B.—Effects of Venom upon the Red Corpuscles.
(1) Hæmolysis.—The hæmolytic properties of venoms, that is to say, their faculty of dissolving the red corpuscles, have been the subject of very important researches on the part of a number of investigators during the last few years (W. Stephens,[45] Flexner and Noguchi,[46] Calmette,[47] Phisalix,[48] Preston Kyes and Hans Sachs,[49] Noc[50]).
The different venoms are all hæmolytic, but in very variable doses. It is possible to make a very precise comparative study of them from this special point of view by taking as a base for each venom, as was done by Noc, the unital dose of 1 milligramme (or one-tenth of a cubic centimetre of a 1 per cent. solution freshly prepared and not filtered, the filtration through porcelain retaining an appreciable part of the active substance), and noting the time strictly necessary for this dose of 1 milligramme to dissolve completely, in vitro, 1 c.c. of a 5 per cent. dilution of red corpuscles of the horse in physiological saline solution.
It is very important, before allowing the venom to act on the red corpuscles, to first wash the latter by means of several successive centrifugings with 8 per 1,000 physiological saline solution.
It is also better to choose the corpuscles of the horse in preference to those of other species of animals, since they exhibit a nearly constant mean sensitivity. The corpuscles of the ox, goat, sheep, and rabbit are less sensitive. Those of man, the guinea-pig, and the rat, on the contrary, are more so.
On experimenting with washed corpuscles, it is found that venom alone is incapable of dissolving them. In order that dissolution may take place, we are obliged to add to the mixture either a small quantity of normal horse-serum, preferably heated, and, consequently, deprived of alexin (Calmette), or ½ c.c. of a 1 in 10,000 solution of lecithin in physiological saline water (P. Kyes).
Venom, therefore, is capable of hæmolysing red corpuscles only when it is quickened, either by heated normal serum, or by lecithin. The solution of lecithin employed for this purpose should be prepared by dissolving 1 gramme of lecithin in 100 grammes of pure methylic alcohol. Taking 1 c.c. of this dilution we add it to 9 c.c. of 8 in 1,000 saline solution, and make a second dilution of 1 c.c. of the foregoing mixture in 9 c.c. of saline water. This latter dilution of 1 in 10,000 is utilised as the reagent.
Let us now see how the serum or lecithin acts. It has been shown by P. Kyes that with either of these substances the mechanism of the hæmolytic action is the same, for the serum quickens the venom only through the agency of the free lecithin it contains. The lecithin takes part in the reaction by combining with the venom to form a hæmolysing lecithide more resistant to heat than its two components, for it may be heated for several hours at a temperature of 100° C., without the loss of any of its properties.
When venom is brought into contact with certain kinds of highly sensitive red corpuscles, those of the rat for example, these corpuscles, although washed and freed from serum, may undergo hæmolysis. This result is due to the fact that these corpuscles contain sufficient quantities of lecithin, which becomes liberated from their protoplasm and, uniting with the venom, constitutes the active lecithide.
It was already known that lecithin is capable of combining with various albuminoid matters and with sugars to form lecithides. We must not, therefore, be surprised to find that such a combination may take place with the proteic substances in venom. The combination in this case is a truly chemical one. Lecithin in its natural state, or that which is normally found in serums which quicken venom, such as horse-serum, even when heated to 65° C., therefore plays the part of complement according to Ehrlich’s theory, or that of alexin according to the theory of Bordet, while venom itself would be an amboceptor or sensitiser.
This is not, however, the way in which the phenomenon should be understood, for it is impossible to admit the identification of heated serum or lecithin with the complementary substances or alexins, seeing that the essential characteristic of the latter is that they are intolerant of heat and become entirely inactive on being raised to a temperature of 58° C., or even by simply being kept for a few days exposed to the air and light. We must therefore suppose, with P. Kyes and H. Sachs, that the red corpuscles themselves contain substances capable of playing the part of complements (endo-complements), and that it is with these that the venom combines when quickened by the presence of lecithin or heated serum, the latter only acting because it contains free lecithin.
All substances that contain lecithin, such as bile, hot milk, or cephalin, are capable of exerting the same quickening action, but do not themselves possess any inherent hæmolytic power.
Cholesterin, on the contrary, represents a kind of antidote to lecithin, as also to normal serums. It prevents hæmolysis of the red corpuscles in a mixture of washed corpuscles and venom, yet it does not in any way modify the properties of true alexins or complements.
Moreover, no correlation exists between lecithides and the neurotoxin in venoms. The combination lecithin + venom possesses hæmolytic action, but is in no way neurotoxic. Conversely, venom can be freed from its groups of molecules combinable with lecithin, and remain neurotoxic.
Lecithide is insoluble in ether and acetone, but soluble in chloroform, alcohol, toluene, and water. Its properties are therefore entirely distinct from those of its two components. It precipitates slowly from its aqueous solutions, without losing its hæmolytic power; it does not show biuret-reaction; it dissolves with equal readiness the red corpuscles of all species of animals, and its effects, like those of venom, are impeded by cholesterin.
Kyes has succeeded in obtaining lecithides with all the hæmolytic venoms that he was able to study: thus he has prepared lecithides from Lachesis lanceolatus, Naja haje, Bungarus, Lachesis flavoviridis, and Crotalus. It is therefore probable that the lecithinophile group exists in all venoms, even when these differ as regards their other properties.
A wide range of difference is exhibited by the various venoms, as regards their hæmolysing power in the presence of normal heated serum or lecithin. The venom of Naja and that of Bungarus are the most active. The action of the venoms of Viperidæ, and especially of those of Crotalus, is very weak. For example, while 1 milligramme of Cobra-venom dissolves in from five to ten minutes 1 c.c. of a 5 per cent. dilution of red corpuscles in the presence of lecithin or normal heated serum, the same dose of the venom of Vipera russellii takes thirty minutes to effect the dissolution, and the venom of Lachesis lanceolatus takes three hours.
P. Kyes and H. Sachs have discovered the apparently paradoxical fact that, if to the red corpuscles of certain species of animals Cobra-venom be added in increasing doses, hæmolysis augments up to a certain point, beyond which the destruction of the corpuscles shows progressive diminution. In a large dose Cobra-venom no longer produces any effect upon the corpuscles of the horse, for example, even when the venom is added in presence of a great excess of lecithin or heated serum. It would seem, then, that, according to the theory of Ehrlich, under the influence of an exaggerated amount of venom-amboceptor there is produced a deviation on the part of the complement (serum or lecithin), and that the latter, instead of fixing itself upon the corpuscles, becomes united with the surplus fraction of the amboceptors, which has remained free in the liquid.
Noguchi,[51] resuming the study of this extremely curious action of strong doses of venom, observed that the red corpuscles of certain species of animals (such as the horse for example), when previously washed and held in suspension in a physiological solution of sea-salt containing 4 per cent. of Cobra-venom, acquire a considerable augmentation of resisting power with regard to various physical and chemical agents. In consequence of this they are no longer hæmolysed by distilled water, ether, or saponin.
Nevertheless, acids or alkalies, except ammonia, destroy corpuscles treated with venom more easily than those in their normal condition.
If corpuscles, previously treated with a strong dose of venom, are subjected to repeated washings in physiological saline solution, the special resistance acquired by them in the presence of the venom disappears; they even become more sensitive to the action of destructive agents, such as water, ether, or saponin.
The principle contained in venom, to which must be attributed the protective action, is not destroyed by heating to 95° C., although at this temperature Cobra-venom becomes partially coagulated. Moreover, the protective substance is contained in the coagulum, while the hæmolysin remains entirely in the filtrate. The agglutinin of venom, on the other hand, is destroyed at a temperature of 75° C. The protective substance, therefore, can be identified neither with the hæmolysin nor with the agglutinin.
It follows that it is impossible to accept the hypothesis of the “deviation of the complement” suggested by Kyes and Sachs to explain the innocuousness of strong doses of venom. Besides, it would be difficult to reconcile this hypothesis with the fact, observed by Noguchi, that venom in a strong dose protects corpuscles, not only against the action of lecithin (complement), but also against distilled water, ether, &c.
Noguchi, seeking more thoroughly to elucidate the mechanism of this protective action, finds that Cobra-venom forms a precipitate with blood-serum, when the latter is relatively poor in salts or when it is dilated with water. It likewise forms a precipitate with the aqueous extract of red corpuscles, and precipitates the globulins, hæmoglobin, or globin of the corpuscle, when treated separately. The precipitates are insoluble in water, but dissolve with the assistance of a small quantity of acid or alkali, and also in a great excess of saline solution.
Noguchi supposes that red corpuscles, when treated with strong solutions of venom, are protected against destructive agents on account of the formation by the venom and certain constituents of the corpuscle (chiefly hæmoglobin) of a compound insoluble in water. When this compound is removed by repeated washings in physiological solution, the corpuscles can easily be hæmolysed afresh by the ordinary destructive agents. Venom, none the less, exerts a noxious influence upon the corpuscles in all cases; but when strong solutions are employed, this effect is masked by the protective action.
All kinds of red blood corpuscles are not equally sensitive to the protective action of strong doses of venom. In this respect all degrees are observed in the action of venom. Thus the corpuscles of the dog are not protected at all by Cobra-venom. But it is interesting to observe that this venom in no way precipitates either the aqueous extract of dog’s corpuscles, or the hæmoglobin, or the globin of this animal.
The venom of Crotalus and that of Ancistrodon likewise possess protective power, which is, however, less marked than in the case of Cobra-venom.
Noguchi finally points out that corpuscles treated with venom are not hæmolysed by fluorescent substances such as eosin. They are also refractory to the hæmolysing action of tetanolysin.
The resistance of the hæmolysins of venom to heat (which, according to Morgenroth, may extend to heating for thirty minutes at a temperature of 100° C.) explains how it is that the serum of horses immunised by means of venoms heated to 72° C. is distinctly antihæmolysing, and capable of perfectly protecting the red corpuscles in vitro and in vivo.
I have been able to prove that the antineurotoxic property of antitoxic serums with regard to the venoms of Colubridæ is pretty much on a par with their antihæmolysing property, so that it is possible to measure in vitro the antitoxic activity of a serum by establishing the degree of its antihæmolysing activity. Thus we see that a serum, which is antitoxic and antihæmolytic with respect to the venom of Naja, is likewise antihæmolytic as regards the other Colubrine-venoms, and even certain venoms of Viperidæ. Here we have a very important fact, for it enables us to measure in vitro the activity of antivenomous serums.
(2) Precipitins of Venoms.—The serum of rabbits treated with increasing doses of Cobra-venom precipitates the latter in more or less concentrated solution. It has no effect as regards other venoms. On the other hand, the serum of a strongly immunised horse, the antivenomous power of which was pretty considerable, gave no precipitate with Cobra-venom; the formation of precipitate is therefore entirely independent of that of antitoxins (G. Lamb).[52]
(3) Agglutinins of Venoms.—Besides their hæmolytic action, it is easy to observe that certain venoms, especially those of Viperidæ, agglutinate the red corpuscles, and that the substance that produces this agglutination is different from the hæmolysing substance; for it acts rapidly at a temperature of O° C., at which hæmolysin manifests its effects only with extreme slowness. Heating to 75° C. destroys this agglutinant property (Flexner and Noguchi).