Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction

Abstract

Objectives: NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA.

Methods: Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced.

Results: Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency.

Conclusion: These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Fig 1

Distribution of human hepatic sulfamethoxazole-hydroxylamine (SMX-HA) reduction activities in 111 human livers. Mean (± SD) activity was 0.52 ± 0.24 nmol/min/mg microsomal protein in 64 females, 46 males, and one individual of unrecorded sex.

Fig 2

Relationship between b5 and b5R immunoreactive protein expression, normalized to β-actin, for 111 individual human liver samples. The expression of both proteins was significantly correlated (r = 0.74, p < 0.0001)

Fig 3

SMX-HA reduction activity (a), b5 protein (b), and b5R protein expression (c), in livers heterozygous for non-synonymous cSNPs in CYB5A and CYB5R3, compared to WT population data. Experimental data for the population is shown as a whisker-box plot with the box representing median and interquartile range, and the whiskers representing 95^th and 5^th percentiles. Outlying values for the WT population are shown as individual points outside the whiskers.

Fig 4

Expression of b5 and b5R immunoreactive protein in three human livers with non-synonymous cSNPs in either CYB5A or CYB5R3. Representative immunoblots shown for assays done in duplicate; 30 µg microsomal protein loaded and probed with rabbit polyclonal anti-human b5 or anti-human b5R antibody. (a) wild-type (WT) CYB5A homozygote, and S5A heterozygote in CYB5A showing absence of b5 expression at 17 kDa. Additional bands (22 and 54 kD) represent as yet unidentified proteins that cross-react with anti-human b5 antibody. (b) wild-type CYB5R3 homozygote, and R59H heterozygote in CYB5R3, showing modest but significantly decreased b5R protein expression (37 kDa), when normalized to β-actin (42 kDa). Separate blot with wild-type wild-type CYB5R3 homozygote, and R297H heterozygote in CYB5R3, showing two bands for the R297H subject.

Fig 5

Enzyme efficiency (V_max/K_m) for SMX-HA reduction from kinetic determinations in a subset of 18 WT human livers, compared to data obtained from livers with CYB5A or CYB5R3 cSNPs. Reduction activities for human livers in the subset were in three defined ranges (>90^th percentile, n=7; 45^th–55^th percentile, n=6; <10^th percentile, n=5). *R59H and *R297H denote separate calculated efficiencies for the high affinity components observed in these heterozygous livers. Individual apparent K_m and V_max values are given in the text.

Fig 6

Fitted velocity versus substrate concentration curves and Eadie-Hofstee plots for SMX-HA reduction in human liver microsomes heterozygous for the R59H (a), T117S (b), and R297H (c) CYB5R3 cSNPs.. Kinetic experiments were performed for individual livers with (□) R59H, (▼) T117S, and (○) R297H CYB5R3 allozymes. Data for the T117S livers was fit to the Michaelis-Menten equation (data from one representative liver shown), while the kinetic constants for the high and low-K_m components of the R59H and R297H livers were derived from Hanes plots.

Fig 6

Fitted velocity versus substrate concentration curves and Eadie-Hofstee plots for SMX-HA reduction in human liver microsomes heterozygous for the R59H (a), T117S (b), and R297H (c) CYB5R3 cSNPs.. Kinetic experiments were performed for individual livers with (□) R59H, (▼) T117S, and (○) R297H CYB5R3 allozymes. Data for the T117S livers was fit to the Michaelis-Menten equation (data from one representative liver shown), while the kinetic constants for the high and low-K_m components of the R59H and R297H livers were derived from Hanes plots.

Fig 6

Fitted velocity versus substrate concentration curves and Eadie-Hofstee plots for SMX-HA reduction in human liver microsomes heterozygous for the R59H (a), T117S (b), and R297H (c) CYB5R3 cSNPs.. Kinetic experiments were performed for individual livers with (□) R59H, (▼) T117S, and (○) R297H CYB5R3 allozymes. Data for the T117S livers was fit to the Michaelis-Menten equation (data from one representative liver shown), while the kinetic constants for the high and low-K_m components of the R59H and R297H livers were derived from Hanes plots.