ascorbic-acid and sulfamethoxazole-hydroxylamine

Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

Topics: Adult; Aged; Antioxidants; Ascorbic Acid; Cell Separation; Cyclic N-Oxides; Drug Hypersensitivity; Female; Glutathione; HIV Infections; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Oxidation-Reduction; Spin Labels; Sulfamethoxazole

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

Topics: Animals; Anti-Infective Agents; Antioxidants; Ascorbic Acid; Ascorbic Acid Deficiency; Cell Proliferation; Cytochrome-B(5) Reductase; Cytochromes b5; Drug Hypersensitivity; Glutathione; Guinea Pigs; Inactivation, Metabolic; Liver; Male; Sulfamethoxazole; T-Lymphocytes

Sulfamethoxazole is metabolized by microsomal CYP2C9 to a hydroxylamine that is thought to be responsible for the relatively high incidence of hypersensitivity reactions associated with the drug. Accurate quantification of the hydroxylamine requires the loss of metabolite through autoxidation to be blocked with ascorbate. In this study, a partly nonenzymatically generated arylhydroxylated derivative of sulfamethoxazole was identified by liquid chromatography/mass spectrometry in incubations of human liver microsomes, and it was found to coelute with the isomeric hydroxylamine under the conditions of three published high-performance liquid chromatography (HPLC) assays. Partial inhibition of the aryl hydroxylation by 1-aminobenzotriazole suggested some involvement of cytochrome P450. However, the formation of this compound was ascorbate-dependent, and it was enhanced by the addition of Fe2+/EDTA and inhibited by desferrioxamine but not by mannitol. These findings are consistent with the phenol being generated via an Fe2+/ascorbate/O2-oxygenating system that does not involve hydroxyl radicals. It was also produced by H2O2/ascorbate. Because the compound shares close chromatographic similarities with the hydroxylamine metabolite, it is possible that previous studies may have inaccurately characterized or quantified sulfamethoxazole metabolism.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Deferoxamine; Edetic Acid; Enzyme Inhibitors; Ferrous Compounds; Humans; Hydrogen Peroxide; Hydroxylation; Kinetics; Microsomes, Liver; Molecular Structure; NADP; Sulfamethoxazole; Tandem Mass Spectrometry; Triazoles

The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions.. Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro.. Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%).. Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.

Topics: Adult; Anti-Infective Agents; Ascorbic Acid; Ascorbic Acid Deficiency; Case-Control Studies; CD4-CD8 Ratio; Cysteine; Dehydroascorbic Acid; Female; Glutathione; HIV Infections; Humans; In Vitro Techniques; Male; Middle Aged; Oxidation-Reduction; Sulfamethoxazole