A CONSIDERATION OF SOME BIOLOGIC TESTS FOR

EPINEPHRIN

R. G. HOSKINS
From the Laboratories of Physiology in the Harvard Medical School and the Starting-
Ohio Medical College

Received for publication, August 22, 1911

In connection with other researches during the past year a series

of investigations has been made of the comparative utility of
certain biologic tests for epinephrin. Of those hitherto described
the frog’s eye, uterus, and intestinal strip methods seemed most
promising and were selected for further investigation.

I. THE FROG’S EYE TEST

The Meltzer-Ehrmann reaction has the disadvantages of lack

of sharpness, and insufficient delicacy for many researches. By
applying the fluid to be tested directly to the iris, however, a
sharpness of reaction was obtained considerably greater than
that following the application of the fluid to the conical surface.
This procedure involves considerable manipulative difficulty but
by the following technique successful results were secured without
undue consumption of time : the frog is decapitated by a quick
cut with strong scissors passing through the cranium near the
angle of the jaw. The eyes are then removed and freed from
adherent tags of tissue. During these manipulations undue pres-
sure upon the eyeball must be avoided. Through the stump of the
optic nerve a fine scissors point is inserted and a small incision
made so that the sclera may be grasped by a pair of forceps;
this grasp is retained while the whole eye is circumsected about
a millimeter posterior to the margin of the iris. The lens now
lies in a cup formed by the anterior half of the eye from which it
must be removed without injury to the pupil. This can be done
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94 BIOLOGIC TESTS FOR EPINEPHRIN

by holding down the eye with a pair of forceps between the tips
of which the lens is grasped by a second pair and gently removed.
The pupils of pairs of eyes so prepared are usually approximately
equal in size7 and one can be used as a control for the other. The
eyes should, of course, be placed in Ringer’s or isotonic saline
solution until ready for use but they should be not kept longer than
fifteen minutes before being used. Receptacles made by cement-
ing segments of glass tube 1 cm. long and 1 cm. in diameter to
ordinary wide microscopic slices are convenient, particularly if
the pupils are to be observed under the microscope. The delicacy
of the test is enhanced by following Schultz” suggestion to keep
the eyes in the dark except during observation.
By this method a mydriasis is secured unmistakably evident
by inspection within five minutes in a dilution of adrenalin,
1 : 5,000,000. By means of camera lucida tracings at a magni-
fication whereby slight differences in the size of the pupils can be
distinguished, it is possible to determine adrenalin in consider-
ably higher dilution ; the upper limit is about one part in a hundred
millions. This dilution requires from ten to twenty minutes’
application. In a trial series of eight determinations of adrenalin,
1: 100,000,000, at a magnification of twenty-five diameters at
the end of fifteen minutes six were positive. The average increase
in diameters was: longitudinal, 11 per cent; transverse, 25 per cent,
greater in cases of the eyes in adrenalin than of control eyes in
isotonic saline solution.
For use in studies of epinephrinemia the pupil reaction is not
sufficiently specific. The mydriatic effect of epinephrin is shared
by pituitary extract,2 parathyroid neucleo-protein and iodothyrin.3
Extract of desiccated thyroid gland also was observed to cause
dilatation.
The chief advantage of the method is that it can be carried
out with a small quantity of fluid.

I Schultz: Bulletin No. 61, Hygienic Laboratory, United States Public Health
and Marine Hospital Service, Washington, 1910.
2 Cramer: Quarterly Journal of Experimental Physiology, 1908, I: 189; Ott
and Scott: New York Medical Journal, 1908, lxxxviii: 1180.
Ott and Scott: Monthly Cyclopedia and Medical Bulletin, 1909: ii: 493.
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R. G. HOSKINS 95

. II. THE UTERUS TEST

Fr#{228}nkel’s statements as to the advantages of his uterus method

were readily confirmed. By his technique reactions to adrenalin
were obtained in dilutions of one to twenty or thirty millions.
There are however, certain material objections to the method.
The uterus is, as Fr#{228}nkel himseff has noted, particularly sensitive
to mechanical stimulation, and even with careful manipulations is
somewhat erratic in its reactions. Its tendency to undergo
spontaneous rhythmic contractions in Ringer’s solution precludes
its use for determination of minimal quantities of epinephrin;
with larger quantities, however, spontaneous and induced con-
tractions are easily differentiated. So far as its use in studies of
epinephrinemia is concerned, a more serious disadvantage is, as
in the case of the frog’s eye, a lack of specificity. Ott has ob-
served that the tendency of adrenal extract to cause contractions
of uterine segments is shared by extracts of mammary, prostate,
parotid, pancreatic and pituitary glands, spleen, thymus, testicle
and ovary, and by spermine and iodothyrin. Moreover, by direct
experiment it was found that rabbit’s blood taken an hour after
epinephrectomy causes a marked contraction. Fr#{228}nkel’s con-
elusion therefore, that any tendency of an experimental blood
to cause contractions in the uterus is due to the presence of
epinephrin is quite unjustified.

III. THE INTESTINE TEST

An attempt to adapt the intestinal strip method6 to the rabbit

led to the discovery that segments of smallintestine serve admirably
as biologic tests for epinephrin. The technique employed was
similar to that of other investigators in this field. Pieces 3 or 4
cm. long are removed from the animal to warm oxygenated Ringer’s
solution in which almost immediately rhythmic contractions begin
and continue for hours. To permit free access of oxygen the

Fr#{228}nkel: Archly f#{252}r

experimentelle Pathologic und Pharmakologie, 1909, lx:
395.
Ott: Journal of Experimental Medicine, 1909, xi: 326.
6 Cannon and de Ia Paz: American Journal of Physiology, 1911, xxviii: 64.
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96 BIOLOGIC TESTS FOR EPINEPHRIN

pieces are suspended by glass hooks through the mesentery.

From time to time as required, segments can be used to obtain
graphic records. The piece is attached to a writing lever and
suspended in Ringer’s solution in a cylindrical container sur-
rounded by water at 37#{176}
C. Oxygen is constantly bubbled into
the cylinder from a supply tube connecting with the base. The
lower end of the tissue is held by a serre-fine attached to a thread
leading out through the supply tube. A second small tube con-
necting with the base of the cylinder and with a piece of rubber
tube at its free end facilitates the changing of fluids in the con-
tamer with a minimal disturbance of the segments. The animal
from which the intestine is taken should not be etherised since the
activity of the segments is thereby much diminished. The ani-
mals can be killed by a blow on the neck or anaesthetized with
urethane per os, 2 grams per kilo. This substance does not share
the depressing effect of ether upon the activity of the intestine.
The intestine with its normal blood supply maintains its vitality
better than if kept in Ringer’s solution and during the long anaes-
thesia following administration of urethane, segments can be
removed as required.
The observations of Ott,7 Magnus, Boruttau,9 Langley,”’
Pal,L1 Elliott,12 and Cannon and de la Paz6 that epinephrin inhib-
its the activities of the intestine was readily confirmed. It was
found indeed that the threshold of this effect is surprisingly low.
In the most favorable instances immediately after removal of
the tissue from the animal, a brief but clear cut diminution of
activity was secure. in dilutions of adrenalin” of from four to five

Ott: Medical Bulletin, 1897, xix: 376.

8 Magnus: Archiv f#{252}r
die gesammte Physiologic, 1905, cviii: 50.
Boruttau: Ibid., 1899, lxxviii: 97.
10 Langley: Journal of Physiology, 1901, xxvii: 249.
“Pal: Wiener medizinische Presse, 1901, xlii: 2023.
12 Elliott: Journal of Physiology, 1905, xxxii: 401.
13 For these experiments a fresh bottle of adrenalin was secured directly from the
Boston agency of Parke, Davis and Company. This brand of epinephrin has been
shown by Schultz’ to test quantitatively true to label. The fact that it caused
reactions in rabbit’s uterus at a dilution of one to twenty or thirty millions as a
minimum indicates that the sample used did not surpass its supposed strength.
 R. G. HOSKINS 97

hundred millions (Cf. Fig. 1). Fig. 2 shows a characteristic rec-

ord of the effect of adrenalin in dilution of 1 : 100,000,000. This
diminution in amplitude of os#{244}illation is usually accompanied by
a decreased tonus. The threshold of this effect varies pan passu
with the vitality of the tissue. In some instances after keeping
the segments three or four hours in Ringer’s solutionreactions were
not secured in higher dilutions than one in five millions. Either
Ringer’s solutionor defibrinated blood may be used as a diluent,
but in the former case, similar blood must of course be used as a
standard. The degree of depression of the tissue depends directly
upon the strength of solution employed. A sensitive tissue can
be brought to a complete stand-still by a dilution of one to twenty
or thirty millions. The sensitiveness of the tissue to epinephrin
diminishes to some extent after each application but a number of
successive records can be secured with one segment. This fact
permits comparing an unknown solution and a known standard,
with some degree of ease. If the order of application of standard
and “unknown” be varied the method is capable of giving val-
uable quantitative results. Segments of cat’s intestine are about
as sensitive as rabbit’s but the amplitude of oscillation being smal-
ler the results are less striking. In cat’s blood, rabbit’s intestine
beats as freely as in that of the rabbit itself. Other bloods were
not tested.
The use of intestinal segments has several distinct advantages.
They are always available and with the possible exception of ar-
tery strips’ are the most sensitive test objects yet described.
The intestine has the advantage over both the uterus and the
artery that it will respond to a number of applications of epine-
phrin, and hence can be used with greater certainty in quantita-
tive work. It has the obvious advantage over the intestinal
strip method that the tissue is subjected to much less manipulative
violence and is consequently in much more sensitive condition.
Both the intestinal methods have the advantage of greater speci-
ficity than either the frog’s eye or the uterus methods; there are
few known substances occurring in blood that cause dilatations

14 Cf. Meyer: Zeitschrift f#{252}rBiologie, 1907, 1: 93.

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98 BIOLOGIC TESTS FOR EPINEPHRIN S

of smooth muscle.’5 It is to be noted, however, that the blood

of an animal dying in asphixia gives the depression reaction with
intestinal segments even if the adrenals have been previously
extirpated. So far as the writer is aware, no other gland shares
with the adrenals their depressing effect upon the gut. Ott and
Scott’6 found that intestinal peristalsis is even increased by iodo-
thyrin, ovary, infundibulin, mammary gland, prostate, thymus,
spleen, parathyroid, pancreas, driedbrainandparotid gland. The
material in each case was extracted and injected in aqueous
solution by jugular. Peristalsis was observed by connecting a
balloon inserted into the intestine with a piston recorder. By
the intestinal segment method these investigators found that
pancreatic extract sometimes showed a tendency to relax the gut
and slow rhythmic contractions, but a repetition of their work has
shown that the relaxing effect is obtained only if the pancreas
has been kept for a time; if the extract is made immediately
after the death of the animal, it increases the contractions. The
depression observed was probably due, therefore, to digestive pro-
ducts. Blood in itself, as Cannon and de la Paz, have noted,
has a marked tendency to increase tonusin the gut, and the depres-
sion reaction, when secured in a blood of unknown composition,
has accordingly the more significance as indicating the presence
of epinephrin. The fact that the segments usually begin activ-
ity immediately after attachment to the writing lever adds not a
little to the satisfaction of the method for routine work. It
seems, therefore, that in point of specificity, promptness and deli-
cacy, the intestinal segments are the most satisfactory test
objects for epinephrin yet described.
I wish to express my appreciation of the permission to make
use of the facilities of the Laboratory of Physiology in the Harvard
Medical School in this research.

“Grutzner: Ergebnisse der hysiologie: 1904, iii (2): p. 66.

1 Ott and Scott: American Medicine, 1911, xvii: 154.
 R. G. HOSKINS 99

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