Isolation and quantitation of steroids from normal human amniotic fluid.

Abstract

Eight steroids have been isolated and quantitated from a pool of 4000 ml of amniotic fluid collected from pregnant women immediately prior to delivery. An ethyl acetate extract of the fluid obtained after enzyme hydrolysis with β-glucuronidase and sulfatase was subjected to celite column, paper and thinlayer chromatography. Preliminary identification of each steroid was achieved by comparison of its chromatographic behavior with known compounds in various gas-liquid chromatographic systems, both as the parent compound and as several derivatives. Final identification of the isolated steroids was accomplished by gas-liquid chromatography-mass spectrometry. The following steroids were found in the amounts indicated: dehydroisoandrosterone (D), 8.3 μg/1; 16α-hydroxydehydroisoandrosterone (16-OH-D), 797.7 μg/1; 16-ketoandrostenediol (16 keto-A), 575.0 μg/1; androstenetriol (Δ5-triol), 48.0 μg/1; estriol (E3), 1572.5 μg/1; and estetrol (E4), 83.8 μg/1. With the exception of pregnanediol and estriol, this is the first reported isolation of these steroids from amniotic fluid. The preponderance of 16α-hydroxylated C18-, C19- and C21-compounds as compared with their precursors suggests that 16α-hydroxylation by the fetus may facilitate the removal of steroids by urinary excretion.