From The Pharmacological Laboratory of The Johns Hopkins University

ON THE PRESENCE OF HISTAMINE IN EXTRACTS OF
THE POSTERIOR LOBE OF THE PITUITARY GLAND

AND ON PRELIMINARY EXPERIMENTS WITH THE
PRESSOR CONSTITUENT
JOHN J. ABEL AND T. NAGAVAMA
From the Pharmacological Laboratory of the Johns Hopkins University
A year ago Abel and Kubota (1) found that histamine is one
of the constituents of the pituitary gland, and thereupon stated
it as their belief that this amine is the plain-muscle-stimulating
and blood-pressure-lowering constituent of the gland. As these
authors had based their conclusions on experiments made with
the entire gland, it seemed advisable to submit their findings to
a re-examination. But, in place of using extracts of the entire
gland, the present authors have employed extracts of the fresh
posterior lobe, or infundibular portion, of the gland only.
We may state at once that infundibular extracts that have
been prepared with care from fresh glands, that is to say, with
avoidance of long boiling or of long exposure to acids, contain
only a small amount of histamine in the free state, while extracts
such as those employed in therapeutics contain much more of
this powerful amine. But even in this latter case the amount
of histamine present probably accounts for only a few per cent of
the total oxytocic strength of the preparation. In view of our
own findings during the past autumn and winter, as also in view
of the work of other investigators which will be considered pres-
ently, we can no longer maintain that histamine itself is the plain
muscle stimulant of the pititary gland. While making this ad-
mission, we nevertheless believe that importance attaches to
histamine as a decomposition product of a constituent of the pos-
terior lobe, whose identity cannot be determined with absolute
certainty at the moment.
347
THE JOUR. OF PH.tRM. AND EXPER. THERAP., VOL. XV. NO. 4

348 JOHN J. ABEL AND T. NAGAYAMA
While we were engaged in our studies, other investigators ap-

proached the subject from different points of view, and have
shown that the amount of free histamine present in extracts of
the infundibular portion of the gland is sufficient to account for .
the plain-muscle-stimulating action of such extracts. L. Popiel-

ski (2) states, in a paper which has just come to hand, that his
examination of the subject leads him to conclude that histamine
is not present in “pituitrin” or “in the hypophysis.” Jackson
and Mills (3) have shown conclusively that histamine is a power-
ful broncho-constricting agent in the case of the dog, while pitui-
tary extracts have no constricting action on the bronchioles unless
histamine has made its appearance in such extracts. These au-
thors conclude ‘ ‘ that certain commercial preparations of the pi-
tuitary gland apparently contain very small and very variable
proportions of histamine, and the amount of this substance is
probably great enough in some samples to exert some therapeutic
action . . . . “ These authors also point out “ that there
are very obvious clinical advantages in favor of the pituitary
extracts, rather than of the very intense broncho-constricting
and blood-pressure-lowering histamine for obstetrical uses.”
The results of recent experiments of our own on the broncho-
constrictor action of pituitary extracts which have been treated
with acids for a brief period, and on other phases of this broad
question, have convinced us of the correctness of the opinion,
held by many at present, that histamine should not be employed
therapeutically in any of those instances in which pituitary ex-
tracts now hold the field.
We may be permitted to point out that Jackson and Mills,
in their pharmacological assay of various commercial pituitary
extracts, have failed to take into consideration the varying
concentrations of such extracts. Thus, it does not follow, if 4
cc. of a preparation of one manufacturer causes a greater broncho-
constriction in the dog than 4 cc. of the extract of a second manu-
facturer, that the former preparation is inferior to the latter.
To be sure, it contains per cubic centimeter more histamine, as
also more histamine-like-substance, than the latter, but the
proportion of histamine to the unaltered oxytocic and blood pres-
PRESENCE OF HISTAMINE IN PITUITARY GLAND 349
sure raising substance may be no greater and may be even less

in the first than in the second preparation. Spaeth (4) has shown
that commercial pituitary extracts vary in oxytocic strength from
1 : 5. Other things being equal, that is to say, method of prep#{225}ra-
tion, exposure to acids, and age of the preparation, the amount of
histamine obtainable by chemical methods appears to be in pro-
portion to the amount of organic matter in the extract. Our
own experience with a combined chemical and pharmacological
method of assaying preparations for histamine has convinced us
that it is incorrect to assay commercial preparations for this
amine, by a pharmacological method alone, comparing the prepa-
rations, volume by volume, without taking into consideration
other items of the kind suggested above. Assays such as those
of Jackson and Mills, have, therefore, a qualitative but not a
quantitative value.
The recently published paper of H. W. Dudley (5) also fur-
nishes conclusive proof that the uterine stimulant and histamine
are not identical, but are two distinct chemical individuals.
Guggenheim’s (6) earlier observation that alkali will destroy
both the pressor and the muscle stimulating activities of a pitui-
tary extract in a concentration which leaves the action of hista-
mine on blood pressure unaltered, is verified by Dudley. But
in addition, other points of difference between the two substances
are brought out in his paper. Thus, the pituitary uterine stimu-
lant is readily extractable from acid solution by butyl alcohol,
while histamine is only very slowly extracted from an acid solu-
tion by this solvent; the pituitary principle is insoluble, while
histamine is soluble in boiling chloroform; the pituitary uterine
stimulant is rapidly destroyed by trypsin, while histamine is
unacted on by this ferment.
We cannot follow Dudley, however, when he states that
“there is no detectable quantity of histamine present in extracts
of the infundibular material.” He bases his opinion on an experi-
ment which was made with only 0.005 gram of an extract which
was treated with sodium carbonate and hot chloroform, as ad-
vised by Abel and Kubota. The chloroform extract from this
small quantity of infundibular extract was dissolved in 20 cc. of
water, and of this amount only 2 cc. was used for a uterine test:
we feel confident that if the entire chloroform residue had been
dissolved in about 1 cc. of an isotonic medium and if this entire
cubic centimeter had been used for the uterine test, a very good
contraction would have been obtained. We think that we have
shown, in the following parts of this paper, that not only do the
most carefully prepared extracts of the infundibulum contain
detectable quantities of histamine, but that the mild hydrolysis
to which commercial preparations have been subjected during
their sterilization greatly increases the amount of this substance.
It was our intention to use the dried infundibular powder of
Burroughs, Wellcome and Company, a preparation which was
used in Dudley’s experiments, but as we were unable to secure
this preparation in this country, we had to use instead the ster-
ilized infundibular extract (“ Infundin”) of this firm. Thern im
mediate drop in blood pressure and the typical bronchoconstric-
tion produced by this extract had already induced Jackson and
Mills to state that “this preparation contains a considerable
proportion of histamine.”
METHOD OF SHOWING THE PRESENCE OF HISTAMINE IN “INFUNDIN”
The contents of eighty 1-cc. phials of infundin were evaporated

nearly to dryness in a shallow bowl on the water bath, under a
rapidly revolving electric fan. Previous experiments had shown
us that this method of concentration, when applied to small vol-
umes of extract does not lower its oxytocic strength. When the
volume had been reduced to about 5 cc., powdered, anhydrous
Na2CO3 was added in excess. [An abundant precipitate of gummy
character is immediately produced at this point. This salting
out also occurs when the extract has first been treated with col-
loidal iron. This gummy precipitate, it may be remarked in
passing, gives the biuret reaction with great intensity, raises the
arterial pressure and stimulates plain muscle tissue. A similar
“salting out” is also effected by saturation, or half-saturation
with ammonium sulphate as was discovered some years ago by
Osborne and Vincent (7)].
In the course of six or eight hours the alkaline mixture can be
dried in vacuo over sulphuric acid, if it is occasionally stirred

as soon as a surface crust begins to form. When it is thoroughly
dry the mixture is reduced to a fine powder. This is repeatedly
extracted (five times) with 20 cc. of hot chloroform at a time.
The residue left behind, after allowing the chloroform to evapo-
rate, weighed, when dry, 0.0058 gram. The residue from 100 cc.
FIG. 1. CAT, 3.3 KGM., ETHER
At A, 0.5 cc. CHC1, extract, Burroughs, Wellcome and Company’s “Infun-

din” injected into femoral vein. At B, injected 1 cc. = 0.1 mgm. histamine
acid phosphate (ergamine acid phosphate, Burroughs, Wellcome and Company).
of chloroform weighed 0.0020 gram, giving 0.0038 as the net

weight of the first chloroform extract of the above 80 cc. of
“Infundin.” The active residue was dissolved in 5.8 cc. of
water and tested for its effect on blood pressure. Figure 1, A,
shows the pronounced effect of the intravenous injection of 0.5
cc. of this solution on the arterial pressure of a cat. At B, in the
same figure, is shown the comparative effect in the same animal

of the intravenous injection of 0.1 mgm. of histamine acid phos-
phate. The two parts of the figure are so nearly alike that we
may safely assume that this first chloroform extraction of eighty
1-cc. phials of infundin yielded not less than 1.16 mgm. hista-
mine, estimated in terms of the acid phosphate of the base.
But this quantity of histamine represents not more than 60 per
cent of that present in the dry, sodium carbonate mixture. A
further 60 per cent or more, of what remains may be extracted
by proceeding as follows, and this was done in the case under
consideration. The powder was freed from all traces of chloro-
form by drying in vacuo, about 5 cc. of water were added and
thoroughly incorporated with the mixture. The mass was again
dried in vacuo over sulphuric acid, again powdered and again
extracted with boiling chloroform. The residue left after evap-
oration of the chloroform was evaluated as to its effect on the
arterial blood pressure of the dog in terms of the acid phosphate
of histamine. The yield of histamine from this second extrac-
tion was not less, and, if anything, more than 0.5 mgm. in terms of
the acid phosphate. Taking into consideration the results ac-
tually obtained above, and bearing in mind that a small fraction
of histamine still remains in the alkaline powder after the second
extraction with chloroform, we may safely conclude that the 80
cc. of sterilized “Infundin” contained an amount of histamine
equivalent to about 2 mgm. of histamine acid phosphate. Ac-
cording to our findings, therefore, a single 1 cc. phial of this
preparation contains 0.025 mgm. of histamine (calculated as the
acid phosphate).
It remains to add that the blood pressure lowering chloroform
extract above described also caused tonic contractions of the
uterus in minute amounts, and gave the Pauly reaction with
great intensity. In view of the actual isolation of histamine by
Abel and Kubota from extracts of the entire pituitary gland, there
can be little doubt that the substance here described is in reality
histamine, and not some similarly acting substance. We have
in our possession a considerable quantity of crude histamine
picrate, which was obtained by treating a large amount of depro-
teinized infundibular extract with hydrochloric acid. This

picrate will be purified and recrystallized at our leisure, and
the acual proof of its histamine character can easily be furnished,
should any doubts be expressed on this point.
HISTAMINE IN PITUITARY EXTRACTS OF AMERICAN MANUFACTURE
Results entirely like those just described were obtained with

the Pituitary Liquid of Armour and Company, and preliminary
experiments with the preparations of other firms force us to be-
lieve that histamine is present in every commercial extract of
the kind at present employed in therapeutics. The method em-
ployed here was entirely like that already described, with the
exception that colloidal iron was used in one experiment as a
clearing agent, as described in Dudley’s paper.
. Method
Experiment 1 The .contents of forty 1-cc. ampoules of Ar-

mour’s Pituitary Liquid’ were cleared with colloidal iron, the
clear filtrate was reduced to a small volume on the water bath,
under the electric fan and was then treated with sodium carbon-
ate and boiling chloroform in the usual way.
Experiment 2. The contents of another forty ampoules were
treated in the usual way with sodium carbonate and boiling
chloroform, but without being cleared with colloidal iron.
The chloroform extract of experiment 1 weighed 2.6 mgm. It
was dissolved in 2.6 cc. of water. 0.5 cc. of this solution injected
into the femoral vein induced the fall in arterial pressure shown at
A in figure 2. The chloroform extract of experiment 2 weighed
3.2 mgm. and was dissolved in 3.2 cc. H,O. 0.5 cc. of this solu-
tion induced the fall of arterial pressure shown at B, figure 2.
It will be seen that the fall in arterial pressure is practically the
same in the two cases, when the difference in concentration of
active substance in the two solutions injected is taken into con-
‘Purchased of the local Armour concern. A label on the package states that
it is not advised to use the contents after March, 1921. The above experiments
were made in February, 1920.
sideration. The colloidal iron precipitate carried down with it

a very small, though distinctly detectable, amount of histamine,
as shown at C, figure 2.
We have here, then, a second instance of a commercial pitui-
tary preparation which, from all that we can learn, is made with
great care, but which nevertheless contains readily detectable
amounts of histaniine. The fact is, one need take no more than
three ampoules of the above-named preparation in order to obtain
a chloroform extract which gives a decided fall in the arterial
pressure of the cat.
FIG. 2. CAT, 2.75 KGM., URETHANE ANESTHESIA
At A, 1 cc. of the CHCI, extract obtained from 40 ampoules Armour’s Pituitary

Liquid, cleared with colloidal iron. Injection made into femoral vein. At B,
injection into femoral vein of 1 cc. of the chloroform extract from 40 ampoules
as above but not cleared with colloidal iron. At C, injection of the chloroform
extract of the colloidal iron precipitate.
IS THE HISTAMINE, WHICH WE HAVE SHOWN TO BE PRESENT IN

COMMERCIAL PITUITARY EXTRAcTS, A PRODUCT
OF BACTERIAL ACTION?
The following experiments force us to give a negative answer

to the above question. A preliminary experiment was made as
follows. From an establishment in which only pigs are slaugh-
tered, and whih is in close proximity to our laboratory, we ob-

tamed a half dozen pigs’ pituitary glands. These were removed
from the skull, under our eyes, immediately after the animals
were killed. That there might be no question in regard to bac-
1erial action, the infundibular portion of the glands was im.medi-
ately ground up in a 2 per cent mercuric chlorid solution con-
taming 0.9 per cent of hydrochloric acid and allowed to stand
verthght. The coagulated proteids were removed by filtra-
tion under pressure, the filtrate was treated with hydrogen sul-
phide in the usual manner, and the mercury-free filtrate was con-
centrated on the water bath under the electric fan. To the con-
centrated solution anhydrous sodium carbonate was added in
excess, the mixture was dried in vacuo over sulphuric acid and
then exhausted with hot chloroform. Even with this very small
amount of material (0.315 gram fresh posterior lobe) we were
able to prove that the chloroform extract contained a blood pres-
sure lowering substance, which, in consideration of our earlier
work, can be no other than histamine. Figure 3, the legends of
which give the necessary details, proves that both histamine and
a histamine-like substance, which is insoluble in chloroform and
about which we shall have more to say later, are obtainable from
fresh infundibular material when treated in the manner described.
In view of the fact, however, that we did not neutralize the
extract after removing the mercury, but concentrated the solu-
tion on the bath while it was still acid, we can only cite this ex-
periment as showing that both histamine and a histamine-like
substance make their appearance when infundibular extracts are
heated on the water bath with very dilute hydrochloric acid.
The precursor of t.he histamine in this experiment cannot be one
of the ordinary coagulable proteids, as these had all been removed
from the field of action by the precipitating agent used-mercuric
chloride. Nor can it be one of the ordinary albumoses present
in all tissues, as these are also usually removed by adsorption
(mercuric sulphide) in the process above employed.
The above experiment, small as was the amount of material
at our disposal, proves once for all that histamine is obtainable
from absolutely fresh material which has been freed of proteids
by means of mercuric chloride. The destruction of the pressor

constituent in this experiment was caused by the hydrochloric
acid and this also occurred in the work of Kubota and Abel-a
fact which is now quite obvious. It was now advisable to test
infundibular extracts for histamine, which had been prepared
#{149}““\\
. \..
A .8
FIG. 3. LARGE CAT, ETHER
At A, injection into the femoral vein of the chloroform extract obtained

from only 0.315 gram fresh posterior lobe of the pig, treated with HgCl2 in 0.9
per cent HC1, as described. At B, injection of the alcohol extract of the material
(Na,CO, powder) that had been extracted with chloroform. Under ordinary
circumstances, that is to say, in the absence of hydrolysis, we should have at B
first a fall and then a rise of blood pressure. The effect of the HC1 on the extract,
after removal of the mercury, is to destroy the pressor substance. In its place
appear histamine (A) and a histamine-like substance (B), insoluble in chloro-
form but soluble in alcohol (93 per cent). A small amount of histamine (40
per cent of the amount used in A) also contributes to the marked fall in pressure
seen in B.
in a manner, in which hydrolysis with acids has been reduced to

the lowest possible minimum. Extracts of this character we
owe to the liberality of Armour and Company. Our thanks are
due to this firm, and especially to Dr. Frederic Fenger, of their
Chemical Research Laboratory, for his hearty cooperation and
for his kind assistance in the preparation of various extracts.
Under Dr. Fenger’s direction pituitary extracts were prepared in

the following manner. The entire glands were removed from the
sella turcica shortly after the animals were slaughtered, and were
placed on ice for a few hours, without coming in contact with ice water.
They were then kept in a “ chill room” over night at a temperature
of 0#{176}C.The next morning the posterior lobes were dissected out
and repeatedly minced, until a smooth mass of uniform consistency
was produced. This finely minced material was macerated at room
temperature for half an hour with nine times its volume of distilled
water containing 0.2 per cent of glacial acetic acid, and was then heated
to boiling and kept at the boil for ten minutes. The liquid was sepa-
rated by filtering the mixture while hot through two layers of filter
. paper. The filtrate was cooled down to 50#{176}C., 1 per cent of chloroform
was added, the mixture was thoroughly shaken, transferred to a jug,
and shipped in a refrigerator car to us at Baltimore.
Immediately after its receipt by us the extract was concentrated
almost to dryness in shallow evaporating bowls on the water bath
under the electric fan, after which it was kept in vacuo over sulphuric
acid. The material as thus dried may be kept indefinitely, apparently
without loss of its blood-pressure-raising or oxytocic power. The
amount of dry residue obtained in a quantitative experiment with
2900 cc. of extract made from 290 grams fresh posterior lobes was 15
grams. The method described above is essentially like that used by
manufacturers of pituitary extracts. It gives the largest possible
yield of active principle or principles, and is certtinly not at all drastic
in its character.
But even in extracts thus prepared, histamine is present in de-

tectable quantity. The amount of the base present in such
extracts is, however, very much less than is obtainable from those
ordinarily employed in therapeutics, such, for example, as have
been assayed and described in preceding pages. We calculate
that the amount of histamine present in these recently prepared
and immediately used extracts is only 20 per cent, or even less,
of that found in the commercial preparations, and naturally it
is a still smaller fraction of that obtainable by means of the mild
hydrolysis with acids.
Method
Experiment 1 . 1 .045 gram dry scales of Fenger’s infundibular

extract was dissolved in 10 cc. of water; anhydrous Na2CO3 (2.25
gram) was added and well stirred in ; the mixture was dried in
vacuo and extracted with chloroform. The chloroform residue
was dissolved in very dilute HC1 and tested for its action on the
blood pressure of the dog. Figure 4, B, shows the effect of the
FIG. 4. DOG, 5.6 KGM., ETHER ANESTHESIA
At B injection into the femoral vein of 0.5 cc. H,O solution of the acidulated
CHCI, residue obtained from 1.045 gram dry infundibular extract. At A 0.5
cc. injection of a similar solution from 1.045 grams dry extract. In this case,
however, the extract had been boiled for two and a half hours with 30 cc. H2O.
No increase in histamine production. At C, injection of 0.1 mgm. of ergamine
acid phosphate.
intravenous injection of 0.5 cc. (the total solution being 1 cc.).

At C is seen the comparative effect of 0.1 mgm. ergamine acid
phosphate in the same animal. This curve is somewhat broader
at the top than it should be, as a clot occurred at + and after
its removal the float was poorly adjusted. Two “doses” of hista-
mine hydrochkwide of the strength shown at B were therefore
obtained from a quantity of dry extract (1.045 gram) equivalent
to 12.7 grams fresh infundibular material. This quantity of

histamine is easily detectable.
Experiment 2. 1 .045 gram of the dry material used in experi-
ment 1 was dissolved in 30 cc. H2O and boiled under a reflux con-
denser for two and one-half hours. The solution was then fil-
tered to remove a little coagulated material and was then concen-
trated to 10 cc. After cooling, 2.25 grams, anhydrous sodium
carbonate was stirred into the solution. This was then dried
and extracted with chloroform. The chloroform extract, as in
experiment 1 weighed
, 0.001 gram, and was dissolved in 1 cc. of
H20. The effect of injecting 0.5 cc. of this solution intravenously
into a dog is shown at A, figure 4. It will be seen that boiling
the solution without the addition of any acid has left its hista-
mine content entirely unaltered. A solution thus treated was
found, however, to have lost about four-fifths of its oxytocic
strength. The acidity of the solution was not determined, but
it was of a low order. Our observation confirms the discovery of
Adams (8) that an infundibular extract with an acidity of ap-
proximately N X 10 which is kept at the boil for an hour loses
nearly four-fifths of its oxytocic strength.2
Further proof that a detectable amount of histamine is obtain-
able from an extract which has been prepared as above described
is given in the following experiment.
2 We find no evidence in his paper that Adams tested the H-ion content of his
extract after having completed the heating at 100#{176}C. It is unlikely that an
extract of such complex character, as the one in question, can be boiled for an
hour without the appearance of small quantities of ammonia or other bases. The
quantity of alkali that is obtainable from the flask and the condenser tube during
an hour’s boiling is also far from negligible, unless these pieces of apparatus are
made of hard glass. We have not made a study of these points in the present
instance. We have, however, observed that a butyl alcohol extract (in H20)
of the active principles of the infundibulum, which is just acid to litmus, can be
boiled for two hours at the reflux condenser without the slightest alteration in
its pressor or oxytocic action. The reaction to litmus was the same after boiling
as before. The solution here employed differed materially in its composition
from that tested by Adams. The flask and condenser used in the experiment had
been repeatedly exposed to hydrochloric acid in hydrolysis experiments. Is it
not possible that in the experiments of Adams, the extracts became alkaline dur-
ing the boiling, and that we have in his work an illustration of an OH ion effect
at 100#{176}C.,rather than an effect of boiling at an H ion concentration of N X 10,
as stated by Adams?
8.14 grams of the dry extract, representing approximately 103

grams of fresh infundibular material, were dissolved in 30 cc. of
water, acidulated with 2 drops of 25 per cent hydrochloric acid,
filtered from a small amount of insoluble material, then extracted
for thirteen hours with butyl alcohol, under diminished pressure,
FIG. 5. CAT, 3.3 KGM., ETHER ANESTHESIA; BEING THE SAME ANIMAL USED FOR
THE EXPERIMENT ILLUSTRATED IN FIGURE 1
At ,i’ intravenous injection of one-sixth of the chloroform residue obtained

from one-fourth of the butyl extract of the pituitary extract described in the
text.
in an apparatus similar to that described by Dudley. The ex-

traction consumed three days, the apparatus being kept in opera-
tion for about four hours each day. After removing all traces
of alcohol from the butyl alcohol extract, the residue was found to
weigh 1.76 grams. This was dissolved in a given quantity of
water, and the histamine content of an aliquot part (one-fourth)
of the solution was determined in the usual manner. Figure 5

illustrates the extent to which the blood pressure of a cat weighing
3.3 kgm. was lowered by injection of one-sixth of the acidulated
chloroform residue which was obtained from this aliquot part.
The entire butyl alcohol extract (1 .76 grams) must, therefore,
have contained 24 such doses, or quantities, of histamine. Dud-
ley has found that histamine is only slowly extracted from an
acid solution, and we may assume therefore that some histamine
still remained in the aqueous solution.
We w’ould mention that the butyl alcohol extract gave the pink
biuret reaction and that it was nearly devoid of oxytocic activity.
The aqueous layer which retained 6.38 grams of the original
extract was also found to be nearly inactive for the guinea-pig’s
uterus. It is not difficult to elucidate the cause of this deprecia-
tion of oxytocic strength, which amounted to more than 80 per
cent. We may be certain that we have here only another illus-
tration of the instability of solutions of the active principle whose
H-ion content lies close to the neutral point. Evidently an in-
sufficient quantity of hydrochloric acid was added to the aqueous
extract before putting it into the extracting apparatus.3 We
might also add that treatment of the inactive butyl alcohol resi-
due with hydrochloric acid increased the histamine content to
only a slight extent, if at all. The above experiment is of inter-
est as proving that histamine is extractable with butyl alcohol
from a freshly prepared infundibular extract, such as was made
for us by Dr. Fenger in the manner described above.
A later experiment proved that this amumption is correct. In this experi-

ment the extract was made up to contain 0.1 per cent HC1 and the butyl alcohol
residue (fifteen hours extraction) was found to have a pure pressor action. The
aqueous layer was evaporated (without first reducing the acidity, unfortu-
nately), under reduced pressure at 40#{176}C.;
water was added to the dry residue and
the process repeated. When the residue was now taken up in water and tested,
it was found to have lost its pressor action entirely, and to give a pronounced
depressor effect instead.
ALTERATIONS IN THE PHYSIOLOGICAL PROPERTIES OF INFUN-
DIBULAR EXTRACTS, INDUCED BY MILD HYDROLYSIS WITH
HYDROCHLORiC ACID
We have seen that infundibular extracts which have been

made by a very simple process can be shown to contain detect-
able quantities of histamine, and reference has occasionally been
made in the preceding pages to certain conditions which cause
the appearance of an increased amount of histamine in such
extracts. In the following pages we shall describe the effects
of mild hydrolysis of infundibular extracts with hydrochloric
acid.
1 . Changes in the blood-pressure effects of extracts after treatment

with dilute HC1
Dilute preparations, such as are used in therapeutics, when

made up to contain 0.25 per cent HC1, may be boiled (reflux
condenser) for half an hour without any appreciable loss of the
pressor or oxytocic activity of the extract. When the HC1 con-
tent of the extract reaches 0.5 per cent, half an hour’s boiling
almost, if not completely, annuls its blood-pressure-raising power,
while, boiling for half an hour, when the HC1 content is 1 per
cent certainly abolishes every trace of pressor activity. The
blood-pressure-raising power of an infundibular extract is also
completely abolished if it is made up to contain 2.5 to 3 per cent
HC1 and is then rapidly evaporated to dryness in a shallow bowl
on the water bath, under a rapidly revolving electric fan. In
making this experiment it is best to use only a few cubic centi-
meters of the extract at a time. This small volume of liquid
can be quickly removed by this method, and the addition of a
little alcohol, when the extract is nearly dry, assists in removing
the last traces of HC1. Naturally, in instances of hydrolysis
described above, the HC1 was nearly neutralized before the solu-
tions were tested.
Treatment with hydrochloric acid, as above described, leads
not only to the complete disappearance of the pressor action of
an extract, but causes a blood-pressure lowering, or depressor
action, to appear in its place. This reversal of action may be

obtained with a single ampoule of a commercial preparation, as
is shown in figure 6, A, A2, B and C, the legends of which give
the necessary explanations. As it might appear to some that
the tracing given in figure 6, B, does not prove that the pressor
substance is entirely destroyed, we present the results of a second
experiment in which similar quantities of an infundibular extract
FIG. 6. Ema’r OF BO1LING AN INFUNDIBULAR EXTRACT (ARIouR’s PITUITARY

LIQUID) WITH ACID
At A, as also at A2 the contents of 1 ampoule (1 cc.) were injected intrave-

nously. At B. the contents of 1 ampoule previously boiled (6 one cc. ampoules in
all) for one-half hour with 1 per cent HC1, were injected. Animal: young dog,
4.25 kgm. Anesthetic: ether. The quantities here injected are small. The
injection of larger quantities of boiled acidulated extract gives a very striking
“histamine effect.” At C’, 1 cc. of the contents out of 6 cc. (6 ampoules), treated
on the water bath with 2.5 per cent HC1; injection made much later than those
at A and B.
TUE JOUR. OF PHARM. AND EXPER. TEERAP., VOL. XV, NO. 4

were boiled for half an hour with 0.5 per cent and with 1 per
cent HC1. In figure 7, at A and B, may be seen the effect of
boiling with these concentrations of HC1, as compared with the
normal action of the extract which is shown at C.
It might be thought that the depressor effect seen in figures
6 and 7 is due to the action of hydrochloric acid on traces of
FIG. 7. EFFECT OF HEATING INFUNDIBULAR EXTRACTS (ARMOUR’S PITUITARY

LIQUID) WITH ACIDS
At A, effect of intravenous injection of 1 cc. extract (5 ampoules 1 cc. each)

boiled with 0.5 per cent HC1 for one-half hour. A, B, injection of 1 cc. extract
boiled with 1 per cent HC1 for one-half hour. At C, injection of 1 cc. extract as
taken from the ampoule (1 cc.). Animal: young dog. 5.13 kgm. Anesthetic:
ether.
coagulahie proteids or of inactive albumoses present in the ex-

tracts used. This cannot be true since the above effects are
produced when an extract which is entirely free from coagulable
proteids, and from the greater part, if not all of the physiologically
inactive albumoses, is similarly treated with hydrochloric acid,
as shown in figure 3 and also below.
The following experiment illustrates the method employed.

Frozen infundibular material was ground up very finely and
stirred into five volumes of a 2 per cent solution of mercuric
chloride in 9 per cent HC1. The precipitated mass of proteid
was separated by filtration at the pump and washed once with
the 2 per cent mercurial solution, and then thoroughly pressed
until free from adherent fluid. The proteid-HgC12-precipi-
tate was suspended in water, a quantity of 10 per cent NaOH
solution was then added, up to a point just short of the appearance
of the yellow oxide of mercury. The mercury was now precip-
itated from the mixture with hydrogen sulphide, and after
removing the excess of hydrogen sulphide with a current of air,
a slightly acid solution was obtained, which has a pure blood-
pressure-raising action. Figure 8, A , illustrates this action,
although in this particular instance the rise of pressure was not
very marked, very probably because the injection was made
too soon after an injection of a pressor preparation which had
induced a larger and more prolonged rise of pressure. When a
pressor solution of this kind is treated on the water bath with
hydrochloric acid, the pressor effect disappears and a depressor
action is found to have taken its place, as is well shown in figure
8, B. We may here remark that the above method yields solu-
tions which are more powerful, both in regard to oxytocic and
pressor activity, than any other form of crude extract which we
have yet been able to prepare. As a rule, the rise in arterial
pressure after an intravenous injection of such a solution takes
place promptly, reaches a high level and is of long duration.
There is no indication of a drop in pressure, either at the begin-
ning or end of such a curve. The method is not an economical
one as at present elaborated, but we intend to examine its possi-
bilities still further.
Abel and Kubota, in their work on the pituitary gland, em-
ployed mercuric chloride as a clearing agent, and as they did
not guard against the harmful action of free hydrochloric acid,
their solutions contained not a trace of the pressor principle of
the gland, but only the depressor substances.
We have found only one reference in the literature in regard

to the action of very dilute acids on pituitary extracts. Guggen-
heim (9) states that dilute acids hardly alter the activity of
“Pituglandol” w-hen acting at ordinary temperatures, but that
concentrated acids completely inactivate the extract in respect
to blood pressure, respirations and uterus. A “Pituglandol”
FIG. 8. YOUNG DOG, 5.4 KGM.; ANESTHETIC, CHLORETONE
Effect of heating an infundibular extract with hydrochloric acid. At A,

injection of 1 cc. of a solution deproteinized with mercuric chloride, etc., but
not subjected to the action of acids during the process of removing the mercury.
At B, effect of 4 cc. of the same solution after treatment with hydrochloric acid
on the water bath.
.
solution which has been completely hydrolyzed with hydro-

chloric acid was, however, found by Guggenheim to have retained
its stimulating power for the surviving intestine.
2. Depreciation of the oxytocic power of infundibular extracts after

treatment with dilute acids
It has been shown that a complete reversal of action on the

arterial blood pressure is easily induced in infundibular extracts
when these are subjected to mild hydrolysis with acids. Exper-
iments show that this treatment also greatly reduces the oxytocic
power of the extracts. It is difficult to give exact ratios here,
but we think that we are justified in stating that at the time when
the hydrolysis has caused a complete reversal of the blood pres-
sure action the loss in oxytocic strength amounts to four-fifths
and possibly more. Boiling an extract for half an hour with 0.25
per cent HC1 causes no appreciable alteration in its oxytocic
strength (fig. 9, A and B). It has been shown that an extract
thus treated also retains its pressor action unimpaired. Boiling
for half an hour with 0.5 per cent HC1 appears to induce the
maximum of reduction in oxytocic value, or at least very nearly
so (fig. 10, A and B). The results of boiling extracts for one-
half hour with 1 per cent HCI are seen in figure 11.
The action of greater concentrations of HC1 was studied as
follows. To 24 cc. of an infunclibular extract (Armour’s) an
equal volume of a 28 per cent solution of HC1 was added, and the
mixture was then boiled at the reflux condenser for six hours.
The hydrochloric acid was then removed on the water bath,
under the electric fan, with the assistance of strong alcohol,
which was added as needed. To the dry residue a little water
was added, the solution was made alkaline with sodium carbonate,
then dried in vacuo, and when dry, the residue was finely pow-
dered and then repeatedly extracted with 95 per cent alcohol.
Toward the end of the extraction the alcohol was used at a tem-
perature of about 50#{176}.After removing the alcohol, the residue
was made up to the original volume of extract, and this was
then matched, in uterine tests, against a known volume of nor-
C)
HE
nJ
‘-C)
‘-
C
‘-C)
‘Ho
.
C)
. ‘ .C)
o .H
. ‘-
Ho
‘H
CI ‘H
C)
C
nJ
‘-H
:
‘H ) H
‘H ‘ -
33
-o
C) C)
C)2
0. ,
PRESENCE OF HISTAMINE iN PiTUITARY GLAND 369
.‘
H
C)C)
.
mO ‘C)
H
El.
,C)
r.i 2-
n
C, fp.C
- #{149}) .
H
H I.
H
..‘!
H
- crC
il
H
-
‘-CI
‘H
0
0
H .
C)
C
“3 . E-’
.ln)
..4.”
H .C
.0 C)
mal infundibular extract, both being diluted to the same extent.

Figure 12 gives the comparative results of this prolonged hydro-
lysis, as far as its influence on the oxytocic value of the extract
is concerned. At A is seen the action on the virgin guinea-pig’s
uterus of the unaltered extract. At B, the action of a comparable
amount of the alcoholic extract obtained from the hydrolyzed
At / 10 drops from the pipette used for the experiment of figure 10, A and B
(32 drops to the cubic centimeter) of pituitary liquid, boiled for half an hour
with 1 per cent HC1, cooled and neutralized. Uterine chamber, as in other
experiments with uterus, contained 24 cc. of Tyrode’s solution. At T, fresh
Tyrode’s solution. The uterus here employed is the one used in figure 10. It
will be seen that it responds only slowly to a five-fold increase in the dosage of
the liquid treated for half an hour with 1 per cent HC1.
infundibular preparation. A remarkable reduction in oxytocic

strength certainly follows the above treatment, but it does not
appear to be greater than that which is produced by the milder
treatment with 0.5 and with 1 per cent HC1, as above described.
We would state at this point that the oxytocic substances con-
tamed in one of the above described hydrolyzed extracts are
FIG. 12. At A, action of 2 drops of a diluted infundibular extract (Armour)

A)fl the virgin guinea-pig’s uterus. At B, action of same quantity of extract,
after it had been boiled with 14 per cent HC1 at the reflux condenser for six hours,
and then treated as described in the text, before testing on the uterus. At T, T,
.changed to fresh Tyrode’s solution.
completely extractable with 93 to 95 per cent alcohol. This

alcohol extract, as will be shown presently, contains histamine
and also a histamine-like substance in the proportion of 1 : 4.
A further step was taken with the oxytocic substances obtained
by the use of alcohol as just described, whose action was shown
in figure 12, B. Only a very small part of the alcoholic extract
was consumed in the above test and the main portion was worked
up for histamine in the usual manner. The amount of histamine
obtained was compared in uterine tests with that which was
obtained from an equal amount of the original, unaltered extract.
The normal extract itself gave off not a little histamine to chloro-
form, as shown by the uterine tests, but that hydrolyzed with
14 per cent HC1 for six hours, yielded up to chloroform at least
five times more histamine as determined by comparative uterine
tests (as 20 : 100).
ACID-TREATED PITUITARY EXTRACTS CONTAIN HISTAMINE AND A
HISTAMINE-LIKE SUBSTANCE
Histamine constitutes only part of the oxytocic material that

was taken up by alcohol from hydrolysed extracts as described
above. Accordingly, after separating the histamine (60 per cent
only), alcohol was employed to dissolve out the remaining his-
tamine-like, oxytocic substance. This was matched up in uter-
ine tests against the histamine obtained with chloroform and, as
near as we can estimate, it had fully four, times the oxytocic
value of the histamine. Allowance was made for the 40 per cent
of histamine which still remained mixed with this histamine-
like substance, after the one treatment with chloroform. In
many other experiments we have convinced ourselves that this
alcohol-o1uble substance, which behaves in respect to both blood-
pressure and uterus, exactly like histamine, is in reality a second
individual. We have always been able to obtain a large amount
of alcohol-soluble material from our hydrolyzed extracts, even
after three separate chloroform extractions. Certainly in such
cases the amount of histamine left in the alkaline powder, after
three treatments with chloroform, must be negligible, yet the
use of 95 per cent alcohol now yields an extract which depresses

the blood pressure very powerfully and is active for the guinea-
pig’s uterus. Abel and Kubota supposed that all of the depressor
material in their pituitary extracts was histamine, and that their
failure to obtain a larger yield of histamine was due to the dif-
ficulty of exhausting the various precipitates. We have now
found that the major part, say 80 per cent of the depressor and
oxytocic material present in pituitary extracts which have been
treated with HC1, as above stated, consists of an alcohol-soluble
substance which, as far as our present knowledge goes, is distin-
guishable from histamine only by its entire insolubility in chloro-
form. Like histamine, it gives the Pauly reaction with great
intensity. We shall have more to say in a later paper about the
relationship of the two compounds. It will be shown by Nag-
ayama, in the paper following this, that hydrolysis of albumoses
in general, yields histamine as also a histamine-like substance
of entirely similar properties, physiologically and chemically
speaking, to that here described.
EFFECT OF THE TRYPSIC DIGESTION OF INFUNDIBULAR EXTRACTS
ON THEIR PRESSOR AND OXYTOCIC FUNCTIONS
A few years ago Dale (10) discovered that an active preparation

of trypsin reduces the action of pituitary extracts on the blood
pressure and urinary flow practically to nil, after a few hours’
digestion. Dudley finds that trypsin rapidly destroys the uter-
ine stimulant, but, in commenting on his experiments, he re-
marks (see curve F, p. 309 of his paper) that “it is obvious that
a small amount of the uterine stimulant was still undecomposed.”
We think that the following two points are worthy of note
in connection with the effects of trypsin on infundibular extracts.
1. In place of the destroyed pressor action there appears a
depressor effect, as may be seen by an examination of Dale’s
curves. We have made this observation repeatedly with ex-
tracts that had been incubated with an active preparation of
trypsin, for periods varying from three to fifteen hours. The
reversal in blood pressure action is well shown in the decerebrate
cat, in which animal, also, we have observed the same increase

in the broncho-constrictor action after trypic digestion, that is
seen after mild hydrolysis with acids. These experiments in
regard to broncho-constrictor effects will be given in greater
detail in a later paper.
2. We estimate that the amount of uterine stimulant which
remains unaffected even after the most prolonged tryptic digestion
amounts to 10, or perhaps, even 20 per cent of that originally
present. In Dudley’s experiments, the time of exposure to the
ferment was only thirty minutes.
Our reason for placing emphasis on these two points-the
appearance of a depressor, in place of a pressor, action, and the
retention of from 10 to 20 per cent of the oxytocic strength,
in extracts treated with trypsin-is the obvious similarity of
these effects to those that appear when extracts are treated with
acids. As after hydrolysis with weak acids, so after tryptic
digestion, the depressant action on the blood pressure and the
oxytocic effect of the uninjured residue appear to be due to two
substances, the one, histamine, present in smaller amount, the
other, a histamine-like substance, insoluble in chloroform but
soluble in alcohol, and present in larger amount than histamine.
We would not be understood as maintaining that all of the de-
pressor and oxytocic material obtained after tryptic digestion is
due to the action of the ferment. Probably all but a small part
of this material exists as such in the extract, as in the case with
extracts of other organs.
DISCUSSION
In the foregoing pages we have described the circumstances

under which histamine, and also a histamine-like substance,
may be obtained from extracts of the infundibular portion of
the pituitary gland. Histamine was isolated as a pure chemical
individual by Abel and Kubota from extracts of the dried whole
gland which had been deproteinized with mercuric chloride. In
the experiments above described the histamine was not thus
isolated and purified, but there can be no doubt, in view of the
work of Abel and Kubota, that the substance which was extracted
by us with chloroform from infundibular extracts is in reality
histamine. The histamine-like substance, on the other hand,
whose physiological properties are so very much like those of
histamine, as far as present researches have determined, has not
yet been isolated by us as a pure chemical individual, though
much work toward this objective has already been done by us.
The problem appears to be one which is capable of solution, and
we shall continue our work on it.
The two substances named, if given together in sufficient quan-
tity, are able to cause a profound and lasting fall in the arterial
pressure. And, as has been repeatedly pointed out, this effect
does not follow the injection of a recently prepared non-hydro-
lyzed, infundibular extract. The effect of a small quantity of
such an extract on the blood pressure, as is well known, is to
cause it to rise rapidly and to maintain itself at a high level for
a considerable time. But such an extract may, nervertheless,
at this time have concealed within itself a considerable amount
of both histamine and a histamine-like substance. This is cer-
tainly true of the ordinary preparations used in medical practice,
as may easily be demonstrated by the injection of a larger amount
of one of these preparations. Thus, in figure 13 is given a tracing
which shows the effect on the arterial pressure of the injection
of the contents of three 1-cc. ampoules of pituitary liquid. As
shown in figures 6 and 7, one ampoule ordinarily causes a rise of
pressure, uncomplicated by a fall, but when three ampoules are
employed, a noticeable drop in arterial pressure at once makes
its appearance. One naturally asks whether the entire amount
of depressor material which makes its appearance in pituitary
extracts that have been treated with 1 per cent hydrochloric
acid for half an hour, or which have been subjected to prolonged
tryptic digestion, was originally present as such in the extracts.
Could so much depressor substance have been antagonized by
the pressor substance and thus rendered incapable of making
itself manifest? Is the depressor substance which appears after
mild treatment with acids identical with that which is present
in untreated commercial extracts? These questions call for
further studies of a quantitative nature.
Let us assume for the present that the histamine-like, depressor

and oxytocic substance, which is soluble in alcohol and insoluble
in chloroform, is one substance and not several, and let us for
convenience name it B, while histamine is to be named C. Both
substances, B and C, as will be shown in a paper by one of us
(Nagayama) can easily be produced by hydrolyzing albumoses
(Witte’s peptone and similar products) and both substances
together, we believe, account for the depressor effects4 of the
various organ extracts and enzymatic products, as erepton,
meat extracts and shoyu sauce, which Abel and Kubota have
FIG. 13. LARGE CAT, ETHER
At / injection of contents of three ampoules of pituitary liquid (Armour).

The drop in the blood pressure proves that the preparation contains histamine
and the histamine-like substance.
studied. Now, in all of the instances just named, the depressor

substances had their origin in some inactive or very little active
precursor. Ferments and similarly acting agents, such as hydro-
chloric acid, are capable, then, of producing B and C in quantity
from relatively inactive tissues, or from pure proteids. It is
natural to assume, therefore, that, when B and C make their
appearance in an infundibular extract which has been treated
The laboratory notes of Abel and Kubota show that they frequently obtained
alcoholic extracts after the use of chloroform, in working with organ extracts,
etc., which were oxytocic and depressant for the blood pressure. There can be
no doubt, then, that B was present in all of their alcoholic extracts.
with hydrochloric acid, they have their origin, in part at least,

in some precursor, or precursors, of similar character to those
present in other extracts. It is admitted that we have not yet
proved that B is one and the same substance in all of the various
tissues and extracts in which it is found. The resemblance be-
tween the behavior of pituitary extracts whose pressor constituent
has been destroyed and the various extracts described by Abel
and Kubota is, however, very striking, to say the least. And
we would not be understood as maintaining that the same amount
of B and C is obtainable per gram of tissue from the various
tissues. It is our impression, however, that both B and C are
present in larger quantities in hydrolyzed infundibular extracts
than in equal quantities of extracts of other organs similarly
treated, but the correctness of this impression waits on further
quantitative researches for its verification.
The above considerations strongly support an opinion already
expressed in earlier papers that B and C should not be regarded
as hormones or substances which are specific to the pituitary
gland. Until B shall have been isolated from the infundlibulum
as a chemical individual, it must remain uncertain whether or
not it exceeds histamine (C) in oxytocic and depressor strength.
As already stated, this problem is now occupying our attention.
PRELIMINARY OBSERVATIONS IN REGARD TO THE BLOOD-PRESSURE-
RAISING CONSTITUENT OF THE PITUITARY GLAND
While B and C, as above obtained, do not appear to us to be

substances specific to the pituitary gland, the case is distinctly
otherwise with the pressor constituent of the gland. This sub-
stance is one of the constituents of the gland for which the claim
of being a specific substance can be upheld. For convenience let
us call this constituent A. We have seen that the action of A
is easily annulled by acids and by trypsin (Dale), and by alkalis
at room temperature (Guggenheim), and it is pertinent to inquire
whether A, when isolated as a chemical individual, will yield
any decomposition products whose action resembles that of B
#{149}and
of C. Without bearing in mind some of the considerations
already presented above in regard to the widespread distribution

of B and C, one might incline to the belief that all of B and C
comes from A . All of B and C cannot be derived from A , for
the very simple reasons that other tissues, which apparently
contain no A , yield a certain amount of B (or a very similar
substance) and also a certain amount of C (histamine). Or it
might be supposed that when the pressor action is annulled, A
is destroyed without the appearance of any physiologically active
residues whatever. Evidently these questions can only be an-
swered by the isolation of A as a single, pure principle.
Inasmuch as circumstances beyond our control will prevent
us from continuing our researches for a number of months, we
may be permitted to give here such facts in regard to A , as at.
present isolated, and its decomposition products as are now in
our possession.
Method 1 for the isolation of A
We have already stated that a highly active pressor solution.

may be prepared by decomposing the so-called proteid-HgC12
-precipitate, which was referred to on page 365. The proteid
and mercury-free filtrate which was obtained from this precipi-
tate was concentrated to a small volume, at a slightly acid re--
action. Into the thickish mixture, anhydrous sodium carbonate
was stirred until a large excess had been added. (Much active
material is salted out in this procedure as we found by blood
pressure and uterine tests, and is thus protected from further
injury by the carbonate.) The alkaline mass was then placed
in shallow bowls and dried in vacuo over sulphuric acid. The
dry material was reduced to a fine powder and extracted with
dry chloroform to remove histamine, after which it was repeatedly
extracted with 93 to 95 per cent ethyl alcohol. The alcohol was
at first applied a few times at room temperature and later the
extractions were carried out on the water bath, with the appli-
cation of gentle heat, well below the boiling point of the alcohol.
A very considerable fraction of the pressor and oxytocic substance,
or substances, can be extracted from the alkaline powder by-
this frequently repeated use of alcohol. The alcohol is removed

at as low a temperature as possible on the water bath, under a
rapidly revolving electric fan. The highly active basic residue
thus obtained is small in bulk, as compared with the original
material. By the application of fractional crystallization, frac-’
tional precipitation, and by the use of several solvents and acids7
more especially picric and phosphoric acid, we finally obtained a
relatively insoluble picrate and a colorless, very soluble non-’
hygroscopic phosphate, both of which showed a high degree of
physiological activity. We shall not here give more than this
bare outline of our method, as we think it capable of great
improvement.
As will be seen from an examination of the accompanying
figures, small quantities of both the picrate and the phosphate
raised the arterial pressure in a decided manner. Both salts
were also possessed of oxytocic power to an extraordinary degree,
being many times more powerful in this respect than histamine.
The results obtained with the guinea-pig’s uterus were, indeed,
so extraordinary that we prefer to withhold the tracings for
corroboration in experiments to be made later. We are here
dealing with salts of the pressor or blood-pressure-raising constit-
uent of the infundibulum. The phosphate is especially adapted
to chemical tests, and we would state that this salt gives the
Pauly reaction with great intensity, so also the pink biuret
reaction. Neither salt has been obtained in an indubitably
crystalline form, and hence we cannot be sure that we are dealing
with a single chemical individual. Their aqueous solutions show
an acid reaction toward litmus, which indicates that the pressor
substance is a very weak base. It is noteworthy that we have
never, at any time, had on hand a preparation, or residue of any
kind, that had the power of raising the arterial pressure promptly
and decidedly, which did not give both the biuret and the Pauly
reaction. On the other hand, neither B nor C gives the biuret
reaction, although both, as above stated, give the Pauly reaction
with great intensity. It is also noteworthy that the biuret
reaction is lost entirely when A, in the form of its salts, is hydro-
lyzed with acids, but the Pauly reaction remains unaltered. This
THE JOUR. OF PHARM. AND EXPER. TRERAP., VOL. XV, NO. 4

point would be of great significance as pointing to a fundamental

relationship between A , B and C, if once we could be certain of
the chemical individuality of A ; all the more so, since treatment
of the pressor substance A with acids, as wifi be shown below,
gives rise to a certain amount of the depressor substances B
nd C.
Method 2. The use of tetranitro-aniline as an agent for the

isolation of A, B and C
The work of Barger and Tutin (11), who showed that amino-
acids and peptides can be combined with $- or with ‘y- trinitro-
toluene (the former attaching themselves to the benzene ring
by an amino-group, in replacement of a reactive nitro-group)
suggested the advisability of using one of these nitrotoluenes in
our own work. While we were attempting to procure one of these
isomers, our friend, Professor W. R. Orndorff, suggested that
we give tetranitro-aniline,
NH2
O2N(NO2
JNO2
NO2
which has one easily replaceable nitro-group (3) a trial, as a

combining agent, in place of the proposed 3- or 7-trinitrotoluene,
and very kindly sent us a supply of the pure compound. This
substituted aniline promises to be of great service, and we have
already found it useful in effecting a preliminary separation of
A and B, after previously removing C with chloroform. The
condensation products, as at present obtained, are still
very
impure; and we are giving these incomplete experiments at this
time, because we find that the A-tetranitro-aniline compound
We are now preparing these isomers from “T N T still residues,” which were
kindly sent to us by Dr. Charles L. Reese, Director of the Chemical Department,
E. I. du Pont de Nemours and Company, and we hope later to test their useful-
ness as isolating reagents in our work on pituitary principles.
reacts to HC1 in exactly the same manner as the pressor phos-

phate of A, as will be shown below. This condensation product
also acts powerfully on the guinea-pig’s uterus.
The condensation product with B, also lowers the arterial
pressure, and stimulates the uterus exactly as does B, when
obtained by the method already described. The chemical re-
actions of these A- and B- and C-condensation products are also
identical with those given by A, B and C, as otherwise obtained,
except in so far as the aniline component of the condensation
product interferes with a given reaction. The Pauly reaction
is given with great intensity by all of them. The biuret reaction
with the A-condensation product does not give the ordinary
pink, but a ripe olive color, the result of the combination of the
pink of the biuret and the yellow of the aniline compound.
ACTION OF HC1 ON THE PHOSPHATE OF A AND ON THE TETRANITRO-
ANILINE COMPOUND OF A
It was of interest to learn whether A , as separated from in-

fundibular extracts by the above methods, would show the same
behavior to acids as do the extracts themselves. An examina-
tion of our figures wifi show that the results obtained in the mild
hydrolysis of A are identical with those obtained in the hydrol-
ysis of infundibular extracts.
Our findings may be summarized as follows:
1. Mild hydrolysis with HC1 completely abolishes the pressor
action of the A-phosphate, as also of the tetranitro-aniline con-
densation product of A, exactly as it does in the case of infundib-
ular extracts. Figures 14, 15, 16, 17, 18 and 19, all furnish
examples of the pure blood-pressure-raising action of varying
doses of the phosphate,6 picrate and tetranitro-aniline compound
of A. In figure 18, A and B, is seen the comparative effects of
0.23 mgm. of pressor picrate and 1 cc. of pituitary liquid. At
The
#{176} salts used in these tests were made at different times and often by
methods that differed very materially. For example, the phosphate tested in
figure 20 was prepared from a precipitate obtained in the fractional salting out
with (NH4)2S04 + Na2CO,, of a product which itself had a high degree of pressor
and oxytocic activity.
4
1’
A
,_____1______ I I I I I I I I I I I 1 1 1 1 1 1 I 1 1
FIG. 14. SMALL DOG, 6.4 KGM., CHLORETONE IN OIL INTRAPERITONEALLY
At A, injection into femoral vein of 1 cc. solution of a pressor phosphate = 2.1

mgm. Long continued rise of pressure. At ++, clots in cannula cleared out.
FIG. 15. DOG, 5.5 KGM., CHLORETONE IN OIL INTItAPERITONEALLY
At / 2 cc. = 2 mgm. of phosphate of the pressor substance A.

382
BC
flnnmnnmm
FIG. 16. YOUNG Dou, 6 KGM., ANESTHETIC NOT NOTED AT TIME
At A, injection into femoral vein of 0.64 mgm. pressor phosphate in 1.1 cc.
water. At B, 1.2 cc. of the same pressor phosphate solution rapidly evaporated
on the water bath under the electric fan with a little concentrated HCI. At C,
0.5 cc. of the same acid-treated solution. At D, which was the first injection given
to the animal, we injected 1 cc. of pressor picrate solution, containing 0.33 mgm.
of the salt in the cubic centimeter. The rise of pressure was prolonged. At +
a clot was cleared out.
B and C, figure 17, is shown the complete annulment of the pres-

sor activity of a small dose of the A salt. This result has been
obtained many times in the course of our work, and is again
shown at B and C, figure 16. By way of comparison, we point
to figure 18, C and D, where is shown the complete disappearance
Fxo.17. 4.9 XGM., CHLORETONE IN UL INTRAPERITONEALLY
At A, 1 mgm. of pressor phosphate into the femoral vein. At B, 1 cc. of a

phosphate solution (= 1 mgm.) after previous hydrolysis with HC1. At C, re-
peated injection B. The phosphate used in B and C was as active as the one
used at A and the concentration of the solution was the same. At D is seen the
effect of the injection of 10 cc. = 10 mgm. pressor phosphate, after hydrolysis
for three hours with strong HO! in a long test tube submerged in a water bath.
The depressor action is quite apparent in this case.
of the blood-pressure effect of a pituitary extract, after a very

brief treatment with HC1 on the water bath, of the contents of
only one ampoule of Pituitary Liquid, without the appearance in
its stead of a very marked fall in pressure. Here the effect is
decidedly less than that shown in figures 6 and 7. Evidently,
the length of time of exposure to HC1, as well as the strength of
acid used, are of importance in bringing out the full depressor

action.
2. A depressor effect, in place of the blood-pressure-raising
action of A, is plainly evident after mild hydrolysis with HC1,
only when about 10 mgm. of the pressor salt are used. This
effect is clearly shown at D, figure 17, and at B, figure 19, where
is seen the result of,heating 20 mgm. of the impure tetranitro-
FIG. 18. YOUNG DOG, 3.95 KGM., ANESTHETIC NOT NOTED
At A, injection into femoral vein of 0.5 cc. containing 0.23 mgm. pressor
picrate. At B, contents of 1 ampoule of pituitary liquid (Armour). At C.
injection of contents of 1 ampoule rapidly evaporated (electric fan) with a con-
tent of 2.5 per cent HC1 on the water bath. At D, content.s of 4 ampoules
treated in the same quick fashion with 2.5 per cent HC1 on the water bath. The
hydrolysis was not so complete at C or at D as in figures 6 and 7.
aniline compound. It is also evident in figure 20, B, where is

shown the effect of HC1 on 10 mgm. of a preparation of A, which
was subjected to a more severe treatment with HC1 than some
of the other preparations.
3. Having found that both of our blood-pressure-raising corn-
pounds (phosphate and TNA derivative) lost their activity on
mild treatment with HC1, we determined to try more vigorous
treatment with the acid, in the hope of obtaining a greater yield
of histamine, and also with the purpose of learning whether any
tA
FIG. 19. DOG, 6.9 KGM., ETHER
At A, injection into the femoral vein of a solution of the tetranit.roanilin

compound (crude) of the pressor body. 1 cc. = 20 mgm. The result was a
long continued rise of pressure. At B, injection of 1 cc., same strength of solu-
tion, but heated on the water bath under the electric fan with 10 per cent HC1.
of the alcohol-soluble and chloroform-insoluble histamine-like

substance would be formed. Accordingly, we dissolved 0.225
gram of pressor phosphate, which gave both the biuret and the
Pauly reactions, in 15 cc. of 25 per cent HC1,7 and boiled the solu-
We have frequently observed that the addition of strong HC1 to the pressor
phosphate causes the appearance of a beautiful violet color which soon passes
into a plum color. We find that Guggenheim has already described this reaction
as appertaining to the commercial extract pituglandol when similarly treated.
Bioch. Ztschr., lxv, 202, 1914.
FIG. 20. \OUNG DOG, 4.2 XGI., ANESTHETIC, CHLORETONE INTRAPERITONEALLY
At A injection into the femoral vein of 2 cc. = 2 mgm. of the pressor phos-
phate, prepared from salted out material. At ++ clots removed. At B injec-
tion of same quantity of phosphate (2 mgm.) after theatment. on the water bath
with 10 per cent HC1.
tion at the refiux condenser for six hours. After driving off the
HC1 on the water bath, under the electric fan, with the help of
alcohol, a crystalline residue was obtained which no longer. gave
the biuret reaction, but which still gave the Pauly reaction with
great intensity. This residue, after the usual preliminary treat-.
ment, was extracted with pure, dry chloroform. The chloro-
form residue weighed 0.004 gram, was partly crystalline, and
contained only a mere trace of fatty material. It gave the Pauly
reaction with great intensity, and the injection of a minute por-
tion of it causeda prompt and noticeable fall in arterial pressure.
A second extraction with chloroform was made, after again moist-
ening the alkaline powder with water and again drying in vacuo.
The two residues were used up in an unsuccessful attempt to
prepare the crystaffine picrate of histamine in a pure condition, in
weighable amounts. We can only say that our results show that
the hydrolysis of the 0.225 pressor phosphate yielded only a few
milligrams (probably 2 to 3 mgs.) of histamine. After the second
extraction with chloroform, the dry alkaline powder was extracted
with 95 per cent alcohol on the water bath, the alcohol removed
from the extract and the residue treated with 25 per cent H2SO4
in a sealed tube for two hours, at 100#{176}to 110#{176}C. This second
hydrolysis yielded only a little more histamine. The Pauly
reaction of the fraction which was insoluble in chloroform and
which was here taken up in alcohol, remained unaltered, show-
ing that we are dealing here with a nucleus which is quite as
resistant as imidazole.
As in the case of ordinary pituitary extracts, so here also, the
employment of alcohol, after the extract has been exhausted
with chloroform, removes a substance which gives the Pauly
reaction. This substance, as we have found, lowers the arterial
pressure and stimulates the uterus. This is the substance which
we have provisionally named B, and which we have shown to
be present in acid-treated infundibular extracts. It was ob-
tained, as in all other cases, in larger amount than histamine.
An accident prevented a quantitative determination of it in
terms of histamine.
PRESENCE OF HISTAMINE IN PITUITARY GLAND . 389
The above experiments therefore show that the blood-pressure-

raising compound, A, as at present isolated, behaves toward
acids in the same manner as pituitary extracts. On hydrolysis
with HC1, the phosphate of A is decomposed, with the loss of
its blood-pressure-raising property, the destruction of its oxy-
tocic power, with the appearance among the decomposition
products of a very small amount of histamine, C, and of a much
larger amount of the histamine-like substance, B. Further
researches must determine whether these preliminary findings are
really characteristic of A , or whether they pertain to an impurity
that is still attached to A.
One point, however, deserves especial notice. It has been
repeatedly noted above that the salts of A give the pink biuret
reaction (1 : 1000, in 1 cc.), and this fact, together with the lack
of crystallizing power shown by its salts, suggest that A may in
fact be something on the order of an albumose. It cannot,
however, be a substance as complex as the ordinary albumoses
produced by digestive ferments, for the reason that all of the
latter, when hydrolyzed with HC1, yield a very large amount of
material which is soluble in chloroform, as will be shown in
Nagayama’s paper, while equivalent amounts of our substance,
A, yield to chloroform after hydrolysis only a few milligrams of
material (C). The difference in this respect is most striking.
If we are to permit ourselves any suggestions in respect to the
nature of A, it will be to say that the substance is probably a
compound of the nature of a slightly basic, unstable, peptamine,
[differing in these respects from thos which Guggenheim has
prepared synthetically] or of the nature of a relatively simple
but unstable, peptid. In either case, we assume that an imidazol
ring is present. A number of authors (Adams (12), Aldrich (13),
FUhner (14)) have suggested that the oxytocic principle might
be a complex amine, and Barger (15) concludes a survey of the
evidence at hand in 1913 with the words: “Possibly, therefore,
the pituitary active principle is a polypeptid-like derivative of
histidine.” Fenger and Hull (16) in a paper which has just
appeared, conclude “that the uterus contract.ing active principle
of the posterior lobe of the pituitary body does not occur in the
fresh gland in a free or crystalline form, but is linked to, or part

of, some protein complex.” Hot 95 per cent alcohol is stated
by these writers to split the protein complex, yielding an
amorphous, highly active split product which is very hygro-
scopic and very sensitive to desiccation. No blood pressure
tests were made with the split product. Crawford (17) in
FIG. 21. DOG, 7.9 KGM. ETHER
Injection at / of 2 cc. containing 46 mgm. of impure T N A (tetranitroani-

line) compound of the deressor substance, B (histamine-like substance). It is
possible that a little histamine was still present. At + artificial respiration
and massage of the heart were applied. The ether bottle was disconnected at
E, at which point also the float was riding on the manometer and could sink
no further, as is evident in the tracing.
a recent number of this journal, gives a full account of the

literature pertaining to the pressor substance, as also some pre-
liminary experiments of his own in reference to this substance.
No statement is made in regard to the plain-muscle-stimulating
property of his amorphous precipitate which, when freshly dis-
solved in water, gave no Pauly reaction.
We stated above that tetranitro-aniline forms a crystalline

compound with histamine, and also that it readily combines
with B, the histamine-like product, which is so readily obtain-
able from infundibular extracts. We do not wish to close our
paper without giving at least one tracing showing the extra-
ordinary lowering of the blood pressure which follows the intra-
venous injection of an excessive dose of this B-tetranitro-ani-
line compound. The compound was made from an infundibular
extract that had been cleared with colloidal iron. We were sur-
prised to obtain only a comparatively small yield of the pressor
TNA addition product (A) and a relatively large yield of the
depressor addition product (B), and can only conclude that a
large part of A was destroyed in the chemical manipulations
involved, the destruction taking place probably in consequence
of the presence of a little HC1 derived from the colloidal iron.
Figure 21 gives the results of the injection of an impure TNA
preparation of B, which was almost entirely crystaffine in char-
acter, gave the Pauly but not the biuret reaction, and was active
for the guinea-pig’s uterus. The compound is capable we hope,
of being isolated as a chemical individual.
Discussion
We have seen that a very brief exposure to hydrochloric acid

completely destroys the pressor action of pituitary extracts, as
also that of the pressor compounds thus far isolated by us. As
is well known, Schafer and Vincent (18) first showed that pituitary
extracts contain two active substances, one pressor, the other
depressor. We have already raised the qtiestion whether our
treatment with acids causes an increase in the amount of actual
depressor substance normally present in pituitary extracts,
while it at the same time destroys the pressor substance. The
question is probably to be answered in the affirmative, and a
part of the increase, though small in amount, is derivable from
the pressor and oxytocic constituent, A, although this point
awaits further research for its verification.
One difficulty encountered here lies in the fact that the intra-
venous injection of a large dose of the pressor substance has the
curious after-effect of greatly lessening, or entirely abolishing,

the effect of subsequent injections, and we suggest, of possibly
even causing a reversal of action, that is is to say, of actually
inducing a fall in arterial pressure. In many of the injections
with our pressor salts, it was noticed that when the injections
were repeated too rapidly, a slight fall of pressure occurred, with-
out any or only the slightest evidence of a rise of pressure. At
the same time, we have numerous tra4ings which show that a
later injection had practically no effect whatever, although the
preceeding injections (two) caused a very great rise of pressure.
At the time we were inclined to the opinion that these observa-
tions proved that our pressor salts were still contaminated with
the depressor substance. We now feel doubtful of the correct-
ness of this assumption, and incline rather to the supposition
that the uninjured depressor substance can itself, under the
proper experimental conditions (previous injections, age of animal
and stage of anesthesia) prepare the way for a decided fall of
pressure in response to later injecUons. This supposition can,
of course, be put to the proof only after the isolation of A as a
pure chemical individual shall have been accomplished.
We ourselves, and others also, have up to the present time
assumed that the depressor constituent of the infundibulum is
the specific plain-muscle-stimulant of the infundibulum. A
closer examination of the subject convincingly shows, however,
that we have no proof at the resent moment that the depressor
substance or substances of the gland that appear after destruc-
tion of the pressor fraction are any more specific in character
than are the depressor constituents of other tissues. We find
that Guggenheim has also raised this question and suggests,
on the basis of his own studies and the work of other continental
investigators, that the depressor constituents of the pituitary
gland, and of tissue extracts in general, consist of some as yet
unisolated and unidentified proteinogenous amines. Neither
Guggenheim nor any of the authors named by him, have, how-
ever, as far as we can learn, isolated from any animal extract,
even in an impure condition, a proteinogenous amine, compar-
able with the histamine obtained by Abel and Kubota. Credit
must nevertheless be given for the suggestion, which appears to

us to be based on sound deductions.
Aronson, (19) in a paper published in 1913, which has just
come to our knowledge, also thinks it very probable that the
active toxic substance of organ extracts in general is either his-
tamine or a similarly constituted organic base.
Abel and Kubota supposed that the depressor substance, wher-
ever found, consists only of histamine, an opinion which is so far
modified by our present findings, and by those of Nagayama, as
described in the following paper, that it takes the following form:
The depressor material, wherever found, gives the Pauly but
not the biuret reaction, stimulates plain muscle, lowers the blood
pressure, and yields two active substances, B, a histamine-like
substance and C, histamine. Until a B substance shall have
been isolated from the pituitary gland, which is specific to the
infundibulum, which is not a decomposition product of A, the
pressor constituent, and which is a more powerful oxytocic sub-
stance than the B, which is extractable from any and every tissue,
we may hold to the opinion that pituitary B is not a specific sub-
stance or hormone.
Abel and his collaborators have in previous papers given a
partial review of the extensive literature relating to “ motilines,”
“peristaltic hormones,” “d#{233}pressine,” “vaso-dilatin,” etc., and
have given their reasons for believing that the depressor action
and the stimulation of plain muscle supposedly characterizing
one or the other of the above-named “principles,” are in reality
properties of one and the same widely distributed material con-
sisting of the B and C of the present paper. In our earlier studies,
our attention was so completely occupied with the relationship
of the “depressor substance” to histamine that we neglected to
mention the pioneer work of Swale Vincent and his collaborators.
Vincent first emphasized the wide distribution of the blood-
pressure-lowering substances without however noting the power-
ful plain muscle-stimulating power of those substances. rfhe
reader will find in one of his papers (20) of recent years, a review
of his work and that of his collaborators in this field, together
with a critical analysis of some of the latter day opinions in
respect to hormones.
The blood-pressure-raising compound of the infundibulum,

(Oliver and Shafer (21), Howell (22)) has been isolated by us,
. in these preliminary experiments, in a form which does not yet
fulfill the demands that must be made upon a pure chemical
individual. It is nevertheless noteworthy that we have had in
hand salts of a very weak base, which not only cause a marked
rise of blood pressure, but which also, as stated above, have an
oxytocic power which is many times greater than that of his-
tamine. We have named this substance A, and have shown
that when its salts or derivatives, in their present state of purity,
are treated with acids, they promptly lose their pressor action,
but retain a certain minimum of oxytocic power, and give rise
to a small but definite amount of histamine, C, and of a larger
amount of the histamine-like substance, B. We repeat that
there is always a chance that both the Pauly and the biuret
reactions are due to an impurity, and that neither B nor C is a
decomposition product of the real A . Nevertheless, we incline
to the belief that A is the specific constituent, or hormone, if
we may use the term, of the infundibular portion of the pituitary
gland. We have already stated in what respects it differs chem-
ically from the higher polypeptids (albumoses), and have sug-
gested that it may be a relatively simple unstable peptid or pep-
tamine containing an imidazole nucleus and giving the biuret
reaction. The latter reaction calls to mind the work of the chem-
ists of the Farbwerke-Hoechst Company, who claimed to have
isolated four crystalline principles from the infundibulum, three
of which gave both the Pauly and the biuret reactions, while
the fourth gave the Pauly and not the biuret reaction. Abel
and Pincoffs (23) have subjected these claims to a critical analysis,
and the present writers can find no experimental evidence that
speaks for the presence of more than one specific active substance,
the A of this paper. While we believe that the Hoechst chemists
have announced more active principles than can be isolated from
the infundibulum, we are nevertheless glad to admit that great
importance attaches to their insistance on the Pauly and biuret
reactions, as characteristics of their pretended active substances.
Quite aside from the chemical work done by us, which has
inclined us to the belief that A is both pressor and oxytocic in
character and that it fully accounts for these two distinguishing
characteristics of pituitary extracts (as compared with the non-
specific depressor and oxytocic effects of pituitary as of other
organ extracts) we may point out that the following facts also
speak for the existence of a single, rather than of a multiplicity
of active principles.
The pressor action of pituitary extracts is entirely destroyed,
(1) when extracts whose pH = 5 are heated to 100#{176}C.for two
hours ; (2) by tryptic digestion (Dale, Dudley) ; (3) by boiling
with 0.5 to 1 per cent HCl for half an hour, or by the briefest
treatment on the water bath with weak HC1; (4) by contact with
2 N NaOH for two hours at room temperature (Guggenheim).
The oxytocic strength of an extract is also rapidly reduced to
one-fifth or less of its original strength by (1) [Adams], (2) and
(3) of the preceding paragraph. In our nomenclature, A , the
active oxytocic and pressor constituent, is destroyed, and. what
is left is the B and C of our paper, augmented by so much of B
and C as is derivable from A .(4) According to Guggenheim,
2 N NaOH destroys both the pressor and the oxytocic constituents
completely.
That all of the agencies named should similarly and simul-
taneously alter both activities of pituitary extracts, speaks for
the unitary hypothesis rather than for dualism. Guggenheim,
indeed, maintained in 1914, that the sensitiveness of “pitu-
glandol” to alkali is alone sufficient to prove that both activities
are properties of one and the same substance. Adams has shown
that when the principle which affects the uterus is destroyed by
boiling (pH = 5), the destruction proceeds in a manner character-
istic of a single substance which is decomposing according to the
law for a monomolecular reaction. This author did not concern
himself with the pressor activity of his boiled extracts, but sug-
gests “the possibility that the substance measured (i.e., the
oxytocic substance) may not be concerned in the characteristic
pressor effect of pituitary solution.” We have stated in the
body of our paper that our pressor salts had an extraordinary
TEE JOUR. OF PHARM. AND EXPER. THERAP.. VOL. XV, NO. 4

396 JOHN .r. ABEL AND T. NAGAYAMA
oxytocic power, and we can hardly believe that their great

activity for the uterus was due merely to an impurity.
If it is argued that the above named four agencies can as easily
destroy two independent substances, one pressor and the other a
plain muscle stimulant, as one substance which possesses two
separate activities (pressor and oxytocic), one may reply with
Guggenheim (24) that in the former case we must assume an
analogous chemical constitution for the two assumed compounds.
The inferences drawn above stand entirely opposed to the
opinion and the findings of Dudley. This author extracts the
pituitary active principles from an aqueous acid solution with
butyl alcohol at a pressure of 10 to 15 mm. Hg, and finds that
the alcohol removes thirty-nine fortieths of the uterine stimu-
lant and 60 per cent of the pressor principle. Dudley’s experi-
ments, as presented in his paper, clearly indicate that two specific
principles are present in the infundibulum. Our own experi-
ments with the compounds which we have prepared, although
these do not yet meet all the requirements of a pure chemical
individual, nevertheless, speak for one specific principle. The
separation of a highly active and specific depressor substance,
which is also a powerful oxytocic agent, would definitely establish
the validity of the dualistic hypothesis.
The questions that arise in connection with histamine call for
a word of comment. it is certainly of interest to find that no
inconsiderable quantities of histamine (these words are permis-
sible in view of the physiological activity of the base) can be
extracted from recently prepared cormnercial pituitary solutions,
which give no external signs of bacterial decomposition, and
which are apparently quite unaltered in respect to their pressor
and oxytocic activities. No doubt the quantity of histamine
increases with the age of an extract, and after a time the quantity
may become large enough to exert some therapeutic action. We
suggest that manufacturers aim to produce a preparation of the
pressor and oxytocic principle which is, as nearly as possible,
free from B and C, and which can be given to the physician in
the form of sterile tablet triturates. A tablet of this kind must,
of course, dissolve in water to a perfectly clear solution.
It is at present not possible to state to what extent the fraction

of histamine which appears in all pituitary extracts on long stand-
ing is derived from A , the pressor principle. Certainly some of
the histamine that makes its appearance when pituitary extracts
are hydrolyzed with acids would appear to arise from some other
source, as is the case when proteid-free extracts of other tissues
are similarly treated. It is evident that only comparative ex-
periments of a quantitative nature can give decisive answers to
these questions. Our experiments with the isolated pressor com-
pounds point, however, to the pressor body A as being in part,
at least, a source of histamine. Of equal or even greater signifi-
cance is the fact (if future investigations prove it to be a fact)
that A yields a second decomposition product, the histamine-
like depressor substance which we have provisionally named B.
. CONCLUSIONS
1. Infundibular extracts that have been made with care from

fresh glands, that is to say, with the avoidance of long boiling
or of long exposure to acids, contain a small, but readily detect-
able amount of histamine.
2. Extracts such as are employed in therapeutics contain
larger quantities of this amine. It was estimated in one experi-
ment, that nearly, if not quite, 2 mgm. of histamine were present
in eighty 1 cc. phials of one of the more powerful commercial
extracts. A 1-cc. phial would therefore contain approximately
0.025 mgm. of this powerful base.
3. The method of preparing the extract, the degree of its
acidity, the time that has elapsed since it was made, and the
method of sterilization employed, are all no doubt concerned as
causes in the appearance of the histamine.
4. Brief treatment of freshly prepared infundibular extracts
with hydrochloric acid on the water bath, or boiling such extracts
at the refiux condenser with low concentrations of. this acid (0.5
per cent HC1 for half an hour) completely abolishes the blood-
pressure-raising action of the extract and causes a marked
increase in the amount of free histamine.
5. Extracts which have been subjected to this treatment with
acids always induce a pronounced fall in the arterial pressure, in
place of the rise of pressure usually observed with normal or

untreated extracts.
6. This fall in the arterial pressure is caused by two substances,
B, a histamine-like substance, which gives the Pauly but not the
biuret reaction, is soluble in alcohol, but insoluble in chloroform
and, C, histamine. About four-fifths of the depressor effect on
the blood pressure appears to be due to B and one-fifth, or a little
more to C, histamine.
7. Neither a brief nor prolonged treatment with hydrochloric
acid, compktely destroys the plain-muscle-stimulating power of
infundibular extracts, in contradistinction to the action of this
treatment on the pressor function, which, as we have seen, is
completely abolished. As shown by tests on the guinea-pig’s
uterus, about 20 per cent or a little less, of plain-muscie-stimu-
lating power is retained.
8. The residual oxytocic activity is due here as in the case of
the depressor effect on the blood pressure, to the two substances
already named, B, the histamine-like substance and C, histamine,
and these two substances are concerned in this residual action
on the uterus in the same ratio as in the blood-pressure-lowering
action, namely in the proportion of 4 to 1, approximately.
9. The histamine-like substance, and histamine as well, as
obtained after mild treatment with acids, are apparently not
specific constituents of the infundibulum. Together they appear
in animal extracts of all kinds and they are easily obtained by
hydrolyzing proteoses as will be shown in the following paper by
Nagayama. This is not to say, however, that they do not vary
in amount in the different animal extracts and preparations
studied in our previous papers. It certainly appears as if in-
fundibular extracts were especially prone to break down and to
yield no inconsiderable amount of histamine. Further quanti-
tative studies must be made to learn what fraction of the his-
tamine-like substance and what fraction of histamine are deriv-
able, if at all, from a specific pressor and oxytocic constituent
of the infundibulum.
10. Preliminary observations in regard to the blood-pressure-
raising constituent of the infundibulum are given. Highly
active pressor salts which are many times more powerful in their
action on the uterus than histamine are described. Preliminary

experiments in regard to tetranitro-aniline as a helpful agent in
the isolation of the pressor and oxytocic constituent of the in-
fundibulum are also described. The action of hydrochloric
acid on these pressor and oxytocic derivatives appears to be quite
like that observed when pituitary extracts are treated with acid.
11 . Tentative opinions are expressed in regard to the chemical
nature of the pressor substance. As far as our own observations
go, they point rather to the conclusion that the infundibulum
contains but one active specific substance, or hormone, and that
this in its uninjured state is not only
a blood-pressure-raising
but also a plain-muscle-stimulating substance. We are here
disregarding for the time other physiological properties of pitul-
tary extracts such, for example, as the diuretic effect. This and
other properties wifi be studied later in connection with a closer
examination of our pressor compounds.
REFERENCES
(1) ABEL, J. J., AND KUBOTA, S. : This Journal, xiii, 243, 1919.
(2) POPIELSKI, L. : Pfluger’s Archiv. f. Physiol., cxxvi, 483, 1909.
(3) JACKSON, D. E., AND MILLS, C. A. : Jour. Lab. an.d Clin. Med., v. 2, 1919.
(4) SPAETII, R. A.: U. S. Hygienic Lab. Bull., no. 115, 39, October, 1918.
(5) DUDLEY, H. W.: This Journal, xiv, 295, 1919.
(6) GUGGENHEIM, M.: Biochem. Ztschr., lxv, 189, 1914.
(7) OSBORNE, W. A., AND VINCENT, S.: Brit. Med. Jour., i, 502, 1900.
(8) ADAIs, H. S.: Jour. Biol. Chem., xxx, 235, 1917.
(9) GUGGENHEIM, M.: Bioch. Ztschr., lxv, 202, 1914.
(10) DALE, H. H.: Bioch. Jour., iv, 427, 1909.
(11) BARGER, GEORGE, AND TUTIN, FRANK: Bioch. Jour., xii, 402, 1918.
(12) ADAS.M8, H. S.: Jour. Biol. Chem., xxx, 235, 1917.
(13) ALDRICH, T. B.: Jour. Am. Chem. Soc., xxxvii, 203, 1915.
(14) FUENER, H.: Munch. med. Woch., lix, 852, 1912.
(15) BAnGER, G.: The Simpler Natural Bases. Longmans, Green & Co., Lon-
don, 1914, P. 110.
(16) FENGER, F., AND HULL, M.: Jour. Biol. Chem., xlii, 153, May, 1920.
(17) CRAWFORD, A. C.: This Journal, xv, 81, March, 1920.
(18) SCHAFER, E. A., AND VINCENT, S.: Jour. Phys., xxv, 87, 1899.
(19) ARONSON, H.: Ben. klm. Woch., 1, 253, 1913.
(20) VINCENT, S., AND SHEEN, W.: Jour. Phys., xxix, 242, 1903.
(21) OLIVER, G., AND SCHAFER, E. A.: Jour. Phys., xviii, 277, 1895.
(22) HOWELL, W. H.: Jour. Exp. Med., iii, 245, 1898.
(23) ABEL, J. J., AND PINCOFFS, M. C.: Proc. NatI. Acd. Sc., iii, 507-517, 1917.
(24) GUGGENHEIM, M.: Bioch. Ztschr., lxxxi, 277, 1917.

From The Pharmacological Laboratory of The Johns Hopkins University

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

From The Pharmacological Laboratory of The Johns Hopkins University

Uploaded by

Copyright:

Available Formats

ON THE PRESENCE OF HISTAMINE IN EXTRACTS OF

THE POSTERIOR LOBE OF THE PITUITARY GLAND

JOHN J. ABEL AND T. NAGAVAMA

From the Pharmacological Laboratory of the Johns Hopkins University

THE JOUR. OF PH.tRM. AND EXPER. THERAP., VOL. XV. NO. 4

While we were engaged in our studies, other investigators ap-

the plain-muscle-stimulating action of such extracts. L. Popiel-

sure raising substance may be no greater and may be even less

METHOD OF SHOWING THE PRESENCE OF HISTAMINE IN “INFUNDIN”

The contents of eighty 1-cc. phials of infundin were evaporated

dried in vacuo over sulphuric acid, if it is occasionally stirred

FIG. 1. CAT, 3.3 KGM., ETHER

At A, 0.5 cc. CHC1, extract, Burroughs, Wellcome and Company’s “Infun-

of chloroform weighed 0.0020 gram, giving 0.0038 as the net

same figure, is shown the comparative effect in the same animal

teinized infundibular extract with hydrochloric acid. This

HISTAMINE IN PITUITARY EXTRACTS OF AMERICAN MANUFACTURE

Results entirely like those just described were obtained with

Experiment 1 The .contents of forty 1-cc. ampoules of Ar-

sideration. The colloidal iron precipitate carried down with it

FIG. 2. CAT, 2.75 KGM., URETHANE ANESTHESIA

At A, 1 cc. of the CHCI, extract obtained from 40 ampoules Armour’s Pituitary

IS THE HISTAMINE, WHICH WE HAVE SHOWN TO BE PRESENT IN

The following experiments force us to give a negative answer

tered, and whih is in close proximity to our laboratory, we ob-

by means of mercuric chloride. The destruction of the pressor

FIG. 3. LARGE CAT, ETHER

At A, injection into the femoral vein of the chloroform extract obtained

in a manner, in which hydrolysis with acids has been reduced to

Under Dr. Fenger’s direction pituitary extracts were prepared in

But even in extracts thus prepared, histamine is present in de-

Experiment 1 . 1 .045 gram dry scales of Fenger’s infundibular

FIG. 4. DOG, 5.6 KGM., ETHER ANESTHESIA

intravenous injection of 0.5 cc. (the total solution being 1 cc.).

to 12.7 grams fresh infundibular material. This quantity of

8.14 grams of the dry extract, representing approximately 103

At ,i’ intravenous injection of one-sixth of the chloroform residue obtained

in an apparatus similar to that described by Dudley. The ex-

of the solution was determined in the usual manner. Figure 5

A later experiment proved that this amumption is correct. In this experi-

ALTERATIONS IN THE PHYSIOLOGICAL PROPERTIES OF INFUN-

DIBULAR EXTRACTS, INDUCED BY MILD HYDROLYSIS WITH

We have seen that infundibular extracts which have been

1 . Changes in the blood-pressure effects of extracts after treatment

Dilute preparations, such as are used in therapeutics, when

action, to appear in its place. This reversal of action may be

FIG. 6. Ema’r OF BO1LING AN INFUNDIBULAR EXTRACT (ARIouR’s PITUITARY

At A, as also at A2 the contents of 1 ampoule (1 cc.) were injected intrave-

TUE JOUR. OF PHARM. AND EXPER. TEERAP., VOL. XV, NO. 4

FIG. 7. EFFECT OF HEATING INFUNDIBULAR EXTRACTS (ARMOUR’S PITUITARY

At A, effect of intravenous injection of 1 cc. extract (5 ampoules 1 cc. each)

coagulahie proteids or of inactive albumoses present in the ex-

The following experiment illustrates the method employed.

We have found only one reference in the literature in regard

FIG. 8. YOUNG DOG, 5.4 KGM.; ANESTHETIC, CHLORETONE

Effect of heating an infundibular extract with hydrochloric acid. At A,

PRESENCE OF HISTAMINE IN PITUITARY GLAND 367

solution which has been completely hydrolyzed with hydro-

2. Depreciation of the oxytocic power of infundibular extracts after

It has been shown that a complete reversal of action on the

mal infundibular extract, both being diluted to the same extent.

infundibular preparation. A remarkable reduction in oxytocic