THE INFLUENCE OF HEAVY METALS ON THE

ISOLATED FROG HEART

WILLIAM SALANT AND HELENE CONNET

Received for publication February 14, 1920

Although no justification for experimental investigation on

the action of heavy metals is necessary, it might be pointed out
by way of explanation that a better understanding of their
behavior in the body is particularly desirable for practical
reasons. Many of the heavy metals frequently occur in various
food products and some of them give rise to industrial poison-
ing, a subject which, thanks to the efforts of those interested in
the improvement of the condition of the working classes, has
within recent years been forced upon the attention of the public
and is rapidly becoming an integral part of social and preventive
medicine. For these reasons the writer has with the assistance
of some of his collaborators planned and conducted for several
years past investigations on heavy metals. Observations were
made on general toxicity, absorption, eliminaticn, distribution in
the tissues
and organs of the body, their effect on carbohydrate
metabolism, on the kidney and on the isolated intestines.
While the pharmacology of heavy metals has for years re-
ceived a great deal of attention at the hands of different work-
ers, some of their effects on different organs are still unknown or
in doubt and is therefore in need of investigation. This is not-
ably the case as regards their action on the circulation and par-
ticularly their effect on the heart. The statements on the sub-
ject in text-books of pharmacology agi#{128}ethat heavy metals are
cardiac depressants, but examination
of the literature shows
that these teachings in standard works on pharmacology are
based on indirect and very frequently, insufficient evidence.
The senior author had therefore planned and carried out experi-
ments with a number of heavy metals on the isolated heart.
Some of the results are presented in the following pages.
27
218 WILLIAM SALANT AND HELENE CONNET

METHODS

The experiments were conducted on the isolated heart of frogs

weighing about 30 to 40 grams and were carried out at room
temperature. The perfusion time with the different metals was
usually one minute, but in some instances, it was one-half, two and
sometimes even three minutes. The method of perfusion was
the same as that previously employed by the writer and his
collaborators. For a detailed description the reader is referred to
the publications by Salant and Livingston (1) and Salant and
Connet (2), Ringer’s solution was used throughout this re-
search. In some experiments the sodium bicarbonate was
replaced by the equivalent amount of sodium chloride. Ringer’s
solution was perfused for some time before the effects of the
different metals were tested. Since the addition of salts of the
metals increased the tonicity of the solution, control tests were
made to observe the effects
of a slightly hypertonic Ringer’s
solution. Sodium actetate 1: 10,000 in Ringer’s solution was
employed for this purpose. This also served as a control for.
some of the metals which were used in our experiments in the
form of acetates. It may be stated in advance that this con-
centration of sodium acetate failed to cause any disturbance of
heart action.

EXPERIMENTS WITH COPPER

The influence of the salts of copper on the heart was studied

by Harnack (3), who observed that cardiac paralysis occurs
soon after the administration of different salts of the metal.
Harnack and Hafemann (4) found in perfusion experiments with
the isolated frog heart that copper was toxic to cardiac muscle
but that it did not affect the nervous mechanism of the heart
since they obtained a reaction upon stimulating the vagus.
This was corroborated by tests with atropine. According to
Schwarz (5) different salts of copper perfused through the iso-
lated frog heart caused retardation preceded by a preliminary
increase in frequency, but the heart finally stopped in systole,
and could not be revived, which indicated complete paralysis.
 INFLUENCE OF METALS ON FROG HEART 219

Schwartz claimed that 0.25 mgm. CuO in 50 cc. fluid produced

arrest of the heart within a few minutes when copper sulfate or
copper sodium tartrate was employed. Copper albuminate was
less toxic than the other salts of copper. He stated that dilute
solutions of this salt may even cause stimulation of the heart.
The experiments of Richet (6) may also be mentioned in this

FIG. 1. FROG HEART PERFUSED FOR ONE MINUTE WITH 1: 10,000 COPPER
SULPHATE IN RINGER SOLUTION MINUS SODIUM BICARBONATE

Marked depression of heart is shown which persisted when the heart was no
longer exposed to the action of copper. The heart also showed irregular action
in the after period. Reduced one-half.

connection. He found that when copper chloride was applied

to the exposed frog heart in situ it caused arrest of heart action
when the concentration of the salt of the metal was above 33
grams per liter.
In our experiments with copper the sulfate was used exclu-
sively. Complete abolition of cardiac activity took place in
220 WILLIAM SALANT AND HELENE CONNET

some experiments with a concentration of 1: 10,000 copper sul-

fate in Ringer’s solution when perfused for 1 minute. In a
number of cases the heart continued to beat during and for some
time after its exposure to copper sulfate, but the heart beats
in these cases were very feeble, sometimes barely perceptible
(see fig. 1). Only in one experiment in which 1: 10,000 copper
sulfate in Ringer’s solution was used did the heart continue to
beat quite vigorously though the force was much below normal.
When the heart was perfused with the same concentration of
copper sulfate for two minutes it was sufficient to produce pro-
longed stoppage of its action. The effect of copper sulfate on
the heart was much more marked when the concentration was in-
creased. A sOlution of 1: 1,000 produced promptly complete
inhibition of the heart.

EXPERIMENTS WITH IRON

Although much was written on the effects of iron in the

body very little attention was paid by experimenters to its
effect on the circulation. Myers and Williams (7) studied the
effect of iron tartrate in cats and rabbits. After the intravenous
injection 10 to 17 mgii. into rabbits and 50 mgm. into cats they
reported a fall of blood pressure. It is doubtful, however,
whether this could be attributed to cardiac injury produced
by the iron for the frequency of the heart was increased and
when death resulted from repeated injections the heart con-
tinued to beat for some time. According to Richet (8) iron
chloride applied to the exposed heart of the frog in situ abolished
its activity when the concentration was above 22 grams per liter.
Iron citrate which we used in our experiments produced a
marked effect on the heart only when used in sufficient concen-
tration. When perfusion was made with Ringer’s solution con-
taining 1: 10,000 iron citrate for one minute the only change we
observed was a moderate increase of amplitude which persisted
for some time after exposure to the salt was discontinued.
Quite different was the action when the concentration of iron
citrate was 1: 1,000 (fig. 2) and perfusion lasted for the same
 INFLUENCE OF METALS ON FROG HEART 221

period. Heart action was depressed, there was a decrease of

amplitude amounting in some cases to about 50 per cent and in
some experiments the force of heart action rapidly diminished
and finally complete cardiac inhibition occurred lasting for sev-
eral minutes. The duration of exposure to the iron exerted a
decided influence on its effect on the heart. When perfusion

:11 IIIlII

i-S,oOO T eJ’.ot.
f.%. %M.QZAS B
I . ‘(‘fa.,.’
t. 1.’ y’

.“‘

cL#{149}.& 3Oc
;1::..

Fio. 2. PERFUSION OF THE FROG HEART WITH 11,000 IRON CITRATE IN

RINGER’S SOLUTION FOR ONE-HALF MINUTE

Heart action became accelerated, but the force was decreased. Note tendency
to improvement when perfusion with iron was discontinued. Reduced one-half.

lasted half a minute the more concentrated solution either caused

a suspension of heart activity for a very brief period or merely
decreased the force of heart action, the frequency being appreci-
ably increased in some experiments. Recovery occurred in all
cases except in one in which the heart was subjected a number
of times to the effect of a concentrated solution of the salt.

THE JOUR. OF PHARM. AND EXPER. THERAP., VOL. xv, NO. 3

222 WILLIAM SALANT AND HELENE CONNET

In this case the arrest of heart action lasted two and a half
minutes. At the end of this time there was a slight attempt at
recovery and only a few very feeble beats were executed.

EXPERIMENTS WITH MANGANESE

The action of, manganese on the heart was studied by Kobert

(9) ,many years ago. According to his observations which were
carried out on the intact animal manganese first accelerated
then retarded and finally paralyzed the heart. But he also tested
the action of manganese on the isolated heart which he sus-
pended in defibrinated ox-blood which contained 10.5 per cent
of manganese. Initial stimulation was likewise noticed in these
experiments and was followed by paralysis.. Observations with
manganese were made later by Wohiwili (1Q) His results,
based on subcutaneous injections into frogs, indicate that man-
ganese and iron cause gradual retardation of the heart and finally
stoppage in diastole. Our results with manganese chloride
showed that its effect n the heart is practically the same as
that of iron citrate. When the heart is perfused for two min-
utes with Ringer’s solution.. containing 1: 10,000 of manganese
chloride there was flrsti moderate decrease of amplitude which
was followed in one test by irregular b,eart. action and consider-
able augmentation in the force with diminution in the frequency
of heart action. A stronger solution completely inhibited heart
action. This was followed by very feeble contractions which
were just strong enough to record a wave on the kymographic
record and remained in this condition for a considerable time.

EXPERIMENTS W1T1 NICKEL AND COBALT

Stuart (11), working in Kobert’s laboratory, tested the action

of cobalt and nickel on the isolated frog heart. From his obser-
vations on intact animals he concluded that these metals are
not cardiac depressants although they caused a fall of blood
pressure, for in his experiments on the isolated frog heart he
failed to notice any diminution of cardiac activity. The ex-
periments of Richet (12) showed, however, that both of these
 INFLUENCE OF METALS ON FROG HEART 223

metals were toxic to the frog heart. More recently Wohiwill

(13) studied the effect of these metals on the frog heart. After
the subcutaneous injection of the citrate of cobalt or nickel

sq’. a.u. 3c tc

A B

Fia 3. PERFUSION WITH 1 10,000 NICKEL ACETATE IN RINGER’S SOLUTION FOR

ONE MINUTE

The effect of the nickel indicates initial depression followed by increased force
with diminished frequency of the heart (A). Two minutes after perfusion was
discontinued heart action was nearly normal (B). Reduced one-half.

the heart continued to beat regularly until the animals died.

Towards the end of the experiment, however, retardation
occurred and steadily increased until the heart stopped in
diastole. It is evident from the observation of previous workers
224 WILLIAM SALANT AND HELENE CONNET

that nickel and cobalt are not very toxic to the heart. It is
equally clear, however, that the subject ought to .be investi-
gated de novo. We, therefore, undertook experiments with
both of these metals. Our results with nickel which was used
in the form of acetate showed that it was a cardiac depressant
when perfused in sufficient concentration. A solution of
1: 10000in Ringer’s solution perfused for one minute produced
in one experiment diminished heart action, both the force and
the frequency being decreased at first (fig. ). The number of
heartbeats in thirty seconds decreased from 20 to 9. #{149}The ampli-
tude was reduced about 40 per cent at the same time. #{149}The
effect was less marked in other experiments. The force of
heart action was diminished by one-fourth to one-third, the
frequency showd only a moderate diminution. In all cases
recovery took place soon after perfusion with nickel acetate
was discontinued. Complete abolition of cardiac action was
observed with 1: 1000 nickel acetate. This was noticed when
the perfusion lasted only half a minute. Even after perfusion
with this concentration recovery of the heart took place within
a few minutes and in one experiment the heart not only recov-
ered soon after butt. the contractions became much stronger,
though less frequent. In some experiments,
‘ it might be added,
the heart became irregular before it stopped.
The results obtained with cobalt chloride were somewhat
differeqt from those with ni#{235}kelacetate. Perfusion with
1: 10,000 in Ringer’s solution produced in some cases a very
marked diminution in force and sometimes also, in frequency of
the heart (fig. 4). This is especially noticeable when the period
of perfusion lasted two minutes but in all cases recovery of the
heart occurred. A concentration of 1: 1000 caused complete
standstill of the heart within a few seconds, and remained in
this condition five minutes and sometimes longer before the
heart resumed its activity.
INFLUENCE OF METALS ON FROG HEART 225

.,.
226 WILLIAM SALANT AND HELENE CONNET

EXPERIMENTS WITH ZINC

All the writers on the action of zinc are agreed that it pro-
duces paralysis of the heart. Letheby (14) stated that the cir-
culation was affected in poisoning with zinc and suggested that
the dilatation of the heart in this condition was probably due
to paralysis. Falck (15) administered a dose of 1 gram of
zinc acetate to medium-sized rabbits by mouth which caused
death in these animals. According to Falck this was due to

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*,. 1k.,1trt’s 3

T(,.%..)

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rj

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....L. ...... . ....

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paralysis of the heart. Blake (16) observed in experiments with

zinc sulphate which he injected into the veins and arteries of dogs
that prompt arrest of heart action followed the introduction of
the salt. Harnack (17) has likewise shown in experiments with
frogs that the heart may be paralyzed after the administration of
 INFLUENCE OF METALS ON FROG HEART 227

small doses of the metal in the form of a double salt of pyrophos-

phate. The action of zinc on the heart was studied later, by
Richet (18) whose results corroborated previous investigations on
this subject A systematic study of the action of zinc on the
isolated heart was made by Sacher (19) who perfused the frog
heart with defibrinated beef-blood which contained zinc albu-
minate or sodium zinc tartrate. When the concentration of zinc
was 1: 50,000 heart action was retarded and if perfusion lasted
five hours cardiac paralysis took place. When the concentra-
tion of zinc was doubled serious injury to the heart could be
noticed in a few minutes and cardiac paralysis occurred within
one hour.
We used various concentrations of zinc malate in our experi-
ments. Tests were made with 1: 1,000, 1: 2,000, 1: 10,000, and
1: 50,(XX). The effects varied somewhat with very dilute solu-
tions. In one experiment in which 1: 50,000 zinc malate was
perfused for one minute a moderate amount of reduction in
amplitude and scarcely any diminution in frequency was ob-
served. But the same concentration of the zinc malate, which
was likewise perfused for one minute, produced very marked
depression in other experiments which persisted for a consider-
able period after perfusion with zinc walate was discontinued
(fig. 5). The contractions were barely strong enough to be vis-
ible five and a half minutes after the perfusion of the zinc salts
was stopped, thus indicating that the injury tended to be per-
manent even when theheart was exposed to a very dilute solu-
tion of zinc salts. It might be observed that in this case heart
action was not very vigorous before perfusion with the zinc?
.

which may account for the marked difference of effect in. these
experiments. These results are suggestive. It is quite possible
that zinc is much more toxic to the heart when it is not in good
condition. The use of higher concentrations is in all cases ulti-
mately followed by total abolition of heart action. The heart
at first became irregular, its frequency decreased and its force
also showed considerable diminution. This condition con-
tinued in nearly all experiments during and for some time after
the perfusion with the zinc salt. The heart then either sud-
228 WILLIAM SALANT AND HELENE CONNET

denly stopped or executed a few weak contractions before ceas-

ing its activity. The tendency in most experiments with higher
concentrations of zinc was toward permanent stoppage of the
heart. The effcct of medium concentrations such as 1: 10,000
zinc malate indicated depression, but not complete abolition of
heart action. In some cases the heart recovered from the
treatment, but repetition of the experiment on the same heart
several minutes later produced complete arrest of its action.

EXPERIMENTS WITH CADMIUM

Marm#{233} (20) stated that cadmium retards the circulation in

man and in animals. Thirty years later Athanasaiu and Lang-
lois (21) reported experiments with cadmium salts on frogs and
on the isolated heart of the turtle. These observers noticed that
heart action became slower and the force of the heart was dimin-
ished after the injection of cadmium sulfate. They also ob-
served that there was a marked prolongation of the pause and a
certain periodicity of the rhythm. The effect was the same on
the isolated heart of the turtle. They also reported that cad-
mium differs somewhat in its action on the heart under changed
conditions of temperature as they noticed that the depressing
effect of cadmium was preceded by acceleration when heated to
28#{176},
or 31#{176}C.That cadmium is injurious to the heart was also
reported by Richet (22).
Our experiments showed that cadmium is very toxic to the
heart. Very dilute solutions perfused for one or two minutes
often produced. injirious effects. In one experiment 1: 1Q00,Q00
cadmium acetate in Ringer’s solution perfused for two minutes
caused a slight diminution in frequency and a wellLmarked de-
crease in force of heart action (fig. 6). The same concentration
produced complete arrest of the heart in about one and one-half
minutes in another experiment in which the perfusion time was,
only one minute. The injurious effect of cadmium acetate was
very marked with a concentration of 1: 100,000 (fig. 7). The heart
was either completely arrested or its action became very feeble
and. remained in this condition for three or four minutes and
 -ioo,oo CcJ a.tita±a..
e Y_4t!O3

r(I-n..)

tjr#{248}J..u sjt. .c.

I.
i.o, (.i.C.)

FIG. 6. EFFECT OF 1: 1,000,000 CADMIUM ACETATE IN RINGER’S SOLUTION MINUS

SoDIUI BICARBONATE WHEN PERFUSED THROUGH THE FROG
HEART FOR ONE MINUTE

Heart action became much weaker continuing ‘in this condition after per-
fusion with the cadmium was discontinued.

i-QO,oooCd. t1i

P4Ai. 0(i -

30 1
dJcp5

.‘.‘ 5kC ....-J O IC

k.

FIG. 7. PERFUSION WITH 1:100,000 CADMIUM ACETATE IN RINGER SOLUTION

MINUS SODIUM BICARBONATE

Note that heart action became very feeble as a result of exposure to the action
of a weak solution of cadmium and showed only a very slight improvement
when the perfusion with cadmium which lasted only one minute, was discon-
tinued. Reduced one-half.

229
 230 WILLIAM SALANT AND HELENE CONNET

sometimes even longer. In both cases, however, recovery was

ultimately observed. But this was not the case with higher
concentrations of cadmium acetate. The effect in these ex-
periments was very prompt; the amplitude decreased very rap-
idly and the heart remained at a standstill either permanently or
for a long time.

EXPERIMENTS WITH URANIUM ACETATE

As far as we could ascertain the literature did not contain

any reports of studies on the action of uranium on t.he circula-

1.1. .-

1 ( (rIk,..)

AS.. LI Ck,.cI .3C .k C

k ....L

Fzo. 8. SHOWS GRADUAL WEAKENING OF HEART ACTION BY 1 1,000 URANIUM

ACETATE WhEN PERFUSED FOR ONE MINUTE THE HEART STOPPED
SHORTLY AFTER PERFUSION WITH URANIUM WAS DISCONTINUED

Reduced one-half.

tion. Although our work on this subject is incomplete it is

nevertheless of interest to state that our observations indi-
cated that uranium acetate in sufficient concentration was a
cardiac depressant. Heart action was completely inhibited by
1: 1,000 uranium acetate in Ringer’s solution (fig. 8). It might
 INFLUENCE OF METALS ON FROG HEART 231

also be observed that the effect persisted for a considerable

length of time after perfusion with the metal was discontinued
while in other instances the injury caused was permanent.
With lower concentrations of uranium acetate the depression
produced was slight but heart action became irregular as a
result of the treatment.

DISCUSSION AND CONCLUSIONS

Analysis of the results obtained in the present report wifi

show first that the effect of closely allied metals on the heart is
practically the same in all with the exception of zinc and cad-
mium. Marked differences in action were observed, however, in
the different groups. Thus the effect of iron and manganese
was approximately the same. Both were equally toxic to the
heart. The depression produced upon the heart was the same
in extent and character when the concentration was 1: 1,000.
With a concentration of 1: 10,000 of the salt of either metal,
heart action became, on the contrary, more vigorous, but showed
irregularity of action. Nickel and cobalt manifested very little
difference in toxicity. Both of them, however, were more depres-
sing to the heart than iron and manganese. The effect of copper
sulfate was even more marked. HeaPt action became very
weak under the influence of a concentration of 1: 10,000; both
force and frequency showed a marked decrease. By far the
most toxic of all the metals we studied were zinc and cadmium.
Zinc malate in 1: 50,000 in Ringer’s solution produced approxi-
mately the same effect as a solution of copper sulfate which was
five times as strong. In the case of cadmium the difference was
still greater. The effect of 1: 100,000 cadmium was about the
same as the more conceiltrated solution of zinc. The comparative
toxicity of the different metals may be represented as follows:
iron, manganese, uranium, < nickel and cobalt <copper <zinc
<cadmium. It will be noticed that uranium is not more toxic
than iron and manganese. The work of Richet may be re-
called in this connection. In his studies on the comparative
toxicity of different metals he used the heart of the frog as a test
232 WILLIAM SALANT AND HELENE CONNET

object. His results likewise showed that cadmium was more

toxic than zinc, that nickel and cobalt were equally injurious to
the heart but their effect was only about one-quarter that of
cadmium. Iron and copper were both toxic bi.tt the action’ of
the latter was the greater of the’ two, which is contrary” to ttie
results we obtained with these metals.

REFERENCES

(1) SALANT AND LIVINGSTON: Am. J. Physiol., 1916, xli, 21.

(2) SALANT AND CONNET: J. Pharm. and Exp. Ther., 1918, xi, 81.
(3) HARNACK: Arch. Exp. Path. u. Pharmakol., 1875, iii, 44.
(4) HARNACK AND HAFEMANN: Arch. Exp. Path. u. Pharmakol., 1883, xvii,
145.
(5) SCHWARZ: Arch. Path.
Exp. u. Pharmakol., 1894-5, xxxv, 437.
(6) RICHET: Comt. Rend.
Ac. Sc., 1882,#{149}xciv, 742.
(7) MEYER AND WILLIAMS: Arch. Exp. Path. u. Pharmak., 1880, xiii, 1370.
(8) RICHET: Comp. Rend. Ac. Sc., 1882, xciv, 742.
(9) KOBERT: Arch. Exp. Path. u. Pharmak., 1883, xvi, 361.
(10) WOHLWILL: Arch. Exp. Path. u. Pharmak., 1907, lvi, 404.
(11) STUART: Arch. Exp. Path. u. Pharmak., 1884, xviii, 151.
(12) RICHET: Comt. Rend. Ac. Sc., 1882, xciv, 742.
(13) WOHLWILL: Arch. Exp. Path. u. Pharmak., 1907, lvi, 404.
(14) LETHEBY: The Lancet, 1850, ii, 123.
(15) FALCK: Schmidt’s Jahrb., 1861, cxii, 18.
(16) BLAKE: Edinb. Med. J., 1841, lvi, 109.
(17) HARNACK: Arch. Exp. Path. u. Pharmakol.. 1875, iii, 44.
(18) RICHET: Comt. Rend. Ac. Sc., 1882, xciv, 742.
(19) SAdlER: Arb. a. d. Pharmak. Inst. zu Dorpat, 1893, ix, 88.
(20) MARME: Zeitsch. f. Rat. Med., 1867, xxix, 125. -‘

(21) ATHANASIU AND LANGLOIS: Arch.. de Phsyioh, 1896, vii, 25.

(22) RICHET: Comt. Rend. Ac. Sc., 1882, xciv, 742.