A Rapid, Sensitive Assay for Analyzing Defects in Metabolism of Steroids in Normal and Diseased Human Tissues

In this report, we describe a simple, rapid biopsy-steroid metabolism assay that is applicable to any steroid tissue system. It consists of mincing the sample, tissue culture incubation, extraction of the steroids and their metabolites from the tissue, and fractionation of the metabolites by high-pressure liquid chromatography (HPLC). Radioimmunoassay is used to verify the elution patterns of certain steroids. Studies of the metabolism of [³H]progesterone in the avian oviduct showed the generation of metabolites that eluted from the HPLC system in a pattern similar to androgens, estrogens, and glucocorticoids. Studies of the metabolism of [³H]testosterone in the human foreskin showed the production of metabolites that eluted from the HPLC system similar to 5α-dihydrotestosterone and 5α-androstane-3,17-dione (androstanedione) from the parent [³H]testosterone. In the production of the metabolite that eluted as androstanedione in samples of foreskin from normal subjects, a significant (P<0.001) correlation was found with the age of the donor. Preliminary studies of patients with hypospadias showed a significant (P<0.005) decrease in the production of “androstanedione” compared with that in normal subjects. Because of the wide range in rates of metabolism of testosterone in the patients with hypospadias, the effect of age does not seem to be the sole determinant of a low rate of metabolism in these patients. Some samples of hypospadias foreskin had a decreased rate of production of a metabolite that eluted as dihydrotestosterone in comparison with normal foreskin, even when the age of the donor was considered. The assay described herein should be applicable to any surgical biopsy specimen and to all steroids. More decisive analyses of the metabolites, such as by radioimmunoassay or mass spectrometry, are necessary for precise identification.