Royer Leandro O, Knudsen Fernanda S, de Oliveira Marcone A, Tavares Marina F M, Bechara Etelvino J H
Departamento de Bioquímica and Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo, SP, Brazil.
Chem Res Toxicol. 2004 Dec;17(12):1725-32. doi: 10.1021/tx049821y.
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 (*CH2COR) 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.
氧化应激被认为在包括糖尿病和先天性代谢缺陷在内的多种疾病的发病机制中起作用。在这些病症中观察到的氧化损伤类型归因于与有毒代谢物积累相关的活性中间体的过量产生。在这些病症中，极具氧化性的过氧亚硝酸盐的产生也可能很高。我们在此研究过氧亚硝酸盐引发的乙酰乙酸乙酯（EAA）和2-甲基乙酰乙酸乙酯（EMAA）的氧化反应，这两种代谢物分别在糖尿病和异亮氨酸血症中积累。耗氧研究证实，过氧亚硝酸盐在磷酸盐缓冲液中促进EAA和EMAA的需氧氧化。这些反应伴随着超微弱发光，这可能源于二氧杂环丁烷中间体热解形成的三线态羰基产物。EAA和EMAA的氧气吸收和化学发光动力学受到磷酸根离子的强烈影响，已知磷酸根离子可催化羰基烯醇化以及对羰基的亲核加成。通过耗氧和化学发光测量获得的反应pH曲线表明，过氧亚硝酸根阴离子是EAA和EMAA需氧氧化的引发剂。用自旋捕获剂3,5-二溴-4-亚硝基苯磺酸和2-甲基-2-亚硝基丙烷进行的电子顺磁共振自旋捕获研究表明，在过氧亚硝酸盐氧化EAA的过程中，甲基和以碳为中心的自由基（*CH₂COR）是中间体。对于EMAA，检测到一个叔碳中心自由基（*EMAA）和一个酰基自由基，后者可能是由三线态羰基产物的裂解产生的。两种底物超化学计量形成乙酸盐证实了氧依赖性链反应的发生，这里提出是由底物烯醇形式的单电子提取引发的。EAA和EMAA氧化过程中产生的自由基和电子激发态可能有助于进一步阐明这些疾病的分子基础。
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