Methotrexate alters carbon flow through the hepatic folate-dependent one-carbon pool in rats

The chemotherapeutic value of methotrexate resides in its ability to perturb folate-dependent one-carbon metabolism and subsequently inhibit DNA synthesis, To assess the functional effect of methotrexate on hepatic one-carbon metabolism, we have developed and applied tracer kinetic techniques in vivo to quantify the carbon flux through the folate-dependent one-carbon pool in rats, Following a 7-day treatment period with methotrexate (0.2 mg/kg body weight), the tracers L-[ring-2-C-14] histidine and L-[methyl-H-3] methionine were simultaneously infused into control and methotrexate-treated rats. Methotrexate treatment decreased hepatic tetrahydrofolate, methyltetrahydrofolate, and formyltetrahydrofolate concentrations by 63, 83, and 58%, respectively, Furthermore, the enzymatic activity of 10-formyltetrahydrofolate dehydrogenase, the terminal enzyme in the catabolism of the ring-2-carbon of histidine to CO2, was diminished by 32% in methotrexate-treated animals, These changes in enzyme activity and folate coenzyme concentrations did not result in a significant decrease in the oxidative flow of carbon from histidine to CO2 in methotrexate-treated rats compared to control animals (2.40 and 3.22 mu mol/h/kg(3/4), respectively), Oxidative carbon flow was reflective of tetrahydrofolate and formyltetrahydrofolate pools when expressed as a percent of total folate: neither coenzyme pool was diminished as a result of methotrexate treatment, In contrast, the reductive carbon flux through the one-carbon pool from histidine to methionine was significantly decreased 59% in methotrexate-treated (7.63 mu mol/h/kg(3/4)) versus control rats (18.73 mu mol/(h/kg(3/4))), Likewise, methyltetrahydrofolate, as a percent of total folate, was reduced 51% in methotrexate-treated rats, Consequently, total measured carbon how (oxidative + reductive) was 54% lower in rats subjected to subchronic methotrexate treatment, These tracer kinetic experiments quantitatively demonstrate the extent to which methotrexate alters the actual carbon how through the hepatic folate-dependent one-carbon pool, primarily directed at diminishing the reductive carbon how towards methyltetrahydrofolate and methionine synthesis.