Epoxidation of trans-4-hydroxy-2-nonenal by fatty acid hydroperoxides and hydrogen peroxide

In this study, we reported that fatty acid hydroperoxides and hydrogen peroxide are capable of epoxidizing 4-hydroxy-2-nonenal, a lipid peroxidation product, to the mutagenic epoxide. The evidence of its formation is provided (i) by trapping with [8-3H]deoxyadenosine for the formation of 7-(1',2'-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine as a pair of diastereomers, (ii) by derivatization with (2,4-dinitrophenyl)hydrazine in acidic methanol, and (iii) by comparing its 1H-nuclear magnetic resonance and mass spectra to those of the authentic standard. After incubating 4-hydroxy-2-nonenal with 9- or 13-linoleic acid hydroperoxide at 37 degrees C for 24 h, the epoxide was produced in 13.4% or 12.5% yield, and with hydrogen peroxide, the yield was 21.5%. In the presence of fatty acid (linoleic acid, gamma-linolenic acid, or arachidonic acid) and lipoxygenase, the epoxide of 4-hydroxy-2-nonenal was formed in 15.3%, 7.2%, or 6.2% yield, respectively. The xanthine/xanthine oxidase/superoxide dismutase system generated the epoxide in 1.2% yield. These yields are estimated on the basis of a standard curve obtained from reactions of deoxyadenosine and epoxide. These results show that 4-hydroxy-2-nonenal is epoxidized by biological oxidants, suggesting a plausible endogenous pathway for the in vivo formation of etheno adducts.     

Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.     

Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer's disease is associated with inheritance of APOE4

Cumulative oxidative damage, including lipid peroxidation, is a central component of cellular aging and is thought to play a role in the pathogenesis of late-onset Alzheimer's disease (AD). Lipid peroxidation produces several cytotoxic aldehydes, one of the most potent being 4-hydroxy-2-nonenal (HNE). We have shown previously that HNE is a potent neurotoxin that covalently modifies and cross-links neuronal cytoskeletal protein in neuroglial cultures, suggesting that HNE may contribute to the pathogenesis of AD. In addition to aging, inheritance of the epsilon 4 allele of APOE is the other major risk factor for development of late-onset AD; however, the mechanisms through which aging and apolipoprotein E isoforms may collaborate in the onset or progression of AD are not known. We tested the hypothesis that HNE may yield a particular type of protein modification, pyrrole adduction, and that this may contribute to the pathogenesis of AD. Our data demonstrated that HNE formed pyrrole adducts with protein. Polyclonal antiserum was raised that specifically recognized HNE pyrrole adducts, and immunohistochemical analysis was performed on hippocampus and temporal cortex of 10 patients with histologically verified AD. Pyramidal neuron cytoplasm was immunoreactive in 4 of 4 APOE4 homozygotes, 2 of 3 APOE3/4 heterozygotes, and none of 3 APOE3 homozygotes (P < 0.05). The pattern of staining was highly suggestive of neurofibrillary tangles as the primary immunoreactive structure. These data suggest that differences in neuronal protein modification by HNE may account in part for the APOE-associated stratification of risk for late-onset AD.     

Malondialdehyde and 4-hydroxy-2-nonenal in plant tissue cultures: LC-MS determination of 2,4-dinitrophenylhydrazone derivatives

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their 2,4-dinitrophenylhydrazone (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 5 pmol (1.2 x 10(-9) g; 1 microM) and 0.1 pmol (3 x 10(-11) g; 20 nM) respectively. Mass spectrometer response was linear in the range from 2-200 microM DNP-MDA and 0.02-10 microM 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.     

Simultaneous determination of acrolein, malonaldehyde and 4-hydroxy-2-nonenal produced from lipids oxidized with Fenton's reagent

Ethyl linoleate, ethyl linolenate, ethyl arachidonate and cod liver oil were oxidized with Fenton's reagent. Acrolein, malonaldehyde and 4-hydroxynonenal formed were derivatized to N-methylpyrazoline, N-methylpyrazole and 5-(1'-hydroxyhexyl)-1-methyl-2-pyrazoline with N-methylhydrazine, respectively. The derivatives were simultaneously analysed by gas chromatograph equipped with a fused silica capillary column and a nitrogen-phosphorus detector. The maximum amounts of acrolein (9.7 +/- 2.11 nmol/ml) and malonaldehyde (61.18 +/- 6.51 nmol/ml) were formed from cod liver oil. The highest amount of 4-hydroxynonenal (6.83 +/- 0.53 nmol/ml) was produced from ethyl arathidonate.     

Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine

Aging and the progression of certain degenerative diseases are accompanied by increases in intracellular fluorescent material, termed "lipofuscin" and ceroid, respectively. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However, little direct evidence currently exists because the structures responsible for the fluorescent, cross-linked nature of this material are not well characterized. In this study, we have identified a fluorescent product formed in the reaction of Nalpha-acetyllysine and 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions [Esterbauer, H., Shaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11, 81-128]. This fluorescent compound, characterized as a 2-hydroxy-3-imino-1,2-dihydropyrrol derivative, appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link. Polyclonal antibody was raised to the Nalpha-acetyllysine-HNE fluorophore and found to be highly specific to the chromophore structure of the compound. This antibody has been used to conclusively demonstrate that the lysine-HNE derivative of this fluorophore forms on protein upon exposure to HNE. The results of this study therefore provide the basis for future investigations on the contribution(s) of HNE-derived fluorophore formation to lipofuscin and ceroid accumulation.     

Relationships between 4-hydroxy-2-nonenal, 2-thiobarbituric acid reactive substances and n-6 polyunsaturated fatty acids in refrigerated and frozen pork

The relationships between 4-hydroxy-2-nonenal (HNE), 2-thiobarbituric acid reactive substances (TBARS) and n-6 polyunsaturated fatty acids (n-6 PUFAs) were investigated for pork stored at 0, -20 and -80 degrees C. A significant linear correlation was apparent between HNE and TBARS for the pork stored at each temperature. However, the n-6 PUFA content remained unchanged during storage.     

Inactivation of alpha-ketoglutarate dehydrogenase during oxidative decarboxylation of alpha-ketoadipic acid

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.     

Identification of novel urinary metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in rats

Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4-3H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of "HNE polar metabolites" originate from omega-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy-HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1, 4-Dihydroxy-2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent omega-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers).     

Selective inactivation of glyceraldehyde-3-phosphate dehydrogenase by vinyl sulfones

Incubation of glyceraldehyde-3-phosphate dehydrogenase with vinyl sulfones resulted in a pseudo first-order loss of enzyme activity. The selective inactivation of the enzyme by vinyl sulfones is suggested from the structural requirement analysis and the enzyme susceptibility test. The enzyme inactivation was strongly reduced in the presence of NAD or glyceraldehyde-3-phosphate, and the prior treatment of the enzyme with 5,5'-dithio-bis-(2-nitrobenzoic acid) prevented the enzyme from the inactivation by vinyl sulfones (> or = 90%). Moreover, the early rapid phase of inactivation was much more responsive to L-cysteine reactivation, compared with the slower phase. Based on these results, it is proposed that vinyl sulfones inactivate the enzyme by inducing the oxidation of cysteine residue and/or covalent binding to cysteine residue in active site.     

5-羟基-2-磺酸基-4''''-硝基苯基重氮氨基偶氮苯的合成及其与镍的显色反应

合成了5-羟基-2-磺酸基-4'-硝基苯基重氮氨基偶氮苯(HSDAAN),并研究了其与镍的显色反应.结果表明,在pH 10的四硼酸钠缓冲溶液中,表面活性剂TritonX-100存在下,镍与HSDAAN形成12的红色络合物,其最大吸收波长位于528 nm,表观摩尔吸光系数为2.52×105 L·mol-1·cm-1;25 mL溶液中,镍在0～15 μg范围内符合比尔定律.方法用于合金中镍的测定,相对标准偏差(n=5)在2.7%～4.3%之间.     

3,5-二(3-羟基-2-吡啶氨基重氮基)-2,4,6-三溴苯甲酸的合成及其与铜的显色反应

本文合成了新显色剂3,5-二(3-羟基-2-吡啶氨基重氮基)-2,4,6三-溴苯甲酸(BH-PADTBBA),并研究了该试剂与铜的显色反应,建立了分光光度法测定微量铜的新方法。结果表明,在pH 10.0的Na2B4O7-NaOH缓冲溶液中,表面活性剂TritonX-100存在下,铜与BHPADTBBA生成络合比为1∶2的红色络合物。该络合物在570 nm处有最大吸收峰。检出限为7.8×10-3μg/mL。表观摩尔吸光系数为2.18×105L.mol-1.cm-1,铜量在0~18μg/25 mL范围内符合比尔定律。所拟方法用于铝合金样品中铜的测定,结果同认定值相符。     

Substrate selectivity and biochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli

4-Hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli was identified as a class I aldolase. The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept alpha-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.     

Antibodies to pyruvate dehydrogenase in primary biliary cirrhosis: correlation with histology

Antimitochondrial antibodies to pyruvate dehydrogenase are the hallmark of primary biliary cirrhosis. Their pathogenic role has not been proven, although antibodies to pyruvate dehydrogenase are bound to biliary epithelium. The aim of this study was to characterize serum IgA antibodies to pyruvate dehydrogenase and to evaluate their response to different treatment regimens. We also compared the level of antibodies with severity of histological lesions and the data of biochemical liver tests. Serum samples were collected at baseline and after 24 months from 61 primary biliary cirrhosis patients, whereas 23 patients were treated with ursodeoxycholic acid, 20 with colchicine, and 18 with placebo. ELISA was used to detect antibodies to pyruvate dehydrogenase. IgA and IgG were separated with jacalin and protein-A, respectively. Capacity of immunoglobulins to inhibit enzymatic activity was detected by spectrophotometric observation of the rate of enzyme reaction. 49 (80.3%) of the 61 patients possessed IgA antibodies to pyruvate dehydrogenase. Significant decrease in IgA antibodies was observed only in the ursodeoxycholic acid group (p<0.05). 15 of 18 IgA preparations and all 24 IgG preparations of patients' sera were inhibitory towards pyruvate dehydrogenase (mean inhibitory percent +/-SD: 42+/-33.4% and 79+/-22.4%, respectively, at a protein concentration of 100 microg/ml). The level of serum antibodies to pyruvate dehydrogenase correlated with several histological parameters (fibrosis, inflammatory infiltrate), but not with biochemical parameters. Our data reveal that IgA antibodies to pyruvate dehydrogenase inhibit enzyme function. The correlation between antibodies to pyruvate dehydrogenase and histological parameters might suggest the pathogenic role of these antibodies.     

2-羟基-3-甲氧基苯基荧光酮与钛显色反应的研究及应用

研究了2-羟基-3-甲氧基苯基荧光酮(HMPF)与钛的显色反应,建立了一种直接测定钢铁中微量钛的光度分析方法。在硫酸介质中,表面活性剂OP作用下,2 羟基3 甲氧基苯基荧光酮与钛形成一种红色络合物,络合物的组成为钛∶HMPF=1∶3(物质的量比),其最大吸收峰位于536nm,表观摩尔吸光系数为1.9×105,络合物在7h内稳定。在25mL溶液中,钛质量在0～3μg范围内符合比尔定律。显色反应有良好的选择性,方法简单、灵敏,可不经分离直接测定钢铁中微量钛,结果满意。     

Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate

The structure of the isocitrate dehydrogenase (IDH) complex with bound alpha-ketoglutarate, Ca2+, and NADPH was solved at 2.7-A resolution. The alpha-ketoglutarate binds in the active site at the same position and orientation as isocitrate, with a difference between the two bound molecules of about 0.8 A. The Ca2+ metal is coordinated by alpha-ketoglutarate, three conserved aspartate residues, and a pair of water molecules. The largest motion in the active site relative to the isocitrate enzyme complex is observed for tyrosine 160, which originally forms a hydrogen bond to the labile carboxyl group of isocitrate and moves to form a new hydrogen bond to Asp 307 in the complex with alpha-ketoglutarate. This triggers a number of significant movements among several short loops and adjoining secondary structural elements in the enzyme, most of which participate in dimer stabilization and formation of the active-site cleft. These rearrangements are similar to the ligand-binding-induced movements observed in globins and insulin and serve as a model for an enzymatic mechanism which involves local shifts of secondary structural elements during turnover, rather than large-scale domain closures or loop transitions induced by substrate binding such as those observed in hexokinase or triosephosphate isomerase.     

1-(2-苯并噻唑偶氮)-2-羟基-3-萘甲酸与镍(Ⅱ)的显色反应研究及应用

用分光光度法研究了新试剂1-(2-苯并噻唑偶氮)-2-羟基-3-萘甲酸(BTAHN)与镍Ⅱ的显色反应性能。在pH4·6~10·4范围内,BTAHN与镍Ⅱ形成2∶1的蓝色络合物,该络合物的最大吸收波长λmax在612nm处,25mL溶液中镍的质量在0~34μg范围内服从比尔定律,测定的表观摩尔吸光系数ε612为3·52×104,所拟方法用于合金钢样品中微量镍的测定,结果满意。