1. Caffeine N3-demethylation, the major pathway of caffeine metabolism
in man, is mediated by P4501A2. The carbon of the methyl group lost d
uring 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 pr
oduced following incubation of human microsomes with [3-C-14-methyl]ca
ffeine. This assay, using solid-phase extraction, enables quantitation
of [C-14]formaldehyde/[C-14]formic acid with acceptable precision (wi
thin 5%) and accuracy (within 10%). 3. Typical K-m and V-max for the N
3-demethylation of caffeine were determined by this assay to be 500 (r
ange 220-1200) mu M, and 250 (range 115-450) pmol.mg protein(-1).min(-
1) respectively. 4. The N3-demethylation activity determined in micros
omes 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 fro
m cell lines transfected with human P4501A2 cDNA.5. This assay, for qu
antitation of [C-14]formaldehyde/[C-14]formic acid in human liver micr
osomes, is suitable for use in in vitro drug interaction studies as a
probe for P4501A2 activity.