| Abstract
| - Oxidative stress is believed to play a role in the pathogenesis of several diseases, includingdiabetes and inborn errors of metabolism. The types of oxidative damage observed in thesepathologies have been attributed to the excessive production of reactive intermediates relatingto the accumulation of toxic metabolites. The production of extremely oxidizing peroxynitritecan also be high in these pathologies. We study here the oxidation initiated by peroxynitriteof the ethyl esters of acetoacetate (EAA) and 2-methylacetoacetate (EMAA), metabolites thataccumulate in diabetes and isoleucinemia, respectively. Oxygen consumption studies haveconfirmed that peroxynitrite promotes the aerobic oxidation of EAA and EMAA in phosphatebuffer. These reactions were accompanied by ultraweak light emission, which probably arisesfrom triplet carbonyl products formed by thermolysis of dioxetane intermediates. The kineticsof oxygen uptake and chemiluminescence by EAA and EMAA was strongly affected by thephosphate ion, known to catalyze carbonyl enolization and nucleophilic additions to carbonyls.The reaction pH profiles obtained by oxygen consumption and chemiluminescence measurements indicated that the peroxynitrite anion was the initiator of EAA and EMAA aerobicoxidation. EPR spin-trapping studies with the spin traps 3,5-dibromo-4-nitrosobenzenesulfonicacid and 2-methyl-2-nitrosopropane showed the intermediacy of methyl and a carbon-centeredradical (•CH2COR) in the oxidation of EAA by peroxynitrite. In the case of EMAA, a tertiarycarbon-centered radical (•EMAA) and an acyl radical were detected, the latter probably resultingfrom the cleavage of a triplet carbonyl product. Superstoichiometric formation of acetate fromboth substrates confirmed the occurrence of oxygen-dependent chain reactions, here proposedto be initiated by one-electron abstraction from the enolic form of the substrates. The freeradicals and electronically excited species generated in the oxidation of EAA and EMAA mayhelp shed further light on the molecular basis of these diseases.
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