhages have been observed without any change
in the overlying mucosa. In a study of a great number of sections, many of
them serial, only one marked instance of submucous hemorrhage was found,
and in this case no change was visible in the overlying mucosa. In several
instances where gastric ulceration and hemorrhage were present, parenchy-
matous hemorrhages into the spleen were also observed.

The possibility of hemorrhage occurring in parts of the small or large intes-
tines was thoroughly studied. The entire gastro-intestinal tracts of about 25
animals were carefully examined. No case of extra-gastric hemorrhage was
observed, except in one case in which a little blood-stained fluid was found in
the small intestine.

The vessels in the vicinity of the ulcer are often thrombosed; and fre-
quently a peripheral rim of leucocytes, due to a gradual slowing of the circula-
tion, was seen in these thrombi. That these thrombi are not the cause of the
ulcer, but are secondary, will be shown later. They seal the vessels and thus
prevent hemorrhage. Frequently the capillaries immediately surrounding the
ulcer are congested.

132 THE VENOM OF HELODERMA.

Errect OF PILOCARPINE AND ATROPINE ON THE PRopuUCcTION OF ULCERS.

On the basis of these anatomical facts, we extended our work in order to
clear up the causation of these ulcers by experimental means. We studied the
influence of gastric secretion, the effect of the neutralization of the latter by
sodium-carbonate solution, the relation of these ulcers to the distribution of the
vessels, and the effect of thrombosis. Inasmuch as pilocarpine is supposed to
increase gastric secretion, and atropine to lessen it, it was of interest to observe
in what way they could modify the ulceration produced by venom.

Added interest was attached to these experiments, as the possibility of the
excretion of venom into the stomach as a cause of the ulceration had to be con-
sidered. We therefore injected three sets of animals, one with venom alone,
one with atropine and venom, and one with pilocarpine and venom, and ob-
served them under identical conditions. We found that those animals which
received pilocarpine in addition to venom showed markedly increased ulcera-
tion and hemorrhage. In fact, the most marked cases of ulceration and hemor-
rhage seen inthis work were observed after the combined use of pilocarpine and
venom. For instance, in the case of guinea-pig 109 the stomach was com-
pletely covered with a great number of ulcers and hemorrhagic areas varying
from 1 to 7 mm. in diameter. Atropine also increased the tendency toward
ulceration, but this was not nearly as well marked as in the instance of pilocar-
pine. The addition of either substance, therefore, but especially of pilocarpine,
to venom, increases the gastric ulceration. These results suggested the ques-
tion as to what might be the effect of these alkaloids on the stomach without
the addition of venom.

We therefore administered lethal doses of atropine and pilocarpine with-
out venom. As much as 3.7 grains of atropine sulphate and 8 grains of pilo-
carpine hydrochloride were given subcutaneously in fractional doses with suffi-
cient frequency to keep the animal in a markedly toxic state for several hours
before death. In the two pilocarpine animals distinct ulceration and hemor-
rhage were present, while in two out of six atropine animals hemorrhagic ero-
sion, and in one of these also a typical small ulcer, was found. Three of the
six guinea-pigs that received atropine died so soon after the injection that the
time was insufficient for an ulcer to form. In these experiments also the
ulcers were formed if the animals had been in a markedly toxic state several
hours before death.

Inasmuch as the ulceration and hemorrhage produced differed only quan-
titatively in these cases, we tried various other poisons, observing the precau-
tion to inject the substance with sufficient frequency to keep the animal in a
markedly toxic state for several hours before producing death. Paraldehyde,
chloroform, 10 per cent phenol, and a saturated solution of magnesium chloride
were all tried, as well as sodium fluoride and copper sulphate. Without going
into detailed description, we may state that, with every poisonous substance, we
were able to obtain, under proper conditions, some gastric lesion. This latter
varied from distinct ulceration in the paraldehyde animals to the occasional
hemorrhagic erosions seen in the chloroform or magnesium-sulphate animals.

EXPERIMENTAL PRODUCTION OF GASTRIC ULCER. 133

At first it seemed doubtful whether any change was demonstrable after admin-
istration of the latter substance, but Doctor Loeb has since succeeded in pro-
ducing lesions. It seems, therefore, certain that this reaction is in no wise spe-
cific for venom and that possibly all poisons, whether organic or inorganic,
which will keep the animal in a state of marked intoxication will produce some
change in the gastric mucosa. Whether this lesion is directly proportional to
the depression caused by the poison we are not prepared to state. Certainly
this is not invariably the case, inasmuch as magnesium-chloride animals,
although markedly affected, showed very few lesions; usually, however, the
severity of the lesion was in direct proportion to the degree of intoxication
manifested by the animal.

SIGNIFICANCE OF THROMBOSIS IN Gastric ULCERATION.

As has been previously stated, thrombi were found in the vessels in the
neighborhood of the ulcer, and as a decrease in the blood-supply had to be con-
sidered as a possible cause of the ulcers, and inasmuch as thrombi might be the
cause of such a diminution in the blood-supply, we had to determine whether
thrombosis was a primary or secondary factor in the formation of the ulcers.
For this purpose we caused experimentally an incoagulability of the blood by
injecting hirudin into the jugular vein, this being followed by the injection of
venom. If ulcers occurred under such conditions, it was certain that throm-
bosis could be ruled out as an etiological factor. After several failures due to
errors in technique, we succeeded in fulfilling all conditions in eight animals,
and in all of them hirudin, instead of preventing ulceration and hemorrhage,
actually augmented the latter. There were just as many ulcers present, and
the hemorrhage, which was formerly in direct proportion to the degree of ero-
sion of the vessels, assumed alarming proportions after the injection of this
substance. Immediately on the death of the animal we were accustomed to
withdraw 5 to 10 c.c. of blood from the heart, to test its coagulability and to
control the action of the hirudin. In several instances the blood was not coag-
ulated in vitro two hours after death. In one interesting case the animal had
been in a very weak condition for some time before death. On opening its
heart we were unable to withdraw more than a few drops of blood; the stomach,
however, was distended with liquid blood and in the wall of the stomach were
found several well-defined ulcers. The animal had practically bled to death
into its stomach. In several animals a hemorrhagic mottling of the spleen was
also observed.

From these observations we may conclude that thrombosis is not a pri-
mary factor in the production of the ulcers; that it is a secondary process due to
changes in the circulation and to the liberation of tissue coagulins in the affected
area; moreover, that it is beneficial, inasmuch as it prevents fatal hemorrhage.

IS THE ULCERATION DEPENDENT UPON PEPTIC DIGESTION ?

Beneke believed that in the cases reported by him the ulcers were primarily
caused by digestion, but no adequate experimental proof was offered for this
interpretation. Bolton concluded that inasmuch as alkali prevented the for-

134 THE VENOM OF HELODERMA.

mation of an ulcer after the injection of gastrotoxic serum, the ulcers must be
primarily digestive in character; and Rosenau and Anderson claim that the
pyloric ulcers produced through the injection of diphtheria toxin were due to
circulatory changes, congestion, and hemorrhage, followed by digestion.

Although according to our observation, macroscopically and microscopi-
cally, hemorrhage is nearly always a concomitant of the ulcers of the stomach,
it is questionable whether the latter are primarily hemorrhagic. In order to
decide this question we introduced into the stomach of a guinea-pig 14 c.c. of a
4 per cent sodium-bicarbonate solution through a stomach-tube, and injected
the venom subcutaneously. This was the method used in the beginning.
Later, we gave two injections of 10 c.c., one hour apart, in order to insure
constant contact of the gastric mucosa with alkaline fluid. This mode of pro-
cedure fulfilled two indications: (1) it completely neutralized the gastric juice,
and (2) it constantly moistened the gastric mucosa with alkaline fluid. We
found that alkali markedly inhibited, and in the great majority of cases actually
prevented, the formation of ulcers. Thus, in 30 animals in which alkali and
venom were administered, 6 showed ulceration, 20 per cent; while over 80 per
cent in the same number of animals in which venom alone and no alkali had
been given showed ulceration. Not only was this the case, but in 4 of the 6 the
ulceration was single, and in the other 2 animals the ulcers were very small,
while in the controls the ulcers were large and multiple. Besides, in 2 of these
6 only 10 c.c. of alkaline fluid had been injected, and most of the fluid had prob-
ably passed over into the duodenum at the time the ulceration took place.
The quantity of alkali was, therefore, insufficient. Traumatisms caused by
the stomach-tube also could not be excluded. In the last series of 6 animals
in which the technique was most perfect, the 3 animals with toxin and alkali
showed absolutely no lesions, while the controls showed marked ulceration and
hemorrhage.

These experiments indicate, therefore, that digestion is the primary factor
in the formation of these ulcers. In favor of this interpretation we may like-
wise cite the observation that in a number of instances, as stated above, be-
ginning necrosis or digestion could be observed microscopically without any
preceding hemorrhage. Occasionally, however, hemorrhage occurred independ-
ently of ulceration; but an intact epithelial layer covering such an area makes
it improbable that such a primary hemorrhage has much significance in the
production of the ulcers. Our experiments do not permit us to make any defi-
nite statement as to the cause of the primary digestion, but we are inclined to
believe that a malnutrition of certain areas of the gastric mucosa is brought
about by a slowing of the circulation, especially as we have observed the ulcer-
ation in discrete areas and with especial frequency in those animals in which
the phenomena of collapse were apparent. It is also possible that toxic sub-
stances may pass directly from the capillaries to the surrounding mucosa,
change the vital processes. of these cells, and lower their resistance to peptic
digestion. Beneke’s view, advocated first by Klebs, that spasm of the vessels
with resulting ischemia is responsible for tissue digestion, seems improbable,

EXPERIMENTAL PRODUCTION OF GASTRIC ULCER. 135

inasmuch as it is questionable whether such a variety of poisons should all cause
a constriction of the vessels, especially if we consider that several of these sub-
stances were vasodilators. That fatty degeneration of the glandular elements,
such as described by Gay and Southard, is not the cause of the ulceration is evi-
denced by our inability to demonstrate any such change in sections through
ulcerated areas stained by osmic acid.

It remains for further experiments to determine whether or not it is pos-
sible to establish a chronic lesion. In one instance an animal which had been
injected with venom several months previously was killed and a somewhat
older ulcer found, which under the microscope revealed a base infiltrated with
round cells; no attempt at healing was observed. It is not unlikely that, if the
animal remains alive, such an acute toxic ulcer may become chronic, especially
if it be perpetuated by a bacterial infection, hyperchlorhydria, or anemia.
Total number of guinea-pigs, excluding those used for testing the strength of the venom, 127.
Of 41 injected with venom alone, 35 showed ulcer (85 per cent). :

Of 30 animals injected with venom and alkali (or with venom + atropine and alkali), 6
showed ulcer (20 per cent).
Pilocarpine, 14 animals:
Control (venom alone), 4 animals, 4 showed ulcer (100 per cent).
Venom and pilocarpine, 8 animals, 8 showed ulcer (100 per cent).
Pilocarpine alone, 2 animals, 2 showed ulcer (100 per cent).
Atropine, 34 animals:
Control (venom alone), 9 animals, 8 showed ulcer.
Venom and atropine, 6 animals, 6 showed ulcer.
Venom, atropine, and alkali, 13 animals, 4 showed ulcer (1 of these cases was doubtful).
Atropine alone, 6 animals, 2 showed ulcer (2 died too early).
MgCl, 4 animals, 1 showed hemorrhagic erosion.
Paraldehyde, 2 animals, 2 showed ulcers.

Phenol, 2 animals, 2 showed ulcer.
Chloroform, 2 animals, 2 showed ulcer and hemorrhagic erosion.

In addition several animals were injected with sodium fluoride, CuSo,, and MgCl with
positive results.

Hirudin experiments, 14 animals:
Hirudin + venom, 8 animals, 8 showed ulcer.
Controls (venom alone), 5 animals, 5 showed ulcer.

CONCLUSIONS.

After subcutaneous injections of the venom of Heloderma into guinea-pigs,
gastric ulcer and hemorrhagic erosions are found in approximately 85 per cent
of all the animals used. These ulcerations are aggravated by atropine as well
as by pilocarpine; it is, therefore, unlikely that ulceration is due to the excre-
tion of the venom through the intact mucosa. Various poisonous substances,
otherwise differing widely in their chemical character and pharmacological
actions, may all produce similar changes in the gastric mucosa. The effect of
these substances is not a specific, but is probably an indirect one, acting either
through a weakening influence on the circulation or through a direct injurious
effect on the cells of the mucosa. The degree of efficacy of these substances is,
on the whole, parallel to their general toxic effect or to the extent to which they
affect the general vitality of the animals. There exist, however, marked differ-
ences in the tendency of these substances to produce ulceration (cf. especially
magnesium chloride and paraldehyde).

136 THE VENOM OF HELODERMA.

In the large majority of cases both hemorrhage and ulceration are found
in the affected areas. The fact that in a large majority of cases the introduc-
tion of alkali into the stomach is able to prevent the occurrence of ulceration
and hemorrhage renders it almost certain that usually digestion is primary and
that in many cases hemorrhage is merely the result of secondary erosion of the
vessels. The results of microscopic observation favor this interpretation.

In the course of ulceration, neighboring blood-vessels become occluded by
thrombi. This is secondary and not primary, inasmuch as we were able to
show that experimentally produced incoagulability of the blood failed to pre-
vent the occurrence of ulcers. Thrombi are, therefore, not the cause of ulcer-
ation, but this secondary thrombosis must be considered as a beneficial process,
inasmuch as it prevents fatal hemorrhage.

These facts throw light on hemorrhagic erosions in man, which are, in all
likelihood, due to primary digestion. They also indicate that such lesions may
be transformed into typical gastric ulcer; at least such is the case in guinea-pigs
subjected to the influence of different toxic substances. Whether chronic
round ulcer can be produced by a perpetuation of such a condition remains to
be proved.

VII.

HISTOLOGICAL CHANGES OF THE CENTRAL NERVOUS
SYSTEM PRODUCED BY HELODERMA VENOM.

By Samvue.t Leoporp.

HISTOLOGICAL CHANGES OF THE CENTRAL NERVOUS
SYSTEM PRODUCED BY HELODERMA VENOM.

By SaMuEL LEopoLp.

The effect of the venom of various snakes upon the cerebro-spinal system
has been studied histologically only since 1900. It will not be necessary to
review the literature, as this has been done in Noguchi’s work on Snake Venoms:
An Investigation of Venomous Snakes with Special Reference to the Phenom-
ena of their Venoms (Pub. No. 111, Carnegie Institution of Washington,
1909). It was of interest to compare the action of snake venom with the effect
of heloderma venom upon the central nervous system.

Dr. Leo Loeb kindly placed at my disposal the brains and spinal cords of
rabbits and guinea-pigs which had been injected with varying doses of the
venom of Heloderma. The inoculations were either given subcutaneously or
into the peritoneal cavity. The dose varied from 25 to 60 mg. Some animals
were given but one injection, others two, and in one instance four celloidin cap-
sules had been introduced intraperitoneally and the poison was allowed to act
during a period of 28 days. The animals lived from 5 minutes to several weeks.
This material for study was placed in 10 per cent formalin or in Muller’s fluid,
and the sections were stained usually with thionin (Nissl method) and with
hemalum. In one animal (rabbit Bur, living 13 days), sections of the spinal
cord were stained by the Marchi method.

In most cases only the medulla and spinal cord were examined (guinea-
pigs 16, 21, 22,18, A‘; rabbit B’). In four cases the cerebral cortex cerebellum,
pons, medulla, and spinal cord were studied (rabbits Nos. 1, B1, and a’). In
one instance (guinea-pig a1) sections were taken from every portion of the brain.

For control a healthy guinea-pig’s brain was sectioned at various levels
from the frontal lobes to the spinal cord, and stained by the Nissl method.

The earliest change noted in our animals occurred after 45 minutes (guinea-
pig 18). Some of the cells in the anterior horn of the spinal cord took the
stain a little more intensely and diffusely. The medulla and spinal cord of
several animals dying at shorter intervals than 45 minutes failed to reveal any
changes in their cells. Lamb and Hunter found with the venom of Naja defi-
nite changes only when the animals lived longer than 2 or 3 hours, but the
venom of Enhydrina produced pronounced chromatolytic changes in 90 min-
utes, while Kilvington found chromatolysis with the venom of the tiger-snake
if the animal survived the injection of the poison for over 3 hours.

In rabbit No. 1, which lived 75 minutes, some of the ganglion-cells of the
spinal cord, medulla, and a few of the Purkinje cells showed an early disinte-

gration of the Nissl bodies. A fine powder was seen around the nucleus, which
139

140 THE VENOM OF HELODERMA.

extended toward the periphery. Some of the cells showed a slight swelling.
This latter feature was present in both acute and chronic cases, but seemed a
more constant feature in the various sections of the chronic cases. This swel-
ling of the cell does not seem to be a characteristic feature in the pathological
findings of others. No mention of it is made by Lamb, Hunter, or Kilvington.

The disintegration of the Nissl bodies was the most constant finding in the
acute cases, but it is not characteristic, being also found in the chronic cases.

In none of our acute cases was an eating-out of the cell seen or the complete
dissolution of its cytoplasm, etc., as described by Lamb and Hunter. The
process after injection of heloderma venom seemed much milder when com-
pared with the effect of some of the snake venoms. In the ganglia-cells of
our animals the nuclei were for the most part centrally placed and could always
be recognized. They were misplaced only in exceptional cases.

Chromatolysis was a constant feature in the animals living from 48 hours
to several days (guinea-pigs Ai, A,; rabbits B! and B’). In none of the cells
was the outline lost; the nuclei were always visible and showed no change in
form. These changes corresponded to those of Kilvington, Bailey, Lamb, and
Hunter, save that in our cases the cells were in some instances swollen.

Another change which we found and which was not described by the above
authors was the presence of minute areas of hemorrhage in two of our cases
(rabbit a? and guinea-pig A’). These hemorrhages were small and scattered
through the entire structure of the medulla (rabbit a*), while in guinea-pig A!
they were noted around the central canal of the cord. These hemorrhages
have not been sufficiently constant in our series to conclude that they are
characteristic for the venom of heloderma. In guinea-pig A! the animal had
convulsions prior to its death, and the convulsions may possibly be responsible
for the hemorrhages.

We observed a round cell infiltration in two of the chronic cases (rabbits
B? and A8); in the former case (B?) the cell infiltration was found around the
posterior septum of the cord, and in the latter animal (a’) around the blood-
vessels. In the cases described by Lamb and Hunter, the cells were clustered
around the disappearing ganglion-cells, the process being not unlike a neuro-
phagia similar to that described by McKinas in poliomyelitis; while the infil-
tration in my cases was purely a chronic inflammatory process. As in the
animals poisoned by snake venoms, we also found, after injection of heloderma
venom, affections of the various cranial nuclei. In contradistinction to Lamb
and Hunter’s observations, we could not observe any special selective action
of the venom on the third and fifth nuclei; in our entire series no preference
could be detected for any one of the nuclei of the cranial nerves.

The changes in the ependymal cells were similar to those seen by previous
authors in the case of snake venoms. A vacuolation of the cells was noted and
the canal contained an excess of granular substance. In those cases in which
hemorrhage occurred in the white substance (guinea-pig A! and rabbit a°) a
similar collection of red blood-corpuscles was observed in the canal.

The results obtained with the Marchi method were negative; no changes
were found in the spinal cord of the animal living for 13 days. No changes in
the myelin or in the axis cylinder were to be noted.

HISTOLOGICAL CHANGES OF CENTRAL NERVOUS SYSTEM. 141

PROTOCOLS.

Guinea-pig No. 16.—25 mg. injected; dead in 8 minutes. Spinal cord and
brain placed immediately in 10 per cent formalin. Sections embedded in cel-
loidin. Stained with thionin (Nissl method). Examination of the medulla
and spinal cord showed no changes in the ganglion-cells. The vessels were
unaltered.

Guinea-pig No. 21.—20 mg. injected; dead in15minutes. Brain and spinal
cord placed immediately in 10 per cent formalin. Sections embedded in cel-
loidin. Stained with thionin. Examination of sections of the medulla and
spinal cord taken at various levels showed no alteration in the cells or blood-
vessels.

Guinea-pig No. 22.—20 mg. injected, dead in 30 minutes. Brain and spinal
cord placed immediately in 10 per cent formalin. Portions of medulla and
spinal cord were embedded in celloidin. Sections were cut at various levels
and stained with thionin. Microscopical examination showed no changes in
the cells or blood-vessels.

Guinea-pig No. 18.—25 mg. injected, dead in 45 minutes. Brain and spinal
cord were placed immediately in 10 per cent formalin. Portions of the tissue
were embedded in celloidin, cut, and stained with thionin. Some of the vessels
in the medulla were dilated and congested. These vessels lie just beneath the
fourth ventricle. Some of the cells of the spinal cord (anterior horn) and
medulla (9th, 10th) took the stain a little more intensely.

Rabbit No. 1—15 c.c. of venom injected; dead in 1 hour 15 minutes.
Brain and spinal cord placed immediately in 10 per cent formalin. Portions of
tissue were embedded in paraffin. The spinal cord, medulla, and cerebellum
and pons were cut longitudinally. Sections were stained with thionin. Slight
changes were noted in the cells of the spinal cord (anterior horn cells), the
Purkinje cells in the cerebellum, and in a few cells in the nuclei of the cranial
nerves. Owing to the manner of cutting the sections the identification of the
cranial nuclei could not be assured, but it was evident that the toxin had no
selective action, for cells were affected in the various groups of ganglion-cells
throughout the medulla and pons. The changes to be noted were an early
disintegration of the Nissl bodies into a fine powder, starting around the nucleus
and proceeding to the periphery, a migration of some of the nuclei to the per-
iphery, and a slight swelling of the cells. These changes were to be noted in
only a few of the cells; the majority were normal.

Rabbit a?.—25 mg. of venom injected; dead in 2 hours. Tissues hardened
and embedded as in rabbit No. 1. The changes in the cerebellum, pons, and
medulla were similar to those noted in rabbit No. 1, save that in the medulla
numerous small hemorrhages were found.

Guinea-pig A;.—January 7, 1909, 1 capsule with 0.5 c.c.; January 8, 1909,
5 p. m., dead. Died in 36 hours. Convulsions were noted, together with
weakness and paralysis. Tissues and sections prepared asabove. Portions
were taken for study from several areas of the cerebrum, mid-brain, pons,
cerebellum, medulla, and spinal cord. Many sections were cut from these
areas. Some were stained with thionin and others with hemalum.

Spinal cord: Sections from the cervical region showed swelling of the cells
in the anterior horn, a loss of staining reaction in the Nissl bodies, and dis-
placement of the nuclei. Hemorrhages were to be noted above and below the
central canal.

Sections from the thoracic region showed similar changes in the cells and
marked congestion of the vessels throughout the white substance.

Medulla: Sections through upper and lower levels. The changes in the
ganglion-cells were chromatolysis, swelling of the cells, and displacement of the
nuclei. The ganglion-cells of the cranial nuclei seemed equally affected.

142 THE VENOM OF HELODERMA.

Pons: Similar changes were noted in the ganglion-cells of this region.

Mid-brain and basal ganglion: The cells in this region showed a similar
picture, though not as many were affected.

Sections studied from various areas of the cerebral cortex showed little
change.

Guinea-pig A,.—24 mg. of venom in capsule. Dead in 48 hours. Sec-
tions of the medulla only studied. Quite a few of the ganglion-cells showed
loss of staining power, indicative of a chromatolysis. The nuclei of the fifth,
seventh, ninth, and tenth were affected alike.

Rabbit B,.—2 capsules each 30 mg. placed in the peritoneal cavity. Sec-
tions fixed and stained as before.

Medulla: Chromatolysis of the Nissl bodies and displacement of the nuclei
were seen in some of the ganglion-cells of the cranial nuclei. As these sections
were cut longitudinally, the orientation of the various cranial nuclei could
not be made out with certainty.

Cerebral cortex: Many of the Betz cells show breaking of the Nissl bodies,
displacement of the nuclei, and shrinkage of the cell-body.

Rabbit B?:—2 capsules, 30 mg. each, in the peritoneal cavity. Killed at
the end of 13 days. Sections fixed in formalin and in Muller’s fluid.

Spinal-cord sections stained with picric acid showed no changes.

Medulla: Sections stained by the Nissl method show the ganglion-cells of
the tenth, ninth, seventh, and fifth cranial nuclei affected. Not all the cells
were involved. There was loss of staining power in these cells, although their
outlines were well defined. Shadow cells were seen with nuclei faintly present.
A slight proliferation of connective tissue was noted in one area around the
posterior septum in the medulla.

Rabbit a?:—4 capsules (1 capsule 50 mg., 3 capsules 0.3 c.c.) given during
a period of 28 days. Tissues placed in formalin 3 hours after death. In the
medulla the cranial-nerve ganglion-cells showed some loss of staining power of
the Nissl bodies. The fifth in this case seemed less involved than the tenth,
ninth, and seventh. A few cells showed vacuolation and displacement of the
nuclei. The vessels in the medulla were distended and congested. Slight
round-cell accumulation was seen in the perivascular areas.

VII.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM.

By ExizapetaH Cooke anp LrEo LOEB.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM.

By Exizaseta Cooxr anp Leo Lorn.

HELODERMA VENOM HEMOLYSIS.

The venom of Heloderma bears in certain respects a resemblance to various
snake venoms in which observers have shown more or less strong hemolytic
properties. Van Denburgh and Wight* in a small series of experiments tested
the hemolytic action of heloderma venom; they used the diluted fresh venom
with washed dog-corpuscles and found that the venom, without any activating
substance, was able to produce hemolysis. We have carried out a large series
of experiments in which we studied the influence of the venom on hemolysis of
various sorts of blood-corpuscles, of the combination of venom with various
substances which are supposed to serve as activators—such as sera of various
animals, lecithin, and sodium oleate—and the influence of various sera in inhib-
iting hemolysis.

HEMOLYTIC INFLUENCE OF HELODERMA VENOM.

Following the discovery of the complex character of snake-venom hemoly-
sis by Flexner and Noguchi,} it was shown by Kyes{ that cobra venom acts
directly on certain blood-corpuscles, while it does not hemolyze certain others.
Thus using twice-washed corpuscles, he found that the corpuscles of the ox,
sheep, and goat were not dissolved by the venom, while those of the guinea-pig,
dog, man, rabbit, and horse were dissolved. Similar results have been obtained
with other snake venoms.

The venom of Heloderma, unlike the venom of Cobra or Daboia, and in
common with certain other snake venoms, does not hemolyze corpuscles of any
of the animals used in our experiments. We tested the corpuscles of the horse,
ox, sheep, dog, rabbit, guinea-pig, turtle, frog, and Heloderma, using both fresh
venom and dissolved dry venom. In the case of the fresh venom we mixed
quantities varying between 0.01 c.c. and 0.25 c.c. with 1 ¢.c. of the corpuscles
(which were always made into a 5 per cent suspension in either 0.85 per cent
or 0.95 per cent sodium-chloride solution, depending upon the corpuscles used).
When the dissolved dry venom was used we mixed quantities of the solution
containing between 0.025 mg. and 0.2 mg. of the venom with the blood-
corpuscles to be tested. The hemolytic test was kept for 2 hours in the
thermostat and then left in the ice-chest over night. A reading of the various
degrees of hemolysis was made on the following morning.

*Van Denburgh and Wight, Trans. Amer. Phil. Soc., 1898, xix, 199; Amer. Journ. Phys., tv, 1900-1901,
{Flexner and Noguchi, Jour. Exper. Med., 1902, v1, No. 3.
{Kyes, Berl. klin. Woch., 1902, Nos. 38 and 39.

145

146 THE VENOM OF HELODERMA.

As stated above, no corpuscles were found to be susceptible to the venom.
An example of one of the experiments with dog corpuscles and fresh venom is
shown in the following protocol:

1c.c. & per cent suspension of dog corpuscles.
Amount of heloderma venom: Amount of heloderma venom:

OL OL C.Ci05 cceenccine 0 0:06 G26 is.< ceca 22a 0
O02. C23 sorea eace 2 os 0 OTe CiCikoecnicnens 0
003. CiGis ies wen uaese 0) Oi VOC iCiss sage cites wed 0
O04 Citic cc awwns eas d 0 2 Co Cie aca ea eonate gees 0
0:05-6:05 os.se05 24008 0 025°C :Cin sang weds 0

In all cases the blood-corpuscles had been washed four times in order to
thoroughly free them of the blood serum.

It may be mentioned that in one case out of seven where guinea-pig cor-
puscles were mixed with a solution of the dry venom containing 0.2 mg. of
venom, a trace of hemolysis appeared. It seems most probable that in this
case we were dealing with some exceptional condition and that either the resist-
ance of the corpuscles was less than normal or that the corpuscles had been
injured in some manner.

INFLUENCE OF THE ADDITION OF LECITHIN TO HELODERMA VENOM ON
THE HEMOLYSIS OF VARIOUS CORPUSCLES.

Kyes has shown that the addition of a solution of different commercial
lecithins to cobra venom leads to the hemolysis of ox, sheep, and goat corpus-
cles, which are normally resistant to hemolysis by venom alone.

We have used three different specimens of lecithin, Agfa lecithin, Kahl-
baum’s lecithin aus eigelb, and a preparation of lecithin obtained through the
kindness of Dr. Waldemar Koch, of Chicago. The lecithin was purified by
dissolving it in ether, from which it was precipitated by the addition of a large
quantity of acetone. This precipitated lecithin was then dissolved in methyl
alcohol in the proportion of 1 gram of lecithin to 100 grams of methyl alcohol.
From this stock solution of lecithin (1 per cent solution) a more dilute solution
was made; 1 c.c. of the 1 per cent methyl-alcoholic lecithin solution was mixed
with 99 c.c. of 0.85 per cent or 0.95 per cent sodium-chloride solution. The
suspension contained 0.0001 gm. of lecithin in every cubic centimeter.

A few preliminary experiments were made to compare the three specimens
of lecithin. In these experiments guinea-pig corpuscles were used.

Hemolysis of guinca-pig corpuscles by lecithin.

[1 c.c. of 5 per cent suspension of guinea-pig corpuscles.)

Amt. of Kahl- . | Amt. of . Agfa | Acta (un-
lecithin, Asia. | baum. ie | iecithin. | Chic920- (gitered)., filtered).
| | i |
i " | i
mg. mg.
0.1 | Total. Total. Total. | 0.05 xX x | x
0.05 xx xx xx 0.025 o | 6
0.025 | 0 x x |

We found the Chicago and Kahlbaum lecithin possessed distinctly stronger
hemolytic properties than the Agfa lecithin. Furthermore, filtering the solu-
tion does not appear to diminish its hemolytic properties.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 147

In another series of experiments we tested the activating properties of the
various specimens of lecithin. A combination of the lecithin and venom was
added to the washed guinea-pig corpuscles. The mixture was kept in the
thermostat for 2 hours and left in the ice-chest over night.

Hemolytic action of heloderma venom and lecithin.

{1 c.c. of 5 per cent suspension of guinea-pig corpuscles, 0.1 mg. of heloderma venom.]

Amt. of Kahl . Amt. of = Agia | Agfa (un-
iecithin. | Al- | baum. | Chicago || fecithin, | Chicae>- |(gitered).| filtered).
|

mg. mq.

0.05 Total. | Total. | Total. || 0.05 Total. | Total. | Total.

0.02 | xx | Total. | Total. |} 0.025 | Total. | xx “x

0.0125 o | x x 0.0125 x 0

0.0085 | 0 x 6 |) 0.00065 ) 0 0
|

We found the combination of lecithin and venom produced hemolysis of
the guinea-pig corpuscles. The venom alone caused no hemolysis, and the
lecithin alone in quantities of either 0.025 mg. of the Agfa lecithin, or 0.0125
mg. of the Chicago or the Kahlbaum product were without effect, yet the
combination of these substances produced a hemolysis which appears to be
analogous to that produced by the cobra-venom-lecithin combination. It may
be noted that in our experiments it was necessary to add to the venom a quan-
tity of lecithin equal to half as much as the hemolytic dose of lecithin when not
mixed with venom, in order to hemolyze the guinea-pig corpuscles, while Kyes
states that 55 of the hemolytic dose of lecithin when added to cobra venom
was sufficient to activate the hemolysin. In many other cases, however, we
found that one-quarter or even one-tenth of the hemolytic dose of lecithin was
sufficient to hemolyze the blood corpuscles when combined with heloderma
venom.

Having found that the combination of lecithin with venom produced
hemolysis, we studied the effects of this combination on eight kinds of blood-
corpuscles, namely, those of the ox, sheep, dog, rabbit, guinea-pig, turtle, frog,
and heloderma. In the majority of these experiments Agfa lecithin was used
as activator.

Ox CorPUSCLES.
In these experiments we used fresh venom as well as the dissolved dry

venom.
[2 c.c. of 5 per cent suspension of ox corpuscles.]

Lecithin + Lecithin +
Amt. of sane s Amt. of saps
lecithin, Lecithin. ues fag fecithin. Lecithin. 0.1 mg.
. venom.
mg. mg.
0.4 Total. Total 0.1 x Total.
0.2 XXX Total 0.05 x xx
0.1 xx Total. 0.02 0 x
0.05 x xXx 0.01 0 0
0.02 0 xX 0.004 0 0
0.0125 0 x 0.002 0 0
0.006 0 0 0.001 0 0

From these experiments it appears that the fresh venom hemolyzes the ox
corpuscles with a trifle less lecithin than the dissolved dry venom. Further-

148 THE VENOM OF HELODERMA.

more, in the experiments with the fresh venom the addition of a quantity equal
to only one-quarter the hemolytic dose of lecithin was needed in order to cause
hemolysis, while with the dissolved dry venom a quantity equal to two-fifths of
the hemolytic dose of lecithin was added.

We also tested the hemolytic doses of venom and lecithin with a constant
quantity of lecithin and variable doses of venom, and found that when minute
quantities of venom were added to a quantity of lecithin equal to two-fifths of
the hemolytic dose, the ox corpuscles were hemolyzed.

2 e.c. of F per cent suspension of ox corpuscles.
[Lecithin, 0.02 mg. added to cach tube.]

Fresh venom. | Dry dissolved venom.
|.
04 Ge...... Total. 0.2 mg
0.2 Cc . Total. 0.1 mg
0.1 cc xXx 0.05 mg
0:09: Gases oss xx 0.02 mg
0.025 cc ........ Kx 0.01 mg
LOU x ee x 0.004 mg
0.006 ce... le. x 0.002 mg .. .......

SHEEP CORPUSCLES.
A similar series of experiments was carried out with sheep corpuscles.

2 c.c. of & per cent suspension of sheep corpuscles.

Lecithin || Lecithin Lecithin
Amt. of eas | Amt. of are Amt. of eyes
pen Lecithin. | +0.05 e.c.!! “ibs Lecithin. |+0.1 mg. woe Lecithin. | +-0.2 mg.
lecithin. venom. | lecithin. venom, |} lecithin. cename
|
mg. | mg. mg.
0.4 XXX | Total | 0.1 0 xX 0.5 XXX | Total.
0.2 xXx Total. 0.05 0 x 0.2 xXx Total.
0.1 xX xxx 0 02 0 0 0.1 x Total
0 05 x xxx 0.01 0 0 0.05 0 xXxXX
0.025 0 x 0.004 0 0 0.02 0 xx
0.0125 0 x 0.002 0 0 0.01 0 x
0.006 0 0 | 0.001 0 0

From these experiments we may conclude that, to lecithin alone, the sheep
corpuscles are more resistant than the ox corpuscles, and that the latter are
more resistant than the guinea-pig corpuscles.

When venom and lecithin are mixed the sheep corpuscles are hemolyzed
by quantities of lecithin smaller than the hemolytic dose of unmixed lecithin.
Thus with fresh venom we obtained hemolysis of the corpuscles with a quantity
of lecithin equal to one-quarter the usual hemolytic dose; with the dissolved
dry venom (0.2 mg.) about one-tenth the usual hemolytic dose of lecithin
was required.

2 c.c. of & per cent suspension of sheep corpuscles, lecithin 0.0.2 mg. added to each tube.

T'resh venom, | Dry venom.

O82 MPS as seed 0
OL. SMBs agar sna 0
0.05 mg........... 0
202° AINE oss sincs agcean 0
0.01 MEwsccsicosces 0
0.004 mg... 0
0.002 mg........... 0

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 149

With a constant relatively small quantity of lecithin relatively very large
doses of venom are required in order to cause hemolysis—much larger doses
than were required for the hemolysis of ox and guinea-pig corpuscles; while
with a constant dose of venom more lecithin is required for the hemolysis of
sheep than of ox corpuscles. In a combination of lecithin and venom more of
either of these two substances is required for the hemolysis of ox than of
guinea-pig corpuscles.

Doc CorpPuscLes.

2c.c. of & per cent suspension of dog corpuscles.

Lecithin+
Amt. of “ays
rie Lecithin. | 0.05 mg.
lecithin. venom.
mg.
0.6 Total. Total
04 Total. Total
0.2 xXx Total
0.1 x Total
0.05 0 XKX
0.025 0 xx
0.0125 0 0

In spite of the relatively small quantity of venom used, the addition of
one-quarter of the hemolytic dose of lecithin to the venom is sufficient to cause
hemolysis of dog corpuscles.

When the quantity of venom used was variable and only small quantities
of lecithin were used, very small quantities (0.006 mg.) of venom were sufficient
to cause partial hemolysis of the dog corpuscles, indeed somewhat less than was
necessary to cause partial hemolysis of ox corpuscles (0.01 mg.), and very much
smaller quantities than caused hemolysis with sheep corpuscles (0.2 c.c. fresh
venom, none with 0.2 mg. dry venom).

2 c.c. of 5 per cent suspension of dog corpuscles.

Venom+ Venom+ Venom-+ Venom+

Amt. of | 0.02 mg. | 0.01mg. || Amt cf | 002mg. | 0.01 mg.

: lecithin. lecithin. : lecithin. lecithin.
0.6 Total. xx 0.025 xx x
0.4 xxx xx 0.0125 x x
0.2 xxXxX xx 0.006 x x
0.1 xx x 0.003 0 0
0.05 xx x 0.0015 0 0

Rassit CorPuscLEs.
2c.c. of 6 per cent suspension of rabbit corpuscles.

The reaction of the corpuscles to the venom and lecithin varied consider-
In certain cases, indeed, in most cases, the corpuscles were resistant to

ably.

Lecithin Lecithin Lecithin
Amt. of wane Amt. of wipe Amt. of sans
aries Lecithin. | +0.2 mg. sate Lecithin. | +0.2 mg. wats Lecithin. | +0.1 mg.
lecithin. L eioti lecithin. venom, || lecithin. oe
mg mg. mg.
0.5 XXX Total. 0.02 0 x 0.2 0 0
0.2 x xxXX 0.01 0 x< 0.1 0 0
0.1 x XXX 0.05 0 0
0.05 0 xx 0.02 0 0
0.01 0 0

150 THE VENOM OF HELODERMA.

the lecithin hemolysis and in such cases we were also unable to produce hemoly-
sis with the venom-lecithin mixture. However, in one case in which the cor-
puscles were less resistant than usual, we found that the combination of venom
and lecithin caused hemolysis of the corpuscles even if a quantity of lecithin
equal to only one-tenth the dose hemolytic for rabbit corpuscles was added.
The lecithin activates the venom for rabbit corpuscles as it does for ox, sheep,
and dog corpuscles.

In testing the hemolytic action of a constant quantity of lecithin and a
diminishing quantity of venom we were unable to overcome the great resist-
ance of the rabbit corpuscles, and were thus unable to carry out satisfactory
experiments.

GuINEA-Pi1G CorRPUSCLEs.

In our experiments, the guinea-pig corpuscles were fairly resistant to
hemolysis by lecithin alone, 0.1 mg. of lecithin being required for partial hem-
olysis. If, however, lecithin was added to 0.1 mg. of heloderma venom the
corpuscles were hemolyzed after addition of only 0.02 mg. of lecithin, only one-
fifth of the usual hemolytic dose.

2c.c. of & per cent suspension of guinea-pig corpuscles.

Amt. of

lecithin. Lecithin. | +0.1 mg.

mg.
0.4 XXX Total
0.2 XxX Total.
0.1 x xXx
0.04 0 xX
0.02 0 x
0 008 0 0
0.004 0 0

With a constant quantity of lecithin and diminishing quantities of venom,
very small quantities of venom were able to cause hemolysis; even as little as
0.01 mg. of venom, added to the same quantity of lecithin, was sufficient to
cause hemolysis. It will be seen that in these experiments the degree of hemo-
lysis depended largely upon the amount of lecithin, while, for instance, 0.4 mg.
of venom plus 0.01 mg. of lecithin caused only moderate hemolysis, a similar
quantity of venom added to 0.1 mg. of lecithin (which of itself causes a trace
of hemolysis) caused complete hemolysis of the corpuscles.

2 c.c. of 5 per cent suspension of guinea-pig corpuscles.

Arnicoe Venom-+ | Venom+ | Venom+
SanO ii lecithin lecithin lecithin
‘ 0.1 mg. 0.04 mg. 0.01 mg.

3
=

q

0.04 Total. xxx xx
0.2 Total. XXX xx
0.1 xxx xx xx
0.05 xx Noe Xx
0.025 xx x x
0.01 xX x x
he x 0 0

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 151

TurtTLE CoRPUSCLES.

Turtle corpuscles were distinctly resistant to the hemolytic action of
lecithin; 20 mg. of lecithin were not sufficient to cause hemolysis of the turtle

corpuscles.
2 c.c. of & per cent suspension of turtle corpuscles.

Lecithin+ | Lecithin+ Lecithin+ | Lecithin+
cae Lecithin. fresh venom,| dry venom, anes Lecithin. |fresh venom | dry venom,
soryaMl: 0.015c.c. | 0.5 mg. ; : | 0.015 c¢.c. | 0.5 mg.
mg. mg.
20 Ot eszawe P| teense 0.25 0 Total. Total
8 0 Totals |) ssesises 0.125 0 Total. Total
6 0 Total. | ...... 0.06 0 Total Total
4 0 Total. |) snesee 0.03 0 Totals. | sieieisis
2 0 Total. | ...... 0.015 0 pa Weer
1 0 Total. | ...... 0.0075 0 OP Sree
0.5 0 Total. Total |

If, on the other hand, much venom (0.5 mg. of dry venom or 0.015 c.c. of
fresh venom) was mixed with lecithin the turtle corpuscles were hemolyzed
with very small amounts of lecithin. Thus the addition of 0.015 mg. of leci-
thin to the fresh venom was sufficient to cause slight hemolysis; while 0.06 mg.
of lecithin, the smallest quantity of lecithin added to dry venom, caused
complete hemolysis of the turtle corpuscles. Simultaneously with these exper-
iments with fresh venom a similar series with guinea-pig corpuscles was carried
out, our object being to compare the susceptibility of turtle and guinea-pig
corpuscles to the mixture of lecithin and venom. While turtle corpuscles were
distinctly more resistant to the action of lecithin alone, we found that the
guinea-pig corpuscles and turtle corpuscles were hemolyzed by nearly the same
amounts of venom and lecithin. (In this experiment turtle corpuscles were
moderately hemolyzed by a mixture containing 0.01 c.c. of fresh venom and
0.06 mg. of lecithin, while guinea-pig corpuscles were practically completely
hemolyzed by a similar mixture of these substances.)

Frog Corpuscles.

Only one experiment was carried out with frog corpuscles, and in this
experiment we tested the influence of 0.1 mg. of venom to which lecithin was
added and found that as little as 0.01 mg. of lecithin would activate this quan-
tity of venom.

1 c.c. of 5 per cent suspension of frog corpuscles.

Lecithin+-
Amt.of | Lecithin. | 0.1 mg. of
‘ venom.

0.05 0 XXX
0.03 0 xx
0.01 0 xx

HELODERMA CORPUSCLES.

Heloderma corpuscles, like turtle corpuscles, are resistant to hemolysis by
lecithin; 45 mg. of lecithin are required to hemolyze 1 ¢.c. of heloderma cor-

152 THE VENOM OF HELODERMA.

puscles. In testing the influence of venom plus lecithin on the heloderma
corpuscles we used corpuscles from normal animals as well as from those whose
venom glands had been removed some time previously. At the same time a
comparison was made with guinea-pig corpuscles.

Venom, 0.1 mg. in each tube.

| Heloderma| Heloderma
Amt. of Guinea-pig corpuscles | corpuscles
lecithin. | corpuscles, (1 ¢.e. nor- | (1 ¢.c. gland-
| mal). less).
mg. |
30 Total. Total.
20 Total. Total.
10 Total very rapid. Total. Total
5 Total xx
1 } xx
1

From these experiments it may be seen that the heloderma corpuscles are
very much more resistant to hemolysis by venom and lecithin than are guinea-
pig corpuscles, and (in one experiment) that the corpuscles of the glandless
animal were somewhat more resistant than those of the normal heloderma.

In another experiment we found the heloderma corpusclesmuch more sen-
sitive to the hemolytic action, by the addition of small quantities of lecithin,
to both fresh and dissolved dry venom. We found the addition of 4) of the
usual hemolytic dose of lecithin to a small quantity of venom (0.001 c¢.c. or 0.1
mg.) was sufficient to cause hemolysis of 1 c.c. suspension of the heloderma
corpuscles. The heloderma corpuscles are not resistant to heloderma venom-
lecithin hemolysis.

1 c.c. of 5 per cent suspension of heloderma corpuscles (normal).

Venom+ Venom+
Amt. of | Jecithin, | lecithin,

0.1 mg. 0.2 mg.
0.003 c.c. Total. Total.
0.002 ¢.c. Total. Total.
0.001 c.c. Total. Total.
0.3 mg. Total. Total.
0.2 mg. Total. Total.
0.1 mg.| Total. Total.

SuMMaARY.

Lecithin activates venom in the case of all corpuscles used in these experi-
ments, namely, corpuscles of ox, sheep, dog, rabbit, guinea-pig, turtle, frog, and
Heloderma. The susceptibility of the various corpuscles toward the venom-
lecithin mixtures varies, but in general the corpuscles of the warm-blooded ani-
mals are hemolyzed if a quantity of lecithin equal to from one-half to one-tenth
of the hemolytic dose of lecithin is added to the venom, while the corpuscles
of the cold-blooded animals are hemolyzed by the addition to the venom of
a quantity of lecithin equal to x to a of the hemolytic dose of lecithin.
Since the quantity of lecithin which must be added to venom in order to cause
hemolysis varies only within a relatively small range in the case of various cor-

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 153

puscles, whether of warmor cold blooded animals, the apparent difference in the
activating power of venom in the case of warm and some cold-blooded animals
is due to the greater resistance of the corpuscles of some of the cold blooded
animals to the lecithin. With a constant quantity of venom an increase in the
amount of lecithin causes an increase in hemolysis, while with a constant quan-
tity of lecithin an increase in the amount of venom causes an increase in
hemolysis.

Tn order to accomplish in both cases the same amount of increase in hemo-
lytic effect it seems to be necessary to increase the amount of venom to a greater
extent if the lecithin is kept constant than to increase the amount of lecithin if
the venom is kept constant. The sensitiveness of various corpuscles of warm
blooded animals toward the venom-lecithin mixture differs somewhat, guinea-
pig and dog and ox corpuscles being somewhat more sensitive than rabbit
and sheep corpuscles. An immunity of the heloderma corpuscles toward the
hemolysis by their own venom does not exist, although the blood-corpuscles of
Heloderma were found in a number of experiments to be somewhat more
resistant than the corpuscles of other animals tested.

INFLUENCE OF THE ADDITION OF SODIUM OLEATE TO VENOM ON THE
HEMOLYSIS OF VARIOUS CORPUSCLES.

Noguchi* and Von Dungern and Cocat have shown that sodium oleate
combined with cobra venom acts in a manner similar to lecithin, in that it is
able to activate the cobra venom. Since we have found that lecithin was able
to activate the heloderma venom for the various kinds of corpuscles, it was of
interest to determine whether sodium oleate would also act as an activator for
this venom.

We used for this purpose a Merck & Co. preparation of sodium oleate in a
0.1 per cent solution in 0.85 per cent sodium chloride.

Ox CoRPUSCLES.

We found that1c.c. of a 5 per cent suspension of ox corpuscles was easily
hemolyzed by 0.002 gm. sodium oleate; 0.01 mg. of sodium oleate when added
to 0.5 mg. of venom was sufficient to cause total hemolysis of the corpuscles.
Addition of venom diminishes, therefore, 200 times the hemolytic dose of
sodium oleate.

1 c.c. of & per cent suspension of ox corpuscles.

. Sodium . Sodium
Quantity of oleate-+ Quantity of oleate
et quantity of Result. pie eens of Result.
: venom. i venom.
gm. mg. gm. mg
0.01 etic Total, 0.00001 1.0 Total
0.005 sahoge Total. 0.00001 0.5 Total
0.002 ence Total. 0.00001 0.25 xxx
0.001 ee XXX 0.00001 0.1 x

*Noguchi. Jour. Exper. Med., 1907, rx, 436. TV. Dungern and Coca. Berl. klin. Woch., 1908, p.348.

154 THE VENOM OF HELODERMA.

SHEEP CORPUSCLES.

Sheep corpuscles which react to sodium oleate in the same manner as ox
corpuscles, being dissolved by 0.002 gm. of sodium oleate, were not hemolyzed
by the mixtures of venom and sodium oleate used in our experiments. We
have tested 0.01 mg. of sodium oleate plus 1 mg. of venom, but found no hemo-
lysis. It is apparent that sheep corpuscles are more resistant to the action of
venom plus sodium oleate than are the ox corpuscles.

Doa CorPUSCLES.

Dog corpuscles are hemolyzed by 0.4 mg. of sodium oleate but not by 0.2
mg. of this substance. The smaller quantity, namely, 0.2 mg., of sodium
oleate is able to produce hemolysis after the addition of venom.

1 c.c. of & per cent suspension of dog corpuscles.

Sodium Venom +
cine Sodium oleate ++ Amt. of sodium
‘olaxte oleate. venom venom. oleate.
. 0.05 mg. 0.2 mg.

mg. mg

1 Total. 0.4 xX

0.7 Total. 0.2 x

0.5 Total. 0.1 0

0.4 Total a4 0.05 )

0.2 9 xx

0.1 0 0

0.05 0 0

With dog corpuscles sodium oleate has only a slight activating power for
the venom, as it is necessary to add half or more than half the hemolytic dose
of sodium oleate to the venom in order to produce hemolysis.

Rassit CorPuscles.

Rabbit corpuscles are hemolyzed by 0.5 mg. of sodium oleate and the
quantity of this substance which must be added to the heloderma venom in
order to produce hemolysis is more than half of this quantity. Again the acti-
vating property of the sodium oleate is very slight.

GuinEA-Pia CorPUSCLES.

Guinea-pig corpuscles are slightly hemolyzed by 0.125 mg. of sodium oleate.
In the mixture of venom and sodium oleate about half as much sodium oleate
is necessary to hemolyze the corpuscles.

1 c.c. of 5 per cent suspension of guinea-pig corpuscles.

. Venom
Amt. of F Sodium Amt. of bel
: Sodium oleate +
sodium oleate venom venom. oleate,
oleate. , 0.125 mgs 0.125 mg.
E mg
mg. 2 Total
1 Total. Total. 1 Total
0.5 Total. Total. ee Total
0.25 XXX Total. ee xx
0.125 x xxx «125 XX
0.06 0 XXX 0.06 Xx
0.03 0 0 0.03 x

Control, 0.125 mg.; sodium oleate, X.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 155

The hemolysis caused by 0.125 mg. of sodium oleate is less than the hemo-

lysis caused by a similar quantity of sodium oleate plus venom.
TurtLe CoRPUSCLES.

The turtle corpuscles react to sodium oleate in a manner similar to the
guinea-pig corpuscles; with 0.1 mg. of sodium oleate there is slightly less hemo-
lysis of the turtle corpuscles than of the guinea-pig corpuscles; as was shown in
parallel series carried out with guinea-pig and turtle corpuscles with similar
quantities of sodium oleate or mixtures of sodium oleate and venom.

1 c.c. of & per cent suspension of turtle corpuscles.

A f ere Al f Sire
mt. 0 . oleate + mt, o} ; oleai
sodium pr venom, 0.02 sodium pie venom, 0.02
oleate. ®- | c.c. (fresh || oleate. * | ee. (fresh
venom). venom).
mg. mg
0.4 Total. Total 0.06 0 x
0.3 Total. Total 0.05 0 0
0.2 Total Total 0.04 0 0
0.1 x xxx 0.03 0 0
0.09 XXX 0.02 i) 0
0.08 0 xXx 0.01 0 0
0.07 i) x

After addition of venom the quantity of sodium oleate necessary to cause
hemolysis was a little morethan half the usual hemolytic dose, whereas a some-
what smaller dose of sodium oleate when added to the venom suffices to hemo-
lyze the guinea-pig corpuscles.

SUMMARY.

Sodium oleate serves as an activator for the heloderma venom, but the
difference between the hemolytic dose of the sodium oleate alone and the acti-
vating dose of sodium oleate is very small. Addition of venom to sodium
oleate diminishes the hemolytic dose of the sodium oleate approximately one-
half in the case of the majority of corpuscles. There exists, therefore, an acti-
vating power of venom when combined with sodium oleate, but it is consider-
ably smaller than in the case of lecithin.

ACTIVATION OF HELODERMA VENOM BY VARIOUS SERA.

Flexner and Noguchi,* who discovered that addition of serum to snake
venom made the latter hemolytic, were inclined to identify the ordinary serum
complement with the activating substance. Later the investigations of Kyest
and Calmettet suggested lecithin as the source of the venom-activating sub-
stance. The subsequent studies of Kyes and Sachs,§$ however, made it very
probable that besides the lecithin some other activating substance was present
in various sera.

We have carried out a large series of experiments in which we tested the
power of the various sera to serve as activators of the heloderma venom, and

*Flexner and Noguchi. Jour. Exper. Med. 1902, v1, No. 3.
tKyes. Berl. klin. Woch., 1902, Nos. 38 and 39.

tCalmette. C. R. Acad. Sci., 1902, 134, 1446.

§Kyes and Sachs. Berl. klin. Woch., 1903, xu, Nos. 1, 2, and 3.

156 THE VENOM OF HELODERMA.

in most cases we tested the action of the combination of the various sera with
venom on the corpuscles of different animals.

We have found that the serum of the guinea-pig, rabbit, sheep, ox, Helo-
derma, or frog will not serve as activators for the heloderma venom.

The guinea-pig serum has been tested with washed sheep, dog, rabbit, and
guinea-pig corpuscles, but failed to hemolyze any of these corpuscles with or
without venom. Guinea-pig serum if added to ox corpuscles, either with or
without venom, caused a minute trace of hemolysis in both series of tubes; no
distinct difference could be detected between the hemolysis in the tubes con-
taining serum alone and the combination of serum and venom; guinea-pig
serum did not therefore serve as an activator for the venom.

Rabbit serum also was tested with rabbit, guinea-pig, ox, sheep, and dog
corpuscles. With rabbit and guinea-pig corpuscles, no, or only a trace of,
hemolysis was observed either with rabbit serum alone or with rabbit serum
and venom. With ox and sheep corpuscles the rabbit serum alone, as well as
the combination of serum and venom, produced slight hemolysis, which was,
however, as marked in the control tubes as in the venom-serum tubes. In one
case rabbit serum combined with the venom produced more hemolysis of dog
corpuscles than the same quantities of the serum alone. In other cases, how-
ever, the rabbit serum, when combined with venom, did not hemolyze the dog
corpuscles, so that in this isolated case, when rabbit serum seemed to serve as an
activator for the venom, some accidental circumstances must have been active.

Sheep serum was tested with guinea-pig, rabbit, dog, sheep, and ox cor-
puscles. The ox corpuscles were slightly hemolyzed by sheep serum alone, as
well as in combination with venom, but no difference was found between the
degree of hemolysis in the two series of tubes. With the other corpuscles
neither the sheep serum alone nor the mixture of sheep serum and venom pro-
duced hemolysis.

Ox serum was tested with the same corpuscles, namely, guinea-pig, rabbit,
dog, sheep, and ox; it showed more marked hemolytic power than other serums,
but did not activate the venom.

Frog serum was tested only with frog corpuscles, but did not cause hemo-
lysis in combination with venom.

Heloderma serum was also tried with guinea-pig or heloderma corpuscles,
and neither alone nor mixed with venom produced hemolysis. When helo-
derma serum was added to rabbit corpuscles, either alone or in combination
with venom, the same degree of hemolysis occurred in both sets of tubes.

We may therefore conclude that the sera of guinea-pig, rabbit, ox, sheep,
frog, or heloderma do not serve as activators for the heloderma venom. Cal-
mette found in the case of cobra venom that unheated sera could not activate
venom, but when sera was heated to a temperature above 62°C. it would
activate venom.

We therefore tested 0.1 and 0.2 c.c. guinea-pig and rabbit serum heated
to 63° C. for 30 minutes in combination with 0.1 and 0.2 mg. venom and 2 ¢.c.
of a 5 per cent suspension of rabbit and guinea-pig corpuscles. In no case did

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 157

the heated serum show any activating power. We may therefore conclude
that serum heated above 62° C. does not become an activator in the case of
heloderma venom.

Dog serum was found to activate the heloderma venom. With ox and
sheep corpuscles the activating property of the dog serum is slight, since the
quantity of serum when combined with venom necessary to completely hemo-
lyze either ox or sheep corpuscles will by itself cause slight hemolysis of both
kinds of corpuscles.

2 c.c. of 5 per cent sus- 2 c.c. of & per cent suspen-
pension of ox corpuscles, sion of sheep corpuscles.
| Venom+ | Venom
| Amt. of | 0.1 c.c. dog | 1 Amt. of 0.1 c.c. de
| MaNOD | serum. { venoms serum.
oo cae Os ee ete |
i | !
| | |
mg. i" | mq.
i 0.2 ' Total. ‘ 0.2 Total.
| O01 | Total. | (0.0 | Total.
: 0.05 | Total. i 0.05 Total.
| 900 | x | I | gep4 | xX
|
1 i}

In the case of dog corpuscles the results are more striking, since a quantity
of dog serum equal to half the hemolytic dose for dog corpuscles was sufficient
to activate the heloderma venom.

2 c.c. of 5 per cent suspen- 2 c.c. of 5 per cent suspen- 1 c.c. of 5 per cent suspension
sion of dog corpuscles. sion of rabbit corpuscles. of heloderma corpuscles.
Venom + | Venom + Venom + Venom +
Bape ot 0.1 ¢.c. dog | 0.2 c.c. dog Amt, oF 0.1 c.c. of Amt. of 0.2 e.c. dog
e serum. serum. * | dog serum. venoms serum.
ma mg ma
0.5 Total. Total. 0.5 Total. 0.3 Total.
0.25 Total. Total. 0.2 Total. 0.2 Total.
0.125 Total. Total. 0.1 Total. 0.1 Total.
0 06 Total. Total. 0.05 0 0
0 02 xx Total. ate 9
faa sed 0 Slight.

Rabbit corpuscles appeared to be slightly more resistant than the dog
corpuscles to the hemolytic effect of venom and dog serum combined. The
rabbit corpuscles, like dog corpuscles, were hemolyzed by 0.2 ¢.c. of dog serum.
When the dog serum and venom were combined 0.1 c.c. of the former was suffi-
cient when added to 0.1 mg. of venom to cause hemolysis. If only 0.05 mg. of
venom was added to the dog serum no hemolysis took place.

Guinea-pig corpuscles were completely hemolyzed by 0.2 c.c. of dog serum
alone. When venom and dog serum were combined 0.1 c¢.c. of the latter and
0.02 mg. of venom were sufficient to cause moderate hemolysis, while 0.1 ¢.c. of
serum and 0.06 mg. of venom caused complete hemolysis of guinea-pig cor-
puscles.

Heloderma corpuscles were also hemolyzed by venom and dog serum.
While 0.2 c.c. of dog serum did not hemolyze these corpuscles, this amount of
serum was able when combined with the venom to cause hemolysis of the helo-
derma corpuscles. Smaller quantities of dog serum did not activate the venom
for these corpuscles.

158 THE VENOM OF HELODERMA.

The hemolytic action of dog serum and venom upon turtle corpuscles is
quite variable. In cases in which large quantities of dog serum were added to
venom, the turtle corpuscles were partially hemolyzed, but the same quanti-
ties of dog serum alone also caused hemolysis, although slightly less marked.

1 c.c. of & per cent suspension of turtle corpuscles.

First experiment. Second experiment.
Dog serum Venom +
Ant. of Amt. of
x Dog serum.| +0.1 mg. 0.1 ¢.c. dog
dog serum. wenonk venom. para:
&.0% mg.
0.4 x xx 0.4 0
0.2 x xx 0.2 0
0.1 x x 0.1 0
0.05 x x 0.05 0

In another experiment with corpuscles from another turtle in which dog
serum was combined with diminishing quantities of venom, no hemolysis took

place.
Horse Serum.

Horse serum as well as dog serum serves as an activator for venom. The
action of venom and horse serum was tested upon guinea-pig, rabbit, and dog
corpuscles, with similar results in each case. Horse serum alone has distinctly
less hemolytic activity than dog serum, but it serves equally as well as an
activator for the heloderma venom.

2c.c. of & per cent suspension of guinea-pig corpuscles.

Horse
Amilo t serum+0.1 Horse
sarira meg. of serum.
‘ venom.
Cc.c |
0.5 Total 0
0.3 xXx 0
O.1 xxx 0
0.05 x 0

TuRTLE SERUM.
Turtle serum, like horse and dog serum, activates the heloderma venom.

This was tested on rabbit and guinea-pig corpuscles. When venom was com-
bined with diminishing quantities of turtle serum these corpuscles showed

1 c.c. of & per cent suspension 1 c.c. of 5 per cent suspension
of rabbit corpuscles. of turtle corpuscles.
Amt. of Turtle Amt. of Turtle
turtle serum-+0.1 guis turtle ae serum+0.1
serum. mg. venom. ‘ serum. . mg. venom.
C.c. Ct
0.4 Total 0 0.4 0 0
0.2 XXX 0 0.2 0 0
0.1 xx vy) 0.1 0 0
0.05 xx 0 0.05 0 0

marked lysis. When the activating influence of turtle serum was tested with
turtle corpuscles no hemolysis occurred. Turtle corpuscles seem to be very
resistant to the combination of venom and turtle (or dog) serum.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 159

COMBINATION OF HEATED SERA AND HELODERMA VENOM.

In testing the influence of heat on the activating properties of the various
sera, especially guinea-pig, rabbit, sheep, or heloderma serum, it was found
that 60° C. for 30 minutes did not transform them into activators.

Dog serum, when heated to 56°, 60°, or 70°C. for 30 minutes, lost its
inherent hemolysins, but did not lose its activating property when combined
with venom. It then hemolyzed guinea-pig, horse, dog, and heloderma; the
last named corpuscles were, however, only slightly hemolyzed by this com-
bination. As in experiments with unheated dog serum the addition of heated
dog serum to venom did not hemolyze frog or turtle corpuscles.

Horse serum, when heated to 60° C. retained its action as an activator, and
combined with venom caused hemolysis of horse, dog, rabbit, or guinea-pig
corpuscles. The heated horse serum alone did not cause hemolysis of any of
these corpuscles.

Turtle serum, when heated, was able to activate venom for guinea-pig and
rabbit corpuscles, but not for turtle corpuscles. The quantities of these three
sera that had to be added to venom to cause hemolysis were approximately the
same before and after heating; the heating did not change the activating
properties of these three sera to any marked extent.

Calmette has shown that in conjunction with cobra venom many sera
heated to 62° C. were better able to cause hemolysis of red cells than the un-
heated sera. He believed that the heating destroyed a natural antihemolysin
which was thermolabile, whereas the activating constituent was thermostable.
This activating constituent he considered a “substance sensibilatrice. ”’

Kyes found that heating changed the properties of the activating sera.
Ox serum heated to 56°C. loses its activating power, but when heated to 65° C.
or higher it regains its hemolytic power and is then more powerful than with
the fresh serum. He also found that horse serum serves as an activator when
fresh as well as when heated to 56° or 100° C., and he believed the activating
substance of horse serum was lecithin. Our results make it certain that the
ordinary serum complement is not concerned in the venom hemolysis, but that
a certain thermostable substance already present in the fresh sera is responsible
for the activating action of the sera.

INHIBITORY ACTION OF VARIOUS SERA ON HEMOLYSIS BY HELODERMA
VENOM AND DIFFERENT ACTIVATORS.

We tested the inhibitory action of different sera (horse, guinea-pig, turtle,
heloderma, and rabbit serum), heated and unheated, upon the hemolysis of
erythrocytes of different species by the combination of venom and different
activating substances.

We used horse, guinea-pig, turtle, heloderma, and rabbit serum and the
corpuscles of guinea-pig, horse, Heloderma, turtle, rabbit, and dog. In the
majority of experiments lecithin was the activator used.

Guinea-pig serum has approximately the same action whether heated or
not heated. It inhibits hemolysis of guinea-pig, horse, heloderma, and turtle

160 THE VENOM OF HELODERMA.

corpuscles, but has no inhibitory influence on rabbit and dog corpuscles. In
the case of dog corpuscles, the lysis of the corpuscles appears more slowly in the
tubes to which heated guinea-pig serum ix added thanin those with fresh serum.

When 0.1 mg. of venom mixed with 0.05 mg. of lecithin was added to 2 e.c,
of a 5 per cent suspension of guinea-pig, horse, or 1 ¢.c. of a 5 per cent suspen-
sion of turtle or heloderma corpuscles, hemolysis was complete or nearly com-
plete, but when sufficiently large quantities of guinca-pig serum were added to
these combinations hemolysis was prevented. When, for instance, 0.1 ¢.c. of
guinea-pig serum, cither heated or unheated, was added to mixtures contain-
ing either horse or guinea-pig corpuscles, no hemolysis occurred. The addition
of 0.4 ¢.c. of serum was sufficient to completely prevent hemolysis with turtle
or heloderma corpuscles, while 0.1 ¢.¢. had some inhibitory effect.

Misrtures of 0.1 mg. of venom and 0.1 mg. of lecithin.

2 c.c. 5 per | : ‘Lee. 5 per
Amt. of ‘cent suspen- , > eee 5 per’ Lec. 5 PEL | cent cise,
guinea-pig | sion guinea- | be irae pCenbauspene ion helo-
serum. | pig cor- ton borse sion turtle | derma cor-
puscles. | I egrpuselesy | | corpuscles, puscles
Hidteery eee ee
wm |
ro ‘ F : Ae 0 t
0 6 : | | 0 |
05 0 ' 0 ' i :
U4 0 ( 0
0.3 | o | 0 ss | =
H . i se ‘ é xXx | 3
00 | x ico leas
ow x XXX ee | a
| Total. | Totat. ! Tota. | Total.
1

t

With dog corpuscles no inhibitory action whatsoever could be observed
when either heated or unheated guinea-pig serum was added. The same was
true in the case of the rabbit corpuscles.

No difference existed between the action of heated or unheated guinea-
pig serum in those cases in which an inhibitory action was demonstrated.
Qualitatively and quantitatively their action was the same.

When heated horse serum was used as an activator instead of lecithin,
guinea-pig serum (both heated and unheated) inhibited hemolysis, very mark-
edly with horse corpuscles and slightly less markedly with guinea-pig corpuscles.

Mixtures of 0.1 mg. of venom and 0.1 ¢.c. Mixtures of 0.1 mg. of venom and 0.1 ¢.c.
of heated horse serum. heated dog serum and 2 c.c. of ad per cent
suspension of guinea-pig corpuscles.
2 2 e¢.e. 5 per |
Amt. of c.c. 5 per cent suspen-,
i cent suspen- : Amt. of
euinaap Pi£ | sion of horse ee » guinea pig | Result.
corpuscles: puscles. serum,
| ee,
a] 4 0 | | et &
0.3 0 x | 04 x
0.1 x x . 0.1 Total.
0.05 xx xx 0.05 ‘Total,
0.02 xxx xXX | 0.02 Total.
aisiats Total Total rca ee

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 161

When dog serum heated to 56° C. was used as an activator, the inhibitory
action was not so marked as when lecithin or horse serum was used as an acti-
vator, and we could only partially prevent hemolysis of guinea-pig corpuscles.
In these experiments only heated guinea-pig serum was used.

With sodium oleate as an activator, the guinea-pig serum also inhibited
the hemolytic action. In this case the inhibiting action of guinea-pig serum
was not so strong as with lecithin, more pronounced than with dog serum and
about equally strong with horse serum as an activator.

SUMMARY.

Guinea-pig serum inhibits the hemolysis by lecithin and venom of guinea-
pig and horse corpuscles, and only a little less markedly that of turtle and
heloderma corpuscles. Hemolysis of horse corpuscles by venom and heated
horse serum is also markedly inhibited, while hemolysis of guinea-pig corpus-
cles by similar mixtures is slightly less markedly inhibited. The action of
venom and sodium oleate on guinea-pig corpuscles is considerably interfered
with by guinea-pig serum but the action of heated dog serum and venom is only
slightly affected. On the other hand, guinea-pig serum does not inhibit the
hemolysis of dog corpuscles by venom and lecithin, nor can any influence of
guinea-pig serum be observed when rabbit corpuscles were mixed with venom
plus lecithin or horse serum. Unheated guinea-pig serum acts in the same
manner as heated guinea-pig serum.

Heloderma serum, like guinea-pig serum, has marked inhibitory power,
the unheated and heated serum acting in a similar manner, but the inhibitory
power of the heated serum is, perhaps, slightly stronger than that of the un-
heated serum, as shown in the following :

Mizture of 0.2 mg. of venom plus Mixture of 0.2 mg. of venom and
0.1 mg. of lecithin and 1 c.c. of :0.1 mg. of lecithin.
a 5 per cent suspension of helo-
derma corpuscles.

Amt. of le.c. of a5) 2c.c.ofa5

heloderrsa, per cent per cent
Amt. of Heated Unheated (heated) | SuSPension | suspension
heloderma serurn acu ee turtle guinea-pig
serum. ‘ rum; corpuscles. | corpuscles.

cc. Ce

0.5 0 0 0.6 0 ad
0.3 0 0 0.5 Eg 0
0.2 0 0 0.3 0 0
0.1 0 0 0.1 xX x
0.05 0 x 0.05 Total. xx
amen Total. Total. ene Total. Total.

It will be noted that heloderma serum exhibits a greater inhibitory power
than guinea-pig serum, since 0.05 c.c. of the former is able entirely to prevent
hemolysis, while 0.1 c.c. of guinea-pig serum allows a trace of hemolysis to take
place, although more venom had been added in the experiment with heloderma
venom.

The action of a mixture of venom and lecithin on turtle and guinea-pig
corpuscles was also inhibited by the heated heloderma serum.

162 THE VENOM OF HELODERMA.

The influence of guinea-pig and heloderma serum are approximately the
same in experiments in which guinea-pig corpuscles were mixed with lecithin
and venom.

When heated dog serum was utilized as activator in place of the lecithin,
the addition of heated helodermaserum in large quantities prevented hemolysis.

Mixture of 0.1 mg. of venom and 0.1 c.c. of heated dog serum and guinea-pig corpuscles
(2 c.c. of a & per cent suspension).

Amt. of
heloderma | Result.

serum.
Et.
0.5 0
0.3 x
0.1 xx
0.05 xXx
3 Total

Here again the inhibitory action of heloderma serum is more marked than
that of guinea-pig serum, since 0.7 c.c. of the latter was not sufficient to com-
pletely prevent hemolysis, whereas 0.5 c.c. of the heloderma serum does prevent
hemolysis by venom and dog serum.

From these experiments we may conclude that heloderma serum pos-
sesses a somewhat greater inhibitory power than guinea-pig serum and that it
protects its own corpuscles best of all. It also seems that guinea-pig corpus-
cles are more strongly protected than turtle corpuscles by heloderma as well
as by guinea-pig serum.

Horse serum, when combined with venom and lecithin, does not prevent
hemolysis. The influence of these combinations was tested on horse, rabbit,
dog, and guinea-pig corpuscles and both heated and unheated horse serum was
used. The results were the same in all cases; no inhibition was evident, and
indeed in most experiments hemolysis appeared more rapidly in the tubes con-
taining serum, lecithin, and venom than in those containing only lecithin and
venom. It is evident that horse serum does not inhibit hemolysis by venom
and lecithin.

Turtle serum appears to have a slight inhibitory action when venom and
lecithin act on turtle corpuscles. This holds good only in the case of the
unheated serum.

Mixture of 0.1 mg. of venom and 0.1 mg. of lecithin.

lec.of 5 | lec. of 5 |2ec.ofad
per cent per cent per cent

Amt. of i alg iv ec eon Bote eect

turtle cor- | turtle cor- | guinea-pig

turtle serum. puscles and | puscles and | corpuscles

heated unheated | and heated

serum. serum. serum,
Cig
0.5 xxXX x xx
0.3 xxx x xX
O14 Total. xX xxx
0.05 Total. xxx XXX
ae xXx xXX xX

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 163

We can state that heated turtle serum does not inhibit hemolysis of turtle
or guinea-pig corpuscles by venom and lecithin; indeed the addition of heated
turtle serum seems to increase the hemolysis. It does appear, however, that
unheated turtle serum inhibits, at least to some extent, the hemolysis of turtle
corpuscles by lecithin and venom. The most plausible explanation for the
action of turtle serum seems to be as follows: In turtle serum both an acti-
vating as well as an inhibiting substance exists. In heated turtle serum the
activating substance prevails to a greater extent than in the unheated serum.
If a combination of venom and lecithin is used the activating substance of
turtle serum can act only to a slight extent, but it is able to neutralize the
inhibiting substance.

Rabbit serum acts in a somewhat irregular manner, its action varying with
different sera and different corpuscles.

With rabbit corpuscles the rabbit serum prevented hemolysis in almost
every case; with guinea-pig corpuscles the rabbit serum prevented hemolysis in
three out of five experiments; while with horse corpuscles hemolysis was pre-
vented in only two out of five experiments.

Mixture of 0.05 mg. lecithin, 0.01 c.c. of venom, and 2 c.c. of suspension of corpuscles.

|
nest: Gt it] Guinea-pig| Horse | Rabbit
BOLT corpuscles. | corpuscles. | corpuscles.
€.c.
0.6 0 x 0
0.4 x x 0
0:2! x xX 0
0.1 x Total. 0
0 05 xx Total. x
Sa Total. Total. Total.

The inhibitory action was quantitatively most marked when rabbit cor-
puscles were used, and least marked when horse corpuscles were used. As a
rule when a certain specimen of rabbit serum did not protect the horse corpus-
cles it likewise had no, or only a very slight, protective action with the guinea-
pig corpuscles.

Heated and unheated rabbit serum acted in the same manner; a certain
serum which when heated inhibited the hemolysis of rabbit corpuscles by
venom and lecithin acted in the same manner when unheated; while another
serum, which did not protect horse corpuscles when heated, showed no protec-
tive power when it was added to the tubes containing horse corpuscles, lecithin,
and venom without having previously been heated.

When heated dog serum was substituted for the lecithin in a mixture with
guinea-pig corpuscles, rabbit serum showed as much protective power as when
lecithin was used.

Rabbit serum which inhibited hemolysis by venom and lecithin, inhibited
also the hemolysis by venom and heated horse serum in mixtures with horse,
rabbit, and guinea-pig corpuscles, and, as with lecithin, the inhibition was most
marked with rabbit corpuscles and least marked with horse corpuscles.

164 THE VENOM OF HELODERMA.

The inhibitory action of rabbit serum was also tested with sodium oleate
as anactivator. In this experiment, in which guinea-pig corpuscles were used,
no inhibitory action was observed.

We tested not only fresh rabbit sera but also rabbit serum on the second
as well as the third day after the blood had been withdrawn.

Two sera, which on the first day showed no inhibitory action with horse
and guinea-pig corpuscles, were markedly inhibitory on the second day. Two
other sera, which on the first day showed slight inhibitory power, remained
unchanged in their action on the second day. All rabbit sera, whatever their
action may have been on the first day, however, showed distinct inhibitory
action on the second day.

On the third day all the sera tested again showed inhibitory action, but
while in no case was there an increase, in several cases there appears to have
been a decrease of the inhibitory power. The action of such a serum, when
tested on three successive days with horse corpuscles, is shown in the following
table. In this experiment the same horse corpuscles as well as fresh horse
corpuscles were used, the results being the same with the various corpuscles.

Mixture of 0.05 mg. lecithin and 0.01 c.c. venom and 2 c.c. of a 6 per cent
suspension of horse corpuscles.

Amt. of oH Second :
cAbbibserui: First day. doy. Third day.

Cc.

0.6 Total. 0 x

0.4 Total. 0 x

0.2 Total. 0 xxx
0.1 Total. 0 Total.
0.05 Total. x Total.
amet Total. Total. Total.

In certain cases the rabbit serum was reheated on the second and third
days, and, in order to determine whether such repeated heating of the serum
might influence the inhibitory action, we made parallel experiments with serum
which was reheated on the second day and a portion of the same serum only
heated on the first day. An experiment was also made with serum reheated
on the third day and a portion of the same serum which was not reheated. The
results in the two parallel series were similar: the reheated serum, and the
scrum which was not reheated, acted quantitatively and qualitatively in the
same manner, so that it is apparent that the reheating had no influence on the
inhibitory power of the rabbit serum.

We may therefore conclude that rabbit serum varies in its inhibitory
power; some sera possess no protective power for horse and guinea-pig corpus-
cles, others show this power slightly, but all show some protective power for
rabbit corpuscles. After standing 24 hours all rabbit sera develop protective
power for horse and guinea-pig as well as rabbit corpuscles, and after standing
24 hours longer this power may be diminished or remain unchanged.

The serum of a rabbit which had been immunized against heloderma
venom was added to mixtures of venom, lecithin, and rabbit corpuscles. We

4

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 165

found that this immune serum inhibited hemolysis in the same degree as did the
serum of a normal rabbit; the immune serum had no greater protective power
than the normal serum.

We also tested the inhibitory action of the serum of arabbit that had been
immunized against dog serum and dog corpuscles. At the same time parallel
experiments were carried out in which normal rabbit serum was used to inhibit
hemolysis.

Mixture of venom 0.1 mg. and lecithin 0.05 mg. and 2 c.c. of a 5 per cent
suspension of rabbit corpuscles.

Serum of
rabbit im-
pon Normal sania?
serum. | against dog
penuh s serum and
corpuscles.
c.c.
0.5 x 0
03 xX 0
0.1 Total x
0.05 Total Total.
ioe Total. Total.

In this experiment the serum of the rabbit immunized against dog serum
and corpuscles was distinctly more protective for the rabbit corpuscles than the
normal serum.

CORPUSCLES OF RABBIT IMMUNIZED AGAINST HELODERMA VENOM.

The corpuscles of a rabbit, immunized against heloderma venom, were
washed; a 5 per cent suspension was prepared in the usual manner and the
hemolytic influence of venom combined with various activators was tested.
At the same time the influence of the venom-activator mixture was tested on
corpuscles of normal animals.

When lecithin was added to venom in graded quantities, or when variable
quantities of venom were added to lecithin, the corpuscles of the immunized
animal showed slightly less hemolysis than those of the normal animal.

Venom 0.1 mg. plus 2 c.c. of a &

per cent suspension of corpuscles. Lecithin, 0.05 mg.
Amt. of Normal | Immunized Amt. of Normal | Immunized
lecithin. | corpuscles. | corpuscles. venom. | corpuscles. | corpuscles.
m: mg
0.05 Total. Total. 0.1 Total. Total.
0.03 Total. xxx 0.05 Total. xXxX
0.01 XXX xx 0,02 xxx xx

With heated dog-serum as an activator, the corpuscles of the immunized
animal showed slightly more hemolysis than the corpuscles of the normal ani-
mal; in view of these slight differences and apparent contradictions it seems
probable that no marked differences exist between the resistance of the corpus-
cles of immunized rabbits and of normal rabbits to the hemolytic action of
heloderma venom and various activators.

166 THE VENOM OF HELODERMA.

COMPARISON OF THE HEMOLYTIC INFLUENCE OF THE VENOM OF THE
HELODERMA SUSPECTUM AND HELODERMA HORRIDUM.

All of the experiments which have been reported so far have been carried
out with venom of the Heloderma suspectum, but since we were able to obtain
venom from the Heloderma horridum it appeared advisable to compare the
activity of these two venoms. We tested the action of the two venoms com-
bined with lecithin or heated dog serum on guinea-pig, rabbit, and turtle cor-
puscles. When lecithin was added to the two venoms with all three kinds of
corpuscles, slightly more hemolysis was observed in the tubes containing the
venom of Heloderma horridum. An example of an experiment with turtle cor-
puscles will serve to show this difference.

1c.c. of a6 per cent suspension of turtle corpuscles and lecithin, 0.05 mg.

Heloderma| Heloderma
srl f suspectum | horridum
; venom. venom.
Ces
0.002 xxx Total.
0 001 xx XXX
0.0005 xx xx

A similar difference was observed when heated dog serum was used as
activator; here again the Heloderma horridum venom tubes showed slightly
more hemolysis.

We also tested the action of heated rabbit and guinea-pig sera as activa-
tors for the Heloderma horridum venom, with guinea-pig corpuscles.

The Heloderma horridum venom was not activated by either of these sera;
its behavior was therefore similar to that of the Heloderma suspectwm venom.

We may conclude that the venom of Heloderma horridum and Heloderma
suspectum are identical in their action.

INFLUENCE OF HEATING ON THE HEMOLYTIC POWER OF
HELODERMA VENOM.

It has been shown that heating the venom of various snakes to high temper-
atures, 100° C., for 30 minutes, does not destroy their hemolytic activity. In
the case of the venom of the Heloderma, we have shown that boiling does not
destroy its toxic property, although it may occasionally lessen it to a very
slight extent. In experiments to determine the heat resistance of the hemo-
lytic substance of heloderma venom, fresh venom and dry, dissolved venom
were used, with lecithin as an activator. The effect of venom-lecithin mix-
tures was tested on guinea-pig, Heloderma, and turtle corpuscles, and in the
main the results were similar with all these corpuscles. The turtle and guinea-
pig corpuscles were hemolyzed to the same extent by the various specimens of
heated venom, while heloderma corpuscles were more resistant to the heated as
well as to the unheated venom.

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 167

We tested venom which had been heated to 100° C. for 10 minutes and 30
minutes, and to 120° C. for 15 minutes. Venom which had been heated to
100° C. for 10 minutes was quite as active as unheated venom; when heated to
100° C. for 30 minutes the venom lost most, if not all, of its power to hemolyze
the various corpuscles (some variations were observed in individual cases, de-
pending probably upon the susceptibility of the individual corpuscles). When
venom was heated to 120° C. all hemolytic power was lost. It should be noted
that unheated venom always produced more rapid hemolysis than did the
venom which had been heated to 100° C. for 10 minutes.

1 c.c. of 5 per cent suspension of turtle corpuscles and 0.1 mg. of lecithin.

t

Amt. of venom. | Unbeated. Loo? Gor’) 100" Cttor 120" C.foe

10 minutes. | 30 minutes. | 15 minutes.

Fresh venom:

0.08 C6. .55 oss Total. Total. xx 0

0.02 c.e........ Total. Total. xX 0

UA i eee ee Total. Total. xX 0
Dry venom:

0.3 mg......... Total. Total. xx 0

O.2 MB. nos a Total. Total. xx 0

O.im'g sa 5s <enas Total. Total. xx 0

The results were in general the same with turtle, heloderma, and guinea-
pig corpuscles.

We also compared the influence of heat on the venoms of the Heloderma
horridum and Heloderma suspectum, finding an approximately similar behavior,
although the venom of the Heloderma horridum was slightly more affected by
the heating than the venom of the Heloderma suspectum; the two venoms pro-
duced the same degree of hemolysis when heated for 10 minutes to 100° C., but
after being heated to 100° C. for 30 minutes the venom of Heloderma horridum
was weaker than that of suspectum.

1 c.c. of 5 per cent suspension of rabbit corpuscles and 0.05 mg. of lecithin.

100° C. for | 100° C. for | 120° C. for
Amt. of venom. | Unheated. | 19 minutes. |30 minutes. |15 minutes.

H. suspectum:

WO MB wenadanes Total. Total. xx 0
0.15 mg........ Total. Total. XOX 0
0.05 mg........ xxKxX XK x 0

H. horridum:
0.3. mg........ Total. Total. xx 0
0.15 mg........ Total. Total. x 0
0.05 mg........ XxKX xXX 0 0

The action of heat upon the two venoms is therefore in the main the same.
The venom of the Heloderma is distinctly heat resistant, approximately equally
if not more resistant than the hemolysins of certain snake venoms. The hemo-
lytic property of the venom of Heloderma is, however, not quite as resistant
as the neurotoxic property.

168 THE VENOM OF HELODERMA.

INFLUENCE OF ACIDIFICATION UPON THE HEMOLYTIC ACTION OF
HELODERMA VENOM.

A small amount of hydrochloric acid was added to a solution of dry venom
which previously gave a neutral reaction, and the hemolysis effected by the
acidified and the normal venom was compared. Heated dog serum served as
activator. We found no difference in the action of the normal and the acidified
venom.

2 c.c. of 5 per cent suspension of dog corpuscles and 0.1 c.c. of dog serum.

Amt. of Neutral | Acidified
venom. venom. venom.

mq.

1.0 Total. Total
05 Total. Total
0.2 Total. Total
0.1 Total Total
0.03 xxx xxx
0.015 XXX XXX
0.003 xx xX

Acidification of the heloderma venom does not interfere with its hemolytic
properties.

INFLUENCE OF THE ADDITION OF ALKALI TO HELODERMA VENOM.

Sodium hydroxide and sodium carbonate have been tested as activators
for the venom of Heloderma. Sodium hydroxide possessed no activating prop-
erties. Alone, sodium hydroxide may cause hemolysis of either dog or rabbit
corpuscles. When diminishing quantities of a decinormal solution of sodium
hydroxide were added to venom, the hemolysis was the same in tubes contain-
ing venom and the alkali as in the tubes containing the alkali alone. The
results were similar when either dog or rabbit corpuscles were used, except that
the rabbit corpuscles were more resistant than the dog corpuscles to the hemo-
lytic influence of the alkali.

1 c.c. of 5 per cent suspension of rabbit corpuscles.

N/10 NaOH
Amt. of
N/10 NaOH. N/10 NaOH. Cee

Cen

0.5 Total. Total
0.2 Total. Total
0.1 xx xx
0.05 0 0
0.025 0 0
0.0125 0 0
0.006 0 0

When 0.1 per cent solution of NaHCO; was used instead of NaOH, the
alkali had not only no activating properties, but the combination of venom
with the alkali appeared even to inhibit the hemolysis caused by the alkali
alone. Thus 0.4 and 0.2 c.c. of the 0.1 per cent solution of NaHCO; caused
slight hemolysis of dog corpuscles,while similar quantities of alkali combined
with 0.5 mg. venom produced no hemolysis. Our experiments were, however,

HEMOLYTIC PROPERTIES OF HELODERMA VENOM. 169

not sufficiently numerous to establish the latter point, and we can at present
only state that alkalis do not activate heloderma venom.

1 c.c. of 5 per cent suspen- 1 c.c. of & per cent suspen-
sion of dog corpuscles. sion of guinea-pig corpuscles.
|
Amt. of 0.1 | 0.1 per cent Amt. of 0.1 | 0.1 per cent |
im. 0) | 0.1 percent NaHCOs+ per cent so- | 0.1 per cent | NaHCO3+
NaHCO. NaHCoOs. 0.5 mg. lution of NaHCoOs;.| 0.5 mg.

a venom. NaHCOs3 venom.
C.t. Cf '
04 Total. 0 0.4 xx 0 :
0.2 XxX 0 0.2 0 0 |

CONCLUSIONS.

I. Neither the venom of Heloderma suspectum nor that of Heloderma hor-
ridum possesses hemolytic power when added to erythrocytes.

II. When lecithin is added to the venom of either H. suspectum or H.
horridum, erythrocytes are hemolyzed. Horse, ox, sheep, dog, rabbit, guinea-
pig, turtle, frog, and heloderma corpuscles may thus be hemolyzed. The
quantity of lecithin which must be added to the venom in order to hemolyze
the corpuscles varies between one-half and one-tenth of the quantity of lecithin
which by itself is able to produce hemolysis. In the experiments with turtle
and heloderma corpuscles, a quantity of lecithin equal to 7 and z respectively
of the hemolytic dose of lecithin is able to activate the venom.

III. Sodium oleate also activates the heloderma venom, but its activating
power is less than the power of lecithin.

IV. Alkalis (sodium hydroxide and sodium bicarbonate) do not activate
heloderma venom.

V. The blood sera of the horse, dog, and turtle, either unheated or heated
to 56°C. or even 70° C., activate the venom of Heloderma. On the other hand,
ox, sheep, rabbit, guinea-pig, frog, and heloderma serum, either heated or
unheated, do not activate the venom.

VI. The blood-corpuscles of Heloderma, although not immune against the
hemolytic properties of heloderma venom in combination with activators, were
occasionally found to be more resistant than other corpuscles tested.

VII. Certain sera, which do not activate the heloderma venom, inhibit the
venom-lecithin as well as the venom-serum hemolysis. Heating does not de-
stroy this property. Heloderma serum shows this property more strongly than.
guinea-pig serum. Rabbit serum varies in its inhibitory power; the serum
from one rabbit may inhibit hemolysis, while that of another may not. We
found that on the second day after the rabbit serum was separated from the
clot its inhibitory power was increased, but on the third day there was again a
slight decrease of the inhibitory power. Guinea-pig serum also inhibits venom-.
sodium-oleate hemolysis, while the rabbit serum tested did not have this inhib-
iting power. The inhibiting power of the sera varies with the various corpus-
cles. In the case of the heloderma and rabbit serum, the protective power was.
greatest in combination with their respective corpuscles.

170 THE VENOM OF HELODERMA.

VIII. Heated horse or turtle serum, both of which activate venom, do not
inhibit venom-lecithin hemolysis, but unheated turtle serum may have a slight
inhibiting action. The turtle serum contains perhaps both activating and
inhibiting substances; in some cases the activating, in other cases the inhibit-
ing substance prevailing.

1X. The corpuscles of a rabbit immunized against heloderma venom were
hemolyzed by venom and lecithin as well as by venom and an activating serum;
its corpuscles were, however, somewhat more resistant than those of a control
rabbit. The serum of a rabbit immunized against heloderma venom does not
activate the venom, but inhibits venom-lecithin hemolysis in the same manner
as normal rabbit serum.

X. The hemolysins of the venom of Heloderma resist heating to 100° C. for
10 minutes. After heating to 100° C. for 30 minutes the hemolytic power of
heloderma venom is markedly injured and usually destroyed; after heating to
120° C. for 15 minutes (in the autoclave) the hemolysins are completely de-
troyed. The heat resistance of the hemolytic substances of heloderma venom
is therefore similar to the heat resistance of certain snake venoms and inferior
to the heat resistance of the neurotoxic property of the heloderma venom.

XI. The addition of a small amount of acid to the normally neutral venom
solution does not change its hemolytic properties.

XII. The venom of Heloderma suspectum and Heloderma horridum act in
the same manner, both being activated by lecithin, or horse or dog serum, and
neither possessing the power to hemolyze red blood-corpuscles unless these
activating substances are added. They are also equally heat resistant.

XIII. The hemolysins of the heloderma venom differ in certain respects
from hemolysins of snake venoms, since they can not be activated by ‘‘com-
plements”’ (such as are contained in guinea-pig serum). It may also be added
that, compared with such powerful hemolytic agents as the cobra venom, the
venom of Heloderma is only weakly hemolytic.

IX,

ACTION OF HELODERMA VENOM ON THE CELLULAR
ELEMENTS OF THE BLOOD WITHIN THE
LIVING ORGANISM.

By M. K. Meyers anp Lucius Turt.e.

14 charts.

ACTION OF HELODERMA VENOM ON THE CELLULAR
ELEMENTS OF THE BLOOD WITHIN THE
LIVING ORGANISM.

By M. K. Meyers anp Lucius Turrie.

There is a paucity of information regarding the reaction of the cellular
elements of the blood in response to the presence within the living body of a
venom. The only reference to the subject we could find in the literature con-
cerned work done by Chateney,* who found in an animal rendered immune to a
lethal dose of cobra venom a rise in the leucocytes from 6,500 to 8,000; three
control rabbits that had received lethal doses of the venom died, showing a
hypoleucocytosis. Although Calmetteft states, presumably on the authority
of Chateney, that injection of a venom in amount sufficient to cause feeble
intoxication is followed by a hyperleucocytosis, it will be seen that the above
rise in the number of leucocytes is present only in a certain number of cases.

The following experiments prove definitely that leucocytosis is one of the
reactions of the organism against the introduction of a specific venom; and it
would seem that in an animal already immune this leucocytosis is greatly
increased.

The dried venom was dissolved in a small quantity of salt solution and
injected subcutaneously, unless otherwise stated. In estimating hemoglobin
the Fleischl-Miescher instrument was employed.

The accompanying protocols and charts are more or less self-explanatory.

Red | Hb. | Wate Weight | Amt. of

Experiment. Day. Time. blood cor-| per ear. of | venom Remarks.
puscles. | 100 c.c. piiscles! rabbit. | injected.
gms. gms. gm.
Rabbit I, expt. 1....| Nov. 19 11" 35™ a.m. | 7,300,009 9.70 7,206 | 1,600 0.005

11 45 a.m. mite Boe agevets
12 00 noon | 6,600,000} 14.30 | 5,400
1 00 p.m. | 6,990,000} 13.28 | 6,170

Venom injected.

3 00 p.m aissipe dente sy ti Rabbit fed.
5 00 p.m. | 6,430,000 | 14.82 | 11,500 Sac arte
Nov. 20} 10 00 a.m. } 6,750,000} 11.24 | 18,000) .... carats Differential count
Nov. 21/10 00 a.m.} 5,136,000 7.82 | 11,900} .... Seba taken; leucocy-
tosis.
Rabbit I, expt. 2....| Nov. 25 | 11 00 a.m. | 5,560,000! 8.16 | 12.700] 1,500 | 0.006
ll 45 am ae Fes Venom injected.

1 30 p.m, | 6,240,000 | 11 71 | 25,400
4 00 p.m. | 4,160,000 | 12.78 | 10,400
Nov. 26 | 10 30 a.m. | 4,540,000 9.18 | 15,000
Nov. 27| 10 30 a.m. | 4,970,000 | 10.22 | 8,600

Leucocytosis.

*Chateney. Les réacitions leucocytaires vis-A-vis de certaines toxines. Thése de Paris, 1894.
{Calmette. Les venins, les animaux venimeux et la sérothérapie antivenimeuse. Paris, 1907, pp. 226, 227.

173

174

THE VENOM OF HELODERMA.

me
Red | Hb. | White Weight Amt. of

Experiment. Day. Time. blood cor-| per plat of | venom Remarks.
puscles. | 100 ¢.c. puscles. rabbit. | injected.
|
gms. gms. | gm.
Rabbit I, expt. 2. Dec. 2 | 10450™ a.m. | 4,710,006 9.18 | 10,800 1,480 | 0.0075
11 30 a.m. ee a or Venom injected.
12 CO noon | 5, 760, 000 9.69 7,900 =|
2 30 p.m. 5,320,000 13.40 5,290 reins
Dec. 3} 9 30 a.m | 5,140,000 9.69 | 14,100 7 tea
Rabbit I, expt. 4....) Dec. 12) 11 J5 a.m. | 4,500,000 8.93 | 10,600 i 1400 | 0.0075
11 40 a.m. hse hans fre el ee Do.
12 00 noon | 6,760,000 | 10.71 | 11,000
; 12 45 p.m. | 5,930,000 | .... 6,600)... ss ci
! Dec. 14 | 10 30 a.m. | 4,500,000 8.92 9,600 sieve eds
Rabbit II, expt.1... ' Nov. 22) 11 15 a.m. | 5,200,000 5.10 | 11,700 | 1,700 0.005
12 00 noon Sats G58 en
12 15 p.m. | 6, 20 0, 000 7.64 9,500 | Do.
115 p.m 4°470,000 7.64) 11,665
5 00 p.m. | 5,700,000 8 16 | 24,500) .... |... Leucocy tosis.
Nov. 23,11 15 a.m. | 5,780,000 4.86 | 12,500 eae
Rabbit IJ, expt.2... | Nov. 25} 11 15 am ate, wot: 3.70 6,993 1,560 , 0.006
11 45 am. : Ree? fie ie cae Venom injected.
4 00 p.m. | 5,310,000 8 + | 11,107 on a
Nov. 26 | 10 30 a.m. | 3,760,000 792) 17,520; .... | ..
Rabbit IT, expt.3..... Dec. 2/10 50 a.m. | 4,410,000 8.16 | 12,240} 1,480 0.0075
11 30 a.m. ee ee a Do.
12 00 noon | 5,710,000 9.18 5,920 hee. I oe
: 4 30 p.m. | 3,440,000)... mee as "B) see
| Dee 3} 2 15 p.m. | 4,190,000 7.48 | 14,640 ee eee
Rabbit III Noy. 29} 10 45 a.m. | 4,860,000 7.14 9,100) 1,720 0.015
| 11 00 a.m. é Sates wesiaa’ sae UN) eee Do.
| 11. 30 a.m. 5,420,000 12.26 | 9,900 D agete
12 15 p.m. | 6,720,000 7.65 | 9,600 |
2 30 p.m. } 6,700,000} 10 88 | §,200 '
4 00 p.m. 13.29 ge Mateo) sees yt S's
| Nov. 30} 10 00 a.m. 4,770,000 % 44 | 195500") ey hata
Rabbit IV... . | Nov. 30} 10 30 a.m. | 5,050,000 8.16 | 12,560; 1,600 0.005
11 30 a.m. vee dusts os wegen "WT fetes Do.
12 00 noon | 5,220,000 4.76 9,500 Bate) cdi
2 15 p.m. | 5,420,000 8 67 5 Bia
4 00 p.m. | 5,700,000; .... 7,440 ease
Dee. 1) 9 45 a.m. | 4,380,000 7 14 9,400 erate mag's
Rabbit V, expt.1....| Dee. 5] 10 00 a.m. | 6,370,006) 10.92 4,500 | 1,440 | 0.020
10 30 a.m. een re ae Sete west Do.
11 00 a.m. | 5,800,000 | 15.35 7,080; ....
12 00 noon | 6,250,000} 14 66 5,100
2 00 p.m. | 8,000,000; 14.02 7,600
Dec. 6)|11 15 a.m. | 5,140,000 | 12 75 7,500 z mss
Dec. 7 | 10 15 a.m. | 5,000,000 | 10.20; 11,000) .... peas
Rabbit V, expt. 2 Dee. 11} 10 90 a.m | 5,330,000 9.56 7,600, 1,420 | 0.020
10° 30) a.m. | 4,070,000 10.20 6.1000 aseecs one Do.
10 45 a.m. | 7,000,000 8.92 2,300 sacs ae Si
2 60 p.m. | 7,000,000 oa seonets sieve nag Rabbit died.
Rabbit VI, expt.1...) Dee. 5/10 20 a.m. | 5,260,000) 13.29 6,200 | 1,540 0.020
10 30 a.m. ee beams aaa pul Venom injected.
11 00 a.m. 7,720,000 17.37 6,240 |
12 00 noon ' 7,040,000} 13 29 5,440 |
Dec, 6} 11 00 a.m. misters 13.29) 11,120 | ae
Dec. 7] 2 15 p.m. 3 10,960, . ashes
Rabbit VI, expt. 2 Dec. 11} 10 00 a.m Foes 12 78 13,640... 0.020
10 20 am.| | 5 aac Do.
11 00) a.m. | 5,570,000) 10 71: 12,080
12 00 noon | 4,860,000} 13 29, 11,520 san bade
Dee 12) 9 45 a.m. | 5,820,000) 10 20 7,800 Les ee
Rabbit VIL. Dec. 1610 30 a.m. | 3,230,000 7 OL 7,600 . 2,600 0 0075
11 00 a.m. | 4,670,000} .... 7,920! 2... | sstyene Do.
12 15 p.m 5,350,000 10 21 | 13,360
2 45 p.m | 4,600,000 8.92 9,400 | rie i ,
Der. 17 | 10 15 a.m. | 4,500,000} 9 35 8,000 eee ea
Rabbit VUT . ..... Dec, 19 | 11 00 a.m | 4,800,060 4.46 | 14,000 1,440 | 0.0075 |
Dec. 21/10 15 am | 460/000} 4 56] r4}o00 | 7. |
10 20 a.m é ah set | Do.
10 45 a.m | 4,830,000 1148 7,200 !
11 450 a.m 4 1020) 4,900) l ‘
4 00° p.m, | 5,880,000 6 34) 16,500 | .. i 24
Dec. 22/10 30 a.m. | 4,060,000 7 00 | 11,500 25 ee |
Dee. 23.) 10 25 a.m. | 4,100,000 7 44 R400! 2: Lawl
Rabbit IX. Dec, 23} 11 20 am. | 3,160,000 5 52 3,860 | 1,940 | 0.0075
it 45 a.m. nie ae oe Higa sa Do.
1215 acm. | 2,980,000] 816) 3,300, |
1 00) p.m. | 3,200,000 7.82 8,600
2 30 p.m. | 3.200.000) 8 45) 3'a00, | Rabbit died
|
|

| shortly after
© 2b 20% p.m.

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 175

Red | Hb. | White | Weight | Amt. of
Experiment. Day. Time. blood cor | per Gor of — | venom Remarks.
puscles. | 100 c.c. puscles vabbit.| injected .
gms. gms, gm.
Rabbit X........... Jan. 4 | 105 40™ a.m. | 4,200,000 9.18 7,400 | 2,160 0.0275
10 45 a.m. sorte eases Venom injected.

10 55 a.m | 6,060,000 9.18 | 7,600
11 15 a.m. | 4,700,000 9.34 | 3,700
12 10 p.m. | 5,430,000 | 10.20] 6,200] .... -

2 30 p.m. | 4,800,000 | 10.65} 4,800] .... oe
Hee oe 8,500 | 1,880 | 0.00564

Rabbit XI ......... Jan. 15/11 00 a.m.
1 30 a.m. oe Do.
11 50 am. 6,400
12 35 a.m. 13,200 a
1 30 p.m. 3,600 a8
Rabbit XIT......... Jan. 17| 9 50 a.m. 3,300 0.0045
10 00 a.m. aie er = 9368 aiaaa Do.
10 25 a.m. dae sens 3,800
10 50 a.m. re Laois 3,590
11 25 a.m. Bases dene: 10,100
10 50 a.m. Ssh ste 2,400) .... or
3 00 p.m. Sets a gs 5,200)| eos: ees
Rabbit XIIT........ Dee 27/12 00 noon | 4,890,000) 10.65 | 13.700 | 1,640 | 0.010 Mesenteric vein.
12 00 noon | 4,950,000| 11.24 | 22,320; ... S ances Ear vein.
12 10 p.m. bie aioe aed ittite ries Venom injected.
2 00 p.m. | 7,200,000) 14.98 9,400 sag oe Mesenteric vein.
2 00 p.m. | 4,275,000 7.44) 0... sleet eee Ear vein.
2 00 p.m vactane Rabbit died
shortly after
2 p.m.
Rabbit XIV........ Dec. 30} 11 (CO a.m. | 4,770,600 9 35 | 16,100} 1,900 0.016 Mesenteric vein
11 00 a.m. | 4,400,000 9.35 | 15,000 | .... seeks Ear vein.
Il 20 a.m. ae eve ers Venom injected.
2 40 p.m. | 6,130,000 722] 9,000; .... eens Mesenteric vein.
2 40 p.m. eee 10.96 | 15,900) .... “ Ear vein.
Rabbit XV... ..... Jan. 8/11 15 a.m | 5,100,000; 8 2 6,200 | 1,340 | 0.0076 | Mesenteric vein.
11. 15 a.m. | 5,200,000 9.5 | 22,800] .... ate Ear vein.
11 32 am cae ace ae Venom injected.
12 15 p.m. ; 5,200,000 &4 4,700 a anes Mesenteric vein.
12 15 p.m. | 5,700,000) 10.00] 38,400) .... = Ear vein.
2 20 p.m. | 4,900,000 8.40 4,600 er etd Mesenteric vein.
2 20 p.m. | 5,440,000 8 40; 9,600 | Ear vein.
Jan. 91/10 30 a.m. | 5,200,000 7.60 4,000 nee aes Mesenteric vein.
10 30 a.m. | 5,020,000 | 138.80 6,320 oo Sedais Far vein.
Rabbit XVI........ Jan. 24) 9 45 a.m. eipee nese 9,800 1,750 | 0 00525 Do

10 10 am. Sosa Venom iniected. |.
10 40 a.m. 19,100 Ear vein; leuco-
cytosis.
10 55 a.m. 14,000 Far vein.
11 18 a.m. 3 aon oe 3,000; .... paveds Mesenteric vein.
1] 220 a.m. seve aks 7.700 ese er Splenic vein.
11 23) a.m. rasa 1624 16,600 ies isch Ear vein.
11 26 a.m. Be gee 7,000} 2... ee Vena cava.
Rabbit XVII, expt.1.) Jan. 25/11 45 a.m. | 4,000,000] 7.52 | 14,200] 1,480 | 0.055 | Venom injected.
12 15 p.m. | 4,080,000 8 69} 11,200} .... ier
2 10 p.m. ; 4,100,000 6.63 | 22,000] .... aside Leucocytosis.
4 20 p.m. ate .... {270,000 | .... ansd Do.
Jan. 26| 9 45 a.m. see sigan 40,300 | .... musta’ Do.
3 00 p.m ope ae 8g, eet easier
Jan. 27| 8 45 am 10,600
Jan. 28 | 8 45 a.m 12,500
Jan. 30] 8 45 a.m. 7,400 er utes
Jan. 31/11 45 am es 83 11,800 “aee scat
Rabbit XVII, expt 2 | Jan. 31) 11 45 a.m. ee eit 11,800 | 1,510 | 0.060
1 00 p.m. ea re shige yetoce ahs’ Venom injected.
1 30 pm. 7,200
3 00 p.m. 7,000
Feb. 1] 9 15 a.m. 9,800
11 00 a.m :
ll 15 am Leucocy tosis.
12 30 p.m Do.
2 45 p.m Do.
4 45 p.m Do.
Feb. 2/ 9 00 am Do.
1 55 p.m had Do.
‘ Feb. 3] 9 00 am ee fos £5
Rabbit XVII, expt.3 | Feb. 11] 9 30 am 1,420 | 0.070 Venom injected.
10 30 a.m, ayeasts seiccud
Feb. 12] 9 10 am
Feb. 13] 9 00 am
Feb. 14] 8 50 a.m.
Feb. 15; 8 40 am

176 THE VENOM OF HELODERMA.
Red Hb. tee Weight |} Amt. of
Experiment. Day. Time. blood cor-| per oe: of | venom Remarks.
puscles. | 100 c.c. puscles. rabbit. | injected.
5 (eee 2| eease ——-|-—— —}- =
gms. gms. gm. |
Rabbit XVII, expt.4 | Feb. 18 5 00™ p.m. 3 pics 1,640 0 080
Feb, 19/11 00 a.m. 13,200] .... aura
3 00 p.m. 3,900 |...
Feb 20/10 00 am. 9,800 | ...,
3 00 p.m. 15,000 mire ;
Feb. 21} 9 20 a.m. 18,000 | .... |
2 20 p.m. 18,500 Fras i
Feb. 22] 8 40 am] ...- | 8,000) 222: oat CF
Feb. 24 | 9 20 a.m. hinaya Bineas 8,300 ee Sarre
DOB so dsesencccisttenen .| Jan. 3110 30 a.m. ; 6,400,000} 10.54 | 13,000 | 4,820 | 0.0289 | Venom iniected,
If given sub-
! cutaneously at
| 115 10™ a.m.,
; half given intra-
venously by sa-
phenous vein at
11° 30™ a.m.
12 15) p.m. | 6,130,000 | 14 05 | 25,200 sis Leucocy tosis.
2 15 p.m. | 4,430,000! 12.77 | 24,000 cto Do.
4 10 p.m. | 8,100,000) 18.90 | 53,000 extn stn Do.
Feb. 1/11 45 a.m. | 5,800,000} 13.62 | 35,000 dasttes Beat Do.
Doak Bids divaiieeanars Feb. 6} 9 30 a.m. | 6,660,000} 17.04 | 19,700 7,760 | 0.0466 | 6 mg. per kilo.
11 18 a.m. gene ore waite oi tian Venom injected
intravenously.
11 58 a.m. | 6,700,000 | 17.37 | 16,000
1 00 p.m. | 7,830,000) 20 43 | 24,200
2 30 p.m. fees 19.76 | 30,480 Leucocytosis.
3 30 p.m. | 7,550,000 zfs cca
4 15 p.m. | 7,900,000 19.92 | 20,000
Feb. 8! 9 40 a.m | 7,000,000 | 19.41 | 32,000 Do.

DIFFERENTIAL COUNTS.

Blood of rabbit [ at period of greatest leucocyte count, 18,000:
Amphophiles
Lymphocytes.
Mononuclears.
Transitional .
Basophiles. .
Eosinophiles

Blood of dog I before injection, 13,000:
Neutrophiles
Lymphocytes.
Mononuclears.
Eosinophiles. .
eae

At period of highest leucocytosis:
Neutrophiles
Lymphocytes.
Mononuclears
Fosinophiles
Pepe.

Per cent. | No.

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 177

Hb, Robo W121 2 3 4 5 JOAMIOAM 11:12 1 2 3 4 ee

al Se AM
36,000--+ per
1
34000) oe. 4fns in 0,006 Gm
32,000 dried Venom dried Venom
: injected injected
a 6 days after
Exp. No. |)
28,000 xp. No
a Exp. Exp. 2

26,000 a -
24,000 5 | :
22,000 S re e 7
20,000 = = ; f ;

18,000 iS a :

16,000 000,00 = <

14,000 5 04 000 qt / ‘i

1: ir — ee iN
12,000 6,000,000 N aon :
10,000 5,000.000
00 Lt
6,000 L—_+— 16 — 4,000,000 L = I
6,000 |__| 45~ 3,000,000 Mes L<ft-5 sk L i i f
rotod al “Las sae ee
4,000} 8 — 2,000,000} i
an
2,000 1-4 — 1,000,000

Fic. 25.—Rabbit 1, Experiment 1. The morning of the day after the injection the number of leucocytes reaches 18,000;
which is a distinct rise in the number of leucocytes in this rabbit. A differential count shows that poly-
morphonuclear amphophilic leucocytes constitute 69.6 per cent of the total number. This is rather high.
The amount of hemoglobin and number of red cells run in fairly parallel curves.

Rabbit 1, Experiment 2. The number of leucocytes rises immediately after the injection to 25,400, then
falls; the next morning the number is again high. Variations in the hemoglobin index seem to fluctu-
ate about a nearly constant mean.

Hb. Robe 11 12 f— 2 9,30 i 12 10.30.
L [ [oe AM AM |
36,000 |—+- per
100
34,0007 —- 7 0.0075 Gm 0.0075 Gm
32,000 dried Venom dried Venom
: injected injected
30,000 (8 days after (6 days after
28,000 Exp. No. 2) Exp. No. 3)
26,000
24,000
22,000
Exp. 3 .f
20,000 u Piped
18,000 a re
a 6
16,000 a 8
14,000 > Zz
12,000 =} | ANI
10,000 000.00 Pt tA N
' ly
8,000 -— 16 ~ f.000,004 =~ iw i
i)
— a 12-3, 000,000 > =
6,000 2 a Ext
4,000 H+ — 2,000,000 Ed h~
2,000 —-L- 4 — 1,000,000

Fic. 26.—Rabbit 1, Experiment 3. No leucocytosis detected.
Experiment 4. No leucocytosis detected.

178 THE VENOM OF HELODERMA.

Hb. Robo. 12 1 2 3 4 5 Wis 12 1 2 3 4 10.3011 12 1 2:3 4 2/15
[| Gm. AM AM PM

36,000 |}-—+— per

100
34,000 -—- 57 0.005 Gm 0.006 Gm 0.0075 Gm
32,000 dried Venom dried Venom dried Venom
‘ injected injected injected
30,000 (3 days after (2 days after
28,000 Exp. No. I) Exp. No. 2)
26,000 |
24,000 E I 5 | 3

i: xp. :
‘22,000 xP. P Exp. 3
20,000 = \
18,000 e J 3
16,000 000,000 2 x
|

| 14,000 4,000,000.2 \ o 9
ineiee We 7 3
12,000 pponod la. ne Zz
10,000 | ~~ 20 ~ $.000.000 x ai 2 44 7 S

fl 7 |
8,000 |—— 16 —4,000,000 t NK

¢ fl ! | ~

6,000 | —7— 12 - 3,000,000

1
4,000 8 — 2,000,000 _ eat L-t+} | _
2,000 LL 4 {oodood = canis

Fic. 27.—Rabbit 2, Experiment 1. Rise in number of leucocytes 5 hours after injection; followed next morning by ap
absence of leucocytosis. .
Experiment 2, The morning after injection number of leucocytes is high.
Experiment 3. The morning after injection number of leucocytes is higher than before injection.

_Hb,_ Rbc. 10 4 12 | 2 3 4 10.00 won 121 2 3 4 945
LT Gm. AM. AM
36,000 }—~ per
34,000 L_4 100 4
j ' cc. | 0.015 Gm 0,005 Gm
32,000 dried Venom dried Venom
‘ injected injected
30,000 7 |
28,000 | iow
26,000 RABBIT 3 RABBIT 4
24,000
22,000
20,000
18,000 ° a
a
16,000 3,000,000 Fy o
i
14,000 7,000,000 —+— z
| z z
6,000,000 =
12,000 f 0,000 7 ~ a at]
10,000 -—-— 20 ~ 5,009,000 ot <
1 Pp
8,000 | + 16 — 4,000,000 4 <
' at
6,000 | —+—12— 3,000,000 7 =: ae
tot i N -T hs ace
4,000 -—+— 8—2,000,000 x t = =
' ~ 4
2,000 —— 4 — 1,000,000 =

Fic, 28.—Rabbit 3. Only very slight rise found on morning of day following injection.
Rabbit 4. No leucocy tosis.

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 179

Hb, R.b.c. 10 U1 12 1 2 W415 10.15 10 1 12 - 2
LT am. “A.M. | ALM. |
30,000 }—+- per
28,000 J 1°? -' 9.990 Gm 0.020 Gm
26,000 |" dried Venom, dried Venom
; injected fi injected
24,000 (6 days after
22,000 Exp. No. 1)
20,000 = Exp. 1 3 |
18,000 ro P
u (=)
16,000 w 2
fe
14,000 7,000,000-—+ = Ss)
to t
12,000 6,000 00 VA z 4
10,000 |_1- 29 —5,000 ood a Ee
' fay
8,000 }—_+— 16 — 4,000,000 L
|b oohood_ LAN aT t= i 7
6,000 + 12 —3,000,000 a Ny FE
4,000 |_1- § —2,000,000 S =
2,000 oleen = 1,000,000 l

Fic. 29.—Rabbit 5, Experiment 1. _A rise to over 10,000 leucocytes occurred 2 days after injection of a relatively large
amount of venom. The animal had a low leucocyte count at beginning of experiment.
Experiment 2. A large quantity of venom was again injected; number of leucocytes fell, and 3 or
4 hours after injection rabbit died. Slight rise in number of leucocytes occurred after first injection,
followed by a distinct fall after second injection of venom.

i re R.b.c. 10. 11 12 3.15 10 11 12 9 9.45
im. xU-6 | P.M. A.M
36,000 LT per 11-7
34,000 + 100
1 cc, 0.020 Gm 0.020 Gm [J
32,000 |__| dried ere dried Venom_| _|
injecte injected
01000 TTA M| (6 days after 7—|
28,000 Exp. No. I)
26,000
24,000 Bai
22,000 Ps Exp. 2
20,000 3 =
18,000 e Sg
16,000 8,000,000} 5 g
POR Tl z/ >
14,000 7,000,000 7 Z
I
12,000 6,000,000-—r
\ t—{ |
10,000 -—-~20-- 8.000.000 pea
1
8,006 -—+— 16 - 4,000,000
i ' rr Rae 4 _
6,006 -—7- 7 — 3,000,000. ~y oN Es
4,000 || & 2,000,000
2,000 ae 4 — 1,000,001 !

Fic. 30.—Rabbit 6, Experiments 1 and 2. Large doses of venom used.

180 THE VENOM OF HELODERMA.

_ Hb. R.b.c. 10 1 12 2 10.15.10 11 12 1 2 3 4 10.30 10.251) 12 1 2 3

1
“yom [ [1 f amy [| AM TAM | [|
30,000 |—7- ns ret | | i
28,000]. 6 77 0.0075 Gm 0.0075 Gm 0.0075 Gm +—
; dried Venom dried Venom dried Venom
26,000 injected injected injected |
24,000
22,000
20,000
RABBIT| 7 a RABBIT 8, RABBIT 9
18,000 w
=
16,000 Oo o4
fa} a m
14,000 7,000,000—+ 4 2 Va =
12,000] ——24— 6,000,000 3 ft A =
10,000 20 ~5.00¢.000 +2 | fe \ L-7) a
: 008 2 RSS TY :
8,000/-1 16 ~4,000,000 ,, g
\
6,000 F-—7— 12 — 3,000,000 ——
rod | P= Sen el a PSY gi ee
4,000 t— 8 —2,000,000 —4->4 7 i — :
feet d ‘ is ican Gael a
2,000 — 4 — 1,000,000

Fic. 31.—Rabbile 7 and 8. Smaller doses of venom were used here than in experiments on rabbit 6; a slight rise in both
occurred. In rabbit 8 the rise is preceded by a fall.
Rabbit 9. This rabbit had an initially low leucocyte count; it succumbed toa rather small dose of venom.
No rise in number of leucocytes.

38,000 Hb. Rbc. 10 11 12 | 2 3 11 12 | 2 10 1112 | 2 3

[—] Gm.
36,0001 + por
100
34,000 -—- 7 0.0275 Gm 0.00564 Gm 0.0045 Gm J
32,000 dried Venom, dried Venom dried Venom
30,000 injected injected injected
28,000
26,000
24,000
22,000 RABBIT 10 RABBIT 11 RABBIT 12
20,000
18,000 a J
uw
16,000 8,000,0 2 a
Kd tl Hy °
14,000 7,000,000 —- bow
Tell e at z oO b A
12,000 |}—-+- 24 — 6,000,000 + + ot
10,000 | 1-20 5,000,000 fw {= 12 I a
fie et) re
8,000 | + 164,000,000 t 5 |
I
6,000 }+— 12 — 3,000,000 \ = 1 z | |
ae z
4,000 | — 8 — 2,000,000 va - \ a : |
2,000 1 4 — 1,000,000

Fia. 32.—Rabbit 10. Large dose; no leucocytosis. Some initial fall in number of leucocytes.
Rabbit 11. Slight fall followed by a not considerable rise in leucocytes.
Rabbit 12. Small number of leucocytes observed in beginning of experiment; followed by inconsiderable
rise after injection.

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 181

H R.b.c. 121 i}. 1212 3 WW 12> 12 10,30
L 2 AIM
| Gm. IM.
36,000 }—- per
34,000 | 10°
cae 0.010 Gm 0.010 Gm 0.0076 Gm
32,000 dried venom dried venom dried venom
30,000 injected injected injected
28,000 | | | |
26,000
: RABBIT 13 RABBIT 14 RABBIT 15
24,000 t EAR
22,000 EAR a f
20,000 2) 5 r
18,000 S a
: 5 Mes.= \
16,000 Fe a= a
14,000 It calomel \
ESENTERV. y \
12,000 te =
10,000} —-~20 — 5.000.000 EAR} — fet
‘g,000/- 1-16 —4,000,000 Se ee
6,000|-—4 123,000,000 EAR : a
4,000 -+~ 8 — 2,000,000 MES" ts cite BS
z roto
2,000 4— 1,000,000

Fic. 33.—Rabbit 18. Simultaneous counts were made from a vein of the ear and one of the mesentery; that of the ear
was high, that of the mesentery lower; the count of the mesenteric vein fell after venom had been injected.
Rabbit 14. An initial high leucocyte count was found in both the blood from the ear and the mesenteric
vein. After injection no striking rise in the leucocytes from the ear-vein was noted, and there was a
marked fall in the number of leucocytes in the blood of the mesenteric vein, followed by a slight rise.
Rabbit 15. A very marked fall in number of leucocytes was observed in blood of ear-vein, associated with
a slight fall in number in mesenteric vein of a rabbit that had a preexisting leucocytosis following injec-
tion of a relatively small dose of venom.

t—_ He. R.b.c. 9.45 9.55 10.05 10.15 10.25 10.35 10.45 10.55 11.05 115 11.25
Gm.
36,000 ++ per

34,000 ee 0.00525 Gm
32,000 dried Venom
injected

30,000
28,000
26,000
24,000
22,000
20,000
18,000
16,000
14,000
12,000
10,000 }—— 20 — 5,000.00!
8,000 -F— 16 4,000,000
6,000 -— i ate
4,000 ae 8 ae cea +
2,000 — 4 — 1,000,000 MES,SPLENIC &VENACAVA

> INJECTED

EARL]

Fia. 34.—Rabbit 16. A rise in leucocytes of blood of ear-vein was found after injection of venom, with a considerably
lower number of leucocytes in successive counts taken from veins of abdominal cavity (mesenteric vein,

splenic vein, vena-cava). In this experiment an initial fall in number of | ytes 1
through repetition of early counts. ea Sele Reet

182 THE VENOM OF,HELODERMA.

Hb. R.b.c. 11 12 1 2 3 4 945 3.00 £27 1-28 129 303
[ Gm. AMTPeM
54,000 F per (1-26)] (I-26)

52,000

50,000
0.055 Gm

48,000
[ dried Venom
46,000 injected

44,000
42,000
40,090
38,000
36,000
34,000 \
32,000 \ |
30,000
28,000 ‘ \ pice
26,000 # \ eds
24,000 he 4
22,000
20,000
18,000
16,000
14,000
12,000 +— 6,000,000 fi
10,000 }-20 — $096,000
8,000 | 16 — 4,000,000 al
6,000 2 008000 |
4,000 -— 8 —2,000,000-—4-=4} + }

|

|

DINJECTED
T—~
Ge ee Tee |
|__f

Sik
l
|

i) | {
2/000: |-—4— ',e00,009 :

L | |

|
ue

Fic. 35.—Rabbit 17, Experiment 1, This rabbit had been immunized against venom by injection of increasing doses.
Figure shows very marked leucocytosis following administration of very large dose of venom that per-
sisted about a day. The rabbit had a relatively high leucocyte count at beginning of experiment.

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 183

212 3 9151 1112 1 2 3 4 900 155 ___9.00_
L it AM. amMy]em] [AM] ]

70,000
68,000
66,000
64,000
62,000 /
60,000

58,000
56,000 /
54,000 /

52,000 /

50,000

48,000 0.060 Gm
46,000 dried Venom
pipes injected _ fi |
i (6 days after \
42,000 Exp. No. 1) \
\
\

L+-T |

40,000 [
38,000
36,000 ii
34,000 |
32,000
30,000
28,000
26,000

24,000 ; |
|

22,000
20,000
18,000 |
16,000
4,000
12,000
10,000
8,000 afl
6,000
4,000
2,000

INJECTED

® 1a. 36.—Rabbit 17, Experiment 2. With only slightly larger dose of venom than i i i i
was much higher, but latent period was longer than feo pic nee NO Ae

D 1 e after preceding injection. In
rabbit had a relatively high leucocyte count at beginning of exporineat. zi Siac

184 THE VENOM OF HELODERMA.

14 W-t9 M-20 U-2)_ 22

U-15, 118-190-202 Ib 24

9.30 1030 5PM.1 3PM.] 3.30 [ 2.20 | 9.20
4 9.20 _| 8.40

I-20
L Ue ote tr-r

a)
36,000 AM. AM. {HAM

34,000; —-_ 0.070 Gm im Gin
32,000 + dried Venom dried Venom
injected injected
30,000 }—Fi4y days after (7 days atter
28,000} + Exp. No. 2) Exp. No. 3)

26,000
24,000
22,000
20,000
18,000
16,000
14,000
12,000 [ \
10,000
8,000
6,000
4,000
2,000

PINJECTED

-—7> INJECTED
Pp
|
‘=

Fic, 37.—Rabbit 17, Experiment 3. Amount of venom slightly larger than in preceding experiment; if there was a
leucocytosis, it occurred at times other than when counts were made, 1 and 2 days respectively after
injection.

Rabbit 17, Experiment 4. Amount of venom administered again increased. Slight rise in number of leuco-
cytes on third day after injection was probably not due to action of venom. It is difficult to explain why
no leucocytosis was found in this case; it may have occurred during the night.

L Hb. Rbc. 10 12 1 2 3 4 11.45 90Nn 21 234 9.30.
Gm. | AM T I 38
54,000 per boo |
100 DOG 1 DOG 2
52,000 K 6. 1 0.0289 Gm
50,000 dried venom i
injected
—o \ 0.046 Gm
46,000 dried venom
44,000 | \ injected
3 |
42,000 2, 1
40,000 ez
38,000 a2 | \
mw \
36,000 3s
Oa
34,000 25 »
=a a
32,000 32 { 3
30,000 gz 3
28,000 ie : 2 \
Le
26,000 + \ 7
24,000 = \
u
22,000 .
; o
20,000 / : rk \/
18,000} + 9.000.000 / : /
16,000 }—+— 8,000,000 A Sa
14,000 L—++— 7,000,000 7
12,000 + 24 -6,000,000 A JIN
“10,000 |- 20 -5.000.000 L i
8,000 | 16 —4.000,000 gia 4--E-}-
\ J ot *
6,000 - 12 — 3,000,000 7 ‘
4,000 KE 4 — 2,000,000
2,000 L4 — 1,000,000

Fra. 38.—Dog 1. Leucocytosis followed injection of venom, part of which was given intravenously.
Dog 2. Leucocy tosis followed intravenous administration of venom, 7

ACTION ON THE CELLULAR ELEMENTS OF THE BLOOD. 185

SUMMARY.

The subcutaneous injection of solutions of heloderma venom in quantities
varying from 5 to 27.5 mg. into a series of 16 non-immunized, apparently
healthy rabbits of an average weight of 1,500 grams, led in many cascs to a rise
in the number of leucocytes within the 24 hours following the injection. This
rise in the number of leucocytes was frequently preceded by aslight fall. This
rise in the number of leucocytes was not noticeable in all cases; it was absent in
several cases, in which relatively large quantities of venom had been injected.
It was also absent in three cases in which the animals died a few hours after the
injection. In one of these rabbits the injection was followed by a marked fall
in the number of leucocytes.

In two healthy, non-immunized dogs, non-lethal doses of venom admin-
istered partly or entirely intravenously were followed by a leucocytosis that
was noticeable as early as 2 hours after the injection and persisted at least a day.
The increase in the number of leucocytes was in all cases due to an increase in
the polymorphonuclear neutrophiles or pseudoeosinophiles. In contradistinc-
tion to ordinary rabbits, a rabbit that had gained a certain degree of active
immunity through frequently repeated injections of gradually increasing doses
of venom showed a very marked leucocytosis following the successive injections
of 55 and 60 mg. respectively of the venom. In this animal there seems to
been a slight but constant increase in the number of leucocytes. After the
injection of 55 mg., an increase in the number of leucocytes appeared 2 hours
30 minutes after the injection, 4 hours 45 minutes after the injection the increase
was greater, and was very high 22 hours after the injection. After administra-
tion of 60 mg. of venom, the leucocytosis was observed a day after the injection
and seemed to persist about 24 hours. On two later occasions, when large
doses of the venom (70 and 80 mg. respectively) were given, repeated examina-
tions of the blood during the succeeding 4 days did not show the presence of a
leucocytosis. There can be little doubt that the injection of large doses of
venom during the process of immunization increased very markedly the leuco-
cytosis following the administration of venom.

Whether this leucocytosis depends on an actual increase in the number of
leucocytes or on an unequal distribution of leucocytes in the various regions of
the body must at present be left undecided.

Injection of venom into rabbits or dogs has no marked effect on the num-
ber of erythrocytes or quantity of hemoglobin in the circulating blood.

THE INFLUENCE OF HELODERMA VENOM UPON
PHAGOCYTOSIS.

By Lucius Turrte.

THE INFLUENCE OF HELODERMA VENOM UPON
PHAGOCYTOSIS.

By Lucius Tutte.

We have tested the influence of the venom of Heloderma upon the pro-
duction of a cellular exudate in the peritoneal cavity of the guinea-pig, upon
the phagocytosis of red corpuscles of the pigeon in the peritoneal cavity of a
guinea-pig, and also upon the phagocytosis of bacteria in vitro by the leuco-
cytes of the dog in vitro.

The influence of venom upon the production of cellular exudate was
studied in the peritoneal cavity of 18 guinea-pigs. On the first day of the
experiment we injected sterile bouillon into the peritoneal cavity of the guinea-
pigs; on the following day, approximately 24 hours after the injection of the
bouillon, we injected into the peritoneal cavities of half of the guinea-pigs small
quantities of venom, usually 4 mg. pro kilo. of body-weight. The other guinea-
pigs received no injection on the second day, except in a few cases in which we
injected 0.85 per cent sodium-chloride solution. At different times, from 1 to
5 hours after the injection of the venom and 25 to 29 hours after the injection
of bouillon, the fluid was drawn from the peritoneal cavity, smears were made,
stained, and examined microscopically.

We found the same characteristic exudate in all cases, both in those in
which venom had been injected and in those in which no venom was injected.
This peritoneal exudate was composed mostly of polymorphonuclear leuco-
cytes, with a few mononuclear leucocytes. The venom therefore seems to
exert no influence on the formation of cellular peritoneal exudate.

In the next experiments we injected venom (4 mg. pro kilo.) and pigeon’s
red corpuscles into the peritoneal cavities of guinea-pigs, making several injec-
tions before removing the peritoneal fluid for microscopic examinations. On
two or three successive days we injected venom, and each injection of venom
was immediately followed by an injection of defibrinated pigeon’s blood. In
the majority of experiments we injected the venom and pigeon blood on two
successive days, and on the third day venom alone. At various periods from’1
to 24 hours after the last injection of venom, fluid was removed from the peri-
toneal cavity and examined microscopically. Control experiments were made
in which pigeon erythrocytes, but no venom, was injected.

In studying the smears of the peritoneal fluid, we noted the number of
phagocytes containing pigeon-blood corpuscles, the number of pigeon erythro-
cytes lying free in the fluid, the number of guinea-pig erythrocytes, and the
amount of granular detritus.

189

190 THE VENOM OF HELODERMA.

Usually there was little granular detritus, which bore no relation to the
phagocytosis and varied from time to time in the various individuals. No
relation existed between the amount of granular detritus and the injection or
non-injection of venom. A few guinea-pig blood corpuscles were observed in
the peritoneal fluid in every case, but in no case was any large number found.

In certain experiments, no pigeon corpuscles were found in the smears.
We have not included these experiments in our series. When venom was in-
jected into the peritoneal cavities of guinea-pigs, we found marked or moderate
phagocytosis in 15 out of 19 cases, while, when no venom was injected, we
found similar conditions in 19 out of 23 cases. It thus appears that the degree
of phagocytosis does not differ materially after the injection of venom.

In one series of these experiments we counted the number of phagocytic
cells containing few pigeon corpuscles and the number containing many cor-
puscles. In each animal of the control as well as of the venom series 100:
mononuclear leucocytes were classified in each case.

Percentage of mononuclear leucocytes containing pigeon corpuscles.

Animals injected with venom. Animals used as controls.

No. of With many} With few No. of With many| With few
phagocytes. | corpuscles. | corpuscles. phagocytes. | corpuscles. | corpuscles.

p. ct p. ct. p.ct p. ct

13 42 16 47

7 39 9 28

12 44 14 33

18 36 12 35

11 27 17 39

Average.. 13 36 Average. 12 37

From this table it will be seen that the leucocytes of the animals which
received venom took up the pigeon’s corpuscles in almost exactly the same
manner as did the corpuscles of the normal animals.

Proportion of leucocytes containing few and many bacteria per hundred leucocytes
not containing any bacteria.

Bacteria with venom. | Bacteria without venom.

Amount of venom.

None. | Few. | Many. | None. |} Few. | Many.

10 mg. (0.5 per cent 100 ie * 108 os rH
solution of venom) 100 69 16 100 67 16
100 110 50 100 60 15

MMO B iss 38 oe Saecbantiie 100 127 45 100 56 19
100 131 62 100 88 19

100 69 22 100 04 15

BME. wien emaasies 100 70 30 100 106 16
100 125 27 100 64 ll

The influence of venom on phagocytosis was tried 7n vitro with the corpus-
cles of a dog. These corpuscles were obtained by injecting a suspension of
aleuronat into the pleural cavity of a dog. The dog was killed about 36 hours
after the injection and the pleural exudate removed. To 1 c.c. of this exudate

INFLUENCE UPON PHAGOCYTOSIS. 191

containing mostly polynuclear leucocytes we added 0.5 ¢.c. of a 24-hour bou-
illon culture of a staphylococcus and to five tubes, 10, 7, 5, 2, or 1 mg. of venom
dissolved in 0.85 per cent NaCl solution, while to five control tubes equal quan-
tities of salt solution were added. These tubes were placed in an incubator
and smears were made and examined 2, 4, and 18 hours later.

We carried out four experiments with the exudate of 4 dogs. No differ-
ence was found between the degree of phagocytosis in most cases. In one
experiment the leucocytes in the tubes containing 7 and 5 mg. had taken up
fewer bacteria than their controls, but in all other experiments neither 10, 7,
nor 5 mg. of venom influenced the phagocytosis.

We may conclude that the venom in no way prevents the phagocytosis of
bacteria. The wide variations observed in these experiments are probably
due to individual variations.

CONCLUSIONS.

The intraperitoneal injection of heloderma venom into guinea-pigs does
not modify the cellular exudate resulting from the intraperitoneal injection of
sterile bouillon.

The injection of heloderma venom into the peritoneal cavity of guinea-
pigs in no way interferes with the phagocytosis of pigeon’s blood.

The addition of heloderma venom to mixtures of dog’s leucocytes and
staphylococci in vitro does not inhibit phagocytosis.

It is therefore apparent that heloderma venom has no injurious influence
on leucocytes and that it does not diminish the phagocytic activity of the
leucocytes.

\
|

XI.

CAN THE PRESENCE OF ANTIBODIES TO THE VENOM OF
HELODERMA SUSPECTUM BE DEMONSTRATED IN THE
BLOOD SERUM OF THIS ANIMAL BY THE METHOD OF
COMPLEMENT FIXATION?

By Eten P. Corson—-Wuire.

CAN THE PRESENCE OF ANTIBODIES TO THE VENOM OF
HELODERMA SUSPECTUM BE DEMONSTRATED IN THE
BLOOD SERUM OF THIS ANIMAL BY THE METHOD OF
COMPLEMENT FIXATION?

By Extiten P. Corson-WHITE.

An animal secreting a poison deadly to itself would be a rare instance of
the preservation of a factor detrimental to the individual and to the race.
Many stories of poisonous snakes were found in olden times and out of their
mists Francisco Redi (1)* first noted the fact that poisonous snakes were harm-
less to themselves. Fontana (2, 3) later gave experimental proof of this immu-
nity by forcing vipers to bite themselves or each other. Phisalix and Bertrand
(4) confirmed Fontana, and were able to show that harmless reptiles, while less
resistant than poisonous snakes, would withstand doses of venom fatal to other
animals. Weir Mitchell (5) had previously demonstrated the same fact in
cases of the rattlesnake, both when bitten and when the venom was injected
subcutaneously or intravenously. Since these observations, auto-immunity
has been described in scorpions (6), vipers, black snakes, cobras, and in prac-
tically all poisonous snakes. Fayrer (7), in his study of Indian snakes, says
that a serpent not only is not poisoned by its own venom, nor even with that of
another individual of its own species, but is only slightly susceptible to the
venom of another species. Its venom, however, often kills an innocuous rep-
tile; the smaller this serpent and the less poisonous it is, the more readily it
succumbs to the poison. Cooke and Loeb (8) have found this same protective
power in the Gila monster. Natural immunity, while generally present in
snakes, is not absolute (9), and can be overcome by sufficiently large doses of
venom (10).

What gives thisimmunity tosnakes? Phisalix and Bertrand (11) thought
that the protective power was due to the presence of venom, which entered the
blood as an internal secretion from the venom glands of the upper jaw. These
glands have been described by many observers—Fontana (2), Leydig (12),
Reicht (13), Blanchard (14), Jourdain (15), ete.—in both harmless and poison-
ous snakes.

Phisalix found that the blood of many snakes was distinctly toxic (16); if
injected subcutaneously or intraperitoneally it produced local and general
effects very similar to that produced by venom. These observers removed
(17) the poison glands from 46 vipers and 167 days later injected the blood

*The figures in parentheses refer to the Bibliography on page 198.
195

196 THE VENOM OF HELODERMA.

from the viper into guinea-pigs. All these guinea-pigs survived, while all the
control animals injected with the blood of normal vipers died. The toxicity of
the blood is destroyed by heating it to 68° C., but if the heated blood is injected
into guinea-pigs it confers an increased resistance to venom.

Calmette (18) also observed this and called attention to the fact that while
the toxicity of blood was lost at 68°C., the venom would resist prolonged heat-
ing at much higher temperatures. He concludes that the toxic substance is
probably not venom but a diastatic substance, physiologically distinct, but
analogous to some constituent of the venom. It may be a forerunner from
which the venom is produced in the process of secretion by the poison gland.

Flexner and Noguchi (19, 20) made no definite statement. concerning this
fact, but distinguished between the two sets of active principles by their capa-
bility to unite with or be activated by their homologous and heterogeneous
complements.

Fraser (21) thinks the immunity depends on changes, similar to artificial
immunization, produced in the serum by constant absorption of venom, which
all snakes secrete in varying amounts. The venom may enter the body through
abrasions, through the mucous membrane of the alimentary tract, or may be
absorbed directly from the venom gland by its lymph channels. The animal
(4) may also receive venom from its own bite or the bite of other animals. In
the artificially immunized animal, definitely antitoxic substances may be dem-
onstrated by the usual methods. Metschnikoff (22), by mixing the blood-
serum and venom of a scorpion, showed an undoubted neutralizing power in the
blood.

Fraser (2) found that the serum of a Hamadryas destroyed the toxie action
of cobra venom; this power was present both when the blood-serum and venom
were injected simultaneously or at intervals of 15 or 30 minutes. This fact
also he found was true of the blood serum of the Australian blacksnake and the
venom of the same snake. His experiments show that definite substances are
present in the serum of poisonous snakes antidotal against their own or alien
venom. Natural antidotal substances are, however, not as strong as those
found in the blood of animals immunized against venom.

In the case of the Heloderma suspectwm, Cooke and Loeb showed that
while the monster possessed an immunity against its venom, its blood was not
toxic, nor did it possess antidotal properties. Notwithstanding this deficiency
in demonstrable antitoxin the serum might perhaps contain certain antibodies
which could lhe demonstrated by the complement fixation test of Bordet and
Gengou (23), especially inasmuch as Cooke and Loeb (8) have shown that the
venom contains an antigen which on injection into rabbits causes the formation
of precipitins.

Two series of experiments were made to test for the presence of antibodies
in the blood-serum or the power of the mixture of serum and venom to fix com-
plement. In one, the active and the inactive serum of the Heloderma was used
with an antihuman hemolytic system and in the other inactive serum and an
antisheep system.

DEMONSTRATION OF PRESENCE OF ANTIBODIES. 197

A preliminary titration of the serum was made with both hemolytic
systems to determine the amount of complement fixed by the serum alone.
Guinea-pig serum was used for the complement and such dilutions of this
serum were made that 0.1 ¢.c. of the dilution would equal respectively 0.01,
0.02, 0.03 ¢.c., ete., up to 0.1 ¢.c.

Active heloderma serum to the amount of 0.5 ¢.c. was incubated for 2
hours with 0.1 ¢.c. of each dilution of complement and likewise two other sets,
II and III, of 0.5¢.c. of inactive sera with 0.1¢.c. of each dilution of guinea-pig
serum. At the expiration of this time, to the first series was added 1 ¢.c. of 5
per cent suspension of washed human cells and 2 units of antihuman ambo-
ceptor, to series IT the same was added, and to series III, 1 c.c. of 5 per cent
washed sheep-cells suspension and 2 units of antisheep amboceptor. After a
second incubation of an hour the result was read. In all, three separate titra-
tions of the serum were made with the different systems. The complement
present in the unheated serum activated the amboceptor and in every tube
there was complete hemolysis. The result with the inactive serum and the
two hemolytic systems were practically the same.

(1) 0.5 e.c. 1-3000 dilution of venom and .05 ¢.c. complement.
(2) 0.5 e.c. 1-6000 dilution of venom and .05 c.c. complement.
(3) 0.5 ¢.c. 1-12000 dilution of venom and .05 c.c. complement.
(4) 0.5 ¢.c. 1-24000 dilution of venom and .05 c.c. complement.
(5) 0.5 ¢.c. 1-48000 dilution of venom and .05 c.c. complement.
(6) 0.5 ¢.e. 1-64000 dilution of venom and .05 c.c. complement.
(7) 0.5 e.c. 1-80000 dilution of venom and .05 c.c. complement.
(8) 0.5 e.c. 1-96000 dilution of venom and .05 ¢.c. complement.
(9) 0.5 ¢.¢. 1-128000 dilution of venom and .05 c.c. complement.

Incubate for 2 hours at 37° C., then add 1 ¢.c. washed erythrocytes and 2 units of
amboceptor to every tube and again incubate for 1 hour at 37° C.

Result: Result:
(1) No hemolysis. (6) Partial hemolysis.
(2) No hemolysis. (7) Partial hemolysis.
(3) No hemolysis. (8) Complete hemolysis.
(4) No hemolysis. (9) Complete hemolysis.

(5) No hemolysis.

In the three titrations using the sheep system the dose absorbed was
between 0.05 + 0.06 c.c. of complement.

Three different titrations with the two systems were then made, using a
fixed dose of 0.05 ¢.c. of complement and 0.5 c.c. of varying dilutions of the
venom.

Another titration was now made of the venom in dilutions between 40,000
and 90,000, fixing the dose causing almost complete hemolysis at 1 to 70,000.

Having decided on the amount of complement fixed by the serum and
venom, four series of fixation tests were made with the two hemolytic systems,
using in every case a complement 18 hours old and made up of the blood of
three guinea-pigs. 0.5 c.c. of serum was added to 0.5 c.c. of 1 to 70,000 dilu-
tion of venom and the sum of the amounts of complement absorbed by each
factor alone added; that is, 0.05 c.c.+0.05 c.c. or 0.1 ¢.c. of complement; con-

198 THE VENOM OF HELODERMA.

trolling this were similar tubes with increasing and decreasing doses of com-
plement.

(1) 0.5 ¢.c. serum and 0.5 ¢.c. venom and .15 ¢.c. complement.
(2) 0.5 ¢.c. serum and 0.5 ¢.c. venom and .12 ¢.c. complement.
(3) 0.5 ¢.c. serum and 0.5 ¢.c. venom and .10 ¢.c. complement.
(4) 0.5 ¢.c. serum and 0.5 c.c. venom and .08 c.c. complement.

(5) 0.5 ¢.c. serum and 0.5 ¢.c. venom and .06 ¢.c. complement.

Incubate for 2 hours at 37° C., then add 1 ¢.c. washed erythrocytes and 2 units of
amboceptor and incubate again for 1 hour at 37° C.

Result: Result: :
(1) Complete hemolysis. (4) No hemolysis.
(2) Complete hemolysis. (5) No hemolysis.

(3) Partial hemolysis.

CONCLUSIONS.

In all four tests made and using both systems, no true fixation of comple-
ment could be determined. In no case was more complement absorbed than
the sum of that fixed by the venom and serum alone. By this method, there-
fore, the presence of an antibody can not be demonstrated.

BIBLIOGRAPHY.

1. Rept, F. Observationes de Viperis scriptae literis ad gener. dominum Laurentium
Majalotti 18 Amsterd., 1675. (Quoted in ‘‘ Researches on Rattlesnakes,” by Weir
Mitchell, Washington, 1860.)

2. Fontana. Recerches fisiche sopra el vileno della vipera. Lucca, 1767. Translated by
Skinner. London, 1787.

3. Fontana, F. Abhandlung iiber das Vipera gift. Berlin, 1787.

4. Patsatix and Bertranp. Compt. rend. Soc. de Biol., 1893.

5. Werr Mitcueiy. Researches upon the venom of rattlesnakes. Smithsonian Contribu-
tions to Knowledge, 1860.

6. Bourne. Proc. Royal Society, London, 1887.

7. Farrer. The Thanatophidia of India, London, 1874.

8. Cooxe and Logs. Proc. Soc. Exp. Biol. and Med., 1908, v, 104.

9. Patsatrx and Bertrand. Compt.rend.Soc. de Biol, 1893; also Compt. rend. Acad.&ci.,
1893.

10. Bernarp, C. Lecons sur les effets des substances toxiques, Paris, 1857.

11. Puisatrx and Bertranp. Archiv de Physiol, 1894.

12. Leypie. Archiv fiir mikrokopisches Anatomie, 1873.

13. Retcut. Morphologisches Jahrbuch., 1883.

14, Buancnarp. Compt. rend. Soc. de Biol., 1884.

15. JourpaIn. Compt. rend. Acad. Sci., 1894.

16. Parsatrx and Berrranp. Compt. rend. Soc. Biol., 1893; also Compt. rend. Acad. Sci.,
1893.

17. Paisatrx and Bertranp. Compt. rend. Acad. Sci., 1894; also Compt. rend. Soc. Biol.,
1894,

18. Catmerre, A. Venoms; trans. E. I. Austen, London, 1908.

19. Frexner and Nocucat: Jour. Path. and Bact., 1903.

20. Noaucnt. Snake Venom, Washington, D. C., 1909.

21. Fraser. Proc. Roy. Soc. Edinb., 1895, June 3.

22. Metscunikxorr. Immunity of infectious disease, Cambridge, 1905.

23. Bropret and Gencou. Ann. de |’Instit. Pasteur, xxv, No. 5.

XI.

ACTION OF CALMETTE’S COBRA ANTIVENIN UPON THE
VENOM OF HELODERMA.,

By Moyer 8. FLeIsHER anp Leo LoeEs.

ACTION OF CALMETTE’S COBRA ANTIVENIN UPON THE
VENOM OF HELODERMA.

By Moyer 8. FLetsHer aNnp LEo Lorn.

The question whether the antivenin prepared by Calmette through injec-
tion of cobra venom into horses exerts any influence upon the venom of Helo-
derma seemed to us of sufficient interest to warrant the carrying out of some
experiments in this direction.

The majority of the investigators in the field of snake venoms have come
to the conclusion that antivenins have a definite specific relation to the venom
which served as antigen. This specific relation is of twofold character. In
the first place, the venoms of various snakes contain apparently several poison-
ous substances, which exert a totally different pharmacologic action, as, for
instance, the neurotoxin, the hemorrhagin, and the substance causing throm-
bosis. The chemical relationship of these substances is uncertain at present,
and very divergent opinions concerning it have been expressed.

As far as the immunizing power of snake-venom antisera is concerned,
all investigators admit that an antivenin prepared from a venom that contains
only neurotoxin is powerless against the hemorrhagin or coagulin, which are
contained principally or side by side with neurotoxin in various venoms. Sera
prepared from hemorrhagins as antigen do not neutralize the neurotoxin of
snake venoms.

There exists, however, possibly a second kind of specificity. Are the
neurotoxins of the various snake venoms identical, or do they differ among each
other? The same question may be asked in regard to the hemorrhagins. Here
also the majority of investigators believe that there is a decided specificity of
the various neurotoxins or hemorrhagins, and consequently a specificity of the
antivenins prepared with certain neurotoxins or hemorrhagins. This view is
to a great extent based upon the results obtained by Lamb and is also upheld
by Noguchi. According to this view the antivenin prepared by the injection
of cobra neurotoxin protects only against cobra neurotoxin, or perhaps, though
very much less, against the neurotoxin of very nearly related snakes, while it is
powerless against all other neurotoxins. Calmette, on the other hand, main-
tains that the neurotoxins of the various snake venoms are identical and that
the antivenin prepared with cobra venom as antigen protects against the neu-
rotoxins of other snake venoms.

Under these conditions it was of interest to determine whether the cobra
antivenin possesses any protecting influence against heloderma venom. Helo-
derma venom exerts its lethal influence exclusively through a neurotoxin;
from a pharmocologic point of view it resembles closely cobra venom, although

201

202 THE VENOM OF HELODERMA.

it is not so poisonous as the latter, if equal amounts of the mixture which is
called venom are compared. Cobra antivenin should therefore exert a powerful
protecting influence against the venomofheloderma. On the other hand, from
the point of view of zoological relationship, Heloderma is further distant from
the Cobra than any snake. According to the prevalent view cobra antivenin
should be without any effect on the venom of heloderma.

In our experiments we used a cobra antivenin which Doctor Calmette
kindly put at our disposal; 1 ¢.c. of this antivenin was sufficient to neutralize
1 mg. of cobra venom (tested by Doctor Calmette) ; 0.01 mg. of cobra venom is
a dose lethal for a white mouse; 1 c.c. of Calmette’s serum neutralizes, there-
fore, 100 lethal doses. We used white mice in our experiments. In all cases a
definite quantity of dry venom and Calmette’s serum were mixed in wtro.
The mixture, having stood 45 minutes at room temperature, was injected into
the mice subcutaneously. In control experiments mice were injected with
mixtures of venom and 0.85 per cent NaCl solution, or rabbit serum, horse
serum, and diphtheria antitoxin. None of these latter substances exerted any
protecting influence. In another experiment less than a lethal dose of helo-
derma venom was used (namely, 0.1 mg.),and it was found that rabbit serum
did not increase the toxicity of heloderma venom. Cobra antivenin, on the
other hand, had a definite though very slight antitoxic effect on heloderma
venom; it is able to neutralize a fraction of a lethal dose. After the addition of
cobra antivenin a certain number of mice died as quickly under the influence of
heloderma venom as the control mice injected with venom plus 0.85 per cent
NaCl solution. In each experiment there were, however, others in which the
antivenin delayed the appearance of the heloderma-venom poisoning; 40 per
cent of the animals injected with one or two lethal doses of heloderma venom
mixed with either 0.5 or 0.75 c.c. of cobra antivenin survived, while all the con-
trols died. If more than one or two lethal doses of heloderma venom were used,
0.5 c.c. of cobra antivenin was without effect. In an experiment in which the
antivenin and the heloderma venom were injected at different places without
having been previously mixed in the test-tube, merely a delay in the lethal
action was noticeable.

In an experiment the mixture was kept in the thermostat, and the venom
underwent some deterioration, in consequence of which some of the control
mice survived. This experiment is, therefore, inconclusive.

Although the action of Calmette’s cobra antivenin on heloderma venom is
very slight, the serum neutralizing only a fraction of a lethal dose, there can be
no doubt that it does exist. The same result was obtained in several experi-
ments, while horse and rabbit serum and the diphtheria antitoxin (namely, the
dissolved globulins) were without effect. We can not hold an unknown acci-
dental factor responsible for these results.

We conclude then that Calmette’s cobra antivenin has a slight neutralizing
action on the venom of Heloderma. The action is, however, several hundred
times weaker than on cobra venom. Our experiments prove, therefore, the
existence of a relative specificity of the snake-venom antitoxins, prepared with

ACTION OF CALMETTE’S COBRA ANTIVENIN. 203

the neurotoxins as antigen, the antitoxin acting strongly on the neurotoxin
used as an antigen, and only very weakly upon the neurotoxin of a related
reptile.

The recent experiment of Bang and Overton,* who found that the cobra
antitoxin had a protecting influence on tadpoles which were kept in a solution
of crotalus venom, to which the cobra antivenin had been added, seems to
point to a similar conclusion as that reached by ourselves. We must take into
consideration the fact that Bang and Overton observed the neutralizing effect
in experiments with crotalus venom, a substance rich in hemorrhagin; but per-
haps in this case also the protecting influence of the antivenin was mainly
directed against the neurotoxin in the crotalus venom.

The following table gives a summary of our result:

Mice . : Mice
Fluids injected. used Died. Survived.| used as
eae controls.

Calmette serum 0.5 ¢.c.. venom 0.15 mg.......... 12 7 4 4
Calmette serum 0.5 c.c.. venom 0.3 mg... 4 3 1 2
Calmette serum 0.5 ¢.c., venom 0.6 mg... 1 1 0 al
Calmette serum 0.5 c.c., venom 1.2 mg... 1 1 0 1
Calmette serum (.5 c.c., venom 1.8 mg... ae 1 1 0 0
Calmette serum 9.5 c.c., venom 2.25 mg.......... 1 1 0 0
Calmette serum 0.75 c.c., venom 0.14 mg.; mixture

kept at room temperature.................... 4 2 2 4
Calmette serum 0.75 c.c., venom 0.14 mg.; mixture

Kept at'37 16° G ccc) ascac ae nots tere sales carers 4 1 3 t4
Calmette scrum 0.5 c.c., venom 0.15 mg.; injected

SPAT Ate ly ie wsesisvescucew avis sex Soy vin atetennvencssrenanere 2 2 0 1
Horse serum 0.75 ¢.c.; venom 0.14 mg... . 6 6 0 4
Diphtheria antitoxin 0.5 c.c., venom 0.15 mg 2 2 0 1
Rabbit serum 0.5 c.c.; venom 0.15 mg...... 7 2 2 0 1
Rabbit serum 1.0 c.c., venom 0.15 mg........... E 2 2 0 1
Rabbit serum 0.75 c.c.; venom 0.1 mg.; 3 affected

as soon as controls; 3 survived (1 contro! died,

UsSUTVAV OO) cesta sie seer ah irs aaa aes 2 2 0 1

*Bang and Overton. Biochem. Zeitschrift, Bd. 34, p. 428, 1911.
tOnly one of these died.

XU.

INFLUENCE OF VARIOUS STAINS UPON VENOM.

By Lucrus Turtte.

INFLUENCE OF VARIOUS STAINS UPON VENOM.

By Lucius Turrie.

In the following experiments we have determined the influence of heat
upon venom, both before and after the addition of certain stains. We have
tested the influence of these stains upon the toxicity as well as upon the pro-
duction of precipitates. In further experiments we have tested the influence
of light upon venom mixed with various stains, comparing these specimens
with similar ones which had been left in darkness.

INFLUENCE OF STAINS AND HEAT UPON VENOM.

In most of these experiments we used a 0.5 per cent solution of eosin
(G. Gribler, water-soluble) made up with 0.85 per cent sodium-chloride solu-
tion, but in a few cases we used a 0.5 per cent solution of neutral red made
up with the same diluent. In experiments testing the toxicity of the various
solutions we injected quantities of the various mixtures into mice, in one exper-
iment only (an experiment in which eosin was used) we used rats to test the
toxicity of the venom. In all cases we heated the venom for 10 minutes to
a temperature of 100° C.

We found that when 0.15 c.c. of the 0.5 per cent eosin solution was added
to 0.85 c.c. of diluted venom (0.5 c.c. of venom diluted with twice its volume of
0.85 per cent NaCl solution plus 0.35 c.c. of NaCl solution) no precipitate
appeared, but if only 0.1 ¢.c. of the eosin solution was added to 0.9 c.c. diluted
venom (0.5 c.c. of diluted venom and 0.4 c.c. NaCl solution) a precipitate
appeared after boiling for 10 minutes. We furthermore noted that when suffi-
ciently large quantities of 0.5 per cent eosin solution were added to fresh undi-
luted, unfiltered venom, which as usual was turbid, it was possible to diminish
but not to prevent altogether the precipitate. Even though we addeda quan-
tity of solid eosin more than sufficient to saturate the undiluted unfiltered
venom (the solid eosin being dissolved on heating) we were unable to prevent
precipitates in this turbid venom. Aron found previously that addition of
eosin to a proteid solution prevented the formation of precipitates.*

With the neutral red we were unable to prevent precipitation even when
0.3 c.c. was added to 0.7 c.c. of diluted venom (0.5 ¢.c. of venom diluted with
twice its volume of 0.85 per cent NaCl solution and 0.2 ¢.c. NaCl solution).

We tested the influence of eosin and heat combined upon the venom in five
experiments. In each series we injected several mice with different quantities
of venom which had been heated, venom mixed with eosin and heated, and

*Aron. Biochemische Zeitschrift, 1907, v, 413.
207

208 THE VENOM OF HELODERMA.

venom which had been mixed with cosin but had not been heated. It has been
previously shown that heloderma venom may be heated to 100° C. without
seriously impairing its toxicity.

In four of the five experiments the animals (in three cases mice, in one case
rats) injected with unheated venom mixed with eosin died most rapidly; those
injected with venom which had been heated died less rapidly than those injected
with the venom mixed with eosin and heated. In one experiment the degree of
toxicity of the various venom mixtures was practically the same, independent
of whether eosin had or had not been added or whether or not they had been
heated. In this experiment the venom was heated to 100° C. for 10 minutes
and also to 120° C. for 10 minutes. In spite of the negative result of this one
experiment it would appear that the addition of eosin to heloderma venom
prevents the slight harmful influence of heat upon the toxicity of the venom,
perhaps by diminishing the precipitate which includes a part of the venom.

Neutral red we found prevented neither heat coagulation nor the slight de-
structive influence of heat upon the heloderma venom. On the contrary, the
addition of neutral red to venom, even if the mixture was not heated, appeared
to lessen the toxicity. We found that mice injected with either heated or
unheated venom died about 50 minutes after the injection, while mice injected
with either heated or unheated venom to which neutral red had been added
died about 1 hour 30 minutes after injection. We have, however, carried out.
too few experiments with neutral red to enable us to draw any definite conclu-
sion as to whether this stain diminishes the toxicity of heloderma venom or not.

COMBINED INFLUENCE OF LIGHT OR DARKNESS AND VARIOUS
STAINS ON VENOM,

In testing the influence of light or darkness upon mixtures of solutions of
venom and various stains we have used eosin, 1: 500, 1: 2000, and 1: 5000
and methylene blue 1: 500 and 1: 5000. The test-tubes containing the venom
were exposed to the diffuse daylight of the room for several days and the con-
trols were wrapped in black cloth and kept in a drawer. We have tested the
toxicity of the solutions, both those left in the light and those left in the dark,
after 2, 4, and 14 or 18 days.

In every experiment we used fresh heloderma venom diluted with twice its
volume of 0.85 per cent sodium-chloride solution and sterilized. To small
quantities of this sterilized solution equal quantities of a previously sterilized
solution of the stain were added and the tubes placed in previously sterilized
tubes, closed with cotton, and sealed with paraffin. In every case duplicate
sets of tubes were mixed, so that one might be left in the light and the other in
the dark.

We found no difference between the action of the various dilutions of the
same stain. Thus, the action of the 1: 500 solution was the same as of either
the 1: 2000 solution or the 1: 5000 solution of eosin. Therefore, in describing
the results of these experiments, we shall describe only the experiments carried
out with the 1: 500 eosin solution.

INFLUENCE OF VARIOUS STAINS UPON VENOM. 209

We found in the majority of experiments that two days after mixing the
eosin and venom the venom left in the dark had lost little if any of the toxicity
when tested by injections into mice. The venom which had been exposed to
the light, however, had lost a considerable part of its toxicity; when a quantity
equal to one lethal dose was injected, the animal usually survived; frequently
even the injection of a quantity equal to two and a half lethal doses produced
no effect, but when a quantity equal to five lethal doses was injected the mice
died.

Four days after the venom and eosin had been mixed, we occasionally
noted a weakening of the venom which had been left inthe darkness; the toxic-
ity was not lost, but the mice lived longer after the injection. In most cases,
however, no change was noted. The venom which had been exposed to the
light had lost somewhat more of its toxicity; when a quantity equal to two and
a half lethal doses was injected the mice did not die, but succumbed when a
quantity equal to five lethal doses was injected.

After 14 or 18 days we usually found no change in the venom which had
been left in the dark, while the mixture of venom and eosin which had been
exposed to the light was not toxic for mice, even though a quantity equal to five
lethal doses was injected. The two different dilutions of methylene blue, like
the various dilutions of eosin, both acted in the same manner.

Two days after mixing the venom and methylene blue the specimens
exposed to light were not lethal for mice, though a quantity equal to one lethal
dose was injected; at this time the injection of two and a half lethal doses of
the venom-methylene-blue solution exposed to light was able to cause the death
of the mouse. The mixtures which had been left in the dark had lost none of
their activity.

After four days no further change was noted. The injection of two and
a half lethal doses of the venom-methylene-blue solution exposed to light was
still lethal for mice.

After 14 or 18 days the mixtures left in the dark had lost little if any of their
toxic properties. The mixtures left in the light had lost their toxicity and the
injection of a quantity of venom-methylene-blue solution which had been
exposed to the light, equal to five lethal doses, did not have any effect upon
mice.

Noguchi* found that eosin affects the hemolytic and toxic properties of
cobra, daboia, and rattlesnake venom when these are mixed with the stain and
exposed to sunlight for 30 hours. The destructive action was most marked
when the photodynamic action of eosin was tested with rattlesnake and least
marked when tested with cobra venom. Indeed, rattlesnake venom lost
almost entirely its toxic properties after 8 hours’ exposure to the photodynamic
action of eosin. Erythrosin possessed a slightly destructive action on the toxins
of these same snake venoms, but its action was much weaker than that of eosin.

It appears that these venoms react to the photodynamic activity of eosin
in much the same manner as they react to heat. Rattlesnake venom, which is

*Noguchi. Jour, Exper. Med., 1906, vu, 252.

210 THE VENOM OF HELODERMA.

more easily destroyed by heat than cobra venom, is also more rapidly and fully
destroyed by the photodynamic action of eosin.

The toxins of heloderma venom, which are relatively thermostable, are
not easily destroyed by the photodynamic activity of eosin, since it requires
several days for the eosin to destroy the toxic properties of this venom.

SUMMARY.

1. The addition of 0.5 per cent solution of eosin to freshly diluted and fil-
tered venom in the proportion of 0.15 c.c. of eosin solution to 0.85 ¢.c. venom-
sodium-chloride solution prevents the formation of a coagulum even when the
venom is heated to 100° C. for 10 minutes.

2. The addition of neutral red to venom does not prevent the formation
of a coagulum.

3. The addition of eosin to venom prevents the slight decrease in the toxic-
ity of venom usually resulting from heating the venom to 100°C. It is prob-
able that neutral red has no similar action.

4. The addition of eosin (1: 500, 1: 2000, or 1: 5000) or of methylene blue
(1: 500, or 1: 5000) to fresh diluted venom does not influence the toxicity of
such venom even after a period of 14 or 18 days if the venom is kept in the dark.

5. If, however, eosin (1: 500, 1: 2000, or 1:5000) or methylene blue
(1: 500 or 1:5000) be added to venom and this mixture be exposed to the
light, the venom gradually loses its toxic properties and after 14 or 18 days
seems to have lost all toxicity.

XIV.

ADSORPTION OF HELODERMA VENOM BY SUSPENSIONS
OF VARIOUS SUBSTANCES.

By Moyer S. FLeIsHer anp Lro Loes.

ADSORPTION OF HELODERMA VENOM BY SUSPENSIONS
QF VARIOUS SUBSTANCES.

By Moyer S. FLeisHer anp Lor Logs.

We have investigated the adsorptive power of various organic and inor-
ganic substances for heloderma venom; and furthermore, we have determined
whether the various organs of one species and the corresponding organs of ani-
mals of different species showed a specific power of adsorption for the venom of
Heloderma.

TECHNIQUE.

In determining the power of adsorption of certain organic and inorganic
substances and of the organs of various animals for the venom of Heloderma,
we used both diluted fresh and dissolved dry venom. Whenever the fresh
venom was used, I ¢.c. of the venom was added to 9 c.c. of 0.85 per cent sodium-
chloride solution; the dried venom was ground in a mortar to very fine powder
and 0.85 per cent sodium-chloride solution was added, in such quantity that
each cubic centimeter of fluid contained 1 mg. of venom. The solutions of
both fresh and dried venom were always filtered before the addition of the
material whose adsorptive power was to be tested.

In most cases we used 7 c.c. of venom solution and a quantity equal to one-
fourteenth part of the solution of the various adsorbent substances, usually
0.5 ¢.c. A-special note is added in those experiments in which the bulk of the
substances added to the venom solution varied. The venom solution and the
adsorbent substances were mixed in a mortar in order to obtain an even sus-
pension. This suspension, in a small, tightly stoppered bottle, was then placed
in a shaker for 2 hours 30 minutes. At the end of this time the mixtures were
either filtered through filter paper or centrifugated in order to separate the
adsorbent material from the fluid part of the mixture. In our earlier experi-
ments all mixtures were filtered; on account of the fact that a certain amount
of the fluid was held back by the filter paper and because of the length of
time required for the filtration; this method was discarded and in all our later
experiments the centrifuge was used to throw down the adsorbent particles
from the suspension. In a few experiments even prolonged centrifugation did
not separate the solution and the suspended particles, and these mixtures were
filtered through Berkefeld filters.

Throughout our experiments mice were used to test the toxicity of the
supernatant fluid and of the residue.* When the fresh diluted venom was used

*The layer which we call residue contained the coarser particles of the adsorbent material.

213

214 THE VENOM OF HELODERMA.

for testing the adsorptive power of the various substances, we injected in each
mouse quantities of the supernatant fluid which should have contained between
0.050 c.c. and 0.005 c.c. of venom, provided no venom had been adsorbed by the
substance tested; it having been shown that the smallest of these doses of
venom was sufficient to kill a mouse.

In the case of dissolved dry venom we injected either 2 c.c., 1 ¢.c., or 0.5
c.c. of the solution after separation from the adsorbent particles. The small-
est dose injected should, therefore, have contained 0.5 mg. of venom, which is
approximately 34 times the lethal dose.

In the first experiments we used the fresh venom; but in all our later
work the dissolved dry venom, which was more uniform in its action, was
employed.

In testing the toxicity of the residue, we used that portion of the mixtures
which had been thrown down by the centrifuge after the supernatant fluid had
been decanted (or in the case of oils, the surface layer). This residue was
shaken up with 0.85 per cent sodium-chloride solution and again centrifuged.
After the second centrifugation the supernatant fluid containing the venom
which had perhaps been retained between the adsorbent particles, was poured
off and the residue added to 7 c.c. of salt solution. Thus the residue was sus-
pended in a quantity of non-toxic fluid equal to the original venom solution,
and any toxic effect of this emulsion could justly be ascribed to the venom
adsorbed by the particles. In each case 2c.c. of this emulsion corresponding to
133 lethal doses, provided the venom had been entirely adsorbed, were injected
into mice.

In each series of experiments some control mice were injected with the
same quantities of the venom solution.

ADSORPTIVE POWER OF CARMINE.

The adsorptive power of carmine was tested with fresh diluted venom.
Venom solution, mixed with a quantity of carmine equal to half its volume,
entirely lost its toxic action. Three mice injected with quantities of the super-
natant fluid, which, had no venom been adsorbed, should have contained from
one to ten lethal doses of venom, survived the injection. In one experiment
the venom solution mixed with a quantity of carmine equal to one-quarter of
its volume was injected into two mice in quantities corresponding to two and
six lethal doses respectively, and both survived the injection, although both
were slightly affected. In another experiment the supernatant fluid derived
from a mixture of carmine and only one-eighth of its volume of venom solution
was injected into mice in quantities corresponding to a little more than two and
six lethal doses. The first survived the injection and the second died.

According to these experiments, carmine, when present in sufficient quan-
tity, adsorbs the heloderma venom completely or almost completely. A
quantity of carmine equal to one-eighth of the volume of the venom solution
adsorbs more than half of the venom.

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 215

ADSORPTION OF VENOM BY ANIMAL CHARCOAL.

In testing the adsorption of venom by animal charcoal, both fresh and dry
venom were used. In the case of the fresh venom we added quantities of char-
coal equal to one-half, one-quarter, and one-sixteenth of the volume of the
venom solution and found, after these mixtures had been shaken for 2 hours 30
minutes, that all the mice injected with the supernatant fluid survived the injec-
tion. In an experiment in which a quantity of charcoal equal to only one
thirty-second of the volume of the venom solution had been added, the adsorp-
tion was incomplete, and two mice injected with the supernatant fluid from
this mixture died as soon as their respective controls. Quantities of charcoal
equal to one-sixteenth of the volume of venom solution are, therefore, sufficient
to adsorb all or almost all of the venom; while a quantity of the charcoal equal
to one thirty-second of the volume of the venom solution adsorbs less than half
of the venom.

In experiments in which the time of contact between the venom and the
charcoal was shortened to 15 minutes, the adsorption was not complete and
animals injected with two or four lethal doses of venom died. Throughout
subsequent experiments we used solutions of dry venom with a quantity of
charcoal equal to one-sixteenth of the volume of the solution. Only one
mouse of the sixteen injected with quantities of this supernatant fluid died as
a result of the injection, even when amounts corresponding to 134, 62, and 34
were administered. This mouse injected with a quantity of the fluid corre-
sponding to 63 lethal doses lived, however, longer than the control mouse,
which had received a similar quantity of the pure venom solution.

Charcoal not only adsorbs all, or almost all, of the venom, but moreover
it holds the adsorbed venom very firmly. Eight mice injected with 2 c.c. of a
suspension containing the residue of the charcoal-venom mixture survived the
injection. It is not probable that the venom was removed from the charcoal
during the washing with salt solution.

The addition of 2 drops of decinormal hydrochloric acid to 7 c.c. of venom
solution interfered with the adsorption of venom by the charcoal in only one
experiment and then only to aslight extent. Nineteen mice injected with the
supernatant fluid of this mixture survived the injection, but two mice injected
with quantities corresponding to 134 lethal doses, two injected with 62 andone
injected with 3% lethal doses, died. These five mice had all been injected
with the same solution, and their death was the only instance in which any of
the mice injected with the supernatant fluid died. Oftheeleven mice injected
with the residue, only two died. The addition of weak acid to the venom-
charcoal mixture may, therefore, cause the bond between the charcoal and the
venom to be more easily broken after the injection of the residue into a living
organism.

In other experiments we added first two drops of decinormal sodium
hydroxide and subsequently the usual volume of charcoal to 7 c.c. of venom
solution. Of 21 mice injected with the supernatant fluid, 1 died as soonas the

216 THE VENOM OF HELODERMA.

control, 3lived longer than the controls, and 17 survived the injection. Sev-
eral of the mice which survived the injection were quite markedly affected by
the venom, showing weakness, as well as the typical changes in the eyes
described in one of the preceding papers. These results suggest that alkaliz-
ation of the venom solution interferes to some extent with the adsorption of
the venom by charcoal. In experiments in which the charcoal residue of the
alkaline suspensions was injected into mice, three survived the injection,
whereas eight died a considerable time after the injection. Besides interfer-
ing with the adsorption of venom by charcoal, the addition of an alkali to the
venom solution seems, therefore, to prevent the charcoal from firmly binding
the adsorbed venom. Identical results were obtained with various venom
solutions.

The addition of 2 c.c. of either dog or rabbit serum to 7 c.c. of venom
solution interfered much more seriously with the adsorption of venom by the
charcoal. All the animals injected with the supernatant fluid from the serum-
charcoal-venom mixture died, 6 as soon as their controls and 12 after longer
periods. Of the animals injected with the residue, 6 died and 4 survived.
The toxic effect of the solid residue shows that although the addition of dog
or rabbit serum to venom solution seems to prevent almost entirely the ad-
sorption of venom by charcoal, still a small quantity must have been adsorbed
and bound rather loosely by the charcoal.

Finally we tested the adsorption of venom by a mixture of equal parts of
charcoal and lecithin (Kahlbaum). In this experiment a suspension of leci-
thin was obtained by rubbing and finely distributing 0.07 gram of lecithin in
7 c.c. of the venom solution. To this emulsion a quantity of charcoal equal
to one-fourteenth of the bulk of the solution was added. Of the mice injected
with the supernatant fluid five survived, and only one, which had been in-
jected with 133 lethal doses of the venom, died. Of the three mice injected
with the residue of the charcoal-lecithin mixture, only one died. This mix-
ture of charcoal and lecithin acts, therefore, in approximately the same man-
ner as the charcoal unaccompanied by the lecithin; the largest part of the
venom is adsorbed and is held by the charcoal-lecithin mixture and in such a
manner that it can not be absorbed by the injected animal. It is furthermore
very probable that the charcoal rather than the lecithin held most of the
venom; it was later found that the lecithin did not bind the venom as tightly
as the charcoal.

ADSORPTION OF VENOM BY KAOLIN.

To the solution of dried venom a quantity of kaolin equal to one-four-
teenth of the volume of the solution was added and the mixture kept in the
shaking apparatus for 2 hours 30 minutes. Of 18 mice injected with this
supernatant fluid 6 survived, 10 lived longer than their controls, and 2 died as
soon as the controls. Of these last 2 mice 1 had received a quantity of fluid
corresponding to 134, the other to 63 lethal doses. These experiments indi-
cate that kaolin does not adsorb as much venom as charcoal, but it may, how-
ever, adsorb from 70 to 80 per cent of the venom.

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 217

Eight mice injected with the residue died. The kaolin seems to hold the
adsorbed venom very loosely and the bond between the kaolin and the venom
is easily broken after the injection of the kaolin into a living organism.

ADSORPTION OF VENOM BY ALUMINIUM OXIDE.

Two different samples of Al,O, were tested. Three mice injected with
the supernatant fluid obtained from the first sample (Eimer & Amend’s pre-
paration) died as soon as their controls. This solution was found to give an
alkaline reaction.

A suspension of a second sample of Al,O, gave a neutral reaction. In
this experiment 12 mice injected wth the supernatant fluid survived while 1
receiving 34 lethal doses died. On the other hand, 7 mice injected with the
residue of Al,O, died; 6 of these had been injected with the residue of the
second, 1 with the residue of the first (alkaline) sample of aluminium oxide.
It appears, therefore, that aluminium oxide adsorbs the venom very well, but
that here, as in the case of charcoal, the alkali interferes with the adsorption.
The venom is, however, only loosely held by the aluminium oxide.

ADSORPTION OF VENOM BY OLIVE OIL.

By prolonged shaking an emulsion was made of one volume of olive oil
in fourteen volumes of venom solution. All of the mice injected with this
fluid after separation from the larger oil droplets died as soon as their respec-
tive controls. It was found that centrifugation alone did not free the fluid
entirely of the finer oil droplets, and inorder to obtain a fluid free from these
droplets the mixture was passed through a Berkefeld filter and the filtrate
injected into mice. All of the 6 mice injected with this filtrate ultimately
died after having survived their controls for considerable periods of time.
Furthermore, of 12 mice injected with the residue (containing both large and
small oil droplets) only one died. It is, therefore, probable that some venom
is adsorbed by the very fine oil droplets which are not separated from the fluid
by the centrifugation, but that the amount of venom adsorbed is very small.

ADSORPTION OF VENOM BY LECITHIN.

In testing the adsorptive power of lecithin, three specimens were used,
namely Merck’s “Ovo,” Kahlbaum’s ‘Aus eigelb,” and Agfa lecithin. In
each case 0.07 gram of lecithin was mixed with 7 c.c. of venom solution. By
rubbing the lecithin with small quantities of venom solution in a mortar a
homogeneous, fineemulsion was obtained. Very fine particles of lecithin were
held in suspension in the supernatant fluid, even after centrifuging. It was
therefore necessary to filter the venom-lecithin mixture through a Berkefeld
filter in order to free the supernatant fluid from particles of lecithin. Both
the supernatant fluid obtained after centrifuging and the filtrate which had
passed through the Berkefeld filter were tested.

Experiments with the supernatant fluid, decanted from the centrifuge
tubes, showed that Merck’s “Ovo” lecithin as a rule adsorbed somewhat less
than 70per cent of the venom; at times, however, it adsorbed somewhat more.

218 THE VENOM OF HELODERMA.

The Agfa lecithin adsorbed approximately the same proportion of the venom,
while Kahlbaum’s lecithin apparently adsorbed somewhat less than either of
the two first-mentioned preparations. This was due to the fact that emul-
sions prepared with Kahlbaum’s lecithin were finer and that consequently the
supernatant fluid contained in this case a larger number of small particles of
lecithin, to which venom adhered.

We find accordingly that the fluid obtained after filtration through a
Berkefeld filter was not more toxic in the case of Kahlbaum’s than in the case
of Merck’s lecithin; in most cases the lecithin adsorbed almost 70 per cent of
the venom and in a few experiments even more.

All the mice injected with 2 c.c. of the washed and reemulsified residue
of lecithin died, irrespective of the kind of lecithin used; one mouse injected
with only 0.5 c.c. of the residue survived the injection. The amount of the
emulsion of the lecithin residue injected in this last-mentioned case should
have contained 3} lethal doses, provided all the venom had been adsorbed.

We may conclude from these experiments that lecithin adsorbs a large
quantity of venom, but that the venom adsorbed exerts its toxic influence if
injected with the lecithin to which it adhered.

The adsorptive power of a mixture of lecithin and cholesterin was also
tested. 0.07 mg. each of lecithin (Kahlbaum) and cholesterin (Merck) were
separately added to 7 c.c. of venom solution. Later a separation of the leci-
thin and cholesterin from this mixture was accomplished by means of a Berke-
feld filter. Three mice, injected with the clear filtrate, survived, while three
mice injected with the supernatant fluid obtained through centrifuging died as
soon as their controls. One mouse injected with the lecithin-cholesterin resi-
‘due died in a very short time. The addition of cholesterin to lecithin appears
to increase the amount of venom adsorbed.

ADSORPTION OF VENOM BY THE ORGANS OF VARIOUS ANIMALS.

We investigated the adsorptive powers of the organs of the heloderma,
turtle, frog, pigeon, guinea-pig, rabbit, and dog. Several organs were tested
in each animal; in every case the kidney and liver and in most cases the brain
also. In a few experiments some other parts were also used. As a rule, the
organs were tested immediately after the death of the animal, before impor-
tant autolytic processes could have taken place. The organs, cut into small
pieces, were rinsed with salt solution, in order to wash out the blood, and then
crushed in a mortar. 7c.c. of venom solution were added to about 0.5 c.c. of
organ pulp. This mixture was rubbed in the mortar until an even suspension
of the organ pulp in the venom solution was obtained.

The further steps were similar to those described above. The solid par-
ticles of the various organs were separated from the solution in most cases by
centrifugation and in a few cases by filtration through a Berkefeld filter.

Heloderma brain was tested with both fresh and dry venom. With the
fresh venom all the animals injected with the supernatant fluid died: one
mouse injected with a volume corresponding to four lethal doses, one injected

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 219

with a volume corresponding to one lethal dose, and two injected with volumes
corresponding to two lethal doses lived a little longer than their controls;
none, however, survived. Two of eight animals injected with supernatant
fluid from the heloderma brain and dry venom mixture lived longer than their
controls, but here also no animals survived. As might be expected from these
results, the two mice injected with the heloderma brain-venom residue sur-
vived. The larger particles, which constitute the mass of the heloderma
brain suspension, adsorb, therefore, very little venom, while to judge from the
experiments with dog brain, to be reported later, a considerable part of the
venom has been adsorbed by the small particles of brain contained in the
supernatant fluid.

Heloderma liver adsorbed a small quantity of fresh venom; all five mice
injected with the supernatant fluid from this mixture lived longer than their
controls, and all but one lived 24 hours after the injection. With the dis-
solved dry venom solution the heloderma liver adsorbed as much as 85 per
cent of the venom, but frequently less than 70 per cent. In some experiments
mice injected with the smaller dose died, while in other experiments animals
injected with a quantity equivalent to 62 lethal doses survived.

Two animals injected with the residue from the heloderma-liver venom
mixture died, and two animals survived. On the whole, therefore, the liver
of heloderma adsorbs a considerable part of the venom.

The adsorptive power of heloderma kidney, when mixed with fresh
venom, was tested in only two animals, both of which died. They both, how-
ever, survived their controls for a considerable time; one had received a
quantity of supernatant fluid corresponding to one lethal dose, the other a
quantity corresponding to two lethal doses.

When heloderma kidney was mixed with a solution of dry venom, as
much as 85 per cent of the venom was adsorbed in some cases, while in others
less than 70 per cent. Four mice were injected with residue from the helo-
derma-kidney venom mixture; one of these died and three survived. In the
cases when the residue was not toxic, the venom had possibly been held more
firmly by the pulp, or it had been washed out with the salt solution used to
wash the residue. The latter interpretation is, however, less probable. From
allthese experiments we may conclude that heloderma liver and kidney adsorb
a considerable amount of venom.

One mouse injected with a mixture of venom solution and heloderma
serum died approximately at the same time as its control.

Turtle brain, like heloderma brain, adsorbed apparently very little
venom. Two mice injected, respectively, with one and two lethal doses of the
supernatant fluid from the turtle-brain and venom mixture, lived only a
little longer than their controls. The majority of the mice injected with the
supernatant fluid from the mixture of turtle brain and dissolved dry venom
died as soon as their controls. Two mice out of nine, however, survived the
injection; one of these had been injected with a quantity of fluid correspond-
ing to 63 lethal doses; but as in this experiment the mouse injected with 34

220 THE VENOM OF HELODERMA.

lethal doses lived only a little longer than the control, it would appear that
some factor other than the adsorption of the venom was the cause for the sur-
vival of this animal. In another experiment one animal, injected with 3}
lethal doses, survived. The residue of the turtle-brain venom mixture in-
jected into mice caused death in one case, while the only other mouse injected
survived. In the case of turtle brain we have to make the same reservation
as in the case of heloderma brain, namely, that a considerable part of the
venom may have been, and in all probability had been, adsorbed by the small
particles suspended in the supernatant fluid. A small part had fixed itself
upon the surface of the larger particles of the suspension.

Only two mice were injected with the supernatant fluid from the fresh-
venom turtle-liver mixture and both of these died.

In experiments in which dissolved dry venom was used, turtle liver seemed
to adsorb the venom better than turtle brain; 5 of 27 mice injected with the
supernatant fluid survived. These five animals had for the most part been
injected with quantities corresponding to 33 lethal doses of venom, in one case
with a quantity equivalent to 62 lethal doses. Turtle liver may, therefore,
adsorb as much as 85 per cent of the venom, and usually adsorbs more than
70 per cent. Three out of four injected with the residue of this turtle-liver
venom mixture survived the injection. Whether venom was held very firmly
or destroyed by the liver pulp or whether the venom was washed off by the salt.
solution we can not state, although it is improbable that the latter explanation
is correct.

Two mice were injected with quantities of the supernatant fluid from the
turtle-kidney fresh-venom mixture, equivalent to one and two lethal doses of
venom respectively, and both of these animals survived; 8 of the 28 mice
injected with the supernatant fluid from the mixture with dry venom survived,
and 2 of the 8 had been injected with quantities corresponding to 63? lethal
doses. The injection of the residue of turtle-kidney venom mixture was lethal
in three out of four cases. Turtle kidney adsorbs approximately the same
amount of venom as turtle liver.

Two mice were injected with the supernatant fluid from a mixture of
venom solution and turtle egg; one of these, receiving 33 lethal doses, died as.
soon as the control mouse, while the other, injected witha quantity correspond-
ing to 62 lethal doses, lived longer than the control. One mouse injected with
venom mixed with turtle serum died as soon as its control. Neither the admix-
ture of turtle egg or turtle serum diminishes the toxicity of the venom to any
marked degree.

Of 10 mice injected with the supernatant fluid from a frog-liver venom
mixture, 6 died as soon as their respective controls, 3 lived longer, and 1 sur-
vived the injection. The only mouse which survived had been injected with a.
quantity of fluid corresponding to 63 lethal doses. Hence it would seem that
the adsorptive power of frog liver is very slight.

Of 3 mice injected with frog-liver venom residue, 1 died and 2 survived.
These results agree with the findings in the experiments in which the super-
natant fluid was injected.

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 221

The action of frog kidney was tested on 9 mice, only 1 of which (an animal
injected with a large quantity of the supernatant fluid), died in as short a time
as the control; 1 which had received a quantity corresponding to only 34
lethal doses of venom, survived, while 7 lived longer than their controls. Frog
kidney adsorbs, therefore, a small quantity of venom, but less than heloderma
or turtle kidney.

Frog-kidney venom residuc was injected into 3 mice, none of which died.
Since frog kidney adsorbs a small quantity of venom, frog kidney, like turtle
liver and kidney and heloderma kidney, either holds the adsorbed venom suffi-
ciently firmly to prevent the venom from being adsorbed very rapidly by the
injected mouse or it destroys the adsorbed venom.

Three animals injected with the supernatant fluid from pigeon-brain
venom mixture died as soon as their respective controls; one injected with the
residue from the pigeon-brain venom mixture survived. Since thesupernatant
fluid from this mixture contained fine particles of brain matter, we can not
exclude the possibility that pigeon brain adsorbs a certain amount of venom;
it seems, however, that the quantity of venom adsorbed is small.

Of 9 mice injected with the supernatant fluid from pigeon-liver venom
mixture, 2 injected with large quantities died as soon as their controls; 3 lived
longer, while 4 survived the injection. Of the 4 surviving animals, 2 had been
injected with quantities of venom corresponding to 34 lethal doses, 1 with a
quantity corresponding to 63 lethal doses, and 1 with a quantity corresponding
to 134 lethal doses. We may therefore conclude that pigeon liver adsorbs a
certain amount, approximately from 70 per cent to 80 per cent of the venom.
This conclusion is confirmed by an experiment in which 3 mice were injected
with the residue from the pigeon-liver venom mixture; 2 of these died and 1
survived.

The supernatant fluid from a pigeon-kidney venom mixture was injected
into 6 mice; 4 died as soon as the controls; 1, which had been injected with a
quantity corresponding to 63 lethal doses, survived the injection. Pigeon kid-
ney, therefore, seems to adsorb somewhat less venom than pigeon liver. Of
the two animals injected with pigeon-kidney venom residue, one survived the
injection.

Of 5 mice injected with the supernatant fluid from the mixture of fresh
venom with the pulp of the whole brain of rabbits, 4 died as soon asthe controls;
the fifth lived a little longer than the control. Of 4 injected with the super-
natant fluid from a mixture of venom with the gray matter of rabbit brain only,
2 lived longer than the controls, while the others died as soon as the controls.

In an experiment in which the white matter of rabbit’s brain was used in
place of the gray matter, 3 animals out of four lived longer than the controls.
Here again a certain amount of venom had probably been adsorbed by the very
small particles of the brain emulsion.

In 2 cases in which supernatant fluid from a fresh-venom rabbit-liver mix-
ture was injected into mice, the animals died as soon as the controls. Of 3
mice injected, 2 with 133 lethal doses, and 1 with 63 lethal doses, respectively,
from this rabbit-liver dry-venom mixture, died as soon as their controls, whereas

222 THE VENOM OF HELODERMA.

1 injected with a quantity corresponding to 34 lethal doses lived somewhat
longer. Very little venom had, therefore, been adsorbed, and accordingly we
find that a mouse injected with 2 c.c. of the residue of this rabbit-liver venom
mixture survived.

Of 3 mice injected with the supernatant fluid from a mixture of fresh
venom and rabbit kidney, 2 lived longer than the controls, and a third died as
soon asthe control. Little or no adsorptive effect of rabbit kidney was there-
fore evident. Similar results were obtained with the supernatant fluid when a
solution of dry venom had been mixed with rabbit kidney, and, furthermore,
one mouse injected with the residue from this mixture survived. Considerably
less than 70 per cent of the venom is adsorbed by rabbit liver and kidney.

Nine mice injected with the supernatant fluid from a venom dog-brain
mixture died as soon as their controls. Since even by prolonged centrifugation
it was impossible to throw down some of the finest particles of the dog-brain
venom emulsion,” we filtered this mixture through a Berkefeld filter in order to
obtain fluid free from brain tissue; 2 animals injected with a quantity of the
filtrate corresponding to 133 lethal doses and 62 lethal doses, respectively,
lived longer than their controls, while 1 injected with a quantity corresponding
to 34 lethal doses survived the injection. These results make it probable that
the lethal effect of the non-filtered supernatant fluid was in part due to the very
fine particles of brain tissue present in this fluid. These fine particles of brain-
tissue which were removed by filtration had evidently adsorbed considerable
quantities of venom and when injected into mice gave up the venom in sufficient
quantities to cause the death of the animal. We may therefore conclude that,
under the conditions of our experiments, dog brain adsorbs almost 70 per cent
of the venom. We found that 2 of 3 mice injected with residue from dog-brain
venom mixture survived; 1 died. It seems, therefore, very probable that the
fine particles which remained in the supernatant fluid had adsorbed very large
proportions of the venom; it still remains to be seen whether some venom
had been removed from the brain-pulp during the process of washing.

Six mice were injected with the supernatant fluid from a venom dog-liver
mixture; 5 died as soon as the controls; 1 injected with a quantity correspond-
ing to 62 lethal doses survived. Six mice injected with the supernatant fluid
from dog-kidney venom mixture all died as soon as their controls. It is there-
fore evident that dog kidney, like dog liver, adsorbed no venom, or almost none.

Two mice were injected with residue from the dog-liver venom mixture
and 2 with residue from dog-kidney venom mixture; all survived. These
results are in accord with the results obtained in the case of the supernatant
fluid and confirm the conclusion that neither of these organs adsorbs appreci-
able quantities of venom.

Two series of six mice each, injected with the supernatant fluid from a
mixture of venom with either washed dog-erythrocytes or the stroma of these
cells, died as soon as their controls. Notwithstanding these results, injection of

*The supernatant fluid from the brain-venom mixtures contained fine particles of brain substance, not only in
the cage of dog, but of all other species as well; although more pronounced in the case of dog brain.

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 223

the residue from these mixtures proved that erythrocytes adsorb a certain
amount of venom. One of the 2 mice injected with the residue from the dog-
erythrocyte venom mixture died and the injection of the residue from the
stroma-venom mixture caused the death of both of the mice injected. It is
possible that a part of the erythrocyte-venom combination had dissolved in the
supernatant fluid, and this dissolved substance may have caused the toxicity of
the supernatant fluid. We may also have to consider the possibility that some-
how dog erythrocytes increase the toxicity of venom. It is, however, quite
clear that erythrocytes and stroma adsorb a certain amount of venom.

Of 9 mice injected with the supernatant fluid from a mixture of guinea-pig
brain and dry venom, only that one injected with a quantity corresponding to
34 lethal doses survived; 2 injected with a quantity corresponding to 63 lethal
doses lived longer than their controls, and the remaining 6 died as soon as the
controls. Of five animals injected with the residue from the venom-brain
mixture, 4 died. It is difficult to draw a definite conclusion from the results of
these experiments, as the supernatant fluid contained particles of brain in sus-
pension, which may have adsorbed venom, and their retention in the superna-
tant fluid may have increased its toxicity. It was thus necessary to test a fluid
entirely free from these particles. We therefore passed the guinea-pig-brain
venom mixture through a Berkefeld filter.

We then carried out a series of experiments comparing the supernatant.
fluid obtained by centrifugation, the Berkefeld filtrate, and also the Berkefeld
filtrate of a guinea-pig-brain venom mixture, which, however, before the fil-
tration, had not been shaken for the usual period of 24 hours, but which had
been filtered immediately after mixing. Through this latter experiment we
wished to determine whether the passage of the venom-brain through a Be:ke-
feld filter caused either a diminution in the toxicity of the venom through
adsorption of venom by the substance of the filter or through clogging of the
pores of the filter by minute particles of the brain substance. We found the
simple separation of the fluid from the solid portion of the venom-brain mixture
by means of the Berkefeld filter did not cause any marked diminution in the
toxicity of the filtrate; 4 mice injected with such a filtrate from a venom-guinea-
pig-brain mixture died as soon as their controls, and 2 injected with quantities
corresponding to 63 lethal doses lived a little longer than the controls.

All 5 mice injected with the filtrate from the guinea-pig-brain venom mix-
ture (after it had been shaken for the usual period of time) lived longer than the
controls, but none survived. In the control experiments in which the super-
natant fluid from the guinea-pig-brain venom mixture was obtained by centri-
fuging, three animals, one injected with a quantity of fluid corresponding to
133 and two with a quantity corresponding to 63 lethal doses, lived longer than
their controls, while three other animals died as soon as the controls. We may
therefore conclude that guinea-pig brain adsorbs a certain amount of venom,
but less than 70 per cent. Here, as in the case of dog erythrocytes and the
brain substance of other species, we must consider the possibility of a toxic
combination of venom and brain partly soluble in the supernatant fluid.

224 THE VENOM OF HELODERMA.

In the experiments in which the residue of guinea-pig-brain venom mix-
ture was injected into mice, 4 of 5 died, an additional proof that guinea-pig
brain adsorbs some venom.

In the experiments testing the adsorptive power of guinea-pig liver 26 ani-
mals were used; 3 injected with quantities of the supernatant fluid correspond-
ing to 34 lethal doses of venom survived the injection, 12 lived longer than the
controls, and 11 died as soon as the controls. Thus it is evident that guinea-
pig liver may adsorb a considerable quantity of venom. That guinea-pig liver
does adsorb venom is substantiated by the experiments in which the residue of
the guinea-pig-liver venom mixture was injected into mice; 4 of 7 mice injected
with this residue died.

Experiments were undertaken with guinea-pig liver similar to those carried
out with guinea-pig brain, comparing the various methods of separation of the
fluid from the solid portions of the mixtures. If the guinea-pig-liver venom
mixture was filtered through a Berkefeld filter without having been placed in the
shaker previously, the filtrate thus obtained was almost as toxic as the pure
venom solution. Mice injection with the Berkefeld filtrate of the guinea-pig-
liver venom mixture (shaken for 23 hours before the filtration), in the majority
of cases did not die as soon as their controls (4 out of 6 lived longer than their
controls), but none survived. Of 6 mice injected with supernatant fluid from
the guinea-pig-liver venom mixture (separated by centrifugation) at the same
time as these last-mentioned animals, 4 also lived longer than their controls;
the remaining 2 injected with supernatant fluid died as soon as the controls.

In the experiments testing the adsorptive power of guinea-pig kidney, 5
of the 21 mice injected with the supernatant fluid, died as soon as the controls;
12 lived longer than the controls, and 4 which had received quantities of the
supernatant fluid corresponding to 33 lethal doses recovered. We may, there-
fore,conclude that guinea-pig kidney adsorbs in most cases as much as 70 to 80
per centofthe venom. After injection of the residue from a guinea-pig-kidney
venom mixture, 2 out of the 3 animals died. It is, therefore, evident that
guinea-pig kidney, like guinea-pig liver, adsorbs venom, but that the venom is
easily liberated from the organ pulp.

Three mice injected with a venom-egg albumen mixture died as soon as
their controls. Egg albumen exerts, therefore, no antitoxic influence on venom.

CONCLUSIONS.

Of the various substances tested, charcoal adsorbs heloderma venom most
completely. A quantity of charcoal equal to one-sixteenth of the volume of
the venom solution adsorbs all or almost all of the venom, and the adsorbed
venom is held firmly by the charcoal when injected into a living organism.

Carmine adsorbs venom completely or almost completely, when pres-
ent in a quantity equal to one-quarter of the volume of the venom solution, but
does not adsorb very much more than half of the venom when present in a
quantity equal to one-eighth of the volume of the venom solution.

Aluminium oxide, when present in a quantity equal to one-sixteenth of the

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES. 225

volume of the venom solution, adsorbs most of the venom, probably as much
or only a little less than charcoal. Unlike charcoal, the aluminium oxide, when
injected into a living organism, gives off the venom.

Kaolin does not adsorb venom as well as the three first-mentioned sub-
stances, and probably adsorbs but little more than 70 per cent of the venom.
Like aluminium, kaolin holds the venom but loosely.

Lecithin adsorbs considerable quantities of venom, but much less than
charcoal; its action appears to vary considerably ; the addition of cholesterin to
the lecithin does not markedly alter the adsorption of venom. The venom
adsorbed by lecithin readily exerts its toxic action after subcutaneous injection
of the residue, due to the rapid absorption of the injected lecithin and the sub-
sequent dissociation of the venom-lecithin combination.

The addition of dog or rabbit serum to the venom solution interferes very
markedly with the adsorption of venom by charcoal; the addition of a small
amount of alkali tothe venom solution interferes considerably less than the addi-
tion of serum. Under the influence of alkali the venom-charcoal combination
is very much more easily dissociated in the body, alkali preventing the strong
fixation of the venom to the charcoal.

In a similar manner an alkaline aluminium-oxide preparation adsorbs the
venom much less strongly than a neutral preparation.

The addition of a small quantity of a weak acid to the venom solution
interferes only slightly with the adsorption of venom by charcoal.

Olive oil adsorbs but little venom, and a considerable part of that adsorbed
is held by the very finest particles, as in the case of the venom adsorbed by
lecithin.

If we turn to the adsorption of heloderma venom by suspensions of organs,
we can state in general that the pulp of organs adsorbs less venom than charcoal,
aluminium oxide, or carmine. The behavior of brain is very similar to emul-
sions of lecithin. In both cases the small particlesthat remain in the fluid after
centrifugation adsorb a certain quantity of venom. Even after filtration
through a Berkefeld filter these particles are not removed thoroughly and the
supernatant fluid retains a certain degree of toxicity. The whole brain adsorbs
less venom than emulsions of lecithin. That a certain amount of venom is
adsorbed by brain pulp is shown by the fact that the unfiltered is more toxic
than the filtered fluid, and, furthermore, by the toxicity of the residue which we
observed incertaincases. The fact that even after filtration the filtrate obtained
has not become harmless and that in a number of cases the mice injected
with the residue survived, shows that much of the venom remained unadsorbed.

The brain of various species does not on the whole adsorb any more venom
than liver andkidney. This is clear in the case of guinea-pig. Inno case did
the brain of any of the species tested adsorb as much venom as the liver and
kidney of heloderma and turtle. In this respect heloderma venom seems to
differ from cobra venom, which, according to Flexner and Noguchi, lost much
more of its toxicity after adsorption by brain than by other organs. Also,
Calmette and Rogers found that brain adsorbs much snake venom.

226 THE VENOM OF HELODERMA.

On the whole, kidney and liver act similarly in the case of the various
species; when a definite adsorbing power is present in the kidney of a certain
species, the liver will behave in approximately the same manner.

We find in certain cases that the residue is not particularly toxic, although
much venom had been removed from the supernatant fluid through adsorption.
In such cases (heloderma and turtle liver and kidney) several possible explana-
tions have to be taken into consideration. The venom may be so strongly
attached to the residue that at a given time very little venom enters into the
circulation—not more than can easily be eliminated or neutralized by the body;
or the venom may in part have been rendered innocuous through chemical
action of certain parts of the organ pulp on the venom; or—a not very probable
assumption—a part of the venom may have been removed from the residue in
the process of washing. Further investigations will have to decide between
these various explanations.

In the case of dog erythrocytes we found both the supernatant fluid and
the residue toxic. This apparently contradictory behavior will also have to be
investigated in further experiments.

In a preliminary manner we may arrange the organs (liver and kidney) of
various species of animals in the order of their decreasing adsorptive power for
heloderma venom: Heloderma, turtle, pigeon, guinea-pig, frog, rabbit, and dog.
This order suggests a connection between the natural relationship of various
species and their adsorptive power for heloderma venom. Heloderma and
turtle, both reptiles, showed in our experiments the greatest adsorptive power.
The natural immunity of Heloderma against its own venom may possibly in
part depend on a specific adsorbing and neutralizing power of certain of its
organs. These results should, however, at present not be regarded as definite,
the number of our experiments being as yet relatively small; but they should
serve as indicating the direction of further experiments, which might yield
definite results of importance.

We may summarize some of our results as follows: In the case of helo-
derma venom no specific adsorptive or neutralizing action of the brain exists;
other organs, like liver and kidney, adsorb in the case of some animals equally
as much, and in other cases even more venom than the brain.

The adsorption of venom by organ pulp is considerably inferior to the
adsorption by certain other materials, like charcoal, carmine, or aluminium oxide.

There is some indication that differences exist in the adsorptive power of
the organs of different species of animals, and that the organs of heloderma and
of animals nearly related to Heloderma adsorb more venom than some other
animals less nearly related, and that the natural immunity of Heloderma to its
own venom may in part depend on this specific adsorptive power. The num-
ber of our experiments is perhaps not yet large enough to establish these con-
clusions; they, however, suggest such an interpretation. It remains for further
experiments to decide definitely the value of this hypothesis.*

*Wolff-[isner also suggested that the liver and perhaps other organs were of importanco in protecting the brain
from the action of certain poisons and in conferring immunity against certain toxic substances. (Centralblatt f. Bact.,
Originale, Bd. 47, 1908; Bd. 48, 1908-09.

ADSORPTION BY SUSPENSIONS OF VARIOUS SUBSTANCES.

Experiments with fresh venom.

227

Material.

Died as soon as controls.

controls.

Lived longer than

Charcoal, 3 bulk
Charcoal, } bulk. .
Charcoal, } bulk
Charcoal, 7s bulk
Charcoal, «x bulk... .
Charcoal, } bulk (mis ed
only 15 minutes)*. .
Carmine, } bulk
Carmine, } bulk
Carmine, } bulk

10

|
neal ae Se ac
; , a
pe lles :

6 | 5

i

Doers

meee

*The controls, charcoal } bulk mixed 23 hours, also died as soon as controls.

Experiments with dissolved dry venom.

Material.

Lived longer than

controls. controls.

Survived.

' Residue, 2c.c.

Died as soon as |
|

Charcoal
Charcoal and lecithin. .
Charcoal and acid...
Charcoal and alkali....
Charcoal and dog serum.
Charcoal and rabbit serum
Kaolin
AlOs, alkaline
AlO3. neutral
Olive oil, centrifuged
Olive oil, Berkefeld..
Lecithin (Merck).

Lecithin (Merck) ‘Berkefeld..

Lecithin (Kablbaum) .

Lecithin and
Berkefeld

stetin.....
cholesterin,

5! men.
a Ces

ete eee POP PT

AIH AD

|

| Died.

Or 0 MOM DOH ONDE:

me meet:

rey

Experiments with fresh venom.

Died as soon as controls.

Lived longer than
controls.

Survived.

Material.

Heloderma:

Ean gray....

roy
me HED

cero
ee

ro corre

Domne

THE VENOM OF HELODERMA.

Experiments wilh dissolved dry venom.

Died as soon as | Lived longer than Survived. ' Residue, 2 ¢.c.
controls. controls. ;
i
Material. —<—- ae arene ——u
1 i : "i
| 134 | 63 3h 133 | 63 | 33 | 133 | 63 | 38 Rio ' Died.
| fe set |
i | 4 :
Heloderma: | | | | I
Brain Be Be otf | Oy CU haa ; 2
ee | 1 6 40) 2h 3 2 2
z 3 Bor F Be Te 2 5 3 1
MeN) sete” | ' ts as
3 fi 8 1] ot ls. t } 2 1 1
2 1 | 4 7 Geel Ce Ro | 1 | 4 3 1
Bogs 2 il ll 8 4, 4 1 2 5 3 1
ani Le hee Pens) Pees Mga eas Lb
a s ‘ ‘ . | sa ais ae
a ae ae et 2 1 | 1 as 2 *1
1 | A 2 oe $2 1 3
aL? 7h) ae ae ile : bo a
22 Joa 1 1 1 2 1 2
y oo Bae, ¥ a a | 1 5a 1 a | 1 a 1
Rabbit: | : |
[vGr dense vose: <2 ugk. 0 1 wee Delage (PS 1
Kidney 1 1 es i 1 Meo Had 1
Guinea-pig brain, not put in | j
shaker, _ but filtered 5
through Berkefeld....... 2 bes 2 | 2 ae | Ih og
Guinea-pig brain, mixed and | |
centrifuged............. 3, 1 2 {2 whey 38M} 1 | 1 4
Guinea-pig brain, Berkefeld ; y
IRGE dash omcanc ahah cs i bBo Ay B84 |
Guinea-pig liver not put in | i !
shaker, but filtered | ‘ '
through Berkefeld...... tae | anes | Abie Hm sesso |
Guinea-pig liver, mixed and | | |
centrifuged.............. 4." 2 a | 8 6 1} 3 | 3 4
Guinea-pig liver, poke ' | :
fillers cena ima de 4 We Abt ag Ul Va 1 21 1 | ee ba
Guinea-pig kidney 2 | 1 2 5 At oR a 43 1 2)
Dog brain centrifuged. 3 1 3 Sieaie poe: ae | 2 ' i oe 1
Dog brain, Beckelele', a 2 all 1 1 | 1 | oe ss
Dog liver. 2: 21 1 jets a ‘ oe on 2 |
Dog kidney. 2 2 2 0 s ea j 2
Dog erythrocytes. 2; 2 P95 | eon | 1 1
Dog stroma..... ...-- te 2 2 ui “e F ; ne 2
Egg albumen...... ..... .. 1 1 1 | yes ee
1 !

*Mixed with bile.

XY.

BIOCHEMICAL STUDIES UPON THE VENOM OF
HELODERMA SUSPECTUM.

By Cart L. ALSBERG.

BIOCHEMICAL STUDIES UPON THE VENOM OF
HELODERMA SUSPECTUM.

By Cari L. ALsBERG.

The most important study of the chemical nature of the venom of Helo-
derma was made by Santesson.* His conclusions were in the main that no
alkaloidal substances were present, but that the toxic principle was part nuclein
and part albumose. He also discovered that boiling a solution of the venom,
even in the presence of acetic acid, did not materially injure its toxicity; and
that the toxic principle was precipitated but not coagulated by alcohol and
similar agents. The purpose of the present investigation was to endeavor to
isolate the toxic principle or, failing that, to extend the observations of San-
tesson. It was expected that these questions might be solved because more
material was available than Santesson had, and because in recent years, through
the work of Faust on cobrat and rattlesnakef venom, our knowledge of the
chemistry of venoms of reptiles has been greatly extended. It was hoped
that it might be possible to isolate the active principle of heloderma venom by
means devised by Faust.

The first step in this investigation was to apply to the dried venom the
various methods used by Faust. To test the toxicity in the course of this chem-
ical work mice were invariably used and the solutions injected subcutaneously.

Faust’s copper method was first tried. 0.2 gm. of finely powdered venom
was suspended in 15 ¢.c. of water and allowed to extract for an hour. A small
portion remained undissolved. It was removed by filtration and thoroughly
washed with water on the filter. It was dissolved on the filter in 2 c.c. of 0.8
per cent sodium-chloride solution rendered weakly alkaline with sodium car-
bonate. 0.5 c.c. was injected into a white mouse of 20 gm. beneath the skin
ofthe abdomen. The following morning the mouse was found dead, lying flat
and relaxed on its abdomen, as though it had died of progressive paralysis with-
out convulsions. The experiment was repeated with the remainder of the
sodium-carbonate solution neutralized with a trace of hydrochloric acid. Neu-
tralization caused the solution to become opalescent; a slight excess of acid
caused a precipitate. The result of the injection, as was to be anticipated, was
entirely the same as with the unneutralized solution, for the amount of sodium
carbonate used was very slight, quite insufficient to produce serious effects by
itself, as control injections of pure sodium-carbonate solutions of the same
strength showed.

*Santesson, C. G. Ueber das Gift von Heloderma suspectum Cope, einer giftigen Eidechse, Nordiskt Medicinisk
Archiv., Festband Axel Key, 1897, Nr. 5.
{E. St. Faust. Ueber das Orphiotoxin aus dem Gifte der Ostindischen Brillenschlange (Naja tripudians), Archiv fiir
experimentelle Pathologie und Pharmakologie, Bd. 56, S. 236 (1907).
tE. St. Faust. Ueber das Crotalotoxin aus dem Gifte der Nord-Amerikanischen Klapperschlange (Crotalus adaman-
teus), ibid., Bd. 64, S. 214 (1911).
231

232 THE VENOM OF HELODERMA.

The part of the venom insoluble in water could not be freed completely
from toxic material without very long-continued washing, though it is likely
that the toxic material is merely adsorbed or otherwise mechanically held in
the insoluble portion. The aqueous solution, which was slightly yellow and
faintly opalescent, was treated with a little chemically pure neutral copper ace-
tate and then with weak potassium hydrate, drop by drop, till the reaction be-
came distinctly alkaline and no further precipitate formed. The liquid and
the precipitate were then separated by centrifugation. The precipitate was
dissolved in water acidulated with acetic acid and then carefully made alkaline
again. The precipitate was again separated by centrifugation and washed
repeatedly. It was finally freed from copper by first washing with 95 per cent
alcohol; then with alcohol containing a little hydrochloric acid, till all the cop-
per was removed. There remained only an insignificant precipitate, which
was washed free from chlorine with alcohol.

Only a few milligrams of material remained. The adherent alcohol was
allowed to evaporate spontaneously and the residue was dissolved in 0.75 ¢.c. of
normal saline solution. Of this 0.5 c.c. were injected into a mouse of 22 gm.
weight beneath the skin of the abdomen. The mouse was uncomfortable for
several hours, but within 24 hours was apparently quite normal again. Hence
either the treatment destroyed the toxic substance or else the copper precipi-
tate did not carry it down. The venom of Heloderma must therefore be differ-
ent from that of the cobra and the rattlesnake. According to Faust’s observa-
tions, the toxic principles of the latter are precipitated with copper hydrate.

Further observations showed that in all probability no appreciable quan-
tities of the venom were destroyed by the copper treatment. The clear solu-
tion decanted from the precipitate of copper hydrate showed a strong typical
biuret reaction. It was carefully acidified with acetic acid. An abundant
white precipitate formed, which was separated by centrifugation from the
liquid. To remove any traces of copper it may have contained it was carefully
washed with a little water containing a trace of acetic acid; then dissolved with
the aid of sodium carbonate and again precipitated with acetic acid and washed.
It was finally washed with an abundance of weak alcohol. The precipitate,
when finally quite free from copper, was dissolved in 10 ¢.c. of normal saline
solution with the help of very little very weak sodium carbonate. 0.25 ¢.c. was
injected into a series of white mice of 20 to 25gm. weight;all died with charac-
teristic symptoms. The protocol of one experiment may serve as an example;

White mouse weighing 20 gm. received 0.25 ¢.c. beneath the skin of the
abdomen at 4% 23™ p. m.; became uncomfortable and sick within a few min-
utes; at 45 41™ p. m. it fell over on one side for a moment and then had convul-
sions, then recovered somewhat. This was followed by dyspnea and paralysis,
the mouse lying on one side. Finally it was quite paralyzed, dying at 5> 15™
p.m.

It is therefore evident that the copper method of Faust is not applicable to
heloderma venom, since the copper precipitate carries down little, if any, of the
toxic principle.

BIOCHEMICAL STUDIES. 233

Thereupon the metaphosphoric acid method was tried. The aqueous
venom solution prepared as in the previous experiment was saturated with
sodium chloride,weakly acidulated with acetic acid, and the vessel containing the
solution immersed for 15 minutes in a water-bath heated to 90t095°C. It was
then quickly chilled by immersion in ice-water and filtered. The precipitate
was washed on the filter with distilled water till the greater part of the chlorine
was removed. It was then dissolved in 10 c.c. distilled water with the help of
a little sodium carbonate and the alkalinity partially neutralized with hydro-
chloric acid. Complete neutralization was not feasible because of the forma-
tion of a precipitate. 0.5 ¢.c. of the faintly alkaline solution, which contained
some sodium chloride, was injected into a mouse of 20 gm. beneath the skin of
the abdomen at 3 p.m. The mouse was uncomfortable and restless at first.
at 3"45™ it became quiet and plainly quite sick; at 355™ it had a convulsion,
and thereafter it became evident that there was the beginning of paralysis with
dyspnea, paralysis being most pronounced in the hind leg; at 6 p.m. the para-
lysis was far advanced, though the mouse still reacted to pinching; it died
between 6 and 8 p. m.

The filtrate obtained from the above precipitate was dialyzed till all the
sodium chloride was removed. Through the addition of wash-water and
through the dialysis the original volume was much increased. It was therefore
concentrated at a pressure of 2 mm. of mercury, barium oxide being used as
drying agent. Within 12 hours it was thus reduced to about the original vol-
ume of 15¢.c. In1c.c. of this solution, which gave a strong biuret reaction,
8 mg. of sodium chloride were dissolved to make it approximately isotonic with
anormal saline solution, and 0.5 c.c. injected into a mouse of l8gm. The injec-
tion was made at 105 30™ a. m.; at 10"50™ the mouse was somewhat affected
but still restless; at 11" 30™ a. m. paralytic effects began to be noted; at 12 noon
the effects were marked; at 12 15™ the mouse was lying on its side; at 12) 45™
it was dead. Evidently heating and dialysis had not destroyed the activity,
though it would appear that the solution had lost somewhat in strength. This
is undoubtedly due, in part, to the occlusion in the precipitate obtained with
acetic acid, sodium chloride, and heat of some of the toxic principle. It is
probably also due in some measure to the concentration of the solution and to
the dialysis. Other experiments have shown that concentrating solutions of
the venom, freed from the greater part of their protein, causes them to lose tox-
icity; and that dialysis also causes some loss of activity. As the investigations.
of Cooke and Loeb have shown, the toxic principle is able to dialyze slowly
through membranes.*

The remaining dialyzed solution was then treated with small quantities of
freshly prepared 10 per cent metaphosphoric acid solution, avoiding an excess;
a slight, very fine, flocculent precipitate formed, which could be removed com-
pletely only with the greatest difficulty. The ordinary, fine, ashless filter paper
used in quantitative analysis was quite ineffective. A clear, non-opalescent
solution was finally obtained by filtering many times through the same small,

*Cf. the paper by Elizabeth Cooke and Leo Loeb.

234 THE VENOM OF HELODERMA.

hardened filter paper. A portion of the filtrate thus obtained was carefully
neutralized with sodium carbonate. The solution was almost colorless and
gave no trace of the biuret reaction. Of the neutralized solution 0.5 c.c. was
injected into a mouse of 20 gm. beneath the skin of the abdomen. The mouse
became restless; after half an hour, some dyspnea appeared with possibly some
weakness; but these passed away, so that on the following day the mouse was
quite well. Asa dose of 0.5 c.c. produced so few effects, 3 hours later 1.6 c.c. of
the same solution were injected into a fresh mouse of 22 gm. beneath the skin
of the abdomen. Such arelatively large amount of liquid always causes a great
deal of discomfort, but for some hours little else was noticeable. The injection
was made at 3 p. m.; at 5 p. m. some paralysis and dyspnea began to appear; at
6 p. m. the mouse was weak and lay flat upon its abdomen as though paralyzed,
but it still reacted to pinching; at 6" 46™ p. m. it no longer reacted to pinching,
but was still able to move about feebly. It was not again observed till the fol-
lowing morning, when it had apparently recovered. By the following evening
it was quite well and vigorous.

It was thought that perhaps the failure of this method might be due to the
quality of the metaphosphoric acid used; therefore the entire experiment was
repeated with metaphosphoric acid prepared from phosphorus pentoxide, as
recommended by Abel and Ford.* This modification did not alter the result.
Evidently this method, which has been so successful in the hands of Faust and
of Abel and Ford, is not applicable to heloderma venom, because so much of the
toxic material is held in the precipitate produced by acetic acid and heat, while
almost all the remainder is carried down with the metaphosphoric acid pre-
cipitate.

The next method tried was the precipitation of the protein with dialyzed
colloidaliron. A solution of 0.2 gm. venom in 15 c.c. of water was prepared as
before. On the addition of the colloidal iron a very heavy and abundant pre-
cipitate was formed. The addition of iron was continued until a precipitate no
longer resulted. The precipitate was removed by centrifugation. The clear
supernatant liquid did not give the biuret reaction and contained but a very
small amount of material in solution. 0.5 c.c. was injected into a mouse of 19
gm. beneath the skin of the abdomen at 1la.m. All day the mouse showed no
symptoms except those due to the injection, and on the following morning was
quite well. A second mouse of 22 gm. received a similar injection of 1.5 ¢.c. at
2p.m. This mouse showed moderate paralytic symptoms from 5 to 8 p. m.;
but on the following morning it had quite recovered. Evidently the toxic
material is carried down with the protein by the colloidal iron.

The toxic principle seems to differ from that of the cobra and of the rattle-
snake in the greater ease with which it is adsorbed by all kinds of precipitates.
Thus a solution of 0.05 gm. venom in 5 c.c. of water was filtered from the insol-
uble portion and freed from coagulable protein. The solution obtained was
very toxic. It was then shaken with a little pure kaolin. The kaolin was

‘*Abel, J. J., and Ford, W. W. On the poisons of Amanita phalloides, Journal of Biological Chemistry, vol. m1, p. 278
(1907).

BIOCHEMICAL STUDIES. 235

removed by filtration. The filtrate was practically devoid of toxicity. The
kaolin on the filter paper was then extracted on the filter with a little very weak
acetic acid. The extract gave a distinct biuret reaction and was quite toxic,
though not as powerful as the original solution.

Since the only precipitate which did not carry down the toxic principle was
the copper precipitate; and since this was formed in alkaline solution, it seemed
possible that a method by which protein is removed from an alkaline solution
might not carry down the toxic principle. Such a method is the uranyl acetate
method used by Kowalewsky,* Jacobyf, Glaessner,t Abel and Ford,§ and
others. The procedure is to render the solution faintly alkaline with sodium
carbonate and then to precipitate the protein with a saturated solution of
uranyl acetate. A solution of venom, prepared as in the first experiment, was
treated in this way. It was rendered weakly alkaline with sodium carbonate,
and uranyl] acetate was added till all the biuret-giving material had been pre-
cipitated. The precipitate was then removed by filtration. The filtrate was
dialyzed till free from uranium and then concentrated in vacuo to the original
volume; 1 ¢.c. injected into a mouse did not produce any characteristic symp-
toms. The precipitate produced by the urany] acetate was then dissolved with
the aid of acetic acid and dialyzed till free from uranium. It was then concen-
trated to a volume of 15 c.c. 0.25 c.c. was injected into a mouse of 18 gm.
The symptoms set in very rapidly and death ensued within an hour. Itisthere-
fore evident that uranyl acetate is not a suitable reagent for purifying the
venom.

Thereupon 0.2 gm. of venom finely powdered was treated for a few hours
with 10 c.c. glacial acetic acid. Only a portion was dissolved. The residue
was removed by filtration. It was gelatinous and semi-translucent. Most of
it dissolved readily in water. The solution was filtered, the filtrate being quite
opalescent. The filtrate was precipitated with twice its volume of alcohol.
A relatively scanty precipitate formed. This was separated by filtration and
thoroughly washed with alcohol. It was then dissolved in 1.5 ¢.c. of normal
saline solution, neutralized with sodium carbonate and 0.5 c.c. injected into a
mouse of 26 gm. beneath the skin of the abdomen. The injection was made at
4p.m. The animal showed no special symptoms that afternoon, but was very
quiet; the next morning it was found dead, lying flat on its abdomen, much in
the position in which it had been last observed on the preceding afternoon.
Evidently while the glacial acetic acid extracted most of the toxic principle,
some of it remained undissolved.

The glacial acetic-acid solution of the venom was treated with twice its
volume of 95 per cent alcohol. The flocculent precipitate formed was allowed
to settle over night and then removed by filtration. After washing thoroughly
with weak alcohol (alcohol 95 per cent 2 parts, water 1 part) it was dissolved
in 1.75 c.c. of normal saline solution. It was slightly acid and gave a power-

* Zeitschr. f. anal. Chem., Bd. 24, S. 551 (1895).

+ Zeitschr. f. physiol. Chem., Bd, 30, S. 135 (1900).

t Beitrage (Hofmeister) z. Chem. Physiol. u. Path., Bd. I., 8. 1.
§ Journ. Biological Chemistry, vol. vm, p. 273 (1907).

236 THE VENOM OF HELODERMA.

ful biuret reaction. After neutralizing with sodium carbonate 0.75 c.c. was
injected into a mouse of 30 gm. at 4"10™ p.m. The effect was noticeable
almost at once. Besides the usual symptoms there seemed to be a good deal of
pain and irritation at the site of injection. At 5" 10™ p. m. the hind legs were
completely paralyzed. At 5" 40™ p. m. respiration was feeble, though the ani-
mal still struggled occasionally. At 6" 30™ p. m. the respiration was still more
feeble. The animal was not observed during the night, but was found dead the
following morning in the position in which it had last been seen the evening
before.

The alcoholic filtrate was treated with an equal volume of 95 per cent alco-
hol. A-second precipitate formed, apparently, about half as voluminous as the
first. This was separated from the alcohol and dissolved in 1 ¢.c. of normal
saline solution. It gave a rather weak biuret reaction. After neutralization
with sodium carbonate 0.5 ¢.c. was injected into a mouse of 21 gm. Symptoms
set in almost at once and ran a rapid course, the mouse dying within an hour.
Paralysis set in so soon there were almost no convulsive symptoms.

The alcoholic filtrate was then treated with an equal volume of ether. A
slight precipitate formed, which was allowed to settle overnight. Itwasthen
removed by filtration and dissolved on the filter in 10 c.c. of normal saline solu-
tion. While the previous fractions obtained by precipitation with alcohol did
not dissolve readily in water, this fraction was very soluble, yielding a perfectly
clear, colorless, and faintly acid solution. This was concentrated in vacuo for
14 hours to a volume of 1.5 ¢.c.; of this, 0.25 c.c. was injected into a mouse of
23 gm. The mouse was affected almost at once; there was no struggle, but a
rapid progressive paralysis, the animal dying in 85 minutes. This experiment
was repeated upon a second mouse with quite similar results. The remainder
of the solution was examined with great care for biuret-giving substances, but
the reaction was quite negative.

The alcohol-ether filtrate was concentrated by anair-current until all the
ether had been removed. Water was added to the alcoholic solution remain-
ing. An appreciable amount of precipitate was formed, soluble in ether and
capable of being extracted from its suspension in weak alcohol by ether. It
was removed in this fashion: the ether evaporated, and the residue suspended
in normal salinesolution. When injected into a mouse it produced no untoward
effects. There seems to be present in the venom an appreciable amount of
fatty, or at least of ether-soluble, material. Whenever a solution of the venom
was precipitated with alcohol an appreciable amount of material remained
dissolved.

The entire experiment with glacial acetic acid was twice repeated with
somewhat larger quantities of venom (0.5 gm.). In each instance the results
were essentially the same. After all the material that could be precipitated
from the glacial acetic-acid solution with alcohol had been removed a further
slight precipitate could be obtained by the addition of ether. This precipitate
was always very small in amount—not more than a few milligrams. In one
of these experiments it did not give the biuret reaction; in the other it did.

BIOCHEMICAL STUDIES. 237

The quantities which had to be used for this reaction were unfortunately of
necessity so small that this does not finally prove the toxic principle to be non-
protein, though it renders it exceedingly probable. The material was exceed-
ingly toxic, though the quantity available was insufficient for quantitative deter-
minations of toxicity. The impression obtained, however, was that it was very
much more toxic than any of the other preparations.

The efforts to purify the venom by this method were not pursued further,
because of the insufficient yields. Since the toxic principle withstood the
action of glacial acetic acid it was thought that it might perhaps be purified by
means of peptic digestion. Therefore 0.1 gram was dissolved in 10 ¢.c. of 0.15
per cent hydrochloric acid. It was filtered and 5 mg. of pepsin added. It was
placed in the thermostat at 38° C. for 19 hours. During this time a few fine
floccules had separated (nuclein?). These were removed by filtration; 0.3 c.c.
of filtrate was rendered neutral and injected into a mouse of 18 gm. without
producing much effect in 2 hours; but on the following morning the animal was
dead. The material was replaced in the thermostat for 5 days. Then 0.3 c.c.
was neutralized and injected into a mouse of 20 gm.; it did not become very
sick and recovered. The experiment was repeated with the same result.

The experiment was repeated with tryptic digestion. After 18 hours of
digestion the injection of 0.25 gm. into a mouse of 28 gm. produced a severe
intoxication with paralysis, but ultimate recovery.

It is therefore evident that while the venom withstands peptic and tryptic
digestion for a considerable time, the resistance is not great enough to warrant
the use of digestion in the process of purification.

The best results, after many trials, were finally obtained by a modification
of one of the methods used by Faust in his recent paper on the venom of the
rattlesnake.* The filtered venom solution was treated with acetic acid as
long as a precipitate formed. This precipitate was removed by centrifugation
and repeatedly washed. There is at this stage a considerable loss of activity,
for, in spite of thorough washing, this precipitate, which is either mucine or
nuclein, retains a high degree of activity. This phenomenon was also observed
by Santesson and led him to conclude that the active agent was in part nuclein.
The filtrate, which was active, was treated very carefully with weak sodium
carbonate till only very weakly acid. It was then rapidly heated to boiling
over a free flame to coagulate the small amount of coagulable protein. It is
very important that the acidity be right. If it is not, clear filtration is exceed-
ingly difficult and tedious. As soon as the solution had come to a boil it was
rapidly chilled with ice-water and the very slight coagulum removed by filtra-
tion. The clear filtrate was then dialyzed till free from chlorine. Dialysis
should not be continued beyond this point, for there is a distinct loss of activity
in the process. The dialyzed solution was then concentrated over sulphuric
acid in vacuo, saturated with ether, and transferred to a centrifuge tube. It
was then put in a centrifuge placed in the cold-storage room at a temperature of
—18°F. The centrifuge wassetinmotion. From time totime it was stopped

*Op. cit.

238 THE VENOM OF HELODERMA.

and the tube examined. Gradually three layers were formed—an upper layer
of fine ice-crystals; a middle layer rather cloudy or even milky from the sepa-
ration of material due tothe chilling; and a sediment consisting of fine precipi-
tate formed by the chilling. This process was allowed to go on until the layer
of ice-crystals had filled about half the tube. Then the tube was removed, a
rift made with a file at the lower limit of the ice layer, and the tube held over a
dish and cracked with a hot wire at the level of the file-scratch. Thepart of
the tube containing the ice was thus removed. As much of the liquid above
the sediment as possible was removed and thesediment rinsed into a fresh cen-
trifuge tube with as little water as possible. It was warmed till the solution
became clear and the freezing and centrifuging repeated. After the fourth
freezing a solution was obtained which was exceedingly active, yet gave not the
slightest trace of biuret reaction. The amount of material in it was very
slight, for when working with small quantities of material, as was unfortunately
necessary, the losses are relatively considerable, due to the imperfect separa-
tion of the active principle by freezing. By using larger quantities these losses
would undoubtedly be lessened. The precipitate obtained in the first freezing
was never free from biuret-giving material. Faust reports that in his experi-
ments this was sometimes the case. The solutions which he subjected to cold
seem to have contained less protein than the heloderma solution similarly
treated, because colloidal iron removes protein better than coagulation. The
heloderma solutions still contained much biuret-giving material and a part of
this is precipitated on freezing. On the second freezing, the dilution of the
biuret material being greatly reduced, less or none is frozenout. Finally, the
yields seem to be less with this method for heloderma venom than for rattle-
snake venom. A good dealof theactive principle is adsorbed in the acetic-acid
precipitate; not all is precipitated in the chilling process; the clear liquid was
always quite toxic. The solution finally obtained contained very little mate-
rial in solution. The doses injected could not have contained more than a
small fraction of a milligram of the toxic principle. The animals were affected
almost immediately. There was usually a short convulsion followed by rapid
progressive paralysis and death in from 14 to 60 minutes.

Though many of the experiments performed in the effort to isolate the
active principle yielded negative results so far as the purification of the toxine
is concerned, it was still thought advisable to describe the typical experiments
in some detail, because they furnish information concerning the behavior of the
venom to various reagents. While this investigation, unfortunately, has not
accomplished all that was expected, it is believed that it has made some con-
tributions to the chemistry of heloderma venom. Many of the observations of
Santesson have been confirmed. It has been shown that the toxic principle is
very readily adsorbed by all kinds of precipitates.* The observation of San-
tesson that the venom contains a good deal of a protein precipitable by acetic
acid has been confirmed. This precipitate, which Santesson classed with the
nucleins, but which may beamucin, is toxic. Santesson believed this “nuclein”

*Cf. the chapter on adsorption for a more detailed account.

BIOCHEMICAL STUDIES. 239

itself to be a part of the toxic principle. In view of the great ease with which
the latter is adsorbed it is not likely that this is the case. If it were it would be
necessary to assume that the venom contained two different active principles
with similar action. That two principles are present, the ‘nuclein” and an
albumose, is actually assumed by Santesson. It was possible to show that the
“albumose”’ is not such, since very active solutions were obtained which were
quite free from substances giving the biuret reaction. Unfortunately through
lack of material it was not possible to obtain the active principle in sufficient
quantity for chemical study or analysis; but methods have been described by
which with larger quantities of material this ought to be possible.

The venom was also studied in another direction. It was examined for
some of the commoner enzymes. For this purpose 1 per cent aqueous solution
of the dried venom was prepared, and used unfiltered because some enzymes
are not soluble in water. This solution with suspended particles in it was used
for all the experiments except those on lipase.

DIASTASE.

The following mixtures were placed in small test tubes:

No. i ae ¢.c. venom suspension + 0.25 ¢.c. 1 per cent boiled starch solution, 1 drop

toluol.

No. 2. The same.

No. 3. The same.

Tubes Nos. 1 and 2 were placed in the thermostat at 37° C. and were stop-
pered to prevent the evaporation of the toluol. Tube No. 3 remained at room
temperature. At intervals a drop was removed from each tube with a clean,
dry glass rod, placed on a white porcelain plate, and tested for starch with
Lugol’s solution diluted fifty times. The following results were obtained:

2 hours. 4 hours. | 12 hours. 24 hours.
No. 1........ Dark blue. Dark blue. Purple. Weak red.
NO, Qeessseavess Dark blue. Dark blue. Purple. Weak red.
No. 82.ce0e0s Dark blue. | Dark blue. | Purple. Strong red.

It may therefore be concluded that the dried venom has weak diastatic
action.
Prprtic FERMENT.
For the detection of peptic digestion the edestin method of Fuld and Levi-
son* was used. A solution of edestin was prepared of 1 part in 1,000 of N/30
hydrochloric acid. The following tubes were prepared:

No. 1. 0.5 ¢.c. of venom suspension, 0.5 ¢.c. edestin solution, 0.1 ¢.c. toluol.

No. 2. The same.

No. 3. The same.

All three tubes were stoppered to prevent escape of toluol and placed in
the thermostat at 37°C. At the end of 4 hours all were filtered and a little
sodium chloride in substance added. No trace of a precipitate could be seen.
The venom therefore contains a peptic ferment.

*Cf. E. Abderhalden. Handbuch der Biochemischen Arbeitsmethoden, Bd. III, S. 18.

240 THE VENOM OF HELODERMA.

Tryptic TREATMENT.

To test the venom solution for tryptic enzyme it is only necessary to give
it the requisite alkalinity and then let it digest itself, since the venom itself con-
tains a mucine-like substance which is precipitable on acidifying with acetic
acid. The following experiments were performed:

No. 1. 0.5 ¢.c. venom suspension, 0.5 ¢.c. 0.4 per cent sodium carbonate solution, 0.1

c.c. toluol.

No. 2. The same.
No. 3. The same.

All the tubes were stoppered and set in the thermostat. After 4 hours
they were filtered and 4 per cent acetic acid carefully added. A good precipi-
tate formed. A second set of similar tubes was prepared and digested 12 hours.
On addition of acetic acid to these a precipitate also formed.

The original 1 per cent venom solution was then treated with just enough
weak acetic acid to precipitate the mucine-like substance. This was filtered
off. The filtrate was carefully neutralized and tested for tryptic enzyme by
the casein method of Gross, Fuld, and Michaelis.* To this end a solution of
0.1 gm. casein in 200 c.c. water containing 10 drops of 10 per cent sodium car-
bonate was prepared. The following tubes were then prepared:

No. 1. 0.5 ¢.c. venom suspension, 0.5 c.c. casein solution, 0.1 c.c. toluol.
No. 2. The same.
No. 3. The same.

These were incubated in the thermostat for 12 hours. They were then
carefully acidified. Casein was precipitated in two of the tubes.

Neither of the two methods may be very reliable. The method by which
the acetic-acid precipitable protein of the venom is used as indicator may have
been negative because there is too much of the protein present. If only asmall
amount of it had been digested this would have escaped detection. The casein
method is exceedingly unsatisfactory because of the difficulty of avoiding an
excess of acidity. Therefore further experiments were tried. Gelatin plates
were poured in Petri dishes with 2 per cent gelatin. Small areas of the gelatin
surface were covered with the venom suspension rendered alkaline with sodium
carbonate. After considerable lengths of time no digestion of these areas
could be detected; they were not depressed below the rest of the surface. Ex-
actly similar experiments were tried, using blood-serum coagulated at 56°C. in
place of the gelatin. In these dishes there seemed to be a slight solution of the
serum surface over the areas covered with venom suspension; the effect was
very slight. It is, therefore, taking all these experiments into consideration,
exccedingly doubtful whether any tryptic enzyme at all occurs in the venom.

INVERTASE.

This experiment was performed by Dr. C. 8. Hudson. He was unable
with the polariscope to detect any effect on the rotation of cane-sugar solution,
the medium being 2 per cent acid with acetic acid.

*Cf, Abderhaldon, E. Op. cit., S. 19.

BIOCHEMICAL STUDIES. 241

LIPASE.

Lipase was determined according to the method suggested by Michaelis.*
Some fresh lecithin was prepared from egg-yolk. The yolks were dissolved in
an equal volume of 10 per cent sodium choloride and the resulting solution
extracted with ether. The ethereal extract was concentrated by means of an
air current. The oily residue was extracted, till white, with acetone. The
undissolved residue was freed from acetone by exposure to air in a thin layer on
paper. Of this preparation a 2 per cent emulsion in water was made by shak-
ing. This emulsion was then used as the reagent.

As lipase is exceedingly sensitive, and as its action is usually com-
paratively limited, it was decided not to use the venom solution, but to use
the finely powered venom in substance. The following tubes were therefore
made up:

No. 1. 5 c.c. lecithin emulsion, 0.1 gm. powdered venom, 0.2 c.c. toluol.

No. 2. The same.
No. 3. The same.

Tube No. 3 was examined at once. 5..c. of absolute alcohol were added
and the mixture filtered. The tube and the filter were then washed with two
successive portions of 5 c.c. of absolute alcohol. Tube No. 1 was treated in
the same fashion after 6 hours’ incubation and No. 2 after 12 hours’ incuba-
tion. The alcoholic filtrates were all titrated with N/10 potassium hydrate
and phenolphthalein as indicator. The following results were obtained:

No. 1. 1.35 ¢.c. N/10 alkali required.

No. 2. 1.85 ¢.c. N/10 alkali required.
No. 3. (control). 0.70 ¢.c. N/10 alkali required.

Subtracting the amount of acidity originally present in the control we obtain:

No. 1. 0.65 e.c. N/10 acid formed.
No. 2. 1.15 c.c. N/10 acid formed.

To make sure of these results a further control was made, bringing the solu-
tion to a boil before incubating. In this experiment 0.8 c.c. N/10 acid, very
little more than the control (No. 3), was required. It seems, therefore, reason-
ably certain that the venom contains some lipase.

The occurrence of lipase in heloderma venom is a matter of considerable
interest. The occurrence of lipase in various venoms has been reported by
Neuberg and Reicher, } and Neuberg and Rosenberg,{t and was by them and by
Noguchi§ brought into relation with hemolysis. Previously Delezenne{ and
Friedemann || had discovered that neutralized pancreatic juice is hemolytic.

*Cfi. Abderhalden, E. Op. cit., S. 22-23.

tNeuberg, C., and Reicher, C. Lipolyse, Agglutination und Hamolyse, Biochemische Zeitschrift, Band 4, S. 281.

}Neuberg, C., and Rosenberg, E. Lipolyse, Agglutination und Hiimolyse, Berliner klinische Wochenschrift, Band
44,5. 54.

§Noguchi, H. Oncertain thermostabile venom activators, The Journal of Experimental Medicine, vol. 8, p. 87.

Noguchi, H. Ueber einer lipolytische forme der Hiimolyse. Biochemische Zeitschrift, Band 6, 8. 184.

“[Delezenne, C. Comp. rend. d. I. Soc. Biol. d. Par., T. 55, p. 171 (1903).

||U. Friedemann. Deutsche Med. Woch. 1907.

242 THE VENOM OF HELODERMA.

Indeed, Noguchi showed that pancreas lipase when freed from fat loses its
hemolytic power, while the venoms of various snakes are activated by lecithin,
triolein, and oleinic acid. Since the venom of Heloderma is hemolytic the occur-
rence of lipase becomes interesting, especially since it has been shown that the
venom contains appreciable quantities of alcohol and ether-soluble material.
It seemed, therefore, worth while to study the lipolytic action of the venom in
detail. Two points seemed important. In the experiments reported above,
relatively large amounts of venom were used. It was therefore necessary to
determine whether positive results could be obtained with smaller amounts.
Furthermore, the thermolability of the venom seemed to demand further inves-
tigation, since this point has not been much investigated by previous authors.
At the same time the effect of the reaction of the medium, the action on oil
instead of lecithin, the effect of manganese, and of bile salts were also considered.

The following series of experiments was set up and all except some of the
controls (Nos. 1, 2, 6) incubated for 60 hours. Then 25 c.c. of alcohol and a
little phenolphthalein were added to each and the acidity titrated. The con-
trols, Nos. 1 and 2, were titrated at once. From the value obtained the acidity
of the control is subtracted and the result represents the acid formed in the
given experiment.

No. 1. 1 ¢.c. 1 per cent venom solution, 10 ¢.c. 2 per cent lecithin emulsion, 1
c.c. water, 3 drops toluol. This served as the control and was titrated
immediately. Cubic centimeters N/10 alkali required for neutrali-
zation, 1.5.

No. 2. The same as No. 1. Number of cubic centimeters of N10 alkali
required for neutralization, 1.8.

No. 3. The same as No. 1. The titration, however, was done after 60 hours
of incubation at 38° C. and required 4.9 c.c. N/10 potassium hydroxid.
Subtracting the average of the controls (Nos. 1 and 2) this leaves
3.25 ¢.¢.

No. 4. 1 c.e. 1 per cent venom solution was heated to 60°C’. for 30minutes. It
was cooled; and 10 c.c. 2 per cent lecithin emulsion, 1 ¢.c. water, and
3 drops of toluol added. The increase in acidity after incubation
corresponded to 2.5 c.c. N/10 potassium hydroxid. Evidently the
lipase was not completely destroyed by the heating, but only weakened.

No. 5. The same as No. 4. Acidity = 2.1 ¢.c. N/10 potassium hydroxid.

No. 6. 1 ¢.¢. 1 per cent venom solution was heated to 100° C. for 10 minutes.
In all other respects the conditions were as in Nos. 4 and 5. Acidity
=1 ¢.c. N/10 potassium hydroxid. Evidently the heating had al-
most completely destroyed the activity.

No. 7. 1 ¢.c. 1 per cent venom solution, 10 ¢.c. 2 per cent lecithin emulsion,
0.5 c.c. of 0.2 per cent sodium taurocholate solution, 0.5 ¢.c. water.
Increased acidity after usual incubation 2.7 c.c. N/10 potassium hy-
droxid. Evidently the bile salt had not increased the lipolysis. ~

No. 8. 1 ¢.c. 1 per cent venom solution, 10 ¢.c. 2 per cent lecithin emulsion,
1c.c. of an 0.8 per cent solution of manganese sulphate. Increased
acidity = 2.8 c.c. N/10 potassium hydroxid. Evidently the man-
ganese salts did not increase the lipolysis.

No. 9. 2 ¢.c, water, 10 c.c, olive oil which had been rendered neutral by standing
in contact with powdered calcium carbonate for some time, 3 drops
toluol. Acidity after incubation =1.5 c.c. N/10 potassium hydroxid.

BIOCHEMICAL STUDIES. 243

No. 10. 1 ¢.c. 1 per cent venom solution, 10 c.c. olive oil, 0.5 ¢.c. of 0.8 percent
magnesium sulphate solution, 0.5 c.c. water, 3dropstoluol. Increased
acidity above No. 9 afterincubation = 1c¢.c. N/10 potassium hydroxid.

No. 11. 1 c.¢. 1 per cent venom solution, 10 c.c. olive oil, 0.5 c.c. of 0.2sodium
taurocholate solution, 0.5 ¢c.c. water, 3 drops toluol. Increased acid-
ity above No. 9 after incubation = 0.7 ¢.c. N/10 potassium hydroxid.
Evidently the lipolysis is far less for olive oil than for lecithin.

No. 12. 1 ¢.c. water, 10 ¢.c. 2 per cent lecithin emulsion, 1 ¢.c. N/10 hydro-
chloric acid, three drops toluol. After incubation acidity =13.5 c.c.

No. 13. 1 ¢.c. 1 per cent venom solution, 10 c.c. 2 per cent lecithin emulsion,
1 c.c. N/10 hydrochloric acid, 3 drops toluol. The acidity after incu-
bation was the same as No. 12.

Evidently lecithin is very easily hydrolyzed by mineral acid, and this
hydrolysis is not influenced by the venom. It may be that the ease with which
lecithin is hydrolyzed by acid accounts for some of the results obtained in the
course of this investigation and by other investigators. It may be one of the
factors upon which the greater lipolysis of lecithin depends, for the small quan-
tities of acids liberated at the beginning by enzyme action probably tend even
without the further help of any enzyme to hydrolyze the lecithin further.

No. 14. 1 ¢.c. water, 10 ¢.c. 2 per cent lecithin emulsion, 1 c.c. N/10 potassium
hydroxid, 3 drops toluol. After incubation acidity = 1.9 c.c. N/10
potassium hydroxid.

No. 15. 1 ¢.c. 1 per cent venom solution, 10 c.c. 2 per cent lecithin emulsion,
1 c.c. N/10 potassium hydroxid, 3 drops toluol. After incubation,
acidity after subtracting the control No. 14 = 1.6 ¢.c.N/10 potassium
hydroxid.

Evidently, while the venom lipolyzes in a solution of this alkalinity, it is
more active in less alkaline ones.

Since these experiments demonstrated that too great an acidity had an
action independent of the enzyme, and since too great alkalinity diminished
lipolysis, a further series of experiments was undertaken under conditions which
should insure continued neutrality in the solution. To attain this end, a solu-
tion of 9 gm. disodic phosphate (Na,HPO,) and 1 gm. of monosodic phosphate
(NaH,PO,) in 100 c.c. of water was employed, as recommended by L. J. Hen-
derson. At the same time the destructive effect of high temperatures was
confirmed. The following experiments were set up:

No. 16. 1 c.c. 1 per cent venom solution, 10 c.c. 2 per cent lecithin emulsion,
1 es water, 3 drops toluol. This was titrated at once. Acidity =
2.4 C.¢.

No. 17. 1 ¢.c. 1 per cent venom solution, 10 ¢.c. 2 per cent lecithin emulsion,
lc.c. water, 3 drops toluol. Acidity after incubating and subtracting
control No. 16 = 1.2 ¢.c._ It is not clear why in this experiment a
lower value was obtained than in No. 3.

No. 18. 1 c.c. 1 per cent venom solution, 10 ¢.c. 2 per cent lecithin emulsion,
2 drops toluol, 1 c.c. phosphate solution. In orderthat the phosphate
might not interfere with the titration, this solution was exhausted at
once with ether, the ether evaporated with an air current, the residue
dissolved in alcohol and titrated. Acidity = 2.1 ¢.c. N/10 potassium
hydroxid.

No. 19. This experiment was in every way identical with No. 18, except that
the mixture was incubated before extraction and titration. Acidity
pa e.c. N/10 potassium hydroxid or, subtracting control (No. 18),

6 ¢.c.

244 THE VENOM OF HELODERMA.

No. 20. The same as No. 19, except that it remained in the incubator 12 hours
longer. Acidity = 4.7 c.c. N/10 potassium hydroxid or, subtracting
control (No. 18), 2.6¢.c. Evidently keeping the medium neutral did
not have a very great effect. ;

No. 21. 1 ¢.c. 1 per cent venom solution heated to 100° C. for 30 minutes, 10
c.c. 1 per cent lecithin emulsion, 1 ¢.c. water, 3 drops toluol. After
incubation and subtraction of control (No. 16) acidity = 0.2 c.c.
N/10 potassium hydroxid. Evidently boiling, as also appears from
the first series of experiments, destroys the lipolytic power.

Dry Venom Heartep To 60°.

All the experiments were performed exactly as in the first series, so that
they need not be redescribed. The following results were obtained:

Diastase: Absent.

Pepsin: Present.

Trypsin: Probably absent.

Lipase: After 6 hours’ digestion, subtracting control, 0.3 ¢c.c. N/10 alkali required.
After 12 hours’ digestion, 0.8 ¢.c. N /10 alkali.

Drizep BLoop or HELopERMA, 1910.

Diastase: Doubtful.

Pepsin: Absent.

Trypsin: Slight. (Casein method only.)

Lipase: After 6 hours, subtracting control, 0.3 c.c. N/10 alkali. After 12 hours, 0.5
c.c. N/10 alkali.

Drizp Bioop or HELopEeRMA (SECOND SPECIMEN).

Diastase: Weak.

Pepsin: Absent.

Trypsin: Present, slight. (Casein method only.)

Lipase: After 6 hours 0.2 ¢.c. N/10 alkali required. After 12 hours 0.6 ¢.c. N10
alkali required.

Tur Venom GLAND (DRIED AND PowDERED).

Diastase: Absent.

Pepsin: Trace.

Trypsin: Doubtful trace, probably absent. (Casein method only.)

Lipase: After 6 hours’ incubation required 0.6 c.c. N/10 alkali. After 12 hours’ incuba-
tion required 1.1 ¢.c. N/10 alkali.

The absence of even a trace of diastase in the dried gland is interesting.
The diastase present in the venom must be supplied by some one of the other
glands pouring their secretion into the mouth.

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