367

J. Physiol. (I943) 102, 367-37I

577.I74.5

A COMPARATIVE BIOLOGICAL ASSAY OF ACTIVITY

IN SIMPLE SOLUTIONS OF ADRENALINE
BY G. B. WEST, From the College of the Pharmaceutical Society,
Bloomsbury Square, London

(Received 24 June 1943)

When a sample of adrenaline is to be tested to see if it has the full activity of
the pure substance, the biological assay is usually performed using the vasopressor action in a spinal cat or dog, or by its action on the isolated rabbit
intestine. Isolated frog hearts prepared by Straub's method have also been
utilized [Gaddum & Kwiatkowski, 1939], and estimates by this method were
found to agree with colorimetric determinations [Shaw, 1938]. Frogs are more
easily obtained in sufficient quantities than cats or dogs at the present time,
and so perfusion of the frog heart was studied as a means of assaying activity
in simple pharmaceutical solutions .of adrenaline.
METHOD
The isolated frog heart was perfused by a modified Symes's method. Having
exposed the heart free from pericardium and having ligatured the anterior
venae cavae, a mammalian venous cannula, full of frog Ringer's solution, was
inserted into the posterior vena cava. The heart was then removed from the
animal and the movements recorded on a kymograph by means of a thread
and a long isotonic lever. The cannula was connected to the perfusion apparatus
(see Fig. 1), which was supported on one retort stand. The frog Ringer's
solution in reservoir A was maintained at a constant level (as shown by an
arrow), the adrenaline dilution in bottle B also being at this level before
perfusion. The heights of the two siphon tubes were equal, being about 5 in.
above the venous cannula. Results were consistent using this apparatus,
although Mariotte bottles could be used to maintain a constant pressure of

perfusion.
PERFORMANCE OF THE ASSAY
A dilution of the standard adrenaline solution was placed in bottle B, and
tap B was opened so that this solution siphoned over by blowing down outlet
tube B. Using a slow drum, a short tracing was taken to show the normal beat,
and then the three-way capillary tap C was turned to allow perfusion of the

368
G. B. WEST
adrenaline solution in place of the Ringer's solution. This perfusion lasted
exactly 1 or 2 min., after which the lever was removed from the drum. The
drum was stopped, and tap C turned to allow the Ringer's solution to reperfuse. The contents of bottle B and of the siphon tube B were removed, and
the liquid remaining in the tube between tap C and tap B was washed out with
Ringer's solution by turning tap C and opening tap B. The dilution of the
test adrenaline solution was then prepared, and this took exactly 4 min.
During this period the heart beat had nearly always returned to normal. The
drum was started, and the test solution perfused for the same fixed time, at
the end of which it was replaced by Ringer's solution. The test adrenaline was
repeated using the same dose, and, finally, the standard so that a group of four

Fig. 1. The perfusion apparatus. The retort stand has been omitted; it supports
reservoir A, bottle B and the three-way capillary tap a.

results was obtained. It was found that the response to a given dose was more
constant in the region of the maximum response than elsewhere, and so solutions were compared in exactly the same manner as that used for the official
assay of pituitary (posterior lobe) extract by using doses which just produced
submaximal contractions. Equality of response was useful only as confirmatory evidence.
The most accurate procedure for obtaining dilutions was to have the
adrenaline solutions supplied in 2 ml. ampoules. Each ampoule was used for
one response only, and dilutions were always carried out using one complete
set of pipettes and three 100 ml. volumetric flasks. One ml. of the solution in
the ampoule was diluted with 99 ml. of freshly prepared boiled and cooled
distilled water in flask 1; of this dilution, 1 ml. was removed and diluted with
99 ml. of freshly boiled and cooled distilled water in flask 2; of this dilution,

369
ADRENALINE ASSAYS ON FROG IJEARTS
aliquot portions were removed and made up to 100 ml. with Ringer's solution
in flask 3. This was the perfusion fluid, and it was used immediately and never
more than once, as over 30 % of the activity is lost in 5 min. On most hearts,
the standard gave an adequate response in a concentration of 1/100,000 (that
is, 1/100,000,000 of adrenaline).
RESULTS

Over forty samples of simple adrenaline solutions have been assayed. For each
experiment, the standard solution was freshly prepared unheated liquor
adrenaline of the British Pharmacopoeia (1/1000). Fig. 2 shows a comparison
between this standard- (S) and a test solution (T). From these tracings,
(a) 1/150,000 of test was stronger than 1/200,000 of standard, or test was

Fig. 2. Effects of standard (8) 1/200,000 compared with test (T) 1/150,000 and 1/200,000.
Each perfusion lasted for 1I min. only.

stronger than 150,000/200,000=0075 of standard; (b) 1/200,000 of test was

weaker than 1/200,000 of standard, or test was weaker than standard. Hence,
the approximate strength of the test solution was 87-5 % of the standard, that
is, 1/1143 of adrenaline. The comparison was then repeated using different
doses of the test solution so as to obtain closer limits.
The results obtained using varying dilutions of the freshly prepared standard are recorded in Table 1. Freshly boiled and cooled distilled water was
used for preparing the dilutions, which were carried out by an independent
worker. The standard error of the test, calculated by the method of Gaddum
[1938] using the true values of the 'unknown' dilutions, was 2-17 %. The
limits of error (P = 0.99), therefore, are 100 + 559 %. It is recognized that
differences of 5-6 % can be accurately measured by the cat method, so that
the frog-heart method is just as accurate.

G. B. WEST

370

TABLE 1. The estimation of unknown dilutions Of the standard adrenaline solution

Dilution of
Individual percentage
standard
Mean of
deviations of the
four results
(as %)
true result
Values found
90
91-3
90-9, 91-7, 91-7, 90-9
1-0, 1-9, 1-9, 10
80
80-1, 80-1, 80-1, 83-3
80-9
0-1, 0-1, 0-1, 4-1
70
70-9
71.7, 71-4, 69-0, 71-7
2-4, 2-0, 1-4, 2-4
60
60-3
60-7, 59-0, 60-7, 60-7
1-2, 1-6, 1-2, 1-2
50
50-0, 51-3, 51-3, 48-8
50-4
0-0, 2-6, 2-6, 2-4
40
40-8, 38-5, 39-3, 40-8
39-9
2-0, 3-7, 1-7, 2-0
30
30-3, 29-6, 29-9, 30-3
30-0
1-0, 1-3, 0-3, 1-0

Comparison with the rabbit-intestine method

Three dilutions were awssayed by the frog-heart method and then by the
rabbit-intestine method. The results are recorded in Table 2.
TABLE 2. Estimation of adrenaline solutions by the two methods

Frog-heart method
(using a concentration of
1/108 of adrenaline)
Dilution of
.
standard
Value obtained
Percentage
(as %)
(as %)
deviation
90
90-9
1-0
60
60-7
1-2
30
29-5
1-7
A

Rabbit-ileum method
(using a concentration of
1/5 x 107 of adrenaline)
A ,
Value obtained
Percentage
(as %)
deviation
91-4
1-6
59-0
1-6
28-6
4-7,

These results show good agreement. The frog-heart method was not only
slightly more sensitive than the rabbit-gut method (as a lower concentration
of the standard adrenaline was sufficient), but it also showed lower percentage
deviations. The sensitivity could be still further increased by cocainization;
when a concentration of 1/333,333,333 of adrenaline was perfused through the
frog heart alone, no stimulatory effect was observed, but when perfused with
1/200,000 of cocaine hydrochloride, a measurable response was shown.

DIscusSION
Repeated perfusion of the same dose. of standard adrenaline produced similar
responses, but after 4 or 5 hr. the response diminished gradually. This did not
upset the assay, the result being based on groups of four tracings, and these
were reproducible. It is important to keep a fixed time interval between doses,
and this, of course, will depend on the output of the heart. Large winter male
frogs have been used throughout. Results were not so consistent with summer
frogs, the hearts of which needed larger doses and were always useless after
about 3 hr. The heart-beat was about 30-40 per min. in nearly every experiment; if the ligature had been placed on auricular muscle, the beat was much
reduced and the preparation was useless for the test.

ADRENALINE ASSAYS ON FROG HEARTS

371

SUMMARY
A biological assay of adrenaline solutions by the effect on isolated perfused
frog hearts is described. The method has the virtue of using the same tissue
for the standardization, so allowing direct comparison between the standard
and test solutions with the opportunity of interchange so as to make comparisons and contrasts. Winter male frogs provide the best test objects. The
limits of error (P = 0.99) of the test were estimated as 100 + 559 %.
I wish to express my indebtedness to Prof. A. St G. Huggett for helpful advice and criticism,
and to Mr G. F. Somers for technical assistance.

REFERENCES

Gaddum, J. H. [1938]. Quart. J. Pharm. 11, 697.

Gaddum, J. H. & Kwiatkowski, H. [1939]. J. Physiol. 96, 385.
Shaw, F. H. [1938]. Biochem. J. 32, 19.