Studies on the Metabolism of Steroids in the Foetus

Abstract

The hydroxylation of androstenedione in the human foetal liver a t midgestation was investigated in vitro. After incubations carried out with 105000 x 9 microsome-preparations of hepatic tissue using a medium supplemented with NADPH-regenerating system the C,,O,-metabolites were isolated by thin-layer and gas chromatography and identified by mass-spectrometry.The presence in the liver microsome preparations of I/?-, 2@-, 2a-,.6a-, 6/?-and 18-hydroxylase activities was demonstrated by the isolation and identification for the first time in the human of six ol,/?-unsaturated C,,O,-steroids. Two of them, 18-hydroxy and 2a-hydroxy-androstenedione have not been previously reported in animal tissue. Three C,,O,-steroids (testosterone, 3or-and 3t9-hydroxy-4-androstene-17-one) have been also isolated in this investigation. MATERIAL AND METHODS SteroidsAndrostenedione and testosterone were obtained from Schering (Berlin) and 6p-hydroxyandrostenedione was purchased from E. R. Squibb & Sons (New Brunswick, U. S.A.). Androstenedione was recrystallised twice before use, its purity was controlled by gas chromatography-mass spectrometry by using SE-30 and QF-1 phases. Epimeric 3/?-and 3a-hydroxy-4-androsten-17-0nes were kindly supplied

Studies on the Metabolism of Steroids in the Foetus

“…Gas chromatography was performed on 270 x 0.4 cm columns of 1.5,/, SE-30 or 3' 1, QF-1 phases a t 220 "C with nitrogen as carrier (25 ml/min), and the relative retention time (tR) was calculated with reference to 5a-cholestane. The steroid derivatives, silylether and methoximesilylether, were formed as previously described [9]. The conditions for electron-impact mass spectrometry were described in a previous paper [ll].…”

“…I n a typical experiments, approximately 2 g adrenal glands from a foetus (20 cm crown to rump length) were homogenized a t 4 "C with a loose-fitting teflon pestle to give a loo/, (wlv) preparation in a 0.25M sucrose. This homogenate was submitted to differential centrifugation for the preparation of microsome fractions from centrifugation a t 105000 x g as described in earlier papers in this series [9,12]. A final microsome suspension was adjusted to a concentration of 1.0 g fresh tissue per ml, corresponding to a concentration of 6.4 mg proteinlml as measured by the biuret reaction according to Lomy et al…”

“…This homogenate was submitted to differential centrifugation for the preparation of microsome fractions from centrifugation a t 105000 x g as described in earlier papers in this series [9,12]. A final microsome suspension was adjusted to a concentration of 1.0 g fresh tissue per ml, corresponding to a concentration of 6.4 mg proteinlml as measured by the biuret reaction according to Lomy et al [f 31.…”

“…The steroid derivatives, silylether and methoximesilylether, were formed as previously described [9]. The conditions for electron-impact mass spectrometry were described in a previous paper [ll].…”

“…Localization of the unmetabolized testosterone and its a$-unsaturated metabolites was done by exposure to a short-wave ultraviolet light (254nm) or by reflectance spectrometry a t 240nm. The silica gel zones scraped off between the starting line and the front were eluted as previously described [9] and analysed by gas chromatography (Pye-Unicam instrument with flame ionisation detector) or gas chromatography-mass spectrometry (LKB-gas chromatographmass spectrometer, model 9000).…”