1 A radiometric high performance liquid chromatographic method is described for the assay of theophylline metabolism in vitro by the microsomal fraction of human liver. 2 Formation of the three metabolites of theophylline (3-methylxanthine, 1-methylxanthine and 1 ,3-dimethyluric acid) were linear with protein concentrations to 4 mg mland with incubation times up to 180 min. 3 The coefficients of variation for the formation of 3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid were 1.2%, 1% and 1.6%, respectively. 4 Theophylline is metabolised by microsomal enzymes with a requirement for NADPH. 5 The mean (n = 7) Km values for 1-demethylation, 3-demethylation and 8-hydroxylation were 545, 630 and 788 FtM, respectively, and the mean Vmax values were 2.65, 2.84 and 11.23 pmol min-' mg-', respectively. 6 There was a high correlation between the Km and Vmax values for the two demethylation pathways suggesting that the demethylations are performed by the same enzyme. 7 Overall the in vitro studies are consistent with the in vivo results which suggest the involvement of two cytochrome P-450 isozymes in the metabolism of theophylline.Keywords theophylline metabolism cytochrome P-450 in vitro

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