3-nitrotyrosine and dityrosine

Early pregnancy exposure to endocrine disrupting chemicals (EDCs) may contribute to poor birth outcomes through oxidative stress (OS)-mediated disruption of the maternal and fetal milieu. Most studies have investigated the effect of single EDC exposures on OS.. Assess the association of uniquely weighted mixtures of early pregnancy exposures with the maternal and neonatal OS markers.. Prospective analysis of mother-infant dyads.. University hospital.. 56 mother-infant dyads.. The association of OS markers (nitrotyrosine, dityrosine, chlorotyrosine) in maternal first trimester and term, and cord blood plasma with maternal first trimester exposure levels of each of 41 toxicants (trace elements, metals, phenols, and phthalates) from 56 subjects was analyzed using Spearman correlations and linear regression. The association of OS markers with inflammatory cytokines and birth outcomes were analyzed by Spearman correlation and linear regression analysis, respectively. Weighted mixtures of early pregnancy exposures were created by principal component analysis and offspring sex-dependent and independent associations with oxidative stress markers were assessed.. (1) An inverse relationship between levels of maternal/cord OS markers and individual EDCs was evident. In contrast, when assessed as EDC mixtures, both direct and inverse associations were evident in a sex-specific manner; (2) the maternal term OS marker, nitrotyrosine, was inversely associated with gestational age, and (3) both direct and inverse associations were evident between the 3 OS markers and individual cytokines.. Provides proof of concept that effects of exposures on OS varies when assessed as EDC mixtures versus individually.

Topics: Adult; Endocrine Disruptors; Environmental Pollutants; Female; Fetal Blood; Gestational Age; Humans; Infant, Newborn; Linear Models; Male; Maternal Exposure; Oxidative Stress; Pregnancy; Pregnancy Trimester, First; Principal Component Analysis; Proof of Concept Study; Sex Characteristics; Statistics, Nonparametric; Tyrosine

Interstitial lung diseases (ILDs) are associated with oxidative stress. Plasma biomarkers that are directly linked to oxidative stress responses in this disease have not been identified. Stable oxidation products of tyrosine residues in proteins may reflect the oxidative microenvironment in the lung or a systemic inflammatory state.. To determine if levels of protein tyrosine oxidation are elevated in plasma of patients with ILD compared with an age- and sex-matched healthy control cohort.. Three tyrosine oxidation products (3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine) were quantified by tandem mass spectrometry in cellular models, a mouse model of injury-induced fibrosis, and in plasma of healthy control subjects and patients with ILD (n = 42 in each group).. Plasma levels of 3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine were markedly elevated in patients with ILD compared with control subjects with receiver operating characteristic curves separating these groups of 0.872, 0.893, and 0.997, respectively. In a murine model of lung fibrosis, levels of all three oxidative tyrosine modifications were increased in plasma and lung tissue. Cellular models support a critical role for a heme peroxidase and enzymatic sources of reactive oxygen species in the generation of these oxidized products.. We demonstrate an increase in oxidized tyrosine moieties within proteins in the circulating plasma of patients with ILD. These data support the potential for development of oxidative stress-related biomarkers in early diagnosis, prognostication, and/or in evaluating responsiveness to emerging therapies for ILD.

Topics: Animals; Biomarkers; Cells, Cultured; Female; Humans; Lung Diseases, Interstitial; Male; Mice; Mice, Inbred C57BL; Middle Aged; Oxidative Stress; Tandem Mass Spectrometry; Tyrosine

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has been indicated in the pathogenesis of various ocular disorders. The PON induces mobilization of intra cellular calcium which plays an important function in structure and activity of lens proteins. Moreover, the amount of calcium increases to the pathogenic level in the cataractous lenses. The aim of this study was to assess the impact of calcium ion on structure and aggregation of PON-modified lens crystallins, using spectroscopic techniques and gel mobility shift assay. The PON modification of lens proteins was confirmed with detection of the significantly increased quantity of carbonyl group, dityrosine, nitrotyrosine and nitrotryptophan. Moreover, the modified proteins indicated high levels of solvent exposed hydrophobic surfaces and markedly elevated proteolytic instability which can be explained with their structural alteration upon this type of modification. The results of UV-vis absorption studies suggest that PON-modified lens crystallins are highly sensitive to aggregation in the presence of both physiological and pathological ranges of calcium ion. Also, the results of thioflavin T fluorescence study indicated absence of any ordered aggregate entity in the calcium-induced aggregate samples. The results of gel mobility shift assay demonstrated the importance of calcium ion in the induction of disulfide and dityrosine covalent cross-linking and formation of the oligomeric structure with relatively larger sizes in the PON-modified crystallins compared to the non-modified protein counterparts. Overall, this study may suggest that a simultaneous raise of calcium ion and PON in the eye ball is an important risk factor for development of cataract diseases.

Topics: Animals; Benzothiazoles; Calcium; Cattle; Crystallins; Hydrophobic and Hydrophilic Interactions; Lens, Crystalline; Oxidants; Peroxynitrous Acid; Protein Aggregates; Protein Carbonylation; Protein Stability; Proteolysis; Spectrometry, Fluorescence; Thiazoles; Tryptophan; Tyrosine

To explore the effects of tyrosine oxidized products (TOP) on the redox state of rats myocardium and investigate the oxidative damage mechanism.. Male SD Rats were randomly assigned to three groups and treated respectively with normal diet (Group C), 440 mg/kg TOP (Group TOP), 440 mg/kg tyrosine oxidized products plus 5 mg/kg lipoic acid (Group TOP + LA). Indicators of oxidative stress in blood and myocardial were measured at the end of 6 weeks, 12 weeks and 24 weeks. RT-PCR was used to detect mRNA expression of Nrf2, Trx, NF-κB and AP-1 in myocardium.. Compared to the control group (Group C), reactive oxygen species (ROS), carbonyl (PC), malondialdehyde (MDA), dityrosine (Dityr), 3-nitrotyrosine (3-NT) in rats blood and myocardium were all significantly increased in the TOP group (P < 0.05). Total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reducing glutathione (GSH) and oxidized glutathione (GSSG) levels all decreased significantly (P < 0.05). The mRNA expression of Nrf2, Trx-1, NF-κB, AP-1 RNA significantly up-regulated (P < 0.05) in the TOP group compared to the control.. Tyrosine oxidized products impairs antioxidant defense system and lead to accumulation of protein oxidation products in rats myocardium. Moreover, lipoic acid performs a positive effect in the protective reaction, which may adjusted by regulation of redox signaling pathway.

Topics: Animals; Antioxidants; Catalase; Glutathione; Glutathione Peroxidase; Heart; Male; Malondialdehyde; NF-kappa B; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxide Dismutase; Thioctic Acid; Transcription Factor AP-1; Tyrosine

Oxidative stress is a key factor regulating the systemic pathophysiological effects associated with periodontitis. Resveratrol is a phytochemical with antioxidant and anti-inflammatory properties that can reduce oxidative stress and inflammation. We hypothesized that resveratrol may prevent the progression of periodontitis and reduce systemic oxidative stress through the activation of the sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) and the nuclear factor E2-related factor 2 (Nrf2)/antioxidant defense pathways. Three groups of male Wistar rats (periodontitis treated with melinjo resveratrol, periodontitis without resveratrol, and control rats with no periodontitis or resveratrol treatment) were examined. A ligature was placed around the maxillary molars for 3 weeks to induce periodontitis, and the rats were then given drinking water with or without melinjo resveratrol. In rats with periodontitis, ligature placement induced alveolar bone resorption, quantified using three-dimensional images taken by micro-CT, and increased proinflammatory cytokine levels in gingival tissue. Melinjo resveratrol intake relieved alveolar bone resorption and activated the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in inflamed gingival tissues. Further, melinjo resveratrol improved the systemic levels of 8-hydroxydeoxyguanosine, dityrosine, nitric oxide metabolism, nitrotyrosine, and proinflammatory cytokines. We conclude that oral administration of melinjo resveratrol may prevent the progression of ligature-induced periodontitis and improve systemic oxidative and nitrosative stress.

Topics: 8-Hydroxy-2'-Deoxyguanosine; AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Antioxidants; Bone Resorption; Cytokines; Deoxyguanosine; Disease Models, Animal; Gingiva; Inflammation; Male; NF-E2-Related Factor 2; Nitric Oxide; Oxidative Stress; Periodontitis; Random Allocation; Rats; Rats, Wistar; Resveratrol; Sirtuin 1; Stilbenes; Tyrosine

Peroxynitrite is a potent oxidant and nitrating agent and has in vivo existence. It is a powerful proinflammatory substance and may increase vascular permeability in inflamed tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of unknown etiology. Since its discovery, numerous self- and non-self, nuclear, and cytoplasmic antigens have been suggested as stimuli for SLE initiation, but the exact trigger is yet to be identified. In this study, an attempt has been made to investigate the binding characteristics of SLE anti-DNA autoantibodies to native DNA and native and peroxynitrite-modified H2A histone to explore the possible role of modified protein antigen(s) in SLE initiation and progression. The nuclear protein (H2A histone) was modified by peroxynitrite synthesized in our laboratory. The peroxynitrite-modified H2A revealed generation of nitrotyrosine, dityrosine, and carbonyls when subjected to investigation by physicochemical methods. Binding characteristics and specificity of SLE anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. A band shift assay further substantiated the enhanced recognition of peroxynitirite-modified H2A histone by anti-DNA autoantibodies. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing SLE characteristic autoantibodies. The preferential binding of peroxynitrite-modified H2A histone by SLE anti-DNA antibodies points out the likely role of oxidatively modified and nitrated H2A histone in the initiation/progression of SLE. Moreover, oxidatively modified and nitrated nuclear protein antigen, rather than nucleic acid antigens, appear to be more suitable as a trigger for SLE.

Topics: Antibodies, Antinuclear; Autoantibodies; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Epitopes; Histones; Humans; Lupus Erythematosus, Systemic; Nitro Compounds; Peroxynitrous Acid; Tyrosine

Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.

Topics: Isoaspartic Acid; Oxidation-Reduction; Protein Carbonylation; Proteins; Sulfhydryl Compounds; Tyrosine

Glycation, oxidation, and nitration of endogenous proteins occur spontaneously and these modifications are also present in foods. Increased levels of these chemical changes are associated with chronic renal failure; however, little is known about acute kidney failure. We measured these modifications of plasma protein and related free adducts in plasma following bilateral nephrectomy and bilateral ureteral obstruction. Advanced glycation end-product (AGE) residues of plasma protein were increased 3 h post-surgery, and thereafter slowly decreased in all groups, reflecting changes in plasma protein synthesis and transcapillary flow post-surgery. Ureteral ligation increased oxidation and nitration adduct residues. There were, however, marked increases in AGE, dityrosine, or 3-nitrotyrosine free adducts in both nephrectomized and ureter-ligated rats compared to rats that had undergone sham operations. There were lower modified adduct concentrations in the ureter-ligated compared to the nephrectomized rats, reflecting residual glomerular filtration and tubular removal. There was no increase in glycated, oxidized, and nitrated proteins. Glyoxal and methylglyoxal were also increased in both renal failure models. Our study shows that the acute loss of renal function and urinary excretion leads to the accumulation of AGE, oxidation, and nitration free adducts in the plasma.

Topics: Animals; Body Weight; Disease Models, Animal; Glycation End Products, Advanced; Glyoxal; Kidney Failure, Chronic; Ligation; Male; Nephrectomy; Oxidative Stress; Pyruvaldehyde; Rats; Rats, Wistar; Tyrosine

The endogenous synthesis of peroxynitrite (ONOO(-)) has been implicated in a number of diseases, but assessments of its cytotoxicity and genotoxicity have been hampered by its extremely short half-life under physiological conditions (<20 ms) and the consequent difficulty in exposing cells to known concentrations of it over at least several hours. Two methods for peroxynitrite delivery to cell cultures were investigated, one involving steady infusion of preformed ONOO(-) and the other based on the continuous in situ synthesis of ONOO(-) from NO and O(2)(-). In the latter, NO was supplied by diffusion through gas permeable tubing and O(2)(-) was generated using the hypoxanthine-xanthine oxidase reaction. The performance of both methods was assessed by measuring the rates of formation of tyrosine derivatives (dityrosine and nitrotyrosine) that are commonly employed as biomarkers for peroxynitrite. Experimental results in the absence of cells were compared in each case with predictions from kinetic models. In the infusion system, the measured dityrosine and nitrotyrosine yields were in excellent agreement with those predicted from the model. To characterize the other system, experiments were performed first to determine the kinetics of hypoxanthine oxidation by xanthine oxidase and uric acid oxidation by uricase. Simulations of the complex reaction network in the complete synthesis system suggested that dityrosine should be the major product there, that the yields of both tyrosine derivatives should be very sensitive to the relative rates of NO and O(2)(-) delivery, and that equal rates for NO and O(2)(-) should maximize those yields. Experiments performed under the predicted optimal conditions showed much lower levels of dityrosine than expected and no detectable nitrotyrosine. The unexpectedly low yields of tyrosine products could be explained largely by the partial inactivation of both xanthine oxidase and uricase by peroxynitrite-derived NO(2) and CO(3)(-) radicals. We conclude that continuous infusion of peroxynitrite is the more promising approach.

Topics: Animals; Cells, Cultured; Dimerization; Drug Delivery Systems; Humans; Hypoxanthine; Models, Chemical; Nitrates; Nitric Oxide; Oxidation-Reduction; Peroxynitrous Acid; Toxicity Tests; Tyrosine; Uric Acid; Xanthine Oxidase

The biological relevance of tyrosine nitration is a subject of much interest, because extensive evidence supports formation of 3-nitrotyrosine in vivo under a variety of different pathological conditions. Several reagents are likely to be responsible for nitration in vivo, among others peroxynitrite and nitrite in the presence of H(2)O(2)/peroxidases. In this work we show that also metmyoglobin and methemoglobin can nitrate free tyrosine in the presence of nitrite and H(2)O(2). The results of these studies are simulated rather well by using a scheme that comprehends all the possible reactions that can take place in the system. Thus, a good understanding of the factors that determine the yields is achieved. Finally, we demonstrate that the system metMb/H(2)O(2)/NO(2)(-) can also lead to the nitration of tryptophan and produces, in particular, 6-, 4-, and 5-nitrotryptophan.

Topics: Hydrogen Peroxide; Methemoglobin; Metmyoglobin; Myoglobin; Nitrites; Peroxidase; Peroxynitrous Acid; Tryptophan; Tyrosine

. Peroxynitrite is a highly potent reactive oxygen/nitrogen species present in the environment and also endogenously in the eye, that causes a variety of disorders. This study was undertaken to look at the oxidative damage that peroxynitrite causes to the proteins of the lens and the functional consequences thereof.. . Peroxynitrite was allowed to react with alpha-, beta-, and gamma-crystallins. The formation of nitrotyrosine and nitrotryptophan, dityrosine, protein covalent cross-links, and chain degradation products were monitored by spectroscopy and SDS-PAGE. Conformational changes occurring in the protein were monitored with circular dichroism spectroscopy. The chaperoning ability of alpha-crystallin was assayed by monitoring its ability to inhibit the self-aggregation of two test proteins: beta-crystallin and insulin.. . Peroxynitrite reaction produced nitrotyrosine, nitrotryptophan, and dityrosine, nondisulfide covalent cross-linked aggregates, and peptide chain degradation. The hydroxyl radicals produced by peroxynitrite caused more chain degradation than did the carbonate radicals. The oxidative reaction caused increased conformational disorder. The yield was highest in gamma-crystallin and least in alpha-crystallin. The chaperoning ability of alpha-crystallin was not affected.. . Peroxynitrite reacts with lens proteins, causing extensive covalent chemical changes. However, alpha-crystallin retains its chaperoning ability, despite the oxidative changes caused by the peroxynitrite reaction, indicating its functional robustness.

Topics: alpha-Crystallins; Animals; Cattle; Lens, Crystalline; Molecular Chaperones; Oxidative Stress; Peroxynitrous Acid; Tryptophan; Tyrosine

We have shown that peroxynitrite (ONOO-) inhibits streptokinase-induced conversion of plasminogen to plasmin in a concentration-dependent manner and reduces both amidolytic (IC5o approximately 280 microM at 10 microM concentration of enzyme) and proteolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstrates a concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin. Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partially protects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggest that nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalytic activity.

Topics: Blotting, Western; Chromatography, Affinity; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Fibrinolysin; Glutathione; Inhibitory Concentration 50; Oxidants; Peroxynitrous Acid; Plasminogen; Spectrophotometry; Streptokinase; Time Factors; Tyrosine

To examine the preventive effect of the lemon flavonoid, eriocitrin (eriodictyol 7-O-rutinoside), on oxidative stress during acute exercise in vivo, levels of N( epsilon )- (hexanoyl)lysine, HEL; o,o-dityrosine, DT; and nitrotyrosine, NT, as oxidative stress markers, were determined by ELISA in livers of trained rats in addition to thiobarbituric acid-reactive substance (TBARS). Eriocitrin administration prior to exercise significantly suppressed the increases in TBARS caused by lipid peroxidation during acute exercise. The contents of HEL, DT, and NT in rat liver increased dramatically by exercise without eriocitrin administration. However, these increases were significantly suppressed by eriocitrin administration before exercise. Moreover, in this study, to clarify whether eriocitrin influences glutathione metabolite system that is considered to be important for a defense against the damage by oxidative stress, the levels of glutathione in rat liver were determined during exercise. The level of reduced glutathione after exercise was maintained by administration of eriocitrin. The increase in the concentration of oxidized glutathione caused by exercise was significantly suppressed by eriocitrin. This result suggested that eriocitrin might play an important role in the control of the change in glutathione redox status in rat liver during exercise. These findings showed that eriocitrin was effective in the prevention of oxidative damages caused by acute exercise-induced oxidative stress.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Flavanones; Glutathione; Hesperidin; Hexanols; Lipid Peroxidation; Liver; Lysine; Male; Oxidative Stress; Physical Conditioning, Animal; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Tyrosine

Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).

Topics: Animals; Cats; Chromatography, High Pressure Liquid; Isotopes; Spectrometry, Mass, Electrospray Ionization; Tyrosine

We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts.. Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts.. A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group.. We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Topics: Animals; Biomarkers; Cholesterol, Dietary; Hyperlipidemias; Male; Malondialdehyde; Models, Animal; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Peroxynitrous Acid; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides; Tyrosine; Xanthine Dehydrogenase

Protein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.

Topics: Biochemistry; Borohydrides; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Iodoacetamide; Isoaspartic Acid; Mass Spectrometry; Oxidation-Reduction; Phenylhydrazines; Protein Carbonylation; Proteins; Reference Standards; Spectrophotometry; Sulfhydryl Compounds; Tritium; Tyrosine

Recent evidence argues strongly that the marked increase in risk for atherosclerotic heart disease seen in diabetics cannot be explained by a generalized increase in oxidative stress. Here, we used streptozotocin to induce hyperglycemia in cynomolgus monkeys for 6 months and tested whether high glucose levels promote localized oxidative damage to artery wall proteins. We focused on three potential agents of oxidative damage: hydroxyl radical, tyrosyl radical, and reactive nitrogen species. To determine which pathways operate in vivo, we quantified four stable end products of these reactants -- ortho-tyrosine, meta-tyrosine, o,o'-dityrosine, and 3-nitrotyrosine -- in aortic proteins. Levels of ortho-tyrosine, meta-tyrosine, and o,o'-dityrosine, but not of 3-nitrotyrosine, were significantly higher in aortic tissue of hyperglycemic animals. Of the oxidative agents we tested, only hydroxyl radical mimicked this pattern of oxidized amino acids. Moreover, tissue levels of ortho-tyrosine and meta-tyrosine correlated strongly with serum levels of glycated hemoglobin, a measure of glycemic control. We conclude that short-term hyperglycemia in primates promotes oxidation of artery wall proteins by a species that resembles hydroxyl radical. Our observations suggest that glycoxidation reactions in the arterial microenvironment contribute to early diabetic vascular disease, raising the possibility that antioxidant therapies might interrupt this process.

Topics: Animals; Aorta; Arteriosclerosis; Diabetes Mellitus, Experimental; Glucose; Glycated Hemoglobin; Hydroxyl Radical; Lipids; Macaca fascicularis; Male; Mass Spectrometry; Oxidation-Reduction; Time Factors; Tyrosine

The flavanol (-)-epicatechin is known to protect against peroxynitrite-induced nitration and oxidation reactions. This study investigated the protection afforded by (-)-epicatechin against both these reaction types on one target molecule, the aminoacid tyrosine, in a hydrophilic milieu as well as with a lipophilic tyrosine derivative, N-t-BOC l-tyrosine tert-butyl ester (BTBE), bound to liposomes. The flavanol efficiently attenuated both tyrosine nitration and tyrosine dimerization (which is based on an initial oxidation reaction) and was active in the hydrophilic and hydrophobic systems at similar IC(50) values, approximately 0.02-0.05 mol (-)-epicatechin/mol peroxynitrite. Related procyanidin oligomers of different chain-length (dimer to octamer) were also tested for their protective properties, and exhibited protection that, on a monomer basis, was in the same order of magnitude as those for (-)-epicatechin.

Topics: Antioxidants; Catechin; Dimerization; In Vitro Techniques; Liposomes; Nitrates; Peroxynitrous Acid; Reactive Oxygen Species; Tyrosine

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.

Topics: Dimerization; Flavin Mononucleotide; Free Radicals; Hydrazines; Hypoxanthine; Kinetics; Nitrates; Nitric Oxide; Nitrogen Oxides; Spermine; Tyrosine; Xanthine Oxidase

The inhibitory effects of various endogenous and synthetic compounds on the nitration and oxidation of L-tyrosine by peroxynitrite were examined. Nitrating and oxidizing activities were monitored by the formation of 3-nitrotyrosine and dityrosine with a HPLC-UV-fluorescence detector system, respectively. Glutathione, serotonin and synthetic sulfur- and selenium-containing compounds inhibited both the nitration and oxidation reaction of L-tyrosine effectively. However, 5-methoxytryptamine, melatonin and alpha-lipoic acid only inhibited the nitration reaction, and enhanced the formation of an oxidation product. This is important evidence that there are different intermediates in the nitrating and oxidizing reactions of L-tyrosine by peroxynitrite. It was suggested that 5-methoxytryptamine, melatonin and alpha-lipoic acid reacted only with the nitrating intermediate of peroxynitrite and inhibited nitration of L-tyrosine. Actually, the DNA strand breakage, which is believed to be a typical reaction of hydroxyl radical-like species, caused by peroxynitrite was not effectively inhibited by 5-methoxytryptamine. 5-Methoxytryptamine, melatonin and alpha-lipoic acid were viewed as useful reagents for investigating the mechanisms of damage by peroxynitrite in vitro.

Topics: Antioxidants; Chromatography, High Pressure Liquid; DNA Damage; Free Radical Scavengers; Indicators and Reagents; Nitrates; Oxidation-Reduction; Plasmids; Spectrophotometry, Ultraviolet; Spin Trapping; Thioctic Acid; Tryptamines; Tyrosine

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.

Topics: Anions; Bicarbonates; Carbon Dioxide; Carbonates; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Free Radicals; Hydrogen-Ion Concentration; Hydroxyl Radical; Hydroxylation; Kinetics; Nitrates; Nitric Oxide; Spectrophotometry, Ultraviolet; Tyrosine

Cystic fibrosis (CF) is associated with chronic pulmonary inflammation and progressive lung dysfunction, possibly associated with the formation of neutrophil myeloperoxidase (MPO)-derived oxidants. Expectorated sputum specimens from adult CF patients were analyzed for MPO characteristic protein modifications and found to contain large amounts of active MPO as well as high levels of protein-associated 3-chlorotyrosine and 3,3'-dityrosine, products that result from MPO activity, compared with expectorated sputum from non-CF subjects. Sputum levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), indicating local production of nitric oxide (NO. ), were not elevated but in fact were slightly reduced in CF. However, there was a slight increase in protein-associated 3-nitrotyrosine in CF sputum compared with controls, reflecting the formation of reactive nitrogen intermediates, possibly through MPO-catalyzed oxidation of NO(2)(-). CF sputum MPO was found to contribute to oxidant-mediated cytotoxicity toward cultured tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of secondary cytotoxic products within CF sputum. Our findings demonstrate the formation of MPO-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF, which collectively may contribute to bronchial injury and respiratory failure in CF.

Topics: Adult; Bronchi; Case-Control Studies; Cell Line; Chromatography, High Pressure Liquid; Cystic Fibrosis; Drug Synergism; Humans; Hydrogen Peroxide; Nitrates; Nitrites; Oxidants; Oxidation-Reduction; Peroxidase; Peroxidases; Proteins; Respiratory System; Sputum; Trachea; Tyrosine

In the NO2-exposure of tyrosine in 70% dioxane/phosphate buffer (pH 7.4), beta-carotene enhanced the degradation of tyrosine and/or 3-nitrotyrosine produced, whereas alpha-tocopherol and ascorbyl palmitate inhibited the transformation of tyrosine into 3-nitrotyrosine. Generation of certain active species in the interaction of beta-carotene with NO2 was suggested. Ascorbyl palmitate effectively and alpha-tocopherol slightly inhibited the transformation of tyrosine in the NO2-exposure in the presence of beta-carotene. In the reaction of tyrosine with ONOO-/ONOOH, beta-carotene enhanced the degradation of 3-nitrotyrosine produced suggesting generation of certain active species, whereas alpha-tocopherol and ascorbyl palmitate completely suppressed the transformation of tyrosine into 3-nitrotyrosine.

Topics: Ascorbic Acid; beta Carotene; Dioxanes; Dose-Response Relationship, Drug; Free Radical Scavengers; Nitrogen Dioxide; Nitrous Acid; Peroxynitrous Acid; Time Factors; Tyrosine; Vitamin E

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acids; Animals; Brain; Chelating Agents; Dopamine Agents; Free Radicals; Gas Chromatography-Mass Spectrometry; Male; Mice; Mice, Inbred C57BL; Nitrates; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Pentetic Acid; Tissue Distribution; Tyrosine

The inhibitory effects of endogenous and synthetic compounds on the nitration and oxidation of L-tyrosine by peroxynitrite were examined. Nitration and oxidation activities of L-tyrosine by peroxynitrite were estimated by monitoring the formation of 3-nitrotyrosine and dityrosine with a high-performance liquid chromatography-ultraviolet (HPLC-UV)-fluorescence detector system. Glutathione and synthetic compounds ((2S,3R,4S)-N-ethylmercapto-3,4-dihydroxy-2-hydroxymethylpyrrolidine, L-N-dithiocarboxyproline) inhibited both the nitration and the oxidation reactions of L-tyrosine effectively. On the other hand, 5-methoxytryptamine and lipoic acid inhibited only the nitration reaction of L-tyrosine, and instead increased the oxidation reaction. It was assumed that 5-methoxytryptamine and lipoic acid reacted only with the nitrating species of peroxynitrite. This is the first report of a selective inhibitor for the nitrating reaction of peroxynitrite.

Topics: 5-Methoxytryptamine; Antioxidants; Chromatography, High Pressure Liquid; Dopamine Agents; Levodopa; Nitrates; Oxidants; Oxidation-Reduction; Proline; Pyrrolidines; Thioctic Acid; Tyrosine

HPLC with electrochemical array detection (HPLC-ECD) was used to quantify 3,3'-dityrosine (diTyr) and 3-nitrotyrosine (3-NO2-Tyr) in four regions of the human brain that are differentially affected in Alzheimer's disease (AD). DiTyr and 3-NO2-Tyr levels were elevated consistently in the hippocampus and neocortical regions of the AD brain and in ventricular cerebrospinal fluid (VF), reaching quantities five- to eightfold greater than mean concentrations in brain and VF of cognitively normal subjects. Uric acid, a proposed peroxynitrite scavenger, was decreased globally in the AD brain and VF. The results suggest that AD pathogenesis may involve the activation of oxidant-producing inflammatory enzyme systems, including nitric oxide synthase.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Brain; Brain Chemistry; Chromatography, High Pressure Liquid; Electrochemistry; Female; Free Radical Scavengers; Humans; Male; Nitrates; Nitrites; Oxidation-Reduction; Tyrosine; Uric Acid