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Published on: February 15, 2016
Methemoglobin oxidation of N-acetylbenzidine to form a sulfinamide
T V Zenser1, V M Lakshmi, F F Hsu
1Veterans Affairs Medical Center, Division of Geriatric Medicine, St. Louis University School of Medicine, St. Louis, Missouri, USA. zensertv@slu.edu
Methemoglobin catalyzes the peroxidatic formation of N-acetylbenzidine (ABZ) sulfinamide adducts, a novel biomarker for aromatic amine exposure. This reaction involves glutathione and is inhibited by various agents, suggesting a specific metabolic pathway.
Area of Science:
- Biochemistry
- Toxicology
- Medicinal Chemistry
Background:
- Aromatic amine sulfinamide adducts of hemoglobin serve as biomarkers for exposure and indicate cytochrome P-450 N-hydroxylation.
- The potential for methemoglobin to catalyze the peroxidatic formation of N-acetylbenzidine (ABZ) sulfinamide adducts warrants investigation.
Purpose of the Study:
- To examine the peroxidatic formation of an N-acetylbenzidine (ABZ) sulfinamide adduct catalyzed by methemoglobin.
- To characterize the reaction conditions and identify the resulting adduct.
Main Methods:
- Methemoglobin-mediated metabolism of [3H]N-acetylbenzidine (ABZ) in the presence of hydrogen peroxide (H2O2) and glutathione.
- Analysis of adduct formation using High-Performance Liquid Chromatography (HPLC) and identification via electrospray ionization/mass spectrometry (ESI/MS).
- Investigation of reaction kinetics, optimal conditions (methemoglobin concentration, glutathione levels, pH), and the effect of various inhibitors.
Main Results:
- A novel ABZ-glutathione (ABZ-SG) adduct was formed, representing 17% of total radioactivity under optimal conditions (3 μM methemoglobin, 0.1–0.3 mM glutathione, pH 5.5).
- The reaction was sensitive to inhibitors like cyanide and azide but not mannitol or superoxide dismutase, supporting a methemoglobin-peroxidatic mechanism.
- The identified adduct, N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide, was susceptible to gamma-glutamyltranspeptidase and hydrolysis, and an ABZ sulfinamide conjugate with hemoglobin was formed.
Conclusions:
- Methemoglobin can catalyze the peroxidatic formation of ABZ sulfinamide adducts, potentially via a diimine monocation intermediate.
- This pathway represents a novel mechanism for aromatic amine adduct formation and may serve as a valuable biomarker.
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