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mise à jour du 24 avril 2003
Int J Vitam Nutr Res
1981; 51(4); 331-41
A lethal hypervitaminosis A syndrome in young monkeys (Macacus fascicularis) following a single intramuscular dose of a water-miscible preparation containing vitamins A, D2 and E
Manuel P Macapinlac , James A Olson
Department of biochemistry Mahidol university, Bangkok, Thaïland


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.


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.