NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
19
conclude that heating in this way completely destroys this constituent of the
poison.
I have shown above that it is not possible, even with minute quantities of
venom, to demonstrate in vitro the negative phase of diminution of blood
coagulability, which negative phase is such a marked phenomenon in chronic
cases of daboia intoxication. I have also shown in the case of cobra venom that a
marked diminution in the blood coagulability produced by this poison is capable
of demonstration both in vivo and in vitro. It was thought that the failure of
daboia venom to cause diminished coagulability of the blood in vitro might
possibly be due to the fact, that the constituent of this poison which causes
clotting is a different substance to that which causes the diminution of coagula-
bility in vivo and that this latter is not able to exert its specific action in
vitro in the presence of the former. Now, we have seen that heating a weak
solution of cobra venom diminishes, but does not destroy, the power which, this
poison has to prevent coagulation of citrate blood in vitro, while heating a
solution of daboia venom completely destroys its power to cause an increase of
coagulability. If the above hypothesis were correct, then it would be
found that by heating a weak solution of daboia venom, thereby completely
destroying the constituent which causes coagulation of citrate blood, the heated
poison would have the effect of completely inhibiting or diminishing the
coagulability of citrate blood. This theory, however, was not supported by
experimental data. For a reference to Table XV of the protocols will show that
2 milligrammes of daboia venom, heated in weak solution, after being in contact
with I c.c. of citrate blood for 3 hours, had no effect of lengthening the coagula-
tion time of the blood, when a quantity of lime solution sufficient to clot the
control in about 2 minutes was added. This result, then, strongly supports
the conclusion I have already put forward on page 17, namely, that the sub-
stance in daboia venom, which causes the diminution of coagulability in vivo,
differs both in nature and in method of action from the substance in cobra
venom, which has such a marked inhibitory effect on the blood coagulability
both in vivo and in vitro.
Further, it is evident that the observations on the action of daboia venom
on the coagulability of the blood in vitro neither support nor disprove Martin's
hypothesis, namely, that the constituent of all snake venoms which acts as
a blood poison is a proteid coagulable by heat. Finally, when we consider the
results of all the observations with cobra and daboia venom detailed above, there
can be no possible doubt that in their action on the cell constituents of the
blood and on the blood plasma, the differences between these two venoms are
fundamental differences, and not only differences in degree of toxicity. These
observations, in short, support my contention, that Calmette's statement,
that all snake venoms are alike in their physiological actions, is quite wrong
and evidently not founded on experimental data carefully worked out.
20
The action of cobra and daboia venoms on the coagulability of
citrate and oxalate plasma.
We may now pass on to consider whether or not results, similar to those
which have been collated above in the case of citrate whole blood, can also be
obtained with citrate or oxalate plasma which has been freed from red cells by
a process of sedimentation. The importance of such an investigation is evi
-
dent in view of the theory put forward by Martin (page 10 ), namely, that the
action of snake venoms on the blood coagulability in vivo probably depends
on the fact that, as a result of a great destruction of cells, especially of the red
cells of the blood, by the poison, a large quantity of nucleo-proteids is set free
and:that it is these nucleo-proteids which affect the blood coagulability.
Some of the experiments detailed below were made along with Professor
Martin, to whom I am indebted for much valuable suggestion and encourage
ment.
In these experiments the same samples of citrate and oxalate plasma were
used throughout, so that it will suffice once for all to give a short description
of the manner in which these were obtained and kept. They were both got
from the horse. In the case of the citrate plasma one litre of blood was drawn
into 5o c.c. of a 20 per cent. solution of citrate of soda (o.75 per cent, saline
solution), that is to say, there was I per cent. citrate in the whole blood. This
was kept in an ice chest (temperature about 14° C.) for 48 hours. Then the
supernatant plasma was syphoned off into small bottles, which were stored in an
ice chest till required.
In the case of the oxalate plasma one litre of blood was drawn into 40 c.c.
of a 5 per cent, solution of oxalate of potash (o.75 per cent, saline solution),
that is to say, there was 0.2 per cent. oxalate in the whole blood. This was
then treated in exactly the same way as the citrate blood. The plasma was
likewise stored in the ice chest till required. Strict aseptic precautions were
observed in the collection and storing of these samples of plasma.
Just before use in both cases the bottles were well shaken up and heated
in an oven at 37° C. for a short time.
The next step was to determine the amount of soluble lime salt which it
was necessary to add to a given quantity of each plasma to procure rapid and
firm clotting. To save repetition it may be stated here that in all these experi-
ments 2 c.c. of plasma was the amount run into each tube, and that a I per cent.
solution of calcium chloride was employed as the lime solution.
The results of this estimation are detailed in Table XVI of the protocols.
From this Table it will be seen that o.5 c.c. of the lime solution clotted 2 c.c.
of citrate plasma in less than 8 minutes , and that 0.4 c.c. of lime solution clot-
ted 2 c.c. of oxalate plasma in to minutes. These, then, were the quantities
of lime solution used in the experiments with cobra venom.
Having by preliminary observation ascertained that cobra venom did
22
of the hydrocele fluid. The addition of a small quantity of soluble lime salt
did not affect this result in any way. Taking these obser vations along with
the others already recorded, into consideration, I can only conclude that cobra
venom owes its anti-clotting power either to some obscure chemical combina-
tion which it effects with the nucleo-proteids, rendering these substances inert as
far as coagulation is concerned, or to an action of a destructive nature on the
hypothetical fibrin ferment contained in plasma. I have been unable, so far,
to obtain nucleo-proteids, except as contained in plasma, which were able to
cause clotting of any sample of hydrocele fluid which I could procure. It is
impossible, therefore, to exclude the action which the venom may have on any
ferment which may be contained in plasma. Further experiments are, however,
in progress and will form the subject of another communication.
I may now pass on to put on record some experiments, which bear on the
action which daboia venom has on the coagulability of citrate and oxalate plasma
in vitro. As the same samples of plasma as were employed for the experiments
with cobra venom were used for these experiments, it is unnecessary to again
detail the method of their preparation.
We have already seen (Tables X to XV) that daboia venom has a marked
action on the coagulability of citrate whole blood. It has been demonstrated
that it can, even without the addition of any soluble lime salt, produce solid
clotting of citrate whole blood and, further, that it contains no substance strictly
analogous to the substance contained in cobra venom, a substance which
has such a remarkable power of completely inhibiting the coagulation of citrate
blood as brought about by the addition of a soluble lime salt.
Three series of experiments were made with daboia venom and citrate and
oxalate plasma.
In the first series varying quantities of the venom alone were added to a
series of tubes each containing 2 c.c. of citrate plasma. A reference to the
protocols (Table XXI) will show that, as in the case of citrate whole blood,
daboia venom causes clotting of citrate plasma without the addition of any
lime. Thus any quantity of venom from 3 to 5 milligrammes clotted 2 c.c. of
plasma in less than 3 hours : with an amount between 0.4 and I milligramme
clotting occurred, but was delayed for about 20 hours, while 0 '2 milligramme
caused only a slight clot after 20 hours.
The second series of experiments (Table XXII) was similar to the above
series, only oxalate plasma was substituted for citrate plasma. The results
obtained show that daboia venom causes very slight clotting of plasma of this
nature, for even 5 milligrammes of venom produced only a feeble clot in 2 c.c. of
plasma after 20 hours ; smaller amounts down to o.6 milligramme had the
same effect ; while o.4 milligramme produced no clotting at all.
The third series of experiments was made with the object of ascertaining
23
the reason of this difference between citrate and oxalate plasma. In this series
(Table XXIII) to each tube of oxalate plasma, at the same time as the daboia
venom was added, there was run in a small amount of soluble lime salt, an
amount, however, not sufficient to clot the control in 20 hours. As a result of
this addition of lime the contents of each tube clotted solidly in about 10
minutes. It is, therefore, apparent that the failure of daboia venom to produce
solid clotting of oxalate plasma, while it does clot citrate plasma, is due to the
fact that the oxalate of lime is a more insoluble salt than the citrate. The
coagulation of plasma by daboia venom does not take place independently of a
certain amount of lime salt in solution. The venom acts, evidently, by increasing
the coagulability of the plasma, but solid formation of clot cannot take place unless
a small quantity of lime in solution, in some form or other, is present as well.
With a view of further searching for a working hypothesis to explain this
action of daboia venom on blood plasma, experiments were made to ascertain
whether or not this poison caused clotting of hydrocele fluid, either with or
without the addition of a soluble salt of lime. The sample of hydrocele fluid
used was clotted solidly on the addition of a small amount of oxalate plasma;
it was, in fact, the same sample which was employed in the experiments with
cobra venom detailed in Tables XIX and XX of the protocols.
The results of both these series of experiments were, however, entirely
negative (Table XXIV of protocols). Even 5 milligrammes of venom produced
no clotting in 2 c.c. of hydrocele fluid either alone or when lime was added. It
is evident, then, that in whatever manner daboia venom does act in increasing
coagulability, it cannot replace the substances in plasma, be they nucleo-
proteid, ferment or both, which are so intimately connected with the pheno-
menon of coagulation.
Further, Martin's hypothesis, namely, that the remarkable intravascular
clotting which some snake venoms produce in vivo is due to the setting free
of a large quantity of nucleo-proteids, the result of the great destruction by the
venom of cells and of red blood corpuscles in particular, receives no support
from the observations with daboia poison which I have collated above. When
we consider that this venom is able to increase markedly the coagulability of
citrate or oxalate plasma, which no doubt contains nucleo-proteids but is free
from cells, it is at once apparent that this' destruction ' theory of Martin does
not furnish a sufficiently definite mental picture of the phenomena, when looked
at in the light of these new facts which I have put forward above. It would
appear, rather, that the ultimate cause of this increase of coagulability produced
by daboia venom is some obscure inter-action between the poison and the
nucleo-proteids, or between the poison and the clotting ferment. It is certainly
not the result of the setting free of normal nucleo-proteids through the
destruction of the cells by the poison.
24
We have seen that cobra venom can prevent the coagulation of citrate
plasma, which takes place on the addition of a soluble salt of lime, and that, on
the other hand, daboia venom has a marked action in increasing the coagula-
bility of this plasma. It is interesting, now, to be able to record a series of
experiments which were made with the object of determining what would be
the result of the addition of cobra and daboia venoms together to citrate plasma.
In this series of experiments 5 milligrammes of cobra venom were added to each
tube of a series containing 2 c.c. of plasma. This, as we have seen; is a
quantity of venom five times greater than the amount which can completely
inhibit the coagulation of citrate plasma that takes place on the addition of a
soluble salt of lime. Half an hour afterwards a different amount of daboia
venom was run into each tube. This amount varied from 5 to 0.2 milligrammes.
When we compare the results obtained in this series of experiments (Table XXV)
with the results obtained in the series of experiments already detailed (Table
XX1) in which daboia venom alone was added to the plasma, it will be seen
that the previous addition of this large quantity of cobra venom had no effect
in even delaying the time in which the various amounts of venom brought about
coagulation of the plasma. In other words, the presence of a large quantity of
cobra venom does not inhibit in the slightest degree the clotting action of
daboia venom.
Although it is hardly justifiable to make deductions from the results of
experiments in vitro, as to what obtains in vivo, it appears to me interesting
and important to draw attention to the following considerations.
I have mentioned in an early part of this communication that, when a quantity
of daboia venom not sufficient to cause intravascular clotting is introduced
directly into the blood stream of an animal, the blood coagulability becomes
markedly diminished. Further, along with Dr. Hanna, I have shown that,
when this negative phase of blood coagulability has once developed, the injection
of even large quantities of daboia venom does not increase the blood coagula-
bility, and that in consequence it is now impossible to cause death from intravas-
cular thrombosis. Again, I have shown in this paper that this negative phase
of blood coagulability is not obtained in experiments with daboia venom and
citrate blood in vitro. When we consider these facts in the light of the ex-
periments, both in vivo and in vitro, with cobra venom, the results of which have
been collated above, namely, that this poison has both in vivo and in vitro
a marked power of preventing coagulation or diminishing the coagulability of
blood, and in the light of the experiments in vitro with cobra and daboia venoms
together, great support is given to the conclusion, which I have already stated,
namely, that the substances contained in cobra venom which affect blood
coagulability are quite different, both in nature and in action, from the substances
contained in daboia venom which affect the coagulability of the blood. While,
25
therefore, superficial and cursory observations might lead one to conclude that
the action of cobra and daboia venoms on blood plasma differed only in
degree and not in nature, I hold that these observations are sufficient to show
that this is not the case, and that in this, as in other respects, these two snake
venoms differ widely from one another.
Finally, I may sum up the conclusions, which the observations detailed in
this paper would appear to justify, as follows :—
(1) Cobra venom and daboia venom have a marked hæmolytic action both
in vivo and in vitro. While the hæmolytic action of cobra venom is better
observed in vitro than in vivo, the reverse holds good in the case of daboia
venom. The hæmolysing constituent of cobra venom is not a proteid coagulable
by heat.
(2) Cobra venom never produces intravascular clotting. Injected either
intravenously or subcutaneously it brings about a certain, but not well marked,
diminution in the blood coagulability. No symptoms, as a rule, result from this
diminution of blood coagulability.
On the other hand, the result of the injection of daboia venom in sufficient
quantity is rapid death, due to an extensive intravascular clotting. Should,
however, the quantity of poison injected not be sufficient to cause this clotting,
a negative phase of marked diminution of blood coagulability supervenes. Symp-
toms of an urgent nature may result from this diminution of blood coagulability.
(3) In vitro, cobra venom completely prevents the clotting of citrate whole
blood, of citrate plasma and of oxalate plasma, which results normally from the
addition of a small quantity of a soluble salt of lime.
In vitro, daboia venom has a marked action in increasing the coagulability
of citrate whole blood, citrate plasma and oxalate plasma. Even in small
amounts it does not produce in vitro any negative phase of blood coagulability
which is such a prominent action in vivo.
The constituent of cobra venom which affects the blood coagulability
is not a proteid coagulable by heat.
(4) Cobra venom and daboia venom differ widely as regards the nature of
their action on the red blood cells and on the coagulability of the blood plasma.
Calmette's statement, therefore, that all snake venoms are alike in their
physiological action, is by this fact alone shown to be incorrect and must be
modified.
(5) Martin's hypothesis, namely, that all snake venoms contain at least
two toxic proteids, A and B A, being a neurotropic poison incoagulable by
heat ; B, a hæmotropic poison coagulable by heat ; and that the venom of one
variety of snakes differs from that of another variety only in the proportionate
amounts of the constituents, A and B, contained therein, is shown to be a theory
which does not fit in with the facts now available. This conclusion was
D 2
26
foreshadowed in a paper on daboia venom which I published along with Dr.
Hanna.35
(6) Martin's other hypothesis, namely, that the action of snake venoms on
the blood coagulability in vivo is due to the setting free of a large quantity of
nuclec-proteids, the result of the destruction of cells, and especially of red blood
cells, by the venom, is not a theory which forms a complete mental picture of
the phenomena in the light of the new facts I have put forward. It is evident
that it will have to be considerably modified to suit these facts.
Protocols.
TABLE I.—Experiments to show the effect of heating solutions of cobra venom
of different strengths on the hœmolysing power of this poison.
A sample of cobra venom was dissolved in o.6 per cent. salt solution. The
strength of this solution was 1 per cent. From this original solution a tenfold
dilution was prepared. Portions of these two solutions were heated for half an
hour at 75° C. and then filtered. An estimation of the hæmolytic
power of the
original unheated venom and of these two heated samples was made. Each tube
in the series contained o.5 c.c. of venom solution, which was half the strength
of the solution in the tube next above it in the series. The dilutions were made
with 0.6 per cent. saline solution. Fresh blood of the same animal, namely, a
rabbit, was used. The amount of blood added to each tube was 0.005 c.c.
The observations were recorded 20 hours after the preparations were made.
Strength of venom solutions.
Unheated.
Heated 1 per cent.
solution.
Heated 0.1 per cent.
solution.
0.5 c.c. = o.5 mgr . . .
C. H.
C. H.
C. H.
0.5 c.c. = 0.25 mgr . . .
C. H.
C. H.
C. H.
0.5 c.c. = 0.125 mgr .
.
C. H.
C H.
Trace H.
0.5 c.c. = 0.0625 mgr
. . .
.
C. H.
Nearly C. H.
Nil.
0.5 c.c. = 0.0312 mgr .
.
.
C. H.
Nearly C. H.
Nil.
0.5 c.c. = 0.01516 mgr .
C. H.
Trace H.
Nil.
0.5 c.c. = 0.0078 mgr .
.
Trace H.
Nil.
Nil
0.5 c.c. = 0.0039 mgr
. .
.
Nil.
Nil.
Nil.
0.5 c.c. = 6 per cent. salt solution.
(control).
Nil.
Nil.
Nil.
Note.—In this and the following tables the following degrees of hæmolysis were recorded :—
Complete Hæmolysis. .
.C. H.
Hæmolysis . . . H.
Slight
Hæmolysis . . Slight H.
Trace Hæmolysis . . Trace H.
No Hæmolysis
.
.
Nil.
27
TABLE II.—Experiments to demonstrate the result of the hæmolytic
action of
daboia venom in vitro on the blood of man, monkey, rabbit, guinea-pig, and
pigeon.
The salt solution used to dissolve the venom in each series was of such a
strength as did not produce of itself any laking. It was about 0.1 per cent.
stronger than the solution which did produce slight laking. The dilutions
were made with the corresponding salt solution in the same way as in the previ-
ous series. The amount of fresh blood added to each tube was 0.005 c.c. The
observations were recorded 20 hours after the preparations were made :—
Strength of
venom solutions.
Human blood
(.6 per cent.
saline).
Monkey's blood
(.6 per cent.
saline).
Rabbit's blood
(.6 per cent.
saline).
Guinea-pig's
blood (.7 per cent.
saline).
Pigeon's
blood (.7 per cent.
saline).
0.5 c.c. = 2 mgr .
. . .
...
. . .
. . .
H.
0.5 c.c. = 1 mgr . Nearly C. H. Slight H
. . .
C. H.
C. H
0.5 c.c.=0.5 mgr . Slight H.
C. H.
Nearly C. H.
C. H.
C. H.
0.5 c.c.=0.25 mgr
C. H.
C. H.
C. H.
C. H.
Trace H.
0.5 c.c. = 0.125 mgr Trace H.
C. H.
C. H.
C. H.
Trace H.
0.5 c.c. = 0.0625
mgr
Trace H.
H.
C. H.
C. H.
Trace H.
0.5 c.c. = 0.03125
mgr
Trace H.
Trace H.
H.
C. H.
H.
0.5 c.c. = 0.0156 mgr
Nil.
Trace H.
Slight H.
Slight H.
Nil.
0.5 c.c. = 0.007
8 mgr
Nil.
Nearly C. H.
Nil.
Trace H.
Trace H.
0.5 c.c. = 0.0039
mgr
Nil.
Nil.
Nil.
Nil.
Nil.
0.5 c.c. sal1t solution
(control).
Nil.
Nil.
Nil.
Nil.
Nil.
28
TABLE III.—Experiments to demonstrate the variability in the hœmolytic action
of daboia venom in vitro.
The same technique as in the previous series of experiments was used :
0.005 c.c. of fresh rabbit's blood was added to each tube. Observations were
recorded 20 hours after the preparations were put up.
Strength of venom solution.
Result.
0. 5 c. c. = 1 mgr .
. . . . .
Trace H.
0.5 c.c. = 0.5 mgr. . . .
.
.
Nil
0.5 c.c. =.0.25 mgr . . . . .
. .
Trace H.
0.5 c.c. = 0.125 mgr . .
. . .
. .
C. H.
0.5 c.c. = 0.0625 mgr . . .
. . .
C. H.
0.5 C.C. = 0.0312 mgr . . . .
. . . .
C. H.
0. 5 c. c. = 0. 0156 mgr . .
. . . . .
C. H.
0.5 c.c. = 0.0078 mgr . . .
. . . .
Trace H.
0.5 c.c. = .6 per cent. saline (control) . . . .
Nil.
TABLE IV.—Experiments to show the effect of heating a weak solution of daboia
venom on the hœmolysing power of this poison.
A 0.1 per cent. solution of daboia venom in 0.6 per cent. saline solution was
divided into two portions. One portion remained unheated and the other portion
was heated for half an hour at 75° C. and then filtered. The dilutions were
made in the same way as the series of experiments detailed in Table I. To
each tube was added 0.005 c.c. of the same rabbit's blood. The observations
were recorded 20 hours after the preparations had been made.
Strength of venom solution.
Unheated solution.
Heated solution.
0.5 c.c. = 0.5 mgr . .
Slight H.
Nil.
0.5 c.c. = 0.25 mgr . .
H.
Nil.
0.5 c.c. = 0.125 mgr .
H.
Nil
0.5 c.c. = 0.0625 mgr . .
C. H.
Nil.
0.5 c.c. = 0.0312 mgr . .
H.
Nil.
0.5 c.c. = 0.0156 mgr . .
Nil.
Nil.
0 5 c.c. = 0.0078 mgr . .
Nil.
Nil.
0.5 c.c. = 0.0039 mgr . .
Nil.
Nil.
0.5 c.c. = 0.6% saline (control).
Nil.
Nil.