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

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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 16α-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 (pH 7; 150 mM), NADPH (0.1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 μM) or [16-3H]dehydroepiandrosterone (100 μM). The enzymatically relased 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 16α-hydroxylated metabolites) and is independent of any further metabolism of the 16α-hydroxylated products.