Assay and characterisation of debrisoquine 4‐hydroxylase activity of microsomal fractions of human liver.

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Abstract
1 A method for the assay of debrisoquine 4‐hydroxylase activity in vitro by microsomal fractions of human liver is described. The assay utilises gas chromatography‐mass spectrometry with d9‐4‐ hydroxydebrisoquine as internal standard. 2 The limit of detection of 4‐ hydroxydebrisoquine was 2 ng ml −1 and the coefficient of variation was 4.4%. 3 Debrisoquine 4‐hydroxylase activity was linear with protein to concentrations above 2.1 mg ml −1 and with incubation times of at least 15 min. 4 Debrisoquine 4‐hydroxylase is a microsomal enzyme with a requirement for NADPH. Activity was inhibited by carbon monoxide. It is concluded that the activity is catalysed by cytochrome P‐450. 5 In three samples of human liver the mean value for Vmax of debrisoquine 4‐ hydroxylase activity was 69.9 +/‐ 14.3 pmol mg −1 min −1 and for Km it was 130 +/‐ 24 microM. 6 The only variable from smoking status, alcohol ingestion, sex of the patients, source of liver sample and presence of liver disease that had a significant effect on 4‐hydroxylation of debrisoquine was the presence of liver disease. This was associated with a decrease in enzyme activity.