_PHYSIOLOGY OF POISONING_ (_continued_).
PROTEOLYTIC, CYTOLYTIC, BACTERIOLYTIC AND VARIOUS DIASTASIC
ACTIONS OF VENOMS: DIASTASIC AND CELLULAR ACTION ON VENOMS.
A.--PROTEOLYTIC ACTION.
The proteolytic action of venoms on gelatine, fibrin, and egg-albumen
has been studied by Flexner and Noguchi,[53] Delezenne,[54] and
subsequently by Noc[55] in my laboratory. It was already known that
_in vivo_ certain venoms exert a manifestly dissolving action on the
endothelium of blood-vessels and on the muscular tissues themselves.
Delezenne, on his part, has established the existence in snake-venoms
of a _kinase_ analogous to the kinase of leucocytes and enterokinase.
Venom alone does not attack egg-albumen coagulated by heat, but it
confers an exceedingly strong digestive power on inert pancreatic
juices.
_Lachesis_-venom has been found to be much the richest in kinase. It
digests gelatine perfectly, and when this substance has been subjected
to its action it is no longer capable of being solidified.
_Lannoy_,[56] on the other hand, experimenting upon albuminoid
substances (casein, albumins of ox-serum) in solution, has shown
that _Cobra_-venom and that of _Vipera_ disintegrate the albuminoid
molecule; but the latter remains soluble after the addition of formol
and is no longer precipitable by acetic acid. The hydrolysis never
leads to the stage of peptone, but only to the formation of albumoses
which give biuret-reaction.
The action of venoms upon fibrin may be demonstrated _in vitro_
by bringing sufficient quantities of venom, 1 centigramme, for
example, into contact with small fragments of non-heated fibrin,
derived from blood clots from an ox, rabbit, or birds, and carefully
washed. These fragments soon separate from each other, and become
dissolved in a space of time which varies according to the venom used.
The VIPERINE-venoms, especially those of _Lachesis_ and
_Ancistrodon_, are the most active. _Viper_-venom is much less so, and
the venoms of COLUBRIDÆ are the slowest.
This proteolytic action of the various venoms corresponds pretty
exactly to their coagulant and decoagulant action on rabbit- or
horse-plasma, so that, as I have already stated, we must suppose that
the property possessed by VIPERINE-venoms of more or less
rapidly dissolving blood which they have caused to coagulate, results
from the fact that these venoms contain, in addition to a coagulant
substance, another substance which is strongly proteolytic.
The latter is destroyed by heating. _Lachesis_-venom, when heated to
70° C., no longer has any dissolving action on either gelatine or
fibrin. Moreover, antivenomous serum furnished by horses vaccinated
against heated venoms does not prevent proteolysis by non-heated
venoms. On the other hand, the serum of animals vaccinated against
VIPERINE-venoms, simply filtered by the Chamberland process
and non-heated, affords perfect protection to gelatine and fibrin
against the dissolving action of these venoms.
B.--CYTOLYTIC ACTION.
Simon Flexner and Noguchi[57] have observed that the venoms of
_Naja_, _Ancistrodon_, _Crotalus_, _Vipera russellii_, and _Lachesis
flavoviridis_, contain substances which possess the property of
dissolving a large number of the cells of warm-blooded and cold-blooded
animals, and that these _cytolysins_ are very markedly resistant to
high temperatures.
They employed for their experiments 5 per cent. emulsions of organs,
spermatozoids, or ova in physiological saline solution. The solution
of venom at a strength of 1 per cent. was kept in contact with the
different kinds of cells for three hours at a temperature of 0° C.; the
liquid was then centrifuged and examined with the naked eye and under
the microscope.
The venoms experimented upon dissolved more or less rapidly the
parenchymatous cells of the liver, kidney and testicle of the dog,
guinea-pig, rabbit, rat and sheep. The most active venoms in this
respect were those of _Vipera russellii_, _Ancistrodon_ and the
_Cobra_; the venom of _Crotalus_ was the least active.
With regard to the nerve-cells, spermatozoids and ova of cold-blooded
animals (frogs, fish, arthropods, worms, and echinoderms) _Cobra_-venom
proved to be the most active; then that of _Ancistrodon_, and lastly
that of _Crotalus_.
These cytolysins are not destroyed by heating for thirty minutes at 85°
C. in a damp medium, nor by dry heating for fifty minutes at 100° C.
C.--BACTERIOLYTIC ACTION.
If we bring into contact with a 1 per cent. solution of _Cobra_-venom,
rendered aseptic by filtration through porcelain, sensitive
micro-organisms, such as the cholera vibrio, or the bacterium
of anthrax in a very young non-sporulated culture, or in its
non-spore-producing variety, we find that these microbes are dissolved
by the solution of venom in varying periods of time.
On making a direct microscopical examination we see that Koch’s vibrios
become immovable, then break up into granulations and disappear in the
liquid. The bacteriolysis is even more distinct in the case of the
bacterium. The enveloping membrane seems to dissolve, and the microbe
appears as though composed of a series of granulations placed end to
end, which finally disperse and disappear.
By my instructions this bacteriolytic property of venom with respect
to different species of micro-organisms was studied by Noc. It was
especially clearly seen with the non-spore-producing bacterium of
anthrax, the cholera vibrio, _Staphylococcus aureus_, the bacillus
of diphtheria, and _B. subtilis_ in a young culture; it is less
distinct with _B. pestis_, _B. coli_, and _B. typhi_, is almost _nil_
with the pyocyanic bacillus and _B. prodigiosus_, and _nil_ with _B.
tuberculosis_.
Investigations have likewise been made by Noc, and subsequently by
Goebel,[58] in order to determine whether cobra-venom dissolves
Trypanosomes. These hæmatozoa are more resistant than bacteria, but
they nevertheless end by being dissolved after twenty to thirty
minutes’ contact in the 1 per cent. solution.
The bacteriolytic substance in venom is distinct from that which
produces proteolysis, for the latter is destroyed at 80° C., while
the former only disappears with a temperature of and beyond 85°
C. maintained for half an hour. It is likewise distinct from the
hæmolysin, for this resists temperatures considerably higher than 85°
C. Moreover, venom which has dissolved microbes until the saturation
point has been reached, is found to have preserved in its entirety
its hæmolytic power upon the red corpuscles of the horse. Neither
does it act upon the microbes owing to the presence of a _cytase_
or _alexin_. The well-known characteristics of alexins are not met
with here--destruction at 55° to 56° C., sensitivity to light, rapid
alteration at ordinary temperatures, &c.
We cannot, again, compare the bacteriolytic action of venom to that
of rat-serum, which dissolves _B. anthracis_ by aid of a substance
distinct from vibrionicide alexin. According to the researches of
Malvoz and Y. Pirenne, the lysin of rat-serum appears to be a basic
substance, the neutralisation of which destroys its activity. Now
_Cobra_-venom in a very active solution is perfectly neutral to
sensitive litmus papers, while these are turned blue by rat-serum.
Moreover, venom acts not only upon microbes of the same kind, but
also on very different species which are not affected by rat-serum,
especially upon _B. pestis_, for which, on the contrary, this serum,
when fresh, proves a favourable culture medium. The bacteriolytic power
of _Cobra_-venom therefore constitutes a special property of venom.
“In their work on the cytolysins of venom, S. Flexner and Noguchi have
shown that animal cells, when heated to 55° C. and rendered inactive,
do not undergo complete dissolution under the influence of venoms which
destroy the fresh cells. The authors in question infer the existence
of cellular receptors (_endo-complements_, according to the theory
of Ehrlich), which fix the amboceptors of venom. Pursuing the same
order of ideas, I have observed that bacteria killed by heating for
one hour at 60° C. do not undergo total disintegration as do living
bacteria. But, while Flexner and Noguchi infer the plurality of the
cytolysins in venom for different animal cells, I have not been able
to prove the same thing with regard to the bacteriolysin; venom
saturated with cholera vibrios to such an extent that vibrios added at
repeated intervals are no longer dissolved, is incapable of dissolving
another highly sensitive species of microbe, such as the asporogenous
bacterium, and _vice versâ_. Besides, it would be difficult to
understand the existence in venom of cytolysins specific for a whole
series of species of micro-organisms” (Noc).[59]
Antivenomous serum, in a dose of 0·01 or 0·05 c.c., neutralizes the
bacteriolytic action of 1 milligramme of _Cobra_-venom, while normal
serum heated, even in larger doses, is without effect. The lysin and
the antivenomous serum appear also to enter into stable combination; by
heating to 80° C., after dilution of the mixture neutral antivenomous
serum + venom, the property of dissolving is not restored to the latter.
Pursuing his researches upon the bacteriolytic actions, Noc has also
shown that the _fresh_ serums of the rabbit, horse, guinea-pig, rat,
and man are capable of destroying them completely. We must conclude
from this that venom has the property of fixing the alexin of these
fresh serums, and in fact it is easy to show that this fixation takes
place by experimenting with hæmolytic alexin, which is much more
easy to study; it is sufficient to eliminate the intervention of the
hæmolysin proper to _Cobra_-venom.
With this object, Noc employed horse-corpuscles (which are readily
dissolved by fresh rat-serum), and neutralised the hæmolysin proper
to the venom by antivenomous serum, which has no effect upon fresh
horse-corpuscles and upon the alexin of rat-serum.
For experimental purposes six tubes are prepared with contents as
follows:--
(1) 0·5 c.c. of fresh rat-serum.
(2) 0·5 c.c. of fresh rat-serum + 0·5 milligramme of Cobra-venom (0·5
c.c. of a solution of 1 in 1,000).
(3) 0·5 c.c. of fresh rat-serum + 1 milligramme of venom (after fifteen
minutes’ contact of the venom with the alexin in tubes 2 and 3 the
venom is neutralised by 1 c.c. of antivenomous serum in the case of
tube 2, and by 2 c.c. in that of tube 3).
(4) 1 milligramme of venom.
(5) 1 c.c. of antivenomous serum.
(6) 0·5 c.c. of fresh rat-serum + 1 c.c. of antivenomous serum.
To each tube 2 drops of defibrinated horse-blood are added, and the
tubes are placed in the stove at a temperature of 35° C.
In tubes 1 and 6, which contain fresh rat-serum alone, and fresh serum
+ antivenomous serum, hæmolysis appears in a few minutes. In tube 4,
which received venom alone, hæmolysis is also produced in one hour. It
is not produced at all in tubes 2 and 3, which received the neutral
mixture of fresh serum and venom, proving that the hæmolytic alexin has
been fixed by the venom. The latter, therefore, here plays the part of
a true fixator or _amboceptor_.
Venom behaves, in short, after the manner of extracts of organs. The
fixation of hæmolytic alexin by extracts of organs, the tissues, and
animal cells (liver, spleen, spermatozoids, &c., &c.), has already been
demonstrated by V. Dungern, P. Müller, Levaditi, and E. Hoke. The same
fact is also observed with solutions of peptone. The fixation of alexin
is therefore a general property of certain albuminoid molecules.
It was interesting to endeavour to reproduce, with _Cobra_-venom, J.
Bordet’s experiments upon alexins and anti-alexins. It was to be hoped
that we had in this substance an anti-alexic body capable of being
preserved for an indefinite time and constant in its activity, which
would enable us easily to measure the dose of alexin contained in a
small quantity of a serum, or other liquid of leucocytic origin.
The experiment proved to Noc that, contrary to the ideas of Ehrlich
and his pupils, and conformably to the results obtained by Bordet
with serums and toxins, the neutralisation of venom takes place in a
variable ratio.
If a dose A of fresh serum is capable of neutralising exactly 5
milligrammes of _Cobra_-venom with regard to a sensitive microbe, on
employing a dose of the strength of 2 A we ought to find a bactericidal
dose, 1 A, in the excess of serum, according to the theory of definite
proportions. No such bactericidal action is seen, however; the serum,
on the other hand, acts in the contrary direction by means of its
nutritive substances, and in the mixture 2 A + _venom_ we obtain a
larger number of colonies of micro-organisms than in the mixture A +
venom.
We see, then, that the property of cells of fixing in excess the active
substance in serums, discovered by Bordet for the hæmolysins (staining
phenomena), is met with again in the case of extracts of organs, at
least with regard to the bacteriolytic substance of _Cobra_-venom.
It results, then, from the foregoing facts that _Cobra-venom contains a
cytolysin, which acts upon micro-organisms and is capable of fixing the
alexin of normal serums_.
The application of these data to the living animal is evidently full
of difficulties, by reason of the complexity of the substances that
come into play. Let us see, however, to what extent they are capable
of serving to explain the phenomena that are produced as the result of
poisoning.
It was observed by Kaufmann that the cadavers of animals which have
died from snake-bite are very rapidly invaded by the bacteria of
putrefaction. Welch and Ewing, referring to these phenomena of rapid
putrefaction in cases of death from venom, explained them as being due
to the loss of the bactericidal power of the serum. In hot countries,
even when snake-bites are not fatal, they are frequently complicated by
local suppuration or gangrene, occasioned by micro-organisms introduced
at the time of the bite. The minute analysis of the phenomena of
poisoning shows, in reality, that the organism undergoes different
modifications according to the quantity of venom injected and its
channel of penetration.
When the dose of venom is rapidly lethal, whether because it penetrates
into the veins or because a larger amount of it is diffused beneath the
skin, it occasions a transient hypoleucocytosis, which is, moreover,
a reaction common to injections of venom, pro-peptone, extracts of
organs, and microbic toxins (Delezenne, Nolf). It follows that blood
collected a short time after the injection may be totally bereft of
its bactericidal power, in consequence of the disappearance of the
leucocytes, which have migrated into the organs.
Thus it was observed by S. Flexner and H. Noguchi that the serum of
a rabbit, treated with 10 milligrammes of _Cobra_-venom, showed,
fifty-seven minutes after the injection, a great loss of bactericidal
properties. But it is impossible to conclude, from the diminution of
bactericidal power in this experiment, that the alexin becomes fixed by
the venom. Since the secretion of alexin is connected with the presence
of leucocytes, the hypoleucocytosis due to the venom is sufficient to
explain the loss of bactericidal power.
Nevertheless, the action of venom is not confined to these
physiological phenomena; in diffusing itself through the organism
it stays more especially in parts where the circulation has become
slower, in the capillaries of the organs where the leucocytes that
have disappeared from the general circulation are already to be found
agglomerated and altered. Here the cytolysins of the venom, continuing
their effects, are capable of neutralising the alexins set at liberty
by the destruction of the leucocytes, and thus the rapid multiplication
of the bacteria of putrefaction, which have come from the intestine or
were carried in with the bite, is easily explained. In the same way, we
can account for the suppuration that is met with as a complication of
non-lethal bites, in spite of the hyperleucocytosis consequent upon the
penetration of a weak dose of venom; immediate neutralisation of the
alexin set at liberty at the level of the wound has sufficed to enable
micro-organisms to multiply.
D.--VARIOUS DIASTASIC ACTIONS OF VENOMS.
So long ago as 1884, de Lacerda, in his “Leçons sur le venin des
serpents du Brésil,” described the results of his researches upon
the diastasic actions of venom. He proved that venom emulsifies
fats, causes milk to curdle, and does not saccharise starch. But the
solutions of venom employed by this author were not sterile, so that
putrefactive phenomena may be believed to have occurred in the course
of his experiments.
The subject has been studied afresh by Wehrmann[60] in my laboratory,
and afterwards by Lannoy.[61] These two investigators have shown that
venoms do not hydrolyse either starch or inulin. _Cobra_-venom and that
of _Vipera_ change saccharose very slightly. They do not modify the
glucosides (amygdalin, coniferin, salicin, arbutin, and digitalin);
they therefore do not contain emulsin.
On the other hand, these venoms possess, as I have already stated,
very interesting _kinasic_ properties, which have been pointed out
by Delezenne.[62] They consist in the fact that while venom alone
is incapable of digesting cooked albumin, we have only to add to a
pancreatic juice, in itself devoid of effect upon albumin, a trace of
venom, to see this albumin immediately become digested.
_Lachesis_-venom is especially active in this respect. In Delezenne’s
experiments it was generally sufficient to add to 1 c.c. of inactive
pancreatic juice, 0·5 to 1 c.c. of a 1 in 1,000 solution, that is 0·5
to 1 milligramme of venom, to obtain the digestion of a cube of albumin
weighing 0·5 gramme in the space of from ten to twelve hours. Much
weaker doses, ⅕, ⅒, sometimes even 1/80 of a milligramme still gave the
same result, with this sole difference that digestion took twenty-four,
forty-eight, and even seventy-two hours to become complete.
_Cobra_-venom was found to be a little less active than the foregoing,
but still its action was usually evident enough when it was employed in
a dose of 0·5 milligramme or even 0·1 milligramme. As for the venom of
_Vipera berus_, it was often necessary to employ it in doses from five
to ten times stronger in order to obtain the same result.
Delezenne has ascertained, on the other hand, that these venoms
entirely lose their kinasic power when they are subjected to ebullition
for fifteen minutes.
This _kinase_ or diastase, capable of quickening the inert pancreatic
juice, must evidently be of very great utility to the reptile in
enabling it to digest its prey. Venom, therefore, is not, as has long
been believed, a purely defensive secretion; it corresponds to a
physiological necessity, like that of the intestinal or pancreatic
juice. Herein is to be found the explanation of the fact that the
non-poisonous snakes, although destitute of organs of inoculation,
possess supralabial or parotid glands which produce venomous saliva.
Experiments have been made by Ch. Féré[63] to determine the effect
upon the development of the embryo of the introduction of venom into
the albumen of the egg of the fowl. He found that 83 per cent. of
the embryos, developed in eggs intoxicated with 0·05 milligramme of
viper-venom and opened after being incubated for seventy-two hours,
exhibited various anomalies in development.
E.--ACTION OF VARIOUS DIASTASES UPON VENOMS.
Venoms are modified or destroyed by certain normal diastases of the
organism. It was shown long ago by Lacerda, Weir Mitchell, Sir Joseph
Fayrer, and Lauder Brunton, that it is possible to introduce without
danger into the stomachs of adult animals amounts of venom many times
greater than the lethal dose. I have repeatedly verified this, but have
nevertheless observed that young mammals, while being suckled, readily
absorb venom by their alimentary canal, and succumb to the ingestion
of doses scarcely larger than those which kill when subcutaneously
injected. Here we have a very important fact, which once more proves
how easily the intestinal mucous membrane of young animals is permeated
by toxins. By my instructions Wehrmann[64] and Carrière,[65] in my
laboratory, have studied the modifications undergone by venoms in the
alimentary canal of rabbits. We have seen that these animals can ingest
without inconvenience doses of venom 600 times greater than the lethal
dose, and that, if we cause these ingestions to be repeated several
times, contrary to the assertion of Fraser[66] (of Edinburgh), we
never succeed in obtaining immunity to the subcutaneous injection of a
simple lethal dose, and no antitoxin is formed in the blood.
The _ptyalin_ of the saliva, _pancreatic juice_, and _bile_ destroy
_Cobra_-venom _in vitro_. We must, therefore, assume that these
diastases are veritable agents of destruction for ingested venom. The
intestinal microbes play no part, any more than the intestinal juice by
itself. The gastric juice has very little effect. Papain is almost as
active as the pancreatic juice.
It had already been proved by Fraser, so long ago as 1895, that bile,
after prolonged contact and in a sufficient dose, has a strongly
destructive effect upon Cobra-venom; but, contrary to the opinion of
this investigator, it is not antitoxic, for it possesses no preventive
or curative property, and its effects are produced only _in vitro_.
We see from what has been stated above, that venoms introduced into a
sensitive organism are capable of producing extremely complex effects
upon the various tissues or humours. They act on the nerve-cells
by their _neurotoxin_, on the endothelium of the blood-vessels by
their _hæmorrhagin_ (Flexner and Noguchi), on the red corpuscles by
their _hæmolysin_, on the fibrin of the blood and muscles by their
_proteolytic diastase_, and on the fibrin-ferment itself by their
_thrombase_.
They also act on the leucocytes, according to the experiments of
Chatenay,[67] performed under the direction of Metschnikoff, and
according to those of Flexner and Noguchi,[68] already cited.
Thus we understand how complex must likewise be the means of defence
that have to be employed in order to afford an effective protection
against such poisons.
The slightly intoxicated organism at first reacts by the intervention
of the leucocytes; a _hyperleucocytosis_ is produced, accompanied by
a more or less considerable rise of temperature. After a few hours
everything returns to its normal condition, and if the injection of a
lethal dose of venom is repeated several times, at intervals of a few
days, it is not long before antitoxic substances are found to appear in
the serum.
When the dose of venom injected is sufficient to cause death, we
observe, a very few moments after the injection, a _lowering of
temperature_ and a _hypoleucocytosis_, which is the more pronounced in
proportion to the nearness of the dose of venom to the minimal lethal
dose. With very strong doses the hypoleucocytosis has not time to
manifest itself.
It is therefore probable that, in intoxication by venoms as in that by
the toxins of micro-organisms, the protective _rôle_ of the leucocytes
is all-important, not only because these cells are capable of digesting
venoms owing to their protoplasmic digestive juices, but also because
they constitute if not the only, at any rate the principal source of
the _antitoxic substances_ or _amboceptors_.