Enantioselective metabolism of trans-4-hydroxy-2-nonenal by brain mitochondria

Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation with many cellular effects. HNE possesses a stereogenic center at the C4 carbon that influences the metabolism and alkylation targets of HNE. We tested the hypothesis that rat brain mitochondria metabolize HNE in an enantioselective manner after exposure to racemic HNE. The study of HNE chirality, however, is hindered by the lack of facile methods to chromatographically resolve (R)-HNE and (S)-HNE. We used a chiral hydrazine, (S)-carbidopa, as a derivatization reagent to form diastereomers with (R)-HNE and (S)-HNE that were separated by reverse-phase HPLC. After exposure to racemic HNE, rat brain mitochondria metabolized HNE enantioselectively with a higher rate of (R)-HNE metabolism. By using the purified enantiomers of HNE, we found that this enantioselective metabolism of HNE was the result of higher rates of enzymatic oxidation of (R)-HNE by aldehyde dehydrogenases compared to (S)-HNE. Conjugation of HNE to glutathione was a minor metabolic pathway and was not enantioselective. These studies demonstrate that the chirality of HNE affects its mitochondrial metabolism and potentially other processes in the central nervous system.     

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner

Protein carbonylation is the covalent modification of proteins by α,β-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC–MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.     

Quantitation of mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites in a smoking cessation study

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.     

Mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites are in vivo markers of oxidative stress

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.     

Detection of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal after gavage of trans-4-hydroxy-2-nonenal or induction of lipid peroxidation with carbon tetrachloride in F344 rats

The 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp-adducts) were quantitated in tissues of rats treated with trans-4-hydroxy-2-nonenal (HNE) or carbon tetrachloride, respectively, using a 32P-postlabeling method. The method development was based on chemically synthesized HNE-1,N2-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by Nuclease P1. They were subsequently reacted with gamma-32P-ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose-TLC and quantitated by autoradiography. The labeling efficiency for the adduct standard was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. Internal standard was used to eliminate methodological variations. The determination of the limit of quantitation in DNA from rat tissues by spiking of HNE-dGp-adduct standard revealed a sensitivity of about 20 HNE-dGp-adducts/10(9) normal nucleotides. Background levels of HNE-dGp-adducts in tissues of rats including liver, kidney, lung, colon and forestomach were found in the range of 18-158 adducts/10(9) nucleotides with relatively high adduct levels in the liver and low adduct levels in kidney, lung and colon. These background levels were statistically significantly increased by the factor of 2 in liver, lung, colon and forestomach after induction of lipid peroxidation by carbon tetrachloride. The finding that background HNE-dGp-adduct levels may be in context with different metabolic activities of the tissues and the increase of HNE-dGp-adduct levels after application of carbon tetrachloride indicate that HNE-dGp-adducts are an endogenous lesion and that they are probably formed from radical initiated lipid peroxidation.     

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.     

Mechanism of destruction of microtubule structures by 4-hydroxy-2-nonenal

A major end product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is an electrophilic alkenal and produces Michael adducts with cellular proteins. It is known that exposure of cultured cells to HNE causes rapid disappearance of microtubule networks. In this study we addressed the mechanism. Immunochemical studies revealed that HNE preferentially modified alpha-tubulin in rat primary neuronal cells, PC12 cells, and rat fibroblast cell line 3Y1 cells. This was morphologically associated with the disappearance of microtubule structures in those cells. In a purified rat brain microtubule fraction, HNE modified unpolymerized tubulin and impaired its polymerizability, with a concomitant increase in insolubilized tubulin. Nevertheless, HNE had a marginal effect on the stability of pre-polymerized microtubules. These results suggest that disruption of microtubule assembly as a result of HNE modification of unpolymerized tubulin, rather than destruction of assembled microtubules, is responsible for the disappearance of microtubule structures in cells exposed to HNE.     

4-hydroxy-2-nonenal as a COX-2 inducer

4-hydroxy-2-nonenal (HNE) activates a variety of signaling pathways. We have recently evaluated the effect of oxidized fatty acid metabolites on cyclooxygenase-2 (COX-2) induction in rat liver epithelial RL34 cells and found that, among the compounds tested, HNE most dramatically induced COX-2. A p38 mitogen-activated protein kinase (p38 MAPK) pathway has been shown to play a key role in the mechanism of the HNE-induced COX-2 expression. It appears that the HNE-induced activation of p38 MAPK leads to the stabilization of COX-2 mRNA.     

Protection by thiols of the mitochondrial complexes from 4-hydroxy-2-nonenal

In the present study, the effects of 4-hydroxy-2-nonenal (HNE) on highly purified pyruvate dehydrogenase complex (PDC) and its catalytic components in vitro and on PDC, alpha-ketoglutarate dehydrogenase complex (KGDC), and the branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells were investigated. Among the PDC components, the activity of the dihydrolipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) only was decreased by HNE. Dihydrolipoamide dehydrogenase (E3) protected the E2-E3BP subcomplex from HNE inactivation in the absence of the substrates. In the presence of E3 and NADH, when lipoyl groups were reduced, higher inactivation of the E2-E3BP subcomplex by HNE was observed. Purified PDC was protected from HNE-induced inactivation by several thiol compounds including lipoic acid plus [LA-plus; 2-(N,N-dimethylamine)ethylamidolipoate(.)HCl]. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities, whereas BCKDC activity decreased to a lesser extent. Lipoyl compounds afforded protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. These findings show that thiols, including lipoic acid, provide protection against HNE-induced inactivation of lipoyl-containing complexes in the mitochondria.     

The stereochemical course of 4-hydroxy-2-nonenal metabolism by glutathione S-transferases

4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues.     

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ? A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA^? Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA^? bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.     

Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.     

Chemotaxis and chemokinesis of rat polymorphonuclear leukocytes in response to 4-hydroxy-2-tetradecenal and 4-hydroxy-2-nonenal

Since a number of experimental evidences suggests that some lipoperoxidation products can affect leukocyte migration "in vitro", we have investigated the chemotactic and chemokinetic properties of two of these products (4-hydroxy-2,3-trans-tetradecenal and 4-hydroxy-2,3-trans-nonenal) using rat neutrophils. The cells were obtained from the pleural cavity after injection of 1.0 ml isologous serum. The granulocytes were suspended in Hanks' plus BSA 2% and the motility determined by means of a modified Boyden chamber. For evaluating the chemotactic properties, the aldehyde were added into the lower compartment, while for detecting the chemokinetic power, the compounds were placed in both the compartments. Our results show that both the chemicals (in a range between nano- and micromolar concentrations) are able to exert -at different degree- a chemotactic activity. In this connection, the more active aldehyde appeared to be the tetradecenal. On the contrary, the same compounds seem uneffective in stimulating the random migration of polymorphonuclear cells.     

LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress in excitable tissues

A sensitive, selective, specific and rapid liquid chromatographic-electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination in skeletal muscle of the Michael adducts between 4-hydroxy-trans-2-nonenal (HNE), one of the most reactive lipid peroxidation-driven unsaturated aldehyde, and glutathione (GSH) and the endogenous histidine-containing dipeptides carnosine (CAR) and anserine (ANS), with the final aim to use conjugated adducts as specific and unequivocal markers of lipid peroxidation. Samples (skeletal muscle homogenates from male rats) were prepared by protein precipitation with 1 vol. of a HClO(4) solution (4.2%; w/v) containing H-Tyr-His-OH as internal standard. The supernatant, diluted (1:1, v/v) in mobile phase, was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9:1:0.01, v/v/v) at a flow rate of 0.2 ml/min, with a run time of 12 min. Detection was on a triple quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were in multiple reaction monitoring (MRM) mode using the following precursor-->product ion combinations: H-Tyr-His-OH (IS): m/z 319.2--> 156.5+301.6; GS-HNE: m/z 464.3--> 179.1+308.0; CAR-HNE: m/z 383.1--> 110.1+266.6; ANS-HNE: m/z 397.2--> 109.1+126.1. The method was validated over the concentration ranges 1.5-90 (GS-HNE) and 0.4-40 (CAR-HNE, ANS-HNE) nmoles/g wet tissue, and the LLOQ were 1.25 and 0.33 pmoles injected respectively. The intra- and inter-day precisions (CV%) were <7.38% (相似文献   

Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes

Intracellular metabolism of 4-hydroxy-2-nonenal (HNE), a major product and mediator of oxidative stress and inflammation, is analyzed in resting and fMLP-stimulated human polymorphonuclear leukocytes (PMNL), where this compound is generated during activation of the respiratory burst. HNE consumption rate in PMNL is very low, if compared to other cell types (rat hepatocytes, rabbit fibroblasts), where HNE metabolism is always an important part of secondary antioxidative defense mechanisms. More than 98% of HNE metabolites are identified. The pattern of HNE intermediates is quite similar in stimulated and resting PMNL - except for higher water formation in resting PMNL - while the initial velocity of HNE degradation is somewhat higher in resting cells, 0.44 instead of 0.28 nmol/(min × 10⁶ cells). The main products of HNE metabolism are 4-hydroxynonenoic acid (HNA), 1,4-dihydroxynonene (DHN) and the glutathione adducts with HNE, HNA, and DHN. Protein-bound HNE and water account for about 3-4% of the total HNE derivatives in stimulated cells, while in resting cells protein-bound HNE and water are 4% and 20%, respectively. Cysteinyl-glycine-HNE adduct and mercapturic acids contribute to about 5%.     

Effect of 4-hydroxy-2-nonenal modification on alpha-synuclein aggregation

Several observations have implicated oxidative stress and aggregation of the presynaptic protein alpha-synuclein in the pathogenesis of Parkinson disease. alpha-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with alpha-synuclein. Incubation of HNE with alpha-synuclein at pH 7.4 and 37 degrees C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of alpha-synuclein resulted in a major conformational change involving increased beta-sheet. HNE modification of alpha-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 mum HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of alpha-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.     

Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to "carbonyl stress" by this RCS, especially in the liver. Using chemoprecipitation based on a solid-phase hydrazine chemistry coupled with LC-MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase β-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC-MS/MS. A network was also created based on the identified protein targets, which showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications.     

Mitochondrial oxidation of 4-hydroxy-2-nonenal in rat cerebral cortex

4-hydroxy-trans-2-nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4-hydroxy-trans-2-nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non-toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time-dependent and dose-dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 micro m HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.     

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC–MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal–glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione–alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d₁₁-4-hydroxy-2-(E)-nonenal (d₁₁-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d₁₁-4-HNE-perfused rat livers, the amount of d₁₁-(S)-4-HNE–GSH released from the rat liver was higher than that of d₁₁-(R)-4-HNE–GSH, and more d₁₁-(R)-4-HNE–GSH than d₁₁-(S)-4-HNE–GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.