Peroxynitrite-initiated oxidation of acetoacetate and 2-methylacetoacetate esters by oxygen: potential sources of reactive intermediates in keto acidoses.

Oxidative stress is believed to play a role in the pathogenesis of several diseases, including diabetes and inborn errors of metabolism. The types of oxidative damage observed in these pathologies have been attributed to the excessive production of reactive intermediates relating to the accumulation of toxic metabolites. The production of extremely oxidizing peroxynitrite can also be high in these pathologies. We study here the oxidation initiated by peroxynitrite of the ethyl esters of acetoacetate (EAA) and 2-methylacetoacetate (EMAA), metabolites that accumulate in diabetes and isoleucinemia, respectively. Oxygen consumption studies have confirmed that peroxynitrite promotes the aerobic oxidation of EAA and EMAA in phosphate buffer. These reactions were accompanied by ultraweak light emission, which probably arises from triplet carbonyl products formed by thermolysis of dioxetane intermediates. The kinetics of oxygen uptake and chemiluminescence by EAA and EMAA was strongly affected by the phosphate 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 aerobic oxidation. EPR spin-trapping studies with the spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid and 2-methyl-2-nitrosopropane showed the intermediacy of methyl and a carbon-centered radical (CH 2 COR) in the oxidation of EAA by peroxynitrite. In the case of EMAA, a tertiary carbon-centered radical (EMAA) and an acyl radical were detected, the latter probably resulting from the cleavage of a triplet carbonyl product. Superstoichiometric formation of acetate from both substrates confirmed the occurrence of oxygen-dependent chain reactions, here proposed to be initiated by one-electron abstraction from the enolic form of the substrates. The free radicals and electronically excited species generated in the oxidation of EAA and EMAA may help shed further light on the molecular basis of these diseases.