野口英世著

Journal of Experimental Medicine

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ON E X T R A C E L L U L A R AND, I N T R A C E L L U L A R V E N O M
A C T I V A T O R S O F T H E BLOOD, W I T H E S P E C I A L
R E F E R E N C E TO L E C I T H I N A N D F A T T Y ACIDS.
AND T H E I R COMPOUNDS. 1
BY HIDEYO NOGUCHI, M.D.
(From the Rockefeller Institute [or Medical Research, N e w York.)

INTRODUCTION.
Snake venom forms a h~emolytic compound with pure lecithinwhen it is shaken with a chloroform solution of this substance. 2
The same is true of the venom of bee. 3 Judging from this important discovery, linked with the fact that the serum as well as the
corpuscles of blood yield upon hot alcoholic extraction a large quantity of lecithin, Kyes was led to deduce that the venom activating
substance of blood is lecithin. This theory has, however, somerather serious difficulties in explaining certain fundamental phenomena which are observed in venom h~emolysis. In spite of thefact that all serums or corpuscles do yield upon extraction nearly
uniform amounts of lecithin, certain kinds of serums possess noactivating property; and the susceptibility of the corpuscles of different bloods exhibits a wide variation according to the species ofanimals. In some instances the corpuscles are completely refractory to the action of venom. ' Speaking quantitatively there is no
relation between the amount of lecithin and that of the venom activating substance of serum or corpuscles. If we are forced to holdlecithin responsible for venom h~emolysis in normal serum or blood
corpuscles we must assume that lecithin exists in different statesin different bloods, the explanation adopted by Kyes to account forthe phenomena observed.
This hypothesis appears to furnish an easy solution of the phenomena, but it requires, in the meanwhile, its verification by experiReceived for publication April I5, 19o7.
2Kyes, Berl. klhz. Woch., 19o3, xi, 956, 982.
8Morgenroth and Carpi, Berl. klin. I/Voch., 19o6, xliii, 1424.
436

I-Iideyo Noguchi.

437

mental evidence. T h e first question will be whether lecithin everexists as an available compound in the activating serum and in thevenom-susceptible corpuscles. T o decide this point it is absolutely
necessary to exclude other activators than lecithin f r o m the activating serum or susceptible corpuscles. T h i s h a v i n g been done,
we ca~ compare the degree of firmness of lecithin-containing compounds of various kinds of serums and corpuscles, including theoriginally activating and non-activating serums and corpuscles.
E v e n if we find that in one set of serums there is present an available lecithin compound, and not in the other, we shall then face a
new problem, namely, whether the fact of availability or the looseness of the lecithin compound in the f o r m e r serums is applicableto the whole blood or is to be attributed to a particular lecithin compound found only in the serum, but not in the blood corpuscles.
T h e part which lecithin m a y play in v e n o m haemolysis caused by
fresh serum is not quite clear, inasmuch as serum contains stillother complementary substances, which, doubtless, play their own
r61es. A sharply differential m e t h o d for v e n o m haemolysis causedby lecithin and by certain other complementary substances is required to clear up the question to what extent lecithin is responsible
for the production of haemolysis by v e n o m in unmodified serums.
B e f o r e recording the results of m y present investigations relating
to the points already r e f e r r e d to above, as well as to some otherquestions to be introduced and considered later in this paper, abrief statement of the recent development of o u r knowledge concerning venom haemolysis will make the necessity and object of thepresent w o r k m o r e obvious.
Snake venom produces h~emolysis only through the aid of a secondary substance. Thus Flexner and Noguchi 4 first demonstrated that the blood corpusclesof certain species of animals undergo venom h~emolysis when there is present
suitable serum, and believed that complement of such serum is the activating
principle. This was, however, found not to be always the case, as Kyes5 discovered that there are instances where the corpuscles are dissolved without theaddition of serum. The cause of the independent susceptibility of these corpuscles was ascribed to the presence of intraeellular activator in these cells.
The general characteristics of intracellular activators agreed with those of serum
complement; Kyes called them endocomplements. At the same time lecithin
4Flexner and Noguchi, Jour. of Exper. Med., 19o2, vi, 277.
Kyes, Berl. klin. Vdoch., I9o2, xxxix, 886, 918.

438

Venom Activc~tors of the Blood.

was found to be capable of activating venom with striking readiness, but Kyes
distinguished this form of venom hmmolysis from that caused by the complementary substances of certain fresh serums or by the endoeomplement of certain
susceptible corpuscles, by their resistance to high temperature and their activity
at o ° C., in which lecithin excelled the other. He also confirmed the important
observation of Calmette 6 of the acquisition of power or the increase in strengthof the venom activating property of various serums after heating to 65 ° C. andhigher. This phenomenon was attributed by Keyes to the liberation of lecithinby the action of heat. While adding still further evidences to the occurrence ofvenom-complement hmmolysis, Keyes and Sachs 7 withdrew the opinion that endocomplement was a thermolabile complement, and presented a new view that itis really lecithin contained in the stroma of the susceptible corpuscles. The
reason why the stroma of venom-resistant corpuscles does not react with venom
was explained by the hypothesis that lecithin exists in an unavailable form inthese corpuscles. In his third communication Kyes s succeeded in preparing acompound of venom and lecithin (lecithid) and threw suspicion on that modeof venom hmmolysis in which complement is thought to take a part.
Kyes suggested the possibility that any injurious substance may modify the
corpuscles so as to render available the lecithin otherwise inaccessible to thevenom. But this assumption has never been proven experimentally. My previous experiments ~ show lecithin to be by no means an inert compound, although
its activity is likely to be underestimated on account of the slow reaction-time.
Further, I was led to consider certain oleic compounds as well as oleic acid asvenom activating agents/° This point has an important bearing on the present
investigation of the venom activating substances of normal serum and susceptible
corpuscles. Although no relationship between the linolvti~- and the hmmolytic
properties of venom and phytogenous toxalbumens nave been established, yet
the discovery by Neuberg and Rosenberg xl of the lipolytic property of various
venoms and bee poison is a fact of great interest. Ricin, which is a powerfullipolyzer, 12 forms a strong hmmolysin when mixed with free lecithin, is a fact
important in that it shows that ricin does not unite with the lecithin in the
integral corpuscles or serum. Apparently lecithin in the native condition inthese substances is unattackable by ricin.

H e n c e it appears that we are a g a i n in the d a r k as to the realnature of venom h~emolysis. We are entirely unable to answer thequestion whether complement and certain complement-like bodies
of serum have any part in venom hmmolysis, and whether lecithin
Calmette, Compt. rend. d. l'Acad, d. Sciences, 19o2, cxxxiv, 1446.
' Kyes and Sachs, Berl. kiln. Woch., 19o3, xl, 21, 57, 82.
Kyes, Berl. klin. ICoch., 19o3, xl, 956, 982.
9 Madsen and N'oguchi, Oversight over det Kongl. Danske Vedenskabernes
Selskabs Forhandlungen, 19o4.
1oNoguchi, Jour. of Exper. Med., 19o6, viii, 87.
11 Neuberg and Rosenberg, Berl. klin. Woch., I9o7, xliv, 54.
~2Pascucci, Ho[meister's Beitriige, 19o6, vii, 457.
18Neuberg and Rosenberg, loc. cit.
'

Hideyo Noguehi.

439

is really present in available form for venom in activating serumand susceptible corpuscles. The sum of what we know at present
is that blood serum and corpuscles yield large amounts of lecithinupon alcoholic extraction, that venom can form a powerful hmmolytic compound with free lecithin, that the activating property ofserum and venom susceptibility of the corpuscles have no direct
relation to their lecithin content, that certain activating serums contain venom activating principles which in some respects closely
resemble complement, that certain chemicals, which may be present
only in certain bloods as normal constituents, can produce a formof venom h~emolysis hardly to be distinguished from that caused
by normal serum.
A ready way to clear up this confusing point is to discover anagent possessing the elective inhibitory action upon one or other ofthe venom activating principles. In course of my study 14 on the
anticomplementary action of various acids, alkalies and salts, I
found, among others, that various salts of the alkali earths inhibit
complementary action without altering the serum amboceptors.
Calcium chloride, 15 when used in a dilute solution, is most suitableto remove the complementary action of serum. This salt was
employed in the present work to inactivate serum complement, as
it has no marked destructive action upon venom amboceptor, whenused in a strength of ~ N, or weaker. On the other hand, calcium
chloride has no anti-activating power against lecithin. Venom
lecithid retains its ha~molytic activity in a medium containing calcium chloride. When the amount of lecithin, or venom lecithid,
is very small the salts retard complete hmmolysis, but have no ultimate effect on the process.
In addition to this differential agent an ethereal extraction ofvenom activators prepared from an active serum or the stroma ofsusceptible corpuscles was employed as a means to distinguish thelecithin-like activator from the complement-like ones. This method
was especially useful for determining the protein-lecithid nature of
the activator of certain serums and all heated serums. I shallreturn later to a discussion of this point.
1, Noguchi, read at the meeting of the American Society of Bacteriologists,
New York, Dee. 28, I9O6.
1~o.85 per cent. of this salt is isotonic.

440

Venom Activators of the Blood.
VENOM

ACTIVATORSOFFRESI-I

SERUM.

To determine whether the venom activating property of fresh
serum is removed by calcium chloride, 0. 5 c.c. of the serum underconsideration was mixed with 0. 5 c.c. of ~ N. solution of this salt,
and the volume made 2 c.c. with 0. 9 per cent. salt solution. The
washed corpuscles were added in proportion of five per cent. Afteran incubation of half an hour at 37 ° C., o.I c.c. of 0.4 per cent.
solution of cobra venom was introduced, and the whole incubatedfor several hours at 37 ° C. and left at room temperature for the
rest of the night. The result was read off, as a rule, within twentyfour hours. The corpuscles and serum came from the same blood;
but sometimes the washed corpuscles of a second blood were employed. This last combination is, of course, possible only when theserum in question has no h~emolytic action upon the corpuscles.
Table I. shows the results obtained with the serum and corpusclesof the same blood.
TABLE I.
Blood of

Man
Horse
Pig
Cat
Guinea-pig
RabbitPigeon
Hen
Goose
Goat
OX

Control.
Fresh Serum o.5 c.c. ) o.* c.c. of o.t ¢+ Corpuscles.
~- Cobra Venom+ o.9 ~ NaC1 z.5 c.c. ) Solution.
complete

CaCI, Addition.
Fresh Serum o.5 c.c. '{ o.* c.c. of-I- Corpuscles.
[ o.x~Cobra+ N/1oCaClao.sc.c. [ V e n o m+ 0. 9 ~ N a C I Lo c.c. J Solution.

hmmolysisno h~emolysis

"
.

.

.

.

.

.

.

.

.

.

.

.

"

"

"

"

"
"
no hm,molysis

"
"
"
~*

The above experiment shows that the majority of the varietiesof blood employed undergo complete h~emolysis by venom and that
their activating substances are completely inhibited by calcium
chloride. The absence of h~emolysis in the presence of CaCI 2 is
not due to destruction of the venom, because the addition of a small
quantity of free lecithin to such mixture produces complete h~emolysis. Or, the presence of a small amount of lecithin, which may
be added purposely at the same time as the CaC12, leads to complete
h~emolysis. There is still another way to prove t h a t the corpuscles
are sensitized by venom in the CaC12 mixture. The corpuscles may

Hideyo Noguehi.

441

be washed with 0.9 per cent. salt solution and finally suspended ina fresh lot of the latter.
If now a small amount of lecithin oro.5 c.c. of any activating serum is added complete h~emolysis occurs.
Whether or not the venom activators of the serums are identicalwith compounds contained in them cannot be determined from thisexperiment, as both are inactivated by CaC12. At all events, leci,
thin is excluded by this test as the activating agent of these serums.
The serum of the dog is extremely rich in venom activating substances and differs from other serums in its relation to inactivation
with calcium chloride.
Of twelve different samples of normal
dog serum complete inactivation by CaC12 was obtained only inthree, marked delay, in five, and slight retardation, in the rest.
In this series homologous and heterogeneous corpuscles were tested
and gave the same result. Thus dog's serum is an example of a
fresh serum, in which lecithin exists in an available form forvenom activation.
The simultaneous presence of certain CaC12
inhibiting venom activators was also demonstrable; and in a few
instances this class of activators alone was present in the dog serum.
VENOM ACTIVATORS OF BLOOD CORPUSCLES.
A wider variation is exhibited by the corpuscles of different
species of animals in their action toward venom. Some kinds ofcorpuscles are promptly, while other not at all dissolved by venom.
The latter class of corpuscles remains undissolved because of theabsence of suitable venom activators although sensitization still
occurs.
The corpuscles of all bloods undergo h0emolysis whensuitable activators are present.
The most susceptible corpuscles
are those of dog and guinea-pig, and entirely refractory are thoseof ox, goat and sheep. The corpuscles of horse, rabbit, rat, pig
and man occupy an intermediary position. Those which approach
the limit of the series of non-susceptibility require a longer incubation for hmmolysis than those which stand near the opposite end.
This incubation time can be greatly shortened by adding certain
activating serums.
The original assertion of Flexner andNoguchi 16 that venom requires complement for its activation was
based on the fact that certain washed corpuscles remained intact~' F l e x n e r and Noguchi, four. of Exper. Med., 19o2, vii, 277.

442

Venom Activators of the Blood.

during a period of experimentation in which the same corpuscles
underwent complete haemolysis when fresh serum was added. In
a later paper Flexner and Noguchi 17 described the presence of complementary substances in various kinds of organic cells.
The cause of destruction of certain corpuscles by venom alone
is ascribed by Kyes is and Kyes and Sachs 19 to the presence ofendocomplement. The thermolabile nature and the inactivity at
o ° C. of endocomplement led them first to classify it with complements. But later they showed that the thermolability is caused by
the simultaneous presence of hmmoglobin and hence concluded that
it was the lecithin of the stroma. To this particular point I will
return a little later.
In my experiment regarding the nature of intracellular venom
activators of the integral corpuscles, I employed calcium chloride
to determine whether this salt can suppress haemolytic action ofvenom upon susceptible corpuscles. The result was rather remarkable. The washed corpuscles of horse, rat, rabbit, cat, guinea-pig,
pig, pigeon, goose, hen and man remained undissolved in ~ N. to~¼~ N. CaC12 medium in the presence of corbra venom. The controls dissolved completely in from fifteen minutes to twenty-fourhours. The protection of the susceptible corpuscles is not due tothe desctruction of venom by CaC12, because by a later addition
as well as by the simultaneous introduction of a small amount of
lecithin, or even by the addition of old dog serum (inactive withage) complete haemolysis can be induced. The least susceptible
corpuscles require the smallest amount of CaC12 for protection.
On the other hand, a trace of hamolysis is often observed with the
corpuscles of guinea-pig or man after twenty-four hours. Different samples of dog corpuscles behave differently. I employed
several samples and with some there is perfect protection, withothers partial protection. Dog corpuscles are liable to spontaneous
hzemolysis within twenty-four hours, which fact may be responsiblefor irregular results.
The removal of intracellular activators by means of digestion~7F l e x n e r a n d N o g u c h i , ]our. of Path. and Bact., I9o5, x, i I I .
is Kyes, loc. cit.
1, K y e s and Sachs, loc. cit.

Hideyo Noguchi.

443

in a calcium chloride solution g a v e variable results. T h e corpuscleso f horse or pig are protected f r o m v e n o m a f t e r digestion in 1
N. CaC12 f o r h a l f an hour, but those of g u i n e a - p i g and d o g are not.
I f we use ~v N. solution these corpuscles are rendered even m o r esusceptible to v e n o m t h a n the undigested samples. T h e mechanicalinjuries b r o u g h t a b o u t d u r i n g the p r o c e d u r e o f w a s h i n g a w a y thecalcium salt with sodium chloride solution m a y h a v e some influence.
P e r h a p s a sudden c h a n g e o f tonicity and b r e a k i n g up of a g g l u t i n a t e dmasses o f corpuscles hasten the destruction.
H e n c e it would app e a r t h a t in the m a j o r i t y of the corpuscles lecithin seems not to beable to play a p a r t as activator.
B e f o r e deciding this m a t t e r finallyo t h e r considerations should be t a k e n up.
T h e presence o f h m m o globin in lecithin containing fluid, or the addition of lecithin to the
corpuscular solution does not p e r m i t o f the inactivation b y calcium
chloride.
Ovovitellin of hen's e g g contains a h i g h p e r c e n t a g e o flecithin.
T h i s lecithin-proteid is not split up w i t h ether, but b y
hot alcohol.
I p r e p a r e d a saline suspension of ovovitellin and exa m i n e d it for v e n o m activation.
I t was f o u n d to be an excellent
activator.
H e n c e the question arose w h e t h e r this proteid c o m p o u n d could not be easily inactivated by calcium chloride.
This
p r o v e d not to be possible.
T h u s lecithin, either in a mechanicalm i x t u r e with h~emoglobin or corpuscular contents, or in a chemicalc o m b i n a t i o n w i t h proteid, is not inactivated by CaC12. T h a t lecithin does not exist in an available f o r m f o r v e n o m hmmolysis insusceptibIe as well as insusceptible corpuscles can be s h o w n b y thef o l l o w i n g experiments.
The washed corpuscles of the guinea-pig were broken up with about threetimes their volume of water. The stroma was separated out by adding 0.9 per
cent. sodium chloride and the precipitated stroma was colIected by centrifugation.
The clear, intensely dark-red supernatant fluid was pipetted off and nsed forsome tests, while the stroma was twice washed in a 0.9 per cent. sodium chloride
solution.
The pink-colored stroma was tested for its venom activating property, forwhich the corpuscles of ox and goat were used. The activation was not rapid,
but progressed rather slowly. The deep-red supernatant fluid was not activating.
The test with calcium chloride showed that the activating property of the stroma
is completely removed by this salt. The shaking of the stroma with a large
quantity of ether also removed this property. On the other hand, the ethereal
extract upon evaporation left a small amount of fatty substances, which contained
chiefly aceton-soluble fats, but almost no lecithin to be detected even with venom.

444

Venom Activators of the Blobd.

I redissolved the oily mass in 0.9 per cent. salt solution, in which it showedturbidity and an acid reaction to litmus. This suspension is activating.

We have thus succeeded in locating the site and identifying theprobable nature of the phosphorus-free fat acting as an intraeellularvenom activator.
It may be added that the activating propertyof this suspension is completely set aside by calcium chloride. As
Kyes and Sachs first showed, lecithin could be isolated from thestroma with hot alcohol. The lecithin obtained from it is activating, but entirely different from the activators of the integral corpuscles, corpuscular solution and ethereal extract of corpuscles in
its relation to CaC12. While lecithin can be extracted with alcoholfrom any of the corpuscles, no matter whether they belong to thesusceptible or the insusceptible class, in nearly equal quantities, theether-soluble venom activators are found only in the corpuscles susceptible to venom. I have tried many times to extract an ethersoluble venom activator from the corpuscles of the ox and goat,
but without success.
These corpuscles are naturally venomresistant, and their corpuscular solutions do not contain a venom
activator. A later experiment with the corpuscles of the dog gavepractically the same result as those of guinea-pig.
Direct evidence that certain fatty acids, soaps and neutral fats
are capable of acting as venom activators can easily be given. The
venom-resistant corpuscles are washed and freed from the serum.
Then the minimal h~emolytic quantities of oleinic acid, sodium
oleate, ammonium oleate, neurin oleate and triolein are ascertained.
The subminimal h~emolytic dose of any of these chemicals is taken
and mixed with the corpuscular suspension of any of the insusceptible bloods.
No h~emolysis occurs after twenty-four hours.
But if an adequate quantity of cobra venom is added at the sametime, complete hmmolysis occurs. The addition of these chemicals
to the corpuscular solution (not suspension) of an insusceptible
blood renders it venom-activating. This artificially prepared activating solution of blood corpuscles behaves in the manner of thesolution of susceptible corpuscles, and becomes inactive upon
ethereal extraction in case of oleinic acid, organic soaps, and triolein.
With the solutions whose activating property is conferred by theaddition of alkaline oleate soaps, ether fails to remove it. Calcium

Hideyo Noguchi.

445

chloride is very effective in depriving these solutions of their acquired venom-activating property. The result with triolein is notsatisfactory, as two preparations from Merck and Kahlbaum gave
entirely different results, as the latter was almost entirely inactive.
The activators of susceptible corpuscles were completely and permanently inactivated by boiling.
From these results it seems justifiable to conclude that lecithin
does not exist in the corpuscles, irrespective of their susceptibility
to venom h~emolysis, in an available form for venom. The degreeof susceptibility of blood corpuscles depends chiefly upon theamount of ether-soluble activators contained in the cells. These
ether-soluble activators are, doubtless, fatty acids, and especiallyoleinic acid. The absence of fatty acids in the insusceptible corpuscles is in perfect harmony with the reaction to venom. If wecompare the amount of fatty acids 2° and the degree of venom susceptibility of different corpuscles we discover that a definite andundeniable parallelism between these two factors exists.
If anyamount of venom activators are present in the stroma after ethereal
extraction it does not necessarily follow that they are lecithin, because the ether-insoluble soaps may be prefixistent or be formed
during the manipulation of the corpuscles with water, and theywould certainly take on a good share in the activation.
VENOM ACTIVATORS OF HEATED SERUM.
Calmette discovered that any serum acquires venom activating
property or has its property increased by heat above 65 ° C. Kyes
found that the maximum effect is reached at IOO° C. and coincides
with the maximum liberation of lecithin. That the activating
property of such heated sera is due to lecithin can be further established by means of chloride of calcium, as the next table shows.
An attempt was made to ascertain whether lecithin exists in afree state or as a proteid compound in heated serum. That a noncoagulable proteid exists in serum heated to 80 ° to ioo ° C. in aneutral or slightly acidified reaction has long been known. Chabri6, =1 who first described this proteid, called it albumon. Howell's 22
20Abderhalden, Zeit. [. Physiol. Chem., 1898, xxv, 65.
Chabri6. Compt. rend. de l'Zcad, de Sciences, 1891, cxiii, 557.
Howell, Amer. Jour. of Physiol., 19o6-7, xvii, 280.

446

Venom

Activators
TABLE

of theBlood.

II.

Serum o 2 c.c.
(Total Volume made to
Corpuscles 5 per cent.
~ 2 e.c. with o. 9 per cent.
Cobra Venom o. i per cent., Solution o. i c,c. (added later).
NaCI Solution.

I
Fresh.

56° C.

Saline. ] C a C I ,,£~/zO O.5

c72j

Man
Horse
Guinea pig
Ox
None
Goat

"

Saline.

!

I. CaCI~-IN"°°sl

65 ° C.
S a ne85 ° C.

. CaCI2

I"'.... ,

Saline.....

Slight H. None Much H. Moderate C;,H.
"
I "
j
"
[Much it.
C. H. ~
"
C.H.
C.H.
None
"
Slight H. Slight H.

]

loo ° C,CaCI2 !Saline

CaCls. iN/,_o o.5 _ _" N:~?o~

Ci,H.

C;,H.

C~H.

recent investigations of this proteid added many new facts. AlbuNon is characterized as soluble in water, indiffusible through collodium membrane, non-coagulable in presence of neutral or acidreaction upon boiling, but easily precipitable by half saturation withammonium sulphate. It contains a large percentage of phosphorus
and iron. The phosphorus represents the lecithin which exists in
its molecule as a component extractable only by warm alcohol.
Howell considers albumon an artificial product formed through theaction of the heat, while some previous investigators considered ita normal constituent, or were uncertain on this point.
In my experiments I employed ox serum. The serum was diluted with threetimes its volume of water and gradually boiled at neutral reaction (neutralization by means of acetic acid). The non-coagulable portion was separated fromthe coagulum by filtration. The clear fluid gave the required characteristics foralbumon, and upon examination it proved to be highly active for venom and not
to be inactivated by calcium chloride. Ether failed to extract any activator fromthe fluid medium or after drying. On the other hand, warm alcohol yieldedmuch lecithin and perhaps some other venom activating substances. The proteid
precipitated out by the process of extraction was inactive. Goat serum yieldeda similar albumon.

Thus the conclusion seems warranted that the lecithin of heatedserum does not exist in a free state, but in a proteid compound,which is capable of reacting with venom. In this case the preexistence of this proteid in unheated serum is conclusively excludedby the fact that neither the fresh serum, nor the coagulum of thesebloods contained any available lecithin compounds.
* Guinea-pig corpuscles contain endocomplement, which accounts for complete h~emolysis. If o x or goat corpuscles are used no h~emolysis results, while
the same corpuscles are readily dissolved by the addition of over-heated serum,and cannot be reduced by CaCh.

H i d e y o Noguchi.
LOCATIONOF

AVAILABLE

LECITHINOFIN

VARIOUS447
PROTEIDFRACTIONSSERUM.

T h e e x i s t e n c e of v e n o m a c t i v a t o r s of l e c i t h i n n a t u r e i n n o r m a ldog

serum has been mentioned.

But the question whetherthea v a i l a b i l i t y o f l e c i t h i n i n this p a r t i c u l a r s e r u m is d u e to looseness o f
a l e c i t h i n - p r o t e i d c o m p o u n d , o r t o t h e p r e s e n c e o f a definite l e c i t h i n
c o m p o u n d p e c u l i a r t o this s e r u m w a s left u n d e c i d e d .
Two different samples of dog serum were dialyzed in collodium sacs against
running water and after seventy-two hours the whitish precipitates were collected by filtration and dissolved in o.9 per cent. sodium chloride solution. The
filtrate was made isotonic with the same salt. The first represents the serum
globulin and the second the serum albumin. These were now tested for venom
activating property. The result shows that the globulin fraction was inactive
and the albumin highly active. The same serums were then fractionated withammonium sulphate. The precipitate produced by half saturation was separated
from the serum by filtration. The filtrate was then completely saturated byfurther addition of this salt. The precipitate was obtained by filtration. The
first represents the globulin and the second the albumin fraction of the serum.
Both were dialyzed in eollodium sacs for seventy-two hours. In the first sac,which contained the globulin fraction, there was a large amount of whitish
precipitate. This was filtered and collected; the filtrate was also preserved fortesting. The content of the second sac was perfectly clear, and was used forthe tests. All three were made isotonic with sodium chloride. The globulinprecipitate dissolved in this medium with slight opalescence. The tests forvenom activation gave the following result: the solution of the globulin precipitate and the albumin fraction were unable to activate venom, while the clear
portion of the first sac was extremely active. This last fraction was noncoagulable in saline solution upon boiling, but precipitable by ammonium sulphate and heat and coagulable by alcohol. It gave a blue-violet Biuret reaction.
The venom activating property of this portion was unaffected by CaCI2 or ethereal
extraction.
Similar tests with ox and pig serums (both non-activating) gave results
differing from those with dog serum, as the globulin lacked the venom activating
property.
T h e n e x t question was whether the globulin and a l b u m i n fract i o n s of t h e s e s e r u m s c o n t a i n a n y lecithin. T h e e x a m i n a t i o n w a s
m a d e b y e x t r a c t i n g the s e r u m s w i t h h o t a l c o h o l ( 7 o ° C . ) .
T h e g l o b u l i n f r a c t i o n of t h e s e r u m s o f dog, horse, o x a n d p i gyielded u p o n hot alcoholic extraction comparatively small a m o u n t sof lecithin.

Ont h e o t h e r h a n d , t h e a l b u m i n f r a c t i o n of t h e s es e r u m s y i e l d e d m u c h m o r e l e c i t h i n t h a n the g l o b u l i n . T h e n o n c o a g u l a b l e p o r t i o n o b t a i n e d f r o m t h e first sac ( c o n t a i n i n g the pre-

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Venom Activators of the Blood.

cipitates by half saturation with ammonium sulphate) contained alarge amount of lecithin.
The globulin as well as albumin fractions did not produce uponboiling, either with or without sodium chloride, as much lecithin(in available form for venom) as the whole serums from which
they were isolated. In some instances even considerable reductions
were observed with the globulin fraction plus the non-coagulable
lecithin proteid of dog serum. I am inclined to ascribe this reduction in activity to the simultaneous presence of cholesterin in
the medium. The problem, which was set at the beginning of this
topic, has been resolved experimentally: the venom activating substance of lecithin nature in dog serum exists as a definite proteid
compound, and does not depend upon a loose combination between
lecithin and globulin or albumin.
The non-venom-activating
serums do not contain a similar lecithin proteid to that found indog serum.
P R E P A R A T I O N OF A R T I F I C I A L V E N O M

ACTIVATING SERUM.

Normal serum may contain two different sets of venom activators, namely, one resembling complement and the other a proteid
compound of lecithin resembling albumon. The latter is occasionally present in normal dog serum, while the former constitute thevenom activating substances contained in all venom activating
serums. Calcium chloride renders the first class of activators
ineffective. The venom activating property of these serums, which
are subject to the calcium inactivation, is completely or in part
removable by ethereal extraction. The ethereal extract contains
fatty acids and neutral fats, but not lecithin. The inactivation ofthese serums by the temperature of 560 C. is rather uncertain,
although more or less reduction in activity is observed. Sometimes
complete loss is obtained. The activating function of this class ofvenom activators is suspended at o ° C. Except in certain herbivorous mammalian bloods (ox, goat and sheep) the majorityof mammalian serums and certain avian serums contain the complement-like venom activators in varying quantities, and no lecithinlike activators are to be found in these serums. Even in dog
serum, which contains an available compound of lecithin, there isHideyo Noguehi.

449

a large amount of the other present. I have already mentionedthat in the ethereal extract of these serums varying quantities offatty acids and fats are contained. But more convincing is thefact that none of the non-activating serums yields more than aminute quantity of activating fatty acids.
By the term " venom activating fatty acids " oleic acid is chiefly
meant. Palmitic and stearic acid are far less effective in this respect. The more oleic acid is present in the ethereal extract, thestronger is the venom activating property.
Apart from the varieties of the fatty acids, the whole amount ofthese acids extractable from dog serum is nearly twice as much as
from the serum of ox, goat or sheep. ~a I have already mentionedthat two samples of triolein which I used acted very differently,
so that I have been put in doubt regarding my earlier experiments. 24
The facts above enumerated clearly indicate that fatty acids havea direct relation to the venom activating property of blood serum.
Leaving aside the question whether fatty acids represent theentire venom activators or only part of them, I will consider next
whether or not certain fatty acids and soaps can confer the venom
activating property upon non-activating serums.
For this experiment two sets of normal ox serum were used:
the one without any modification, and the other after being shaken
with a large volume of ether. To both sets oleinic acid in amountsof o.12 25 to IOO grams of the serum was added. In still otherseries, and with ox serum, sodium oleate was used instead of oleinicacid. Oleinic acid and sodium oleate are highly h~emolytic if theseare dissolved in this concentration (or emulsified in case of oleinicacid) in a o. 9 per cent. sodium chloride solution; but in ox serum
they remain completely inactive. When these mixtures are usedas venom activators they display very powerful h~emolytic action
upon insusceptible corpuscles (ox and goat). Their action is not
so prompt as that of dog serum or any heated serum, but more likethat of guinea-pig serum, except that it is prompter than that. By
Abderhalden, loc. cit.
Noguchi, Jour. of Exper. Med., 19o6, viii, 87.
This proportion was taken from the data given by Abderlhalden for dog
sertlrn.

450

Venom Activators of the Blood.

reducing the quantity of the mixture a descending scale of activation can be secured until no effect whatever is obtained. There isno marked difference between the acid and soap in the mode ofaction. Calcium chloride completely inactivates these mixtures.
The temperature of 5 60 C. has a marked reducing power upon thesoap and serum mixture, but hardly any upon the acid and serum
mixture. Ethereal extraction removes the activating property of
the latter, but not of the former. At o ° C. the latter is still slightly
active, but not the former. 26
These artificially prepared venom activating serums are not easily distinguishable from certain natural serums.
The second type of venom activating serum, in which lecithin
is present in an available form, can be artificially prepared, by adding pure lecithin, or the non-coagulable p roteid of heated serum,
to non-activating serum. When the mixture is made its activating
property cannot be stopped by calcium chloride or the temperatureof o ° C. Cholesterin inhibits the effect of this mixture. Ether
cannot remove much of the lecithin when once mixed with theserum; this perhaps may be due to the fact that lecithin can enter
into combination with some of the serum componentsYT H E PROTECTIVE ACTION OF CALCIUM CHLORIDE AGAINST VENOMCYTOLYSIS.

The powerful cytolytic property of various kinds of venoms upon
the cells of liver, kidney, nerve, testis, and ova of different animals has been demonstrated by Flexner and Noguchi3 sThemechanism of the cytolysis was found to be essentially the same asthat of h~emolysis and that intracellular complements played an important part. Knowing no other means to eliminate the intracellular complement at that time, Flexner and Noguchi employed heat(temperature of 55 ° C. maintained for thirty minutes).
Theyfound that this temperature rendered the cells insusceptible tovenom, unless fresh serum was added to the mixture. In this way
the similarity of venom cytolysis and serum cytolysis was established.
**No suppression of action without serum constituents.
Mayer and Terroine, Compt. rend. d. I. Soc. d. Biol., I9o7, lxii, 398.
"SFlexner and Noguchi, Jour. of Path. and Bact., I9o5, x, III.

Hideyo Noguehi.

451

Since I found in calcium chloride a powerful anti-complementary
haemolytic substance it was natural to expose the somatic cells to
the influence of this chemical to establish the nature of the complementary body present in them. In this series of experiments
the cells of liver, kidney, testis and brain of the guinea-pig and rat
were employed. It was difficult to obtain many ganglion cells ingood condition, while the testicular cells were easily obtained intact.
Uniform emulsions (5 per cent.) in salt solution of these cells were
measured into small test-tubes to which daboia venom was addedin proportion of I : IOO. The mixture was placed in a water thermostat at 37 ° C. for five hours and examined microscopically. To
test the action of calcium chloride the salt was added to the suspensions of these cells in ratio of ~ N., and half an hour later thevenom was introduced.
The results are briefly as follows: the cells of the liver, kidney
and testis are well preserved in the saline solution after five hours.
The ganglion cells are less distinct in outline, but apparently havenot disintegrated (controls).
In the venom solution the testicular
cells are nearly all dissolved, but the spermatozoa minus heads
remain. The clearing up of the cellular elements is distinctly
visible in vitro. The liver cells become more or less swollen andthe outlines indistinct. The granules disappear and the nuclei become more distinct. Agglutination of free cells is marked. The
kidney cells and tubules become gradually indistinct and a generaldisintegration of the former occurs. The number of cells is lessthan in the controls; agglutination occurs. The ganglion cells
have disappeared wholly, and a general solution of granular elements has taken place. These alterations are much more pronounced in the emulsions of guinea-pigs' than of rats' brains. Not
only the microscopical, but also the macroscopical appearances ofthe venomized emulsions is at once recognized by a marked clearing up of the original turbid suspensions. The addition of calcium chloride completely prevents the destructive action of venom
upon the cells. The protection afforded by this salt is greater thanis obtained by heating the cells to 55 ° C.
From these facts the complement-like nature of the venom activators contained in the somatic cells is once more established.

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Venom Activators of the Blood.
SUMMARY.

In normal serums of the majority of mammalian and avian blood
there exists certain substances capable of activating venom hmmolysin. They are extractable from serum by means of ether, and arecapable of conferring upon the originally non-activatlng serum a
power to activate venom, when mixed with the latter. The ethereal extract consists of fatty acids, neutral fats and possibly also
some ether soluble organic soaps. The fatty acids and soaps, especially of the oleinic series, acquire certain characteristics of complements in general, when they are mixed with serum. They areinactive without the venom in the mixture; they are inactivablewith calcium chloride; they exhibit a tendency to go off in activity
with age; they are inactive or only weakly active at o ° C., and they
are extractable by ether. In testing the serum from which theether soluble substances are removed, it is found that no venom
activating property is left. W a r m alcoholic extraction of such
serum yields, however, a large quantity of lecithin. In the caseof non-activating serums no venom activating fats appear in theethereal extract. Lecithin exists in such serum in no less quantity
than in the activating kind.
The addition of oleinic acid or its soluble soaps to a non-activating
serum, in a ratio which corresponds to the percentage of fatty
acids or soaps contained in some of the easily activating serums,
will make the serum highly active in regard to venom.
In normal serum of dog there exists, besides the group of activators already mentioned, another kind of venom activators which
has been identified as a lecithin compound acting in the manner offree lecithin.
A very sharp differentiation of the h~emolysis produced by thisactivator and by the other groups of activators is obtained by meansof calcium chloride, which is powerless against lecithin or lecithincompounds, but effective in removing the action of the latter. Thislecithin containing proteid can l~e precipitated by half saturation
with ammonium sulphate, but is perfectly soluble in water, and is
not coagulated in neutral alkaline salt solutions upon boiling. Alcohol precipitates a proteid-like coagulum and extracts lecithin fromit; ether does not extract lecithin from this compound.

tIideyo Noguehi.

453

Non-activating serums do not contain any such lecithin compound.
Lecithin contained in other serum proteids, mainly as lecithalburain, and perhaps as contained in globulin, is not able to activate
venom. This is true of all the serums with which I worked; itmatters not whether these fractions (obtained with ammonium
sulphate) belong to the most activating serum (dog) or to the
non-activating serum ( o x ) .
The non-coagulable portion of all heated serum contains a venom
activator of the nature of lecithin. This activator is contained ina non-coagulable proteid described by Howell which is identicalwith Chabri~'s albumon. .As there is no ether-extractable lecithinin this portion of the serum, the activating property of heatedserum must be due to this proteid compound of lecithin. That thislecithin proteid does not pre-exist in normal serum but is producedby the action of high temperature is true of all serums except thatof the dog. In venom activation we know now that lecithin becomes reactive with venom when it is transformed from other proteid compounds into the non-coagulable form, the albumon. Howell's view of the non-existence of the non-coagulable proteid innormal serum seems to receive a biological support from venom
h~emolysis.
Ovovitellin derived from hen's egg is one of the best venom
activators of the lecithin proteid type.
The cause of venom susceptibility of various kinds of blood corpuscles does not depend upon the existence of lecithin in the corpuscles, but solely upon the amount of fatty acids, and perhaps, also,soaps and fats, contained in the corpuscles. The protection which
calcium chloride gives against venom h~emolysis is proof of theabsence of lecithin activation. F r o m the stroma of susceptible
corpuscles fatty acids or some fats can be extracted with ether.
.After ethereal extraction the stroma becomes non-activating, while
the extract contains fatty acids and some soaps or fats, which when
added to venom-resistant corpuscles render the latter vulnerable tovenom. The corpuscular solution of non-activating corpuscles does
not contain enough fatty acids. The larger the amount of fatty
acids and soaps in the corpuscles, the easier the cells undergo

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Venom Activators o[ the B[ood.

venom haemolysis. Lecithin exists in the stroma of all kinds ofcorpuscles, but in a form unavailable for venom activation.
The somatic cytolytic processes caused by venom requires intracellular complements. The experiments performed on the cells of
liver, kidney, testis and brain of the guinea-pig and rat indicatethat the substances which act as complements are inactivable bycalcium chloride.