Validation of bupropion hydroxylation as a selective marker of human cytochrome P4502B6 catalytic activity

The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activit y. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activ ity varied 80-fold when assayed at 500 mu M substrate and significantly cor related with CYP2B6 blotting density (r(2) = 0.99) and S-mephenytoin N-deme thylase activity (r(2) = 0.98). Kinetic analysis of BUP hydroxylation was p erformed in a subset of seven HLMs representative of the 80-fold range in a ctivity. Sigmoidal kinetics suggestive of allosteric activation was observe d in five HLMs exhibiting low or high activity; the mean apparent K-m for B UP hydroxylation in these HLMs (130 mu M) was similar to the K-m for cDNA-e xpressed CYP2B6 (156 mu M). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 iso -forms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylati on at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The rela tive contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MA B-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhi bition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 mu M BUP. The se results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 m u M BUP, thereby validating its use as a diagnostic in vitro marker of CYP2 B6 catalytic activity.