James C. Sacco

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

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.

Evaluation of polymorphisms in the sulfonamide detoxification genes NAT2, CYB5A, and CYB5R3 in patients with sulfonamide hypersensitivity.

Objective To determine whether polymorphisms in the sulfonamide detoxification genes, CYB5A (encoding cytochrome b5), CYB5R3 (encoding cytochrome b5 reductase), or NAT2 (encoding N-acetyltransferase 2) were over-represented in patients with delayed sulfonamide drug hypersensitivity, compared with control patients who tolerated a therapeutic course of trimethoprim–sulfamethoxazole without adverse event. Methods DNA from 99 nonimmunocompromised patients with sulfonamide hypersensitivity who were identified from the Personalized Medicine Research Project at the Marshfield Clinic, and from 99 age-matched, race-matched, and sex-matched drug-tolerant controls, were genotyped for four CYB5A and five CYB5R3 polymorphisms, and for all coding NAT2 SNPs. Results CYB5A and CYB5R3 SNPs were found at low allele frequencies (<3–4%), which did not differ between hypersensitive and tolerant patients. NAT2 allele and haplotype frequencies, as well as inferred NAT2 phenotypes, also did not differ between groups (60 vs. 59% slow acetylators). Finally, no difference in NAT2 status was found in a subset of patients with more severe hypersensitivity signs (drug reaction with eosinophilia and systemic symptoms) compared with tolerant patients. Conclusion We found no evidence of a substantial involvement of these nine CYB5A or CYB5R3 polymorphisms in sulfonamide hypersensitivity risk, although minor effects cannot be completely ruled out. Despite careful medical record review and full resequencing of the NAT2 coding region, we found no association of NAT2 coding alleles with sulfonamide hypersensitivity (predominantly cutaneous eruptions) in this adult Caucasian population.

Dapsone‐Associated Methemoglobinemia in a Patient With Slow NAT2*5B Haplotype and Impaired Cytochrome b5 Reductase Activity

Dapsone (4,4′-diaminodiphenylsulfone; DDS) is a sulfone antimicrobial used to treat leprosy, malaria, and inflammatory dermatoses and to prevent and treat opportunistic infections in immunocompromised patients, particularly those who cannot tolerate sulfonamide antimicrobials.1 However, dapsone can lead to dose-dependent hematologic toxicity and is the most commonly recognized cause of acquired methemoglobinemia in human patients.2 The hydroxylamine metabolite of dapsone oxidizes the iron moiety of hemoglobin,3 which leads to increased levels of methemoglobin, impaired oxygen delivery, and cyanosis. Dapsone hydroxylamine is generated by CYP2C9 and CYP2C19, with minor contributions from other P450s.4,5 A rapid activity variant for CYP2C19 (CYP2C19*17) has been reported6 but not for CYP2C9. Dapsone hydroxylamine is detoxified by cytochrome b5 (b5) and its electron donor, cytochrome b5 reductase (b5R),7 which are expressed in blood, liver, and other tissues. This reduction pathway converts the hydroxylamine back to the parent dapsone. Dapsone itself is inactivated and eliminated following N-acetylation by the enzyme NAT2 in the liver,8 and impaired N-acetylation activity is a risk factor for hematologic and neurologic toxicity from dapsone.9,10 However, genotypic analysis of the CYP2C19, NAT2, and b5/b5R pathways has not been performed in the setting of dapsone-associated methemoglobinemia. This report describes a patient who developed methemoglobinemia at prophylactic dosages of dapsone, for which we characterized wild-type alleles at the CYP2C19*17 loci, but low activity of the b5/b5R pathway along with low expression of the CYB5A gene encoding b5, and a homozygous slow NAT2*5B haplotype, both of which may have contributed to defective dapsone detoxification.

Congenital Methemoglobinemia in a Dog with a Promoter Deletion and a Nonsynonymous Coding Variant in the Gene Encoding Cytochrome b5

The reduction of methemoglobin back to hemoglobin is catalyzed primarily by methemoglobin reductase, now recognized as the 2 enzyme system cytochrome b5 (b5) and cytochrome b5 reductase (b5R) (Fig 1). A negligible amount of methemoglobin is reduced by another enzyme, reduced nicotinamide adenine dinucleotide phosphate (NADPH) methemoglobin reductase. Treatment with methylene blue (MB) decreases methemoglobin concentrations dramatically by increasing the activity of the NADPH methemoglobin reductase pathway roughly 5to 10-fold. This report describes the diagnosis of a dog with congenital methemoglobinemia with normal b5R activity, 2 potentially deleterious variants in the cytochrome b5 gene (CYB5A), and the preoperative treatment of this dog with MB. A 4-year-old male castrated Pit Bull mix breed dog was referred for evaluation of markedly cyanotic mucous membranes and poor oxygen saturation, noted at the time of anesthetic induction for planned orthopedic correction for a right cranial cruciate ligament rupture. The surgery was aborted, but the dog reportedly experienced a prolonged anesthetic recovery. Results of a preanesthetic CBC and serum chemistry panel, as well as an electrocardiogram reportedly detected no abnormalities. Thoracic radiographs revealed a mildly enlarged right heart and mild interstitial pulmonary infiltrates. There was no history of coughing, sneezing, collapse, syncope, exercise intolerance, or respiratory distress at home, and the dog had no known access to toxins including acetaminophen. He was fed a commercial adult canine food diet. The dog was adopted approximately 2–3 years before presentation and had suffered only chronic skin allergies until the cranial cruciate ligament rupture occurred. The owner reported that the dog’s tongue and gums had always had a bluish discoloration. The patient was receiving carprofen, monthly milbemycin oxime—lufenuron, and spinosad. A course of antibiotic treatment with cefpodoxime had recently been discontinued. Relevant physical examination findings included cyanotic mucous membranes including the tongue, gums, and penis (Fig 2). There was no noted improvement in the color of the mucous membranes with administration of supplemental oxygen. Thoracic auscultation was normal with no evidence of a heart murmur or arrhythmia and clear lungs sounds. The dog was panting but eupneic. A lameness of the right pelvic limb was noted. A CBC, serum chemistry panel, urinalysis, and coagulation profile with D-dimer concentration were submitted for analysis. Abnormalities included erythrocytosis (HCT = 58%, reference interval 37–55%), increased total hemoglobin concentration (21.6 g/dL, reference interval 12–18 g/dL), mild hypokalemia (3.7 mEq/L, reference interval 4.0–5.6 mEq/L), hypercholesterolemia (412 mg/dL, reference interval 112– 328 mg/dL), elevated aspartate aminotransferase (115 U/L, reference interval 5–55 U/L), and increased creatine kinase (890 U/L, reference interval 10–200 U/L). Coagulation parameters and D-dimer concentration were within normal limits. Arterial and venous blood From the Department of Emergency and Critical Care, Advanced Critical Care, Emergency & Specialty Services – Los Angeles, Culver City, CA (McKenna, Son); the Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI (Sacco, Trepanier); the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA (Callan); the Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL (Harvey); and the Department of Cardiology, Advanced Critical Care, Emergency & Specialty Services – Los Angeles, Culver City, CA (Arndt). The work contributing to this case report was performed at Advanced Critical Care, Emergency & Specialty Services, and the veterinary schools of the University of Florida, University of Wisconsin and University of Pennsylvania. Corresponding author: J.A. McKenna, DVM, Advanced Critical Care, Emergency & Specialty Services – Los Angeles, 9599 Jefferson Boulevard Culver City, CA 90066; e-mail: jmckenna@accla.net. Submitted December 31, 2013; Revised May 1, 2014; Accepted June 23, 2014. Copyright

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis

AIMS Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b(5) (b5) and cytochrome b(5) reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens. METHODS Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly. RESULTS There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µm vs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r= 0.39, P= 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg(-1) min(-1)) than controls (18.9 µmol mg(-1) min(-1), P= 0.008). After 3-4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash. CONCLUSIONS Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.