A radiometric assay for debrisoquine 4-hydroxylase in human liver microsomes

A radiometric method to assay debrisoquine 4-hydroxylase activity in human liver microsomes was established. Following incubation with 14C-labelled debrisoquine, unreacted debrisoquine was extracted with chloroform; 4-hydroxydebrisoquine was derivatized with hexafluoroacetylacetone, extracted, and subjected to high performance thin-layer chromatography (HP-TLC) followed by liquid scintillation counting. The Km values for debrisoquine 4-hydroxylase were 70 and 120 microM and the Vmax values were 8 and 24 pmol per milligram microsomal protein per minute. By application of this assay, it was possible to show that 4-hydroxydebrisoquine formation was competitively inhibited by sparteine. Antipyrine up to a concentration of 4 mM had no effect.