Measurement of progesterone and pregnenolone 16alpha-hydroxylase activities by a tritium-exchange method

A new isotropic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group, has been developed for the measurement of progesterone and pregnenolone-16alpha-hydroxylase activity. The incubation medium consists of a phosphate buffer (pH 7; 150 mM), NADPH (1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween-80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]progesterone (200 micronM) or [16-3H]-pregnenolone (50 micronM). The microsomal fraction isolated from a male rat liver is used as the enzymic source. The enzymatically released tritium is recovered in the incubation medium as molecules of tritiated water which are distilled under reduced pressure. The amount of radioactivity present in the water exactly reflects the 16alpha-hydroxylase activity. The method is easy to perform and is completely independent of any further metabolism of the 16alpha-hydroxylated products.