GLC/MS identification of new pregnenolone metabolites in confluent embryonic rat fibroblast cultures

Four new metabolites and their fatty acid derivatives have been identified as components of the steroid metabolic pathway in rat embryo fibroblasts grown during the confluent phase in the presence of [4-14C]-pregnenolone and [4-14C]-progesterone. These compounds are 3 alpha, 20 alpha and 3 beta, 20 alpha-dihydroxy-5 alpha-pregnanes, 4-pregnene-3 alpha, 20 alpha-diol and 5 alpha-pregnane-3 beta, 20 alpha, 21-triol. The labelled free and esterified steroids synthetized in the cells were released into the culture medium. The purpose of this study was to demonstrate that, besides t.l.c. enzymatic and chemical reactions, g.l.c./mass spectrometry coupling is a powerful tool for identifying the structures of sub-microgram amounts of radiolabelled steroids. The consequent metabolic features are discussed in terms of steroid cellular biosynthesis and kinetics.