Assay of estosterone progesterone and 17α-hydroxyprogesterone in human plasma by radioimmunoassay after separation on hydroxyalkoxypropyl sephade

Abstract

A method for the determination of testosterone, progesterone and 17α-hydroxyprogesterone in 1–2 ml samples of male and female blood plasma is described. After extraction of the unconjugated steroids, they are chromatographed on a column of a highly lipophilic Sephadex derivative, hydroxyalkoxypropyi Sephadex, in light petroleum-chloroform, 95:5. The final measurement of the individual steroids is made by radioimmunoassay using dextran-coated charcoal in the separation of bound and unbound radioactivity. Using 1–2 ml plasma samples the limit of sensitivity for progesterone and testosterone determination was about 0.1 ng/ml and for 17α-hydroxyprogesterone about 0.15 ng/ml. The coefficients of variation in the determination of these steroids ranged from 7.9–15.4%. The blank values, obtained by analysis of quartz-distilled water by the method described, were negligible in the tesosterone and 17α-hydroxyprogesterone determinations. Values below 10 pg were sometimes observed in the progesterone analysis. Plasma concentrations of these three steroids in a group of females both in the follicular and luteal phase of the menstrual cycle and in connexion with the intake of oral contraceptive of the combination type are presented. In addition, values obtained in a group of normal males are also given. The concentration values can be obtained within two days of drawing the blood samples and one technician can analyze 20 plasma samples in a 5-day period.