Naturally occurring folates in the blood and liver of the rat

Abstract

All of the folate activity in rat plasma appears to be in the monoglutamate form. The major component, active only for L. casei, appears to be N 5-methyltetrahydrofolate and is stable when the plasma is stored frozen. A minor component, active for P. cerevisiae, appears to be tetrahydrofolate and is unstable, even when the plasma is stored frozen, unless protected by ascorbate. A carefully prepared heated extract of whole rat blood has very low activity for L. casei and P. cerevisiae. Treatment of this extract with pH 7.8-active conjugase gives the maximum L. casei activity while treatment with pH 4.7-active conjugase gives the maximum P. cerevisiae activity, both representing several-fold increases in activity, and both higher than can be obtained from the same blood by autolysis. The maximum folate activity for L. casei cannot be obtained for rat blood or rat liver which have been frozen, with or without ascorbate, before treatment with conjugase. Chromatography of a heated extract of fresh rat liver indicates the presence of at least seven folate derivatives, all but two of which are polyglutamates responsive to the action of conjugases. Freezing and thawing rat liver has a conjugase-like effect in that it causes the release of monoglutamates from much of the folate activity originally present as polyglutamates. Autolysis of fresh rat liver causes the splitting of all folate polyglutamates and the accumulation of N 10 - and N 5 -formyltetrahydrofolates and N 5 -methyltetrahydrofolate. Autolysis of frozen liver results in further oxidative changes and the accumulation of only N 10 - and N 5 -formyltetrahydrofolates. Frequently used preparatory treatments of blood and liver which mask the original folate forms present and alter assay results are pointed out. Procedures are outlined which allow naturally occurring folate forms in blood and liver to be defined more accurately.