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Folate-dependent one-carbon metabolism and methylation reactions have been implicated in the secretory function of the pancreas. Because vitamin B-12 deficiency perturbs folate metabolism, we determined the effects of nitrous oxide inactivation of methionine synthase on the compartmentation of folate metabolism in rat pancreas. Rats were exposed to an atmosphere of nitrous oxide and oxygen (80 and 20%, respectively) for 18 h; control rats breathed air. Folate coenzyme concentrations were determined by HPLC and Lactobacillus casei microbiological assay of the cytosolic and mitochondrial fractions of pancreas, which contained 62 and 46%, respectively, of the total folate. In pancreas of control rats, cytosolic folates were 5-methyltetrahydrofolate (31% of total folates), tetrahydrofolate (54%) and 5- and 10-formyltetrahydrofolate (6 and 8%, respectively). In the rats exposed to nitrous oxide, cytosolic 5-methyltetrahydrofolate concentrations were significantly greater (59% of total folates) and tetrahydrofolate concentrations were significantly lower (32%) than in controls; however, total cytosolic folate levels were unaffected by nitrous oxide exposure. In controls, mitochondrial folates were composed of 5-methyltetrahydrofolate (9% of total folates), tetrahydrofolate (60%) and 5- and 10-formyltetrahydrofolate (22 and 10%, respectively). Exposure to nitrous oxide led to significantly lower total mitochondrial folates (1.49 [+ or -] 0.18 vs. 0.75 [+ or -] 0.29 nmol/g, control vs. nitrous oxide, P < 0.05). This was due to a significantly lower concentration of tetrahydrofolate and 5-formyltetrahydrofolate, but not of 5-methyl- or 10-formyltetrahydrofolate. The activity of methionine synthase was 85% lower (P < 0.001) in pancreatic extracts of rats exposed to nitrous oxide than in controls. These results show that cytosolic folates accumulate in pancreas as the 5-methyl derivative at the expense of other reduced folates, as happens in liver. However, in contrast to results in liver, the mitochondrial folate concentration was lower in the pancreas of rats exposed to nitrous oxide, and this decline was limited to the 5-formyl- and tetrahydrofolate derivatives. KEY WORDS: folic acid; nitrous oxide; vitamin B-12; rats; pancreas mitochondria

The coenzyme 5,10-methylenetetrahydrofolate (CH2-THF) follows two forms in catalyzing the same reaction of purine synthesis. The NADP-dependent process is interchangable with the NAD-dependent process when the flow of one carbon units is in the oxidative direction. However, the NAD-dependent CH2-tetrahydrofolate dehydrogenase is responsible for the CHO-THF production, marking its catalytic role in the oxidation of one-carbon units. The NADP-dependent process plays a catalytic and non-catalytic role in purine synthesis.

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent K(sub m) = 2.80 micron mol.L to the -1 and V(sub max) = 3.49 pmol.min to the -1.mg protein to the -1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria. J. Nutr. 122: 2204-2209, 1992.

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