Peroxynitrite-Mediated Decarboxylation of Pyruvate to Both Carbon Dioxide and Carbon Dioxide Radical Anion Jeannette Va ´ squez-Vivar, Ana Denicola, † Rafael Radi, † and Ohara Augusto* Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo, CxP 26077, 05599-970 Sa ˜ o Paulo, SP, Brazil, and Department of Biochemistry, Facultad de Medicina, Universidad de la Repu ´ blica, 11800 Montevideo, Uruguay Received February 25, 1997 X There has been a recent renewal of interest in the antioxidant properties of pyruvate which are usually attributed to its capacity to undergo oxidative decarboxylation in the presence of hydrogen peroxide. The interaction of pyruvate with other oxidizing biological intermediates, however, has been scarcely considered in the literature. Here we report that peroxynitrite, the oxidant produced by the reaction between superoxide anion and nitric oxide, reacts with pyruvate with an apparent second-order rate constant of 88 ( 7M -1 s -1 at pH 7.4 and 37 °C. Kinetic studies indicated that pyruvate reacts with peroxynitrite anion (k ) 100 ( 7M -1 s -1 ), peroxynitrous acid (k ) 49 ( 7M -1 s -1 ), and a highly oxidizing species derived from peroxy- nitrous acid. Pyruvate decarboxylation was proved by anion exchange chromatography detec- tion of acetate in incubations of peroxynitrite and pyruvate at pH 7.4 and 5.5. Formation of carbon dioxide radical anion was ascertained by EPR spin-trapping studies in the presence of GSH and the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The use of pyruvate labeled with 13 C at the 1-position led to the detection of the labeled DMPO carbon dioxide radical anion adduct. In the absence of GSH, oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways, the latter being preponderant. Hydrogen peroxide-mediated pyruvate oxidation does not produce detectable levels of carbon dioxide radical anion except in the presence of iron(II)-ethylenediamine- N,N,N′,N′-tetraacetate (EDTA). The apparent second-order rate constant of the reaction between pyruvate and hydrogen peroxide was determined to be 1 order of magnitude lower than that of the reaction between pyruvate and peroxynitrite. The latter process may contribute to the antioxidant properties of pyruvate. Introduction Pyruvate is an ubiquitous cellular metabolite which affects a variety of physiological and biochemical func- tions. When consumed or infused at supraphysiologic concentrations in man and animals, pyruvate can induce a multitude of beneficial metabolic effects ranging from normalization of plasma hypoglycemia in non-insulin- dependent diabetes (1) to inhibition of malignant growth (2). Although it is doubtful that pyruvate induces this myriad of responses through only one mechanism, there has been renewed interest in the antioxidant properties of pyruvate as a possible mechanism underlying some of its physiological effects (3). Recently, pyruvate has been shown to protect against oxidative damage promoted by various insults such as denaturation of lens proteins induced by xanthine/xanthine oxidase (4), myocardial injury after ischemia-reperfusion (5, 6), renal injury mediated by hydrogen peroxide (7), and peroxisomal proliferation induced by clofibrate (8). The antioxidant properties of pyruvate and other R-keto acids are considered to be primarily dependent on their capacity of undergoing nonenzymatic decarboxyla- tion in the presence of hydrogen peroxide, a reaction first described in 1904 (9). Through this reaction, the R-keto acid is converted to a carboxylic acid, and carbon dioxide is produced while hydrogen peroxide is detoxified to water (eq 1): Reactions between pyruvate and oxidizing biological intermediates other than hydrogen peroxide, however, have been scarcely considered in the literature. One oxidant of emerging biological importance is peroxyni- trite 1 which is formed by the fast combination reaction (k ) 4-7 × 10 9 M -1 s -1 )(10, 11) between nitrogen monoxide and superoxide radical anion (eq 2): Since peroxynitrite is a stronger oxidant than both nitric oxide and superoxide anion, it has been suggested to be the oxidant responsible for some of the pathological conditions associated with an overproduction of these radicals (12). Indeed, peroxynitrite appears to be formed in vivo (13-15) and is a versatile oxidant that reacts with * Address correspondence to this author. Telephone: 55-11-818- 3873. Fax: 55-11-8187986 and 55-11-815-5579. E-mail: oaugusto@ quim.iq.usp.br. † Universidad de la Repu ´ blica. X Abstract published in Advance ACS Abstracts, June 1, 1997. 1 The term peroxynitrite is used to refer to the sum of all possible conformers of peroxynitrite anion (ONOO - ) and peroxynitrous acid (ONOOH) unless otherwise specified. IUPAC-recommended names for peroxynitrite anion, peroxynitrous acid, and nitric oxide are oxoper- oxynitrate(-1), hydrogen oxyperoxynitrate, and nitrogen monoxide, respectively. CH 3 COCOO - + H 2 O 2 f CH 3 COO - + CO 2 + H 2 O (1) O 2 •- + • NO f ONOO - (2) 786 Chem. Res. Toxicol. 1997, 10, 786-794 S0893-228x(97)00031-3 CCC: $14.00 © 1997 American Chemical Society