A microbiological assay system for naturally occurring folates

Abstract

For the first time nonformylated tetrahydrofolic acid is shown to be a major form of folic acid in rat liver, along with N5-methyltetrahydrofolic acid. A system of differential microbiological assays that will identify the folates occurring naturally in tissues is presented. It involves rapid heat inactivation of natural enzymes, cleavage of folate polyglutamates with conjugase, and assay with three microorganisms under standardized conditions that allow identification and quantitation of both free and conjugated forms of four folate derivatives. Evidence is presented that the procedures outlined produce a minimum of change in the naturally occurring folate derivatives.

FOLATE LOSSES IN BEEF LIVER DUE TO COOKING AND FROZEN STORAGE1

“…(2) The homogenate was incubated at 37°C for 3 h to allow the endogenous conjugase to hydrolyse the polyglutamates and then heated at 95°C for 5 min to minimize enzyme activity during storage of the extracts (Bird et al 1968). …”

“…One of the few studies available on the effects of cooking on beef liver folates (Cheldelin et al 1943) reported a 15% loss due to frying beef liver compared with a 95% loss for pork loin. More recent studies have dealt mainly with folate losses during canning and processing of vegetables and legumes.…”

Determination of Folate Derivatives in Rat Tissues during Folate Deficiency.

“…The quantitation of each folate is difficult, because folates in tissue exist at very low lev els and are unstable. Early studies of folate determina tion employed bioassay using microorganisms such as Lactobacillus casei, Streptococcus faecalis and Pediococcus cerevisiae (3)(4)(5)(6)(7). However, this method is time consum ing, and cannot specifically determine each folate deriv ative.…”

Evaluation of ascorbic acid in protecting labile folic acid derivatives.

“…Early studies employed the differential growth requirements of Lactobacillus casei, Streptococcus faecalis, and Pediococcus cerevisiae (3)(4)(5)(6)(7). Subsequently these procedures were improved by prior separation of extracts by ion-exchange column chromatography (8)(9)(10)(11)(12)(13)(14)(15).…”