1. The composition of guinea-pig liver folates and the biochemical route of formation of liver folates from injected tritium-labelled pteroylglutamic acid (folic acid) have been studied.

2. Endogenous folate was measured by microbiological assay with Lactobacillus casei and Streptococcus faecalis, with and without deconjugation of whole liver pteroylpolyglutamates (conjugated folates). Individual folate compounds were identified by microbiological assay after fractionation of liver folates by DEAE cellulose ion-exchange column chromatography.

3. Liver folate in the guinea-pig consists of about 84–87% reduced pteroylpolyglutamates with more than three glutamate moieties/molecule, about 12–15% reduced pteroyltriglutamates, about 1% reduced pteroyldiglutamates and only traces of reduced pteroylmonoglutamates.

4. About 53% of the liver folate consists of methylated derivatives.

5. Injected pteroylglutamic acid was first rapidly reduced and formylated or methylated. Glutamate moieties were then added, probably singly, to form di-, tri- and poly-glutamates. This was a relatively slow process with a hold-up at the triglutamate stage.

6. The proportion of the labelled pteroylglutamic acid in the polyglutamate form approximated to the proportion of endogenous folates in this form after 3–4 days.

7. The amount of radioactive folate in the liver increased progressively from 1 to 84 h after injection of a standard amount of radioactive pteroylglutamic acid.

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