3-nitrotyrosine and Cystic-Fibrosis

Endogenous Nitric Oxide (NO) plays a key role in the physiological regulation of airway functions. In response to various stimuli activated inflammatory cells (e.g., eosinophils and neutrophils) generate oxidants ("oxidative stress") which in conjunction with exaggerated enzymatic release of NO and augmented NO metabolites produce the formation of strong oxidizing reactive nitrogen species, such as peroxynitrite, in various airway diseases including asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis and acute respiratory distress syndrome (ARDS). Reactive nitrogen species provoke amplification of inflammatory processes in the airways and lung parenchyma causing DNA damage, inhibition of mitochondrial respiration, protein dysfunction and cell damage ("nitrosative stress"). These effects alter respiratory homeostasis (such as bronchomotor tone and pulmonary surfactant activity) and the long-term persistence of "nitrosative stress" may contribute to the progressive deterioration of pulmonary functions leading to respiratory failure. Recent studies showing that protein nitration can be dynamic and reversible ("denitration mechanisms") open new horizons in the treatment of chronic respiratory diseases affected by the deleterious actions of "nitrosative stress".

Topics: Animals; Asthma; Cystic Fibrosis; Humans; Nitric Oxide; Oxidative Stress; Pulmonary Disease, Chronic Obstructive; Reactive Nitrogen Species; Reactive Oxygen Species; Respiratory System; Tyrosine

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Eosinophils; Humans; Nitrates; Nitric Oxide; Pneumonia; Tyrosine

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Topics: Amino Acid Sequence; Animals; Antibodies, Immobilized; Binding Sites; Chromatography, Affinity; Cystic Fibrosis; Eosinophil Granule Proteins; Humans; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Tyrosine

3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5-17 years) were enrolled in this study, including children with asthma (n=12), cystic fibrosis (n=12), and healthy controls (n=18). Additionally, 14 healthy non-smoking adults (aged 18-59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54-6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97-2.49) in healthy adults, 2.51 (1.22-3.51) in healthy children, 1.46 (0.88-2.02) in children with asthma, and 1.97 (1.37-2.35) in CF children, respectively (p=0.24, Kruskall-Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC-NICI-MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.

Topics: Adolescent; Adult; Asthma; Biomarkers; Breath Tests; Child; Child, Preschool; Cystic Fibrosis; Exhalation; Female; Humans; Male; Middle Aged; Oxidative Stress; Tyrosine

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.

Topics: Adult; Aged; Analysis of Variance; Cystic Fibrosis; Female; Humans; Male; Middle Aged; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Reference Values; Respiratory Mechanics; Respiratory System; Tyrosine

Exhaled nitric oxide (ENO), a marker of inflammation in airway diseases is decreased in cystic fibrosis (CF) patients, perhaps because nitric oxide (NO) is metabolized to oxidative end-products. A stable product, 3-nitrotyrosine, may indicate local formation of reactive nitrogen species. Whether NO metabolites in exhaled breath condensate may be increased in CF patients was investigated. The fractional concentration of ENO (Feno), nitrotyrosine and oxides of nitrogen in exhaled breath condensate from 36 stable CF patients were compared to 14 normal subjects using an enzyme immunoassay and fluorescence assay. Nitrotyrosine levels in breath condensate were increased significantly in stable CF patients, compared with normal subjects (25.3 +/- 1.5 versus 6.3 +/- 0.8 ng x mL(-1), p<0.0001). There was an inverse correlation between the levels of nitrotyrosine and the severity of lung disease. Feno levels were significantly lower in CF patients than in normal subjects (4.4 +/- 0.3 versus 5.6 +/- 0.4 (parts per billion), p<0.05). No correlation was found between nitrotyrosine and Feno levels in CF. There was no significant difference in the levels of nitrite and nitrate between CF patients and normals. The elevation in nitrotyrosine may reflect increased formation of reactive nitrogen species such as peroxynitrite or direct nitration by granulocyte peroxidases, indicating increased oxidative stress in airways of cystic fibrosis patients.

Topics: Adult; Breath Tests; Cystic Fibrosis; Female; Humans; Lung Volume Measurements; Male; Oxidative Stress; Reactive Nitrogen Species; Reference Values; Tyrosine

Nitric oxide (NO) is increased in the exhaled air of some patients with inflammatory lung disorders, but not in others. NO may combine with superoxide to form peroxynitrite, which lowers NO gas concentrations, increases formation of nitrate, and increases nitration of tyrosine residues on proteins. We hypothesized that superoxide released from neutrophils in the lower respiratory tract of cystic fibrosis (CF) results in increased nitrate and nitrotyrosine levels in sputum. In order to test this hypothesis, exhaled NO was collected from 5 stable adult CF subjects and from 5 nonsmoking normal controls. Consistent with previous reports, exhaled NO concentrations were not increased in CF exhaled air (22.6 +/- 1.5 ppb vs. 28.6 +/- 1.5 ppb in normals, P > 0.05). Sputum was collected from 9 adult CF subjects and the same 5 normal controls and evaluated for nitrite, nitrate, and nitrotyrosine. Nitrate and nitrotyrosine levels, but not nitrite, were significantly elevated in CF. Recently, myeloperoxidase has also been implicated as a mechanism of nitrotyrosine formation. Therefore, myeloperoxidase was measured and found to be elevated in the CF sputum (64.2 +/- 35.9 vs. 0.73 +/- 0.16 U/mL, P < 0.001), and was found to correlate with concentrations of nitrotyrosine (r = 0.87, P < 0.05). However, in vitro studies with myeloperoxidase and murine lung epithelial cells did not demonstrate a reduction of NO gas with nitrotyrosine or an increase in nitrate formation. These data demonstrate that nitrate and nitrotyrosine are elevated in the sputa of CF subjects and suggest increased production of NO in the lower respiratory tract of CF patients, despite the relatively low exhaled NO levels. Pediatr Pulmonol. 2000; 30:79-85. Published 2000 Wiley-Liss, Inc.

Topics: Adolescent; Adult; Cystic Fibrosis; Female; Free Radicals; Humans; Inflammation; Male; Nitrates; Nitric Oxide; Sputum; Superoxides; 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