Side-chain cleavage as related to steroid hormone synthesis

Abstract

The mechanism of side-chain cleavage during the course of steroid hormone biosynthesis by rat testicular and adrenal enzyme preparations has been investigated. 17α-Hydroxypregnene-C-17-C-20 lyase in testicular tissue required molecular oxygen in addition to NADPH. Progesterone and 17α-hydroxyprogesterone, under an 18O 2-enriched atmosphere, and 17α- 18O-hydroxylated progesterone under an 16O 2 atmosphere were incubated with a rat testicular enzyme preparation, and the products were analyzed by high-resolution mass spectrometry. Progesterone-17α-hydroxylase required molecular oxygen, which was incorporated in the 17α-hydroxyl group of 17α-hydroxyprogesterone. However, in the case of the side-chain cleavage of 17α-hydroxyprogesterone, the molecular oxygen required was not incorporated into the direct steroid product, androstenedione, nor into the further product testosterone. It was thus established that the oxygen atoms of the 17-ketone and 17β-hydroxyl groups of androstenedione and testosterone, respectively, which were derived by testicular enzymes from progesterone, originated from the 17α-hydroxyl group of 17α-hydroxyprogesterone. The side-chain cleavage of cholesterol, in the synthesis of pregnenolone, was also studied, by incubation of 20α, 22 R-dihydroxycholesterol with rat adrenal preparation under an 18O 2 atmosphere. The immediate product pregnenolone and its further metabolite progesterone were isolated, and analysed by mass-spectrometry. No incorporation of molecular oxygen into the metabolites was observed, suggesting that the oxygen in the 20-ketone group of pregnenolone and progesterone is derived from the oxygen of the 20α-hydroxyl group introduced into cholesterol prior to the side-chain cleavage.