A.B. Makar

Methanol poisoning I. The role of formic acid in the development of metabolic acidosis in the monkey and the reversal by 4-methylpyrazole

Abstract The administration of methanol (3 g/kg) to rhesus and pigtail monkeys produced signs and symptoms similar to those described for methanol poisoning in man. These were a mild central nervous system depression, a latent period of 8–12 hr when no signs were observed, followed by a severe metabolic acidosis leading to coma and death 12–33 hr after the initial administration. The gradual development of metabolic acidosis coincided with the accumulation of formic acid in the blood, and the decrease of bicarbonate in the plasma. There was an increase in the anion gap during the period of metabolic acidosis, and formic acid concentration accounted for about one-half of the increase observed. Therefore, formic acid was a major, but not the only, determinant of the metabolic acidosis. Administration of 4-methylpyrazole (50 mg/kg), a potent inhibitor of monkey hepatic alcohol dehydrogenase, produced a 75% inhibition of the rate of [ 14 C]methanol metabolism to 14 CO 2 in the monkey. During the first 36 hr following the administration of 4-methylpyrazole and methanol, no metabolic acidosis developed, no formate accumulated in the blood, and no signs of toxicity were observed. After a single dose of 4-methylpyrazole and methanol, the toxic syndrome was delayed by about 36 hr in the monkey, after which time the onset of metabolic acidosis and the accumulation of formic in blood was noted. The use of the monkey as a model for the study of methanol poisoning is presented, and the possible use of 4-methylpyrazole in the treatment of methanol poisoning is implicit.

Methanol poisoning. V. Role of formate metabolism in the monkey.

The accumulation of formic acid is an important factor in the production of the metabolic acidosis after methanol administration to the monkey. Formic acid accumulation following methanol administration does not occur in the rat, which oxidizes formate at rates that are markedly higher than those seen in the untreated monkey. The objectives of this study were to determine the pathway responsible for formate oxidation in the monkey and to compare the disposition of formate in the monkey with that of the rat. This information was used to increase and decrease the rate of accumulation of formate in the monkey and to increase or decrease the sensitivity of the monkey to methanol poisoning. Results show that a folate-de-pendent pathway is the major route of formate metabolism in the monkey as has been shown previously in the rat. Folate administration to the monkey increases formate oxidation. 4-Methylpyrazole treatment, after the production of metabolic acidosis and formic acidemia in the monkey, reverses the acidotic state and formic acidemia. Although the folate-deficient monkey is the most sensitive animal towards methanol poisoning, formaldehyde did not appear in the blood, body fluids, or tissues. Since formaldehyde does not accumulate in the presence of high methanol and high formate, and since reversal of formate accumulation leads to reversal of the features of the methanol poisoning syndrome, it is suggested that formate rather than formaldehyde is the major determinant of methanol poisoning in monkeys and probably in man.

Formate assay in body fluids: application in methanol poisoning.

Abstract A sensitive and specific assay for formic acid in body fluids has been developed. The assay is based on the reaction of formate with bacterial formate dehydrogenase coupled to a diaphorase-catalyzed reduction of the nonfluorescent dye resazurin to the fluorescent substance resorufin. Formate concentrations of 0.5 μg/ml of reaction mixture can be accurately measured. Small volumes of body fluids can be used for the analysis of both methanol and formate. The procedure described is simple and allows for the economical and rapid determination of formate. It can be used in studies concerned with the disposition of formate, as it relates to methanol metabolism. Also, it may be useful in studies where formate might exist as a metabolic intermediate of certain drugs or chemicals.

The regulation of one-carbon oxidation in the rat by nitrous oxide and methionine

Abstract Formate is oxidized to CO2 in the rat by folate-dependent reactions. Nitrous oxide treatment inhibited hepatic methionine synthetase activity, reduced hepatic S-adenosyl- l -methionine (Ado-Met) and tetrahydrofolate (H4 folate) concentrations and decreased the rate of formate oxidation in the rat. The administration of methionine to nitrous oxide-treated rats increased hepatic Ado-Met concentrations and restored hepatic H4folate levels and formate oxidation to control values but did not reverse the inhibition of methionine synthetase. Positive correlations were observed between hepatic Ado-Met levels and H4folate concentrations and between hepatic H4folate concentrations and formate oxidation. These results suggest that alterations in hepatic H4folate concentrations may profoundly influence the oxidation of one-carbon compounds. They confirm the importance of the methionine synthetase reaction as a major source of regeneration of H4folate. These findings also indicate that methionine acts at a site other than the methionine synthetase reaction to restore hepatic H4folate concentrations and formate oxidation to control values in nitrous oxide-treated rats.

Inhibition of monkey liver alcohol dehydrogenase by 4-methylpyrazole.

Abstract Pyrazole and 4-methylpyrazole competitively inhibited monkey liver alcohol dehydrogenase (E.C.1.1.1.1), and the Ki value obtained for 4-methylpyrazole (8 μ m ) was one-fourth that of pyrazole. These values for monkey liver ADH were 100–600 times higher than those reported for horse liver ADH. Although hepatic catalase activity is inhibited after treatment of rats with pyrazole, 4-methylpyrazole had no effect on hepatic catalase activity in vivo or in vitro. Since methanol poisoning in the monkey is dependent on its oxidation through alcohol dehydrogenase, 4-methylpyrazole may be a useful adjunct to therapy in the toxicity produced by that alcohol.

THE MONKEY AS A MODEL IN METHANOL POISONING

Methanol poisoning in man is characterized by a mild central nervous system depression, metabolic acidosis and ocular toxixity followed by coma and death. Previous work from this laboratory has described the production of metabolic acidosis, coma and death in rhesus and pigtail monkeys without the demonstration of definitive ocular lesions. Since animals used in those studies died rapidly after methanol administration a prolonged and less intense state of intoxication was deemed necessary for the production and recognition of ocular toxicity. Thus, methanol was administered at a dose of 2 g/kg followed by subsequent doses of 0.5 g/kg until signs of ocular toxicity were observed: usually at 48 hours, or later, after the first dose of methanol. Ocular toxicity was characterized as optic disc edema with dilated pupils and a slow reaction of the pupillary reflex to light. A rapid intraarterial perfusion of appropriate fixatives was used in order to minimize autolysis of tissues. Histopathologic changes included intracellular swelling and mitochondrial disruption in the area of the optic disc but otherwise retinal histology was normal. Clinical symptoms appeared to be similar to those described in man and may provide a basis for our understanding of the mechanism of methanol toxicity with respect to the ocular lesions observed in man.