4-hydroxy-2-nonenal and 1-4-dihydroxy-2-nonene

Our previous work in perfused rat livers has demonstrated that 4-hydroxynonenal (HNE) is catabolized predominantly via β oxidation. Therefore, we hypothesized that perturbations in β oxidation, such as diet-altered fatty acid oxidation activity, could lead to changes in HNE levels. To test our hypothesis, we (i) developed a simple and sensitive GC/MS method combined with mass isotopomer analysis to measure HNE and HNE analogs, 4-oxononenal (ONE) and 1,4-dihydroxynonene (DHN), and (ii) investigated the effects of four diets (standard, low-fat, ketogenic, and high-fat mix) on HNE, ONE, and DHN concentrations in rat livers. Our results showed that livers from rats fed the ketogenic diet or high-fat mix diet had high ω-6 polyunsaturated fatty acid concentrations and markers of oxidative stress. However, high concentrations of HNE (1.6 ± 0.5 nmol/g) and ONE (0.9 ± 0.2 nmol/g) were found only in livers from rats fed the high-fat mix diet. Livers from rats fed the ketogenic diet had low HNE (0.8 ± 0.1 nmol/g) and ONE (0.4 ± 0.07 nmol/g), similar to rats fed the standard diet. A possible explanation is that the predominant pathway of HNE catabolism (i.e., β oxidation) is activated in the liver by the ketogenic diet. This is consistent with a 10-fold decrease in malonyl-CoA in livers from rats fed a ketogenic diet compared to a standard diet. The accelerated catabolism of HNE lowers HNE and HNE analog concentrations in livers from rats fed the ketogenic diet. On the other hand, rats fed the high-fat mix diet had high rates of lipid synthesis and low rates of fatty acid oxidation, resulting in the slowing down of the catabolic disposal of HNE and HNE analogs. Thus, decreased HNE catabolism from a high-fat mix diet induces high concentrations of HNE and HNE analogs. The results of this work suggest a potential causal relationship to metabolic syndrome induced by Western diets (i.e., high-fat mix), as well as the effects of a ketogenic diet on the catabolism of lipid peroxidation products in liver.

Topics: Aldehydes; Alkenes; Animals; Biomarkers; Diet, High-Fat; Diet, Ketogenic; Dietary Fats, Unsaturated; Glutathione; Lipid Peroxidation; Liver; Male; Mass Spectrometry; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar

Free radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively.

Topics: Acetylcysteine; Aldehydes; Alkenes; Animals; Biomarkers; Bromotrichloromethane; Chromatography, Liquid; Cross Reactions; Free Radicals; Immunoenzyme Techniques; Lipid Peroxidation; Male; Rabbits; Rats; Rats, Wistar; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trinitrobenzenesulfonic Acid

Oxidative stress has been implicated in numerous degenerative diseases of aging, including heart diseases. However, there is still a need to identify biomarkers of oxidative stress-related events, such as protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in these diseases in humans. The objective of this study was to assess if circulating levels of HNE-protein adducts (i) can be assessed with precision by GCMS and (ii) vary with disease progression and aging in a model of cardiomyopathy that displays enhanced oxidative stress, namely the spontaneously hypertensive rats (SHR). We modified a previously published isotope dilution GCMS method that quantifies HNE and its inactive metabolite, 1,4-dihydroxynonene (DHN), bound to thiol proteins following treatment with NaB(2)H(4) and Raney nickel, to increase its sensitivity (20-fold), precision, and robustness. Levels of these adducts were measured in blood and plasma collected from SHR and control Wistar rats at 7, 15, 22, and 30 weeks of age. Levels of protein-bound HNE, which were quantitated with good precision in the nanomolar range in blood, but not in plasma, were significantly increased by disease (SHR) and age (P < 0.0001 for both). Compared to Wistar rats, SHR showed greater blood levels of HNE-protein adducts at 22 and 30 weeks. Levels of protein-bound DHN, which were detected in blood and in plasma, were not affected by disease or age. Collectively, the results of this study conducted in an animal model of cardiomyopathy demonstrate that changes in blood HNE-protein thioether adducts with disease progression and aging can be assessed with good precision by the described GCMS method. This method may prove to be useful in evaluating the occurrence and impact of oxidative stress-related events involving bioactive HNE in heart diseases and aging in humans.

Topics: Aging; Aldehydes; Alkenes; Animals; Blood Proteins; Disease Progression; Gas Chromatography-Mass Spectrometry; Hypertension; Male; Rats; Rats, Inbred SHR; Rats, Wistar; Sulfides

An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated.. Rat lens or HLECs were incubated with 30 nmol (5 x 10(5) cpm/ micromol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37 degrees C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS).. On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA.. The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.

Topics: Aldehydes; Alkenes; Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelial Cells; Gas Chromatography-Mass Spectrometry; Glutathione; Humans; Lens, Crystalline; Lipid Metabolism; Male; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization

The human aldo-keto reductase AKR1C1 (20alpha(3alpha)-hydroxysteroid dehydrogenase) is induced by electrophilic Michael acceptors and reactive oxygen species (ROS) via a presumptive antioxidant response element (Burczynski, M. E., Lin, H. K., and Penning, T. M. (1999) Cancer Res. 59, 607-614). Physiologically, AKR1C1 regulates progesterone action by converting the hormone into its inactive metabolite 20alpha-hydroxyprogesterone, and toxicologically this enzyme activates polycyclic aromatic hydrocarbon trans-dihydrodiols to redox-cycling o-quinones. However, the significance of its potent induction by Michael acceptors and oxidative stress is unknown. 4-Hydroxy-2-nonenal (HNE) and other alpha,beta-unsaturated aldehydes produced during lipid peroxidation were reduced by AKR1C1 with high catalytic efficiency. Kinetic studies revealed that AKR1C1 reduced HNE (K(m) = 34 microm, k(cat) = 8.8 min(-1)) with a k(cat)/K(m) similar to that for 20alpha-hydroxysteroids. Six other homogeneous recombinant AKRs were examined for their ability to reduce HNE. Of these, AKR1C1 possessed one of the highest specific activities and was the only isoform induced by oxidative stress and by agents that deplete glutathione (ethacrynic acid). Several hydroxysteroid dehydrogenases of the AKR1C subfamily catalyzed the reduction of HNE with higher activity than aldehyde reductase (AKR1A1). NMR spectroscopy identified the product of the NADPH-dependent reduction of HNE as 1,4-dihydroxy-2-nonene. The K(m) of recombinant AKR1C1 for nicotinamide cofactors (K(m) NADPH approximately 6 microm, K(m)(app) NADH >6 mm) suggested that it is primed for reductive metabolism of HNE. Isoform-specific reverse transcription-polymerase chain reaction showed that exposure of HepG2 cells to HNE resulted in elevated levels of AKR1C1 mRNA. Thus, HNE induces its own metabolism via AKR1C1, and this enzyme may play a hitherto unrecognized role in a response mounted to counter oxidative stress. AKRs represent alternative GSH-independent/NADPH-dependent routes for the reductive elimination of HNE. Of these, AKR1C1 provides an inducible cytosolic barrier to HNE following ROS exposure.

Topics: 20-Hydroxysteroid Dehydrogenases; Aldehydes; Alkenes; Dehydroascorbic Acid; Enzyme Induction; Glutathione; Glutathione Transferase; Humans; NAD; NADP; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Recombinant Proteins

Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [(3)H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60-65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography-chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25-30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.

Topics: Aldehyde Reductase; Aldehydes; Alkenes; Animals; Aorta; Cells, Cultured; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Glutathione; Imidazoles; Imidazolidines; Lipoproteins, LDL; Male; Mass Spectrometry; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The metabolic fate of this aldehyde was investigated in hepatocytes as a cell type with a rapid HNE degradation. The experiments were carried out in rat hepatocytes at 37 degrees C at initial HNE concentrations of 1 microM-that means in the range of physiological and pathophysiologically relevant HNE levels-, 5 microM or 100 microM, respectively. About 95% of 100 microM HNE was degraded within 3 min of incubation. At 1 microM HNE the physiological level of about 0.1 to 0.2 microM was restored already after 30 sec. As primary products of HNE in hepatocytes the glutathione-HNE- 1:1-adduct, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynon-2-ene, were identified. In contrast to previous reports, the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene, was not the main HNE metabolite by far. The sum of these three primary HNE products accounts for about two-thirds of the total HNE degradation after 3 min of incubation. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins, contrary to its great functional importance, is low with about 3% of total HNE consumption after 3 min incubation. The glycine-cysteine-HNE, cysteine-HNE adducts, and the mercapturic acid from glutathione-HNE adduct are not formed. In total, almost 90% of HNE degradation could be balanced by the formation of different HNE metabolites. The fast metabolism underlines the role of HNE degrading pathways in hepatocytes as one important part of the antioxidative defense system in order to protect proteins from modification by aldehydic lipid peroxidation products.

Topics: Aldehydes; Alkenes; Animals; Cells, Cultured; Free Radicals; Glutathione; Kinetics; Lipid Peroxides; Liver; Male; Oxidation-Reduction; Protein Binding; Rats; Rats, Wistar

The metabolism of the cytotoxic lipid peroxidation product 4-hydroxynonenal was studied in perfused rat kidney. We investigated the total capacity of the rat kidney to metabolize 4-hydroxynonenal (HNE) and quantified the metabolites in the venous effluents as well as in the excreted urine. A rapid utilization of HNE was demonstrated, due to its immediate reactions with cellular compounds and its metabolism. During the first 3 min more than 80% of the infused HNE was metabolized in the perfused kidney. Glutathione-HNE conjugate (GSH-HNE: 35%), the corresponding alcohol 1,4-dihydroxynonene (1,4-DHN: 12%), HNE-mercapturic acid conjugate (HNE-MA: 4%), 4-hydroxynonenoic acid (HNA: 7%), tricarboxylic acid (TCA-cycle metabolites), and water (32%) were identified as primary and secondary metabolic products. We postulated that the total capacity of rat kidney to metabolize 4-hydroxynonenal with about 160-190 nmol/g wet wt/min. (initial influent concentration was 100 nmol/ml HNE) and other aldehydic products of lipid peroxidation is in the same range as that in other organs, e.g., intestine with 22 nmol/g wet wt/min (initial 70 nmol/ml HNE) (Siems et al. 1995. Life Sci. 57: 785-789) and heart with about 50 nmol/g wet wt/min (initial 10 nmol/ml HNE) (Grune et al. 1994. Cell Biochem. Funct. 12: 143-147). Compared to other organs, liver and kidney seemed to be the most important organs for the elimination of the final products of metabolism. The importance of the kidney in the formation of HNE-mercapturic acid conjugate was demonstrated (Alary et al. 1995. Chem. Res Toxicol. 8: 34-39). The selective excretion of this final metabolite of aldehyde metabolism may be of central importance in the detoxification of a number of lipid peroxidation products.

Topics: Aldehydes; Alkenes; Animals; Glutathione; Infusions, Intra-Arterial; Kidney; Lipid Peroxidation; Male; Perfusion; Rats; Rats, Wistar

It has previously been reported that isolated rat hepatocytes rapidly and completely metabolize high concentrations of 4-hydroxy-2,3-(E)-nonenal (4-HNE). However, until this report, the degree to which oxidative-reductive and nonoxidative metabolic pathways function in the depletion of 4-HNE by isolated rat hepatocytes has been speculative. The objective of the present study was to quantitate the extent to which cellular aldehyde dehydrogenases (ALDH; EC 1.2.1.3.), alcohol dehydrogenase (ADH; EC 1.1.1.1.), and glutathione S-transferases (GST; EC 2.5.1.18) function simultaneously during hepatocellular metabolism of 4-HNE. Hepatocytes were incubated with varying concentrations of 4-HNE (50, 100, 250 microM) and reversed-phase HPLC was used to quantitate 4-HNE and the oxidative and reductive metabolites, 4-hydroxy-2-nonenoic acid and 1,4-dihydroxy-2-nonene, respectively. Conjugative metabolism of 4-HNE was determined from the depletion of cellular reduced glutathione (GSH) and concomitant formation of a GSH-4-HNE adduct detected as 2,4-dinitrofluorobenzene derivatives measured by reversed-phase HPLC. Hepatocellular elimination of 4-HNE was estimated at rates of 1.666, 0.902, and 0.219 nmol min-1 10(6) hepatocytes-1 for 50, 100, and 250 microM aldehyde, respectively. At aldehyde concentrations of 50, 100, and 250 microM the maximal concentrations of oxidative (acid) metabolites formed were 5.9, 12.7, and 28.9 nmoles 10(6) hepatocytes-1, whereas the concentrations of the reductive (diol) metabolite were 0.4, 12.6, and 42.3 nmoles 10(6) hepatocytes-1, respectively. The presence of 4-methylpyrazole or cyanamide abolished formation of the reductive metabolite 1,4-dihydroxy-2-nonene or the oxidative metabolite 4-hydroxy-2-nonenoic acid in hepatocyte suspensions. At all 4-HNE concentrations evaluated, hepatocellular glutathione was not completely depleted by the aldehyde and the depletion of cellular reduced GSH corresponded to the production of the GSH-4-HNE conjugate. Metabolism by the alcohol/aldehyde dehydrogenase pathways accounted for approximately 10% of the 4-HNE elimination, while bioconversion by GST represent 50-60% of the total 4-HNE removal by hepatocytes. The enzymatic pathways responsible for the remaining 40% of 4-HNE metabolism remain to be identified. Taken together these results describe the quantitative and dynamic importance of oxidative, reductive, and nonoxidative routes in the metabolism and detoxification of 4-HNE.

Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Aldehydes; Alkenes; Animals; Carboxylic Acids; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Glutathione; Glutathione Transferase; Hydroxy Acids; Liver; Male; Rats; Rats, Sprague-Dawley