Abstract

NADPH-cytochrome P450 reductase (CPR) and cytochrome-b₅ (b₅) together with NADH-b₅ reductase (b₅R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes 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. α-Lipoic acid and 6-propyl-2-thiouracil 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 β-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.