Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cri-du-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH(4)(+), and H(2)O(2) has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH(4)(+). Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-(*)OH adduct as a decomposition product from the DMPO-O(2)(*)(-) adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O(2)(*)(-) radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-(*)OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA(*) enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O(2), leading directly to H(2)O(2) generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O(2)(*)(-) and AA(*) enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H(2)O(2) and O(2)(*)(-), triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.

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.

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.

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Hereditary tyrosinemia type I (HT1) is an inborn metabolic error characterized by hepatorenal dysfunction. Affected patients excrete large quantities of succinylacetone (SA), a tyrosine catabolite believed to be involved in the pathogenesis of HT1. A growing body of evidence relates the oxidative stress observed in metabolic disorders to free radicals generated from accumulated metabolites. In this context, oxidation of SA by peroxynitrite or cytochrome c yielding reactive intermediates and products was investigated here. Both peroxynitrite and cytochrome c were able to initiate oxygen consumption by SA, which was followed by polarimetric and chemiluminescence measurements. The light emission arises from triplet carbonyls formed by the thermolysis of dioxetane intermediates, as indicated by energy transfer experiments. EPR spin-trapping studies with 2-methyl-2-nitrosopropane revealed the intermediacy of two different carbon-centered radicals, one of them originating from cleavage of the triplet carbonyl product. The pH profiles obtained by oxygen consumption, chemiluminescence, and stopped-flow spectrophotometry point to the peroxynitrite anion as the initiator of SA aerobic oxidation. Overstoichiometric formation of organic acids based on added peroxynitrite confirms the occurrence of an oxygen-dependent chain reaction, here proposed to be initiated by one electron abstraction from the enolic form of SA. The results obtained may help shed light on the role of both SA and oxidative stress in the pathogenesis of HT1.