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Pes OME AR TMENE OF AGRICULTURE.
BUREAU OF PLANT INDUSTRY—BULLETIN NO. 112.

B. T. GALLOWAY, Chief of Bureau.

THE USE OF SUPRARENAL GLANDS IN
THE PHYSIOLOGICAL TESTING
OF DRUG PLANTS

BY

ALBERT C. CRAWFORD,

PHARMACOLOGIST. DRUG-PLANT INVESTIGATIONS.

IssuEp Avucust 10, 1907.

f 7 cor

SSS

WASHINGTON:
GOVERNMENT PRINTING OFFICE.
£9 OG.

BUREAU OF PLANT INDUSTRY.

Pathologist and Physiologist, and Chief of Bureau, Beverly T. Galloway.

Pathologist and Physiologist, and Assistant Chief of Bureau, Albert F. Woods.

Laboratory of Plant Pathology, Erwin F. Smith, Pathologist in Charge.

Investigations of Diseases of Fruits, Merton B. Waite, Pathologist in Charge.

Plant Life History Investigations, Walter T. Swingle, Physiologist in Charge.

Cotton and Tobacco Breeding Investigations, Archibald D. Shamel, Physiologist in Charge.

Corn Investigations, Charles P. Hartley, Physiologist in Charge.

Alkali and Drought Resistant Plant Breeding Investigations, Thomas H. Kearney, Physiologist in Charge.

Soil Bacteriology and Water Purification Investigations, Karl F. Kellerman, Physiologist in Charge.

Bionomic Investigations of Tropical and Subtropical Plants, Orator F. Cook, Bionomist in Charge.

Drug and Poisonous Plant Investigations and Tea Culture Investigations, Rodney H. True, Physiologist
in Charge.

Physical Laboratory, Lyman J. Briggs, Physicist in Charge.

Crop Technology Investigations, Nathan A. Cobb, Expert in Charge.

Taxonomic Investigations, Frederick V. Coville, Botanist in Charge.

Farm Management Investigations, William J. Spilman, Agriculturist in Charge.

Grain Investigations, Mark A. Carleton, Cerealist in Charge.

Arlington Experimental Farm, Lee C. Corbett, Horticulturist in Charge.

Sugar Beet Investigations, Charles O. Townsend, Pathologist in Charge.

Western Agricultural Extension Investigations, Carl S. Scofield, Agriculturist in Charge.

Dry Land Agriculture Investigations, E. Channing Chilcott, Agriculturist in Charge.

Pomological Collections, Gustavus B. Brackett, Pomologist in Charge.

Field Investigations in Pomology, William A. Taylor and G. Harold Powell, Pomologists in Charge.

Experimental Gardens and Grounds, Edward M. Byrnes, Superintendent.

Vegetable Testing Gardens, William W. Tracy, sr., Superintendent.

Seed and Plant Introduction, David Fairchild, Agricultural Explorer in Charge.

Forage Crop Investigations, Charles V. Piper, Agrostologist in Charge.

Seed Laboratory, Edgar Brown, Botanist in Charge.

Grain Standardization, John D. Shanahan, Expert in Charge.

Subtropical Laboratory and Garden, Miami, Fla., Ernst A. Bessey, Pathologist in Charge.

Plant Introduction Garden, Chico, Cal., August Mayer, Expert in Charge.

South Texas Garden, Brownsville, Tex., Edward C. Green, Pomologist in Charge.

Cotton Culture Farms, Seaman A. Knapp, Lake Charles, La., Special Agent in Charge.

Editor, J. E. Rockwell.
Chief Clerk, James E. Jones.

DRUG-PLANT INVESTIGATIONS.

SCIENTIFIC STAFF.

Rodney H. True, Physiologist in Charge,

Albert C. Crawford, Pharmacologist.

W. W. Stockberger, Frank Rabak, A. F. Sievers, Experts.
Alice Henkel, Assistant.

G. Fred Klugh, T. B. Young, 8S, C. Hood, Scientific Assistants,

112
2

siairtaeen rere

LETTER-OF TRANSMITTAL.

U.S. DEPARTMENT OF AGRICULTURE,
BUREAU oF PLant INDUSTRY,
OFFICE OF THE CHIEF,
Washington, D. C., May 24, 1907.

Str: I have the honor to transmit herewith, and to reeommend for
publication as Bulletin No. 112 of the series of this Bureau, the
accompanying technical manuscript entitled ‘“‘The Use of Suprarenal
Glands in the Physiological Testing of Drug Plants.” This paper was
prepared by Dr. Albert C. Crawford, Pharmacologist in Drug-Plant
Investigations, as one of a series of publications on the subject of
drug testing, and has been submitted by Dr. Rodney H. True,
Physiologist in Charge, with a view to its publication:

This paper is preliminary to a consideration of the subject of the
testing of ergot, one of the most valuable and variable of vegetable
drugs. It has been proposed by recent investigators that the most
acceptable means of measuring the activity of ergot is to standardize it
against a known preparation of the active principle of the suprarenal
glands. In order, therefore, to enable us to carry out the ergot test,
the presentation of a means of standardizing the active principle of
suprarenal glands is a preliminary step.

Among the great advance steps taken by medicine in later years,
the attempt to bring medicinal agents to a known and, when possible,
uniform standard of action is one of the most important. Many
drugs are now standardized by chemical methods and can be admin-
istered by the physician in full confidence that his remedy is capable
of exerting the desired degree of action. In the case of others in
which the active principles are not as yet known or in which the
principle will not admit of isolation, testing by physiological means
has come to be recognized as a prime necessity. Since this phase of
drug investigations is still young, a considerable diversity in methods
exists. It is hoped that this paper, which treats of methods of
testing the active principle of suprarenal glands, may contribute
toward greater uniformity in this important matter and make more
generally available than is now the case the essentials of an important
means whereby physiology may serve medicine.

Respectfully,
B. T. GaALLoway,
Chief of Bureau.
Hon. JAMES WILSON,

Secretary of Agriculture.
112

CONTENTS.

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Src) oem gee eH On Be ee | rade nian Suis Parke a ctu oan Ses oo Sa eae

B. P. 1.—294.

THE USE OF SUPRARENAL GLANDS IN THE
PHYSIOLOGICAL TESTING OF
DRUG PLANTS.

INTRODUCTION.

It has long been recognized that many of the important pharma-
copeeal preparations can not be accurately standardized by any
known chemical processes, so that for this purpose physiological
methods have been employed.¢

There have been some inquiries as to where information concerning

this subject could be obtained, and as most of the data occur in
.sources which are not usually accessible it was deemed wise to abstract
this literature and present the methods in some detail. It must be
remembered that as our knowledge increases these tests are sure to
suffer change, a fate which has been the lot of chemical assay proc-
esses. A few years ago gravimetric assay methods were used almost
entirely, but now only when titration methods are unavailable.
These methods have become an. essential to all analytical pharma-
ceutical laboratories, and thousands of dollars are spent every year
in this country for carrying out these tests.
While often mechanically simple in their execution they require
considerable experience to interpret them properly, and for this
reason some of the large drug firms err in employing inexperienced
persons to perform them.

These tests can be used not only with preparations in which the
active principle is little known, but also to control chemical processes
where the active principle is well recognized. Thus the writer has
controlled the assay for atropin by noting the minimum quantity
necessary to cause dilatation of the pupil and standardizing this with
a known solution of atropin.

a Standardization of pharmaceutical preparations. Brit. Med. Jour., vol. 2, p. 583,
1906.

4
4

Cae

8 SUPRARENAL GLANDS.

No one would now think of using any of the aconitins in medicine
without first determining their toxicity, whatever the result of the
chemical assay.

Kobert has called attention to this creased importance of the
pharmacologist and has claimed that these tests should be an essential
for all medico-legal cases, and he has shown that the physiological
test may respond where the chemical one is not sufficiently delicate.¢

Certain simple physiological tests, such as the dilatation of the pupil
with atropin, the production of a tingling sensation in the tongue by
aconite, and the correct tasting of preparations, have been recognized
by the Pharmacopeia, and the testing of diphtheria antitoxin has
at last obtained recognition. ?

It must be remembered that animals respond differently according
to various conditions. Thus Dixon’ has cited the influence of cere-
bral development in animals as influencing the response to cocain, as
follows:

| Dose of co-

Went oe cain per
Grams ot kilo of ani-
Animal. = | mal] neces-
kilo of ‘sary to pro-

* | duce con-

vulsions.

Various other illustrations could be given, so that the animals in
most cases should correspond as nearly as possible in species, sex, age,
and weight.

SUPRARENAL GLANDS.

SEPARATION OF THE ACTIVE PRINCIPLE.

Attention was recently called to the marked blood-pressure-raising
properties of the suprarenal glands in the work of Oliver and Schaefer,‘

@ Kobert, R. Ueber d. Bedeutung d. biologisch. Giftnachweis f. d. gerichtl. Med.
Ber. d. Deutsch. Pharm. Gesells., vol. 13, p. 325, 1903.
Scholtz, K. Wertbestimmung d. Jequiritols u. d. Jequiritol-Heilserums durch
Tierexperimente. Arch. f. Augenheilkunde, vol. 55, p. 209, 1906.
6 Otto, R. Die staatliche Priifung d. Heilsera. Arbeit. a. d. Kénigl. Institut f. Exper.
Therapie z. Frankfurt, 1906.
¢ Dixon, W. 1. Bio-chemical standardization of drugs. Pharm. Jour., vol. 75, p. 156,
1905.
d Oliver, G., and Schaefer, E. A. Physiological effects of extracts of suprarenal cap-
sules. Jour. Physiol., vol. 18, p. 230, 1895.
On tke physiological action of extract of the suprarenal capsules. Proc. Physiol.
Soc., p.1, Jour. Physiol., vol. 16, 1894; Proc. Physiol. Soc., p. ix, Jour. Physiol.,
vol. 17, 1894-95.
112

SEPARATION OF ACTIVE PRINCIPLE. 3

and later by Cybulski, Szymonowicz, Boruttau, and others.? Vul-
pian ? had in 1856 noted the presence in them of certain principles
giving peculiar color reactions, and from this time these color reactions
were believed to be due to the presence of pyrocatechin or a derivative
Orit”

The chemical work—at least that which has been done in this coun-
try—has been carried out mainly from the influence of Professor
Abel’s laboratory. Abel himself isolated a body to which he gave
the name epinephrin, and calculated the empirical formula to be
C,,H,;NO,, but he was compelled to change this to C,,H,,NO, by the
withdrawal of one benzoyl group, and later to C,,H,,NO,4H,O.2

@ Szymonowicz, L. Die Functiond. Nebenniere. Arch. f. Gesam. Physiol., vol. 64,
p- 97, 1896.
Boruttau, H. Erfahrung. tiber d- Nebennieren. Arch. f. Gesam. Physiol., vol. 78,
Page S00
6 Vulpian, A. Note sur quelques reactions propres a la substance des capsules surré-
nales. Comp. Rend. Acad. des Sci., vol. 43, p. 663, 1856.
Cloez, 8.. and Vulpian, A. Note sur l’existence des acides hippurique et choléique
dans les capsules surrénales des animaux herbivores. Comp. Rend. Acad. des
Sci., vol. 45, p. 340, 1857.
¢ Krukenberg, C. F. W. Die farbigen Derivate der Nebennierenchromogene. Arch.
f. Path. Anat., vol. 101, p. 542, 1885.

Brunner, H. Zur Chemie d. Lecithine u. d. Brenzcatechins, Bestandtheile der
Nebennieren. Schweiz. Woch. f. Chem. u. Pharm., vol. 30, p. 121, 1892.
Miihlmann, M. Zur Physiologie der Nebenniere. Deutsch. Med. Woch.,; vol. 22,

; p. 409, 1896.
Fraenkel, S. Beitr. z. Physiol. u. physiol. Chemie d. Nebenniere. Wien. Med.
Blatter, vol. 19, pp. 211, 228, 246, 1896.

@ Abel, J. J., and Crawford, A. C. On the blood-pressure-raising constituent of the

suprarenal capsule. Johns Hopkins Hospital Bul., vol. 8, p. 151, 1897.
Abel, J.-J. Ueber den blutdruckerregenden Bestandtheil d. Nebenniere, das
Epinephrin. Zeits. i. Physiol. Chemie, vol. 28, p. 318, 1899.
Further observations on epinephrin. Johns Hopkins Hospital Bul., vol. 12, p. 80,
1901.
On epinephrin and its compounds. Amer. Jour. Pharm., vol. 75, p. 301, 1903.
Weitere Mittheil. i. d. Epimnephrin. Ber. d. Deutsch. Chem. Gesells., vol. 36,
p- 1839, 1903.
The function of the suprarenal glands. Contrib. to Med. Research, dedicated to
V. C. Vaughan, 1903, p. 188.
On the phenylcarbamic esters of epinephrin. Proc. Amer. Physiol. Soc., 1899,
p- xvii, Amer. Jour. Physiol., vol. 3, 1900.
On a simple method of preparing epinephrin and its compounds. Johns Hopkins
Hospital Bul., vol. 13, p. 29, 1902.
Abel, J. J., and Taveau, R. de M. On the decomposition products of epinephrin
hydrate. Jour. Biol. Chem., vol. 1, p. 1, 1905.

Norre.—Full literature on the suprarenals may be found in Moller, 8., Kritisch-
exper. Beitr. z. Wirkung d. Nebennierenextraktes, Dissert., Berlin, 1906.

472—No. 112—07 2

10 SUPRARENAL GLANDS.

Von Firth? obtained a principle which he named suprarenin and
gave the formula C,H,NO, or C,H,NO,, but later changed this to
Csr NO,.

Takamine? simplified the method of isolation and made it commer-
clally available, giving his preparation the name adrenalin, with the
formula C,,H,,.NOg.

Simultaneous with Takamine’s paper, Aldrich, Abel’s former asso-
ciate, published his results.° His body was evidently much purer than
Takamine’s, as he purified before precipitating, but his method
was not commercially available on account of the necessary puri-
fication from the lead. Aldrich adopted Takamine’s name adrenalin,
although his formula C,H,,NO, differed by CH, from that of Taka-
mine. These two preparations are often confused. Aldrich pointed
out that if the benzoyl group was removed from Abel’s original
formula, the resultant formula was close to his. All three investi-
gators—Abel, Takamine, and Aldrich—were dealing with the same
body, but in varying degrees of purity.

Abel has compared the analytical data furnished by Aldrich and
Takamine, and declares that the analyses do not bear out the empir-
ical formule deduced.? The formula of Aldrich has been corrobo-
rated by Bertrand in France, ¢ and adopted by Pauly, von Firth, Stolz,
Abderhalden, and Bergell, in Germany’ The two latter investiga-
tors used Abel’s purification method, but came to different conclu-

@ Von Fiirth,O. Zur Kenntnissd. brenzkatechinahnlich. Substanz in d. Nebennieren. .

Zeits. {. Physiol. Chemie, vol. 24, p. 142, 1898; vol. 26, p. 15, 1898-99; vol. 29,
p. 105, 1900.
Zur Kenntniss desSuprarenins. Beitr.z. Chem. Phys. u. Path., vol. 1, p. 248, 1902.
Zur Kenntniss des Suprarenins (Adrenalins). Sitz. d. Kaiserl. Akad. d. Wissen.
Wien, Math.-natur. K1., vol. 112, pt. 3, 1903. ;
Zur Kenntniss des Suprarenins (Adrenalins). Monats. f. Chem., vol. 24, pp. 261-
290, 1903.
6Takamine,J. Adrenalin, the active principle of the suprarenal glands. Amer. Jour.
Pharm’) voles/3- pr o2o. 190iIe
The blood-pressure-raising principle of the suprarenal glands. Therap. Gaz., vol.
Zo pree2 L9OI=
¢ Aldrich, T. B. Preliminary report on the active principle of the suprarenal gland.
Amer. Jour. Physiol., vol. 5, p. 457, 1901.
Adrenalin, the active principle of the suprarenal glands. Jour. Amer. Chem.
Soc., vol. 27, p. 1074, 1905.
d Abel, J.J. On epinephrin and its compounds... Amer. Jour. Pharm., vol. 75, p. 309,
1903.
é Bertrand, G. Sur la composition chimique et la formule de l’adrénaline. Comp.
Rend. Acad. d. Sci., vol. 139, p: 502, 1904.
/ Pauly, H. Zur Kenntniss des Adrenalins. Ber. d. Deutsch. Chem. Gesells., vol. 36,
pt. 3, p. 2944, 1903; vol. 37, pt. 2, p. 1388, 1904.

Abderhalden, C.,and Bergell, P. Zur Kenntniss d. Epinephrins. Ber. d. Deutsch.

Chem. Gesells-, vol. 37, pt. 2, p. 2022, 1904.
Ueber d. Epinephrin. Miinch. Med. Woch., vol. 51, p. 1003, 1904.

112

SEPARATION OF ACTIVE PRINCIPLE. 1G

sions from Abel. It was also adopted by Jowett and by Barger and
Ewins in England.* These latter authors are especially emphatic in
support of Aldrich’s formula. These differences in results have not
yet been finally adjusted.2 The difficulty may be due to the fact
that there is in adrenalin a series of chemically similar bodies,’ as it is
well known that blood-pressure-raising properties and the chemical
reactions shown by adrenalin are given by other pyrocatechin
derivatives.‘

The active principle resides largely in the medullary portion of the
suprarenal glands, although the cortex also contains some. Accessory
suprarenal glands which are found in various portions of the abdom-
inal cavity also contain principles having blood-pressure-raising
properties. Blood-pressure-raising principles are also claimed to
be present in other organs, pituitary bodies, ete./

a Jowett, H. A. D. The constitution of epinephrin. Jour. Chem. Soc. Trans., vol. 85,
p- 192, 1904.

Barger, G., and Ewins, A. J. Note on the molecular weight of epinephrin. Chem.

News, vol. 93, p. 90, 1906.
Norsr.—For a review of the relation of the early chemical workers, see Maben, T.,

Adrenalin: the active principle of the suprarenal gland, in Pharm. Jour., 1907,
p. 388. <A reply to Maben is found in Martin, W., Epinephrin or adrenalin, in
Pharm. Jour., 1907, p. 447.

b Aldrich, T. B. Is adrenalin the active principle of the suprarenal gland? Amer.
Jour. Physiol., vol. 7, p. 359, 1902.

c Halle, W. L. Ueber d. Bildung d. Adrenalins im Organismus. Beitr. z. Chem.
Physiol. u. Pathol., vol. 8, p. 277, 1906.

Elliott, T. Action ofadrenalin. Jour. Physiol., vol. 32, p. 462, 1905. Elliott writes
as follows: ‘“‘By bubbling oxygen through adrenalin solution (Parke-Davis’s
0.1% HCl solution, diluted to 1:2,000 and exactly neutralised with Na,CO,) I
obtained a brown liquid which contained no adrenalin, but was fairly potent to
cause vaso-constriction. In this respect it had one-twentieth of the power of
adrenalin. But even4c.c. of the solution, which correspond to an original
content of 2 mgm. adrenalin, and in respect of ability to raise blood pressure
to 0.1 mgm. of adrenalin, caused no movements of iris or nictitating membrane.
In the same test cat 0.03 mgm. adrenalin gave maximal rise of blood pressure and
typical eye movements. Four c. c. of the oxydised solution were then injected
beneath the skin of a rabbit, and caused neither glycosuria nor prostration.”’

Moore B., and Purington, ©. On the chromogen of the suprarenal medulla. Proc.
Amer. Physiol. Soc., 1899, p. xvi; Amer. Jour. Physiol., vol. 3, 1900.

@ Dakin, H.D. On the physiological activity of substances indirectly related to adre-
nalin. Proc. Royal Soc. London, ser. B, vol. 75, p. 498, 1905.

€ Salvioli, I., and Pezzolini, P. Sur le différent mode d’agir des extraits médullaire
et cortical des capsules surrénales. Arch. Ital. de Biol. vol. 37, p. 380, 1902.

f Schmid, J. Ueber d. blutdrucksteigernde Substanz d. Niere. Med. Klinik, 1906,
p. 976.

Livon, C. Sécrétions internes: Glandes hypertensives. Comp. Rend. Hebd. Soc.
de Biol., vol. 50, p. 98, 1898.

Brown, O. H., and Joseph, D. R. Effects of intravenous injections of extracts of
the bone marrow. Amer. Jour. Physiol., vol. 16, p. 110, 1906.

112

1} SUPRARENAL GLANDS.
COLOR TESTS.

Adrenalin is described as a microcrystalline powder which possesses
basic properties and acts as a reducing agent. Strictly speaking, this
body can not be classed with the alkaloids.

Freshly prepared solutions are colorless, but become rose colored
under the influence of light and air, especially in dilute solutions.
After long standing these solutions finally become brown and lose
their activity. This oxidation especially occurs in an alkaline medium,
so that commercial solutions are usually made acid. @

The active principle when in solution gives a green color on the
addition of ferric chlorid. This green passes into a purple and finally
into a carmine on the addition of ammonia. A dilute solution of
iodin turns adrenalin solution to a rose color.

This production of a green color with ferric chlorid has been utilized
by Battelli ® as a method of estimating the amount of active principle
present. The formation of this color is, however, influenced by acidity
and appears badly in very acid solutions ,° but the main difficulty
with this method is that the delicate green is hard to recognize in great
dilution. :

Cameron ‘states that this is a rough method for assaying solutions
of over 1—40,000 and that it fails for brown or solutions more dilute
than 1—40,000.

Von Firth? has also proposed a color test with iron chlorid, sodium
carbonate, and potassium sodium-tartrate, but this does not offer any —
advantages and is subject to the same criticism as other color reactions.

Abelous, Soulié, and Toujan’ have proposed using the 1odin reac-
tion for the same purpose, judging not by the amount of 1odin used
but by the shade of rose color produced. This is ascertained by con-

@Livon, C. Action des vieilles solutions d’adrénaline. Comp. Rend. Hebd. Soc. de
Biol., vol: 1, p. 125, 1904.
Battelli, F. Transformation de l’adrénaline ‘“‘in vitro.’? Comp. Rend. Hebd. Soc.
de Biol., vol. 54, p. 1485, 1902.
6 Battelli, F. Dosage colorimétrique de la substance active des capsules surrénales.
Comp. Rend. Hebd. Soc. de Biol., vol. 54, p. 571, 1902.
¢ Boulud, R., and Fayol. Sur le dosage colorimétrique de Vadrénaline. Comp.
Rend. Hebd. Soc. de Biol., vol. 55, p. 358, 1903.
@ Cameron, 1. D. On the methods of standardising suprarenal preparations. Proc.
Roy. Soc. Edinburgh, vol. 26, p. 157, 1906.
€ Von Firth, O. Zur Kenntniss der brenzcatechinahnlich. Substanz d. Nebennieren.
Zeits. f. Physiol. Chem., vol. 29, p. 115, 1900.
Zur Kenntniss des Suprarenins. Beitr. z. Chem. Physiol. u. Path., vol. 1, p. 244,
1902.
f Abelous, J. E., Soulié, A., and Toujan, G. Dosage colorimétrique par l’iode de
Vadrénaline. Comp. Rend. Hebd. Soc. de Biol., vol. 1, p. 301, 1905.
Sut un procédé de controle des dosages chimique et physiologique de l’adrénaline.
Comp. Rend. Hebd. Soc. de Biol., vol. 60, p. 174, 1906.

112

PRINCIPAL PHYSIOLOGICAL TESTS. 1h

trasting it with a standard solution of adrenalin freshly prepared

_ which has also been treated with iodin. By this method they claim

that the suprarenal glands (sheep) contain 1.47 mg. in every gram,
while they state that Batelli by his method found 1.45 mg.*

Details of this method can be found in English in C. E. Vande-
kleed’s ‘Method for the preparation of the active principle of the
suprarenal gland’’ (Pharmaceutical Era, 1906, vol. 36, p. 478).

PRINCIPAL PHYSIOLOGICAL TESTS.

Toujan,’ one of the originators of the iodin color test, states
that the physiological response is more delicate than the chemical,
although he objects to the former as being too inconstant for vigorous
results. One of the difficulties with color reactions as a quantitative
test is the fact that nothing is known as to whether in decomposition
of the active principle the change in color reactions runs parallel
with the loss in blood-pressure-raising properties; in fact, the evi-
dence rather makes this doubtful. Battelli claims that certain supra-
renal glands on removal from the body lose their reaction to iron
chlorid, but retain their blood-pressure-raising properties.° When
injected into the vein of an animal an extract of these glands or
a solution of the active principle will cause a marked rise in the
general blood pressure associated with a temporary slowing of the
heart, owing to an action on the vagus centers, but if the vagi are
cut or atropin is given before the injection of the suprarenal prep-
aration, instead of the cardiac slowing there will be a marked
acceleration of the heart beats and the maximum blood pressure
effect will be obtained.? This rise in blood pressure is mainly due
to a local action on the small blood vessels, or rather on the sym-
pathetic nerve terminals in their walls. In fact, Elliott considers
that a ‘‘positive reaction with adrenalin is a trustworthy proof of
the existence and nature of sympathetic nerves in any organ.”’

@Compare Battelli, F., and Ornstein, 8S. La suppléance des capsules surrénales au
point de vue de leur richesse en adrenaline. Comp. Rend. Hebd. Soc. de
Biol., vol. 61, p. 677, 1906.

b Toujan, G. Recherches expér. sur l’adrénaline. These, Toulouse, 1905, p. 40.

¢Ornstein, S. Suppléance des capsules surrénales. These, Geneve, 1906, p. 12.

d Elliott, T. R. Action of adrenalin. Jour. Physiol., vol. 32, p. 447, 1905.

Mathieu, X. Action de l’adrénaline sur le coeur. Jour. de Physicl. et de Path.
Gen., vol. 6, p. 435, 1904.

Kahn, R. H. Beob. itiber d. Wirkung d. Nebennierenextractes. Arch. f. Anat. u.
Physiol., Physiol. Abtheil., 1903, p. 522.

Plumier, L. Action de l’adrénaline sur le circulation cardiopulmonaire. Jour. de
Physiol. et de Path. Gen., vol. 6, p. 655, 1904.

é Brodie, T. G., and Dixon, W. E. Contributions to the physiology of the lungs.
Jour. Physiol., vol. 30, p. 476, 1904.

See also von Frey. Beitr. z. Kenntniss d. Adrenalinwirkung. Sitzb. d. Phys. Med.
Gesells. z. Wurzburg, 1905, p. 43:50.

112

14 SUPRARENAL GLANDS.

Besides this action on the sympathetic nerve terminals there is
some action on the heart itself.¢

ACTION ON THE EYE.

The action on the small blood vessels is well shown by dropping
a dilute solution into the conjunctival sac of an animal, when the
conjunctiva becomes pale and bloodless and the pupil dilates.’
This action on the conjunctiva occurs even after the use of solutions
as dilute as 1-120,000. The action on the pupil of the excised frog
eye has been advocated by Ehrmann” as a method for determining
the amount of active principle present in unknown solutions, but
the objection to this method is the difficulty in exactly measuring the
size of the pupil and the uncertainty as to absorption through
the eye membranes. In Ehrmann’s hands, 0.000025 mg. could be
thus determined with certainty, although 0.0000001 gram produced
a distinct dilatation, while 0.00000005 gram was inactive. Cameron’s
results with this method were unsatisfactory. |

ACTION ON ANIMALS.

A second method of showing this vaso-constrictor action is by per-
fusion of the blood vessels in frogs. Lawen? has obtained a response
showing a decided constriction of the vessels with 0.2 per million
adrenalin, but the method is tedious and frogs vary much in their
response. Cameron obtained a feeble reaction with 0.1 per million. |
Details of this method may be found in English inCameron’s paper.
Meyer® placed sections of the beef subclavian arteries in Ringer’s
solution with the addition of adrenalin. He found that most of them
responded with under 1-100,000,000, but not all. With increasing
amounts of adrenalin the contraction was greater—usually 1-50,000

a Gottheb, R. Ueber d. Wirkung d. Nebennierenextracte auf Herz u. Blutdruck.
Arch. tahxp. Path vokrssp..99, leo
b Meltzer, S. J., and Auer, K. M. Ueber d. Einfluss d. Nebennierenextraktes auf d.
Pupille d. Frosches. Cent. f. Physiol., vol. 18, p. 317, 1904.
Kahn, R. H. Ueber d. Beeinflussung d. Augendruckes durch Extrakte chromaffi-
nen Gewebes (Adrenalin). Zent. f. Physiol., vol. 20, p. 33, 1906.
Lewandowsky, M. Ueber d. Wirk. d. Nebennierenextractes auf d. glat. Muskeln,
im besond. des Auges. Arch. f. Anat. u. Phys., Physiol. Abtheil., 1899, p. 360.
¢ Ehrmann, R. Ueber eine physiol. Werthbestimmung des Adrenalins. Arch. f.
Exper. Path. u. Pharmakol., vol. 53, p. 97, 1905.
Zur Physiol. u. experiment. Path. der Adrenalinsekretion. Arch. f. Exp.
Path. u. Pharmakol., vol. 55, p. 39, 1906.
Ueber die Wirkung des Adrenalins auf die Hautdrtisensekretion des Frosches.
Arch. f. Exp. Path: uu. Pharmakol:, vol) 53s pear, 1905:
d@dLawen, A. Quantitative Untersuchungen iiber d. Gefisswirkung von Suprarenin.
Arch. f. Exp. Path. u. Pharmakol., vol. 51, p. 422, 1904.
€Meyer,O.B. Ueber einige Eigenschaft. d. Gefiissmuskulatur. Zeits. f. Biol., vol.
48, p. 365, 1906.

112

PRINCIPAL PHYSIOLOGICAL TESTS. 1

gave the maximum, although some gave the maximum contraction
with 1-100,000. This method has not yet been controlled as a quan-
titative Deeedure by any other observer, but deserves investigation.

Various synthetic adrenalins have Peed made, but while many give
the chemical reactions of this body yet they do not have its physio-
logical action.* Others have almost the same blood-pressure-raising
properties as adrenalin. Thus the compound made by Dakin®
caused a definite rise in blood pressure in rabbits with cut vagi, when
injected in doses of 0.000001 gram.

The field of usefulness for the suprarenal glands in therapy is
increasing,” but these preparations are being used almost with reck-
lessness. Reports of toxic symptoms and secondary hemorrhage
following their use are now appearing.?

In the case of animals it has been shown that the repeated intra-
venous injections of small doses of adrenalin will cause changes in the
heart muscle (myocarditis)* and degeneration of the arterial walls
resembling, if not identical with, arterio-sclerosis.

D’Amato’ has made the interesting observation that arterial
degeneration occurs from repeated use of small doses of suprarenal

aBarger, G., and Jowett, H. A. D. Synthesis of substances allied to epinephrin.
Jour. Chem. Soc. Trans., vol. 87, pt. 2, p. 967, 1905.
Stolz, F. Ueber Adrenalin und Alkylaminoacetobrenzcatechin. Ber. d. Deutsch.
Chem. Gesells., vol. 37, pt. 4, p. 4149, 1905.
Friedman, E. Konstitution des Adrenalins. Beitr. z. Chem. Physiol. u. Path.,
vol. 8, p. 95, 1906.
Loewi, O., and Meyer, H. Ueber d. Wirkung synthetischer dem Adrenalin ver-
wandter Stoffe. Arch. f. Exp. Path. u. Pharmakol., vol. 53, p. 213, 1905.
6 Dakin, H. D. Synthesis of a substance allied to adrenalin. Proc. Roy. Soc. Lon-
fons ser. B, vol. 76, p. 491, 1905.
On the ihe siological activity of substances indirectly related to adrenalin. Proc.
Roy. Soc. London, ser. B, vol. 76, p. 498, 1905.
¢ Kreuzfuchs, S. Einige Erfahrung. tiber innere Adrenalindarreichung. Wien. Med.
Presse, vol. 47, p. 922, 1906.
Oppenheim, R., and Loeper, M. La médication surrénale. Paris, 1904.
d Burnett, C. H. Results of a mistake in putting up a prescription for adrenalin
chloride to be used as a nasal spray. Internat. Clinic, vol. 4, p. 25, 1902.
Roberts, L. M. Antidote for suprarenal preparations. Jour. Amer. Med. Assoc.,
vol. 47, p. 2159, 1906.
Potts, B. H. Danger from the careless use of the alkaloid of the suprarenal gland.
Jour.-Amer. Med. Assoc., vol. 47, p. 1188, 1906.
ePearce,R.M. Experimental myocarditis: A study of the histological changes follow-
ing intravenous iniections of adrenalin. Jour. Exper. Med., vol. 8, p. 400,
1906.
*D’Amato, L. Weitere Untersuch. iiber d. von den Nebennierenextrakten bewirk-
ten Verinderungen d. Blutgefiisse. Berl. Klin. Woch., 1906, pp. 1100, 1131.
Elliott, T. R., and Durham, H. E. Onsubcutaneous injections of adrenalin. Jour.
Physiol., vol. 34, p. 498, 1906.

Norr.—Data on the effects on the urinary secretion can be found in‘Bardier, E.,
and Frenkel, H., Action de ]’extrait capsulaire sur la diurése et la circulation
rénale, Jour. de Physiol. et de Path. Gén., vol. 1, p. 950, 1899.

112

16 SUPRARENAL GLANDS.

preparations by mouth, which by this method of administration fail
to produce any rise in blood pressure. Metabolic changes, shown by
the presence of glycosuria, and histological changes, especially in the
involuntary muscle fibers of the stomach and intestine, and changes
in the liver cells have been noted in experimental work on animals,@
so that it becomes imperative to exercise more care in the use of these
preparations and to determine accurately the amount of the active
principle used.

MEASUREMENT OF THE RISE OF BLOOD PRESSURE IN HIGHER ANIMALS.

At present the most satisfactory method of standardizing these
preparations is by measuring the actual rise in blood pressure which
follows the intravenous injection into animals of definite amounts
of a solution of the pure active principle and comparing this rise
with that produced by the same amount of the solution to be tested.
The method is based on the fact that in the same animal the same
amount of the active principle will produce the same rise in blood
pressure, provided the conditions are unaltered.2 The vagi nerves
should first be cut or atropin injected to secure the full pressor
effects of the injection. These measurements are usually made by

_ @Blum, F. Nebennierendiabetes. Deutsch. Arch. Klin. Med., vol. 71, p. 146, 1901.
~ Loeper, M.,and Crouzon,O. L’action de Vadrénaline sur le sang. Arch. d. Méd.
Expér., vol. 16, p. 83, 1904.
Paton, D.N. Effect of adrenalin on sugar and nitrogen excretion in the urine of birds.
Jour. Physiol., vol. 32, p. 59, 1905.
Citron, J. Ueber d. durch Suprarenin experiment. erzeugt. Veranderungen. Zeits.
f. Exper. Path. u. Ther., vol. 1, p. 649, 1905.
Drummond, W. B. Histological changes produced by the injection of adrenalin
chloride. Jour. Physiol., vol. 31, p. 81, 1904.
Erb, W. Exper. u. histol. Studien tiber Arterienerkrankung nach Adrenalininjec-
tionen. Arch. f. Exp. Path. u. Pharmakol., vol. 53, p. 173, 1905.
Herter, C. A., and Richards, A. N. Note on the glycosuria following experimental
injections of adrenalin. Med. News, vol. 80, p. 201, 1902.
Wolownik, B. Exper. Untersuch. tber d. Adrenalin. Arch. fi. Path. Anat., vol. 180,
p- 225, 1905.
Papadia,G. Arteriosclerosi da adrenalina. Rev. di Pat. Nerv., vol. 11, p. 113, 1906.
[Contains bibliography. |
Biland,J. Ueberd. durch Nebennierenpraparate gesetzten Gefass- und Organveran-
derungen. Deutsch. Arch. f. Klin. Med., vol. 87, p. 413, 1906. =
b Houghton, E. M. Pharmacologic assay of preparations of the suprarenal glands.
Amer. Jour. Pharm., vol. 73, p. 531, 1901; National Standard Dispensatory, Hare,
Caspari, and Rusby, 1905, p. 1732.
Pharmacology of the suprarenal gland and a method of assaying its products. Jour.
Amer. Med. Assoc., vol. 38, p. 150, 1902. [A solution of 1-10,000 was used. |
Franz, F. Aus exper. Arbeiten tiber Adrenalin, seine physiol. Werthbestim-
mung u. seine Wirkung. Sammelreferat. Med. Klinik, 1907, p. 99.
Battelli, M. F. Quantité d’adrénaline existant dans les capsules surrénales de
Vhomme. Compt. Rend. Hebd. Soc. de Biol., vol. 54, p. 1205, 1902.

112

PRINCIPAL PHYSIOLOGICAL TESTS. EY

means of the mercury manometer recording on the drum of a
kymograph.

Anmals preferred—Dogs, on account of their greater resisting
powers and their closer resemblance to man in responding to drugs,
are usually preferred for this class of work, although rabbits are very
serviceable. Cats are not so sensitive but are preferred by Elliott.

Principal reference literature.—Details regarding the preparation of
the animal as to the mechanical carrying out of the experiment can be
found in Essentials of Experimental Physiology, by T. G. Brodie, 1898,
p. 168; Klein, Burton-Sanderson, and’ Brunton, Handbook for the
Physiological Laboratory; T. Sollmann, Textbook of Pharmacology,
2d ed., 1906, pt. 3; Pembry, Beddard, Edkins, Hill, McLeod, and
Pembry, Practical Physiology; E. Cyon, Methodik der physiolo-
gischen Experimente und Vivisection; O. Langendorff, Physiolo-
gische Graphik; Edmunds and Cushny, Laboratory Guide in Experi-
mental Pharmacology; Hermann, Experimental Pharmacology; H.
C. Wood, jr., Description of the Methods of Investigating the Action
of Drugs, International Clinic, vol. 4, p. 12, 1902; D’Arsonval,
Gabriel, Chaveau, and Marey, Traité de Physique Biologique; R.
Tigerstedt, Lehrbuch der Physiologie des Kreislaufes.

Data concerning the anatomy of the dog, the rabbit, and the cat
can be found in Ellenberger and Baum, Anatomie des Hundes; W.
Krause, Anatomie des Kaninchens; St. George Mivart, The Cat.

The large Handbuch der experimentellen Pathologie und Phar-
makologie of Heinz deals with results rather than with details of
methods. There are, however, a few points which are not usually
mentioned.

Apparatus.—We have been dissatisfied with the ordinary kymo-
graph driven by a spring, on account of the variation in speed and
the necessity fer frequent winding. In the case of the expensive
Hirthle machine this objection does not hold. For this reason the
writer has used an electric kymograph—the one described by him in
American Medicine, 1904, volume 8, page 405. This kymograph can
be easily made in most machine shops, as the wheels are stock cut.
The machine requires very little attention and the paper winds regu-
larly and does not sag. The kymograph made by Blix® also over-
comes many of the usual difficulties. The speed of the kymograph
should be kept uniform and should be noted.

The time markers which have given the most satisfactory results
are those described by Marvin? under the name ‘‘Magnet for record-
ing sunshine and rainfall.’ This by shght modification can be made

aBlix,M. Neue Registrirapparate. Arch. f. Gesam. Physiol.. vol. 90, p. 405, 1902.
bMarvin, C.F. Anemometry. U.S. Dept. Agr., Weather Bureau, Circ. D, Instru-
aent Div., 2d ed., 1900, p. 47.

112

18 UPRARENAL GLANDS.

to write in a vertical position, and it records the seconds in steplike
groups so that they can be counted off at a glance. .

A very convenient manometer is the ordinary one of glass made
into the form of a U. The internal diameter should be as near as pos-
sible to 4 mm., as at this width the least error occurs. To the hori-
zontal portion of the tube a small right-angular tube is sealed, and
this is connected with the pressure bottle. At the end of the hori-
zontal portion a small flexible lead tube is connected by means of
thick-walled rubber tubing, and the opposite end of the lead tube
connects with a wash-out cannula.

The most convenient form of wash-out cannula is the one which has
been used in Professor Howell’s laboratory for ten or fifteen years and
consists of a metallic T tube. The long arm of the T is divided in its
lower half by a longitudinal partition, aie separates this part of the
tube into two sections, one continuing on through the full length, the
other communicating ith the side arm of the T.2. When this lbs
is inserted into the glass cannula and connected with the carotid artery
a continuous stream of fluid will wash out any clots which may form.

In recording respiration the writer uses, when necessary, the chest
tambour connected with an ordinary Marey tambour, writing directly
upon the drum of the kymograph. The secret of abheinnte good
respiratory tracings lies in the use of extremely thin rari Der. For
this purpose rubber about as thin as tissue paper is best. For accu-
racy in reading pressures, a running base line is used. In commercial |
work where it is necessary to save time, the paper is further divided
by a series of equidistant lines running parallel to the base line to
facilitate reading off measurements. One adjusted to run at the level
of normal pressure is especially desirable for rapid work.

As to the respiration apparatus, at present the most suitable one
seems to be that of Meyer* because of the alternate force and suction
pumps, but that of Hoyt,’ which is merely a force pump, is very
serviceable.

Preparation of animals for testing.—The animal should be carefully
anzsthetized both for humanitarian reasons and to render it motion-
less, as any motion on the part of the animal would vitiate the results.
If necessary, curare with morphin may be used to secure immobility;
then artificial respiration will be required. While the use of chlore-

a@Schaefer, E. A. Textbook of Physiology, vol. 2, p. 78.

0 Rcnmaria. L. Exper. Pharmacology, 1883, p. 101.

¢Meyer, H. Zwei neue Laboratoriumsapparate. Arch. f. Exp. Path. u. Pharmakol.,
vol. 47, p. 426, 1902.

dHoyt, J. T. Apparatus for artificial respiration. Jour. Physiol., vol. 27, p. 48,
1901.

112

PRINCIPAL PHYSIOLOGICAL TESTS. 19

tone has disadvantages, it is generally used for this class of work in
dogs where the animal is not allowed to recover (0.2 gram per kilo is
usually given in alcoholic solution after the hypodermic injection of
morphin, or chloretone may be used alone). This does away with the
use of a volatile anesthetic.¢ After the use of large doses of chlore-
tone alone the writer noted that while the animal may be merely drowsy
after one hour, a few whiffs of ether will secure good anesthesia, which
will continue. Sometimes as anesthesia sets in the animal ceases to
breathe, but a few strokes of the respiration machine will soon restore
the breathing, which will then continue of itself. In the case of rab-
bits, 3 grams of urethane may be given in solution by mouth, or 1.50
erams subcutaneously,? while cats should receive 1.25 grams per kilo.
With this anesthetic and kept on a warm table, the blood pressure in
cats will remain at 130-140 mm. for hours.° Urethane is not suitable
for dogs. Before connecting the animal to the kymograph it should
be weighed, so that the necessary dose of adrenalin can be calculated.

Results obtained by various investigators.—Cameron * has shown that
the smallest dose which gives a ‘‘definite and invariable rise”’ in blood
pressure in rabbits weighing about 2,000 grams is 0.00062 mg., or
0.0003 mg. per kilo, or 0.00000031 gram per kilogram; that is, 0.5 c. ce.
of a 0.125 per cent solution of an adrenalin solution of 1 to 1,000,
or 0.5 ¢. c. of a solution of 1 to 800,000 (1 ¢. c. of a 1-1,000 solution
diluted to 800 ¢. ¢.).

In cats, which are more resistant than rabbits, Ehrmann’ found
that the intravenous injection of 0.1 mg. gave a rise which was just
appreciable.

Abel’s epinephrin bisulphate in 0.00013 gram caused a rise of
14 mm. Hg in a small dog with cut vagi/ and von Fiirth’s suprarenin
iron compound in doses of 0.000017 gram per kilo (dog) caused the
maximum rise, while 0.000075 gram caused a marked rise, 24 mm. Hg
in rabbits (2 kilos).9

aImpens, E. Chlorétone. Arch. Internat. de Pharmacodynamie, vol. 8, p.77, 1901.
Houghton, E. M., and Aldrich, T. B. Chloretone. Jour. Amer. Med. Assoc., vol.

Sos De Gl (el S00.

b Schmiedeberg, O. Ueber d. pharmakol. Wirkungen u. d. therap. Anwend. einiger
Carbaminsaure-ester. Arch. f. Exper. Path., vol. 20, p. 203, 1886.

e Elliott, T.R. Actionofadrenalin. Jour. Physiol., vol. 32, p. 449, 1900.

d Cameron, I. D. On the methods of standardising suprarenal preparations. Proc.
Roy. Soc. Edinburgh, vol. 26, p. 161, 1905.

e Ehrmann, R. Ueber eine physiol. Werthbestimmung des Adrenalins. Arch. f.
Exp. Path. u. Pharmakol., vol. 53, p. 106, 1905. [Weight of animal not given. |

f Abel, J. J. Ueber d. blutdruckerregenden Bestandtheil der Nebenniere, das
Epinephrin. Zeits. f. Physiol. Chem., vol. 28, p. 339, 1899.

gVon Firth, O. Zur Kenntniss d. brenzkatechinihnlich. Substanz d. Nebennieren.
Zeits. f. Physiol. Chem., vol. 29, pp. 115 and 112, 1900.

112

20 SUPRARENAL GLANDS.

Epinephrin sulphate intravenously injected into atropinized dogs
previously narcotized with morphin and ether produced a rise as
follows: 3

0.083 millionth of a gram per kilo body weight, 5 mm. Hg.
0.23 millionth of a gram per kilo body weight, 7 mm. Hg.
0.49 millionth of a gram per kilo body weight, 15 mm. Hg.

0.69 millionth of a gram per kilo body weight, 20 mm. Hg.
1.7 millionth of a gram per kilo body weight, 24 mm. Hg.
5.7 milhonth of a gram per kilo body weight, 66 mm. Hg.

Unfortunately, in these experiments the interval of time between
the injections is not specified.¢

In a dog weighing 4.5 kilos with a carotid pressure of 140 mm. H¢
the intravenous injection of 0.02 mg. of adrenalin was followed by a
rise of 46 mm. After the injection of atropin the same dose caused
a rise of 50 mm.?

The normal blood pressure in rabbits as measured from the carotid
artery is about 90 mm. Hg, but varies from 80 to 100.

Josué * noted that while frequently repeated intravenous injections
caused a permanent rise in blood pressure, yet these animals still
gave the characteristic abrupt rise in blood pressure on fresh injec-
tions—in other words, there was no immunity—and Elliott and
Durham in three experiments with cats failed to find the presence
of an anti-body.¢

The normal blood pressure for dogs, as measured from the carotid
artery, is 140 mm. and in rabbits 70 mm. of mercury,’ while the
maximum pressure according to Elliott is +300 mm. for dogs,
180 mm. for rabbits, and 240 mm. for cats.

Oliver and Schaefer’ have claimed that 0.015 gram per kilo of the
fresh glands would cause the maximum rise in dogs (10 kilos).

In non-narcotized and non-curarized rabbits Pruszynski9 noted a
rise of 90 mm. Hg with 0.000047 gram per kilo of adrenalin. With

a Hunt, R. On the effects of intravenous injections of minimal doses of epinephrin
sulphate upon the arterial blood pressure. Proc. Amer. Physiol. Soc., p. vii;
Amer. Jour. Physiol., vol. 5, 1901.

6 Baylac, J. Recherches expér. sur les propriétés physiol. et toxiques de l’adrénaline.
Arch. Med. de Toulouse, vol. 12, p. 265, 1905.

c Josué, O. La pression artérielle chez le lapin a4 la suites d’injections répétées
d’adrénaline dans les veines. Comp. Rend. Hebd. Soc. de Biol., vol. 59, pt. 2,
p. 319, 1905.

d Elliott, T. R., and Durham, H. E. On subcutaneous injections of adrenalin.
Jour. Physiol., vol. 34, p. 490, 1906.

é Langendorff, O. Physiolog. Graphik., p. 204.

J Oliver, G., and Schaefer, E. A. Physiological effects of extracts of suprarenal cap-
sules. Jour. Physiol., vol. 18, p. 235, 1895.

g Pruszynski, J. Influence of adrenalin on the circulatory system. Medicine, vol.
Li p. 924, 1905.

112

PRINCIPAL PHYSIOLOGICAL TESTS. |

an intravenous injection of 1 c. c. of a 1-100,000 solution, Takamine

obtained a rise of 30 mm. Hg in the case of a dog weighing 8 kilos,

while in a dog weighing 15.5 kilos 0.000016 gram induced a rise
Ol Semin. ©

With a similar injection of 1 ¢. c. of a 1-10,000 solution, John”
obtained a rise of 50 mm., registered by a Hirthle manometer, in a
dog weighing 35 kilos when the vagi were not cut. These results
remained constant on repeating. If one vagus nerve was cut the
pressure rose in one case 75 mm., and on repeating rose 60 mm. If
both nerves were cut the pressure rose much higher—about 150 mm.

According to Toujan ° 0.001 mg. of adrenalin will cause an appre-
clable rise in atropinized dogs weighing 10 kilos, a sensibility of
1—-1,000,000, and Sollmann reports a rise of 14 mm. after the injec-
tion of 0.001 mg. per kilo. Carnot and Josserand obtained a maxi-
mum rise of 17.5 cm. Hg in a dog weighing 15 kilos with 0.000016
gram per kilo of adrenalin.4

A dog weighing 11.29 kilos anesthetized with 0.3 gram of morphin
intravenously after an injection into the vein of 0.001 gram of adrenalin
gave a rise from 38 mm. normal to 128 mm. Hg. A second injec-
tion gave a rise from 36 mm. to 132 mm.¢ In Dupuis and Van den
Eeckhart’s hands an intravenuos injection of 1 c. c. of a 1-4,000 solu-
tion of adrenalin increased the blood pressure in the femoral artery
4 cm. Hg in a dog weighing 23 kilos, under morphin and atropin.
This rise persisted 14 minutes. Onec. c. of a 1-1,000 solution produced
arise of 14 cm. This returned to normal in five minutes. The rise
was followed by a hypotension of 1 cm. below normal.’

Sudden death may at times occur in dogs even from 0.12 mg. of
adrenalin, although the rise in blood pressure may have been only
small (82 mm.). In these cases the action seems to fall directly on
the heart, as the respiration may continue after the heart stops.9

@ Takamine, J. Adrenalin, the active principle of the suprarenal glands. Amer.
Jour. Pharm., vol. 73, p. 530, 1901; Therap. Gaz., vol. 25, p. 223, 1901.

b John, K. Nebennierenpraiparate. Dissert., Leipzig, 1906, p. 17.

¢ Toujan, G. Recherches expér. sur l’adrénaline. Thése, Toulouse, 1905, p. 36.

d Carnot, P., and Josserand, P. Les différences d’action de Vadrénaline sur la pres-
sion sanguine suivant les voies de pénétration. Comp. Rend. Hebd. Soc. de
Biol., vol. 54, p. 1473, 1902.

e Reichert, E. T. Adrenalin, the active principle of adrenal extract. Univ. Pa.
Med. Bul., vol. 14, p. 53, 1901.

f Dupuis and Van den Eeckhart. L’adrénaline. Ann. de Méd. Vét., vol. 52, p. 484,
1903.

Norr.—Other figures may be found in Neujean, V., Contrib. a 1 étude expér. de Padre-
naline, Arch. Internat. de Pharmacodynamie, vol. 13, p. 45, 1904.

g Elliott, T. R. Action of adrenalin. Jour. Physiol., vol. 32, p. 465, 1905.

Livon, ©. Danger du principe actif des capsules surrénales dialysé. Comp. Rend.
Hebd. Soc. de Biol., vol. 54, p. 1501, 1902.

112

LLL DOLL LL LOL ALLL

A SUPRARENAL GLANDS.

In cats under urethane, 0.03 mg. of adrenalin produced the maxi-
mum rise.@

Subcutaneous injections® or administration per os usually pro-
duced no rise in blood pressure. The slight rise seen at times may be
due to the local action on the stomach walls. Intramuscular injections
are, however, followed by some rise,’ and the effects vary with the
mode of injection as to whether it passes through the hepatic circu-
lation or through the muscles, etc.?

Hf the injection of small doses is rapidly repeated the rise in blood
pressure with the same amount of active principle is slightly less but
more persistent.© With medium doses the rise and its duration may be
greater than from the first, but if a proper interval of time elapses
between the injections the second rise will correspond to the first, an
observation which is confirmed by Baylac’ For a dog of 8 kilos, five
minutes’ time is sufficient when using small doses—0.006 to 0.001 mg.9
Josserand says there is no accumulation with 0.000016 mg. per kilo.”

If large doses are used, the pressure may stay high for a long time,
then fall below normal,‘ and serious disturbances with the heart, shown

by pericardial effusions, may occur and vitiate the result.’ Weiss and

Harris* have claimed that adrenalin can still be found in the blood
even when the blood pressure has fallen to normal; thus after inject-
ing 0.0034 gram of adrenalin into a cat weighing 3,000 grams and
waiting thirty minutes, the transfused blood caused a rise of 15 mm.
in a second cat. No control experiments with normal blood seem to

have been made. Elliott, however, says that when the blood pressure —

a Elliott, T. R. Action of adrenalin. Jour. Physiol., vol. 32, p. 448, 1905.

b Reichert, E. T. Adrenalin. Univ. Pa. Med. Bul., vol. 14, p. 51, 1901.

¢ Meltzer, 8S. J., and Auer, J. Ueber d. Resorption aus den Muskeln. Zent. f. Phy-
siol., vol. 18, p. 689, 1904.

@ Carnot, P., and Josserand, P. Des différences d’action de Vadrénaline sur la pres-
sion sanguine suivant les voles de pénétration. Comp. Rend. Hebd. Soc. de
Biol., vol. 54, p. 1472, 1902.

é Toujan, G. Recherches expér. sur l’adrénaline. Thése, Toulouse, 1905, p. 37.

J Baylac, J. Recherches expér. sur le propriétés physiol. et toxique de l’adrénaline.
Arch. Méd. de Toulouse, vol. 12, p. 252, 1905. [Used 10-minute interval for
rabbits. |

g Toujan, G. Recherches expér. sur Vadrénaline. Thése, Toulouse, 1905, p. 38.

h Josserand, P. Contrib. a étude physiol. de l'adrénaline. These, Paris, 1904, p. 33.

Notrre.—See also Gioffredi, C., La distruezione dell’ adrenaline nell’ organismo,
Archiv. di Farmacol. Speriment., vol. 6, pp. 127, 145, 1907.

¢Battelli, F. Transtormation:de l’adrénaline dans Vorganisme. Compt. Rend.
Hebd. Soc. de Biol., vol. 54, p. 1518, 1902.

jElhott, T. R. Action of adrenalin. Jour. Physiol., vol. 32, p. 444, 1905.

k Weiss, O., and Harris, J. Zerstérung des Adrenalins im lebenden Tier. Arch. f.
Gesam. Physiol., vol. 103, p. 510, 1904.

Harris, J. Dissert., Kénigsberg, 1904.
Battelli, F. Transformation de l’adrénaline dans l’organisme. Compt. Rend.
Hebd. Soc. de Biol., vol. 54, p. 1519, 1902.

112

PRINCIPAL PHYSIOLOGICAL TESTS. 23

has returned to normal in the cat, the substance has then disappeared
almost entirely from the blood. Elliott’s table showing the effects
of injecting smaller doses gives the different results obtained:

CAT (VAGI CUT).

Time. Treatment. Results.
11.45. Received 0.18 mg. adrenalin. Blood pressure rose 150 to 220
mm. Hg.

11.48. Received 0.30 mg. adrenalin. Blood pressure rose 130 to 230

mm. Hg.

11.56. Blood contained no demonstrable

adrenalin.

12.05. Received 0.3 mg. adrenalin. Blood pressurerose 145 to 220

mm. Hg.

12.08. Received 0.6 mg. adrenalin. Blood pressure rose 110 to 210

min. Hg.

12.14. Received 0.6 mg. adrenalin. Blood pressure rose 130 to 220

mm. Hg.
12.22. Received 0.6 mg. adrenalin. Blood pressure rose 120 to 206
mm. Hg.

12.26. Blood contained no adrenalin.
12.40. Received 1 mg. adrenalin. Blood pressure rose 110 to 200
| mm. Hg.

12.46. Received | mg. adrenalin. Blood pressure rose 140 to 178

mm. Hg.

12.50. Pupils still dilated. Blood pres-

sure 150 mm. Hg.
1.17. Received 1 mg. adrenalin. Blood pressure rose 95 to 170
mm. Hg.

‘1.21. Received 1 mg. adrenalin. Blood pressure rose 130 to 156
mm. Hg.

1.24. Received | mg. adrenalin. Blood pressure 140; no rise.

1.25. Adrenalin found in blood. Five

c. c. contained 0.02 mg.

1.45. Received | mg. adrenalin. Blood pressure rose 60 to 115
mm. Hg.

1.48. No demonstrable adrenalin.

Control cat showed 0.01 mg. in 5 c. c.

In comparing the strengths of solutions it should be remembered
that because 1 c. c. of a solution gave a rise of 17 mm. Hg, 2 ¢. ¢. will
not necessarily double this rise; in fact, while there is a general
increase with increasing doses up to a certain limit, we are unable to
find any mathematical relationship between the rise in blood pressure
and the dose. Thus Toujan im a dog narcotized with chloralose and
atropinized (legm. per 7 kilos), injected 1 c. c. of a suprarenal extract
containing 0.001-mg. per cubic centimeter and obtained a rise of 17
mm. After five-minute intervals—

. and obtained a rise of 19.5 mm.
. and obtained a rise of 25.0 mm.
.and obtained a rise of 23.0 mm.
. and obtained a rise of 25.0 mm.

Injected 2 c.
Injected 3 c.
Injected 4 c.
Injected 5 c.

(Pl (ea) Tee ater!

112

24 SUPRARENAL GLANDS.

The persistency of the rise gives a better idea as to the intensity of
the action; thus, using a drum which traveled at a rate of 0.28 cm.
per second, after injecting 1 ¢. c. of the solution specified the drum
traveled30mm. before the blood pressure returned to its original level—

After 2 c. c. it traveled 40 mm.

Aiter 3 c. c. it traveled 102 mm.

Aiter 4 c. c. it traveled 130 mm.

After 5 c. c. it traveled 150 mm.

Young dogs are claimed to. be more responsive to adrenalin than
older dogs having the same weight.?

The quantitative work which has been carried on with adrenalin
has been performed with the adrenalin of commerce. This was shown
to contain extraneous matter (phosphates)’, as one would expect
from the method of isolation described by Takamine, so that it does
not seem rational to standardize against a body which is not chem-
ically pure and which the manufacturers themselves do not label as
c. p. The standard used should be the highest grade of active prin-
ciple known. Abel’s latest method, as acknowledged by Pauly‘, or
adrenalin as prepared by Aldrich’s method seems to answer this
demand. This adrenalin (Aldrich’s) can perhaps be obtained from
the manufacturers, but at a higher price. A supply of this high
gerade adrenalin (epinephrin) should be kept on hand in a vacuum
desiccator and preserved in the dark. When the test is to be made,
a few milligrams of this are accurately weighed off and dissolved

in water with a little over the calculated amount of HCl 5p; con-

stantly shaking,’ and made up in a proportion of 1 to 50,000, the
solution placed in a Florence flask and gently heated on the bath
to body temperature (374° C.).. One-half of a cubic centimeter of
this solution would correspond to 0.001 mg. per kilo for a dog of 10
kilos. One-half of a cubic centimeter of this warm solution is then

@Josserand, P. Contrib.41’étude physiol. de ladrénaline. Thése, Paris, 1904, p. 31.
6 Abel, J.J. Onasimple method of preparing epinephrin and itscompounds. Bul.
Johns Hopkins Hospital, vol. 18, p. 30, 1902. Prof. Abel says: “I Judge from
the bulkiness of the phosphomolybdate precipitate that a quantitative experi-
ment would result ina high percentage of impurity. x Commercial adre-
nalinwas * * * purified * * * and more than thirty analyses oi various
fractions have been made, but it has been found impossible to secure uniformity
of composition among the various products.”’
Function of the suprarenal glands. Contributions to Med. Research, dedicated
to V. C. Vaughan, p. 155.
Compare also Gunn, A., and Harrison, E. F. A new characteristic reaction of
adrenaline. Pharm. Jour., 1907, p. 718.
c Abderhalden, E., and Bergell, P. Miinch. Med. Woch., vol. 51, p. 1003, 1904.
@ Takamine says ‘‘100 parts of adrenalin needs nearly 19 parts of hydrochloric acid
in forming a neutral salt.””. (Amer. Jour. Pharm., vol. 73, p. 527, 1901.)

112 “7

PRINCIPAL PHYSIOLOGICAL TESTS. 25

injected by means of a small calibrated syringe and connections into
the saphenous vein. The time of this injection should be registered
and should occupy about five seconds, and should be gently per-
formed. This is then rapidly followed from the same syringe with
1 ¢. c. of the normal salt solution to wash into the circulation any of
the solution that may be in the syringe and connections. This injec-
tion should occupy the same length of time and should always be
noted. It is important that the standard solution be made fresh at
each test, as otherwise it gradually loses its activity. The pupils
should be examined to see if there is any action, as there may be
bodies present in the extract which might neutralize the blood-
pressure-raising action. During the experiment the animal must
be kept warm. Not over 2 ¢. c. of fluid should be injected at any one
time, to avoid the pressor action of the large volume of fluid. The
action of the comparatively large amounts of fluid used in this form
of injection may be overcome by injecting the fluid into the vein
without the use of a syringe. For this injection a pipette of 1 ¢. ¢.
capacity accurately divided into tenth cubic centimeters is closely
connected to a fine, clean, sharp hypodermic needle by means of a
rubber tube, and after gently pushing the needle through the wall of
the vein toward the heart the fluid is let run into the circulation and
the amount controlled by the finger or, better, a burette may be
connected with the cannula and the injection thus made. By this
method it will not be necessary to use sodium chlorid solution to
wash the adrenalin solution into the vein.

The most satisfactory method is to connect the syringe or, prefer-
ably, a burette containing the standard solution with one saphenous
vein and the solution to be tested with the corresponding vein of the
other leg, so that alternate injections of standard and unknown solu-
tion can be made under the same conditions. The writer’s own prefer-
ence is for the burette method.

The maximum rise in blood pressure from this injection, 0.001 mg.
per kilo, which is about 14 to 28 mm. Hg, although this will vary in
various dogs, is now noted, and also the distance traveled by the paper
and the time which elapses before the blood pressure returns to nor-
mal. A line running on a Jevel with the base of the blood pressure
curve will aid in rapid calculation. The method of measuring the
blood pressure can be found in W. Stirling’s Outlines of Practical
Physiology, third edition, 1898, page 305.

Very dilute solutions are purposely used, so that there will be no
accumulation of the active principle in the animal. After five or
six minutes a very dilute solution, one which would approximate in
strength that of the normal, and diluted to the same bulk as the con-
trol, is similarly injected, with all the above precautions, and the

112

26 SUPRARENAL GLANDS.

maximum blood pressure and the time which elapses before the blood
pressure returns to normal is likewise noted and compared with the
standard. At intervals of five or six minutes various amounts of this
fluid are injected, until an amount is injected which gives a rise which
corresponds to the 1 c. c. of the standard solution. This injection
should be repeated several times, changing the order of the injection,
and a’mean taken, and finally the correct solution and the standard
one injected into a fresh dog which has had no fluid injected. By
comparing the strength of the two solutions the actual number of
milligrams present can be determined. Thus, ifin1c.c. of the standard
solution there were 0.01 mg. and this gave a rise of 14 mm. of Hg, and
if 2 c. c. of a similar dilute solution of the unknown gave the same
rise, we would naturally argue that the second solution was one-half
the strength of the first and contained 0.005 mg. per c. ce. The
solutions of the unknown can then be adjusted, so as to use the same
amount of fluid of the standard and the test controlled, reversing
the order of the injections. It is advisable to repeat the injections
several times, checking these results repeatedly.

Elliott” advises against using over 0.03 mg. Dale, using cats
according to Elliott’s method, is said to measure epinephrin solu-
tions within about 5 per cent.?

Cameron,’ struck with the work of Marshall on the antagonism of
the members of the digitalis series with the nitrites, extended these
observations to the antagonism between the nitrites and adrenalin ©
and found that 0.6 mg. Gt> gr.) of nitroglycerin would require
0.0075 mg. of adrenalin to neutralize its vasodilator action in a rabbit
weighing 2,000 grams anesthetized with ether. This method he
considers even more satisfactory than the simple blood pressure
measuring, especially if there is not a reliable standard preparation to
standardize against. One example from Cameron will illustrate.
The minimal effective dose of a 1 per cent solution was 0.5 c.c. The
minimal effective dose of an adrenalin solution had been found to be
0.00062 me. for rabbits of 2,000 grams weight; hence, 0.005 c. c. of the
solution = 0.00062 mg. adrenalin, or 1 ¢.c.=0.12 mg. By the nitrite
method 0.6 mg. is neutralized by 0.7 c. c. of 10 percent; this amount
of nitroglycerin is neutralized by 0.5 c.c. of 1.5 per cent adrenalin
solution (0.0075 mg.); therefore, i c.c.=0.107 mg. This solution was
one-tenth stronger than adrenalin chlorid, 1—-1,000.

aElhott, T. R. Action of adrenalin. Jour. Physiol., vol. 32, p. 448, 1905.

6 Barger, G.,and Ewins, A. J. Note on the molecular weight of epinephrin. Chem.
News, vol. 93, p. 90, 1906.

¢ Cameron, 1. D. On the methods of standardising suprarenal preparations. Proc.
Roy. Soc. Edinburgh, vol. 26, p. 170, 1906.

112

a Lo nearer ear CS i ne,
PRINCIPAL PHYSIOLOGICAL TESTS. DT

Ott and Harris % had tried this antagonistic action of these two
bodies, but did not push the matter as far as Cameron. <A similar
antagonism with cholin had been pointed out by Lohmann.’ After
having determined the amount of active principle on the dog, rabbit,
or cat by the simple blood pressure method, as a check to this method
it may be advisable to inject the amount of the unknown solution cor-
responding to 0.0075 mg. adrenalin mixed with a solution of 0.6 mg.
nitroglycerin into the jugular vein of a rabbit. I do not consider
this additional test necessary, but the results of Cameron are cer-
tainly worth controlling.

Giirber ° pointed out the presence of a depressor body in the supra-
renal extracts. A portion of this action may be due to cholin and a
portion to unknown bodies.? Extracts containing these depressor
bodies are especially toxic. In crude extracts the depressor action
| of these bodies, and also of certain inorganic salts, should be kept in

mind.
ce At the end of the experiment the animal should be killed by inject-
ing chloroform into the veins, so that the most humane person could
find no objection on the score of cruelty.

In the case of a mixture of adrenalin and peptone the action of one
follows that of the other, and it is therefore unsuited for antagonistic
study.°

a Ott, I.,and Harris,§8.B. Physiological action of adrenalin chloride. Therap. Gaz.,
vol. 27, p. 378, 1903.

bLohmann, A. Cholin, die den Blutdruck erniedrigende Substanz der Nebenniere.
Arch. f. Gesam. Physiol., vol. 108, p. 222, 1907.

¢Metzger,L. Zur Kenntniss d. wirksam. Substanzen d. Nebenniere. Dissert., Wiirz-
DUES OT epi.

@ Hunt, R. Further observations on the blood pressure lowering bodies in extracts
of the suprarenal glands. Proc. Amer. Physiol. Soc., Amer. Jour. Physiol.,
vol. 5, p. vi, 1901.

Marino-Zuco, F., and Dutto, U. Chem. Untersuch. ti. die addison’sche Krank-
heit. Untersuch. z. Naturlehre. Herausg. von J. Moleschott, vol. 14, p. 617,
1892.

Lohmann, A. Cholin, die den Blutdruck erniedrigende Substanz der Nebenniere.
Arch. f. Gesam. Physiol., vol. 108, p. 215, 1907.

Pari, G. A. Azione locale dell’ adrenalina sulle pareti dei vasi, ed azione delle
minime dose di adrenalina sulla pressione del sangue. Arch. di Farmacol.
Speriment., vol. 4, p. 175, 1905.

Vincent, S. Nature of the physiologically active substances in extracts of nervous
tissues and blood, with some remarks on the methods of testing for cholin.
Jour. Physiol., vol. 30, p. 148, 1904.

eHamburger, W. W. Action of intravenous injections of glandular extracts and
other substances upon the blood pressure. Amer. Jour. Physiol., vol. 11,
p- 282, 1904.

112

28 SUPRARENAL GLANDS.

In the report made to the Council on Pharmacy and Chemistry of
the American Medical Association, no attempt was made to check up
the results with the rise obtained from a definite amount of pure
adrenalin, but only to compare the activity of the preparation on the
market. Sollmann and Brown wisely remark that their results give
no idea as to the activity of the preparations before leaving the
manufacturers’ hands.?

An interesting commercial problem is to see by what means the
yield of adrenalin can be increased. Toujan has claimed that in the
products of auto-digestion of pancreas there is a body (not tyrosin)
which added to the ground-up suprarenals does this,? while Halle has
found that the addition of tyrosin to the ground-up suprarenals caused
in some cases an increase in the adrenalin yield.° Others claim that
the addition of ground-up muscle added to suprarenal extract does
the same.4

TOXICITY OF THE ACTIVE PRINCIPLE.

The following data on the toxicity of adrenalin may be of use:

The lethal dose for frogs is over 0.50 mg. per kilo. The relatively
large amount necessary to kill is due to the fact that pulmonary
respiration in frogs is not indispensable. In animals death is usually
due to pulmonary oedema and an action on the heart.

The subcutaneous injection of 0.01 gram per kilo kills guinea pigs,
but 0.004 gram rarely. :

In rabbits 0.02 gram per kilo given hypodermically is fatal.¢
Baylac gives the immediate toxicity by this method as 1 cg. per kilo
for rabbits and guinea pigs. One milligram adrenalin subcutaneously
injected in rabbits weighing 400 grams rapidly kills’ On the intra-
venous injection into rabbits, 0.0006 gram per kilo is always fatal,9
while three out of four die from 0.0004 gram per kilo. The imme-

aSollmann, T., and Brown, E. D. Comparative physiologic activity of some com-

mercial suprarenal preparations. Jour. Amer. Med. Assoc., vol. 47, p. 792, 1906.
Notr.—Compare also Hunt, R., Comparative physiologic activity of some commer-
cial suprarenal preparations, in Jour. Amer. Med. Assoc., vol. 47, p. 790, 1906.

bToujan, G. Recherches expér. sur adrénaline. These, Toulouse, 1905, p. 67.

¢ Halle, W. L. Ueber d. Bildung d. Adrenalins im Organismus. Beitr. z. Chem.
Physiol. u. Path., vol. 8, p. 276, 1906.

d Abelous, G. E., Soulié, A., and Toujan, G. Influence des extraits des organes et
des tissus animaux soumis a l’autolyse sur la production de l’adrénaline. Comp.
Rend. Hebd. Soc. de Biol., vol. 60, p. 16, 1906.

éTaramasio, P. Etude toxicol. de l’adrénaline. Thése, Geneve, 1902, p. 30.

7Paton, D. N. Effect of adrenalin on sugar and nitrogen excretion in the urine of
birds. Jour. Physiol., vol. 32, p. 62, 1905.

9 Battell, F. Toxicite de l’adrénaline en injections intraveineuses. Comp. Rend.
Hebd. Soc. de Biol., vol. 54, p. 1247, 1902.

112

PRINCIPAL PHYSIOLOGICAL TESTS. 29

diate toxicity by vein in rabbits (2 kilos) is 0.06 milligrams per kilo.¢
In dogs from 0.1 to 0.2 mg. per kilo intravenously injected kills,
while in cats from 0.5 to 0.8 are sufficient.2 Twenty milligrams
adrenalin subcutaneously injected in cats caused no disturbance until
the following day (Elliott).

aBaylac, J. Recherches expér. sur les proprietes physiol. et toxiques de l’adréna-
line. Arch. Méd. de Toulouse, vol. 12, p. 247, 1905.
b Lesage, J. Recherches expér. sur ladrénaline. Arch. Internat. de Pharmacodyn.,
vol. 13, p. 273, 1904.
Note.—See also Amberg, S., Toxicity of epinephrin, in Proc. Amer. Physiol.
Soc., p. xxxu, Amer. Jour. Physiol., vol. 8, 1903.

112

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Active principle of suprarenal glands. See Adrenalin.

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32 : INDEX.
Page
Depressor bodies. .... 4. 55-a282 cates oe ee ee ee ee 27
Dogs, effect of active principle. --22 224... 2 ee
Epinephrin, the name adopted by Abel for active principle.................. 9
Hye, action of active principle]. 2 = hess 2s en 14
Frogs, effect of active principle..... sus SS Sake oe 14-15, 28
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Injection; method 0-23 eee ful See. ie eer 24-26
sub Cutameous: 2352-5. ee ee 2 oboe be oe ee Se eee 22
Injections, effects when repeated —5.- 5.222222. 2) ee eee 22
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Nolutions-for testing,-preparationm 2.22. > :242 <2: ee ee 24
puprarenal glands; medicinal tuse_ 2. -=5-22 2253 22nse- 25. eo SoS
Poisonous action :.5.-352 -S5s- ee es ee ee 15

See Adrenalin for active principle. °

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112

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