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Journal of Experimental Medicine

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THE RELATION OF PROTEIN, LIPOIDS AND SALTS TO
THE WASSERMANN REACTION.1
BY HIDEYO NOGUCHI, M.D.
(From the Rockefeller Institute for Medical Research, New York.)

In the present work on the Wassermann reaction in syphilis andgeneral paralysis several points have been especially considered,namely: (I) the quantitative estimation of the active substancesin syphilitic blood sera and paralytic cerebro-spinal fluids; (2) the
effects of physical and chemical agents upon the active substances;(3) the quantitative estimation of the globulin content of syphilitic
blood sera and paralytic spinal fluids; (4) the alcohol-extractibilityof the active substances of syphilitic sera and paralytic spinal fluids;and (5) the antigenic lipoids and salts entering into Wassermann's
reaction.
THE QUANTITATIVE ESTIMATION OF THE ACTIVE SUBSTANCES OFSYPHILITIC BLOOD SERA AND PARALYTIC CEREBRO-SPINAL FLUIDS.

The blood sera from fifty-five syphilitic patients have beenstudied with the view of determining the minimal quantity of serum
just sufficing to produce complete fixation 2 of complement, theamount of which was contained uniformly throughout the entire
work in o.I c.c. of fresh serum of the guinea-pig. Of eight casesof primary syphilis, five gave positive reactions, the titers ranging
from o.I to a little over 0.2 c.c. The majority of the titers were
0.2 C.c. Of forty-two cases of secondary syphilis, six have been
untreated and gave positive reactions in titers of o.ooo6, 0.002,
0.003, 0.004, o.oI and 0.04 c.c. respectively. Seventeen cases had'Received for publication September , 9o8.
2In each tube 3 hemolytic units of anti-sheep amboceptor (rabbit serum)
were employed, the quantity being o.003 c.c. The alcoholic extracts of theblood and liver of man or rabbits were used as "antigens" in quantities of o.1
to 0.2 c.c. of a o.x per cent. or 0.2 per cent. suspension. Purified preparations oflecithin were also employed as "antigens."

84

Hideyo Noguchi.

86

been slightly treated and gave positive reactions in titers of o.o to
0.2 c.c., or more frequently of 0.02 to o.o6 c.c. This group and thegroup of untreated cases of secondary syphilis gave the highest
titers of all the cases examined. Seven cases were moderately
treated and gave positive reactions in titers of 0.2 or a little morethan 0.2 c.c. Twelve cases were in the late secondary stage understeady and prolonged treatment, of which only two gave positive
reactions in a titer weaker than 0.2 c.c. The remaining ten cases
gave negative results. Five cases of tertiary syphilis, which had
been under steady and prolonged treatment, gave negative reactions
excepting one in which the test was positive and the patient had
lesions of the nervous and osseous systems.
Thirty-four control sera were examined; II were from normal
persons and were uniformly negative; 23 were from persons suffering with other diseases than syphilis; of these five reacted positively in titers from 0.05 to 0.2 c.c. I could not exclude abso-,
lutely a syphilitic infection in the latter.
The cerebro-spinal fluid from cases of general paralysis gavein the majority of instances titer of o.I to 0.2 c.c. It was rarelythat a sample with a titer of 0.02 c.c. was met with. I have not
met an instance in which O.oI c.c. produced complete inhibitionof hemolysis.
Hence the highest titer of any syphilitic serum was o.ooo6 c.c.,and of any cerebro-spinal fluid, 0.02 c.c.
THE

EFFECTSOF PHYSICALAND CHEMICALAGENTS UPON TIHEACTIVE SUBSTANCES.

The active substances of unfractionated syphilitic serum or of
the globulin fractions obtained by half-saturation with ammonium
sulphate, or through dialysis, are inactivated at 700 to 760 C. intwenty minutes. I may add that the activity of the euglobulin
fraction obtained through dialysis disappears more easily whenheated in distilled water than when heated in o.9 per cent. saline
solution. It was almost completely destroyed by a temperature of
650 C.
Drying syphilitic serum at 24 ° C. does not affect its activity inregard to the Wassermann reaction, and heating in a dry state to86

Relation of Protein to Wassermann Reaction.

temperatures below Ioo0 C. for twenty minutes leaves its activity
unimpaired. The temperatures of I200 and 350 C. produce partial insolubility of the dry serum and render it almost inactive.
The cerebro-spinal fluids from cases of general paralysis were,in my experience, which agrees with the findings of Marie andLevaditi, 3 rendered inactive in ten to twenty minutes by temperatures of 700 to 80 ° C.
Concerning the deterioration of the active substances with age,
I may state that a sample of serum which gave a titer of 0.004 c.c.
after the first twenty-four hours was reduced to a titer of o.oI c.c.
after fifty-six days in the refrigerator at 20 C. Some spinal fluids
obtained from cases of general paralysis and almost a year old
gave a titer of o.I to 0.2 c.c., no quantitative observations having
been made when they were first collected. Bruck and Stern 4 statethat the active principle in syphilitic sera will retain its strengthwith little deterioration when kept in the refrigerator, no lengthof time being assigned.
The activity of cerebro-spinal fluids from patients suffering fromgeneral paralysis is destroyed by acids or alkalies. When 0.2 c.c.
of a fairly active sample of spinal fluid of a paralytic is mixed with
i.6 c.c. of a o.9 per cent salt solution with an acidity or an alkalinity corresponding to N/4o concentration, the fluid becomes so
modified that after removal of acid (HCl) or alkali (NaOH) by
neutralization, at the end of one hour at 370 C., it no longer gives
the Wassermann reaction. A concentration above N/40 of hydrochloric acid or sodium hydroxide in the same amount causes marked
reduction of haemolysis. This antihsemolytic phenomenon may possibly be due simply to increase in the salt tonicity after the neutralization.
Trypsin (Griibler's Trypsin sicc.) in neutral or a weakly alkaline(0.5 per cent. Na2 CO 3 ) medium caused complete inactivation of
the syphilitic sera or cerebrospinal fluids. The ferment was allowed to act at 370 C. for three hours before the tests were made.
It was customary to subject 0.2 c.c. of the serum or spinal fluid tothe digestion and then to test the entire mixture, after neutralization in case of alkaline reaction, for the Wassermann reaction.
'Marie and Levaditi, Annales de l'lnst. Pasteur, 1907, xxi, 138.
'Bruck and Stern, Berl. klin. Woch., 908, xlv, 30I.

Hideyo Noguchi.

87

Pepsin (Griibler's Pepsin puriss.) was found to be without effectwhen added to the serum or spinal fluids of syphilitics and paralytics in neutral edium. In the presence of a 0.2 per cent. hydrochloric acid it destroyed the activity of these fluids very promptly andcompletely. The acid alone had been found to be injurious whenused in that strength, although a more complete disappearance of theactive substances in the presence of pepsin conclusively demonstrated
the effect of this ferment. It seems to be of some interest to recall
here that, in spite of the pronounced antienzymotic properties ofblood serum, the active substances of the serum and spinal fluid ofsyphilitics and paralytics are easily destroyed by tryptic and peptic
digestion.
The activity of the syphilitic sera and paralytic cerebro-spinal
fluids disappears when they are exposed to the combined effects ofa photodynamic substance and direct sunlight. Eosin "rein" in
dilution of 0.03 per cent. destroys the substances within three hours
in the presence of direct sunlight. No visible precipitation occurs
in the exposed eosinized mixtures. A three hours' exposure of noneosinized controls to direct sunlight produces a perceptible diminution of the activity. On the other hand, no deteriorating effect isexerted by the dye in the dark. Whether the destructive action ofeosin in direct sunlight is, in part or in toto, due to the acid formation in the mixture has not been determined.
THE QUANTITATIVE ESTIMATION OF THE GLOBULIN CONTENT OFSYPHILITIC SERA AND PARALYTIC CEREBRO-SPINAL FLUIDS.

Klausner5 found that by adding adequate quantities of distilledwater to syphilitic sera a precipitate would form, and Sachs andAltmann0 observed a similar precipitate when a weak alcohol is
employed instead of distilled water. The differences between syphilitic and non-syphilitic sera in these two tests are quantitative andbecome indistinguishable upon prolonged standing. KIlausner
thought his reaction due to an increase in globulins in syphilitic sera.
Porges and Meier 7 ,' discovered that the sera of syphilitic patients'Klausner, Wien. klil. Woch., 908, xxi, 214, 363.
'Sachs and Altmann, Berl. klin. Woch., 1908, xlv, 522.
'Wassermann, Berl. klilt. Woch., 1907, xliv, 1599, 634'Porges, Wien. klin. Woclh., I9o8, xxi, 206.

88

Relation of Protein to Wassermann Reaction.

can be differentiated from normal sera by the appearance of aquicker and more abundant precipitation of lecithin emulsion upon
addition of these sera.
The diagnostic value of the Wassermann reaction and the PorgesMeier reaction for syphilis has been found to be, to a great extent,
similar. Considering the r6le which lecithin plays in both reactions,
it would seem not improbable that the active substances entering
into these reactions are identical. Many chemicals to which antigenic properties had been assigned are also found to produce theprecipitation phenomenon with syphilitic fluids. Thus sodium oleateand sodium glycocholate were shown by Nobl and Arzt 9 and Fritz
and Kren' ° to give a similar reaction to lecithin. According toElias, Neubauer, Porges and Salomon"l the active substances belong to globulins and are present both in normal and syphilitic sera,
differing only in the quantities which are greater in the latter thanin the former.
No quantitative estimation of the serum globulin content of thesyphilitic sera has yet been systematically made.
The presence in the spinal fluid of general paralytics and tabeticsof the active substances has repeatedly been shown by previous workers. 1 2, 13, 14 Long before the Wassermann reaction had been applied
to these cases an increase in proteins in the cerebro-spinal fluid had
been known to be characteristic to these diseases.1 5
In my present investigations thirty-three cases of syphilis andtwenty cases of other diseases than syphilis, including five normal
individuals, have been subjected to simultaneous tests for the globulin content and for the Wassermann reaction. The method of estimating the quantity of serum globulins was a relative one and consisted of weighing the precipitate obtained by half-saturation of thesera with ammonium sulphate (or I part of serum + 4 parts of a
'Nobl and Arzt, Wien. klin. Woch., 190o8, xxi, 287.
"'Fritzand Kren, Wien. klin. Woch., I903, xxi, 386.
"Elias, Neubauer, Porges and Salomon, Wien. klin. Woch., I908, xxi, 748, 83 i .
"Wassermann and Plaut, Deutsch. med. Woch., 190o6, xxxii, 1769.
' Marie and Levaditi, Annales de l'Inst. Pasteur, 1907, xxi, I38.
"Morgenroth and Sterz, Virchow's Archiv, 1907, clxxxviii, 66.
"'The serum must not contain hemoglobin.

Hideyo Noguchi.

89

half-saturated solution of (NH,4 ) 2SO4 ). The precipitate was firstcentrifugalized at a rate of 5,ooo revolutions per minute, during
twenty minutes, decanted, the clear supernatant fluid poured off,
the residual moisture absorbed with soft filter paper, and the solidparts then weighed in the centrifuge tube, whose own weight had
been previously known. Beside this direct estimation, I found thefollowing indirect method to be of some use in avoiding tedious
weighing of the globulin deposit. This indirect estimation is based
upon the precipitation of serum globulins by weak solution of acids.
Take I c.c. of serum and mix it with 4 c.c. of half-saturated solution of ammonium sulphate, centrifugalize, pour the supernatantfluid away, then dissolve the deposit (globulins) in IO c.c. of o.9
per cent. salt solution. The clear globulin solution so prepared isthen mixed. with an equal volume of 10-I5 per cent. butyric acid"'solution containing o.9 per cent. sodium chloride, shaken well andleft at room temperature. The test is observed from time to time.
With the globulin solution from normal serum there is, as a rule,no, or at most, slight opalescence even at the end of two hours fromthe time of acidification. With the globulin solution from syphilitic
serum, which gives positive Wassermann test, a dense cloudinessarises promptly and within thirty minutes or later it becomes flocculent, and finally a deposit in the bottom of the test tube occurs.
In the following table some of my experiments on this point are
given. The ciphers given under the column "Weight of serumglobulins " denote the weight of the moist deposit of the globulins
precipitated from I c.c. of serum and approximately correspondwith eight to nine times the real weight as obtained by proper processes (coagulation with alcohol, hot water, removal of salts, extraction of fatty substances, and drying to constant weight).
The foregoing experiments show that in the blood sera of untreated or slightly treated cases of primary and secondary syphilis
there is a definite increase in the globulin content. The results obtained by means of the direct and indirect methods of globulin estimation indicate that an increase in the globulin content is paralleledby the positive results of the Wasserman test, the only difference be" Various acids other than butyric have been found to be less satisfactory
for this purpose.

90

Relation of Protein to Wassermann Reaction.

tween the two being the slightly higher percentage of positive
results with the indirect globulin method. There was no quantitative parallelism between the globulin content and the titers of theactive substances of syphilitic sera. A syphilitic serum can have a
low titer or be active for the Wassermann reaction without showing
a correspondingly high globulin content. Among the controls thesera from the normal individuals and gonorrheal patients gave inTABLE I.
Syphilitic Sera.
=-

Stage.

I
2

I

3

4

4
5
6
7
8
9
10
II
12

13
14
15

cc

II
II
'4

iI
4'

dc

19
20
21
22
23
24
25

c4

'4

4

44

27

28
29

32

33

CI
CC

0o.348 gram
0o.321
0.355
0.280
0.200

0

0.553 (!)
0 395
0.260
0.250
0.257

X
X
X
XXX
X
X
X
X
XX
XX
XX
XX
XXX
XXX
XXX
XXX

XXX
XXX
XXX
X
XXXX
XXXX

)XXX

++
++
++

X

XXX
XXX
XXX
XXX

26

30
31

0
0
0
0

X

16
'7
18

Indirect Estimation.
Direct Estimation.
Flocculent Precipitation
WeightoftheGlobulinsof Globulin Solution
Treatment.
Weighed as Moist
with o per cent.
Deposit.
Butyric Acid.

0o.4110o.4020.353
0.279
0o.300
0.425
0.298
0.365
0.393
0.251
0.343

+++
+
+
++
+++

Wassermann
Reaction.

+>0.2
+

0.2

+

0.2

.c.

+
0.04
+
0.02
+
0.2
+>0.2
+
+

0.02
0.2

-+- 0.05+ > 0.2+ >0.2
+
±0.I

0.2

+

0.2

+
+4+

o.16I

+>0.2

o. 49

+

0.200
o. 141

0.210
0o.40
0.253

0.230

++

0.252

0.450

+

O.110

+_

0. 05

0.I

(!)

Under column " Treatment " o = no treatment, X = slight treatment, X X =moderate treatment, XXX =much treatment, XX X X treatment extending
over two years.

91

Hideyo Noguchi.
TABLE II.
Non-syphilitic" and Normal Sra.
Nature of Disease.

Direct Estimationof Globulins.

Gonorrhea.

Indirect Ethnatlonll WL"ermntil Reacton wih theof Globulilns,
Serum.

o.205

-

0.175

-

_

-

-

0.110

-

_
-

Rheumatic fever.

0.183

-

-

Gastric ulcer.

0o.170
0.350
0.305

c

.135

o0.130sc~0.140

Cirrhosis of liver.
Chronic valvular disease, etc.
Tuberculosis.
Pneumonia lobar.
Chronic interstitial nephritis.

o.283
o0.66
o. 251

Malaria.

0.312
0o.321

Tuberculosis.
Normal.
tit"c-________________________

o. 65
o0.163
_

0.184
0.205
s 0.204

-

++- +
++
+

- 0.05 c.c.

+ 0.2
+ 0.2

-

++

++

-t 0.05

-

-

_
-

_

variably negative results and no increase in or abnormally large
globulin content could be found by direct estimation. Of the restof the controls four sera showed an increase of globulin and reacted
positively to the Wassermann test.
My experiments on cerebro-spinal fluids from forty-three casesof general paralysis'8 showed that there is a closer relation between
the increase of leucocytes and that of globulins than between the increase of globulins and the positive reaction to the Wassermann
test. In other words, the results obtained by cytological diagnosis
and globulin estimation are in good harmony, while the Wassermann reaction was in some instances absent although the globulin
was increased. In forty-three cases, thirty-seven positive results
were obtained. In two of forty control cases, where the globulin
tests were all negative, the Wassermann reaction was faintly positive.
"The possibility of a previous syphilitic infection could not be excluded insome of the sera giving the positive reaction for the Wassermann test.
'SA fuller account of this series and of still other cases will be publishedelsewhere in conjunction with Dr. J. W. Moore.

92

Relation of Protein to Wassermaonn Reaction.

The method which I employed for detecting an increase of theprotein in the cerebro-spinal fluids may be mentioned here, as it
affords a much sharper and more enduring means to differentiate
normal from paralytic spinal fluid than the Nonne method of halfsaturation with ammonium sulphate.19 It is very simple and canbe made with small quantities of material in less than half an hour.
Its diagnostic value has been found to be equivalent, if not superior,
to that of cytological examination. The technic is as follows:
To o.I c.c. of spinal fluid 20 add o. 5 c.c. of the Io per cent. butyricacid, 21 boil briefly over a flame, then add quickly o.I c.c. of normal
solution of sodium hydroxide and reheat briefly. Allow the test
tube to stand in a frame and observe the reaction. Read the reaction for diagnosis within three hours; longer standing, extending to twenty-four hours, offers no advantage, and the characteristic appearance of the reaction becomes less obvious after the lapseof this time. In the spinal fluid from cases of general paralysis
a coarse granular or flocculent precipitate appears, which gradually
settles down to the bottom of the test-tube leaving the fluid above
clear. In the majority of cases of general paralysis, tabes dorsalis
and cerebro-spinal lues, the precipitate becomes granular within ten
to twenty minutes and settles in a short time afterwards. In the
spinal fluid from cases of alcoholic psychosis, dementia proecox, andepilepsy as well as from normal subjects, there occurs a slight anduniform opalescence only, and no coarse precipitate forms even after
several hours. In case of an ambiguous reaction a test with 0.2
c.c. of the spinal fluid must be made which usually decides the point.
In determining the quantities of proteins, especially those precipitable by half-saturation of ammonium sulphate in cerebro-spinal"This method consists of mixing 2 c.c. of spinal fluid with an equal volume
of saturated solution of ammonium sulphate (strictly neutral) and reading theresult within three minutes. The differentiating value gradually diminishes astime elapses beyond three minutes.
'Specimens which had been preserved in the refrigerator for about one
year, or those from which all cellular elements have previously been removedby centrifugalization, are equally good for the test. It is entirely to one's individual taste whether one or a multiple of the quantities indicated above will
have to be employed.
'A number of inorganic and organic acids have been tried, but were foundto be less satisfactory.

Hideyo Noguchi.

93

fluid, I found that the ones which are peculiarly rich in the paralytic
spinal fluid, are chiefly globulins or globulin-like substances which
are the main substances causing the characteristic coarse or flocculent precipitate in the above test as well as the ones entering into the
Wassermann reaction. The average quantities of these proteins
contained in I c.c. of fluid ranged from o.oI to 0.02 gram 2 2 weighed
as a moist deposit of constant density. A similar estimation with nonparalytic 2 3 and normal spinal fluids, showed that the amount of theprotein never exceeded one-tenth of that found in the specimensfrom paralytics.
CAN THE ACTIVE SUBSTANCES OF SYPHILITIC SERA AND PARALYTIC CEREBRO-SPINALFLUIDS BE EXTRACTED?

and Yamanouchi 2 4 claim that the reacting substances of

Levaditi
syphilitic sera and paralytic and tabetic spinal fluids can be extractedwith alcohol. By alcoholic extraction they obtained a certain
amount of lipoids and of salts. Both sets of substances were inherently antihaemolytic when used in large enough quantity. Mixedin smaller quantity with liver extract or other " syphilis antigens "they gave increased inhibition of hemolysis. Apparently Levaditi
and Yamanouchi consider the Wasserman reactions as a summationof the anticomplementary effect of two sets of lipoids and salts.
They give no quantitative details.
My own experiments in this respect failed to convince me that the
Wassermann reaction is caused by alcohol-soluble constituents ofserum and spinal fluid.
By means of dialysis of the serum in a celloidin sac the cnglobulin
fraction of the serum was obtained free from dialysable salts. The
euglobulin fractions were redissolved in o.9 per cent. salt solution
and tested for the Wasserman reaction. Three samples of syphilitic
and two samples of normal sera examined showed that the euglobulin obtained from syphilitic sera possesses the property of giving the
Wassermann reaction while that from normal sera did not. The
titers of the globulin fractions were the same as those of the corresponding sera.
' To obtain an approximate absolute weight the ciphers must be divided
by 8 to 9.
"Cases of tabes dorsalis are not included.
Levaditi and Yamanouchi, Compt. rend. de la Soc. de Biol., I9o8, Ixiv, .

94

Relation of Protein to Wassermann Reaction.

The above experiment shows that the active substances binding
complement with lipoids remain in the celloidin sac on dialysis. The
removal of dialysable salts from the serum does not affect the activity of the specific substances which are precipitated with the euglobulin fraction. A qualitative difference is shown to exist between normal and syphilitic serum-globulins.
The lipoidal bodies of syphilitic sera and paralytic spinal fluids
are alleged by Levaditi and Yamanouchi 2 5 to represent the specificactive syphilitic substance of these fluids. In studying this question
I have undertaken numerous quantitative estimations of alcohol-extractable constituents of blood sera, blood coagula and spinal fluidsof normal, syphilitic and paralytic subjects, in order to ascertain if
there exist qualitative or quantitative difference between the lipoids
derived from normal and from syphilitic materials. Two syphilitic
sera and one normal serum were extracted with ten volumes of
95 per cent. alcohol for five days at 370 C. The weights of thealcohol soluble matters were, after drying, 0.205, 0.210 and 0.202
gram, respectively, per i I.5 c.c. of the serum (ca. 1.79 per cent. average). The sample which yielded 0.205 gram of alcohol-extractable
substances had a titer of 0.004 c.c., that which yielded 0.210 gram
had a titer of o.oI c.c., and that which yielded 0.202 gram was
normal and had no effect on complement in Wassermann's test.
The alcoholic extracts were dissolved in 0.9 per cent. saline solution( I 1.5 c.c., the original volume of serum) respectively, and tested forthe property of " syphilitic antibody." When used in quantities over0.3 c.c. these solutions were slightly antihoemolytic, but there wasno regular complement fixation even when used in combination
with antigenic substances (lecithin, alcoholic extract of liver, etc.).
There was no difference between the solutions obtained from normal and from syphilitic sera, both being entirely devoid of the"antibody " property which characterizes the whole syphilitic sera.
Three samples of blood coagula of syphilitic subjects (manifest
secondary stage) anrd three of normal persons were extractedwith ten volumes of 95 per cent. alcohol at 370 C. for five days.
The quantities of the coagula and of the lipoids and salts extractedwith alcohol may be stated as follows:
Loc. cit.

95

Hideyo Noguchi.
Syphilitic coagula I 20 grams.
II 17 grams.
III 15 grams.

Extract 0.230 gralm.
Extract 0.219 gram.
Extract o.19o gram.

Normal coagula I 32 grams.
II 20 grams.
III 28 grams.

Extract 0.330 gram.
Extract 0.210 gram.
Extract 0.299 gram.

The average content of syphilitic and normal blood coagula in alcohol-extractable matters is approximately I.I per cent., of whichsalts make up a large percentage.
The experiments in regard to the existence of specific active
syphilitic substance in these extracts have been negative. No positive reaction for the Wassermann test was obtained with any ofthem.
Similar extraction experiments were made with three samples
of general paralytic spinal fluids and one of a non-paralytic patient.
None of the extracts gave a positive Wassermann test conclusive
enough for establishing a "syphilitic antibody" nature. In asimilar way to Levaditi and Yamanouchi, I observed in one ofthe paralytic extracts more or less marked inhibition of haemolysis
in combination with lecithin, but I am not convinced that this reaction is to be regarded as the regular Wassermann reaction.
ANTIGENICLIPOIDS

AND

SALTS

FOR WASSERMANN S REACTION.

2

Landsteiner, Muller and P16tzl
discovered the alcohol solubilityof the antigenic principles of syphilitic and normal tissues. Porgesand Meier 2 7 28 found lecithin to act as antigen, while Lcvaditi and
Yamanouchi 2 9 place sodium glycocholate, sodium taurocholate,
protagon and cholin among the syphilitic "antigens," and foundlecithin to be less active than sodium glycocholate. Sachs andAltmann30 state that sodium oleate has a similar action. Cholesterin and vaselin were effective as antigens in the hands of Fleisch'"Landsteiner, Miller and Ptzl,
Wien. klin. Woch., 907, xx, 1421,'Wassermann, Berl. klin. Woch., 907, xliv, 599, 1634.

565.

Porges, Wien. klin. Woch., 90o8, xxi, 2o6.
and Yamanouchi, Compt. rend. de la Soc. de Biol., 907, xiii, 740;
1908, lxiv, 349.
80Sachs and Altman, Berl. klin. Woch., 1908, xlv, 494.
2-

2. Levaditi

96

Relation of Protein to Wassermann Reaction.

mann31 and cholesterin was found inactive by Levaditi and Yamanouchi.
My study of this topic was directed to the ascertainment whether
qualitative or quantitative differences existed between extracts ofnormal and syphilitic blood and liver. The extraction was madewith eight volumes of 95 per cent. alcohol at 42° C. for six days.
The liver was made into a pasty mass by grinding and straining
before extraction. The sera and coagula from syphilitic and normal bloods were extracted with ten volumes of 95 per cent. alcohol
at 37 C. for five days. Before testing "the antigenic value" ofthe extract of each sample I removed salts by taking up thelipoids with ether. The ether soluble substances were then dried
and fractionated with acetone. 3 2The following table gives the results:
Acetone Insoluble.
s3

Acetone Soluble.

Liver of a congenital syphilitic child.
Liver of a non-syphilitic child.
Liver of a normal rabbit.

15 grams
35 grams
20 grams

0.105 gram

0.665
0.540

0.195 gram
0.250
0.140

Syphilitic coagulum.

I
II
III

20 grams

0.054

0o.07017 grams
15 grams

0035
0.015

0.050
0.050

I
II
III

32 grams

0.059

0.070

20 grams

0.026

0.042

28 grams

I

11.5 c.c.

II

11.5 c.c.

0.035
0.058
Alcoholic extract without fractionation 0.205 gram.
Alcoholic extract without fractionation 0.210 gram.

I

11.5 c.c.

Normal coagulum.

Syphilitic serum.

Normal serum.

Alcoholic extract without fractionation 0.202 gram.

The lipoidal content of different samples of blood coagula varied
rather irregularly, but this may be due to variable consistence ofthe coagula. In general it appears to be somewhat lower than withthe sera, and many times less than with the livers.
38 Fleischmann, Berl.
in. Woch., 908, xlv, 490.
' For redistilled acetone employed in the experiment I am obliged to Dr.
P. A. Levene.
'Theratio between the acetone-insoluble and acetone-soluble portion in thissample of syphilitic liver is very different from the normal specimens, but I

found it impossible, at the time, to secure a second syphilitic liver to confirm
this result.

Hideyo Noguchi.

07

The ratio between the acetone-insoluble and acetone-soluble portion is quite the reverse in the liver and the blood coagula. In
the liver the acetone-insoluble lipoids exceed the acetone-soluble
lipoids, while the contrary is the case with the coagula.
Two cubic centimeters of the cerebro-spinal fluids derived fromthree cases of general paralysis and one non-paralytic patient yielded
upon extraction 0.003 to 0.004 gram of lipoids each.
In determining the antigenic value of these extracts solutions of
uniform concentration were made with the acetone-insoluble andacetone-soluble fraction of each coagulum or liver: thus o.I
gram of the substance in question was dissolved in a few cubic
centimeters of methyl alcohol and then emulsified in Ioo c.c. of o. 9
per cent. salt solution. For the standard of antigenic value lecithinfrom two different sources (Lecithin "Agfa" Merck and one of myown preparations from beef blood corpuscles), were used. A syphilitic serum with the titer of 0.004 c.c. was employed as " antibody"in the amount of o.I c.c.
It was found that the antigenic strength of the acetone-soluble
fraction of the extract of blood coagula, irrespective of theirsources, was somewhat stronger than the acetone-insoluble fraction. With the extracts of livers the acetone-insoluble fraction wasslightly more effective than the acetone-soluble fraction.

It was also found that the lipoids extracted from syphilitic sera or
blood coagula with alcohol can act as "antigens" for the "antibody" found in corresponding native sera. There was no evidence that any qualitative or quantitative difference exists between
the lipoids extracted from syphilitic and those extracted from normal liver, blood sera and blood coagula.
Just which lipoids are responsible for the powerful antigenic
properties of the acetone-soluble portion has not been ascertained,
but I can exclude cholesterin from the possible agents; for I tested negatively cholesterin preparations from sheep's brain, and gallstones and a sample prepared by Merck. Protagon, cholin andneurin did not exhibit in my hands convincing antigenic properties. I can confirm Levaditi in the finding that sodium glycocholate
and sodium taurocholate can act as "antigens" but I found the

98

Relation of Protein to Wassermann Reaction.

quantities required very large compared with lecithin and other antigenic lipoids. Sodium cholate acted in a similar way to the othertwo bile salts.
SUMMARY.

I. The high value in respect to complement-binding exhibitedby blood sera from syphilitics and spinal fluids from general paralytics is associated with an excessively high content of globulin, butthere does not exist a direct quantitative relation between the two.
Cases of secondary syphilis which have been under prolonged andproper medication do not exhibit the globulin increase and usually
fail to give the Wassermann reaction. The active substances entering into the Wassermann reaction are precipitable with the globulin and chiefly with the euglobulin fraction of the fluids.
2. Temperatures of 70° to 760 C. destroy the active substances.
Exposed to sunlight the active substances deteriorate slowly. A
photodynamic substance such as eosin, under the direct influenceof the sun, brings about their complete and rapid destruction.
This effect does not occur in the dark. The active substances are
subject to tryptic and peptic digestion and are destroyed by weakacids and alkalies.
3. The active substances in the blood sera and spinal fluids cannot be separated from them or from the globulin precipitate byalcohol.
4. There are contained in the alcoholic extracts of normal andsyphilitic blood and organs certain acetone-soluble lipoids which
possess high antigenic values for the Wasserman reaction. Cholesterin is inactive and the bile salts less active than the lipoidal bodies.
5. Sodium cholate is about as active as sodium taurocholate andglycocholate, but neurin and cholin are inactive.
I wish to express my cordial thanks to Dr. Victor C. Pedersen(House of Relief), Dr. MacFarland (Sloane Maternity Hospital),Dr. Draper (Presbyterian Hospital), Dr. Sutton and Dr. Murphy(City Hospital), Dr. Lapowski (The Good Samaritan Hospital)and Dr. Mannheimer (The Lebanon Hospital) for the courtesies
and assistances extended to me in securing the materials for thisHideyo Noguchi.

99

study. For the cerebro-spinal fluids I am under deep obligation
to Dr. Moore of the Manhattan State Hospital in permitting me towork on the materials collected by him. To Dr. Wollstein I am
indebted for the livers of syphilitic and normal children.
It gives me pleasure to acknowledge the invaluable assistancerendered me by Dr. R. V. Lamar during the execution of thepresent work.