Biosynthesis of [³H]7α-hydroxy-, 7β-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [³H]-labeled 7α-hydroxy-DHEA (7α-OH-DHEA), 7β-hydroxy-DHEA (7β-OH-DHEA), and 7-oxo-DHEA substrates from 50 μM [1,2,6,7-³H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7α-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2 h/2 ml, respectively; 82% conversion), while in the presence of 1 mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP⁺), more 7-oxo-DHEA than 7α-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP⁺, a mixture of 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10 mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.