NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
21
Effect of heat on daboia venom.
It is well known that heat affects all snake venoms in two ways :—
(1) It causes coagulation of some of the proteid substances present in
the poison.
(2) It either completely destroys or lessens the toxicity of the poison, the
complete destruction of the toxic power depending upon the degree of heat
employed and the time of exposure to this heat. In the case of cobra venom
it has been shown by many observers that moderate heating has only the effect
of coagulating the contained albumins, and, unless the temperature is above 75° C.
and its application is greatly prolonged, has only a slight effect in diminishing
the power of the uncoagulable albumose to which this venom owes its toxicity.
With daboia poison we shall see that heat has a different effect.
The coagulation of proteids.
The temperature at which coagulation of the coagulable proteid substances
takes place was found in the case of daboia venom to be between 70° and 75°C.
A short exposure to the latter temperature causes all the proteids coagulable
by heat to be thrown down as a light flocculent sediment. Two experiments
were made by us with the view of estimating the amount of proteid which in
the case of daboia venom is coagulated at this temperature. For the purpose
of comparison a similar experiment was made with cobra poison. As the tech-
nique employed was the same in all these experiments, one description of the
method employed will suffice for them all.
A considerable quantity of the dried venom, viz., about too milligrammes,
having been carefully weighed, was dissolved in a measured quantity of double
distilled water. The strength of this solution in the case of the cobra venom ex-
periment and in the case of one of the experiments with daboia venom was
1 per cent., while in the case of the other experiment with the latter poison it was
0.1 per cent. It may be noted in passing that, while this difference in the strength
of the heated solutions did not, or only slightly, influence the amount of proteid
coagulated, a marked difference was observed as regards the toxicity of the
two filtrates of the daboia venom.
The solution was then heated in a water bath for half an hour at from 73° to
75° C. It was then thrown on a weighed filter paper, which had been previously
dried in a hot water oven at 100° C., till it reached a constant weight. The
clear filtrate was caught in a thoroughly cleaned and weighed platinum basin.
The filter was well washed through with small quantities of distilled water.
It was then, along with the coagulated proteid, again dried in a water oven at
100° C., until a constant weight was recorded.
The filtrate in the platinum basin was evaporated to dryness on a water
22
bath at 60° C. After thorough desiccation over lime, the basin with the residue
was again weighed.
The following were the results obtained :—
Daboia venom.
Experiment I—
Amount of venom.
... . . .
0.091 grm.
Strength of solution
1 per cent.
(a ) Weight of filter paper and coagulated proteid.
=0. 3103 grm.
Weight of filter paper alone...
=0.2793 grm.
[ i l l e g a b l e ] W e i g h t o f c o a g u l a t e d p r o t e i d .
. . .
. . .
. . .
=0.0310 grm.
(b) Weight of platinum dish and dried residue. =57.8118 grm.
Weight of platinum dish alone
... . . .
. . .
=57.7438 grm.
Weight of dried residue...
. . . .
. . .
=0.0 680 grm.
Thus we recovered from 91 milligrammes of venom, 31 milligrammes of
coagulated proteids and 68 milligrammes of incoagulable residue, a total of 99
milligrammes. We can only explain this gain of 8 milligrammes on the supposi-
tion that water had been taken up either by the coagulable proteids in the pro-
cess of coagulation, the most likely explanation, or by the incoagulable proteids
while heating and evaporation were taking place. Assuming, then, that the same
proportion of water was taken up by all these substances, we arrive at the fol-
lowing percentage estimate :—
Coagulable proteids
...
. . . 3 1 . 3 %
Incoagulable residue . . .
. . .
. . .
6 8 . 7 %
If, on the other hand, water was taken up by the coagulable proteids only,
the percentage composition would be—
Coagulable proteids
... ... ... ...
25.5%
Incoagulable residue ... . . .
. . .
74.7%
Experiment 2 —
Daboia venom.
Amount of venom
... . . .
. . .
. . .
0.097 grm.
Strength of solution
... . . .
0.1 per cent.
(a) Weight of filter paper and coagulated proteid ... . . .
=0.7115 grm.
Weight of filter paper ...
...
. . .
= 0 .6873 grm.
[ i l l e g a b l e ] W
e i g h t o f c n a m i l a t e d n r n t e i d -
...
. . .
= 0 . 0 2 4 2 g r m .
(b) On account of the large bulk of the filtrate two platinum dishes were employed
in this experiment.
(1) Weight of basin A-+-residue . . .
. . .
. . .
=56.6974 grm.
Weight of Basin ... ...
=56.6507 grm.
[ i l l e g a b l e ] W e i g h t o f r e s i d u e
. . .
. . .
. . .
=0.0467 grm.
(2) Weight of basin B +residue.
. . .
=57.7733 grm.
Weight of basin .. .
. . .
=57.7422 grm.
[ i l l e g a b l e ] W e i g h t o f r e s i d u e
. . .
. . .
. . .
. . .
=0. 0311 grm.
[ i l l e g a b l e ]
T o t a l w e i g h t o f
i n c o a g u l a b l e r e s i d u e
. . . .
. .
=0.0778 grm.
23
We thus recovered from 97 milligrammes of dried venom, 24 . 2 milligrammes
of coagulated proteid and 77.8 milligrammes of incoagulable residue, a total of
102.0 milligrammes, i.e., a gain of 5 milligrammes.
Making the same assumptions as in the previous experiment, we arrive at
the following percentage estimate : —
(a) If water was taken up in the same proportions by all the substances,
both coagulable and incoagulable
—
Coagulable proteids
...
...
23.8 %
Incoagulable residue
. . .
. . .
. . .
76.2 %
(b) If, on the other hand, water was taken up only by the coagulable pro-
teids—
Coagulable proteids
. . .
. . .
. . .
. . .
20 %
Incoagulable residue
. . .
. . .
. . .
80 %
As the same s tock of venom was used and as, with the exception of
the different strength of the solutions heated, the same technique was employed
in both these experiments, the slight difference between the results obtained
can be explained only as resulting from experimental error.
Taking, then, the average of the results obtained, we can calculate the per-
centage composition of daboia venom to be
—
Coagulable proteids
. . .
. . .
about 25 %
Incoagulable residue...
. . .
. . .
,,
75 %
Cobra venom.
Experiment 3—
Amount of venom
. . .
. . .
. . .
=0.0925 grm.
Strength of solution... ... . . .
=1 per cent.
(1) Weight of filter paper +coagulated proteid... . . .
=0.3081 grm
Weight of filter paper...
. . .
=0.2784 grm.
•
Weight of coagulated proteid....
. . .
=0.0297 grm.
(2) Weight of platinum basin +residue
... . . .
=56 . 7225 grm.
Weight of platinum basin
. . .
. . .
. . .
=56. 6515 grm.
Weight of incoagulable residue... . . .
=0.0710 grm.
We thus recovered from 92.5 milligrammes of dried venom, 29.7 milli-
grammes of coagulated proteid and 71 milligrammes of incoagulable residue, a
total of 100.7, i.e., a gain of 8.2 milligrammes.
Calculating the percentage composition on the same hypotheses as in the
two similar experiments with daboia venom, we arrive at the following
results :—
(1) If water was taken up in the same proportions by both coagulable and
incoagulable substances
—
Coagulable proteid... . . .
2 9 . 5 %
Incoagulable residue
... ... ... ...
70.5 %
24
(2) If water was taken up by the coagulable proteids only
Coagulable proteid
.
. .
.
23 3%
Incoagulableresidue
.
.
.
76.7 %,
It would appear from these experiments that cobra venom contains practi
- c a l l y t h e s a m e a m o u n t o f p r o t e i d s c o a g u l a b l e b y h e a t a sd
a b o i a
p o i s o n c o n t a i n s .
As regards cobra venom, our estimation is widely different from the estimations
made by Weir Mitchell and Reichert and quoted by Martin in Clifford Allbutt's
" System of Medicine." 20I n t h e t a b l e p u b l i s h e d i n t h a t w o r k c o b r a v e n o m i s
stated to contain only 1.75% of proteid coagulable by heat. As regards daboia
poison we can find no record of any estimation of the amount of coagulable
proteids which it contains. This amount, however, is much the same as the
quantity of coagulable proteid contained in Crotalus venom2 1
( 2 4 . 6 p e r c e n t . ) .
Effect of heat on the toxic power of daboia venom.
It is well known that viperine poisons in general are very sensitive to heat
when in solution. We shall see that daboia venom is no exception to this rule.
The effect of heat, however, apparently depends on the degree of concentration
of the solution heated. Further, the comparison which we shall make between
the effect of heat on daboia venom and on cobra venom distinctly points to the
conclusion that the former venom contains no albumose which acts on the central
nervous system and which is the principal toxic constituent of cobra poison.
We have seen that rapid death following either the subcutaneous or
the intravascular injection of daboia venom is due to an extensive thrombosis ; we
have also detailed experiments which go to show that in the chronic cases of daboia
intoxication the principal pathological condition is a deficiency of the blood
coagulability. We have , therefore, now to consider what the effect of heat
is—
( 1 ) o n t h e s u b s t a n c e w h i c h c a u s e s t h e i n t r a v a s c u l a r c l o t t i n g ; a n d
(2) on the substance which causes the deficiency of blood coagulability.
It is probable, however, that this is one and the same substance.
Further, we have to consider what difference, if any, the degree of the con
- c e n t r a t i o n o f t h e s o l u t i o n h e a t e d h a s o n t h e e f f e c t o f h e a t o n t h e s e s u b s t a n c e s .
For this latter purpose solutions of 1 per cent. strength and of 0.1 per cent,
strength were employed. We have four series of experiments which bear on
this question to bring forward.
(1) Effect of heating a solution of 1 per cent. strength as regards
intravascular clotting.
The material used for these experiments was the dried incoagulable residue,
obtained as detailed in experiment No. 1 above. This, it will be remembered,
25
was obtained in the following manner. A one per cent. solution of daboia venom
was heated at 73º to 75º C. for half an hour. It was then filtered to remove
the coagulated proteids. The clear filtrate was evaporated over a water bath
at 60º.C. The drying was completed over lime. The dried residue was then
collected, powdered and thoroughly desiccated over sulphuric acid.
For our experiments a solution of 0.2 per cent. strength of this powder was
made in sterile normal saline solution. Rabbits were the animals used, and all in
- j e c t i o n s w e r e m a d e i n t o t h e m a r g i n a l v e i n o f t h e e a r . T h e w e i g h t s o f v e n o m
refer to the dried residue.
Rabbit 9—Weight 1,750 grammes.—Received intravenously 7 milligrammes,
i . e., 4 milligrammes per kilo.
Gasping and general convulsions soon set in, and death took place in
about 2 minutes after the injection.
Post-mortem examination.— This showed the same extensive intravascular
clotting which we have already described as present in the rapidly fatal cases of
poisoning with the unheated venom.
Rabbit 10—Weight 1,745 grammes,—Received intravenously 1.75 milli-
grammes, i.e., 1 milligramme per kilo. For a short time after the injection no
symptoms were noticed. In 14 minutes, however, the animal became restless :
this was soon followed by general convulsions and gasping, which after
1 minute ended in death.
Post-mortem examination.—In this case there was also found intravascular
clotting, although to a much less extent than in the case of rabbit 9.
Rabbit II—Weight 1,750 grammes.—Received intravenously I '2 milli-
grammes, i.e., 0. 75 milligramme per kilo.
This animal showed no symptoms for a long time. About 3½ hours after
the injection it was seized with violent convulsions and died immediately.
On the post-mortem examination no clots could be found in the porta
system of veins, in the pulmonary arteries or in the left heart. In the right
ventricle a few small fragments of clot were discovered. The blood taken from
the heart remained uncoagulated. It is probable that in this case some clotting
took place at the time of injection in the right ventricle, at a point where the blood
is not directly subjected to the pressure of the full stream, and that pieces of
this clot got broken off and caused embolism of the larger branches of the
pulmonary arteries, which emboli were not discovered on post-mortem examina
-tion. This is the only possible explanation of the sudden onset of the convul
-sions, which ended in death after a lapse of 3¹ /³ hours from the time of injecting
the poison.
Rabbit 12—Weight 1,660 grammes.—Received intravenously 0 16
milli-
gramme, i.e ., 0 1 milligramme per kilo. No symptoms of any kind followed this
injection.
D
26
These observations are tabulated as follows :—
TABLE v.
Intravascular injection of incoagulable residue obtained by heating
a 1 per cent. solution of daboia venom.
Animal.
Weight in grammes.
Amount of residue
in milligrammes per
kilo.
Result.
Post-mortem examination.
Rabbit 9
. .
1 , 7 5 0
4
Death in 2 minutes. Extensive intravascular
clotting.
Rabbit 10
.
1 , 7 4 5
1
Death in 15 minutes. Extensive intravascular
clotting.
Rabbit 11
.
.
1,750
0.75
Death in 3½ hours.
Clots in right ventricle.
Rabbit 12
. .
1 , 6 6 0
0 . 1
No symptoms.
(2) Effect of heating a solution of 0.1 per cent. strength as regards intra-
vascular clotting.
The material used for these experiments was the dried incoagulable residue,
obtained as detailed in experiment No. 2 above. It is necessary to recall the
method by which this was procured.
A solution of daboia venom of 0.1 per cent. strength was heated at 73° to
75° C. for half an hour. It was then filtered. The clear filtrate was evaporated
to dryness over a water bath at 60° C. The drying was completed over lime.
This dried residue was pounded, and the powder thoroughly desiccated over
sulphuric acid before it was weighed.
Rabbits were the animals employed. A 0.2 per cent, solution of this
powder was used for the injections, which were made intravenously. The
weights of venom refer to the dried residue. As no symptoms were observed in
any of the rabbits injected, it is unnecessary to detail this series of experiments,
which are tabulated as follows :—
TABLE VI.
Intravascular injection of incoagulable residue obtained by heating
a 0.1 per cent. solution of daboia venom.
Animal
Weight in grammes.
Amount of residue in milli-
grammes per kilo.
Result.
Rabbit 13 .
.
.
.
1,550
1
No symptoms.
Rabbit 14 .
. .
1,67o
4
No symptoms.
Rabbit 15
.
1,570
8
No symptoms.
Rabbit 16 .
.
1,770
12
No symptoms.
27
The material used in the above experiments was obtained in exactly the
same manner as that employed in the previous series, with the exception that in
this instance the solution heated was Io times weaker than that used in the first
series of experiments (Table V).
The two following series of experiments (3 and 4) were made with a view
to ascertain whether heating daboia venom in solutions of the same strength as
used in the two series just detailed, affected in a similar manner the power which,
as we have seen, the unheated venom possesses of producing a marked deficiency
in the blood coagulability.
In both these series the solutions of venom of I per cent. and 0.1 per cent.
strength, respectively, were heated in a water bath at 73º to 75º C. for half an hour.
They were then centrifugalised, until the coagulated proteids had completely
settled at the bottom of the tubes. The perfectly clear supernatant fluid was
then decanted off and used for the experiments. Monkeys were, as in the similar
series of experiments with unheated venom, the animals employed. All obser
-vations on the blood coagulability were made with Wright's coagulometer tubes.
(3) Effect of heating a solution of 1 per cent. strength as regards deficiency of
blood coagulability.
Monkey17—16-7-1901, 12 noon.—The blood coagulation time being 2½
minutes, received subcutaneously in left pectoral region 4 milligrammes of
heated venom, equivalent to 2 . 8 milligrammes of dried incoagulable material.
4-15 P.M.—Is quite well. No hæmorrhagic extravasation nor œdema at
site of inoculation.
Blood coagulation time, 2 minutes.
17-7-1901.—There was a certain amount of swelling at site of inoculation,
but no hæmorrhage.
Blood coagulation time, 2½ minutes.
18-7-1901.—Quite well. There was still considerable swelling at the place
of inoculation.
Blood coagulation time, 3½ minutes.
Monkey 18—16-7-1901.—At 12 noon, the blood coagulation time being 3
minutes, received subcutaneously in pectoral region 12 milligrammes of heated
venom, equivalent to 8.4 milligramme of the dried incoagulable material.
4-30 P.M.—Quite well. There was neither bloody extravasation nor cedema
at site of inoculation.
Blood coagulation time, 2¾ minutes.
17-7-1901.—An oedematous swelling of large size had developed over the
lower part of the chest and upper part of abdomen. No general symptoms.
Blood coagulation time, 2¼ minutes.
18-7-1901.—The swelling had considerably diminished in size. The animal
now quite well and lively.
D 2
28
Blood coagulation time, 3¾ minutes.
Monkey 19.—This was a smaller monkey than the other two.
16-7-1901.—At 12-15 P.M., the blood coagulation time being, 2½ minutes;
received subcutaneously in left pectoral region 20 milligrammes of heated venom,
equivalent to about 14 milligrammes of the dried incoagulable material.
4-30 P.M.—Slight œdema at site of inoculation. No hæmorrhagic extra
-vasation.
Blood coagulation time less than 2 minutes.
17-7-1901.—This animal was found dead in the morning. A post-mortem
examination showed that there was neither hæmorrhage nor œdema around the
place of inoculation, nor were there any bloody extravasations in any of the
internal organs.
The heart contained clotted blood, as did also the larger veins.
For purposes of comparison with experiments in Table I V, this series is
tabulated as follows :—
TABLE VII.
Blood coagulability in monkeys following injection of heated
venom (1 per cent. solution).
Animal.
Dose of
heated venom
in milli-
grammes.
COAGULATION TIME OF BLOOD IN MINUTES.
REMARKS.
Before injec-
tion.
About 4 hours
after.
ist day.
2nd day; 3rd day.
Monkey 17
.
4
2½
2
2 ½
3½
. . .
Some swelling at
site of inoculation.
Monkey 18
..
12
3
2¾
2 ¼
3 ¾
. . .
Considerable swell-
ing at site of
inoculation.
Monkey 19
.
20
2½
2
. . .
...
Died within
24
hours; clots found
in heart, etc.
(4) Effect of heating a solution of 0 .1 per cent. strength as regards the
deficiency of blood coagulation.
Monkey 20—12-7-1901.—At 12-50 P.M., the blood coagulation time being 3
minutes, received subcutaneously in pectoral region 6 milligrammes of heated
venom, equivalent to about 4. 2 milligrammes of dried incoagulable substances.
4-30 P.M.—Quite well; neither extravasation of blood nor œdema at site
of inoculation. Blood coagulation time, 3¼ minutes.
13-7-1901.—Quite lively and well; a slight thickening to be felt at site of
inoculation. Blood coagulation time, 3¼ minutes.
14-7-1901.—No symptoms. Blood coagulation time, 3 minutes.
15-7-1901.—No symptoms. Blood coagulation time, 3½ minutes.
Monkey 21—12-7-1901.—At 1-5 P.M., the blood coagulation time being
29
3 minutes, received subcutaneously in pectoral region 6 milligrammes of heated
venom, equivalent to 4.2 milligrammes of dried incoagulable material.
4-45 P.M.—Quite well. No swelling or hæmorrhage at site of inoculation.
Blood coagulation time, 3 minutes.
13-7-1901.—Quite well and lively; a slight swelling on the front of the
chest, but no hæmorrhagic extravasation. Blood coagulation time, 3 minutes,
14-7-1901.—No symptoms. Blood coagulation time, 2¼ minutes.
15-7-1901.—No symptoms. Blood coagulation time, 2½ minutes.
Monkey 22— 12-7-1901.—At 1-10 P.M., the blood coagulation time being
minutes, received subcutaneously in pectoral region 6 milligrammes of heated
venom, equivalent to 4.2 milligrammes of dried incoagulable material.
5 P.m.—Quite well; no swelling or hæmorrhage at the site of inoculation.
Blood coagulation time, 2¾ minutes.
13-7-1901.—A very slight thickening marked the place of injection.
No general symptoms. Blood coagulation time, 2½ minutes.
14-7-1901.—No symptoms. Blood coagulation time, 2 minutes.
15-7-1901.—No symptoms. Blood coagulation time, 2 minutes.
Monkey 23—15-7-1902.—A rather small monkey. At 1-45 P.M., the blood
coagulation time being 2½ minutes, received subcutaneously in pectoral region 18
milligrammes of heated venom, equivalent to 12.6 milligrammes of dried inco-
agulable material; 9 milligrammes were injected on each side.
5 P.M.—No local sign. Appeared quite well. Blood coagulation time, 2
minutes.
16-7-1901.—No symptoms, local or general. Blood coagulation time, 2½
minutes.
17-7-19o1.—No symptoms of any kind. Blood coagulation time, 2½
minutes.
This series of observations is tabulated as follows :—
TABLE VIII.
Blood coagulability in monkeys following injection of heated
venom (0.1 per cent solution).
Animal.
Dose of
heated venom
in milli-
grammes.
COAGULATION TIME OF BLOOD IN MINUTES.
Before in-
jection.
About 4
hours after. 2nd day. 3rd day. 4th day.
REMARKS.
Monkey 20
.
6
3
3 ¼
3¼
3
3 ¼
No symptoms.
Monkey 21
.
6
3
3
3
2¼
2 ½
Very slight swelling
at site of inocula-
tion.
Monkey 22
.
6
3
2 ¾
2½
2
2
No symptoms.
Monkey 23
.
18
2 ¼
2
2¼
2½
. . .
No symptoms.
30
Further, if the poison, heated in the same manner as was done for the
last series of experiments, is injected intravenously, no deficiency of the blood
coagulability follows. The account of a single experiment, among many which
we performed, will serve to illustrate this point.
Rabbit 17 —Weight 1,670 grammes. 3-20 P.M.—The blood coagulation
time being 2¼ minutes, received intravenously 6.6 milligrammes of heated venom
(0. 1% solution), i.e. , 4 milligrammes per kilo.
3-25 P.m.—Blood coagulation time, 2 minutes.
4-15 P.M.—Blood coagulation time, 2½ minutes.
No symptoms of any kind were noticed.
It would be well now to consider the data collated above, in order to
determine what conclusions are justified.
We have already dealt with the physiological action of daboia venom in
causing, under certain conditions, rapidly fatal results from extensive intravas-
cular clotting, and in causing, under other conditions, a serious deficiency in the
blood coagulability.
In the first place, then, as regards the production of intravascular thrombosis,
it would appear that the effect of heating solutions of this poison varies consider-
ably, according as the solution heated is a concentrated or dilute solution.
This is evident from the following considerations :
—
(a) We have seen (Table III) that 0.1 milligramme per.kilo. of unheated
venom injected intravenously into a rabbit is sufficient to cause
intravascular clotting resulting in rapid death.
(b) Now, a glance at Table V will show that it requires 1 milligramme
per kilo. of venom, heated in solution of 1 % strength for half an
hour at 73° to 75° C., injected intravenously to cause death in a
rabbit from intravascular thrombosis.
(c) And, a dose of 12 milligrammes per kilo. of venom, heated in solution
of 0.1 % strength for half an hour at 73° to 75° C., causes no symp-
toms in the rabbit when injected intravenously (vide Table
VI).
It is evident, then, that while heating a concentrated solution of 1 per cent.
strength in the manner which we employed has the effect of only diminishing,
by about tenfold, the power of daboia venom to cause death from intravascular
clotting, heating in a similar way a weak solution of 0.1 per cent, strength has
the effect of completely destroying this toxic property of the poison.
The following fallacy may, however, suggest itself :—
In the first series of experiments, for which the material was obtained by
t he heat i ng of a 1 per cent . s ol ut i on, t he f i l t r at e whi ch had t o be evapor at ed was
of small amount, viz., 9 c.c., while in the second series of experiments, for which