Determination of P4501A2 activity in human liver microsomes using [3-¹⁴C-methyl]caffeine

Abstract

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 [¹⁴C]formaldehyde/[¹⁴C]formic acid produced following incubation of human microsomes with [3-¹⁴C-methyl]caffeine. This assay, using solid-phase extraction, enables quantitation of [¹⁴C]formaldehyde/[¹⁴C]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) μM, 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 [¹⁴C]formaldehyde/[¹⁴C]formic acid in human liver microsomes, is suitable for use in in vitro drug interaction studies as a probe for P4501A2 activity.