- Introduction : Hypervitaminosis A,
which affects man and most vertebrates studied,
can be classified as either acute or chronic.
Acute toxicity is caused by the ingestion of
one, or a limited number, of very large doses of
vitamin A within a few hours or days, whereas
chronic toxicity results from recurrent, often
dally, intakes of sizable doses over periods of
weeks, months or years. The most dramatic cases
of acute toxicity in man involve infants given
large doses of vitamin A and arctic explorers or
fisherman who have ingested large amounts of
polar bear, scal or fish liver. Among laboratory
species, acute or chronic toxicity has been
noted in rats, mice, guinea pigs, rabbits, pigs,
cats, dogs, chickens and ducks.
Early signs of acute toxicity in most animals
and man include malaise, drowsiness, nausea and
vomiting, poor balance, muscular incoordination,
increased cerebrospinal fluid pressure, headache
and blurred vision. In most cases these signs
are transient and disappear within a few hours
or days. In experimental animals given very
large doses, loss of appetite, weight loss,
convulsions and paralysis often precede death.
Among humans, however, no fatal cases of acute
hypervitaminosis A have been recorded, although
in some cases it may be an important
contributing factor. Thus, the lethality of
vitamin A for primates is not known. In fact an
LD50 value for a single dose of vitamin A has
only been determined in young rats.
Most concentrated vitamin A preparations
commercially available also contain significant
quantities of vitamin D, and vitamin E. Thus it
is not surprising that many clinically reported
cases of hypervitaminosis A have been produced
by preparations also containing tbese other fat
soluble vitamins. Whereas vitamin D causes a
specific toxic syndrome, vitamin E is considered
to be innocuous, except possibly at very high
doses (> 4 g/day) in humans. Since the acute
clinical manifestations of persons receiving
pure vitamin A and those receiving preparations
containing other fat soluble vitamins are very
similar, vitainin A is probably responsible for
most toxic signs. On the other hand, the
concomitant administration of vitamin D, E or K
has been reported to reduce the severity of
toxic reactions to vitamin A.
In the present paper, we have described the
sequence of clinical manifestations resulting
from the intramuscular injection of a single
large dose of a water-miscible preparation of
vitamins A, D and E into young monkeys. With the
preparation used, an LD50 for hypervitaminosis A
in monkeys bas been estimated to be 560 000 IU.
retinyl acetate (168 mg retinol) per kg body
weight.
Experimental :
Twenty-four young monkeys
(Macacus fascicularis), purchased locally, and
weighing 1.045-1.775 kg were housed individually
in wooden cages and fed a commercial monkey
ration. Their diet was liberally supplemented
with oranges and bananas during their first few
days in the laboratory and at least thrice
weekly thereafter. An injectable fat-soluble
vitamin preparation containing 500 000 IU. of
all-trans retinyl acetate (equivalent to 150 mg
retinol 5, 50 IU of vitamin E, and 50 000 IU of
vitamin D2 per ml in a hydrophilic solution was
used. The retinyl acetate content was verified
spectrophotometrically by diluting a suitable
aliquot in isopropanol and by using an
extinction coefficient ( E ) of 1525 at 325 nm.
Twenty-one monkeys were given single
intramuscular injections of the vitamin
preparation with doses of retinyl acetate
containing from 100 to 500 mg retinol (333
333-1666 667 IU) per kg body weight. These same
doses contained 33 to 167 IU vitamin E and 33
333 to 166 667 IU vitamin D. per kg body
weight.
Two monkeys were given two
successive injections. Monkey 1 was initially
injected, per kg body weight, with the
equivalent of 30 mg of retinol (+Io IU of
vitamin E and 10 000 IU. of vitamin D) followed
two days later with a second dose equivalent to
250 mg retinol per kg (+83.3 1. U. of vitamin E
and 83 333 1. U. of vitamin D2). Monkey 19 was
injected initially, per kg body weight, with the
equivaient of 200 mg of retinol (+66.7 IU of
vitamin E and 133 333 IU of vitamin D2),
followed one day later with the equivalent of
100 mg of retinol per kg (+33.3 1. U. of vitamin
E and 33 333 IU. of vitamin D2). Monkey 14,
which did not receive any injection, was used as
a control. Vitamin injections were given at the
mid-portion of the left posterior thigh muscle.
Following injection, the animals were observed
continuously for acute signs of toxicity during
the ensuing 1-2 hours and were inspected several
times daily thereafter. The monkeys were weighed
periodically. Some animals which died during the
day were observed carefully for terminal
manifestations of death. An autopsy was
performed on the monkeys for gross pathological
changes, particularly at the site of injection,
brain, and visceral organs. Aliquots of samples
of thigh muscle (from both the injected and
uninjected leg), of brain, and of liver frorn
monkeys which received the equivalent of 500 mg
retinol per kg body weight were obtained and
analyzed for vitamin A content using the
trifluoroacetic acid method.
Clinical signss and manifestations
Following injections of retinyl acetate
equivalent to 200-500 mg retinol/kg body weight,
the first signs of acute toxicity appeared
within 3 to 35 mn. The most frequent signs
were yawning and droopiness of the eyelids,
which gave the animal a drowsy or tired
appearance. Yawning was particularly striking
and recurrent. One animal (monkey 10), for
example, yawned 15 times during the first hour
following injection. Drowsiness characterized by
transient closure of the eyes seemed
uncontrollable and occurred repeatedly, a sign
that contrasted sharply with the apprehension
usually shown by an uninjected monkey when under
constant surveillance.
Hyperextension of the neck, with or without a
rapid, jerky shaking of the head, was
particularly promment in one animal (monkey 7),
in which this phenomenon was observed nine times
during the first two hours. Flits of nausea and
vomiting were consistently observed at the
highest dose level tested (500 mg retinol/kg).
Three animals (monkeys 2, 3, 13) apparently
attempted to obtain relief from the nausea by
drinking water. In some monkeys motor
hyperactivity, consisting of incessant,
purposeless pacing or of repeated climbing up
and down the side of the cage, was a major
manifestation (e. g. monkey 6). Ataxia, as
manifested by swaying of the body and the
failure to maintain an erect posture,
incoordinate muscle movements, such as fumbling
or inept attempts to grasp a cage bar and
slipping while climbing or walking, was seen
only at the higher dose levels (400-500 mg
retinol/kg).
In six monkeys injected with the equivalent
of 200 mg retinol/kg, drowsiness was the only
early manifestation in two animals, and yawning
was in one animal. None of the animals which
received the equivalent of 100 mg retinol/kg
exhibited any early or late signs of toxicity.
These early toxic effects generally subsided or
disappeared within 1/2 to 1 hour after their
onset. Even monkeys which received the highest
dose level (500 mg retinol/kg) became relatively
active and seemingly well within 2 hours after
injection.
Following this apparent recovery, however,
signs of toxicity gradually reappeared.
Decreased activity and malaise became obvious
within the first 24 hours in all animals given
300-500 mg retinol/kg, but appeared somewhat
later (3rd to 6th day) in monkeys given 200 mg
retinol/kg. Two animals in this latter groups
(monkeys 16 and 18), although exhibiting no
early signs of toxicity, showed late toxic signs
at 3-6 days and subsequently died.
Drowsiness was the major presenting late
manifestation, which became increasingly
apparent in those animals which eventually
succumbed. Drowsiness was often accompanied by
inappetence, progressive weight loss and
recurrent vomiting. In some animals (monkeys 8,
10, 16, 18) with survival times ranging from
51/2 to 151/2 days, increased lacrimation was
observed on the 4th or 5th day, followed after a
day or two by a mucoid watery discharge from the
nose. The skin round the eyes and nose was
apparently itchy and often became reddened and
thickened from scratching. These latter
manifestations were transient and subsided after
a few days.
The terminal manifestations and manner of
death were observed in several monkeys. Four
animals (monkeys 2, 3, 5, 6) that had been given
the higher dose levels became increasingly weak
and eventually lapsed into coma. Progression
into a deep coma was slow, and simple reflexes
like the palpebral, corneal, and pupillary light
reflexes were lost only minutes before death.
During the two hours prior to death, monkey 6
showed hyperactive deep tendon reflexes of the
extremities and occasional nystagmus. In three
of these monkeys, respiratory fallure preceded
cessation of heart contractions. Respiratory
abnormalities consisted of spasmodic or jerky
inspiratory movements and uneven breathing. In
two animals (monkeys 11 and 12), which were
given lower doses of retinvl acetate, death was
preceded by a generalized convulsive seizure.
Convulsions probably occurred ln monkey 17, as
well, who was found dead with one arm tightly
flexed, her fist clenched, the face distorted
from spastic facial muscles, and the tongue
protruding and bitten.
-
- Mortality, survival time and weight
changes
- All 11 monkeys niected with retinyl acetate
at dose levels of 300 to 500 mg retinol per kg
body weight and 4 of 6 animals injected with a
dose of 200 mg retinol per kg died. On the other
hand, all 4 monkeys injected with 100 mg retinol
per kg surv'ved (Table II). A conventional plot
of log dose vs. mortality yielded an LD., of 168
mg retinol/kg body weight. The survival time for
fatal cases ranged from one to 151/2 days. The
survival times of monkeys which died at doses of
500 and 400 mg retinol per kg were generally
shorter than those which died at doses of 300
and 200 mg per kg. Interestingly, among the
monkeys which died at the different dose levels,
the females tended to succumb earlier than the
males. Except for monkey 3, all fatal cases lost
weight. On the other hand, monkeys which
received 100 mg retinol per kg and those which
survived the dose equivalent to 200 mg retinol
per kg, ate well and either maintained or gained
weight after dosing.
Of the two monkeys (both males) which
received two injections, monkey 1 survived while
monkey 19 died. Monkey 1 showed no signs of
toxicity 2 days after recelving the equivalent
of 30 mg retinyl acetate per kg. Following a
second dose of 250 mg retinol per kg, however,
manifestations similar to those described above
were seen, namely yawning, nausea,
inappetence and drowsiness. These signs
disappeared gradually and the animal appeared
fully recovered 14 days after the second
injection. Monkey 1 weighed 1260 g on arrival in
the laboratory, 1325 g at the time of the first
injection (5 days after arrival), and 1353 g 30
days after the second injection. Monkey 19,
which received a total of 302 mg retinol per kg
given in two doses (231 mg and 115 mg) on
consecutive days, died 2 days after the last
injection.
-
- Autopsy Findings
Thirteen monkeys were autopsied. The site of
injection showed no pathology except for a
slight swelling in those animals who died within
2 days. No gross evidence existed of an
inflammatory or abnormal tissue reaction in the
injected thigh muscle. Hair growth and skin
histology appeared normal, both over the site of
the injection and elsewhere, even in monkeys who
died 15 days after injection. Serial sections of
the brain were normal. Hemorrhages were absent.
In two animals (monkeys 10 and 11), however, the
brain surfaces appeared particularly shiny and
moist, possibly indicative of an increase in
cerebrospinal fluid pressure or of edema.
The livers of three anirrials (monkeys 4, 8
and 9) had small, pinhead-sized, yellowish white
nodules on the surface as well as in the
parenchyma. These small nodules were fairly firm
in consistency, revealed no purulent material,
and probably were chronic lesions unrelated to
the hypervitaminosis syndrome. With the
exception of monkey 3, the stoinach of autopsied
ammals was generally empty, an expression of the
anoi-exia observed in the majority of the
animals. In a few, the stomach was slightly
bloated with gas, presumably resulting from
terminal respiratory difficulties.
Monkey 12, which showed severe weight loss
and anorexia, had an oval ulcer with a diameter
of 11/2 cni located at the upper third of the
greater curvature of the stomach. The serosal
side of the ulcer was adherent to the diaphragm,
which suggested a chronic condition unrelated to
the hypervitaminosis. No grossly visible
parasites were found in the intestinal tract and
no hemori-hages were noted in the mucosa. The
rest of the visceral organs and lungs showed no
remarkable findings. As expected in young
animals, testes in male monkeys were
undescended, and the uterus in females was
infantile.
Tissue Retinol Concentrations
The vitamin A concentrations in several
organs of the three monkeys receiving the
highest dose of retinyl acetate, equivalent to
500 mg retinol/kg, were analyzed after death. As
expected by other studies on the rapid
mobilization of water-miscible vitamin A from
muscle injection sites, little residual vitamin
A was present in the injected muscle. The liver,
which is the usual repository of vitamin A,
contained only a tiny fraction of the dose
(0.6-3%), even in monkey 2, which survived for
over 2 days. The concentration of vitamin A in
the brain of all three monkeys, on the other
hand, greatly exceeded the normal level of 1
µtg/g.
-
- Discussion
Three distinct stages were noted in the toxic
syndrome induced in young monkeys upon the
intermuscular injection of a single, very large
dose of a water-miscible vitamin A-D-E
preparation. Early clinical manifestations,
which appeared as early as 3 minutes after
injection and usually subsided within an hour,
included yawning, drowsiness, nausea and
motor hyperactivity or incoordination. Not all
monkeys showed all early signs, however, and the
severity of a given manifestation varied
greatly, even within a specific dosage
group.
Late signs, which appeared within 24 hours of
dosage, consisted of drowsiness, general
malaise, physical inactivîty, and lack of
appetite with resultant weight loss. Finally,
animals which had recelved lethal doses slowly
lapsed into a coma, experienced respiratory
difficulties, and in several carefully observed
cases, died of respiratory failure. Convulsive
seizures were also a common terminal event.
Although the variance in each treatment group
was large, some trends in response were evident.
Both the time of onset of the first sign and the
survival time were inversely proportional to the
dose. Thus, from a linear regression plot of the
time of onset data, the first sign was noted at
about 18 minutes at a dosage of 200 mg
retinol/kg and at about 9 minutes at 500 mg
retinol/kg. Similarly thus average survival
times from a linear regression plot was around 8
days at 200 mg/kg and about 1 day at 500mg/kg.
Interestingly, females seemed to succumb more
quickly than males to lethal doses, although
there was no apparent difference in LD50 For
example, the mean survival times of females (n =
7) and males (n = 8) were 2.31 + 1.55 and 7.30
± 6.51 days, respectively, independeli, of
dosage. Most but not all monkeys that ultimately
succumbed also showed early toxic
manifestations. No relationship existed,
however, between the rapidity of onset of the
first sign and the survival time in individual
monkeys (n = 13, R2 = 0.02).
Biochemical events can be nicely correlated
with different stages of toxic manifestations.
Vitamin A in a water-miscible form is very
rapidly mobilized into the plasma from
intramuscular injection sites. Consequently,
plasma vitamin A levels, essentially in the form
of the injected ester, reach very high levels
within minutes of injection. The initial stage
of hyper-vitaminosis thus relates closely to
this surge of injected retinyl ester into the
plasma. Within several hours of injection,
plasma retinol levels are already falling
whereas the concentrations in other organs, such
as the kidney, brain and liver, are steadily
rising. Although the liver ultimately stores
much of the dose retained in the body, even at
these high levels, maximal storage usually
requires 3-7 days. In the meantime much of
injected vitamin A is distributed aniong various
body organs. It is noteworthy that monkeys
recelving 500 mg retinol/kg only stored 0.6-3.3%
of the dose in the liver within 1-2 days,
whereas the brain concentration was
approximately 80-fold higher than normal. Thus,
late manifestations of the toxicity syndrome
seem to be associated with the localization of
the injected vitamin A in specific organs.
Clearly the toxic signs of greatest concern
are elther neuromuscular in nature or relate to
basic physiological processes controlled by the
central nervous system. That vitamin A reaches
such high concentrations in brain relative to
its normal level suggests that the ester itself,
or some metabolite of it, is disrupting brain
function. Both retinol and retinoic acid can
induce hypervitaminosis A, by the way, and the
concentration of the normal plasma carrier,
retinol binding protein, which tends to be
protective does not increase in the
hypervitaminotic state. Although retinol at high
concentrations is known to labilize membranes of
red blood cells and intracellular organelles, it
is noteworthy that no evidence of hemolysis was
seen in the skin or tissues of these monkeys at
autopsy. It is of course possible that the
increased cerebrospinal fluid pressure
characteristic of hypervitaminosis A is giving
rise to some if not all of these abnormal
nervous reactions.
The LD50 value for monkeys of 168 mg
retinol/kg body weight is an unexpectedly high
value. Nonetheless, an LD50 value for rats, the
only other reported in the literature, is 3 g
retinol/kg, an even larger number. In preschool
children given a single oral dose of 300 000 IU.
retinyl palmitate in a water miscible form,
transient hypervitaminosis A was noted in about
4 % of those treated. Assuming a mean weight of
10 kg in affected children, the dosage level was
about 9 mg retinol/kg, less than one-tenth of
that which produced no signs in injected
monkeys. Inasmuch as the early signs observed in
children at a much lower dosage are essentially
identical w th the stage one signs observed in
monkeys, humans do seem to be more sensitive to
vitamin A toxicity.
Finally the possible effects of other
components of the preparation should be
considered. VitaminD produces a toxic syndrome,
but its nature is quite different from that
reported here. VitaminE, as already indicated,
is not known to produce toxic manifestations at
the doses used. Both compounds may offset in
part the effects of hypervitaminosis A, however,
and consequently might raise the LD50 value. The
non-ionic detergent used in the preparation is
of the sorbitan group, which can be hemolytic
when injected intravenously but is benign when
given orally or intramuscularly in the doses
used. Furthermore, no evidence of hemolysis, the
major toxic action of these detergents, was
noted in these experiments. It therefore seems
most likely that the major, if not sole,
constituent of the preparation causing the toxic
manifestations described in this paper is
vitamin.
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