NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
3
In further pursuing the study of this complemental action of lecithin for
cobra venom Kyes was able to prepare a new compound, a cobra venom lecithide.
This substance acts as a powerful hæmolysing agent for all cells against which
it was tested. Further, in the course of the preparation of this new compound
the neurotoxic element of cobra venom was separated from the hæmolytic
element, which latter completely joined on to the lecithin. He examined the
properties of this lecithide and pointed out various differences between it and
lecithin, and also between it and cobra venom amboceptor. Kyes was also able
to prepare lecithides not only for several other snake poisons but also for the
poison of the scorpion, the hæmolytic action of which poison is analogous to
that of cobra venom.
With this short introduction, which sets forth the present position of the
various problems of snake venom hæmolysis, we may pass on to the consideration
of some further points which I have recently investigated. In the first place
various other venoms were investigated as regards their action on the red cells
of different species of animals, ( a) on the washed cells alone, ( b ) with the addition
of the homologous serum as complement, and (c) with the addition of free
lecithin as complement. The poisons used were for the most part those of
Indian snakes, both of the colubrine and viperine families. In the tables they
are denoted by the initials of the genus and species as follows :—
I.—Colubridæ.
Naja tripudians (cobra)=N. T. V.
Naja bungarus (king cobra) =N. B. V.
Bungarus cæruleus (krait)=B. C. V.
Bungarus fasciatus (banded krait)=B. F. V.
Hoplocephalus curtus (tiger snake)=H. C. V.
Enhydrina valakadien (sea-snake) = E. V. V.
II.—Viperidæ.
Daboia Russellii (daboia)=D. R. V.
Echis carinata (phoorsa)=E. C. V.
Trimeresurus gramineus (green pit viper)=T. G. V.
Crotalus adamanteus (rattlesnake)=C. A. V.
The red cells employed were those of the dog, of the ox and of the goat. For
the lecithin I have to thank Professor Ehrlich ; it was prepared from the yolk of
egg, was perfectly pure and was designated " Agfa" lecithin.
The following is a short account of the technique employed in all instances :
—
The blood having been gently defibrinated was centrifugalised. The
supernatant serum was carefully pipetted off and put aside for use as serum
4
complement. The red cells were then thoroughly washed 4 or 5 times with sterile
salt solution (0.85 per cent.), being centrifugalised between each washing.
Finally, a 5 per cent. suspension of the washed cells was made with 0.85
per cent. steri l e sal t sol uti on ; 1 c. c. of such a suspensi on was the amount used i n
each preparation of all the experiments.
The venom solutions were made up fresh each time as required, 0.85
per cent. sterile salt solution being the solvent.
A stock solution of lecithin of a strength of one per cent. was made in the
purest methyl alcohol (Merck) : from this solution a ten-fold dilution with
sterile 0.85 per cent. salt was made as required. In all experiments 0.2 c.c. of
this 0.1 per cent. solution was the amount added to each tube.
When serum was used as the activating agent, the amount added to each
preparation was 0.5 c.c. of a 2-fold dilution of fresh serum. In each instance
the total contents of each tube were made up with salt solution to 2 or 2.5 c.c.
After the mixtures were prepared the tubes were kept at 37°C. for one hour and
then placed in the ice chest overnight, when the results were recorded.
The first observations were made with the cells of the dog. Table I
contains the results of these experiments with the venoms alone, that is to
say, without the addition of any complemental substance. From this table
it is seen that we can divide snake poisons into two groups according as
they have or have not any hæmolytic action on dog's cells without the
addition of free activating agent. Further, it is seen that the venoms which
make up the first group, that is, those poisons which can hæmolyse
Table I.
Amount of
venom in
milli-
grammes.
DOG ' S WASHED CELLS: 1 C.C. OF 5 PER CENT. SUSPENSION.
N. T. V.
N. B. V. B. C. V. B. F. V. H. C. V. E. V. V. D. R. V. E. C. V. T. G. V. C. A. V.
5
C. H.
No H.
C. H.
No. H.
Slight.
No H.
C. H.
C. H.
No H.
No H.
1
C. H.
No H.
Trace
No H.
Slight.
No H.
C. H.
Nearly
C. H.
No H.
No H.
0 . 5
C. H.
No H.
No H.
No H.
Trace.
No H.
C. H.
Trace.
No H.
No H.
0 . 1
C. H.
No H
No H.
No H.
No H.
No H.
C.H.
No H.
No H.
No H.
0 . 0 5
C. H.
No H.
No H.
No H.
No H.
No H.
C. H.
No H.
No H.
No H.
0 . 0 1
Nearly
C. H.
No H.
No H.
No H.
No H.
No H.
Nearly
C. H.
No H.
No H.
No H.
0 . 0 0 5
Trace.
No H.
No H.
No H.
No H.
No H.
Well
marked.
No H.
No H.
No H.
0 . 0 0 1
No H.
No H.
No H.
No H.
No H.
No H.
Mere
trace.
No H.
No H.
No H.
0.0005
No H.
No H.
No H.
No H.
No H.
No H.
Mere
trace.
No H.
No H.
No H.
C. H. = Complete hmmolysis.
5
dog's cells without the addition of extracellular complement, vary considerably
in the degree of this action. Thus, cobra venom and daboia venom even in
small amount have a complete hæmolysing effect ; the venoms of Bungarus
cæruleus and Echis carinata have a complete action only when used. in large
quantity, while the poison of Hoplocephalus curtus has only a slight effect even
when present in comparatively large amount. Group 2, namely, those venoms
which have no hæmolytic action on dog's washed cells, consists of the poisons
of Naja bungarus, Bungarus fasciatus, Enhydrina valakadien, Trimeresurus
gramineus and Crotalus adamanteus.
Turning now to table II, in which are set forth the results obtained with the
same quantities of the same poisons as in the experiments recorded in table I but
with the addition of serum complement, the homologous serum being used for
this purpose, we find that all venoms have now a marked hæmolytic action and
that with the exception of the venom of Enhydrina valakadien the quantitative
Table II.
Amount of
venom in
milli-
grammes.
DOG ' S WASHED CELLS : 1 C.C. OF 5 PER CENT. SUSPENSION. DOG'S SERUM : 0.5 C.C. OF 2-FOLD DILUTION.
N. T. V. N. B. V. B. C. V. B. F. V. H. C. V. E. V. V. D. R. V. E. C. V. T. G. V. C. A. V.
5
C. H.
C. H.
C. H.
C. H.
C. H.
Nearly
C. H.
C. H.
C. H.
C. H.
C. H.
1
C. H.
C. H.
C. H.
C. H.
C. H.
Nearly
C. H.
C. H.
C. H.
C. H.
C. H.
0.5
C. H.
C. H.
C. H.
C. H.
C. H.
Nearly
C. H.
C. H.
C. H.
C. H.
C. H.
0.1
C. H.
C. H.
C. H.
C. H.
C. H.
Nearly
C. H. I C. H.
C. H.
C. H.
C. H.
0 . 0 5
C. H.
C. H.
Nearly
C. H.
C. H.
C. H.
Well
marked.
C. H.
C. H.
C. H.
C. H.
0 . 0 1
C. H.
C. H.
Nearly
C. H.
C. H.
Nearl y
C. H.
Trace.
C. H.
Marked.
C. H.
C. H.
0 . 0 0 5
Nearly
C. H.
Marked. Marked
Nearly
C. H.
Nearly
C. H.
Trace.
C. H.
Mere
trace.
C. H.
C. H.
0 . 0 0 1
Marked.
Mere
trace.
T r ace.
No H.
Slight
No H.
Nearly
C. H.
No H.
Slight.
Nearly
C. H.
0 . 0 0 0 5
Slight.
No H.
No H.
No H.
Trace.
No H.
Slight.
No H.
Trace. Mere trace.
C.H.=Conplete hæmolysis.
differences to be made out between the individual poisons are not great.
It is also to be noted that smaller amounts of both cobra and daboia poisons
6
cause complete hæmolysis when serum complement is present than when it is
absent.
In Table III are recorded the results of the experiments with the same
venoms, dog's washed cells and lecithin as activating agent. From this table it
is seen that all venoms have the power, more or less, of acting as amboceptors
for these cells and free lecithin, but that this power varies considerably in degree,
the poison of Crotalus adamanteus being so weak that even when a large amount
is used complete hæmolysis does not take place. In other respects the table
explains itself and requires no further comment.
Table III.
Amount of-
venom in
milligrammes.
DOG ' S WASHED CELLS: 1 C.C. OF 5 PER CENT. SUSPENSION. LECITHIN : 0.2 C.C. OF 0.1 PER
CENT. SOLUTION.
N. T. V.
N. B. V.
B. C. V.
B. F. V.
E. V. V.
D. R. V.
E. C. V.
C. A. V.
5
C. H.
C. H.
C. H.
Well
marked.
C. H.
C. H.
C. H.
Well
marked.
1
C. H.
Nearly
C. H.
C. H.
C. H.
Nearly
C. H.
C. H.
C. H.
Marked
0.5
C. H.
Marked
C. H.
C. H.
N early
C. H.
C. H.
C. H.
Slight
0.1
C. H.
Slight
C. H.
C. H.
Trace
C. H.
C. H.
Trace
0.05
C. H.
Trace
C. H.
C. H.
Trace
C. H.
Nearly
C. H.
Mere trace
0 . 0 1
Nearly
C. H,
Trace
C. H.
C. H.
Mere
trace.
C. H.
Marked.
No H.
0 . 0 0 5
Well
marked.
No H.
Marked
Nearly
C. H.
No H.
C. H.
Trace
No H.
0 . 0 0 1
Slight
No H.
Mere
trace.
Trace
No H.
Well
marked.
No H.
No H.
0 . 0 0 0 5
Sight
No H.
No H.
No H.
No H.
Slight
No H.
No H.
C. H. = Complete hæmolysis.
The next observations were made with the red cells of the ox. In this
case not one of the venoms used had any action either alone or when fresh
ox serum was added. It is unnecessary, therefore, to publish the details of
these experiments. The details of the observations when free lecithin was used
as activating agent are given in table IV. From this table it is seen that the
7
poisons of the cobra, of the krait, of the daboia and of the phoorsa have a well
marked hæmolysing effect, especially well marked in the case of daboia and cobra
venoms, and that the poisons of the king cobra and of the rattlesnake cause only
a slight, or trace of, hæmolysis even when present in large amount. The pheno-
menon known as Complement-Ablenkung' is well illustrated in the case of the
venom of Bungaius cæruleus.
Table IV.
Amount of
venom in
milligrammes.
OK WASHED CELLS : 1/C.C. OF 5 PER CENT. SUSPENSION. LECITHIN : 0.2 C.C. OF 0.1 PER
CENT. SOLUTION.
N. T. V.
N. B. V.
B. C. V.
B. F. V.
E. V. V.
D. R. V.
E. C. V.
C. A. V.
5
C. H.
Slight
Trace
Slight
Well
marked.
C. H.
C. H.
Mere trace
1
C. H.
Trace
C. H.
Well
marked.
Well
marked.
C. H.
C. H.
No H.
0.5
C. H.
Mere
trace.
C. H.
C. H.
Mere
trace.
C. H.
C. H.
No H.
0 . 1
C. H.
No H.
C. H.
Well
marked.
No H.
C. H.
C. H.
No H.
0 . 0 5 C. H.
No H.
Nearly
C. H.
Slight
No H.
C. H.
Nearly
C. H.
No H.
0.01
Slight
No H.
Marked
No H.
No H.
C. H.
Mere
trace.
No H.
0.005
Mere
trace.
No H.
Trace
No H.
No H.
Marked
No H.
No H.
0 . 0 0 1
No H.
No H.
No H.
No H.
No H.
Mere
trace.
No H.
No H.
0 .
0 0 0 5
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
C. H. = Complete hæmolysis.
Finally, the same venoms were tested in the same manner with the washed
cells of the goat. With these cells, also, there was no hæmolytic action in any
instance either with the poisons alone or on the addition of fresh goat serum.
In table V are put down in detail the results of the observations made when
lecithin was used as the complemental substance. Considerable quantitative
differences between the individual venoms are again noticeable ; thus daboia venom
has a well marked hæmolytic effect, while the poison of Crotalus adamanteus
8
Table V.
Amount of
venom in
milligrammes.
GOAT' S WASHED CELLS : 1 C.C. OF 5 PER CENT. SUSPENSION. LECITHIN : 0.2 C.C. OF 0.1 PER
CENT. SOLUTION.
N. T. V.
N. B. V.
B. C. V.
B. F. V.
E. V. V.
D. R. V.
E. C. V.
C. A. V.
5
Nearly
C. H.
Slight
Mere
trace.
Trace
Well
marked.
C. H.
C. H.
No H.
1
Nearly
C. H.
Mere
trace.
Nearly
C. H.
Trace Mere trace
C. H.
C. H.
No H.
0.5
Well
marked.
No H.
Nearly
C. H.
Trace
No H.
C. H.
C. H.
No H.
0 . 1 Marked
No H.
Well
marked.
Trace
No H.
C. H.
Well
marked.
No H.
0 . 0 5 Marked
No H.
W ell
marked.
Trace
No H.
C. H.
Slight
No H.
0 . 0 1
Trace
No H.
Mere
trace.
No H.
No H.
C. H.
No H.
No H.
0 . 0 0 5
Mere
trace.
No H.
Mere
trace.
No H.
No H.
Slight
No H.
No H.
0 . 0 0 1 No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
0 . 0 0 0 5
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
C. H. = Complete hæmolysis.
has no hæmolysing action whatsoever. The other poisons come in between
these two extremes.
These observations, then, confirm and extend the observations of the previous
workers mentioned above. They, also, offer a simple explanation of the dis-
crepancies which seemed to exist between the American and the German results.
For, it will be remembered that Flexner and Noguchi made the statement that
no venom used by them had any hæmolytic action on the washed cells of any
species with which they worked ; Kyes and Sachs, on the other hand, showed that
cobra venom alone, using the lecithin contained in the cells, had a marked action
on the washed cells of various species, but had no effect on those of certain
other species. It is apparent, therefore, that Flexner and Noguchi had not experi-
mented with any of the combinations of venoms and washed cells in which
hæmolysis takes place without the addition of complemental substance. Their
experiments had evidently been confined to those venoms which have little affinity
for lecithin, such as the venom of Crotalus adamanteus, and which, therefore,
are not able to withdraw the lecithin from its binding to the stroma of the
9
cells even in the case of those cells, such as the cells of the dog, in which this
binding is evidently loose.
In previous papers 4 I have pointed out that working with three pure
antivenomous sera the cytophile groups of the amboceptors of snake venoms
differ greatly amongst themselves ; we have now proof of differences in the lecithin
affinities of the various poisons, some poisons evidently being well provided with
lecithinophile groups while others have few or none.
The question of the failure of both ox serum and goat serum to act as
complements for any venom with the homologous cells is a question which has
already been dealt with by Kyes and Sachs. It need not, therefore, be further
discussed here.
While the experiments which have been tabulated above are in themselves of
a certain interest and importance, they were in reality undertaken with an ulterior
motive, namely, as a necessary preliminary to ascertaining what combinations
of cells, venom and complement were suitable for further research into the con-
ditions which underlie the union of these bodies. In the case of hæmolytic
sera it is well known that under certain conditions the receptors of the red cells
take up and fix the amboceptors of the serum. Ehrlich and Morgenroth 5
have shown that red cells might in certain instances take up many times the
minimum hæmolytic dose of immune body, and that this union takes place at oºC.
in presence of complement, which remains free. Thus a method is afforded for
separating immune body from complement. Muir 6 has confirmed these obser-
vations. Further, working with ox corpuscles and rabbits' immune serum, which
had been inactivated by heat, Muir found that the number of hæmolytic doses
taken up by the cells varied from 6 to 10 when the mixtures were allowed to stand
at 37ºC. for 2 hours. All these experiments, whether made at oºC. with unheated
serum or at 37ºC. with heated serum, are of necessity of such a nature that
either complement or a modification of complement is present. In the case
of the experiments at oºC. with unheated serum free unmodified complement is
present, and in the case of the experiments at 37ºC. with heated serum the
modification of complement which Ehrlich has designated complementoid still
exists. It is, therefore, evident that all experiments which have had as their
object the fixation of amboceptor to red cell have been made in the presence
of either complement or modified complement. Now, it is quite conceivable that
both these substances might act in such a way as to favour the combination between
red cell receptor and cytophile group of immune body without themselves entering
into the union. I know of no similar experiments in which the complement had
been previously neutralised with an anti-complement, contained in a serum
which had no complement for the combination of immune body and red cell
which was being used ; but even if such experiments did exist, the objection
might be raised that the anti-complement had only occupied the haptophore
group of the complement, but had not altered its action as a stimulus to the
union of red cell receptor and immune body.
Now, a moment's consideration will show that in the case of venom
hæmolysis the objections which have been mentioned above cannot be brought
forward. For in venom we have a pure amboceptor, which can be manipulated
free from complement or any modification of complement. We have, therefore,
to select only those instances in which the venom alone has no action on the
washed cells, but has a marked hæmolytic effect on the addition of either
serum complement or lecithin, in order to obtain a series of experiments in which
the question of fixation of amboceptor can be investigated under the best condi-
tions. Furthermore, with dried venoms, which retain their power indefinitely in
this condition, and with a complemental substance such as lecithin, which can
be kept without change in solution in methyl alcohol, we have conditions of
experiment which allow of quantitative observations being made at intervals,
which observations will be strictly comparable amongst themselves.
The combinations which were chosen for the present experiments were as
follow:—
(1) Dog's cells—King cobra venom—Dog's serum complement.
(2) Dog's cells—Bungarus fasciatus venom—Dog's serum complement.
(3) Dog's cells—Crotalus adamanteus venom—Dog's serum complement.
(4) Dog's cells—Bungarus fasciatus venom—Lecithin.
(5) Ox cells—Cobra venom—Lecithin.
(6) Ox cells—Daboia venom—Lecithin.
(7) Goat's cells—Daboia venom—Lecithin.
(8) Goat's cells—Echis carinata venom—Lecithin.
A reference to the Tables 1 to- 5 will show that in all these instances the
venom alone had no action on the washed cells, but that on the addition of the
complemental substance, serum or lecithin as the case might be, a complete hæmo-
lytic effect was obtained, even when a relatively small amount of poison was used.
The question, then, which was set to be answered was this:—How many hæmolytic
doses of venom can a definite amount of red cells take up and fix in the absence
of complemental substance ? Eight series of experiments with the different
combinations of cells, venom and activating agent, as mentioned above, were
made with the object of solving this question. As the technique employed was
exactly the same in each series, the description of only one observation, namely,
that with ox cells, daboia venom and lecithin, will be given in detail.
The blood was gently whipped ; it was then centrifugalised, and the red
cells carefully and thoroughly washed to remove any trace of serum. A 5 per
cent. suspension of the washed cells was then made in 0.85 per cent. sterile salt
11
solution ; of this suspension 1 c.c. was the amount used in all preparations.
In the first instance the minimum complete hæmolysing dose of the venom was
accurately determined for this quantity of red cell suspension when a definite
amount of lecithin was added. This amount was 0.2 c.c. of a 0.1 per cent.
solution, which was made with salt solution (0.85 per cent.) as required from
t h e s t o c k 1 p e r c e n t . s o l u t i o n i n me t h y l a l c o h o l . T h e c o n t e n t s o f e a c h t u b e
were made up to 2 c.c. with salt solution. The tubes were then placed in the
incubator (37°C.) for one hour and overnight in the ice chest. The results were
then appraised. Such an estimation is shown in table VI. In this case it will
be seen that the minimum complete hæmolysing dose was 0.008 milligramme
of venom.
Table VI.
Amount of daboia venom in milligrammes.
Ox washed cells : 1c . c . o f 5 p e r c e n t . s u s p e n s i o n . L e c i t h i n :0
' 2
c . c .
of 0.1 per cent. solution.
Result.
0 . 0 1
.
.
.
.
.
.
. Complete hæmolysis.
0 . 0 0 9
.
.
.
.
.
.
.
Ditto.
0 . 0 0 8
.
.
.
.
.
.
.
Ditto.
0 . 0 0 7
.
.
.
.
.
.
.
Nearly complete hæmolysis.
0 . 0 0 6
.
.
.
.
.
.
. Well marked
”
0 . 0 0 5
.
.
.
.
.
. . Marked
”
0 . 0 0 4
.
.
.
.
.
. . Slight
”
0 . 0 0 3
.
.
.
.
.
.
. Trace
”
0.002
.
.
.
.
.
.
. Trace
”
0 . 0 0 1
.
.
.
.
.
.
.
Mere trace
”
Nil (Control)
.
.
.
.
.
.
No hæmolysis.
Minimum complete hæmolysing dose=0.008 milligramme.
This amount being determined, the next procedure was as follows :—
To a number of tubes each containing 1 c.c. of a 5 per cent. suspension
of ox washed cells were added increasing multiples of the minimum com-
plete hæmolysing dose of venom, namely, 1, 2 , 4, 6, 8, 10 and 20 doses.
The contents of each tube were made up to 2 c.c. with salt solution. The
tubes were then placed in the incubator at 37°C. for 2 hours, the contents
being well shaken up from time to time. They were then thoroughly centrifu-
galised. (1) The supernatant fluid was carefully pipetted off from each tube into
a fresh tube. To each of these latter tubes were added 1 c.c. of the 5p e r c e n t .
12
suspension of ox cells and 0.2 c.c. of 0.1 per cent. solution of lecithin. The tubes
were then placed in the incubator for one hour and overnight in the ice chest.
(2) The sedimented red cells were thoroughly washed six times with salt solution,
being centrifugalised between each washing. Then a suspension of these
washed cells was made in each instance with 2 c.c. salt solution. Finally 0.2 c.c.
of a 0.1 per cent. solution of lecithin was added to each tube. The tubes were
kept as usual for one hour at 37°C. and overnight in the ice chest. The results of
this experiment are given in Table VII.
Table VII.
Amount of daboia
venom in
milligrammes.
Number of complete
hæmolysing
doses.
Supernatant fluid + 1 c.c. of
5 per cent. ox cells + 0.2 c.c.
of 0.1 per cent. Lecithin.
Sedimented red cells + 0.2 c.c.
of 0.1 per cent. Lecithin.
0 . 1 6 .
.
.
20
Complete hæmolysis .
. No hæmolysis.
0 . 0 8 .
.
.
1 0
Di t t o
. .
Ditto.
0 . 0 6 4 .
.
.
8
Di t t o
.
.
Ditto.
0 . 0 4 8 .
.
.
6
Di t t o
.
.
Ditto.
0 . 0 3 2
.
.
.
4
Di t t o
. .
Ditto.
0 . 0 1 6 .
.
.
2
Di t t o
.
.
Ditto.
0 . 0 0 3 .
.
.
1
Di t t o
.
.
Ditto.
Nil (control) .
.
Nil.
No hæmolysis .
.
.
Ditto.
The study of this table will at once show that in not a single preparation
had any fixation of the amboceptor of the venom by the receptors of the red
cells taken place. That this result cannot be explained on the supposition
of the removal of the amboceptor from the cells by the repeated washing to
which the latter were subjected, is evident from the fact that the supernatant
fluid of the tube containing only a single hæmolysing dose was able to com-
pletely hæmolyse the same amount of fresh corpuscles. It is also noteworthy
that even when a fairly large mulitiple, namely 20 times of the hæmolysing
dose was used, still the receptors of the cells did not take up and fix any
amboceptor.
The other experiments were made in exactly the same manner as that des-
cribed above. In the case of those observations in which serum complement was
used the amount of serum added to each tube was 0.5 c. c. of a two-fold dilution.
In all these observations a similar result was obtained to that mentioned above,
that is to say, in no case was there any anchoring of the venom amboceptor to the
red cell. The following Table VIII shows at a glance the various combinations,