Selective quantification of free 3-nitrotyrosine in exhaled breath condensate in asthma using gas chromatography/tandem mass spectrometry.
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 microl(-1) sample injected) and the method was found to give linear results (r2 > 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.
Lärstad
Mona
M
Department of Occupational Medicine, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden. mona.larstad@amm.gu.se
Söderling
Ann-Sofi
AS
Caidahl
Kenneth
K
Olin
Anna-Carin
AC
D000328:Adult; D000368:Aged; D001249:Asthma; D015415:Biomarkers / Q000032:analysis; D016022:Case-Control Studies; D045853:Exhalation; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007249:Inflammation / Q000175:diagnosis; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D015415:Biomarkers; C002744:3-nitrotyrosine; D014443:Tyrosine