LNCaP, a human prostate cancer cell line, metabolizes testosterone into a variety of 5 alpha-reduced C19 steroids, such as dihydrotestosterone (DHT), androstane-3 alpha, 17 beta-diol (3 beta, 17 beta-DIOL), and androsterone (ADT). Recent reports also suggest that 5 alpha-reduced C19 steroid glucuronides can be detected in the medium. The purpose of this work was to characterize by liquid chromatograph ion spray mass spectroscopy (LCMS) the metabolites formed by LNCaP during incubation with testosterone and its 5 alpha-reduced C19 steroids. Time course studies using 10 nM labeled testosterone, 3 alpha-DIOL, 3 beta-DIOL, or ADT showed that a large proportion of polar steroids were produced by LNCaP. Identification of metabolites produced by LNCaP was carried out by LCMS using 1 microM substrates. Analysis of testosterone metabolism indicated that testosterone glucuronide was formed at 77 +/- 2% after 96 h of incubation. Using DHT as substrate, 3 alpha-DIOL-G and DHT-G were the major metabolites, accounting for 46 +/- 4% and 38 +/- 3%, respectively, of the total radioactivity in the medium; ADT-G accounted for 8 +/- 1%. Further analysis by LCMS also indicated that the glucuronide group in 3 alpha-DIOL-G was at position 17-carbon, 3 alpha-DIOL-G (86 +/- 3%) was the prominent metabolite formed from 3 alpha-DIOL, a minor product was detected at 7 +/- 1% and identified by mass spectrometry to correspond to a trihydroxylated C19 steroid.(ABSTRACT TRUNCATED AT 250 WORDS)