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

Xenobiotica. 1995 Sep;25(9):917-27. doi: 10.3109/00498259509046663.


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) 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.

MeSH terms

  • Caffeine / analogs & derivatives*
  • Carbon Radioisotopes
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 Enzyme System / analysis*
  • Formaldehyde / analysis
  • Formates / analysis
  • Humans
  • Microsomes, Liver / enzymology*
  • Oxidoreductases / analysis*
  • Radioligand Assay


  • Carbon Radioisotopes
  • Formates
  • formic acid
  • Formaldehyde
  • Caffeine
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Cytochrome P-450 CYP1A2