Received for publication July 17, 2008.
Revised September 30, 2008.
Accepted for publication October 2, 2008.

Role of NADPH cytochrome P450 reductase and cytochrome b5 / NADH b5 reductase in variability of CYP3A activity in human liver microsomes

Abstract

NADPH-cytochrome P450 reductase (CPR) and cytochrome b₅ (b₅) together with NADH b₅ reductase (b₅R) play important roles in cytochrome P450 (CYP) 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contribute to the known interindividual variability in CYP3A activity. CPR and b₅ were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (>46 years) male donors (n = 11) averaged 27% (P = 0.034) and 41% (P = 0.011) lower CPR levels than young (<45 years) male donors (n = 21) for spectrophotometric and immunoblot values, respectively. Similarly, HLMs from elderly male donors averaged 43% (P = 0.034) and 47% (P = 0.011) lower b5 levels than young male donors for spectrophotometric and immunoblot values, respectively. -Lipoic acid and 6-propyl-2-thiouracil (PTU) were evaluated for selectivity of inhibition of CPR and b₅R activities (respectively) using recombinant enzymes and HLMs, as well as for effects on CYP3A-mediated triazolam hydroxylation in HLMs with either NADH or -NADPH. The results indicate that both compounds are relatively nonselective inhibitors of CPR and b₅R activities. Finally, we used multivariate regression analysis and showed that variability in CPR or b₅ expression between HLMs does not contribute significantly to variability in CYP3A-mediated midazolam hydroxylation. Consequently, while aging is associated with decreased CPR and b₅ expression in human livers, this effect does not contribute to CYP3A variability.

Key words: CYP3A, cytochrome b5, cytochrome P450, cytochrome P450 catalyzed oxidations, cytochrome P450 function, liver microsomes, NADPH cytochrome P450 reductase, reductases