Exhaled Breath Condensate Collection Research Articles

Variability of nitric oxide metabolites in exhaled breath condensate

The collection of exhaled breath condensate (EBC) is simple and non-invasive, however, there are few data on the methodological aspects affecting concentrations of compounds in EBC. The aim of this study was to investigate methodological issues for measuring nitric oxide metabolites (NO(x)) in EBC. Twenty-five healthy adults (12 females, age range 23-55 years) and 22 children (11 females, age range 7-6 years) were recruited for studies investigating inter- and intra-day repeatability, repeatability with controlled expiratory flows and temperature, flow dependence, and analytical variability of EBC NO(x). Both intra- and inter-day repeatability was poor with a coefficient of repeatability of 103.4% of the mean difference between intra-day (15 min) measures and 118.6% of inter-day (24 h) differences. Repeatability was not improved when expiratory flow and temperature of the collection device were controlled. However, some of the variability (approximately 50%) may be accounted for by variability in the analytical technique (analytical variability) and this may result from difficulties in controlling for contamination. NO(x) levels were not affected by different expiratory flows in either adults or children but there was still significant variation within individuals. Levels of NO(x) in EBC seem to be highly variable and this needs to be considered if EBC NO(x) is to be used in clinical studies.

Adenosine level in exhaled breath increases during exercise-induced bronchoconstriction

In asthmatic patients, airway obstruction provoked by exercise challenge is accompanied by an increase in plasma adenosine level. In this study, the current authors investigated if exercise-induced bronchoconstriction was associated with local changes of adenosine concentration in the airways. Oral exhaled breath condensate (EBC) collection (5-min duration) and forced expiratory volume in one second (FEV1) measurements were performed at rest (baseline) and 4-8 times after treadmill exercise challenge in healthy and asthmatic subjects. Adenosine concentration in EBC was determined by HPLC. Observations indicated that physical exercise results in bronchoconstriction together with a significant increase of adenosine level in EBC in asthmatic patients (mean+/-sd maximal fall in FEV1 27+/-13%; associated increase in adenosine 110+/-76% as compared to baseline), but not in healthy control subjects. Exercise-induced changes in adenosine concentration correlated significantly with the fall in FEV1 values in asthmatic patients. In conclusion, the observed increase in adenosine concentration of oral exhaled breath condensate most probably reflects changes in the airways during exercise-induced bronchoconstriction. Due to its known bronchoconstrictor property in asthma, adenosine may contribute to the development of bronchospasm.

Clinical Use of Noninvasive Measurements of Airway Inflammation in Steroid Reduction in Children

The use of noninvasive methods of monitoring airway inflammation, such as exhaled nitric oxide (eNO) and induced sputum, has been shown to improve asthma monitoring and optimize treatment in adult patients with asthma. There is a lack of comparable data in children. Forty children with stable asthma eligible for inhaled steroid reduction were reviewed every 8 weeks, and their inhaled steroid dose halved if clinically indicated. eNO, sputum induction combined with bronchial hyperreactivity testing, and exhaled breath condensate collection were performed at each visit to predict success or failure of reduction of inhaled steroids. Thirty of 40 (75%) children tolerated at least one dose reduction, 12 of 40 (30%) were successfully weaned off, and in total, 15 of 40 (38%) children experienced loss of asthma control. Treatment reduction was successful in all children who had no eosinophils in induced sputum before the attempted reduction. Using multiple logistic regression, increased eNO (odds ratio, 6.3; confidence interval, 3.75-10.58) and percentage of sputum eosinophils (odds ratio, 1.38; confidence interval, 1.06-1.81) were significant predictors of failed reduction. These findings suggest that monitoring airway inflammation may be useful in optimizing treatment in children with asthma.

Collection of exhaled breath condensate and analysis of hydrogen peroxide as a potential marker of lower airway inflammation in cats

The objective of this study was to describe a standardised and non-invasive method for exhaled breath condensate (EBC) collection in cats and to test whether determination of hydrogen peroxide (H 2O 2) in EBC might be used as marker of lower airway inflammation. The technique of barometric whole body plethysmography for cats was combined with a system to condense the effluent air from the plethysmograph, allowing simultaneous EBC collection and respiratory pattern measurement. H 2O 2 was determined spectrophotometrically. Eighteen experimental cats were used to investigate the impact on EBC volume and EBC H 2O 2 of plethysmograph ventilation rate, collection duration, sample stability, within-day and day-to-day variability. After determination of a standardised EBC collection procedure, correlation analyses between EBC H 2O 2 and bronchoalveolar lavage (BAL) cytology of healthy and allergen-challenged Ascaris suum (AS)-sensitised cats were performed. A significant and positive correlation between EBC H 2O 2 and bronchoalveolar lavage (BAL) neutrophil% was found in healthy cats ( P<0.001, r=0.55), whereas in AS-sensitised cats, correlation with BAL eosinophil% was significant ( P<0.005, r=0.61). H 2O 2 was increased after an allergen challenge in AS-sensitised cats ( n=6, 0.56 ± 0.12 versus 1.08 ± 0.35 μmol/L, P<0.05). This study proposes a non-invasive, well tolerated and repeatable method of EBC collection for cats and suggests that EBC H 2O 2 might be used as non-invasive biomarker for monitoring lower airway inflammation.

Expired diagnosis?--the potential of exhaled breath analysis

Analysis of breath condensate is an innovative approach to biochemical information from the lung. It provides a new tool to estimate and characterize the burden of oxidative and inflammatory processes in the airways/lung. Clinical applications in asthma, COPD and CF can be envisioned as well as determining organ-specific inflammation in mechanically ventilated patients or monitoring patients with transplanted lungs. However, besides inflammation other important areas have begun to be evaluated, such as the demonstration of p53 mutations in NSCLC patients or an increased ratio of EBC nitrite to tidal volume in mechanically stressed lungs. Of course a careful validation of each and every parameter is paramount to the use in clinical applications. The comparison to BAL is oftentimes called for but at the same time is not the comparison to a gold standard because of the well-known problems of BAL. The scope of this review is a summary of facts and theories concerning exhaled breath condensate generation, collection and analysis but at the same time the representation of the many aspects that remain to be resolved.

Evaluation of exhaled nitric oxide and nitric oxide metabolites in exhaled breath condensate during asthma

Rationale The collection of exhaled breath condensate (EBC) represents a potentially important non-invasive diagnostic tool to sample non-volatile substances from the lower airways. The purpose of the present study was to evaluate whether there exists a relationship between exhaled nitric oxide (ENO) and NO metabolites in EBC in asthmatic patients. Methods We measured ENO (off-line and on-line) as well as nitrite (NO 2 −) and nitrate (NO 3 −) concentrations in exhaled breath condensates (EBC) from 10 normal individuals, 11 steroid-naïve, and 8 steroid-treated asthmatic patients. In all subjects, EBC was collected during tidal breathing using the Jaeger Ecoscreen system and nitrite/nitrate concentrations in EBC were measured using a chemiluminescence detector. Results ENO was elevated in steroid naïve asthmatic patients compared to healthy individuals (32 ± 23 vs 10 ± 6 ppb) and it was statistically significantly reduced in steroid treated individuals (11 ± 5 ppb, p<0.05). Nitrate but not nitrite concentrations showed an increase in steroid naïve and a reversal in steroid treated patients. Consistent with these findings, there was a positive linear correlation between ENO and nitrate but not nitrite. Conclusions Our results suggest that determination of nitrate but not nitrite might represent a relevant marker in asthma. In addition, there was a trend in increased interleukin-8 and decreased interferon-γ levels in steroid naïve asthmatic individuals. Overall, the utilization of EBC solutes as relevant diagnostic markers will rely on the rigorous standardization of sampling procedures.

Exhaled breath condensate in children: pearls and pitfalls.

Exhaled breath condensate (EBC) is a rapidly growing field of research in respiratory medicine. Airway inflammation is a central feature of chronic lung diseases, like asthma, cystic fibrosis, bronchopulmonary dysplasia and primary ciliary dyskinesia. EBC may be a useful technique for non-invasive assessment of markers of airway inflammation. The non-invasive character of EBC "inflammometry" and the general lack of appropriate techniques makes it particularly interesting for paediatrics. We provide a detailed update on the methods currently used for EBC collection and measurement of mediators. We emphasize on paediatric data. The apparent simplicity of the EBC method must not be overstated, as numerous methodological pitfalls have yet to overcome. Comparison and interpretation of data on this rapidly growing field of research is mainly hampered by the lack of standardization and the lack of specific high-sensitivity immunochemical or colorimetric assays. The initiative of the European Respiratory Society to institute a task force on this topic is a first step towards a uniform technique of EBC. Meanwhile, when using this technique or when interpreting research data, one should be fully aware of the possible methodological pitfalls.

Passive smoking does not increase hydrogen peroxide (H2O2) levels in exhaled breath condensate in 9-year-old healthy children

Environmental tobacco smoke, also called passive smoking, was shown to have adverse effects on the health of children. Hydrogen peroxide (H2O2) is proposed as a sensitive marker of oxidative injury and inflammatory processes in the airways, being increased in adult active cigarette smokers. We tested whether passive smoking had an influence on H2O2 exhalation in healthy children. Thirty healthy passive smoking and 24 nonexposed healthy children aged 9 years were included in the study. Exhaled breath condensate (EBC) was obtained by spontaneous tidal volume breathing with EcoScreen (Jaeger, Germany). All subjects underwent flow-volume measurements immediately after EBC collection. Levels of H2O2 were measured fluorimetrically with the homovanillic acid method. Lung function did not differ between the passive smoking and nonexposed children groups. In the passive smoking group, EBC H2O2 concentration (median and range) was 0.32 (0.00-1.20) microM, and did not differ significantly (P >0.05) from that found in the nonexposed group, i.e., 0,22 (0.00-0.68) microM. Exhaled H2O2 did not correlate with spirometric parameters (FEV1, FEV1%FVC, and MEF50%FVC) in either group. We conclude that passive smoking does not increase H2O2 exhalation in healthy children.

Markers of Inflammation in Exhaled Breath Condensate of Young Healthy Smokers

Although a strong correlation exists between long-term cigarette smoking, pulmonary inflammation, and COPD, efforts to identify populations at risk of acquiring COPD have so far been unsuccessful. To this end, noninvasive detection and monitoring of biomarkers of pulmonary inflammation in young healthy smokers may assist in this task. The purpose of this study was to determine the concentrations of total protein, nitrites, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha, and neutrophil chemotactic activity in exhaled breath condensate (EBC) collected from healthy college student smokers and nonsmokers. EBC was collected from 20 volunteers (9 nonsmokers and 11 smokers) during tidal breathing for 20 min. EBC was also collected from smokers 30 min after smoking one filtered cigarette. The concentrations of total protein, nitrite, IL-1beta, and TNF-alpha in EBC was determined by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity in EBC was determined in vitro using the blind-well technique. The concentrations of total protein and nitrite, and neutrophil chemotactic activity were significantly higher in EBC of smokers in comparison to nonsmokers (p < 0.05). The concentrations of total protein and nitrite in the condensate of smokers did not change significantly after smoking one cigarette. The concentrations of IL-1beta and TNF-alpha in EBC were similar in nonsmokers and smokers. Concentrations of certain inflammatory mediators and neutrophil chemotactic activity are increased in EBC of young healthy smokers. Collection and analysis of EBC may assist in early detection of cigarette smoke-induced pulmonary inflammation and identifying populations at risk for acquiring COPD.

Assessment of exhaled gases in ventilated preterm infants

Hydrogen peroxide (H2O2) production in exhaled air was measured in ventilated preterm newborns at 5, 24 and 48 hours after delivery, using originally designed method of exhaled breath condensate (EBC) collection. H2O2 production in expired gas was 812+/-34 pmol/20 min during the first measurement and then declined to 389+/-21 at 24 hours and 259+/-26 pmol/20 min at 48 hours.

ReviewBiomarkers in Breath Condensate: A promising New Non-invasive Technique in Free Radical Research

Oxidative stress is associated with a range of inflammatory lung diseases including asthma, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, lung transplantation, chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis and lung cancer. Increased concentrations of reactive oxygen species (ROS) in the airways of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples, or examine the compartments. As a consequence, there is a need for less invasive approaches to measure oxidative stress. Analysis of breath hydrocarbons has partly fulfilled this need, however only gas phase volatile constituents can be assessed by this approach. The collection of exhaled breath condensate (EBC) is a simple, non-invasive approach, which comprehensively samples the lower respiratory tract. It is currently used as a research and diagnostic tool in the free radical field, yielding information on redox disturbance and the degree and type of inflammation in the lung. With further technical developments, such an approach may ultimately have a role in the clinic, in helping to diagnose specific lung diseases. EBC can be exploited to assess a spectrum of potential biomarkers, thus generating a “finger print” characteristic of the disease. By assessing the nature of oxidative stress in this manner, the most appropriate therapy can be selected and the response to treatment monitored.

Increased Exhaled 8-Isoprostane in Childhood Asthma

To quantify lung oxidative stress in asthmatic children by measuring concentrations of 8-isoprostane, a marker of oxidative stress, in exhaled breath condensate (EBC), which is a noninvasive method of sampling airway secretions. Secondary objectives were as follows: (1) to measure levels of exhaled prostaglandin (PG) E(2), since impaired PGE(2) production has been implicated in the pathogenesis of asthma in adults; and (2) to measure levels of fractional exhaled nitric oxide (FeNO), which is a marker of airway inflammation. Single-center, cross-sectional study. Twelve healthy children, 12 steroid-naïve asthmatic children, and 30 children in stable condition with mild-to-moderate persistent asthma who were being treated with inhaled corticosteroids (ICSs) [average dose, 300 micro g per day] were studied. Subjects attended the outpatient clinic on one occasion for the collection of EBC and FeNO measurements. 8-Isoprostane and PGE(2) concentrations in EBC were measured with specific radioimmunoassays. FeNO was measured online by a chemiluminescence analyzer. 8-Isoprostane was detectable in the EBC of healthy children (mean [+/- SEM], 34.2 +/- 4.5 pg/mL), and its concentrations were increased in both steroid-naïve asthmatic children (mean, 56.4 +/- 7.7 pg/mL; p < 0.01) and steroid-treated asthmatic children (mean, 47.2 +/- 2.3 pg/mL; p < 0.05). There was no difference in exhaled 8-isoprostane concentrations between the two groups of asthmatic children (p = 0.14). By contrast, exhaled PGE(2) concentrations were similar among the three study groups (p = 0.56). FeNO levels were higher in steroid-naïve children with asthma (49.2 +/- 9.6 parts per billion [ppb]; p < 0.05) and, to a lesser extent, in steroid-treated asthmatic children (37.8 +/- 6.6 ppb; p < 0.05) compared with healthy children (15.2 +/- 1.7 ppb). Lung oxidative stress is increased in children who are in stable condition with asthma, as reflected by increased exhaled 8-isoprostane concentrations. This increase seems to be relatively resistant to treatment with ICSs. Decreased PGE(2) lung production is unlikely to play a pathophysiologic role in childhood asthma.

Safety and success of exhaled breath condensate collection in asthma

Background: Exhaled breath condensate (EBC) is a rapidly expanding area of research to study airway inflammation through the detection of volatile and non-volatile substances in the airways. Aims: To determine...

Validation of leukotriene B4 measurements in exhaled breath condensate.

To qualitatively validate an enzyme immunoassay to measure leukotriene B4 in exhaled breath condensate. Exhaled breath condensate is a new non-invasive method to monitor airway inflammation. Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. Samples were pooled together and purified by reverse-phase high-performance liquid chromatography. The fractions eluted were assayed for leukotriene B4 by enzyme immunoassay. A single peak of leukotriene B4-like immunoreactivity co-eluting with leukotriene B4 standard (retention time: 24 min) was identified by enzyme immunoassay. Reverse phase-high performance liquid chromatography peak of leukotriene B4 was clearly separated from those of 6-trans-leukotriene B4 (retention time: 14 min) and leukotriene B5 (retention time: 18 min) for which the antiserum used in the enzyme immunoassay had the highest cross-reactivity. Leukotriene B4 recovery was 64%. This study provides evidence for the presence of leukotriene B4 in the exhaled breath condensate and the specificity of the enzyme immunoassay used.

Quantitative analysis of 8-isoprostane and hydrogen peroxide in exhaled breath condensate.

Exhaled breath condensate (EBC) provides a noninvasive means of sampling the lower respiratory tract. Collection of EBC might be useful in the assessment of airway oxidative stress in smokers. The aim of this study was to determine 8-isoprostane and hydrogen peroxide levels in EBC, and, in addition, to investigate the reproducibility of these measurements. EBC samples were collected from 12 healthy male smokers at three time points within 1 week. 8-isoprostane and H2O2 were measured in nonconcentrated EBC using immunochemical and colorimetric assays, respectively. 8-isoprostane and H2O2 were detected in only 36 and 47% of all EBC samples, respectively. It was not possible to calculate the within-subject variation in a reliable manner since only three of the 12 smokers exhibited detectable 8-isoprostane concentrations on all three occasions (mean 4.6 pg x mL(-1); range 3.9-7.7 pg x mL(-1)), whereas H2O2 could not be detected on all three occasions in any of the smokers. Spiking experiments revealed a recovery of 83.5-109.5% for 8-isoprostane and 69.9-129.0%, for H2O2 in fresh EBC samples. It was concluded that levels of 8-isoprostane and hydrogen peroxide cannot be reproducibly assessed in exhaled breath condensate from healthy smokers because of their low concentration and/or the lack of sensitivity of the available assays.

HYPOXIA AND REOXYGENATION INCREASE H 2 O 2 PRODUCTION IN RATS

To test the effect of transition from sustained hypoxia to normoxia on production of reactive oxygen species (ROS) in lungs, the authors measured hydrogen peroxide (H 2 O 2) output in the expired air of rats breathing hypoxic, normoxic, and hyperoxic gas mixtures at the end of exposure to 72 hours of hypoxia. Twenty-one male Wistar rats (200 to 280 g) were randomly assigned to 1 of 3 groups. First two groups (experimental) were kept for 3 days in normobaric hypoxic chamber (F 1 O 2 0.1), rats of the third group (controls) breathed air. The rats were then anesthetized, intubated, placed in the plethysmograph, and their ventilation measured. Two periods of exhaled breath condensate (EBC) collection, each lasting 1 hour, were then performed to assay H 2 O 2 output. The controls breathed during both samplings air, the first experimental group breathed during first sampling period hypoxic mixture (F 1 O 2 0.1; SH-H measurement) and then, during second period, air (SH-H-A measurement), the second experimental group breathed first air (SH-A measurement) and then hyperoxic mixture (F 1 O 2 1.0; SH-A-O 2 measurement). Concentration of H 2 O 2 in the EBC was assayed by chemiluminescence. H 2 O 2 production in the control group was low and similar in both measurements (20 ± 10 and 13 ± 5 pmol/h, mean ± SEM). Exposure to 72 hours of hypoxia increased the H 2 O 2 production to 105 ± 18 pmol/h (SH-H). Transition from hypoxia to normoxia resulted in an increase in the H 2 O 2 production (SH-A 421 ± 24 pmol/h, and SH-H-A 366 ± 19 pmol/h). Following transition from air breathing to hyperoxia did not affect the H 2 O 2 production (SH-A-O 2 373 ± 25 pmol/h). The results showed that sustained hypoxia and transition from sustained hypoxia to normoxia increased H 2 O 2 formation in the lungs.

Exhaled leukotrienes and prostaglandins in asthma

Background: Most of the studies investigating the role of leukotrienes (LTs) and prostaglandins (PGs) in asthma have used invasive (eg, bronchoalveolar lavage fluid) or semi-invasive (eg, sputum induction) techniques. Others have measured eicosanoids in plasma or urine, probably reflecting systemic rather than lung inflammation. Collection of exhaled breath condensate (EBC) is a noninvasive method to collect airway secretions. Objective: We sought to investigate whether eicosanoids are measurable in EBC, to show possible differences in their concentrations in asthmatic patients and healthy subjects, and to investigate whether exhaled eicosanoids correlate with exhaled nitric oxide (NO), a marker of airway inflammation. Methods: Twelve healthy nonsmokers and 15 steroid-naive patients with mild asthma were studied. Subjects attended on one occasion for pulmonary function tests, collection of EBC, and exhaled NO measurements. Exhaled LTB4-like immunoreactivity, LTE4-like immunoreactivity, PGE2-like immunoreactivity, PGD2-methoxime, PGF2α-like immunoreactivity, and thromboxane B2-like immunoreactivity were measured by means of enzyme immunoassays. Results: LTE4-like immunoreactivity and LTB4-like immunoreactivity were detectable in EBC in healthy subjects, and their levels in asthmatic patients were increased about 3-fold (P < .0001) and 2-fold (P < .0005), respectively. Exhaled NO was increased in asthmatic patients compared with healthy subjects (P < .0001). There was a correlation between exhaled LTB4 and exhaled NO (r = 0.56, P < .04) in patients with asthma. When measurable, prostanoid levels were similar in asthmatic patients and control subjects. Conclusions: Exhaled LTE4 and LTB4 are increased in steroid-naive patients with mild asthma. EBC may be proved to be a novel method to monitor airway inflammation in asthma. (J Allergy Clin Immunol 2002;109:615-20.)

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Collection and analysis of exhaled breath condensate in humans.

Traditional methods of sampling secretions from the lower respiratory tract include sputum collection, sputum induction, and bronchoscopy with bronchoalveolar lavage (BAL). By far, bronchoscopy with BAL has become the preferred method for sampling the lining fluid of the lower respiratory tract. The invasive nature of bronchoscopy has led to a constant search for less intrusive methods that are easier to implement in ambulant individuals, particularly children. Recently there has been an increasing interest in using exhaled breath as a simple, noninvasive means to sample the lower respiratory tract in humans. The lining fluid of the lower respiratory tract contains various nonvolatile and over 200 volatile substances (1, 2). Although initial attempts were aimed at identifying volatile substances, particularly nitric oxide (NO), studies are now being conducted to detect nonvolatile macromolecules present in exhaled breath, including proteins, lipids, oxidants, and nucleotides. Analysis of these nonvolatile substances requires cooling of the expired breath, which results in condensation. These macromolecules represent biomarkers of various pathological processes in the lungs. In this review, we present the current body of knowledge on exhaled breath condensate as published in peer-reviewed journals in the English language. We focus on the use of this modality in determining host inflammatory response to injury in the lung as well as possible future applications, particularly its potential use as a single, noninvasive sampling method for point-of-care real-time analysis.

Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke

BACKGROUNDCigarette smoking reduces the level of exhaled nitric oxide (NO) in healthy subjects, although the mechanism is unclear. NO is a highly reactive molecule which can be oxidised or complexed...