Chem. Res. Toxicol. 2004, 17, 1725-1732
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Peroxynitrite-Initiated Oxidation of Acetoacetate and 2-Methylacetoacetate Esters by Oxygen: Potential Sources of Reactive Intermediates in Keto Acidoses Leandro O. Royer,† Fernanda S. Knudsen,† Marcone A. de Oliveira,‡ Marina F. M. Tavares,‡ and Etelvino J. H. Bechara*,† Departamento de Bioquı´mica and Departamento de Quı´mica Fundamental, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, 05513-970 Sa˜ o Paulo, SP, Brazil Received July 2, 2004
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.
Introduction Reactive carbonyl compounds and oxidative stress have been implicated as mediators in the pathogenesis of different diseases, including diabetes mellitus (1-4) and inborn metabolic errors, Alzheimer’s disease, and other neurodegenerative diseases (5). The carbonyl stress induced by these compounds has also been proposed to promote uremic toxicity (6) and the aging process (7). Specifically, recent studies suggest that β-diketones are related to the pathogenesis of hereditary tyrosinemia type I (8) (an inborn metabolic error) and diabetes mellitus (9) through mechanisms that involve the generation of reactive intermediates. The suggestion that elevated blood levels of ketone bodies (6-10 mM in blood) (10) commonly encountered in diabetic patients may relate to the observed oxidative damage in diabetes (9, 10) was supported by recent studies showing that acetoacetate can generate oxygen radicals (11), increase the oxidizability of low density and * To whom correspondence should be addressed. Tel: 55-11-30913869. E-mail:
[email protected]. † Departamento de Bioquı´mica. ‡ Departamento de Quı´mica Fundamental.
very low density lipoproteins (12), and cause glutathione depletion and promote lipid peroxidation in monocytes and human endothelial cells (11, 13). In addition to oxidative damage, acetoacetate (AA) also affects cell signaling pathways, as demonstrated by the increased secretion of tumor necrosis factor-R in cultured monocytes (14) and the activation of mitogen-activated protein kinase (MAPK)1 signaling pathways in primary cultured rat hepatocytes (15). Both effects are thought to be mediated through an oxidative stress mechanism. On the other hand, the oxidizability of AA may confer antioxidant properties on it. Accordingly, Squires et al. reported that acetoacetate increases contractile performance and potentiates β-adrenergic inotropism in stunned myocardium by its antioxidant properties (16, 17). In this study, we investigate the oxidation of ethyl acetoacetate (EAA) by the biological oxidant peroxynitrite in normally aerated phosphate buffer. Peroxynitrite was 1 Abbreviations: DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid; EAA, ethyl acetoacetate; EMAA, ethyl 2-methylacetoacetate; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; MNP, 2-methyl2-nitrosopropane; MOPS, 4-morpholinepropanesulfonic acid; TRIS, 2-amino-2-hydroxymethyl-1,3-propanediol.
10.1021/tx049821y CCC: $27.50 © 2004 American Chemical Society Published on Web 11/19/2004
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Chem. Res. Toxicol., Vol. 17, No. 12, 2004
Royer et al.
chosen as the oxidant since it was recently demonstrated that its production in blood, platelets, and renal cortex is increased in diabetes mellitus (18-22). A strong and versatile oxidant, peroxynitrite is capable of reacting with a wide variety of biomolecules, including DNA, lipids, thiols, and amino acids (23, 24) by different mechanisms (one- and two-electron oxidations, nitrations, nitrosations, and nucleophilic addition to carbonyls (25-29)). For comparison, the oxidation of ethyl 2-methylacetoacetate (EMAA) was also investigated here. Large amounts of 2-methylacetoacetate are excreted in the urine (8.0 mol/mol of creatinine) of patients with 2-methylacetoacetyl-CoA thiolase deficiency (30, 31), one of the enzymes of the isoleucine catabolic pathway. We hypothesize that, analogously to other inborn metabolic errors (32-34), the accumulation of 2-methylacetoacetate leads to excessive free radical production and oxidative stress. Our results indicate that peroxynitrite can initiate the aerobic oxidation of EAA and EMAA, generating carboncentered radicals, including the acetyl and methyl radicals. More strikingly, the reactions are chemiluminescent, which probably arises from triplet carbonyls, which are species known to possess a reactivity pattern similar to that observed in alkoxyl radicals (35).
Materials and Methods Chemicals. 2-Amino-2-hydroxymethyl-1,3-propanediol (TRIS), Chelex-100, horseradish peroxidase (HRP) type VI, 4-(2-hydroxymethyl)piperazine-1-ethanesulfonic acid (HEPES), 4-morpholinepropanesulfonic acid (MOPS), sodium nitrite, and succinic acid were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium acetate, sodium bicarbonate, and hydrogen peroxide were supplied by Merck (Darmstadt, Germany). EAA, EMAA, and 2-methyl-2-nitrosopropane (MNP) dimer were obtained from Aldrich (Milwaukee, WI). 3,5-Dibromo-4-nitrosobenzenesulfonic acid (DBNBS) was synthesized as previously described (36). Peroxynitrite was synthesized from sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) in a quenchedflow reactor and quantified spectrophotometrically at 302 nm ( ) 1670 M-1 cm-1). Both EAA and EMAA were distilled before use and stored in a nitrogen atmosphere. Each solution was prepared in distilled water treated in a Millipore Milli-Q system, and the buffers were pretreated with Chelex-100. Oxygen Consumption. Oxygen uptake studies were followed in an oxygen monitor (Yellow Spring Instruments model 53 at 37 °C). The saturating oxygen concentration at this temperature was taken to be 220 µM (37). Low-Level Chemiluminescence Measurements. Lowlevel chemiluminescence was measured either with a Hamamatsu TVC 767 photon counter equipped with a red-sensitive Thorn EMI (9658AM) photomultiplier cooled to -12 °C by a Thorn EMI (model Fackt MKIII) thermoelectric cooler or with a luminometer (Biorbit model 1251). EPR Spin-Trapping Experiments. EPR spectra were recorded at room temperature using a Bruker EMX spectrometer. In the experiments with peroxynitrite, all the spectra were recorded 1 min after the addition of the oxidant, while the pH (7.2) was checked at the end of the experiment to detect eventual changes caused by the addition of alkaline stock solutions of peroxynitrite. Computer simulation analyses of some spectra were performed using the software described by Duling (38). Capillary Electrophoresis. The acetate was analyzed with an instrument (Beckman Instruments model P/ACE system 5510) equipped with a UV detector set at 254 nm. The column consisted of a fused-silica capillary (75 cm × 75 µm i.d.); the electrolytic solution was 10 mM 3,5-dinitrobenzoic acid and 0.2 mM cetyltrimethylammonium bromide at pH 4.43, and separation was performed at -20 kV. Samples were injected by the
Figure 1. Total oxygen consumption by EAA (0) or EMAA (b) at different substrate (A) or peroxynitrite (B) concentrations in the presence of 0.5 mM peroxynitrite in 500 mM phosphate buffer (pH 7.2) at 37 °C: (A) 0.5 mM peroxynitrite, (B) 5 mM EAA and 2.5 mM EMAA. hydrodynamic method, at 3.5 kPa for 1 s. Sodium acetate standard was used in the quantitative analyses.
Results Oxygen Consumption and Chemiluminescence. Addition of peroxynitrite to phosphate-buffered EAA or EMAA solutions led to consumption of the dissolved oxygen within seconds. The extent of oxygen uptake was dependent on the concentration of the two reagents, ketoester and peroxynitrite, as shown in Figure 1. Easily enolizable β-diketones (Kenol ) 0.19-0.21 for acetoacetone in water) and β-ketoesters (Kenol ) 0.07 for EAA in water) (39) with low anodic potential (Ep ) 1.99 V vs NHE, for 3-methylacetoacetone) (29) have previously been found to undergo fast oxidation by peroxynitrite/oxygen, whereas poorly enolizable carbonyl compounds (Kenol < 10-5) (40) such as acetaldehyde and 2-pentanone, in addition to 2,2dimethylpropanal (not enolizable), were found to consume little oxygen under the same experimental conditions (29). It is therefore expected that the enol forms of EAA and EMAA are the actual one-electron reductants in the peroxynitrite/oxygen sustained reactions studied here. Although the millimolar concentrations of EAA used throughout this work are higher than the blood concen-
β-Ketoester Oxidation by Peroxynitrite
Figure 2. Ultraweak chemiluminescence curves monitored during the reaction of 2.5 mM EAA (s) or 5 mM EMAA (---) with 0.5 and 1 mM peroxynitrite, respectively, in 100 mM phosphate buffer (pH 7.2) at 37 °C. The arrows indicate the moment of peroxynitrite addition. Inset: effect of EAA concentration on the chemiluminescence of the reaction of EAA with peroxynitrite (0.5 mM) in 100 mM phosphate buffer (pH 7.2) at 37 °C.
tration of ketone bodies found in normal people (
