Characterisation of theophylline metabolism in human liver microsomes.

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 ml‐1 and 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 microM, respectively, and the mean Vmax values were 2.65, 2.84 and 11.23 pmol min‐1 mg‐1, 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. 1987 The British Pharmacological Society