The objective of this project is to reveal the mechanism of methanol poisoning in humans. To our knowledge only humans and monkeys display characteristic methanol toxicity whereas lower animals, mice and rats, do not. Previous work in this laboratory has shown that the rate of formate oxidation is slower in monkeys than in rats and that hepatic tetrahydrofolate levels which are important for formate oxidation are much lower in monkeys than in rats. The current proposal is designed, first, to examine steady-state levels of hepatic folate derivatives and intermediates of the folate biochemical pathway. Human tissues will be provided by organ transplant donors first and later by patients undergoing upper gastrointestinal surgery. Procedures will include rapid freezing of livers followed by HPLC separations and quantification by microbiological bioassay. Low concentrations of human hepatic tetrahydrofolate and possibly low total hepatic folate is expected. This would lend support to the hypothesis that formate oxidation depends, in part, on hepatic levels of tetrahydrofolate. These experiments will be compared to studies on folate levels in mouse liver. Mice metabolize formate at rates three times the rates observed in rats. We postulate that hepatic tetrahydrofolate levels may be much higher in mouse liver. A second series of projects will examine the enzymatic regulation of formate oxidation to CO2 as catalyzed through the folate system; specifically the mediation led by formyltetrahydrofolate synthetase and formyltetrahydrofolate dehydrogenase. Human liver enzymes will be studied quantitatively and qualitatively in cytosolic fractions and in the homogeneous state. Kinetic experiments and immunoquantitative experiments will be performed using rat, mouse and human liver preparations. Later, molecular biological studies will be performed to examine the possible genetic regulation of synthesis of these proteins. Studies are also proposed to evaluate the regulation of tetrahydrofolate levels in liver. Enzymatic studies on methylene tetrahydrofolate reductase, methionine synthetase and serine hydroxymethyltransferase are described for human liver. These studies should provide insights into the regulation of formate and folate metabolism in animal species, especially in humans.
