3-nitrotyrosine and 2-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

To assess the degree, source, and patterns of oxidative damage to bronchoalveolar lavage proteins as a modification of amino acid residues in patients with acute respiratory distress syndrome (ARDS).. Prospective, controlled study.. Adult intensive care unit of a postgraduate teaching hospital.. Twenty-eight patients with established ARDS were studied and compared with six ventilated patients without ARDS and 11 normal healthy controls.. Supportive techniques appropriate to ARDS.. Evidence of oxidative modification of bronchoalveolar lavage fluid protein, indicative of the production of specific reactive oxidizing species, was sought using a high-performance liquid chromatography technique. Bronchoalveolar lavage fluid samples from patients with ARDS, ventilated intensive care controls, and normal healthy controls were analyzed. Concentrations of orthotyrosine were significantly higher in the ARDS group than in either control group (7.98 + 3.78 nmol/mg for ARDS, 0.67 + 0.67 for ventilated controls, and 0.71 + 0.22 for healthy controls; p < .05). Chlorotyrosine concentrations were also significantly increased in the ARDS group over either control group (4.82 + 1.07 nmol/mg for ARDS, 1.55 + 1.34 for ventilated controls, and 0.33 + 0.12 for healthy controls; p < .05). Nitrotyrosine concentrations were similarly significantly increased in the ARDS groups compared with each control group (2.21 + 0.65 nmol/mg for ARDS, 0.29 + 0.29 for ventilated controls, and 0.06 + 0.03 for healthy controls; p < .05). Chlorotyrosine and nitrotyrosine concentrations showed significant correlations with myeloperoxidase concentrations in bronchoalveolar lavage fluid, measured using an enzyme-linked immunosorbent assay in patients with ARDS. These findings suggest a possible relationship between inflammatory cell activation, oxidant formation, and damage to proteins in the lungs of these patients. Overall, our data strongly suggest heightened concentrations of oxidative stress in the lungs of patients with ARDS that lead to significantly increased oxidative protein damage.

Topics: Adolescent; Adult; Aged; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyl Radical; Hydroxylation; Hypochlorous Acid; Linear Models; Male; Middle Aged; Neutrophil Activation; Neutrophils; Nitrates; Oxidants; Oxidative Stress; Proteins; Respiration, Artificial; Respiratory Distress Syndrome; Statistics, Nonparametric; Tyrosine

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

Many lines of evidence implicate oxidative damage in aging. Possible pathways include reactions that modify aromatic amino acid residues on proteins. o-Tyrosine is a stable marker for oxidation of protein-bound phenylalanine by hydroxyl radical, whereas 3-nitrotyrosine is a marker for oxidation of protein-bound tyrosine by reactive nitrogen species. To test the hypothesis that proteins damaged by hydroxyl radical and reactive nitrogen accumulate with aging, we used isotope dilution gas chromatography-mass spectrometry to measure levels of o-tyrosine and 3-nitrotyrosine in heart, skeletal muscle, and liver from young adult (9 mo) and old (24 mo) female Long-Evans/Wistar hybrid rats. We also measured these markers in young adult and old rats that received antioxidant supplements (alpha-tocopherol, beta-carotene, butylated hydroxytoluene, and ascorbic acid) from the age of 5 mo. We found that aging did not significantly increase levels of protein-bound o-tyrosine or 3-nitrotyrosine in any of the tissues. Antioxidant supplementation had no effect on the levels of protein-bound o-tyrosine and 3-nitrotyrosine in either young or old animals. These observations indicate that the o-tyrosine and 3-nitrotyrosine do not increase significantly in heart, skeletal muscle, and liver in old rats, suggesting that proteins damaged by hydroxyl radical and reactive nitrogen species do not accumulate in these tissues with advancing age.

Topics: Aging; Animals; Antioxidants; Biomarkers; Copper; Female; Free Radicals; Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Hydroxyl Radical; Nitrates; Nitrogen; Oxidation-Reduction; Oxidative Stress; Proteins; Rats; Serum Albumin, Bovine; Tyrosine

Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors may have therapeutic potential as psychotropic agents. A series of mononitro- and dinitro-2- and 3-hydroxyphenylalanines was prepared, and their activity compared with willardiine, 5-nitrowillardiine, AMPA, and 2,4,5-trihydroxyphenylalanine (6-hydroxydopa) as inhibitors of specific [3H]AMPA and [3H]kainate binding in rat brain homogenates. The most active compounds were highly acidic (pKa 3-4), namely, 2-hydroxy-3,5-dinitro-DL-phenylalanine (13; [3H]AMPA IC50 approximately equal to 25 microM) and 3-hydroxy-2,4-dinitro-DL-phenylalanine (19; [3H]AMPA IC50 approximately equal to 5 microM). Two other dinitro-3-hydroxyphenylalanines, and 3,5-dinitro-DL-tyrosine, were considerably less active. Various mononitrohydroxyphenylalanines, which are less acidic, were also less active or inactive, and 2- and 3-hydroxyphenylalanine (o- and m-tyrosine) were inactive. Compounds 13 and 19, DL-willardiine (pKa 9.3, [3H]AMPA IC50 = 2 microM), and 5-nitro-DL-willardiine (pKa 6.4, [3H]AMPA IC50 = 0.2 microM) displayed AMPA >> kainate selectivity in binding studies. Compound 19 was an AMPA-like agonist, but 13 was an antagonist in an AMPA-evoked norepinephrine release assay in rat hippocampal nerve endings. Also, compound 13 injected into the rat ventral pallidum antagonized the locomotor activity elicited by systemic amphetamine.

Topics: Animals; Binding, Competitive; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Isomerism; Models, Chemical; N-Methylaspartate; Phencyclidine; Quinoxalines; Radioligand Assay; Rats; Receptors, AMPA; Structure-Activity Relationship; Tyrosine