In Vitro Bioassay of LH in Human Serum: The Rat Interstitial Cell Testosterone (RICT) Assay

Abstract

A sensitive bioassay for serum LH and hCG has been developed by utilization of the testosterone response of collagenase-dispersed rat Leydig cells to gonadotropic stimulation in vitro. Testosterone production by dispersed interstitial cells is stimulated by human, ovine, bovine, porcine, rat and rabbit LH, and by hCG and pregnant mare serum gonadotropin. The rat testis interstitial cell assay gives parallel dose-response curves for all steroidogenic gonadotropins tested, and thus permits cross-species comparison of the intrinsic biological activities of native and modified gonadotropins. The sensitivity of the rat interstitial cell bioassay is equal to or higher than that of radioimmunoassay, with detection limits of 50 muU for hMG and 20 muU for hCG. The optimum conditions for bioassay of serum gonadotropins were provided by incubation of dispersed interstitial cells in the presence of 1-methyl 3-isobutyl xanthine with the addition of gonadotropin-free serum or 5% BSA to ensure a constant proportion of serum protein in all assay samples and standards. Assays performed under these conditions gave parallel dose-response curves and identical maximum responses to both standards and serum samples containing endogenous LH or hCG. All responses to human gonadotropin standards and serum samples in the bioassay were abolished by incubation in the presence of antisera to LH or hCG. This sensitive and specific method permits bioassay of basal levels of LH in male and female serum, and of the higher gonadotropin levels in postmenopausal and pregnant subjects after appropriate dilution of the serum samples. For normal men and women, serum samples of 25-100 mul are employed, while volumes of 1-20 mul are adequate for assay of LH in postmenopausal or hypogonadal subjects. Serum LH values in hypopituitary and oral contraceptive-treated subjects were usually undetectable, while LH levels in normal subjects were always measurable with good precision. The within-assay coefficient of variation for measurement of a normal male plasma pool (30 mU/ml) was +/- 10%, and the between-assay variation was +/- 15%. The precision of the assay (lambda) was 0.035 +/- 0.015 (n = 72). In 42 normal females, the bio: immuno (B:I) ratio for serum LH was 1.20 +/- 0.40 (SD), and no consistent change in ratio was observed throughout the menstrual cycle. By contrast, significantly higher B:I ratios were observed in normal males (2.5 +/- 0.4), postmenopausal females (2.6 +/- 0.6) and patients with Turner's syndrome (2.6 +/- 0.6). These findings suggest that the LH circulating in normal female plasma is similar in biological and immunological activity to hMG, whereas the LH present in normal male plasma and in states of increased gonadotropin secretion has a relatively higher biological activity. The rat interstitial cell bioassay provides for the first time a practical and precise assay for measurement of the biologically active LH levels in serum of normal biologically active LH levels in serum of normal human subjects.