An improved method for evaluating testosterone biosynthetic defects.
A double-label, double-substrate incubation technique has been developed and used to study the conversion of progesterone to testosterone in testes extracts from incompletely virilized males. The procedure involves separation of the microsomes from a testicular homogenate, incubating the microsomes with 1 microM [7-3H] progesterone, 1 microM 17-hydroxy[4-14C]progesterone, and 0.25 mM NADPH in pH 7.4 phosphate buffer at 37 degrees C. Steroid precursors and products are separated by column chromatography on Sephadex LH-20 with a solvent system of isoctane:ethyl acetate:methanol (4:1:1 by volume). These procedures can be completed in 2 days, and thus the method represents an improvement in time, reproducibility, and simplicity when compared to techniques based on thin layer or paper chromatography. The method has been used to distinguish the biochemical abnormality in three cases with XY sex chromatin, posterior labial fusion, clitoromegaly, and hypospadias. The abnormalities identified were: Case 1, no defect in testosterone synthesis (probable androgen insensitivity); Case 2, 17-ketosteroid reductase deficiency; and Case 3, steroid-17, 20-lyase deficiency.