the estimation of digitalis by pigeon-emesis

THE ESTIMATION OF DIGITALIS BY PIGEON-EMESIS

AND OTHER METHODS

J. H. BURN

From the Pharmacological Laboratory, Pharmaceutical Society of Great Britain,

London, W.C.1

Received for publication March 20, 1930

In a recent number of this JOURNAL, Hanzlik (1) has described

a new method of estimating the potency of digitalis preparations

in which the pigeon is used. The procedure is simple. An injec-tion of the diluted tincture of digitalis is made directly into awing vein, and the pigeon is then watched for ten to fifteen min-

utes to see whether it vomits. Hanzlik suggests that the potencyof tinctures of digitalis be estimated by determining the minimalemetic dose, and says “In determining the minimum emeticdose, a series of pigeons is injected and the just effective dose

determined, which is the just effective dose causing emesis in 2out of 3 pigeons.” Furthermore he says “Considerable experi-

ence with the method in this labo�athry during the past twoyears indicate that from 10 to 12 pigeons are use in the assaywhen starting with an unknown preparation.” Finally Hanzliksuggests that “The minimum emetic dose . . . . might becalled a pigeon-unit, following the expression of standards invogue with insulin, pituitary, etc.”

Since the correct estimation of digitalis is a problem ever

present in this laboratory, I have investigated the proposed new

method, with the more interest because, as described, it entirely

disregards two principles of biological assay on the observance of

which increasing reliance is being placed by many workers.

In the first place it proposes the determination of a “minimal

emetic dose.” Now Trevan (2) showed three years ago that the

expression “minimal lethal dose” is probably never an accurate

expression. Substances as dissimilar as cocaine and diphtheria

toxin, when tested on mice, and digitalis, when tested on frogs,

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222 J. H. BURN

have no “minimal lethal dose.” Trevan showed that for these

substances there is a wide gap between the dose which kills none

and the dose which kills all of the animals when large groups are

taken. If his conclusions were generally applicable it followed

that the term minimal lethal dose should disappear entirely frompharmacological language, to be replaced by the “average” or

“median lethal dose” which is the dose which kills 50 per cent of

animals. Now further work has fully supported the view that

Trevan’s results are generally applicable. Winton (3) found that

the susceptibility of rats to squill poisoning followed the behavior

predicted, and Durham, Gaadum and Marchal (4) have found

that the susceptibility of mice to neoarsphenamine also follows it.

Trevan himself showed that the phenomenon was not restricted to

the determination of lethal doses, for, together with Boock (5)

he demonstrated a great variation in the incidence of convulsions

in mice resulting from administration of insulin. Coward and

Burn (6) found an enormous variation in the sensitiveness of

ovariectornised rats and mice to injections of the oestrus pro-ducing hormone.

It became therefore immediately a matter of interest to seewhether the production of emesis in pigeons by digitalis was an

exception to this hitherto generally obeyed rule. Was it true that

a minimal emetic dose could be determined as was claimed? Or

was the response of pigeons to digitalis a biological property

showing what the botanist is familiar with as continuous

variation?

METHODS

The pigeons used on any one day were kept without food from

5 p.m. on the evening before. Each bird was weighed before

injection and the volume injected was adjusted according to the

body weight. The weights of the birds were almost always

between 300 and 400 grams. The small feathers covering thewing vein were plucked, and the bird was held by an assistant

while the injection was made. Provided that a sharp needle wasused no difficulty was found in making the injection. The bird

was then placed in an empty cage measuring 21 by 16 by 12 inches

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ESTIMATION OF DIGITALIS 223

and, the time of the injection having been noted, the bird was

watched for fifteen minutes. Not more than three birds were

placed in a cage together, and care was taken that during the

period of observation the birds were not scared by noise or move-ments near the cage. No difficulty was ever experienced indeciding whether or not vomiting occurred, as the movements

were well characterized.

Three tinctures were examined ; one prepared from the inter-national standard digitalis powder, S; two, A and B, were tinctures

prepared according to the British Pharmacopoeia. Each tincture

was diluted before injection with 0.9 per cent saline; usually the

dilution was 1 in 2, as suggested by Hanzlik, but for tinctures

S and A, dilutions 1:3 and 1:4 were also prepared.A pigeon was given an injection not more often than once a

week.

EXPERIMENTAL RESULTS

The minimum emetic dose

The description of the minimum emetic dose as the “just effec-tive dose causing emesis in 2 out of 3 pigeons” was first ex-amined. The description implies that if a dose be found which

causes emesis in 2 out of 3 pigeons, then if other groups of 3

pigeons be taken, in each group 2 will vomit when the same dose

is given and one will not. The following experiment is a test of

the implication.

Experiment 1. Twenty-four pigeons were used, and each was in-jected with a dose of 0.15 cc. per kilogran� of tincture A. When thepigeons were injected, they were put into cages by threes, so that the

first cage held the first three pigeons, the second cage the next three, and

so on. The results were as follows.In the first cage two out of three birds vomited. This result may be

expressed shortly as 2/3. The remaining results were, in order,

0/3, 2/3, 2/3, 3/3, 2/3, 1/3, 1/3

Hence it is seen that the eight groups, each of three pigeons,

gave quite dissimilar results. If eight different workers had each

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224 J. H. BURN

had one of these groups, and had applied the criterion proposed by

Hanzlik, four of them would have concluded that 0.15 cc. perkilogram was the minimum emetic dose of tincture A. The other

four would have disagreed; one would have said that 0.15 cc. perkilogram was much below, two would have said that, if not

much below, it was certainly below, and one would have said that

it was above the minimum emetic dose.

Now lest it be thought that the result quoted is in any wayunusual, two more may be given.

Experiment 2. To each of a group of 24 pigeons (not the same birds

as those used in the above experiment), 0.3 cc. per kilogram of tinctureB was given. The results in each cage of three birds were, in order,

1/3, 0/3, 0/3, 2/3, 0/3, 2/3, 0/3, 0/3

The quarrel among eight workers, each with one of the groupsof three pigeons, would, this time, have been even worse than

before. Two would have said that 0.3 cc. per kilogram was theminimum emetic dose of tincture B, while five would have scoutedthe idea that such a dose had any effect at all:

Experiment 3. A group of 21 pigeons (again different birds from thoseused in the first two experiments) was given a dose of 0.2 cc. per kilogramof tincture S. The results were:

2/3, 3/3, 2/3, 1/3, 1/3, 1/3, 1/3

In all, nineteen experiments have been carried out, in each ofwhich the same dose of a tincture has been given to a number of

birds varying from 20 to 25. In every experi.ment there has beenthe same lack of uniformity between the results in different groups

of three as is illustrated above.

The error of the minimum emetic dose

While the results so far described show that a definition of the

minimum emetic dose as the “just effective dose causing emesis

in 2 out of 3 pigeons” is one which will yield different results with

the same tincture when different pigeons are used, they do not

give a measure of the inaccuracy which may actually arise. I

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have not made an exhaustive enquiry into this point, but results

were obtained which show that it exceeds 100 and approaches 300

per cent ; it may quite possibly be greater still.

Experiment 4. A dose of 0.1 cc. per kilogram of tincture A was givento each of 24 pigeons divided into 8 groups. Out of one of the groupsof 3 pigeons, 2 birds vomited.

A dose of 0.2 cc. per kilogram of the same tincture, administeredon another occasion to the same pigeons, caused vomiting in 2 out of 3birds in three of the eight groups of birds. In three other groups only

one bird vomited.A dose of 0.3 cc. per kilogram of the same tincture, administered on

another occasion to the same pigeons caused every bird to vomit.

In the experiment described a worker might have determined

the minimum emetic dose to be either 0.1 or 0.2 cc. per kilogram,or as more than 0.2 cc. per kilogram, but as less than 0.3 cc. per

kilogram. Hence the variation exceeds 100 per cent and ap-proaches 300 per cent. Since only 24 pigeons were used it is

almost certain that the extreme variation in pigeons which

actually exists was not encountered. To determine this, at least

100 and preferably 500 pigeons should be used.

The average emetic dose

The experiments recorded show conclusively that the reactionof pigeons to digitalis by vomiting is another example of “con-

tinuous variation” in a biological property, and that Trevan’s

findings apply here just as elsewhere. There is in fact a wide gapbetween a dose of any one tincture which will cause every pigeon

in a large group to vomit and the dose of the same tincture which

will fail to cause emesis in arty pigeon out of the same large group.

It will, however, probably occur to anyone reading the resultsquoted in experiment 4, that as the dose increases the proportion

of birds which vomit also increases. Thus the results referred

to in experiment 4 may be supplemented by others obtained with

the same pigeons and the same tincture (A), and presented some-

what differently in table 1.

Table 1 shows that, apart from the first two doses, the effect of

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which will be considered later, the percentage increases as the doseincreases. The figures at once indicate that the term “minimum

emetic dose” is meaningless, and that the emetic potency should

be described in terms of the percentage of pigeons in which the

emesis is to be produced. It seems reasonable to adopt here the

suggestion of Trevan that the percentage should be 50, and then to

speak simply of the average or median emetic dose, which means

the dose causing emesis in 50 per cent of pigeons.

TABLE 1

Experiments with tincture A on one group (the second) of pigeons at weekly intervals

DOSENUMBER OF PIGEONS

INJECTED

NUMBER WHICH

VOMITED

PERCENTAGE WHICH

VOMITED

cc. per kgm.

0.1 7 300.15 6 260.2 15 62.50.3 22 100

TABLE 2

Experiments with first group of 25 pigeons

DOSE TINCTURBPERCENTAGE IN WHICH VOMIT-

ING WAS OBSERVED

cc. per kgm.

0.150.20.30.40.5

AABBB

54

7737.54891

Comparison of different tinctures

By carrying out experiments similar to those recorded in table

1, the three tinctures 5, A and B have been compared. Three

groups each of about 25 pigeons were used at different periods.The first two groups were used for comparing A and B, while the

third group was used for comparing S and A. On each occasion

that the pigeons were injected the whole of the group received onefixed dose per kilogram of one tincture. Table 2 gives the results

on the first group of birds, table 3 and table 1 the results on the

second group, and table 4 the results on the third group.

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The results on the three groups of pigeons are plotted as graphs

in figures 1, 2 and 3, though for the abscissae, instead of the

doses, the logarithms of the doses have been substituted. This

method, which is the method first used by Krogh (7) for insulin,

enables the relative potencies of the tinctures to be compared by

drawing parallel straight lines through the points obtained, and

noting the abscissae at the points on the lines corresponding to

50 per cent mortality. The antilogarithms of these abscissae

TABLE 3

Experiments with second group of 25 pigeons

Note that the results with tincture A are given in table 1.

DOSE TINCTIJREPERCENTAGE IN WHICH VOMIT-

INO WAS OBSERVED

cc. per kgm.

0.30.40.40.5

BBBB

21482275

TABLE 4

Experiments with the third group of pigeons

D0�E TINCTUREPERCENTAGE IN WHICH VOMIT-

INO WAS OBSERVED

cc. per kgm.

0.150.20.20.20.20.3

AAASSS

3052

59385274

give the average emetic doses of the tinctures. Where two ob-

servations on the effect of a given dose of one tincture have been

made (e.g., 0.4 cc. per kilogram of tincture B in table 3) the mean

of the two percentages has been used in plotting the graph.

Figure 3, in which tincture A is compared with tincture S, showsthat the average emetic dose of tincture A is 0.188 cc. per kilo-

gram and that of tincture S is 0.214 cc. per kilogram. Hence Ais more potent than S, and if S, which is a tincture prepared from

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228 3. H. BURN

the international standard digitalis powder, be given the value100 then, A has the value 114. In figure 1 the average emetic dose

of tincture A is 0.143 cc. per kilogram and that of tincture B is0.355 cc. per kilogram. Since tincture A .is 114 per cent of the

strength of tincture 5, then tincture B is 0.143x 114 per cent of

01 ‘ O.Z 13 �. �.5 -

Fio. 1 FIG. 2

FIG. 1. The experiments shown were performed on the first group of 25 pigeons.Ordinates are the percentage vomiting, and abscissae are the logarithms of thedoses. (Against each point on the abscissa the figure for the dose itself in cubiccentimeters per kilogram has been written.) Parallel lines are then drawn throughthe points obtained, for each tincture. The line on the left is that for tincture A,and that on the right is the line for tincture B. The abscissa corresponding to50 per cent vomiting is a measure of the potency.

FIG. 2. Similar to figure 1. Experiments on second group of pigeons withtinctures A and B. Tincture A on the left, tincture B on the right.

tincture 5, which is 46 per cent of tincture S. Similarly in figure

2 the average emetic dose of tincture A is 0.178 cc. per kilogram,

and that of tincture B is 0.41 cc. per kilogram, hence tincture B is

49 per cent of tincture S. The mean of the two figures for B in

terms of S, which are 46 and 49, is 47.5 per cent.

Hence A is 114 per cent, and B is 47.5 per cent of S. In this

way a fairly accurate comparison of the three tinctures has been

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obtained, though more labor has been expended than indicated inthe original paper as necessary to achieve this end; “10 to 12pigeons” are not enough.

The impossibility of a pigeon unit

Hanzlik proposed that the minimum emetic dose might becalled a pigeon unit. The evidence makes it clear that this con-ception cannot be employed even when, in place of a group ofthree, a group of 25 pigeons is employed. The average emetic

80

6o

40

20

01

Fzo. 3. Similar to figures 1 and 2. Experiments on third group of pigeons withtinctures A and S. Tincture A on the left.

dose of tincture A was 0.143 cc. per kilogramin the first group ofpigeons, while it was 0.188 cc. per kilogram in the third group ofpigeons, and these figures differ by more than 30 per cent.

Secondly the same group of 25 pigeons changes in sensitiveness

at different times. In table 1 a dose of 0.1 cc. per kilogram of

tincture A produced vomiting in 30 per cent on one occasion.

On another occasion a dose half as large again produced vomiting

in a smaller percentage of the same birds. Similarly in table 3

it is seen that a dose of 0.3 cc. B produced vomiting in 21 per

cent (5 out of 24) whereas 0.4 cc. B produced vomiting on another

occasion in 22 per cent (5 out of 23) and the same dose on a third

occasion in 48 per cent (11 out of 23).

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Consequently in view of differences in sensitiveness between

one group of birds and another, and between the same group of

birds at different times, it is clearly impossible to speak of a pigeonunit as though this were an absolute measure of digitalis potency.

In spite of these differences in sensitiveness however, pigeons

can be used for the assay of digitalis provided that the assay

be made comparative, so that the strength of an unknown prep-aration is stated in terms of a known standard.

The assay of digitalis by the pigeon method

The general lines which should be followed will be clear from the

method by which tinctures A and B have been compared with the

TABLE 5

Results with tincture A

Figures are percentage of birds which vomited

DOSEFIRST GROUP

PIGEONS

SECOND GROUP

PIGEONS

THIRD GROUP

PIGEONSAVERAGE FOR ALL

cc. per kgm.

0.1 30 300.15 54 26 30 37

0.2 77 62.559�52J

62.6

0.3 100 100

standard as shown above. The information obtained however,

may be used to simplify the assay of other tinctures.

Tincture A was examined on about 75 pigeons in all, and the

results obtained with it are summarised in table 5.

The figures for the dose in table 5 are plotted in figure 4 against

the average percentages. The two middle points, determined on

75 pigeons, naturally are much more reliable than t� two

extreme points. In figure 4, however the figures for the doses on

the abscissa have been omitted and an arbitrary scale has been

substituted. A perpendicular has been drawn through the curve

at the point corresponding to 50 per cent of birds vomiting, and

the point where the perpendicular cuts the abscissa has been called

4. Half way between 4 and the origin then becomes 2, and the

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point 6 is of course the same distance to the right of 4, that 2

is to the left.

Figure 4 may now be used for the assay of an unknown tinctureof digitalis. Either the same birds must be injected with both

the standard and the unknown, when the assay will require atleast a day or two between the two sets of injections, or double the

number of birds must be used. The former, of course, will be themore accurate method. Thus a group of either 25 or 50 pigeons

Fio. 4. This curve is an approximation to the characteristic for pigeons in-jected with digitalis. The curve is drawn from the figures in table 5, the doses(not logarithms of doses) being plotted as abscissae. The figures shown as ab-scissae are however arbitrary figures obtained as explained in the text.

is required. In what follows it will be assumed that one group of

25 receives the standard, and the other group of 25 receives the

unknown. The dilutions of the two tinctures (either 1:2 or

1:3) with physiological saline are then prepared. The 25 pigeonsto be injected with the standard are each given the same dose

(reckoned in terms of undiluted tincture), namely, 0.2 cc. perkilogram. The 25 pigeons to be injected with the unknown are

also each given the same dose. In the absence of information

to the contrary, it may be assumed the unknown tincture will be

of the same strength as the standard (for in Great Britain the

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average strength of a large number of commercial tinctures has

been found to be equal to this standard. (See Wokes (8).)

Hence the dose of the unknown tincture may also be 0.2 cc. per

kilogram. Let us suppose that 15 pigeons injected with the

standard vomit, and that 8 pigeons injected with the unknownvomit; that is, 60 and 32 per cent respectively. In figure 4 we

see that the abscissa corresponding to 60 per cent is 4.45, and that

corresponding to 32 per cent is 3.2. Hence the potency of equal

doses of standard and unknown is related by the proportion of 4.45

to 3.2. Or the unknown has 72 per cent of the activity of the

TABLE 6

The figures are the lethal doses for each cat expressed in cubic centimeters

per kilogram of tincture diluted 1: 20.

TINCTURE S TINCTURE A TINCTURE B

14.4 13.3 30.0

15.37 12.4 28.016.45 11.4 18.2

12.0 14.6 19.7

8.8 18.1 16.712.35 12.9 20.714.519.37

14.8

15.416.9

17.9

Average 14.35 13.8 22.2

standard. The error of this result may be calculated by the

usual methods. (See Burn (9).)

THE RELATION OF RESULTS BY THE PIGEON METHOD TO RESULTS

OBTAINED BY THE FROG METHOD AND THE CAT METHOD

Results by the cat method

Before accepting the pigeon method as a proper method for

estimating digitalis, it is necessary to show that when applied todifferent tinctures it gives similar results to other methods which

are based on an action on cardiac muscle, such as the cat method

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and the frog method. The tinctures have accordingly been

compared by these two methods.The method used has been the Hatcher-Magnus method, in

which the cat is artificially respired with a mixture of air andether, and in which the tincture is administered in a 20-folddilution in saline. The blood pressure was recorded, and the

fall of blood pressure to zero was taken as the end point. Theresults with the different tinctures are given in table 6.�

Every experiment performed has been included in table 6, andthe figures show a satisfactory range of variation, indicating that

reliance may be placed upon the averages obtained. The impor-

tance of this range of variation will be considered later. From the

averages of the lethal doses the relative strengths of A and B may

be stated in terms of S, giving S the value 100. These strengths

are 108 and 67 for A and B respectively.

Results by the frog method

The tinctures were compared on Rana temporaria, using an

eighteen-hour lethal dose method, the tinctures being injectedinto the lymph sac after partial removal of alcohol and appro-

priate dilution with 0.6 per cent saline. The method recom-

mended by Trevan of injecting a large number of frogs with one

dose to determine the percentage mortality was followed. Thefollowing experiments were performed.

Comparison of tincture A and tincture S

a. First comparison. A dose equivalent to 0.5 cc. per 100 grams frog

of tincture A was injected into 20 frogs. The same dose of tincture S wasinjected into 20 more frogs. Mortality from tincture 5, 13 out of 20 =

65 per cent. Mortality from tincture A, 19 out of 20 = 95 per cent.

From Trevan’s characteristic curve (See Burn (9)),

Potency of S - 4.25 - 100

Potency of A - 5.25 124

b. Second comparison. The same experiment was repeated, except

that the dose of tincture A was reduced to 0.35 cc. per 100 grams.Mortality from tincture S = 13 out of 20 = 65 per cent. Mortality

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from tincture A = 12 out of 20 = 6J per cent. From the charac-

teristic curve,

Potency of 0.5 cc. S - 425

Potency of 0.35 cc. A - 4.2

4.2Hence 0.35 cc. A = X 0.5 cc. S, or A = 139 per cent of the potency

of S. The .value of A in terms of S is therefore the mean of the tworesults, 124 per cent and 139 per cent, that is 131 per cent.

Comparison of tincture B and tincture S

a. First comparison. Doses: 0.5 cc. per 100 grams frog of tinctureB to each of 20 frogs; 0.5 cc. per 100 grams frog of tincture S to each of20 frogs. Mortality from tincture B, 2 out of 20 = 10 per cent. Mortality

from tincture 5, 11 out of 20 = 55 per cent. From the characteristic

curve,

Potency of B = 2.7 - 63

Potency of S 4.1 - 100

b. Second comparison. Doses: 0.75 cc. per 100 grams of tincture B

to each of 20 frogs. 0.5 cc. per 100 grams of tincture S to each of 20frogs. Mortality from tincture B, 11 out of 20 = 55 per cent. Mor-

tality from tincture S, 4 out of 20 = 20 per cent. From the charac-

teristic curve,

Potency of 0.75 cc. B = 4.1

Potency of 0.5 cc. S 3.25

4.1Hence 0.75 cc. B = X 0.5 cc. S or B = 83 per cent of the potency

of S. The value of B in terms of S is therefore the mean of the tworesults, 63 per cent and 83 per cent, which is 73 per cent.

COMPARISON BY THREE METHODS

It is now possible to compare the results by the three methods,

and the comparison appears in table 7.

I have previously taken part in a thorough investigation of two

other tinctures made in comparison with the same standard used

here (Trevan, Boock, Burn and Gaddum (10)). The results

then obtained by four methods, using very large numbers of

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animals displayed exactly the same features as those seen in

table 7. One tincture was stronger than the standard and the

different values obtained for it were not far apart. So it is with

the results for tincture A, for the difference between the figures

108 and 131 is about 20 per cent, and the agreement between the

different methods cannot be said to be bad. The other tincture

was weaker than the standard, and the values obtained for it were

very far apart; so it is with tincture B; it is true that the cat and

frog results are close together but the pigeon result differs

strikingly.

It should be understood, however, that the variation in the

results by different methods is not an indication that the methods

are inaccurate to the extent of this variation. The variationmeans that the different methods do in fact give different results.

TABLE 7

METHOD TINCTURE S TINCTURE A TINCTURE B

Pigeon 100 114 47

Cat 100 108 67

Frog 100 131 73

Wokes (8) has made an examination of a large number of tinctures

of digitalis by the cat method and the frog method, using the

technique here described, and he found that in freshly prepared

tinctures the relation to the international standard by the frog

method was uniformly higher than by the cat method. Whereas

with older tinctures the reverse was true. Nor is the difference in

the result by different methods peculiar to two methods of which

one is an intravenous and the other a hypodermic method, for

similar differences were shown to exist between the cat method

and the guinea-pig method (10), using the intravenous guinea-pig

technique of Knaffl-Lenz (11). The variation will be further

considered.

DISCUSSION

The experimental results illustrate the inaccuracy which isbound to attach to tests wherein it is laid down that “two out of

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three” animals must fulfill a particular requirement in order thata minimal emetic, or a minimal lethal dose may be determined.

Another example of this kind of test is that laid down in the United

States Pharmacopoeia X for Tincture of Aconite in which two of

three guinea pigs must die when a particular dose is injected.

That these tests are bound to give different results when differ-ent groups of animals are used follows logically from the veryrequirement of the test, and really does not need such an experi-

mental demonstration as has been given here. Thus the stipula-

tion that the minimal emetic dose must cause two out of three

pigeons to vomit may be conaidered as follows. Let us take threepigeons, of which two vomit and one does not (when each receivesthe same dose), and put them into a big cage. Let us add to themthree more which behave in the same way, and again three more,until the cage holds thirty groups of three pigeons. Now if

every one of the pigeons be injected, two-thirds will vomit and

one-third will not. But, from the cage of 90 birds, let us take

them three at a time. Is it possible to be sure that out of the

first three we take, two will vomit and one will not? Of coursethere is no certainty. There are 30 birds in the cage which do

not vomit when the dose is injected, and it is easily possible

that three of these may be chosen. Only in a certain proportion

of groups taken out of the cage will two birds vomit and one failto vomit. In the other groups, vomiting wifi occur in 3 out of 3,

or in 1 out of 3, or in none out of 3.

As has been shown by Durham, Gaddum and Marchal (4) the

different results in different groups of animals may be foretold bya simple mathematical calculation. If we have a large group of

pigeons of which exactly 66 per cent will vomit on injection, then

the probability that one of these pigeons, selected at random from

the group, will vomit is �. If, then, pigeons are taken three at a

time, the proportion of tests in which each of the possible results

will occur is given by the terms of the expansion of (* + �)3,

which is � + � +44 + �. That is to say if we withdraw 27

groups, each of 3 birds, then in 8 of the groups all 3 birds will

vomit, in 12 of the groups 2 will vomit, in 6 of the groups only

one wifi vomit, and in one of the groups none will vomit. Note

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however that this prophecy depends on the assumption that theinitial large group of birds is so chosen that, if all were injected,

exactly two-thirds would vomit.The same arguments apply to the frog test for digitalis. There

are workers who consider that they determine the minimal

lethal (or minimal systolic) dose very carefully because they usefive frogs for each dose, and require four deaths (or systolic arrests)

out of five. But let us suppose one hundred frogs were availableof which 80 per cent would be killed if a particular dose of digitaliswere injected into each, and that these frogs were injected five at atime. Would exactly four frogs die out of each five? Theanswer is no, just as certainly as it is to the question “when coinsare drawn from a bag will they come out heads and tails alter-nately?” If five groups each of five of the frogs were taken,

only in two of the groups would four frogs die. (This is shown by

the last term but one in the expansion of (� + t)�)

In dealing with any biological test it should be realized that ifdifferent doses are given to different small groups of animaLs,

in the effort to determine a minimal dose, then the fundamental

canon of all experimental work is violated. The rule is that, inmaking an experiment, only one condition at a time must bechanged. But if I first give a dose of 0.5 cc. to five animals and

then a dose 0.3 cc. to five other animals I have changed, not one,

but two conditions. The dose has changed and a group of

animals of different sensitiveness has been taken. There is a

very simple way to avoid this, which is to use a large group ofanimals and to give every one the same dose. The potency of the

dose is then estimated by the percentage of animals in which agiven effect follows. It has been shown in this paper how thismay be done for pigeons and how Trevan first recommended thatit be done for frogs.

The same difficulty with the cat method appears in a different

form. Edmunds, Lovell and Braden (12) have recently used thisand other methods, and speaking of the cat method they say with

reference to the examination of a sample of strophanthin (No. 8)

“three cats which died in approximately the same time gaverather widely divergent figures . . . . it is irregular results suchas these which are so discouraging in the use of the cat method.”

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238 J. H. BURN

It is regarded as disturbing that different cats show so muchvariation in the lethal doses. It is in fact no more disturbing

than that different men are of different height. Height in men

shows “continuous variation” just as does weight in kidney

beans, and sensitiveness to digitalis in cats. But variation in

height and in weight are obvious, while variation in sensitivenessto digitalis is not. The important lesson to be learnt however is

that this variation does not make the business of assay difficult.

It does not mean that a large number of cats need be used to getthe true result. The average value of a few cats will usually befound in practice to be surprisingly close to the average of onehundred cats. It is in this approximation, indeed, that the

beauty of the law of averages lies. Many workers however dis-

trust the average from a few cats unless the different figures

happen to be close together, and in this they err. If the figures

for three cats are dissimilar, there seems more chance that they

will give an average close to the average of 100 cats than if they

are similar. The dissimilar cats appear to b� a better “sample”of cats as a whole. The point has been illustrated recently in a

paper from this laboratory by Elphick (13), which should be

consulted by those interested.

The pigeon method if properly used appears to be as good a

method of estimating digitalis as the frog method or the catmethod. All these methods may give different results for a tinc-

ture, and, as already shown by Trevan, Boock, Burn and Gaddum,

(10) the differences appear to be due to the different sensitiveness

of different species to the different glucosides. It is curious how-ever that the results for tincture A, the strong tincture, are notfar apart, while those for tincture B are. The same greateruniformity in the assay of a strong tincture by different methods

was observed by Trevan, Boock, Burn and Gaddum.

SUMMARY

1. It has been shown that the assay of digitalis by emesis in

pigeons gives very different results when different groups of pigeons

are used. If the group contains only three birds the error may

approach 300 per cent, and if it contains 25 birds, it may be 30

per cent.

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2. Providing however that due allowance is made for theindividual variation in different birds, by injecting a group of 25with the same dose to determine the percentage in which emesisfollows, digitalis may be assayed satisfactorily by means of acharacteristic curve relating the percentage having emesis to the

potency of the preparation.3. The assay should be comparative, stating the strength of an

unknown sample in terms of the strength of a standard. The

accuracy is considerably increased if the strength of the standard

is determined on the same group of birds as that used for theunknown.

4. The pigeon method so used, gives results resembling closely

enough those obtained by the cat or the frog method.

5. The errors which follow from injecting different groups of afew animals each with different doses are fully discussed.

REFERENCES

(1) HANZLIK: Jour. Pharmacol. and Exper. Therap., 1929, xxxv, 363.(2) TREVAN: Proc. Roy. Soc., Ser. B., 1927, ci, 483.(3) WINTON: Jour. Pharmacol. and Exper. Therap., 1927, xxxi, 126.

(4) DURHAM, GADDUM, AND MARCHAL: Med. Res. Counc., Spec. Rep. Ser. No.128, 1929.

(5) TREVAN AND BoocK: League of Nations Publications. III. Health, 1926,

iii, 7 (C. H. 398).(6) COWARD AND BURN: Jour. Physiol., 1927, lxiii, 270.

(7) KROGH: League of Nations Publications. III. Health, 1926, iii, 7 (C. H.398).

(8) Wo�s: Quart. Jour. Pharm. Pharmacol., 1929, ii, 48.(9) BURN: Methods of Biological Assay, Humphrey Milford, London, 1928.

(10) TREVAN, BoocK, BURN AND GADDUM: Quart. Jour. Pharm. Pharmacol.,

1928, i,6.

(11) KNAFFL-LENZ: Jour. Pharmacol. and Exper. Therap., 1926, xxix, 407.(12) EDMUNDS, LOVELL AND BRADEN: Jour. Amer. Pharmaceut� Assoc., 1929,

xviii, 338.(13) ELPHICK: Quart. Jour. Pharm. Pharmacol., 1929, ii,507.

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the estimation of digitalis by pigeon-emesis

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