Measurement of Testosterone and Dehydroepiandrosterone 16α‐Hydroxylase Activities by a Tritium Exchange Method

Abstract

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16alpha-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-3H]progesterone and [16-3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH7; 150mM), NADPH (0.1 mM), nicotinamide (10mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 micron) or [16-3H]dehydroepiandrosterone (100 micron). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16alpha-hydroxylated metabolites) and is independent of any further metabolism of the 16alpha-hydroxylated products.