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Oxidative stress-induced lipid peroxidation leads to the formation of cytotoxic and genotoxic 2-alkenals, such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). Lipid-derived reactive aldehydes are subject to phase-2 metabolism and are predominantly found as mercapturic acid (MA) conjugates in urine. This study shows evidence for the in vivo formation of ONE and its phase-1 metabolites, 4-oxo-2-nonen-1-ol (ONO) and 4-oxo-2-nonenoic acid (ONA). We have detected the MA conjugates of HNE, 1,4-dihydroxy-2-nonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), the lactone of HNA, ONE, ONO, and ONA in rat urine by liquid chromatography-tandem mass spectrometry comparison with synthetic standards prepared in our laboratory. CCl 4 treatment of rats, a widely accepted animal model of acute oxidative stress, resulted in a significant increase in the urinary levels of DHN-MA, HNA-MA lactone, ONE-MA, and ONA-MA. Our data suggest that conjugates of HNE and ONE metabolites have value as markers of in vivo oxidative stress and lipid peroxidation. Lipid peroxidation (LPO)2 products are breakdown products of fatty acids formed under conditions of oxidative stress. 4-Hydroxy-2-nonenal (HNE) is a well established LPO product that has been shown to contribute to the development and progression of age-related diseases such as Alzheimer and atherosclerosis (1-4) in addition to being cytotoxic and genotoxic (5, 6). The mechanism of formation for HNE from linoleic acid via 4-hydroperoxy-2-nonenal (HPNE) has been previously demonstrated (7). Once HNE is formed, it can be further metabolized by cytochrome P450, aldehyde dehydrogenase, aldo-keto reductase (AKR), and conjugated by glutathione S-transferase (GST). Certain isoforms of murine and human P450s (8) and aldehyde dehydrogenase (9) can catalyze the oxidation of HNE to 4-hydroxy-2-nonenoic acid (HNA). When conjugated, HNA can undergo spontaneous intramolecular condensation, resulting in lactone formation (10). Aldo-keto reductase 1B1 has been shown to reduce HNE to form 1,4-dihydroxy-2-nonene (DHN) (11). Glutathione (GSH) can form conjugates with HNE and other LPO products (6) via a Michael-type addition mediated by . The GSH can then be further metabolized in the liver and in the kidney to form mercapturic acid (MA), resulting in the conjugates shown in Fig. 1. A number of studies have examined HNE and its metabolites in vivo (10,(15)(16)(17)(18) and have demonstrated the formation of the MA conjugates HNE-MA, DHN-MA, HNA-MA, and HNA-MA lactone in vivo (10). In addition, histidine-1,4-dihydroxynonane (His-DHN) and His-HNA have been found in the urine of obese Zucker rats, a model of metabolic syndrome (19).Not only can HPNE break down into HNE, but it can also form 4-oxo-2-nonenal (ONE) as shown in vitro (20) and in cultured cells (21). ONE can be reduced at the C-4 position by carbonyl reductase to form HNE (22), but it can also be reduced at the C-1 position by aldo-keto reductase to form 4-oxo-2-nonen-1-ol (ONO) (21,23,24) or oxidized by aldehyde dehydrogenase (human aldehyde dehy...

Abstract4-Hydroxy-2(E)-nonenal (HNE), a reactive aldehyde derived from oxidized lipids, has been implicated in the pathogenesis of cardiovascular and neurological diseases, in part by its ability to induce oxidative stress and by protein carbonylation in target cells. The effects of intracellular ascorbic acid (vitamin C) on HNE-induced cytotoxicity and protein carbonylation were investigated in human THP-1 monocytic leukemia cells. HNE treatment of these cells resulted in apoptosis, necrosis and protein carbonylation. Ascorbic acid accumulated in the cells at concentrations of 6.4 mM or 8.9 mM after treatment with 0.1 mM or 1 mM ascorbate in the medium for 18 h. Pretreatment of cells with 1.0 mM ascorbate decreased HNE-induced formation of reactive oxygen species and formation of protein carbonyls. The protective effects of ascorbate were associated with an increase of the formation of GSH-HNE conjugate and its phase-1 metabolites, measured by LC-MS/MS, and with increased transport of GSH conjugates from the cells into the medium. Ascorbate pretreatment enhanced the efflux of the multidrug resistant protein (MRP) substrate, carboxy-2',7'-dichlorofluorescein (CDF), and it prevented the HNE-induced inhibition of CDF export from THP-1 cells, suggesting that the protective effect of ascorbate against HNE cytotoxicity is through modulation of MRP-mediated transport of GSH-HNE conjugate metabolites. Formation of ascorbate adducts of HNE was observed in the cell exposure experiments, but it represented a minor pathway contributing to the elimination of HNE and to the protective effects of ascorbate.

The breakdown of polyunsaturated fatty acids (PUFAs) under conditions of oxidative stress results in the formation of lipid peroxidation (LPO) products. These LPO products such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) can contribute to the development of cardiovascular and neurodegenerative diseases and cancer. Conjugation with glutathione, followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the effects of these LPO products in disease development by facilitating their excretion from the body. We have developed a quantitative method to simultaneously assess levels of 4-oxo-2-nonen-1-ol (ONO)-MA, HNE-MA, and 1,4-dihydroxy-2-nonene (DHN)-MA in human urine samples utilizing isotope-dilution mass spectrometry. We are also able to detect 4-hydroxy-2-nonenoic acid (HNA)-MA, 4-hydroxy-2-nonenoic acid lactone (HNAL)-MA, and 4-oxo-2-nonenoic acid (ONA)-MA with this method. The detection of ONO-MA and ONA-MA in humans is significant because it demonstrates that HNE/ONE branching occurs in the breakdown of PUFAs and suggests that ONO may contribute to the harmful effects currently associated with HNE. We were able to show significant decreases in HNE-MA, DHN-MA, and total LPO-MA in a group of seven smokers upon smoking cessation. These data demonstrate the value of HNE and ONE metabolites as in vivo markers of oxidative stress.

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