Folate Distribution in Cultured Human Cells

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Abstract
We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH2-H4PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH3-H4PteGlu, 10-CHO-H4PteGlu, and 5-CHO-H4PteGlu with smaller fractions, which included 5-CH3-H2PteGlu, 10-CHO-PteGlu, and H4PteGlu. Evidence that the 5-CHO-H4PteGlu may have been derived from 5,10-CH=H4PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH3-H4PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH3-H4PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH3-H4-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH3-H4PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH2-H4PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH3-H4PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.