Simultaneous determination of five sex hormones in human serum by radioimmunoassay after chromatography on Lipidex-5000.

Abstract

We describe a method for determination of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, testosterone, and 5alpha-dihydrotestosterone in 1-2 ml of serum from male or female. Using microcolumns of Lipidex-5000 (hydroxyalkoxypropyl Sephadex, 0.5 g) and light petroleum/chloroform (97/3) as the solvent during chromatography, we resolved these five steroids into four fractions, with pregnenolone and 5alpha-dihydrotestosterone eluting together. By use of selected antibodies, the latter two steroids were also determined specifically. Use of microcolumns allowed minimization of solvent volumes and sample transfers. Consequently, blank values for all the five steroids were negligible. Lowest measureable concentrations (in ng/liter) were: pregnenolone 100, progesterone 25, 17alpha-hydroxyprogesterone 50, testosterone 25, and 5alpha-dihydrotestosterone 25. Intra-assay and inter-assay coefficients of variation ranged from 5 to 9% and 10 to 15%, respectively, for the five steroids. Serum concentrations of these steroids are given for women in the follicular and luteal phases of the menstrual cycle and for women on oral contraceptives of the combination type, as well as for normal men.

hCG‐STIMULATION OF TESTICULAR STEROIDOGENESIS DURING INDUCED HYPER—AND HYPOPROLACTINAEMIA IN MAN

“…The sera were stored at -20°C until the hormone analyses. For the steroid analyses, a I-ml serum sample was extracted with diethyl ether:ethyl acetate (90: 10, by vol) and fractionated by Lipidex-5000TM chromatography as described previously from this laboratory (Apter et al, 1976;Hammond et a/., 1977a). The chromatographic fractions were analysed by radioimmunoassays for pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, and 5a-dihydrotestosterone.…”

Concentrations of androgens in human benign prostatic hypertrophic tissues incubated for up to three days

“…In four cases (nos. [14][15][16][17], the levels of 5a-DHT were 49 to 77% of the starting levels after 6 h at 37°C. These were then left at room temperature for 3 days, by which time the levels of 5a-DHT were 41-70% of the starting levels.…”

“…The solvent extracts were dried under nitrogen and then dissolved in 200 p1 of a solvent mixture of light petroleum ether:chloroform (98:2, v/v) before being applied to chromatography columns of Lipidex-5 ,OOO (hydroxyalkoxypropyl Sephadex; Packard-Becker, Groningen, The Netherlands) as described previously [ 15,161. Eluate fractions containing the steroids of interest were collected and stored at 4°C as described by Hammond [3].…”

“…Duplicate 20% portions (10% for testosterone) of each chromatography fraction were taken for the various steroid radioimmunoassays. Details of these assays have been published previously [3,[15][16][17][18]. In the assay of 5a-androstane-3cr, 170-diol, however, the antiserum used in the androsterone assay [ 181 was used, by virtue of the 40% crossreactivity of 5a-androstane-3a, 176-diol in this assay.…”

Body iron stores in relation to growth and pubertal maturation in healthy boys

“…Total body iron was estimated as the sum of the individual's RBC iron and iron stores. Serum testosterone concentrations were determined by radioimmunoassay (Apter et al, 1976). In this study we also had access to the serum IGFBP-3 concentrations reported by Anttila et al (1994).…”