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The subcellular distribution of folate coenzymes in the brain is unknown. Brain folate concentrations are low and hence require a sensitive assay to determine the subcellular distribution. Rat brain was fractionated by differential centrifugation into cytoplasmic, mitochondrial and crude synaptosomal fractions. The compositions of the folate pools in these subcellular fractions were determined by differential conversion of one-carbon forms enzymatically to 5,10-methylenetetrahydrofolate (5,10CH2H4PteGlu(n)) followed by reaction of the 5,10CH2H4PteGlu(n) with thymidylate synthetase and [3H]fluorodeoxyuridylate to form ternary complexes, which were then separated as a function of polyglutamate chain length by isoelectric focusing, visualized by fluorography and quantified by densitometry. The distribution of the pteridine derivatives in brain was very similar to the distribution of these derivatives in liver. Cytoplasm contained primarily 5-methyltetrahydropteroylpolyglutamates with smaller amounts of unsubstituted tetrahydropteroylpolyglutamates, whereas mitochondria contained approximately equal concentrations of unsubstituted and formyl-substituted tetrahydropteroylpolyglutamates. The subcellular distribution of polyglutamate derivatives in brain, however, was different from that in liver. In the brain, the mitochondrial folates exhibited longer polyglutamate chains than did the cytoplasmic folates, a pattern opposite to that in the liver. Whereas the brain cytoplasmic pteroylpolyglutamates were primarily penta and hexa glutamates, the brain mitochondrial pteroylpolyglutamates were primarily hexa and hepta glutamates. The brain also contained small but measurable levels of oxidized folates, which were seen in crude synaptosomal fractions but not in cytoplasmic or mitochondrial fractions.

The ternary complex method for the determination of folylpolyglutamates was combined with procedures for interconverting folate derivatives to measure 28 different folate derivatives in the subcellular fractions of rat liver. Folates in the homogenate showed a typical distribution with nearly equal quantities of penta- and hexaglutamates and pteridine derivatives in decreasing order as follows: 1) methyl substituted folates [5-methyltetrahydropteroylglutamates], 2) unsubstituted folates [tetrahydropteroylglutamates + 5,10- methylenetetrahydropteroylglutamates], 3) formyl substituted folates [5-formyltetrahydropteroylglutamates + 10- formyltetrahydropteroylglutamates + 5,10-methenyltetrahydropteroylglutamates], and 4) oxidized folates [dihydropteroylglutamates]. In the homogenate the methyl substituted folates exhibited a higher hexa:pentaglutamate ratio than did the other pteridine derivatives. As the fractionation proceeded toward purer subcellular components, the methyl substituted folates were found almost exclusively in the soluble fraction, and this fraction also contained the higher hexa:pentaglutamate ratio characteristic of the methyl substituted folates. The plasma membrane, the microsomal and the nuclear fractions did not contain appreciable folate. The mitochondrial fraction contained primarily formyl substituted and unsubstituted folates, and these folates exhibited the lower hexa:pentaglutamate ratios. These data support the hypothesis that folate-dependent one-carbon metabolism is compartmentalized in the eukaryotic cell.