THE EFFECTS OF CAFFEINE AND SODIUM BICARBONATE

UPON THE TOXICITY OF ACETANILIDE’

WORTH HALE

Frcnn the Hygienic Laboratory, U. S. Public Health and Marine-Hospital Service,

Washington, D. C.

Received for publication, May 25, 1909

Although antipyrine was introduced as an antipyretic two years

before acetanilide the latter quickly outranked it in popularity. This
was largely owing to its cheapness and even in the years immediately
following the general medicinal use of these drugs this was spoken of
by physicians as a reason for prescribing acetanilide2. At the same
time certain reports appeared indicating that antipyrine was the
more toxic3 and the less efficient4.
The immediate popularity of these compounds arose from their
marked antipyretic action but it was soon observed that they were
also very efficacious in relieving various more or less obscure pains,
generally neuralgic in character as facial neuralgia, hemicrania,
and tabes dorsalis.5 At the present time enormously large amounts
are used for the relief of symptoms of this sort and comparatively
little as a means of reducing fever.
Owing to the popularity with the general public of all drugs used
for the relief of pain the legitimate use of the antipyretics was quickly
made subservant to an indiscriminate use especially in the treatment
of headache and Siefert6 as early as 1888 pointed out the great danger
of allowing the apothecaries to dispense these preparations directly
‘A more complete report upon the toxicity of acetanilide and antipyrine as affected
by certain drugs will appear in Bulletin No. 53 of the Hygienic Laboratory.
2 Hinzelman: Munch. med. Wochenschr., xxxiv, 36, 1887.
Barr: Pharm. J. and Tr., Lond., xviii, 170, 1887.
Faust: Deutsch. med. Wochenschr., xiii, 575, 1887.
‘Chomjakow u. Ljwow: Wratsch, vi, 887, 1885. Ungar: Cetitralbi. f. klin. Med.,
vii, 777, 1886. Secretan: Rev. de. Med., Par., vii, 29, 1887.
‘Siefert: Munchen Med. Wochenschr., xxxv, 867, 1888.
186 WORTH HALE

to the general public. Despite this early warning their use has become
more and more common and at the present time they are sold directly
to the laity over the counters of every drug store and at almost every
soda fountain. In nearly every case too these drugs are dispensed
with other drugs, most generally with sodium bicarbonate and caffeine
but also with alkaloids of the opium series, the salicylates and the
bromides.
Two reasons may be suggested for the addition of caffeine to ace-
tanilide mixtures. It has been known for a long time that caffeine
in itself was useful in the treatment of certain forms of headache and
it appeared in several formuhe combined with the bromides a nurn-
ber of years before the introduction of the coal tar remedies. The
natural inference therefore is that it was a direct transfer of caffeine
from the old to the new type of headache remedies. The other explan-
ation of its presence is to be found in the literature relating to the
treatment of cases of acetanilide poisoning. Lepine7 seems to have
first suggested caffeine as an antidote, having reported that the
cyanosis of acetanilide poisoning disappeared after large doses of
this drug. In 1889 Mahnert8 suggested that the excitants be used
and in treating three cases made use of ether injections, wine and
powdered caffeine, the latter being especially recommended by him.
Hartge9 treated a case of poisoning with caffeine and brandy and later
with camphor and ether injections. Falk’#{176}
used caffeine but thought
that alcohol would be harmful owing to the increased solubility of
acetanilide in an alcoholic menstruum and therefore its more rapid
absorption.
Whether caffeine was introduced as an adjuvant of acetanilide or
as an antidote for its poisonous properties it is impossible to say but
the generally prevailing idea at the present time is that caffeine is
added to prevent the deleterious effects of acetanilide upon the
heart.” Although this does not seem to have been the original rca-

Lepine: Rev. de. Med., Par., vii, 531, 1887.

‘Mahnert: Memorabilien, Heilbr., xxxiv, 321, 1889.
‘Hartge, St. Petersb. med. wochenschr., vii, 69, 1890.
10 Falk: Therap. Monatsh., iv, 257, 1890.

“McFarline: Canad. Pharm. J., Toronto, xxxix, 360, 1906, in speaking of head-
ache powders says: “It will be noted that in most cases the depressant effect upon
the heart is thought to be counteracted by the addition of caffeine, bicarbonate of
soda and other drugs of like character.”
 THE TOXICITY OF ACETANILIDE 187

son for its administration no direct observations of its antidotal value

seem to have been made but on purely theoretical grounds it would
apparently be useful as a stimulant to the respiratory center, which
becomes markedly embarrassed from the formation of methama-
globlin, and to a lesser degree to the heart. It does not seem probable
that it would have any influence upon the cyanosis unless indirectly
through increased respiratory activity.
The combination of alkali carbonates with acetanilide became
popular about 1890 but the reason for their presence in such mix-
tures is largely a matter of conjecture. Herczel’2 in 1887 carried out
some experiments upon dogs and was able to show that the exhibi-
tion of acetanilide definitely decreased the alkalinity of the blood and,
although all methods for estimating the alkalinity of the blood are
unreliable, this fact would afford some experimental basis for their
presence. In this connection it is important to remember that Kobert’3
pointed out that the alkalies prevent the formation of methama-
globin and that their exhibition serves to aid in the regeneration
of oxyhamaglobin, an alkali-methamaglobin being formed which
changes the blood from a brown to a red and from this as an inter-
mediary product oxyhamaglobin is formed.
Another reason for the use of alkalies in acetanilide mixtures seems
to have been based upon the belief that their solubility was thereby
increased. Hall’4 in 1891 gave as a reason for the supposed greater
activity of Antikamnia its finely powdered state and the presence of
sodium bicarbonate which he said made it more soluble. This idea
prevailed until in 1906 when Puckner’5 in a series of experiments
proved in direct contradiction not only no increased solubility but
that acids rather than alkalies were the best solvents.
The official recognition of caffeine and sodium bicarbonate as
ingredients of acetanilide mixtures together with the large number
of cases of poisoning arising especially from the use of the so-called
headache remedies suggested the experimental determination of the
effect of these and the other drugs commonly found in such mixtures
upon the action of acetanilide.
12 Herczel: Wien. med. Wochenscher., xxxvii, 1022, 1887.
u Kobert: Lehrbuch der Intoxikationen, pp. 73-74, 1902.

“Hall: Druggist Circular, xxxv, 99, 1891.

“Ptickner: Jour. Am. Med. Ass., xlvii, 1206, 1906.
188 WORTH HALE

DETERMINATION OF THE HEART EFFECT

The popular belief in the value of caffeine in preventing the poison-

ous heart action of the coal tar remedies suggested the experimental
determination of any modification of the action upon this organ
which might occur when it was exhibited in combination with ace-
tanilide. Experiments were carried out upon warm and cold blooded
animals, using the myocardiograph method to record the changes in
the dog’s heart, the perfusion in estimating the changes in the frog’s
heart.
Action Upon the Frog’s Heart

The perfusion method was adopted as being the most suitable for
determining the changes occurring in the frog’s heart after acetanilide
and combinations of this drug with caffeine citrate. By this method
the comparatively small amounts of the relafively insoluble acetanilide
which go into solution in water are sufficient to produce profound
changes in the isolated heart. At the same time the removal of the
heart from the body excludes all secondary effects due to the action
of the drug upon the blood and nervous system, any effect being
limited to an action upon the intrinsic nerves of the heart or to the
cardiac muscle.
In perfusing the heart. one-seventh to one-fifteenth of one per cent
solutions of acetanilide in Ringer’s solution were used as a control
and to compare the effect of caffeine citrate 1/2000 to 1/3000 of one
ler cent of the latter drug’6 were added to the above percentages of
acetanilide. The control experiments demnonstrated that acetanilide
is markedly depressant producing a very slow heart rate and decreas-
ing even more definitely the amount of fluid pumped through the
heart. As a preliminary effect there was occasionally some slight
increased heart action but with the above strengths of solutions the
heart rate and output usually dropped to about half the normal
within a few minutes and in an occasional experiment ceased to beat
entirely. Another peculiar feature of acetanilide poisoning is that

“In a series of experiments it was shown that 1/1500 to 1/5000 of one per cent
caffeine citrate possessed a stimulant action to the heart of the frogs used in these
experiments. In amounts greater than 1/1500 per cent depression of the heart
followed, both rate and output becoming markedly less.
 THE TOXICITY OF ACETANILIDE 189

the effect of the drug appears to grow less in certain instances so that
the rate and sometimes output were secondarily augmented although
never was a normal or even nearly normal action regained. This
latter effect of the drug made the determination of slight differences
between the action of the simple drug and a combination of drugs
difficult.
In one series of experiments it had been shown, using one-seventh
of one per cent solutions of acetanilide, that the heart ceased to beat
after the following intervals 37, 17, 9 and 30 minutes or an average
of 23.1 minutes after the perfusion of the drug was begun. Following
this series a one-seventh of one per cent solution of acetaniuide to
which had been added one-two thousandth per cent caffeine citrate
was perfused using frogs of approximately the same weight. The
intervals between the introduction of the combined drugs and the
stoppage of the heart were as follows: 25, 3, 15 and 8 minutes or an
average of 12.7 minutes after the drug was started. These experi-
ments serve to demonstrate the marked irregularity with which the
heart reacts to the poison. The results as they stand, however, indicate
considerably greater toxicity to the heart from the mixture than
from acetanilide alone.
In a later series smaller amounts of acetanilide were used with the
idea of noting the changes in the rate and output upon the substitu-
tion of an acetanilide caffeine mixture. These experiments were
somewhat disappointing because of their failure to show striking
modifications in action. In five experiments there was clearly a
lessened toxicity from using the drugs in combination but in fourteen
others the results were exactly the reverse, the toxicity being increased.
A protocol illustrating this is to be found in the following table:

Perfusion of the isolated frog’8 heart with acetanilide and a mixture of acetanilide
and caffeine citrate

Protocol 40 10/23/08.

Output
Time Rare per minute

9:20 28
9:25 36 23
9:30 35 22
 oc

190 WORTH HALE

Acetanilide 1/8 % plus Caffeine 1/3000%

9:31 0
9:32 Ringer solution on
9:35 26 10
9:40 33 19

Acetanilide 1/8 per cent

9:42 19 -

9:45 13 8

Acetanilide 1/8 per cent plus Caffeine 1/3000 per cent

9:46 0

It must be admitted that these changes are not invariably so defi-

nite but they confirm those of the first series and indicate that these
drugs are more toxic in the above amounts when given in combina-
tion than when given alone. Smaller amounts of acetanilide were
used in further perfusion experiments and in this series some antag-
onism was demonstrated but again there were a number of instances
in which the mixture showed greater toxicity and were of sufficiently
frequent occurrence to indicate how incomplete the antagonism
between caffeine and acetanilide really is.
Acetanilide and sodium bicarbonate were perfused through frog
hearts in the same manner to determine what effect the alkalies would
have upon the toxicity of acetanilide on the heart. Acetanilide was
used only in one-eighth per cent solution but the amount of bicarbonate
was varied from three-hundredths to one-twentieth per cent. In all
cases there proved to be a considerable antagonism but this was
not at all sufficient to abolish or even prevent quite marked slowing
from the toxic action of the acetanilide. The results of these experi-
ments are well illustrated by the following protocol.

Perfusion of the isolated frog’s heart with acetaniide and with a mixture of acetani-
tide and sodium bicarbonate in Ringer’s solution

Protocol 35
Output
Time Rate per 5 minulee

3:20 34
3:25 46 27
3:30 40 30
 THE TOXICITY OF ACETANILIDE 191

Acetanilide 1/8 per cent plus Na HCO3 1/100 per cent

3:32 32 -

3:35 18 19
3:40 17 15
3:45 17 12
3:50 17 13
Acetanilide 1/8 per cent
3:52 14
3:55 10 11
4:00 7 3
4:03 0 -

Action on the Dog’s Heart

To check the results obtained in the perfusion experiments a

series of experiments were carried out using dogs as experimental
animals. After anlesthesia was complete the heart was exposed and
the right ventricle attached to a modified form of the Roy-Adami
myocardiograph. Blood-pressure tracings were also taken using the
ordinary mercury manometer to record the changes in the pressure.
On account of its slight solubility in water it was necessary to sus-
pend the acetanilide in a small amount of mucilage of acacia. Injec-
tions into the saphenous vein of small amounts of acetanilide were
usually followed by a momentary increase in the strength of the heart,
the systole being more complete and the relaxations only slightly
lessened. This effect lasted for a few seconds and was then succeeded
by a rapid and marked decrease in efficiency, the contractions grow-
ing quickly less complete while the relaxations were scarcely affected
at all. In some instances the amplitude (efficiency(?)) was slightly
greater after recovery from or as a late effect of the drug. As in the
perfusion experiments it was also noted that following the primary
injections the later injections caused very much less weakening.
For example a preliminary dose of 1 gram lessened the amplitude
70 millimeters, a second injection only 38; a third injection of 1.5
gram, 36 and a fourth also of 1.5 gram only 8 millimeters. These
irregularities make the determination of the action of mixtures con-
taining caffeine or sodium bicarbonate somewhat difficult.
As has been noted the effect of acetanilide is to lessen the contrac-
tile power of the heart and caffeine if an antagonist should prevent
this symptom. Injections of a mixture of these drugs were made
192 WORTH HALE

but in no case was the decrease in the systolic phase prevented.

Whether it may have been less pronounced is of course difficult to
determine since, as has been noted, the effect of the poison appears
to become less at subsequent injections.
Further experiments were carried out in which caffeine citrate was
injected as soon as the acetanilide effect became pronounced with
the hope that the action of the caffeine upon the acetanilide heart
would be demonstrated in this way. This did not prove to be the
case, however, at least in so far as an increased contraction was con-
cerned. In certain experiments there was possibly a slight increased
contraction coincident with the injection of the caffeine but in many
instances immediately following its use the contractions became
manifestly less complete. In conclusion therefore it may be said
that in so far as the contractile power of the heart is concerned caf-
feine has little or no value as an antidote to acetanilide poisoning and
often further weakens the heart.
No increase in the heart rate was observed following the injection
of acetanilide in the doses used in these experiments but on the other
hand there was a slowing of from 10 to 20 beats per minute. The
slowing was evidently due to a direct action on the heart muscle,
since paralysis of the vagi had no effect upon the course of the pois-
oning. As regards rate caffeine proved to be decidedly antidotal;
if it were injected at the same time usually preventing the appear-
ance of any slowing and, if injected later but during the depressant
period after acetanilide, restoring the rate to normal.
There was a marked fall in blood-pressure immediately following
the use of acetanilide which is probably in a large measure due to
the inefficiency of the heart. The injection of caffeine, although
restoring a normal rate, had hardly any effect upon the blood-pressure
curve of acetanilide poisoning. In general the injection of caffeine
caused a secondary fall in pressure or at least appeared to check the
return to normal. The changes in the blood-pressure and efficiency
of the heart (amplitude(?)) are shown in the following table:
 #{149}#{149}orr

THE TOXICITY OF ACETANILIDE 193

Determination of the action of a mixture of acetanilide and caffeine citrate upon the
circulation as recorded by a myocardiograph and a mercury manometer. January
19, 1909. Dog, weight 11 kilograms. Antssthetic, morphine and chioretone.
Vagi paralyzed by atropine.

TIME RATE B. P. SYSTOLE DIABTOLE AMPLITUDE

1000 mg. acetani1id 1:10 120 46 20 155 135

1:10’ 105 16 87 144 57
100 caf. citrate 1:11 129 no effect 60

1:112 132 9 152 143

1000 mg. acetanilide 1:34 109 50 19 162 143

1:342 112 24 52 157 105

100 mg. oaf. citrate. 1:35 112 secondary 28

1:36 112 fall 13 163 150

1500mg. acetanilide 1:53 115 46 15 160 145

1:532 108 26 43 152 109

38
100 mg. caf. citrate. 1:53’ 112 secondary
fall
1:542 126 14 159 145

1500 mg. acetanilide 2:04 112 32 27 154 127

2:04’ 102 24 37 156 119

200 mg. caf. citrate. 2:04’ 123 secondary 36

fall
2:05’ 120 24 154 130

1500 mg. acetanilide 2:18 115 28 35 158 123

2:182 112 22 49 160 111
2:18’ 112 35 158 123
2:192 120 33 159 126

Sodium bicarbonate was also used in conjunction with acetanilide

to determine whether the deleterious effects of acetanilide upon the
dog’s heart could thereby be lessened. The carbonate was given
after the heart had been poisoned by acetanilide and caused as a
momentary effect slight weakening of the heart, but this was followed
almost at once by a rather marked increase in the systole and by a
greater amplitude. The rate did not seem to be altered.
 .,.

194 WORTH HALE

DETERMINATION OF GENERAL TOXICITY

Although often obscured by the popular idea of the role the depres-
sion of the heart plays in acetanilide poisoning a number of other
vital functions are probably equally or more depressed. Because of
this a series of experiments was carried out to determine what modi-
fications in the general toxicity of acetanilide could be secured by
administering it to intact animals with the drugs commonly found in
acetanilide mixtures. In these experiments the drugs were admin-
istered to white mice by hypodermic injection, and to white mice and
guinea-pigs by the stomach.
In the first series of experiments acetanilide and an acetanilide-
caffeine mixture were given to white mice by hypodermic injection.
The mice were obtained from the same lot for each series of experi-
ments and after being weighed they were placed in separate jars
and the dose calculated in grams of body weight. Acetanilide is so
slightly soluble in water that it was found necessary to dissolve it in
dilute alcohol (55 per cent) in order to make the hypodermic method
available. This unfortunately introduces an additional factor and
to reduce the amount of alcohol as much as possible 200 milligrams
of the drug was dissolved in each cubic centimeter of solvent, using
heat to prevent precipitation. The minimum lethal dose by this
method was found to be 0.0013 gram per gram of body weight. The
minimum lethal dose of caffeine was also determined for mice, 0.0007
gram per gram body weight being just sufficient to cause death.
It was believed that if there were any antagonism between caffeine
and acetanilide that a just fatal dose of acetanilide plus a very small
amount of caffeine would prevent death. But in all cases such doses
were fatal. A further series of experiments using smaller amounts
of acetanilide showed not only no antagonism to exist but that death
was caused by amounts of these drugs representing half the sum of
the fatal doses of the two drugs. In other words the sum of the
above fatal doses equaled 0.0020 and a mixture of caffeine and acet-
anilide in a dose amounting to 0.0010 was invariably fatal. Another
series using still smaller amounts demonstrated not only summation
but some synergistic action even.
 THE TOXICITY OF ACETANILIDE 195

Feeding Experiments

In order to give these drugs in a manner more nearly approaching

the method of administering them in therapeutics, a series of white
mice and guinea-pigs were given the drug or the mixture of drugs
by the stomach.
The mice were fed, after the method of Ehrlich, upon cakes to
which the drugs to be tested had been added and the time noted
when the animal died. Whenever mixtures were fed other mice
were given cakes containing the uncombined drugs as a means of
control. The results of a large number of experiments showed that
just as in the hypodermic method caffeine added materially to the
toxicity of acetanilide. A brief summary of these experiments appears
in the following table, the figures representing general averages of
the number of days the animals lived.

Summary

CONTROLS. ACETANILIDE MIXTURES.

SERIES.
Acetanilide Acetanilide Acetanilide
Plain cakes. Caffeine 0.040. o oso 0.050. 0.020.
Caffeine Caffeine0.050. 0.010.

I 43.50 43.25 8.75 5.25 6.50

II 30.50 8.00 4.25
III 7.00 4.75 6.00

Average 43.50 36.87 7.91 4.75 6.25

As will be noted the figures in the above summary indicate that an

acetanilide-caffeine mixture is about twice as toxic as acetanilide
when given by itself. Also caffeine by itself, in double to four times
the dose given with acetanilide is scarcely toxic at all.

Toxicity of Acetanilide and Sodium Bicarbonate

Other experiments were carried out in which white mice were fed
upon cakes containing a mixture of acetanilide and sodium bicar-
bonate. In one series also caffeine citrate was added to the above
mixture, thus giving a preparation quite closely simulating the corn-
196 WORTH HALE

pound acetanilide powder of the United States Pharmacopoeia. In

all of the experiments of this series the feeding of such preparations
indicated that the bicarbonate acted as an antagonist to acetanilide,
the mice fed upon the mixture living longer than those fed upon
plain acetanilide cakes. In the case of the mice fed upon the above
mixture plus caffeine, death appeared more quickly than with the
controls, but also more quickly than when the mice received only
acetanilide and sodium bicarbonate.
This may best be illustrated by the following chart. The black
bars indicate the degree of toxicity. The longer the bar the greater
the poisonous action of the drug.

Acetanilide ____________________________

NaHCO3.

Acetanilide.

Acetanilide
Caffeine
NaHCO3.

Acetanilide
Caffeine.

In a series of experiments mice were fed upon cakes containing

acetanilide and morphine, codeine and heroin. Such mice died more
quickly than the control mice fed on cakes containing acetanilide
alone, thus indicating that such mixtures are also more toxic although
not to the degree that acetanilide-caffeine mixtures are.
Mice were fed upon cakes containing acetanilide and salicylic acid,
but such mixtures failed to change the toxicity of acetanilide in any
way, the mice living the same length of time whether fed upon acet-
anilide cakes or upon acetanilide and salicylic acid cakes. Feeding
experiments with the bromides also were negative as far as changes
in the toxicity of the mixture over the simple drug were concerned.
Experiments were also carried out using guinea-pigs as experi-
mental animals. The drugs were given by the stomach, pills of
suitable size being formed by the use of a small amount of acacia and
arrow-root starch as excipients. In the experiments with an acet-
anilide mixture this was shown to be more toxic than acetanilide
alone-a smaller dose of the acetanilide causing death in the ratio
 THE TOXICITY OF ACETANILIDE 197

of 10 to 13 when given with a small amount of caffeine citrate. These

results serve to confirm therefore the results obtained by all the other
methods of experimentation.
Sodium bicarbonate and acetanilide mixtures were also exhibited
in pill form and fed to guinea-pigs. In this series the control pigs
died as a result of 0.0011 gram acetanilide; those given sodium bicar-
bonate in addition after a dose of 0.0014 gram per gram body weight.

CONCLUSIONS

The result of the experiments upon the heart of both warm and
cold blooded animals indicates that caffeine is of little or no benefit
in acetanilide poisoning in so far as the energy is concerned and in
some cases apparently exerts a harmful effect. The same is also true
in the case of the blood-pressure. On the other hand there appears
especially in the dog to be a well-established antagonism upon
the heart rate. This antagonism however would probably be insuffi-
cient to be of any value in cases of poisoning in man.
The feeding experiments further demonstrate the absence of antag-
onism between acetanilide and caffeine, in all cases the addition of
the latter drug causing death more quickly or with a smaller dose.
This in connection with the imperfect antagonism to the heart action
makes the use of caffeine in acetanilide mixtures especially question-
able.
Sodium bicarbonate in contrast lessens the toxicity of acetanilide,
both in its action upon the heart and upon the intact animal, increas-
ing both the rate and the efficiency of the heart and in the intact
animal increasing the duration of the life or making the use of a larger
dose of acetanilide necessary to cause death.