A RADIOIMMUNOASSAY FOR THE REGULATORY ALLYLIC STEROID, 3-ALPHA-HYDROXY-4-PREGNEN-20-ONE (3-ALPHA-HP)

The allylic steroid, 3-alpha-hydroxy-4-pregnen-20-one (3-alpha-HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3-alpha-HP was developed using [H-3]3-alpha-HP and an antiserum raised against 3-alpha-HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3-alpha-allylic configuration of 3-alpha-HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3-alpha-HP such as progesterone) was less then 0.9%. A Scatchard plot indicated a K(a) of 1.56 x 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3-alpha-HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3-alpha-HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3-alpha-HP from other interfering steroids prior to RIA.