DETERMINATION OF P4501A2 ACTIVITY IN HUMAN LIVER-MICROSOMES USING [3-C-14-METHYL] CAFFEINE

1. Caffeine N3-demethylation, the major pathway of caffeine metabolism in man, is mediated by P4501A2. The carbon of the methyl group lost during N3-demethylation is eliminated as carbon dioxide in vivo, or as formaldehyde and formic acid in vitro. 2. A simple and sensitive assay was developed to quantify the [C-14]formaldehyde/[C-14]formic acid produced following incubation of human microsomes with [3-C-14-methyl]caffeine. This assay, using solid-phase extraction, enables quantitation of [C-14]formaldehyde/[C-14]formic acid with acceptable precision (within 5%) and accuracy (within 10%). 3. Typical K-m and V-max for the N3-demethylation of caffeine were determined by this assay to be 500 (range 220-1200) mu M, and 250 (range 115-450) pmol.mg protein(-1).min(-1) respectively. 4. The N3-demethylation activity determined in microsomes from a range of human livers correlated significantly with other P4501A2 activities (p<0.001) and was inhibited (>95%) by furafylline. In addition, caffeine N3-demethylation was catalysed by microsomes from cell lines transfected with human P4501A2 cDNA. 5. This assay, for quantitation of [C-14]formaldehyde/[C-14]formic acid in human liver microsomes, is suitable for use in in vitro drug interaction studies as a probe for P4501A2 activity.