Genetic variability of cytochrome b5 and NADH cytochrome b5 reductase: SNP discovery and characterization

Abstract

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX‐HA) to the parent drug sulfamethoxazole. SMX‐HA and a spontaneous nitroso metabolite are thought to contribute to sulfonamide hypersensitivity. Variability in hydroxylamine reduction could therefore contribute to drug toxicity. The aim of this study was to characterize variability in SMX‐HA reduction in 111 human livers, and investigate the functional significance of non‐synonymous polymorphisms (SNPs) in b5R and b5 cDNA. Hepatic SMX‐HA reduction displayed a 19‐fold variability (0.09‐1.69 nmol/min/mg protein), with the Vmax for the reaction varying 5‐fold between low‐activity and high‐activity outliers. The expression of immunoreactive b5 and b5R were significantly correlated to each other (p<0.0001). SMX‐HA reduction activity increased with increasing b5 and b5R protein content (p=0.003, p=0.0239). A novel S5A SNP in b5 was associated with very low activity and protein expression. Two novel b5R SNPs (R59H, R297H) displayed biphasic SMX‐HA reduction kinetics, with the mutant enzyme contributing to the lower affinity component, and, in the case of the R59H SNP, contributing to a reduction in activity. The b5 S5A and b5R R297H SNPs are associated with reduced SMX‐HA reduction in human liver and work is ongoing to clone, express, and characterize these mutant b5 and b5R proteins.Supported by National Institutes of Health grant GM61753