Exhaled Breath Condensate Concentrations Research Articles

Oxidative stress and airway inflammation after allergen challenge evaluated by exhaled breath condensate analysis

Allergen exposure may increase airway oxidative stress, which causes lipid membrane peroxidation and an increased formation of 8-isoprostane. The aim of the study was to investigate oxidative stress induced by allergen challenge in mild asthmatics, by measuring 8-isoprostane in exhaled breath condensate (EBC), and to examine their relationship with mediators derived from arachidonic acid. Methods 8-isoprostane, cysteinyl leukotrienes (cys-LTs) and prostaglandin E2 (PGE(2) ) concentrations in EBC were measured at baseline and after allergen challenge in 12 patients with mild allergic asthma sensitized to cat allergen. At 24 h after allergen challenge, compared with baseline values, EBC 8-isoprostane increased [48.64 pg/mL (44.14-53.61) vs. 21.56 pg/mL (19.92, 23.35), P<0.001], cys-LTs increased [27.37 pg/mL (24.09-31.10) vs. 13.28 pg/mL (11.32, 15.57), P<0.001] and PGE(2) decreased [18.69 pg/mL (12.26, 28.50) vs. 39.95 pg/mL (34.37, 46.43), P<0.001]. The trend of increasing 8-isoprostane after allergen challenge was significantly correlated with the trend of increasing cys-LTs (R(2) =0.85, P<0.001) whereas the trend of decreasing PGE(2) after allergen challenge was significantly correlated with the trend of increasing cys-LTs (R(2) =0.52, P=0.001). The increase in EBC 8-isoprostane observed after allergen challenge indicates that allergen exposure increases airway oxidative stress in allergic asthma. The strict correlation between cys-LTs and 8-isoprostane underlines the relationship between allergic inflammation and oxidative stress. A shift of arachidonic acid metabolism towards lipoxygenase pathway is induced by the allergen challenge. Airway oxidative stress occurs after allergen challenge even in patients with mild intermittent allergic asthma.

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

BackgroundAirway 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.MethodsEBC 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.ResultsIn 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 (FENO) 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.ConclusionsMeasurements of eotaxin-1 in the EBC of asthma patients may provide another useful diagnostic tool for detecting and monitoring airway inflammation and disease severity.

Markers of Fibrosis and Inflammation in Exhaled Breath Condensate (EBC) and Bronchoalveolar Lavage Fluid (BALF) of Patients with Pulmonary Sarcoidosis: A Pilot Study

Sarcoidosis is a disease of unknown etiology. Little is known of predictive factors of fibrosis. It was suggested that PAI-1, uPA, TGF-beta1, VEGF, IL-8, TNF-alpha influence this process. To assess airway inflammatory and fibrosis markers in EBC in sarcoidosis and the effect of fiberoptic bronchoscopy (FOB), bronchoalveolar lavage fluid (BALF), transbronchial lung biopsy (TBLB) and bronchial mucosa membrane biopsy on their production in the airways. The study group consisted of 11 patients (5 women, 6 men; mean age 40 +/- 9 yrs, mean +/- SD) with sarcoidosis stage I-III. PAI-1 (ng/ml), uPA (ng/ml), TGF-beta1 (pg/ml), VEGF (pg/ml), IL-8 (pg/ml), TNF-alpha (pg/ml) levels were measured in BALF and EBC collected before and 48 hours after FOB. No significant changes in EBC levels of VEGF, PAI-1, TGF-beta1, TNF-alpha (respectively: 8.02 +/- 4.97 pg/ml; 1.1 +/- 1.2 ng/ml; 2909.7 +/- 206.6 pg/ml; 10.7 +/- 19.9 pg/ml) after FOB were observed when compared to baseline. In contrast, IL-8 concentration in EBC (pg/ml) decreased after FOB (0.073 +/- 0.13 v. 0.061 +/- 0.1, p = 0.006) and was significantly lower than in BALF (BALF 0.95 +/- 0.62, p < 0.05). Also, mean level of VEGF was higher in BALF than in EBC both pre- and post- FOB (BALF 66.38 +/- 36.95, EBC pre-FOB 6.75 +/- 3.67 and EBC post-FOB 8.02 +/- 4.97). Significant relationship between TNF-alpha in post-FOB EBC and BALF was also shown (b = 0.63, p = 0.04). FOB does not significantly affect levels of airway inflammation and fibrosis markers present in EBC before and after FOB; they were also comparable to the concentrations marked by BALF. The lack of correlation between markers levels in EBC and BALF indicates that these methods are not equivalent. Due to repeatibility, and less invasive, simple method of EBC test it seems reasonable to continue the research on the larger number of patients.

Wątrobowy czynnik wzrostu w kondensacie powietrza wydechowego i popłuczynach oskrzelowo-pęcherzykowych w sarkoidozie

Hepatocyte growth factor (HGF) is a strong mitogen stimulating lung epithelial cell growth. Elevated levels of HGF have been reported in various biological materials of patients with acute respiratory distress syndrome and in patients recovering from pneumonia or pneumonectomy. Sarcoidosis may be complicated by lung fibrosis. Consequently, HGF could be considered a new biomarker identifying patients with a higher risk of lung fibrosis. The aim of the study was to verify whether: 1. HGF is measurable in bronchoalveolar lavage fluid (BALF) and exhaled breath condensate (EBC); 2. HGF in BALF or EBC is impaired in sarcoidosis; and 3. HGF correlates with chosen activity and prognostic markers. Sixty-four EBC and 30 BALF of sarcoid patients, and 15 and 9 of healthy controls, respectively, were collected for the measurement of HGF using an ELISA test. HGF was detectable in 62% of EBC samples (56% sarcoidosis and 87% of controls) and in all the BALF samples. EBC and BALF concentrations were not different in comparison to the controls. Moreover, no correlation was found between EBC/BALF concentrations and radiological stage, lung function tests, duration of disease, number of relapses, BALF lymphocytes, serum ACE, or serum and urine calcium concentrations. HGF is detectable in BAL and EBC. However, it does not distinguish sarcoidosis patients from healthy subjects. The above, as well as the lack of correlations with various parameters of disease activity and severity rule out EBC/ /BALF HGF as a biomarker for sarcoidosis monitoring.

Biological effect markers in exhaled breath condensate and biomonitoring in welders: impact of smoking and protection equipment

The objective of this study was to investigate the effect of welding as well as the impact of smoking and protection measures on biological effect markers in exhaled breath condensate. Additionally, biomonitoring of chromium, aluminium and nickel in urine was performed to quantify internal exposure. Exhaled breath condensate (EBC) and urine samples of 45 male welders and 24 male non-exposed control subjects were collected on Friday pre-shift and after 8h of work post-shift. In EBC, biological effect markers such as malondialdehyde, nitrite, nitrate, 3-nitrotyrosine, tyrosine, hydroxyproline, proline, H(2)O(2) and pH-value were measured while aluminium, nickel, and chromium were measured in the urine samples. Although internal exposure to aluminium, nickel and chromium in this study was low, welders showed significantly increased concentrations of all these parameters at baseline compared to non-exposed controls. Moreover, welders had higher nitrate concentrations in EBC at baseline and after shift. Nitrate concentration was considerably lower after shift if personal protection equipment was used. H(2)O(2) was increased only when subjects smoked during shift. It has been shown that welding-associated long-term and short-term health effects could be detected in a population of welders. The results also showed that using personal protection equipment is of high importance and H(2)O(2) may be an effect marker associated with smoking rather than with welding fumes, while nitrate in EBC seems to be sensitive to welding fume exposure.

Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects

Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I® (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck® ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H2O2. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L−1. At a resting ventilation of 11.5 ± 3.4 L min−1, the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L−1, and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min−1. After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L−1 and the ventilation increased to 111.6 ± 21.4 L min−1. The EBC collection time decreased to 3.9 ± 1.9 min, and 1.20 ± 0.44 mL EBC were obtained in the recovery period after termination of exercise. The lactate concentration in EBC increased to 40.3 ± 23.0 µmol L−1, and the lactate release in EBC increased to 13.6 ± 8.6 nmol min−1 (p < 0.01). Assuming a volume of 4.3 mL water in 100 L of exhaled air (saturated with water at 37 °C), we calculated a lactate release at rest of 11.5 ± 4.3 nmol min−1 and 48.6 ± 30.7 nmol min−1 (p < 0.01) after exhausting exercise. Detectable releases of lactate in exhaled breath condensate were found already under resting conditions. During exhausting external load on a bicycle spiroergometer, an increase in the lactate concentration was found in arterialized blood along with an increased lactate release in EBC. The correlation between expiratory lactate release via EBC and lactate concentration in arterialized blood is studied in pursuing investigations.

Using the exhaled breath condensate as a tool for non-invasive evaluation of pollutant exposure

The aim of the present study was to investigate whether Exhaled Breath Condensate (EBC) can be employed for a better risk assessment among human exposure to heavy metals. The objective was to develop a human bioindicator to be applicable for professional exposure that, compared with other common indicators, presents important advantages: it is non-invasive and it is easily and quickly collected. EBC was sampled in industrial workers and in a control group consisting of healthy volunteers working in offices. Heavy metal concentrations in EBC were determined by Total Reflection X-Ray Fluorescence (TXRF) in parallel low-volume samplers, and the techniques Particle Induced X-ray Emission (PIXE) and Instrumental Neutron Activation Analysis (INAA) were used to characterise air particulate matter in the factory and in the offices. Results revealed that Pb and Cu were significantly higher in industrial workers EBC, raising the possibility of using this new matrix in occupational settings in order to assess the workers' exposure to toxic agents.

Endogenous Levels of Five Fatty Acid Metabolites in Exhaled Breath Condensate to Monitor Asthma by High-Performance Liquid Chromatography: Electrospray Tandem Mass Spectrometry.

Airway inflammation characterizing asthma and other airway diseases may be monitored through biomarker analysis of exhaled breath condensate (EBC). In an attempt to discover novel EBC biomarkers, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was used to analyze EBC from ten control non-asthmatics and one asthmatic individual for five fatty acid metabolites: 9,12,13-trihydroxyoctadecenoic acid (9,12,13-TriHOME), 9,10,13-TriHOME, 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 12-hydroxyeicosatetraenoic acid (12-HETE), and 12(13)-epoxyoctadecenoic acid (12(13)-EpOME). The method was shown to be sensitive, with an on-column limit of quatitation (LOQ) in the pg range (corresponding to pM concentrations in EBC), and linear over several orders of magnitude for each analyte in the calibrated range. Analysis of EBC spiked with the five fatty acid metabolites was within 81%-119% with only a few exceptions. Endogenous levels in EBC exhibited intra- and inter-assay precision of 10%-22%, and 12%-36%, respectively. EBC from the healthy subjects contained average analyte levels between 15 and 180 pM with 12-HETE present above the LOQ in only one of the subjects at a concentration of 240 pM. Exposure of the asthmatic subject to allergen led to increased EBC concentrations of 9,12,13-TriHOME, 9,10,13-TriHOME, 12,13-DiHOME, and 12(13)-EpOME when compared to levels in EBC collected prior to allergen exposure (range =40-510 pM). 12,13-DiHOME was significantly increased (Student's t-test, p < 0.05). In conclusion, we have developed a new HPLC-ESI-MS/MS method for the analysis of five fatty acid metabolites in EBC, which are potential biomarkers for asthma monitoring and diagnosis.

Concentration of C-reactive protein, magnesium and calcium in children with acute bronchoconstriction

Introduction: In childhood, bronchoobstruction is mostly caused by respiratory viral infections. The aim of this study was to find out possible changes in serum concentration of magnesium and calcium (participants in bronchoconstriction), and concentration of C-reactive protein (marker of inammation) in children with moderate and severe bronchoconstriction, caused by viral respiratory infections. Materials and methods: The study included 32 children with acute bronchoconstriction caused by a viral respiratory infection. Inhalation of salbutamol was administered according to the severity of bronchoconstriction. Blood sampling was done on admission and the third day of salbutamol administration. Results: Therapy with salbutamol led to a relief of dyspnea within 36 hours, and symptoms of viral infection have relieved. During follow-up period, magnesium concentration was higher in children with moderate (but not in severe) bronchoconstriction than in healthy children, with consequently higher magnesium to calcium ratio. Concentration of C-reactive protein decreased spontaneously by gradual disappearance of signs and symptoms of viral infection. Conclusion: Determination of serum magnesium and calcium concentrations and determination of their ratio are not sufficient enough to follow-up on effects of therapy. Additional studies of ionized forms of magnesium and calcium, their intracellular content, as well as concentration in exhaled breath condensate are needed.

RANTES in Exhaled Breath Condensate of Allergic Asthma Patients with Exercise-Induced Bronchoconstriction

Background: The response of asthmatics to exercise differs from that of healthy subjects, and the mechanisms responsible for exercise-induced bronchoconstriction (EIB) remain to be elucidated. Objectives: The aim of this study was to evaluate changes in RANTES levels in exhaled breath condensate (EBC) following intensive exercise in allergic asthmatics. Methods: The study was conducted in a group of 19 asthmatics (11 with EIB and 8 without EIB) and 7 healthy volunteers. Changes in the concentrations of RANTES in EBC induced during the 24 h after intensive exercise were determined. Moreover, these measurements were tested for possible correlations with the results of other tests commonly associated with asthma as well as with changes in airway inflammation after exercise. Results: In contrast to asthmatic patients without EIB and healthy controls, in asthmatics with EIB RANTES concentrations were statistically significantly increased in EBC collected during the first 24 h after an exercise test. There was a statistically significant correlation between the maximum increase in RANTES concentrations in EBC after exercise and either baseline exhaled nitric oxide (F_ENO) or bronchial hyperreactivity to histamine and an increase in serum eosinophil cationic protein or F_ENO 24 h after exercise in the EIB asthmatics. Conclusions: The increase in RANTES in asthmatic airways, promoting the migration and activation of inflammatory cells including eosinophils, may play an important role in the upregulation of airway inflammation after EIB in asthmatic patients.

Increased H2O2 level in exhaled breath condensate in primary breast cancer patients

This study was designed to assess exhaled hydrogen peroxide (H(2)O(2)), blood serum antioxidant capacity, and tumor necrosis factor-alpha (TNFalpha) in primary breast cancer (PBC). The study included 34 consecutive, non-smoking PBC patients (aged 62.5 +/- 13.5 at surgery) prior to the treatments, qualified for modified radical mastectomy and not undergoing any adjuvant systemic therapy, and 33 healthy controls. The post surgery pathological assessment included tissue expression of estrogen (ER) and progesterone (PR) receptors, and epidermal growth factor receptor type 2 (HER-2/neu). Exhaled H(2)O(2) was determined fluorometrically in the exhaled breath condensate (EBC). Blood serum antioxidant capacity and TNFalpha levels were assessed with ferric reducing ability of plasma (FRAP) and ELISA immunoassay, respectively. In PBC patients, 10 ER, 11 PR, and 9 HER-2/neu positive tumors were identified and HER-2/neu score was 2+ in 20% of all tumors. Median (Me) H(2)O(2) was increased up to 0.44 microM (interquartile range IR: 0.20-1.25 microM) compared with healthy control of 0.36 microM (IR: 0.12-0.48 microM; p < 0.05). The H(2)O(2) concentration in EBC was significantly correlated (tau = 0.27; p = 0.03) and increased in cases with nodal metastases (n = 12; p = 0.04). Serum TNFalpha was increased up to 51.7 +/- 21.0 pg/ml compared with controls 17.2 +/- 3.65 pg/ml (p < 0.05). FRAP was increased to 1.41 +/- 0.37 mM Fe(2+) compared with control 1.19 +/- 0.17 mM Fe(2+); (p = 0.006). This is the first study to demonstrate increased H(2)O(2) in exhaled breath condensate in patients with localized breast malignancy and its relation with clinical severity.

EIA and GC/MS analysis of 8-isoprostane in EBC of children with problematic asthma

Asthmatic airways are characterised by enhanced oxidative stress, which can be studied by measuring biomarkers, such as 8-isoprostane. The aims of the present study were: 1) to measure the concentrations of 8-isoprostane in exhaled breath condensate (EBC) and urine of children with problematic and well-controlled asthma; 2) to compare the concentrations of 8-isoprostane measured by gas chromatographic/negative ion chemical ionisation mass spectrometry (GC/NICI-MS) and by an enzymatic immunoassay (EIA). We recruited 20 asthmatic allergic children, 13 with well-controlled asthma and seven with problematic asthma. They underwent exhaled nitric oxide measurements and spirometry, and both EBC and urine samples were collected. 8-isoprostane was measured in EBC by GC/NICI-MS and EIA. 8-isoprostane concentrations in EBC were significantly higher in children with problematic asthma than in children with well-controlled asthma (p = 0.01). An acceptable reproducibility emerged between GC/NICI-MS and EIA (coefficient of reproducibility 11.5 pg x mL(-1)). 8-isoprostane levels measured in urine did not correlate with those measured in EBC. We showed that 8-isoprostane in EBC was significantly increased in children with problematic asthma, suggesting a role for oxidative stress in this asthma phenotype. In addition we found an acceptable reproducibility of EIA compared to GC/NICI-MS, even if the latter method had higher accuracy.

Apocynin decreases hydrogen peroxide and nirtate concentrations in exhaled breath in healthy subjects

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases. Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells. In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6 ml of 0.5 mg/ml) was administered by nebulization and its effects on H 2O 2, NO 2 − and NO 3 − generation were assessed after 30, 60 and 120 min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO 2 − serum level, cough scale control and blood pressure as safety parameters. Apocynin caused reduction of H 2O 2 concentration in EBC as compared to placebo, after 60 min. of inhalation (0.18 μM vs. 0.31 μM, p < 0.05) as well as after 120 min. (0.2 μM vs. 0.31 μM, p < 0.05). Similarly, apocynin significantly decreased concentration of NO 3 − as compared to placebo, after 60 and 120 min. (6.8 μM vs. 14.4 μM and 6.5 μM vs. 14.9 μM respectively, p < 0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study. Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.

Assessment of Airway Inflammation by Exhaled Breath Condensate and Impedance Due to Gastroesophageal Reflux Disease (GERD)

Avoiding oxidative stress in the airways is important for the treatment of respiratory disease associated with gastroesophageal reflux disease (GERD). It is often difficult to decide whether GERD is causing airway inflammation or whether an airway disease is complicated by GERD. Measurement of exhaled breath condensate (EBC) is performed by cooling and collecting the airway lining fluid contained in exhaled air. A decrease of pH and an increase of the 8-isoprostane concentration in EBC have been observed in patients with mild to moderate asthma accompanied by GERD. There are still problems to be overcome before EBC can be used clinically, but pH and 8-isoprostane may be promising objective markers of airway inflammation due to GERD. The disease concept and diagnosis of GERD are constantly advancing, including the development of impedance methods. It is expected that treatment will be based on the latest diagnostic knowledge of GERD associated with respiratory disease and on monitoring of airway inflammation.

Effects of Inhalation of Thermal Water on Exhaled Breath Condensate in Chronic Obstructive Pulmonary Disease

Background: Inhalation of thermal water (TW) is traditionally used as part of the treatment of chronic obstructive pulmonary disease (COPD), but its benefit and mechanisms are controversial. We previously observed a reduced proportion of neutrophils in induced sputum after treatment with TW. Objectives: The aim of this study was to determine whether inhalation of TW in COPD patients is associated with biochemical changes of airway lining fluid, including a reduction in the neutrophil chemoattractant leukotriene B₄ (LTB₄). Methods: Thirteen COPD patients were randomly assigned to receive a 2-week course of TW and normal saline inhalation in a cross-over, single-blind study design. Exhaled breath condensate (EBC) was collected before and after treatments. LTB₄ concentrations in EBC were determined by ELISA, and EBC pH was measured before and after argon deaeration. Results: No significant differences in LTB₄ concentrations in EBC were detected with either treatment. A significant decrease in pH of non-deaerated EBC was observed after a standard course of TW (median 7.45, interquartile range 6.93–7.66, vs. median 6.99, interquartile range 6.57–7.19; p = 0.05), which disappeared after argon deaeration. Conclusions: There is no evidence that TW treatment affects LTB₄ concentration in EBC. The results of EBC pH measurements suggest that TW inhalation induces an imbalance of volatile components of the buffer system in airway lining fluid.

Bronchial nitric oxide is related to symptom relief during fluticasone treatment in COPD

High levels of exhaled nitric oxide (NO) predict favourable response to inhaled corticosteroids in asthma, but the ability of exhaled NO or inflammatory markers in exhaled breath condensate (EBC) to predict steroid responsiveness in chronic obstructive pulmonary disease (COPD) is not known. We measured alveolar and bronchial NO output, levels of leukotriene B(4) (LTB(4)), cysteinyl leukotrienes (cysLTs) and 8-isoprostane in EBC, spirometry, body plethysmography and symptoms in 40 subjects with COPD before and after 4 weeks of treatment with inhaled fluticasone (500 microg b.i.d.). Five subjects (12.5%) with COPD had significant improvement in lung function during fluticasone treatment, whereas 20 subjects (50%) had significant decrease in symptoms. High baseline bronchial NO flux was associated with higher increase in forced expiratory volume in 1 s to forced vital capacity ratio (r = 0.334, p = 0.038) and more symptom relief (r = -0.317, p = 0.049) during the treatment. Baseline EBC levels of LTB(4), cysLTs or 8-isoprostane were not related to response to fluticasone treatment. Inhaled fluticasone decreased bronchial NO flux but not alveolar NO concentration or markers in EBC. High levels of bronchial NO flux are related to symptom relief and improvement of airway obstruction during treatment with inhaled fluticasone in COPD. Markers of inflammation or oxidative stress in EBC are not related to steroid responsiveness in COPD.

Levels of cysteinyl-leukotrienes in exhaled breath condensate are not due to saliva contamination

Saliva contamination has been suggested to be a major contributor to levels of cysteinyl leukotrienes in exhaled breath condensate (EBC). The aim of this study was to compare the levels of cysteinyl-leukotrienes (CysLT) and alpha-amylase activity in EBC to induced sputum and saliva collected from the same subjects (asthmatics and control). We thereby aimed to find out whether saliva contamination could be a plausible explanation to the levels found in EBC or not. EBC, saliva and induced sputum were collected from 11 asthmatic and 19 healthy adults. These samples were analyzed for CysLT concentration and alpha-amylase activity. No significant correlation was found between CysLT concentration and alpha-amylase activity in EBC, saliva or sputum. In addition, we show that the saliva contamination (measured as alpha-amylase activity) was negligible, as the relative amount of saliva CysLT was only 0.6% of that found in EBC. The amount of CysLT correlated between all three compartments (EBC, saliva and sputum), but no similar correlation was seen for the alpha-amylase activity in EBC compared to saliva and sputum. The levels of CysLT were higher in asthmatic patients compared to healthy controls in EBC, saliva and sputum. We conclude that the amount of CysLT in EBC cannot be explained by saliva contamination. Please cite this paper as: Tufvesson E, van Weele LJ, Ekedahl H and Bjermer L. Levels of cysteinyl-leukotrienes in exhaled breath condensate are not due to saliva contamination.

Evidence for Airway Acidity and Oxidative Stress in Exhaled Breath Condensates from Grain Workers

In their article, Dr. Do and colleagues described exhaled breath condensate (EBC) studies that they interpret to indicate airway acidity and oxidative stress (8-isoprostane) in grain workers (1). Unfortunately, neither of these conclusions can be drawn on the basis of the data they have collected. With regard to pH, the species NH4 1 and HCO3 2 represent approximately 90% of the acids and bases found in most samples of EBC (2). Generation of approximately 90% of the NH4 1 is in the mouth rather than the airways, much of it from bacterial degradation of urea (3). Low concentrations of volatile organic acids (e.g., acetic acid) may be found in EBC, but these may also represent oral contamination. Abnormal EBC pH probably reflects oral rather than airway metabolism (3), and the pH and constituents of saliva should have been measured. Furthermore, the investigators did not measure amylase concentrations in the EBC to detect direct salivary contamination, which is more likely because the condenser they used is close to the mouth during collection. Filtering the EBC is not well justified in the article and had no effect. Furthermore, exposure of the samples to argon leaves behind much of the CO2/HCO3 2 and may cause loss of other volatile acids and bases. Interpretation of EBC pH is problematical because the buffering capacity of the EBC was not measured. Condensates should not be used to collect volatile constituents such as NH3 since recovery of these solutes in the aqueous phase can be influenced by local pH, temperature, airflow, and turbulence in airways and condensers. With regard to isoprostane, EBC concentrations of isoprostane cannot be reliably used to estimate either concentrations or changes in concentrations of isoprostane in the airways unless a dilutional indicator is also measured (e.g., conductivity of lyophilized samples, which is readily measured with simple equipment) (4). Increased 8-isoprostane concentrations may reflect the production of more respiratory droplets in the airways rather than increased concentrations in the airway fluid. EBC concentrations of nonvolatiles are approximately 1% those in bronchoalveolar lavage (BAL) and 0.01% those in respiratory fluids. It is therefore puzzling that EBC 8-isoprostane levels in the normals of Do and coworkers (z10 pg/mL) are nearly the same as those reported in BAL by others (5). The EBC approach holds the promise that airway constituents may be measured noninvasively, but oral contamination with volatile solutes and extreme and variable dilution by water vapor from the airways must be experimentally addressed in all studies of this sort.

Non‐invasive biomarker sampling and analysis of the exhaled breath proteome

Exhaled breath condensate (EBC) is a biological fluid that contains trace amounts of secreted pulmonary proteins, and is emerging as a potentially valuable and non-invasively obtained source of disease biomarkers. Proteome analysis of these samples could lead to the identification of prognostic indicators of airway diseases. The objective of this study was to develop a protocol for proteome analysis of EBC samples. In this report, an improved procedure for EBC sample preparation and concentration is presented, together with a method for comparison of the protein profiles between two groups. The presented approach enabled to study the condensed exhaled breath proteome for biomarker analysis, and revealed proteins not previously identified in an EBC proteomics approach. In a comparative pilot study, EBC protein profiles obtained from smokers and non-smokers showed distinct differences and are illustrative for its potential in clinical studies. EBC from smokers contained higher concentrations of the more abundant proteins, such as cytokeratins, compared to non-smokers, and calgranulin B was identified uniquely in EBC samples from smokers.

Exhaled breath condensate—site and mechanisms of formation

Exhaled breath condensate (EBC) analysis is a promising tool for diagnosis and management of pulmonary diseases. Its clinical usefulness is still limited however due to unresolved issues around e.g. reproducibility, anatomical site of origin of EBC solutes and mechanisms of EBC formation. To gain some knowledge on these issues, three different airway deposition patterns of an aqueous aerosol containing technetium-99m were studied in eight healthy non-smoking subjects. EBC was collected 20 min after each radioaerosol administration and analyzed for gamma radiation and electrolytes. Radioaerosol deposition in preferentially central lung compared with preferentially peripheral lung resulted in 3.8 times higher EBC radioactivity. EBC concentrations of Na+ and K+ correlated significantly indicating dilution by water vapor to be a major source of variability. Since Na+/K+- and Na+/S2−-concentration ratios, but not Na+/Cl−- or Na+/Ca2+-, were comparable to those previously reported for alveolar lining fluid (ALF), some mechanisms other than dilution are likely also to be involved. In conclusion, our findings indicate that EBC derives mainly from the central airways, that the electrolyte composition of EBC does not consistently reflect that of ALF, and that EBC concentrations of electrolytes are determined not only by ALF dilution with water vapor but also by other mechanisms.