The goal of the proposed work is to combine the ESR spin trapping and the electrochemical-HPLC techniques (EC-HPLC) for analysis of radical intermediates produced by cell organelles, intact cells and in vivo. The biological systems, however, is limited by the low stability of the spin adducts (nitroxides) formed in the presence of cellular reductants. This proposal addresses the hypothesis that the main metabolic transformation of the ESR-active nitroxides is their reduction to ESR-undetectable hydroxylamine derivatives that can be quantified by EC-HPLC.
Specific Aim 1 - optimization of EC-HPLC protocols for analysis of a series of toxicologically relevant nitroxides and their hydroxylamine derivatives. Radicals e.g. superoxide, hydroxyl, methyl, glutathionyl and 1-hydroxyethyl will be generated by appropriate chemical or enzymatic systems and trapped with DMPO, POBN or PBN. The stability of the nitroxides in the absence and presence of biological reductants e.g. ascorbate, thiols, NADH or NADPH with or without cellular organelle electron transfer systems will be determined. HPLC protocols will be developed to assay for the ESR-detectable nitroxides and their ESR-silent hydroxylamine derivatives.
Specific Aim II will assess the effectiveness of the EC-HPLC protocols for detection of hydroxyl, glutathionyl and 1-hydroxyethyl radicals in intact HL60 and HepG2 cells subjected to oxidative stress.
Specific Aim III will assess the EC-HPLC detection of reduced spin-adducts in liver, blood and bile of rats treated acutely or chronically with iron and/or ethanol. The ESR spin-trapping technique has been applied to biological systems with less success than with in vitro systems mostly because of the low stability of the nitroxides in cellular milieu. Recent studies, however, suggest that a major metabolic transformation of the nitroxides is their reduction to the corresponding ESR-silent hydroxylamines. The proposed combination of the ESR spin trapping and EC-HPLC techniques may overcome this experimental difficulty by simultaneous analysis of both the nitroxide and the hydroxylamine forms of the spin adducts. The method is expected to display several orders of magnitude higher sensitivity than then traditional ESR spin trapping technique and may be a new approach in assays of oxidative stress.
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