The conversion of labeled
formate to
methionine and
serine, as a measure of remethylation of
homocysteine to
methionine and
folate coenzyme cycling, has been studied in control and mutant human fibroblasts. Fibroblasts in monolayer culture were incubated with [14C]
formate, and labeled
methionine sulfone and
serine were determined in hydrolysates of oxidized cell
proteins. In control cells,
methionine and
serine were clearly measurable (n = 21, 1.7-5.5 and 2.4-9.7 nmol/mg
protein/16 h, respectively). In contrast,
methionine formation was reduced in cells from patients with
methylenetetrahydrofolate reductase (MR) deficiency (MR mutant, n = 11, 0.05-0.44), combined
methylmalonic aciduria/
homocystinuria [
cobalamin(cbl)C/D mutant, n = 12, 0.014-0.13), and
methionine synthase deficiency (MS mutant, n = 3, 0.04-0.23). Furthermore,
serine formation was low in cblC/D mutant (0.08-0.98) and MS mutant (0.17-0.94) cells, but normal or high in MR mutant cells (5.2-11.4). Growth of cblC/D mutant cells in medium supplemented with high concentrations of hydroxo-cbl resulted in significant increases of both
methionine and
serine formation. Taken together these findings provide clear evidence for the existence of the
formate to
serine pathway described by W. B. Strong and V. Schirch in cultured fibroblasts and indicate that disturbed MS function due to a specific
genetic disorder is associated with reduced
serine formation in vitro, which reflects availability of reduced
folate coenzymes. The correction of this defect by
vitamin B12 alone, in cblC/D mutant cell lines, correlates well with the clinical response in the patients and fits in well with the idea that reduced availability of
folate coenzymes occurs in functional MS deficiency, in agreement with the methyl trap hypothesis.