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Vol. 26, Issue 4, 299-304, April 1998
Department of Pharmacology, Toxicology and Therapeutics, Center for
Environmental and Occupational Health, University of Kansas Medical
Center (A.J.D., A.M., A.P.) and
XenoTech L.L.C. (K.S.)
The major pathway of testosterone oxidation by human liver
microsomes is the formation of 6
-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 6-hydroxylation as a function of incubation time, the
amount of microsomal protein, and the concentration of substrate (which
yielded identical apparent Km and
Vmax 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
6-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.
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