Radioimmunoassay of Plasma Pregnenolone

A radioimmunoassay for the measurement of plasma unconjugated pregnenolone (5Δ-P) is described. The 3-hemisuccinate of 5Δ-P was coupled to human serum albumin using carbodiimide. One adult ewe was immunized with 2 mg of this conjugate. The antiserum obtained after 7 months of immunization was used in the assay. One to two ml of plasma or serum, with 3H-5Δ-P added for recovery was extracted with ether and chromatographed on a microcolumn of celite. Incubation of the dried 5Δ-P fraction was carried out at 4 C in the presence of 3H-5Δ-P and diluted antiserum at 4 C in phosphate buffer with 0.1% gelatin. The mean recovery of 3H-5AΔ-P after extraction and chromatography was 62%. The sensitivity varied between 0.05 and 0.1 ng 5Δ-P. Recovery experiments of 5Δ-P added to plasma confirmed accuracy. Concentrations of 5Δ-P in plasma from men averaged 0.75 ± 0.55 (SD) ng/ml. Using single determinations during each phase of the menstrual cycle, premenopausal women had levels of 0.71 ± 0.45 (sd) and 1.38 ± 0.64 (sd) respectively during the follicular and luteal phases. However, when six premenopausal women were studied during a complete menstrual cycle, wide daily fluctuations in plasma 5Δ-P were observed with somewhat higher levels than during less frequent sampling. It is possible that the stress of daily venipuncture contributed to this increase in plasma 5Δ-P. Postmenopausal women showed a wide range of levels from 0.5 to 10.7 ng/ml with a mean of 2.7 ng/ml. The levels of 5Δ-P found by the present assay compared favorably with levels obtained under similar physiological conditions by other published methods.