title: 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 Km and Vmax for the N3-demethylation of caffeine were determined by this assay to be 500 (range 220-1200) microM, 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.
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, Frythe, Welwyn, UK.
authors.forename: J C
authors.initials: JC
authors.lastname: Bloomer
authors.forename: S E
authors.initials: SE
authors.lastname: Clarke
authors.forename: R J
authors.initials: RJ
authors.lastname: Chenery
mesh_terms: D002110:Caffeine / Q000031:analogs & derivatives*; D002250:Carbon Radioisotopes; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D005557:Formaldehyde / Q000032:analysis; D005561:Formates / Q000032:analysis; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis*; D011869:Radioligand Assay
chemical_list: D002250:Carbon Radioisotopes; D005561:Formates; C030544:formic acid; D005557:Formaldehyde; D002110:Caffeine; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2