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Assay and characterisation of debrisoquine 4‐hydroxylase activity of microsomal fractions of human liver.
Author(s) -
Kahn GC,
Boobis AR,
Murray S,
Brodie MJ,
Davies DS
Publication year - 1982
Publication title -
british journal of clinical pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.216
H-Index - 146
eISSN - 1365-2125
pISSN - 0306-5251
DOI - 10.1111/j.1365-2125.1982.tb01430.x
Subject(s) - debrisoquine , microsome , chemistry , microsoma , hydroxylation , cytochrome p450 , enzyme , enzyme assay , cytochrome , medicine , biochemistry , cyp2d6
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.