Identification of cardiac oxidoreductase(s) involved in the metabolism of the lipid peroxidation-derived aldehyde-4-hydroxynonenal

Abstract

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.

Aldose Reductase Mediates Mitogenic Signaling in Vascular Smooth Muscle Cells

“…As a result, AR does not display predominant specificity for a unique substrate-product pair; rather, it can reduce several types of aldehydes with nearly equal efficiency. Such behavior confers ideal properties to AR for the reduction of multiple aldehydes, such as the aldo-keto sugars and the aldehydes generated by lipid peroxidation (3)(4)(5)(6)(7)(8). The broad substrate specificity of the enzyme precludes its ready implication in a unique metabolic pathway and suggests that it may be recruited for tissue-specific use under a variety of metabolic contexts.…”

“…The reduction of glucose by AR is particularly significant during hyperglycemia, and increased flux of glucose via AR has been etiologically linked to the development of secondary diabetic complications (1,2). However, recent studies showing that AR is an excellent catalyst for the reduction of lipid peroxidation-derived aldehydes and their glutathione conjugates (3)(4)(5)(6)(7)(8) suggest that in contrast to its injurious role during diabetes, under normal glucose concentration, AR may be involved in protection against oxidative and electrophilic stress. The antioxidant role of AR is consistent with the observations that in a variety of cell types AR is up-regulated by oxidants such as hydrogen peroxide (9), lipid peroxidation-derived aldehydes (10,11), advanced glcosylation end products (12), and nitric oxide (13).…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Once formed, HNE is rapidly degraded by three major reactions: reduction to 1,4-dihydroxy-2-nonene by alcohol dehydrogenases (172), oxidation to 4-hydroxy-2-nonenoic acid by aldehyde dehydrogenase (173)(174)(175), or formation of the glutathione-conjugate (GS-HNE) catalyzed by glutathione-S-transferases as shown in equation <2>. The majority of HNE is metabolized through forming GS-HNE (172).…”

Metabolism of the Lipid Peroxidation Product, 4-Hydroxy-trans-2-nonenal, in Isolated Perfused Rat Heart

“…Identification of the Metabolic Pathways-Our previous investigations (30) show that HNE and GS-HNE are excellent substrates of the polyol pathway enzyme AR. Since the heart is particularly rich in AR, we examined the effect of the AR inhibitor-sorbinil on HNE metabolism in the heart.…”

“…This conclusion is based on the following observations: (a) the formation of GS-DHN is completely suppressed in heart perfused with HNE in the presence of the AR inhibitor sorbinil, (b) in homogenates of human, rat and bovine hearts, the major catalytic activity associated with GS-HNE was NADPH-mediated reduction, which could be attributed to AR, and (c) homogenous AR purified from heart efficiently catalyzed the reduction of GS-HNE with high affinity. Although AR also catalyzes the reduction of unconjuagted HNE (to DHN) (30), it appears that in the presence of high GSH concentration (ϳ3 mM in cardiac sarcoplasm) (38) and GST activity, conjugation with GSH is favored. Thus, in GSH-competent cells, reduction of GS-HNE (rather than free HNE) may be the primary activity of AR associated with HNE metabolism.…”