RESUMEN
Peroxynitrite is shown here to promote the aerobic oxidation of isobutanal (IBAL) and 3-methyl-2,4-pentanedione (MP) in a pH 7.2 phosphate buffer into acetone plus formate and biacetyl plus acetate, respectively. These products are expected from dioxetane intermediates, whose thermolysis is known to be chemiluminescent (CL). Accordingly, the extent of total oxygen uptake by IBAL at different concentrations parallels the corresponding CL maximum intensities. The pH profile based on oxygen uptake data for the MP reaction matches the titration curve of peroxynitrous acid (pK(a) approximately 7), indicating that peroxynitrite anion is the oxidizing agent. Energy transfer studies with IBAL and the 9, 10-dibromoanthracene-2-sulfonate ion, a triplet carbonyl detector, indicates that triplet acetone (tau = 19 micros) is the energy donor. It is postulated that IBAL- or MP-generated triplet carbonyls are produced by the thermolysis of dioxetane intermediates, which are formed by the cyclization of alpha-hydroperoxide intermediates produced by insertion of dioxygen into the IBAL or MP enolyl radicals, followed by their reduction. Accordingly, EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) revealed the intermediacy of carbon-centered radicals, as expected for one-electron abstraction from the enol forms of IBAL or MP by peroxynitrite. The EPR data obtained with IBAL also reveal formation of the isopropyl radical produced by competitive nucleophilic addition of ONOO(-) to IBAL, followed by homolytic cleavage of this adduct and beta-scission of the resulting Me(2)CHCH(O(-))O(*). Superstoichiometric formation of fragmentation products from IBAL or MP attests to the prevalence of an autoxidation chain reaction, here proposed to be initiated by one-electron abstraction by ONOO(-) from the substrate. This work reveals the potential role of peroxynitrite as a generator of electronically excited species that may contribute to deleterious and pathological processes associated with excessive nitric oxide and aldehyde production.
Asunto(s)
Aldehídos/química , Nitratos/química , Oxidantes/química , Pentanos/química , Mediciones Luminiscentes , Detección de SpinRESUMEN
Production of free radicals from acetaldehyde oxidation by enzymes and cellular fractions is a well-known process. The toxic effects of acetaldehyde, however, are usually attributed to its reactions with biomolecules to produce adducts. Here, we demonstrate that hypothetical adducts produced from attack of acetaldehyde by two important biological oxidants, peroxynitrite and hydrogen peroxide, decompose to produce acetate, formate, and methyl radicals. Acetate, formate, nitrate, and nitrite were characterized and quantified by capillary electrophoresis. Radicals were detected and quantified by the EPR spectra produced in the presence of spin traps 3, 5-dibromo-4-nitrosobenzenesulfonic acid and 5,5-dimethyl-1-pyrroline N-oxide. Kinetic studies and product analysis were performed at different pHs. The results demonstrate that production of methyl radicals during oxidation of acetaldehyde by hydrogen peroxide was strictly dependent on the presence of iron(II) and occurred via two routes. One involved acetaldehyde attack by the hydroxyl radical to produce the acetyl radical that decomposes to methyl radical and carbon monoxide. The other route involved acetaldehyde attack by deprotonated hydrogen peroxide to produce a hypothetical intermediate that reductively cleaves via the action of present iron(II) to produce radicals. The latter mechanism predominates in the case of peroxynitrite, but radical formation does not require metal ions. Most of the hypothetical adduct produced from acetaldehyde and peroxynitrite (k = 680 M(-)(1) s(-)(1) at pH 7.4 and 37 degrees C) decays to nitrate and regenerates the aldehyde [Uppu, R. M., et al. (1997) Chem. Res. Toxicol. 10, 1331], but about 30% of it produces acetate, formate, and methyl radicals. Part of these oxidized products result from beta-scission and 1,2-shift reactions of the 1-hydroxyethoxyl radical which, together with nitrogen dioxide, freely diffuses from the adduct (20% yields). The results provide yet another example of the metal-independent free radical reactivity of peroxynitrite and may be relevant to the toxic effects associated with heavy drinking and diabetes.