3-nitrotyrosine and 3-bromotyrosine

Quantification with satisfactory specificity and sensitivity of free 3-Nitro-l-tyrosine (3-NT), 3-Chloro-l-tyrosine (3-CT), and 3-Bromo-l-tyrosine (3-BT) in biological samples as potential inflammation, oxidative stress, and cancer biomarkers is analytically challenging. We aimed at developing a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for their simultaneous analysis without an extract purification step by solid-phase extraction. Validation of the developed method yielded the following limits of detection (LOD) and quantification (LOQ) for 3-NT, 3-BT, and 3-CT: 0.030, 0.026, 0.030 ng/mL (LODs) and 0.100, 0.096, 0.098 ng/mL (LOQs). Coefficients of variation for all metabolites and tested concentrations were <10% and accuracy was within 95-105%. Method applicability was tested on colorectal cancer patients during the perioperative period. All metabolites were significantly higher in cancer patients than healthy controls. The 3-NT was significantly lower in advanced cancer and 3-BT showed a similar tendency. Dynamics of 3-BT in the early postoperative period were affected by type of surgery and presence of surgical site infections. In conclusion, a sensitive and specific LC-MS/MS method for simultaneous quantification of free 3-NT, 3-BT, and 3-CT in human plasma has been developed.

Topics: Aged; Biomarkers; Chromatography, Liquid; Colorectal Neoplasms; Female; Humans; Inflammation; Male; Metabolomics; Middle Aged; Nitrosative Stress; Oxidative Stress; Postoperative Complications; Postoperative Period; Prospective Studies; Reactive Oxygen Species; Reproducibility of Results; Sensitivity and Specificity; Surgical Wound Infection; Tandem Mass Spectrometry; Tyrosine

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N=3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 +/- 11.0 (mean +/- S.D.) in 10(5) tyrosine and 4.4 +/- 3.9 (mean +/- S.D.) in 10(3) tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R(2) = 0.55, p = 0.0065, n=23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.

Topics: Calibration; Chromatography, Liquid; Humans; Radioisotope Dilution Technique; Sensitivity and Specificity; Tandem Mass Spectrometry; Tyrosine

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI GC/MS) is 100-fold more sensitive than liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.

Topics: Artifacts; Gas Chromatography-Mass Spectrometry; Humans; Male; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tyrosine

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

Topics: Eosinophil Peroxidase; Eosinophils; Free Radicals; Humans; Immunohistochemistry; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Oxidants; Oxidation-Reduction; Peroxidases; Phenylpropionates; Proteins; Reactive Oxygen Species; Status Asthmaticus; Tyrosine