ON HISTAMINE AND A HISTAMINE-LIKE SUBSTANCE

AS DECOMPOSITION PRODUCTS OF ALBUMOSES

T. NAGAYAMA

From the Pharmacological Laboratory of the Johns Hopkins Univer8ity

Since Schmidt-M#{252}lheim (1) first made his fundamental experi-

ments with Witte’s peptone, in Carl Ludwig’s laboratory, in
1880, and showed that a shock-like condition is produced by the
intravenous injection of so-called peptones (albumoses), toxic
proteoses have been the subject of many investigations. Abel
and Kubota (2), in discussing certain phases of this subject,
have given a brief account of some of the investigations that
have been carried out since Schmidt-M#{252}lheim’s day.
Abel and Kubota allowed a considerable quantity of Witte’s
peptone to remain for thirteen months in contact with absolute
alcohol, containing a small amount of acetic acid. After remov-
ing the#{149} alcohol by filtration, and evaporating it, they obtained
a residue which yielded to chloroform a basic substance which
depressed the blood pressure, stimulated plain muscle tissue,
gave the Pauly reaction, and which appeared to be in every way
identical with the histamine which these authors isolated from
the hypophysis cerebri, from the intestinal mucosa and other
tissues. While they did not go to the trouble of preparing the
substance as a crystalline picrate, they had no doubt as to its
identity with histamine, and felt that it was simply a matter
of routine to isolate it satisfactorily as a chemical individual.
It has been shown in the preceding paper by Abel and Na-
gayama that the depressor action of pituitary extracts which
have been treated for a very short period of time with a little
weak hydrochloric acid, is due to two depressor substances, one
of which, histamine, or “C” as they have called it, is present in
small amount; the other, a histamine-like substance, which is
401
402 ‘r. NAGAYAMA

present in larger quantity and which they have named “B.’

I would refer to this paper in regard to these two depressor
substances.
It is of very great interest, certainly, to inquire whether both
of these substances, “B” and “C,” which appear to be so closely
related, can be obtained from the ordinary albumoses and so-
called peptones of commerce. At Professor Abel’s suggestion,
I have undertaken to investigate this question. It is well known
that albumoses vary in toxicity, as is shown by their action on
arterial pressure ; also that they vary in their power to stimulate
plain muscle tissue, as is shown by their action on the isolated
uterus. I shall leave out of consideration here other physiological
properties of these albumoses, and confine myself to a consider-
ation of the two functions just mentioned.
It is of interest to note that a secondary albumose is easily
prepared, which has very little action in the way of lowering
the arterial pressure, as compared with the similar action of
Witte’s peptone. An innocuous, or partially innocuous, prepa-
ration of this sort is the thyroid secondary albumose which was
used below, and which was prepared for me by Drs. Abel and
Rohde. This albumose was prepared some years ago from
absolutely fresh thyroid glands, under aseptic conditions, by
means of peptic digestion, and was pretty well purified by Abel
and Rohde by repeated treatment with ammonium sulphate.
As wifi be shown in the figures given below, it had no action on
the blood pressure in doses in which other preparations, Witte’s
peptone, for example, causes very great arterial depression.

I. RELATIVE ACTIVITY OF PROTEOSES

Experiments were first made as to the relative blood-pressure-

lowering power of (1) Witte’s peptone, (2) the peptone manu-
factured by Fairchild, (3) the peptone of the Digestive Ferments
Company, and (4) the pure thyroid albumose mentioned above.
‘The quantities used for intravenous injections in these compari-
sons varied from 0.2 to 0.235 gram, the injections being made
into a young dog. Figure 1 and figure 2, A, B, C and D, show
 DECOMPOSITION PRODUCTS OF ALBUMOSES 403

in a striking manner the very marked difference of the four

preparations named, in their action on the arterial pressure.
Witte’s peptone is by far the most toxic of the four proteoses.
It was in order, also, to compare these albumoses in their
action on the guinea-pig’s uterus. Figure 3, A, B and C, gives
the results of the comparative uterine tests. Here also it is
seen that the preparation which causes the greatest fall in the
blood pressure also stimulates the uterus most powerfully. It

FIG. 1. YOUNG DOG, 5.55 KGM., ETHER

At / injection into femoral vein of 1 cc. = 0.200 gram Witte’s peptone.

is to be noted, therefore, that there is a sharp gradation in our

preparations in respect to their action on blood pressure and on
the uterus. Witte’s peptone is the most active of all the prep-
arations, while there is but little difference in the behavior of
the thyroid albumose and the peptone of the Digestive Ferments
Company.
It may be thought that bacterial action accounts for the
greater activity of Witte’s peptone, and this is a point on which
404 T. NAGAYAMA

FIG. 2. ANIMAL AS IN FIG. 1

At A / injection into femoral vein of 1 cc. = 0.200 gram Fairchild’s peptone.
At B 7 injection of 1 cc. = 0.200 gram of the Digestive Ferments Company’s
peptone. At C / injection of 1 cc. = about 0.25 gram of a secondary albumose
prepared by Abel and Rhode from fresh thyroid glands by peptic digestion. At
D / injection of 1 cc. = 0.235 gram of this thyroid albumose. The injections
at C and D were made at. a different time. Animal used: Dog, 6 kgm., anaesthetic
not noted but presumably chioretone.
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405
406 T. NAGAYAMA

we can give no definite information. We may state, however,

that it is not difficult to prepare crude albumoses from various
tissues, under aseptic conditions, all of which have the phar-
macological properties of Witte’s peptone. This question has
been discussed by Abel and Pincoffs (3).

II. RELATIVE AMOUNTS OF HISTAMINE OBTAINABLE ON HYDROLYSIS

OF THE DIFFERENT PROTEOSES

In order to learn whether the more toxic albu.mose, as repre-.

sented by the Witte preparation, would yield a larger amount of
both histamine and of the histainine-.like substance on hydrolytic
decomposition with acids, I proceeded as follows: A weighed
amount of desiccator-dried material, 2 grams approximately, of
each preparation, was boiled with 15 per cent HC1, for six hours,
at the reflux condenser. The HC1 was then, in each case, driven
off on the waterbath, under the electric fan, with the help of
repeated additiofls of absolute alcohoL After this, the method
of extracting the histamine was exactly likethat employed by
Professor Abel and myself, as described in the preceding paper.
Equal, or proportionate, quantities of NaCO3, and of CHC1,,
according to circumstances, were used in each case-in other
words, the extractions were carried out in all cases in as nearly
a quantitative manner as is possible in work of this kind.
We may note at this point that the amount of material that
passes into chloroform from the dry-Na2CO3-powder, after the
hydrolysis of these albumoses, is extraordinarily large. Abel
and Kubota have already called attention to the unusually large
yield of chloroform-soluble material which was obtained in the
hydrolysis of crystallized edestin and other proteids. Thus,

grams
The chloroform extract from 2.0885 grams of Witte’s peptone
weighed 0.6621
The chloroform extract from 2.5158 grams of Digestive Ferments
Company weighed 0.7698
The chloroform extract from 2.0750 grams of pure thyroid albu-
mose weighed 0.6056
 DECOMPOSITION PRODUCTS OF ALBTJMOSES 407

As compared with the very small quantity of chloroform

extract which was obtainable by us from the hydrolysis of 0.225
gram of the pressor phosphate, as described in the foregoing
paper, the results here given are most striking. Evidently this
large amount of chloroform-soluble material, only a small part
of which consists of the histamine-like substance and of histamine,
demands further investigation. It is hot unlikely that other
proteinogenous amines than B and C, with different pharma-
cological properties, or, such as are quite inactive, will here be
found.

A B

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urttlttuhtttttllllIllllhttllUIIIIIIII1BLIIU1IIIIIIIllttiiiiJ tlllfltIftlltIIlIlIHItItItHtIItfHUIItIftftl

FIG. 4. DoG, a, 9.2 KGM.

Paraldehyde by gastric tube, and anaesthesia was imperfect, tracheotomy

and ether from bottle and later in the experiment, aqueous solution of paralde-
hyde via the femoral vein. At A 7 4 cc. (out of 20 cc.) of the solution of the
CHCI, extract after hydrolysis of 2 grams Witte’s peptone as described. At B 7
injection of 5.4 cc. (out of 20 cc.) of solution of CHCI, residue obtained from
hydrolysis of 2 grains thyroid albumose. Although the quantity injected here
is larger than that of the CHCL. extract from the (Witte’s peptone) injected
at A the depressor effect is less pronounced. Irregularity of the anaesthesia
made it impossible to test the CHCL, residue obtained by the hydrolysis of
the Digestive Ferments Company.
408 T. NAGAYAMA

The chloroform extract obtained in the above hydrolytic

experiments were now tested in respect to their action on the
circulation and on the guinea-pig’s uterus, in the usual way.
Figure 4, A and B, shows the comparative b1ood-pressure-lower
ing action of the chloroform extract obtained from the hydrolysis
of 2.0885 grams of *itte’s peptone and 2.0750 grams of the
thyroid albumose. It will be seen that the Witte peptone extract

FIG. 5. ANIMAL AS IN FIGURE 3

About. half an hour before making the injections here recorded a consider-
able quantity of a saturated aqueous solution of paraldehyde was injected into
the femoral vein. Xow the aqueous solution of the chloroform extract, which
as state(l in the text contained other Constituents, caused a rise in arterial
pressure. At A / injection into the femoral vein, 4 cc. (out of 20 cc.) of the
solution of the chloroform residue obtained from the hydrolysis of 2 grams
thyroid albumose. At. B 7 injection of 4 cc. (out of 20 cc.) of the solution of
the chloroform residue obtained from the hydrolysis of 2 grams Witte’s peptone.
It will be noted that the rise of blood pressure is more pronounced in the case
of the extract from theWitte’s peptone, just as the fall of pressure is also more pro-
nounced \!ith this extract, than it is with the extract from the thyroid albutnose.
 DECOMPOSITION PRODUCTS OF ALBUMOSES 409

causes a noticeably larger depression of the blood pressure,

quantity for quantity, than does the thyroid extract.’ In this
connection I may call attention to a striking reversal in blood-
pressure effect which was obtained with these two extracts when
the animal was completely under the influence of a combination
of ether and paraldehyde. At a certain time in the experiment,
as is explained in the legends accompanying the figure (see fig. 5),
a further injection of the chloroform extracts caused a rise of
blood pressure instead of a fall, and, singularly enough, the rise
of blood pressure was greater in the case of the. Witte peptone
extract, just as the fall of the arterial pressure had been greater
with this preparation. It may be added that no such reversal
was obtained when a small quantity of the histamine salt was
injected, as this was followed by the usual depression of the
blood pressure.
Further examples of the difference shown by the chloroform
extracts of the hydrolyzed proteoses are given in the following
figures. The chemical data are given in the following table:
grams
The chloroform extract from 2.0004 grams of Witte’s peptone
weighed
. The chloroform extract from 2.0140 grams of Digestive Ferments
Company weighed 0.6242
The chloroform extract from 2.0092 grams of pure thyroid extract
weighed 0.6648
a it is thought that an error in weighing was made in this determination, as

the quantity here given is practically twice that obtained in three other hydroly-
es. Compare also the amount of CHCI, extract reported on page 000.

Figure 6, A , B and C, shows very clearly the comparative

action of proportionate amounts of the chloroform extracts of
the three proteoses. The three injections were repeated after
a brief interval and the comparative effects on the blood pressure
which are given in figure 7, A , B and C, are practically like those
given in the preceding figure (fig. 6). The rise of pressure at
A , figure 7, coincided with a brief period of respiratory embar-
rassment-slow and shallow respirations.

S Trouble with the anaesthesia prevented the accurate estimation of the

third proteose.
410 T. NAGAYAMA

The action of the chloroform extracts of the three proteoses

on the guinea-pig’s uterus is in every way comparable with the
effects on the blood pressure ; that is to say, the chloroform
extract obtained from Witte’s ‘peptone is far more active than

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FIG. 6. YOUNG DOG, 3.2 KGM., ETHER

Comparative action of equal quantities of chloroform extract, after hydrolysis

of Witte’s peptone, thyroid albumose and Digestive Ferments Company’s
peptone. In each case 3 cc. out of a total of 10 cc. aqueous solution of the extract
was injected into the femoral vein.

FIG. 7. SAME ANIIAL AS IN FIcURE 6

Repetition of the injections in the same quantities and in the same order
as in figure 6. At 1? the respiratory movements became slow and feeble for a
time and a rise of blood pressure occurred at this time.
 DECOMPOSITION PRODUCTS OF ALBUMOSES 411

that from the other alburnoses. Figure 8, A B and

, C, bears
out this assertion. It will be noted that the effect of the chioro-
form extract. (fig. 8, A) was so marked as to cause a maximal
contraction of the guinea-pig’s uterus.

FIG. 8. EFFECTs OF CHLOROFORM EXTRACFS ArFER HYDROLYSIS OF THE PRO-

TEOSES ON THE VIRGIN GUINEA-PIG’S UTERUS

At A 5 drops (30 drops = 1 cc.) out of 10 cc. of Witte’s peptone were added
to the uterus chamber. At B same amount of the thyroid albumose. At C
same amount of the Digestive Ferments Company’s peptone. At W a weight
had to be attached to the lever to keep it from leaving the drum. At T, T, T,
change to fresh Tyrode’s solution.

III. RELATIVE AMOUNTS OF THE HISTAMINE-LIKE SUBSTANCE

OBTAINABLE ON HYDROLYSIS OF THE DIFFERENT PROTEOSES

The second depressor substance, the histamine-like substance,

“B,” which bears a close resemblance to histamine in its phar-
macological and chemical properties, was next examined. As
stated in the paper of Abel and Nagayama, it is a very simple
matter to obtain this substance. One has only to extract the
412 T. NAGAYAMA

dry alkaline (NaCO) powder repeatedly with 93 to 95 per cent

alcohol to obtain this depressor substance in quantity. It must
be stated, however, that. if the extraction is made by boiling the

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FIG. 9. CAT, 9, 2.63 KGM., ETHER

Effects of alcohol extract of hydrolyzed proteoses on blood pressure. At A

injection into femoral vein of 4 cc. out of 12 cc. from Digestive Ferments Corn-
.pany’s peptone extract. At B injection of 4 cc. out of 10 cc. extract from Witte’s
peptone. At C proportionate quantity of extract from the thyroid albumose.
At + formation of clot.
 DECOMPOSITION PRODUCTS OF ALBUMOSES 413

powder with 93 per cent alcohol, at the reflux condenser, and this
process is repeated a number of times, the product finally ob-
tained has no depressor activity, except in so far as it still contains
a little histamine which was left behind after the chloroform
extraction. I have only one figure to offer as to the effect of
this alcohol-soluble body on the blood pressure and on the
uterus, because of the injury which was sustained by the depressor
body in other experiments in boiling it with alcohol at the reflux
condenser, as just stated. The following table gives the amount
of alcohol-soluble material which was obtained in one of my
experiments following the usual extraction with chloroform:
grams
The alcohol extract from 2.0885 gram of Witte’s peptone weighed. 1.0759
The alcohol extract from 2.5158 grams of Digestive Ferments
Company weighed 1.5514
The alcohol extract from 2.0750 grams of pure thyroid extract
weighed 1.3056

Proportionate quantities, reckoned in terms of the original

weights of the proteos#{233}s, of the alcohol residues from the three
proteoses were tested for their action on blood pressure, and the
results are given in figure 9, A, B and C. Figure 10 also ifiustrates
the action on the virgin guinea pig’s uterus of proportionate
amounts of these alcohol extracts. It will be seen that the Witte
extract caused a maximal contraction of the uterus.
Appant1y the relative proportion of the alcohol-soluble
histamine-like substance to histamine is less in the case of these
hydrolyzed proteoses than in the case of hydrolyzed pituitary
extrac1s.
CONCLUSIONS

1. The proteoses of commerce vary in toxicity, as shown by

comparative tests on the blood pressure and on the uterus. A
secondary albumose prepared from fresh thyroid glands by Abel
and Rohde, by means of peptic digestion, was less active in its
effects on the blood pressure and the uterus than the commercial
preparations tested.
2. Hydrolysis with hydrochloric acid, under the conditions
1escribed in this paper, causes the appearance of an appreciable
414 T. NAGAYAMA

quantity of histamine, and also of a histamine-like substance,

similar if not identical in character with that described by Abel
and Nagayama.
3. Attention is called to the large amount of chloroform-
soluble matter that is obtainable in the hydrolysis of the proteoses
studied.

FIG. 10. EFFECT OF EQUAL QUANTITIES OF THE AicoHoL EXTRACT OF THE THREE
PROTEOSES ON THE VIRGIN GUINEA-PIG’S UTERUS

The same pipette was employed in each case. It will be seen that the Witte
peptone gave the most active alcohol extract. At W a weight had to be attached
to the lever to keep it from leaving the drum. The contraction in this case was
maximal. At T, T, T, change to fresh Tyrode’s solution.

I desire to express my sincere thanks to Prof. John J. Abel

for aid and suggestions throughout this work.

REFERENCES

(1) SCHMIDT-MULHEIM: Du Bois Raymond’s Archiv., 1880.

(2) ABEL AND KUBOTA: Jour. Pharmacol. and Exp. Therap., xiii, no. 3, June, 1919.
(3) ABEL AND PINc0FF5: Proc. Natl. Acad. of Sc., iii, 1917.