The metabolism of 4-hydroxy-trans-2-nonenal (HNE), an ␣,-unsaturated aldehyde generated during lipid peroxidation, was studied in isolated perfused rat hearts. High performance liquid chromatography separation of radioactive metabolites recovered from [ 3 H]HNE-treated hearts revealed four major peaks. Based on the retention times of synthesized standards, peak I, which accounted for 20% radioactivity administered to the heart, was identified to be due to glutathione conjugates of HNE. Peaks II and III, containing 2 and 37% radioactivity, were assigned to 1,4-dihydroxy-2-nonene (DHN) and 4-hydroxy-2-nonenoic acid, respectively. Peak IV was due to unmetabolized HNE. The electrospray ionization mass spectrum of peak I revealed two prominent metabolites with m/z values corresponding to [M ؉ H] ؉ of HNE and DHN conjugates with glutathione. The presence of 4-hydroxy-2-nonenoic acid in peak III was substantiated using gas chromatography-chemical ionization mass spectroscopy. When exposed to sorbinil, an inhibitor of aldose reductase, no GS-DHN was recovered in the coronary effluent, and treatment with cyanamide, an inhibitor of aldehyde dehydrogenase, attenuated 4-hydroxy-2-nonenoic acid formation. These results show that the major metabolic transformations of HNE in rat heart involve conjugation with glutathione and oxidation to 4-hydroxy-2-nonenoic acid. Further metabolism of the GS-HNE conjugate involves aldose reductasemediated reduction, a reaction catalyzed in vitro by homogenous cardiac aldose reductase.
4-Hydroxy-2-trans-nonenal, the most abundant and toxic unsaturated aldehyde generated during membrane lipid peroxidation, was synthesized starting from fumaraldehyde dimethyl acetal. In the first step of the synthesis, the fumaraldehyde dimethyl acetal was partially hydrolyzed using amberlyst catalyst to obtain the monoacetal. The 4-hydroxy-2-trans-nonenal was synthesized by the Grignard reaction of the fumaraldehyde monoacetal with 1-bromopentane. 4-Hydroxy-2-trans-nonenal, obtained as its dimethylacetal, was oxidized to its corresponding 4-keto derivative using pyridinium chlorochromate buffered with sodium acetate as the oxidizing agent. 4-(3H) 4-Hydroxy-2-trans-nonenal was obtained in one step by the sodium borotriteride reduction of the 4-keto derivative.
The aim of this study was to identify the cardiac oxidoreductases involved in the metabolism of 4-hydroxy-2-trans-nonenal (HNE), an alpha,beta unsaturated aldehyde generated during the peroxidation of omega-6 polyunsaturated fatty acids. In homogenates of bovine, human and rat ventricles the primary pyridine coenzyme-linked metabolism of HNE was associated with NADPH oxidation. The NADPH-dependent enzyme catalysing HNE reduction was purified to homogeneity from bovine heart. The purified enzyme displayed kinetic and immunological properties identical with the polyol pathway enzyme aldose reductase (AR), and catalysed the reduction of HNE to its alcohol 1,4-dihydroxynonene (DHN), with a Km of 7+/-2 microM. In the presence of NADP the enzyme did not catalyse the oxidation of DHN. During catalysis, HNE did not cause inactivation of AR. Nevertheless when the apoenzyme was incubated with HNE a dissociable complex was formed between the enzyme and HNE, followed by irreversible loss of activity. Inactivation of the enzyme by HNE was prevented by NADP. Partial modification of the enzyme with HNE led to a 17-fold increase in the KHNEm and Kglyceraldehydem, and the HNE-modified enzyme had a 500-fold higher IC50 for sorbinil than for the reduced enzyme, whereas the IC50 for tolrestat increased 25-fold. Incubation of the enzyme with radiolabelled HNE resulted in the incorporation of 2 mol of the aldehyde per mol of the enzyme. Sequence analysis of the radiolabelled peptides revealed modification of Cys-298 and Cys-187. The amino acid sequence of the HNE-modified peptides confirmed that the HNE-reducing cardiac enzyme is AR and not a related protein such as the fibroblast-growth-factor-regulated protein FR-1 or the mouse vas deferens protein MVDP. These results indicate that AR represents the only major oxidoreductase in the heart capable of utilizing HNE. The high affinity of the enzyme for HNE, the lack of inactivation during catalysis, and the lack of significant alcohol dehydrogenase activity of the protein suggests that AR-mediated catalysis of HNE is unlikely to be limited by substrate/product inhibition. Thus AR might constitute an antioxidative enzyme involved in myocardial protection against endogenous and exogenous cytotoxic aldehydes and against oxidative stress.
Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 microM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in Km for NADPH and Km fordl-glyceraldehyde, whereas the Km for the lipid peroxidation product, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP(H)-binding site of the enzyme.
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