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%.     

Inactivation of the proteasome by 4-hydroxy-2-nonenal is site specific and dependant on 20S proteasome subtypes

The proteasome represents a major intracellular proteolytic system responsible for the degradation of oxidized and ubiquitinated proteins in both the nucleus and cytoplasm. We have previously reported that proteasome undergoes modification by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and exhibits declines in peptidase activities during cardiac ischemia/reperfusion. This study was undertaken to characterize the effects of HNE on the structure and function of the 20S proteasome. To assess potential tissue-specific differences in the response to HNE, we utilized purified 20S proteasome from heart and liver, tissues that express different proteasome subtypes. Following incubation of heart and liver 20S proteasome with HNE, changes in the 2D gel electrophoresis patterns and peptidase activities of the proteasome were evaluated. Proteasome subunits were identified by mass spectrometry prior to and following treatment with HNE. Our results demonstrate that specific subunits of the 20S proteasome are targeted for modification by HNE and that modified proteasome exhibits selective alterations in peptidase activities. The results provide evidence for a likely mechanism of proteasome inactivation in response to oxidative stress particularly during cardiac ischemia/reperfusion.     

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.     

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.     

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.     

In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification

In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in tau marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that tau phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal tau and induces the Alz50 epitope in tau. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of tau but also requires tau phosphorylation because neither methylated, recombinant, nor dephosphorylated tau reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament-tau isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the tau conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of tau and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.     

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease

In the last decade an important role for the progression of neuronal cell death in Alzheimer's disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimer's disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.     

An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds

The facile one step synthesis of 4-hydroxy-2(E)-nonenal and its dimethyl acetal via a cross-metathesis reaction between commercially available octen-3-ol and acrolein or its dimethyl acetal is reported. The method was extended to the synthesis of C₆ and C₁₂ 4-hydroxy-2(E)-enals, their dimethyl acetal and of the 4-hydroxy-2(E)-nonenoic acid (4-HNA).     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

4-Hydroxy-2-nonenal upregulates endogenous antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells: protection against overt oxidative and electrophilic injury

This study was undertaken to determine if 4-hydroxy-2-nonenal (HNE) could upregulate antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells, and if the upregulated defenses led to cytoprotection against oxidative and electrophilic injury. Incubation of H9c2 cells with HNE at noncytotoxic concentrations resulted in significant induction of cellular catalase, glutathione (GSH), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), as determined by enzyme activity and/or protein expression. HNE treatment caused increased mRNA expression of catalase, γ-glutamylcysteine ligase, GST-A1, and NQO1. Pretreatment of H9c2 cells with HNE led to significant protection against cytotoxicity induced by reactive oxygen and nitrogen species. HNE-pretreated cells also exhibited increased resistance to injury elicited by subsequent cytotoxic concentrations of HNE. Taken together, this study demonstrates that several antioxidants and phase 2 enzymes in H9c2 cells are upregulated by HNE and that the increased defenses afford protection against overt oxidative and electrophilic cardiac cell injury.     

Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma

Abstract

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of HNE with pentafluorobenzylhydroxylamine–HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d₁₁) as internal standard. This excellent stable and precise GC–MS method was carefully validated for HNE, and showed good linearity (r² = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4–6.1% and between-day precisions were 5.2–10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5–250 nmol/L) of HNE.

To examine the versatility of this modified GC–MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats.

Taken together, the GC–MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Short-term ascorbic acid deficiency induced oxidative stress in the retinas of young guinea pigs

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.     

A method for detection of 4-hydroxy-2-nonenal adducts in proteins

We developed a procedure to measure 4-hydroxy-2-nonenal (HNE)-amino acid adducts using the fluorescent probe 2-aminopyridine (2-AP). The method is based on the fact that HNE forms Michael addition-type amino acid adducts possessing an aldehyde functionality, which upon reaction with 2-AP in the presence of NaBH₃CN can be converted to their pyridylaminated derivatives. The HNE-amino acid adducts, namely Michael addition-type HNE-cysteine, HNE-histidine, and HNE-lysine adducts, after pyridylamination were resistant to conventional acid-hydrolysis conditions for protein (6 N HCl/110 °C/24 h) and could be detected by HPLC with a fluorescence detector. The reductive amination-based fluorescent labeling of HNE adducts is a simple and accurate technique that may be widely used to reveal increased levels of covalently modified proteins with HNE and its related aldehydes during aging and disease.     

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.     

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.     

Lysine-derived fluorophores formed by autoxidation of linoleic acid

To obtain an insight into fluorophores formed in proteins during lipid peroxidation, a lysine residue analogue (N(alpha)-hippuryllysine) was exposed to autoxidation of linoleic acid catalyzed by iron(III)-EDTA and L-ascorbic acid. The reaction predominantly produced two fluorescent products, N,N'-bis[5-(N-benzoylglycylamino)-5-carboxypentyl]-2-hydroxy-2-pentyl-3-imino-l,2-dihydropyrrole (II) and N,N'-bis[5-(N-benzoylglycylamino)-5-carboxypentyl]-2-hydroxy-2-(7-carboxyheptyl)-3-imino-1,2-dihydropyrolle (I).     

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.     

Malondialdehyde and 4-Hydroxy-2-Nonenal in Plant Tissue Cultures: LC-MS Determination of2, 4-Dinitrophenylhydrazone Derivatives

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their2, 4-dinitro-phenylhydrazone (DNP) derivatives in plant tissue cultures using LC-MS. This paper reports, for the first time, the use of LC-MS methodology to definitively identify 4-hydroxy-2-nonenal in plants. Limits of detection for the two derivatives are approximately 5pmol (1.2 × 10^-9g; 1μM) and O.1pmol (3 × 10^-l1g; 20nM) respectively. Mass spectrometer response was linear in the range from 2-200μM DNP-MDA and 0.02-10μM DNP-HNE.

This methodology has been used to assess the formation of aldehydic secondary lipid peroxidation products in dedifferentiated callus cultures of Daucus carota. The finding that profiles of MDA and HNE can be correlated with embryogenic competence is of considerable interest as oxidative status has already been implicated as a regulatory factor in animal development.