Selective quantification of free 3-nitrotyrosine in exhaled breath condensate in asthma using gas chromatography/tandem mass spectrometry

• Published in: Nitric oxide 2005-09, Vol.13 (2), p.134-144
• Main Authors: Lärstad, Mona, Söderling, Ann-Sofi, Caidahl, Kenneth, Olin, Anna-Carin
• Format: Article
• Language: English
• Subjects: Adult; Aged; analogs & derivatives; analysis; Asthma; Biological Markers; Biomarkers - analysis; Case-Control Studies; diagnosis; Exhalation; Exhaled breath condensate; Female; Fysiologi; Gas Chromatography-Mass Spectrometry; Humans; Inflammation; Inflammation - diagnosis; Male; Mass spectrometry; Medicin och hälsovetenskap; Middle Aged; Nitrotyrosine; Oxidative Stress; Physiology; Reproducibility of Results; Sensitivity and Specificity; Tyrosine; Tyrosine - analogs & derivatives; Tyrosine - analysis
• ISSN: 1089-8603; 1089-8611
• DOI: 10.1016/j.niox.2005.05.009

Reactive nitrogen species can cause oxidative modifications of certain amino acid residues in proteins, notably the modification of tyrosine to 3-nitrotyrosine (3-NT), which is a potentially useful marker of oxidative stress. Since lung diseases are associated with airway inflammation and oxidative stress, quantification of 3-NT in exhaled breath condensate (EBC) may provide a non-invasive means for monitoring ongoing inflammatory processes. 3-NT-like immunoreactivity has previously been detected in EBC, but no definitive evidence for the presence of 3-NT in EBC is available. Here, a method based on gas chromatography/negative ion chemical ionization/tandem mass spectrometry was established for the quantification of free 3-NT in EBC. The detection limit was 0.56 pM (corresponding to 3.0 amol μl −1 sample injected) and the method was found to give linear results ( r 2 > 0.999) in the concentration range of 0–5.0 nM. The coefficient of variation (CV) for within-day and between-day precision were 11 and 12%, respectively. No artifactual nitration was observed during sample processing. The method was applied to study subjects with asthma ( n = 8), and healthy subjects ( n = 10), but only a slight non-significant increase in 3-NT levels was found in the former group (median [interquartile ranges]; 99 [50–547] amol s −1 vs. 75 [35–147] amol s −1). No correlation with exhaled nitric oxide (NO), pulmonary function or EBC levels of total protein was observed. The 3-NT levels were much lower compared to previously reported levels, based on immunochemical measurements. The method does not allow the simultaneous quantification of tyrosine in samples.