Pregnenolone metabolism in Y-1 mouse adrenal cells: HPLC analysis and identification of metabolites by gas chromatography-mass spectrometry.
The Y-1 mouse adrenocortical tumor cells secrete into the culture medium some metabolites of pregnenolone. The long term basic steroid secretion was studied for 24 h by high performance liquid chromatography (HPLC). After 6 h incubation, only one chromatographic peak was shown to be quantitatively important. When the cells were incubated with tritium labeled pregnenolone and carrier, the analysis of radioactivity of each HPLC fraction indicated a rapid incorporation of the exogenous precursor and successive secretion/reabsorption kinetics of the metabolites. The final product, accumulated in the medium, was characterized by gas chromatography-mass spectrometry as 11 beta,20 alpha-dihydroxy-4-pregnen-3-one.