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.     

Electrochemical detection of free 3-nitrotyrosine: application to microdialysis studies

3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1-500 nM (r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 microM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 microM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.     

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX^® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.     

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX^® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.     

Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls,asthma and COPD patients

Background

Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying the acidity (pH) of airway secretions in patients with inflammatory lung diseases.

Aim

To assess the reproducibility of EBC pH for two commercially available devices (portable RTube and non-portable ECoScreen) in healthy controls, patients with asthma or COPD, and subjects suffering from an acute cold with lower-airway symptoms. In addition, we assessed the repeatability in healthy controls.

Methods

EBC was collected from 40 subjects (n = 10 in each of the above groups) using RTube and ECoScreen. EBC was collected from controls on two separate occasions within 5 days. pH in EBC was assessed after degasification with argon for 20 min.

Results

In controls, pH-measurements in EBC collected by RTube or ECoScreen showed no significant difference between devices (p = 0.754) or between days (repeatability coefficient RTube: 0.47; ECoScreen: 0.42) of collection. A comparison between EBC pH collected by the two devices in asthma, COPD and cold patients also showed good reproducibility. No differences in pH values were observed between controls (mean pH 8.27; RTube) and patients with COPD (pH 7.97) or asthma (pH 8.20), but lower values were found using both devices in patients with a cold (pH 7.56; RTube, p < 0.01; ECoScreen, p < 0.05).

Conclusion

We conclude that pH measurements in EBC collected by RTube and ECoScreen are repeatable and reproducible in healthy controls, and are reproducible and comparable in healthy controls, COPD and asthma patients, and subjects with a common cold.     

Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled Breath Condensate pH Levels in Atopic and Healthy Children

Endotoxin, a component of the cell walls of gram-negative bacteria, is a contaminant in organic dusts (house dust) and aerosols. In humans, small amounts of endotoxin may cause a local inflammatory response. Exhaled nitric oxide (eNO) levels, an inflammation indicator, are associated with the pH values of exhaled breath condensate (EBC). This study evaluated seasonal changes on indoor endotoxin concentrations in homes and the relationships between endotoxin exposure and eNO/EBC pH levels for healthy children and children with allergy-related respiratory diseases. In total, 34 children with allergy-related respiratory diseases and 24 healthy children were enrolled. Indoor air quality measurements and dust sample analysis for endotoxin were conducted once each season inside 58 surveyed homes. The eNO, EBC pH levels, and pulmonary function of the children were also determined. The highest endotoxin concentrations were on kitchen floors of homes of children with allergy-related respiratory diseases and healthy children, and on bedroom floors of homes of asthmatic children and healthy children. Seasonal changes existed in endotoxin concentrations in dust samples from homes of children with allergic rhinitis, with or without asthma, and in EBC pH values among healthy children and those with allergy-related respiratory diseases. Strong relationships existed between endotoxin exposure and EBC pH values in children with allergic rhinitis.     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Oxidative stress in cystic fibrosis patients with Burkholderia cenocepacia airway colonization: relation of 8-isoprostane concentration in exhaled breath condensate to lung function decline

The association between oxidative stress and neutrophilic inflammation in cystic fibrosis (CF) lung disease is well recognized. 8-Isoprostane is a product of non-enzymatic oxidation of arachidonic acid. The aim of the present study was to examine the relationship between lung function decline and 8-isoprostane concentrations in exhaled breath condensate (EBC) in CF patients with Burkholderia cenocepacia airway colonization. Concentrations of 8-isoprostane in EBC were measured in 24 stable CF patients with B. cenocepacia airway colonization. The median (interquartile range) age of the cohort was 23.9 (22.0; 26.6) years. All patients underwent clinical examinations and pulmonary function tests at the time of EBC collection and in 1-, 3-, and 5-year intervals. 8-Isoprostane concentrations in EBC correlated to 1- and 3-year declines of forced expiratory volume in 1 s (FEV₁) with r _S values of ?0.511 (p?=?0.0011) and ?0.495 (p?=?0.016), respectively. In multiple regression analysis, 8-isoprostane concentrations in EBC were the only independent predictor for 1-year FEV₁ decline (p?=?0.01). When the median value of 8-isoprostane concentration in EBC (10.0 pg/mL) was used as a cutoff, subgroups of patients with lower and higher level of oxidative stress had significantly different median (interquartile range) FEV₁ declines in 1-year interval, ?2.4 % (?5.3; 0.8) and ?7.3 % (?10.3; ?5.8) predicted (p?=?0.009). In conclusion, 8-isoprostane concentrations in EBC correlated to short-term lung function decline in CF patients with B. cenocepacia airway colonization. This correlation reflects the role of oxidative stress in CF lung pathogenesis and contributes to prediction of prognosis in these patients.     

Analysis of the cough sound frequency in adults and children with bronchial asthma

It is well known that the frequency distribution of cough sound varies in different pathological conditions. Its identification could have diagnostic value. In this study the cough sound frequency in adults (n=20, 51.7 +/- 11 yrs), children (n=21, 11.8 +/- 0.4 yrs) asthmatics and healthy volunteers (n=25, 21 yrs) was explored. All patients were suffering from bronchial asthma. They were on a stable therapeutic regime and in a quiet status. Voluntary cough sound was recorded by a microphone and a tape recorder an ddigitally processed. Overlapping technique and Fast Fourier Transform were used to estimate the sound spectra. The records were smoothed by the method of Pascal triangle. They demonstrate the mean values of cough sound spectra. The registered pseudo three-dimensional plots of cough sound frequency (1 K spectra as function in time) of adults showed that the intensity of frequencies increased from 100 to 900 Hz in 3-4 waves. These frequencies afterwards decreased and between 1 to 2 kHz a smaller elevation was present. The spectra of children resembled to the spectrum of adults but had a smoother course. The spectra of asthmatics had some specificity and differed from the spectrum of healthy volunteers.     

Eotaxin-1 in exhaled breath condensate of stable and unstable asthma patients.

Background

Airway eosinophilia is considered a central event in the pathogenesis of asthma. Eotaxin plays a key role in selective eosinophil accumulation in the airways and, subsequently, their activation and degranulation. The study was undertaken to evaluate eotaxin-1 levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity and to establish the possible correlation of these measurements with other recognized parameters of airway inflammation.

Methods

EBC was collected from 46 patients with allergic asthma (14 with steroid-naïve asthma, 16 with ICS-treated, stable asthma, 16 with ICS-treated unstable asthma) and 12 healthy volunteers. Concentrations of eotaxin-1 were measured by ELISA.

Results

In the three groups of asthmatics, eotaxin-1 concentrations in EBC were significantly higher compared with healthy volunteers (steroid-naïve asthma: 9.70 pg/ml ± 1.70, stable ICS-treated asthma: 10.45 ± 2.00, unstable ICS-treated asthma: 17.97 ± 3.60, healthy volunteers: 6.24 ± 0.70). Eotaxin-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of eotaxin-1 in EBC and exhaled nitric oxide (F_ENO) or serum eosinophil cationic protein (ECP) in the three studied groups of asthmatics. We also discovered a significantly positive correlation between eotaxin-1 in EBC and blood eosinophil count in the groups of patients with unstable asthma and steroid-naïve asthma.

Conclusions

Measurements of eotaxin-1 in the EBC of asthma patients may provide another useful diagnostic tool for detecting and monitoring airway inflammation and disease severity.     

Breath Formate Is a Marker of Airway S-Nitrosothiol Depletion in Severe Asthma

Background

Children with severe asthma have poor symptom control and elevated markers of airway oxidative and nitrosative stress. Paradoxically, they have decreased airway levels of S-nitrosothiols (SNOs), a class of endogenous airway smooth muscle relaxants. This deficiency results from increased activity of an enzyme that both reduces SNOs to ammonia and oxidizes formaldehyde to formic acid, a volatile carboxylic acid that is more easily detected in exhaled breath condensate (EBC) than SNOs. We therefore hypothesize that depletion of airway SNOs is related to asthma pathology, and breath formate concentration may be a proxy measure of SNO catabolism.

Methods and Findings

We collected EBC samples from children and adolescents, including 38 with severe asthma, 46 with mild-to-moderate asthma and 16 healthy adolescent controls, and the concentration of ionic constituents was quantified using ion chromatography. The concentrations of EBC components with volatile conjugates were log-normally distributed. Formate was the principal ion that displayed a significant difference between asthma status classifications. The mean EBC formate concentration was 40% higher in samples collected from all asthmatics than from healthy controls (mean = 5.7 µM, mean±standard deviation = 3.1−10.3 µM vs. 4.0, 2.8−5.8 µM, p = 0.05). EBC formate was higher in severe asthmatics than in mild-to-moderate asthmatics (6.8, 3.7−12.3 µM vs. 4.9, 2.8−8.7 µM, p = 0.012). In addition, formate concentration was negatively correlated with methacholine PC₂₀ (r = −0.39, p = 0.002, asthmatics only), and positively correlated with the NO-derived ion nitrite (r = 0.46, p<0.0001) as well as with total serum IgE (r = 0.28, p = 0.016, asthmatics only). Furthermore, formate was not significantly correlated with other volatile organic acids nor with inhaled corticosteroid dose.

Conclusions

We conclude that EBC formate concentration is significantly higher in the breath of children with asthma than in those without asthma. In addition, amongst asthmatics, formate is elevated in the breath of those with severe asthma compared to those with mild-to-moderate asthma. We suggest that this difference is related to asthma pathology and may be a product of increased catabolism of endogenous S-nitrosothiols.     

Evaluation of oxidative stress, 3-Nitrotyrosine,and HMGB-1 levels in patients with wet type Age-Related Macular Degeneration

BackgroundThis study aims to compare serum HMGB-1, 3-nitrotyrosine (3-NT), TAS, TOS, and OSI levels in Wettype Age-Related Macular Degeneration (wAMD) patients and healthy controls to determine the correlation of these parameters with each other.MethodsThirty patients with Wet-type Age-Related Macular Degeneration (wAMD) and 27 healthy adults, as controls were enrolled in the study. We determined the TAS and TOS levels in serum samples of both groups using commercial kits on a microplate reader. Serum HMGB-1 and 3-NT levels were measured with the enzyme-linked immunosorbent assay method.ResultsHMGB-1 levels were significantly higher in the patient group (137.51 pg/mL, p=0.001), while there was no difference between the two groups in serum 3-NT levels (p=0.428). A statistically significant difference found in the levels of TOS and OSI (p=0.001 and p=0.045, respectively) between the patients and controls, however, no significant difference was observed between the groups in terms of TAS levels (p=0.228).ConclusionsOxidative stress and HMGB-1 levels were increased in wAMD patients and enhanced oxidative stress may be associated with increased tissue necrosis and inflammation. Thus administration of antioxidant treatment in addition to routine therapy should be considered in wAMD.     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

Mitral and Tufted Cells Are Potential Cellular Targets of Nitration in the Olfactory Bulb of Aged Mice

Olfactory sensory function declines with age; though, the underlying molecular changes that occur in the olfactory bulb (OB) are relatively unknown. An important cellular signaling molecule involved in the processing, modulation, and formation of olfactory memories is nitric oxide (NO). However, excess NO can result in the production of peroxynitrite to cause oxidative and nitrosative stress. In this study, we assessed whether changes in the expression of 3-nitrotyrosine (3-NT), a neurochemical marker of peroxynitrite and thus oxidative damage, exists in the OB of young, adult, middle-aged, and aged mice. Our results demonstrate that OB 3-NT levels increase with age in normal C57BL/6 mice. Moreover, in aged mice, 3-NT immunoreactivity was found in some blood vessels and microglia throughout the OB. Notably, large and strongly immunoreactive puncta were found in mitral and tufted cells, and these were identified as lipofuscin granules. Additionally, we found many small-labeled puncta within the glomeruli of the glomerular layer and in the external plexiform layer, and these were localized to mitochondria and discrete segments of mitral and tufted dendritic plasma membranes. These results suggest that mitral and tufted cells are potential cellular targets of nitration, along with microglia and blood vessels, in the OB during aging.     

Oxidative stress and neurological disorders in relation to blood lead levels in children

Oxidative stress plays a pivotal role in the pathogenesis of neurological disorders. Free radical generation appears to be the mode of lead toxicity. We evaluated the effects of blood lead levels on oxidative stress parameters in children suffering from neurological disorders. Thirty children (aged 3-12 years) with neurological disorders (cerebral palsy [n = 12], seizures [n = 11], and encephalopathy [n = 7]) were recruited in the study group. Sixty healthy children (aged 3-12 years) from similar socio-economic environments and not suffering from any chronic disease were taken as the controls. Blood lead levels and oxidant/antioxidant status were determined. Mean blood lead level was significantly higher while delta-aminolevulinic acid dehydratase (delta-ALAD) activity, a biomarker for lead exposure, was significantly lower in the study group as compared to the control group (P < 0.05 for each). Malondialdehyde (MDA) levels, an end-product of lipid peroxidation, were significantly higher while the antioxidant glutathione (GSH) levels were significantly lower in the study group as compared to the control group (P < 0.05 for each). Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were significantly higher in the study group than those of the control group (P < 0.05 for each). There were significant negative correlations of blood lead levels with delta-ALAD (r = -0.35; P < 0.05) and GSH (r = -0.31; P < 0.05), and positive correlations with MDA (r = 0.37; P < 0.05), SOD (r = 0.53; P < 0.05), and CAT (r = 0.31; P < 0.05). In turn, delta-ALAD had significant negative correlations with MDA (r = -0.29; P < 0.05), SOD (r = -0.28; P < 0.05) and CAT (r = -0.34; P < 0.05), but positive correlation with GSH (r = 0.32; P < 0.05). Although a causal pathway can not be determined from the present study, our findings indicate lead-induced oxidative stress in blood of children with neurological disorders. Lead-induced oxidative stress as an underlying mechanism for neurological diseases in children warranted further investigation.     

Biomarkers in Exhaled Breath Condensate Are Not Predictive for Pulmonary Exacerbations in Children with Cystic Fibrosis: Results of a One-Year Observational Study

BackgroundCystic Fibrosis (CF) is characterized by chronically inflamed airways, and inflammation even increases during pulmonary exacerbations. These adverse events have an important influence on the well-being, quality of life, and lung function of patients with CF. Prediction of exacerbations by inflammatory markers in exhaled breath condensate (EBC) combined with early treatment may prevent these pulmonary exacerbations and may improve the prognosis.AimTo investigate the diagnostic accuracy of a set of inflammatory markers in EBC to predict pulmonary exacerbations in children with CF.MethodsIn this one-year prospective observational study, 49 children with CF were included. During study visits with an interval of 2 months, a symptom questionnaire was completed, EBC was collected, and lung function measurements were performed. The acidity of EBC was measured directly after collection. Inflammatory markers interleukin (IL)-6, IL-8, tumor necrosis factor α (TNF-α), and macrophage migration inhibitory factor (MIF) were measured using high sensitivity bead based flow immunoassays. Pulmonary exacerbations were recorded during the study and were defined in two ways. The predictive power of inflammatory markers and the other covariates was assessed using conditionally specified models and a receiver operating characteristic curve (SAS version 9.2). In addition, k-nearest neighbors (KNN) algorithm was applied (SAS version 9.2).ResultsSixty-five percent of the children had one or more exacerbations during the study. The conditionally specified models showed an overall correct prediction rate of 55%. The area under the curve (AUC) was equal to 0.62. The results obtained with the KNN algorithm were very similar.ConclusionAlthough there is some evidence indicating that the predictors outperform random guessing, the general diagnostic accuracy of EBC acidity and the EBC inflammatory markers IL-6, IL-8, TNF-α and MIF is low. At present it is not possible to predict pulmonary exacerbations in children with CF with the chosen biomarkers and the method of EBC analysis. The biochemical measurements of EBC markers should be improved and other techniques should be considered.     

Prediction of Asthma Exacerbations in Children by Innovative Exhaled Inflammatory Markers: Results of a Longitudinal Study

Background

In asthma management guidelines the primary goal of treatment is asthma control. To date, asthma control, guided by symptoms and lung function, is not optimal in many children and adults. Direct monitoring of airway inflammation in exhaled breath may improve asthma control and reduce the number of exacerbations.

Aim

1) To study the use of fractional exhaled nitric oxide (FeNO) and inflammatory markers in exhaled breath condensate (EBC), in the prediction of asthma exacerbations in a pediatric population. 2) To study the predictive power of these exhaled inflammatory markers combined with clinical parameters.

Methods

96 asthmatic children were included in this one-year prospective observational study, with clinical visits every 2 months. Between visits, daily symptom scores and lung function were recorded using a home monitor. During clinical visits, asthma control and FeNO were assessed. Furthermore, lung function measurements were performed and EBC was collected. Statistical analysis was performed using a test dataset and validation dataset for 1) conditionally specified models, receiver operating characteristic-curves (ROC-curves); 2) k-nearest neighbors algorithm.

Results

Three conditionally specified predictive models were constructed. Model 1 included inflammatory markers in EBC alone, model 2 included FeNO plus clinical characteristics and the ACQ score, and model 3 included all the predictors used in model 1 and 2. The area under the ROC-curves was estimated as 47%, 54% and 59% for models 1, 2 and 3 respectively. The k-nearest neighbors predictive algorithm, using the information of all the variables in model 3, produced correct predictions for 52% of the exacerbations in the validation dataset.

Conclusion

The predictive power of FeNO and inflammatory markers in EBC for prediction of an asthma exacerbation was low, even when combined with clinical characteristics and symptoms. Qualitative improvement of the chemical analysis of EBC may lead to a better non-invasive prediction of asthma exacerbations.     

Plasma and exhaled breath condensate nitrite-nitrate level in relation to environmental exposures in adults in the EGEA study

This study evaluated the associations between biological markers in the nitrate-nitrite-NO pathway and four environmental exposures among subjects examined in the second survey (2003-2007) of the French Epidemiological study on Genetics and Environment of Asthma (EGEA). Total nitrite and nitrate (NO(2)(-) /NO(3)(-)) levels were measured both in plasma and in exhaled breath condensate (EBC) in 949 adults. Smoking, diet and exposure to chlorine products were assessed using standardized questionnaires. Exposure to air pollutants was estimated by using geostatistical models. All estimates were obtained with generalized estimating equations for linear regression models. Median levels of NO(2)(-)/NO(3)(-) were 36.3μM (1st-3rd quartile: 25.7, 51.1) in plasma and 2.0μmol/mg proteins (1st-3rd quartile 0.9, 3.9) in EBC. After adjustment for asthma, age, sex and menopausal status, plasma NO(2)(-)/NO(3)(-) level increased with leafy vegetable consumption (above versus below median=0.04 (95%CI: 0.001, 0.07)) and decreased in smokers (versus non/ex-smokers=-0.08 (95%CI: -0.11, -0.04). EBC NO(2)(-)/NO(3)(-) level decreased in smokers (-0.08 (95%CI: -0.16, -0.001)) and with exposure to ambient O(3) concentration (above versus below median=-0.10 (95%CI: -0.17, -0.03)). Cured meat, chlorine products, PM(10) and NO(2) concentrations were not associated with NO(2)(-)/NO(3)(-) levels. Results suggest that potential modifiable environmental and behavioral risk factors may modify NO(2)(-)/NO(3)(-) levels in plasma and EBC according to the route of exposure.     

Nitrate in exhaled breath condensate of patients with different airway diseases.

There is an increasing interest in the measurement of nitric oxide (NO.) in the airways. NO. is a free radical that reacts rapidly with reactive oxygen species in aqueous solution to form peroxynitrite which can then break down to nitrite (NO(2)(-)) and nitrate (NO(3)(-)). NO(3)(-) is considered a stable oxidative end product of NO. metabolism. The aim of this study was to assay NO(3)(-) in exhaled breath condensate (EBC) of normal nonsmoking and smoking subjects, asthmatics, patients with obstructive pulmonary disease (COPD), and patients with community-acquired pneumonia (CAP). EBC was collected using a glass condenser and samples were assayed for NO(3)(-) by ion chromatography followed by conductivity measurement. NO(3)(-) was detectable in EBC of all subjects. NO(3)(-) was elevated in smokers [median (range)] [62.5 (9.6-158.0) microM] and in asthmatics [68.0 (25.8-194.6) microM] compared to controls [9.6 (2.6-119.4) microM; p=0.003 and p=0.006, respectively], whereas NO(3)(-) was not elevated in COPD patients [24.1 (1.9-337.0 microM]. The concentration of NO(3)(-) in patients with CAP [243.4 (26.1-584.5) microM] was higher than that in controls (p=0.002) and NO(3)(-) values decreased after treatment and recovery from illness [40.0 (4.1-167.0) microM, p=0.009]. This study shows that NO(3)(-) is detectable in EBC of healthy subjects and it varies in patients with inflammatory airway diseases.     

Insulin-like growth factor-1, leptin, body composition, and clinical status interactions in children with cystic fibrosis

BACKGROUND/AIMS: Children with cystic fibrosis (CF) are of increased risk of reduced fat body mass (FBM) and lean body mass (LBM). Serum concentrations of insulin-like growth factor-1 (IGF-1)and leptin could be markers of LBM and/or FBM depletion. To evaluate the relationships between disease activity, body composition, IGF-1 and leptin concentrations in CF children. METHODS: A cross-sectional study with 26 CF children aged 5.0-15.5 years and 33 healthy controls, mean age 9.4 years. Body composition was evaluated by dual-energy X-ray absorptiometry. Fasting blood samples were analyzed for leptin, IGF-1 and IGFBP-3. RESULTS: FBM standard deviation score (SDS; CF boys -0.02 +/- 0.88 vs. 0.78 +/- 0.65, p < 0.01; CF girls -0.37 +/- 1.15 vs. 0.70 +/- 0.97, p < 0.05), leptin concentration (CF boys 2.07 +/- 0.79 vs. 3.07 +/- 1.28 ng/ml, p < 0.05; CF girls 2.71 +/- 0.86 vs. 5.00 +/- 2.95 ng/ml, p < 0.05) and IGF-1SDS (CF boys -1.43 +/- 1.50 vs. -0.32 +/- 0.88, p < 0.05; CF girls -0.66 +/- 1.66 vs. 0.64 +/- 0.57, p < 0.01) were lower in CF children compared to controls. Shwachman score was the strongest predictor of lean body mass (R = 0.63). Leptin levels explain 60% of the variability in FBM. CONCLUSION: Serum concentrations of IGF-1 and leptin are decreased in children with CF and are associated with clinical conditions and body composition.