2-4-dinitrophenylhydrazine and 4-hydroxy-2-nonenal

Topics: Aldehydes; Animals; Antioxidants; Bromobenzenes; Bromotrichloromethane; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Chemical Phenomena; Chemistry; Chromatography, Thin Layer; Endoplasmic Reticulum; Fatty Liver, Alcoholic; Free Radicals; Glucosephosphate Dehydrogenase; In Vitro Techniques; Lipid Peroxides; Liver; Malondialdehyde; Mice; Microsomes, Liver; Phenylhydrazines; Rats; Spectrophotometry, Atomic; Sulfhydryl Compounds; Tissue Distribution

Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling.

Topics: Aldehydes; Animals; Cells, Cultured; Dinitrochlorobenzene; Glutaredoxins; Humans; Male; Malondialdehyde; Mercaptoethanol; Oxidative Stress; Peroxiredoxins; Phenylhydrazines; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Sulfhydryl Compounds; Thioredoxins

Topics: Aldehydes; Alzheimer Disease; Analysis of Variance; Biomarkers; DNA Damage; Glycation End Products, Advanced; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Histocytochemistry; Humans; Iron; Membrane Proteins; Neurodegenerative Diseases; Neurofibrillary Tangles; Neurons; Nitrates; Oxidation-Reduction; Oxidative Stress; Oxygen; Phenylhydrazines; Plaque, Amyloid; Tyrosine

Free radical-mediated injury is believed to play a key role in the pathogenesis of acute pancreatitis (AP). Therefore, oxidative damage of proteins may be an important event in the development of AP. The present study was performed to investigate oxidative protein modification, quantified as 2,4-dinitrophenylhydrazine-reactive protein-carbonyls, during the time course of taurocholate-induced pancreatitis of the rat and to analyze oxidatively modified proteins by Western blotting. Protein modification in pancreatic homogenates was found as early as 30 min after induction of severe AP with 3% taurocholate preceding the elevation of serum amylase activity and the increase of malondialdehyde in the tissue. A correlation of protein-carbonyl contents to a score of pancreatic macroscopic alterations (r = .69) and to the wet weight/dry weight ratio (r = .65) was found. Infusion of 5% taurocholate resulted in fulminant AP with high lethality during the 24 h of the experiment. However, rats surviving showed significantly lower level of protein-carbonyls than animals that died between 20-24 h after AP induction. The quantitative data were confirmed by the intensity of immunostained protein-carbonyls. The present data show a rather uniform increase in the staining pattern not revealing single, selectively damaged proteins. The aldehydic product of lipid peroxidation 4-hydroxynonenal (HNE) is known for its reactivity towards proteins. Interestingly, an antibody raised against protein-bound HNE did not indicate an increased protein modification by this aldehyde. In conclusion, experimental AP is characterized by an early oxidative protein modification, possibly contributing to functional impairment of the pancreas. This protein alteration may not be mediated by HNE.

Topics: Acute Disease; Aldehydes; Amylases; Animals; Blotting, Western; Kinetics; Lipid Peroxidation; Male; Malondialdehyde; Oxidation-Reduction; Pancreatitis; Phenylhydrazines; Proteins; Rats; Rats, Wistar; Taurocholic Acid

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lag-phase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.

Topics: Aldehydes; Animals; Antibodies; Blotting, Western; Chromatography; Fatty Acids; Glutathione; Kinetics; Lipid Peroxidation; Mitochondria, Liver; Oxidative Stress; Phenylhydrazines; Proteins; Rats; Rats, Wistar; Spectrophotometry; Thiobarbituric Acid Reactive Substances

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Topics: Aldehydes; Animals; Chromatography, Thin Layer; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Macrophages, Alveolar; Male; Nitrogen Dioxide; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Respiratory Burst

4-Hydroxynonenal (HNE) as an indicator of lipid peroxidation was determined in rat jejunal mucosa. HNE was extracted as the dinitrophenylhydrazone derivative from the tissue, partially separated from other carbonyl compounds by thin-layer chromatography and measured by HPLC. During reperfusion of the small intestine following an ischemic period of 60 minutes a marked increase of the tissue concentration of HNE was observed. The mucosal HNE level passed a maximum value of 3.0 +/- 0.5 microM 10 min after the onset of reperfusion in comparison with 0.7 +/- 0.2 microM as initial value. The increased tissue level of the highly cytotoxic 4-hydroxyalkenal is suggested to be involved in the reperfusion induced morphological and biochemical changes of the small intestine.

Topics: Aldehydes; Animals; Chromatography, High Pressure Liquid; Glutathione; Glutathione Disulfide; Hypoxanthine; Hypoxanthines; Intestine, Small; Ischemia; Jejunum; Lipid Peroxidation; Male; Phenylhydrazines; Rats; Rats, Wistar; Reperfusion; Thiobarbituric Acid Reactive Substances

Incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE) results in a pseudo first-order loss of enzyme activity. The pH dependence of the inactivation rate exhibits an inflection around pH 10, and the enzyme is protected from inactivation by glucose 6-phosphate. Loss of enzyme activity corresponds with the formation of one carbonyl function per enzyme subunit and the appearance of a lysine-HNE adduct. The data presented in this paper are consistent with the view that the epsilon-amino group of a lysine residue in the glucose 6-phosphate-binding site reacts with the double bond (C3) of HNE, resulting in the formation of a stable secondary amine derivative and loss of enzyme activity. We have described a mechanism by which HNE may, in part, mediate free radical damage. In addition, a method for the detection of the lysine-HNE adduct is introduced.

Topics: Aldehydes; Amino Acids; Binding Sites; Glucosephosphate Dehydrogenase; Hydrogen-Ion Concentration; Kinetics; Leuconostoc; Lysine; Phenylhydrazines

Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc = 0.999). The maximum absorbance measured after 2,4-DNPH-staining (epsilon 367 = 21,000) were 1.893 +/- 0.072 (P less than 0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.

Topics: Aldehydes; Animals; Carcinoma, Ehrlich Tumor; Diazonium Compounds; DNA; Histocytochemistry; Liver; Microchemistry; Naphthols; Phenylhydrazines; Photometry; Proteins; Rats

When carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely 4-hydroxynonenal) are incubated with liver microsomes, a substantial amount of these products can be recovered bound to the microsomal protein, by allowing the microsomal protein to react with 2,4-dinitrophenyl-hydrazine. Concomitantly with the binding a decrease in the "exposed" and total -SH groups is stechiometrically accounted for by the amount of carbonyl compounds bound to the microsomal protein. Still in concomitance with the binding, the G-6-Pase activity of the microsomes markedly decreased. Therefore a mechanism similar to that described (12) for the binding of 4-hydroxyalkenals to -SH groups of protein and enzymes can be postulated.

Topics: Aldehydes; Glucosephosphate Dehydrogenase; Lipid Peroxides; Microsomes, Liver; Phenylhydrazines

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.

Topics: Aldehydes; Animals; Glucose-6-Phosphatase; Kinetics; Lipid Peroxides; Male; Mass Spectrometry; Microsomes, Liver; NADP; Phenylhydrazines; Rats; Spectrophotometry; Structure-Activity Relationship