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 (
CH
2COR) 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.