Abstract

The major pathway of testosterone oxidation by human liver microsomes is the formation of 6β-hydroxytestosterone, which is catalyzed by CYP3A4/5 and which accounts for 75–80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-³H]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in no extraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6,7-³H]testosterone paralleled rates of testosterone 6β-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent K_m andV_max values). The sample-to-sample variation in tritium release from [1,2,6,7-³H]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6β-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-³H]testosterone, and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-³H]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-³H]testosterone.