Breath biomarkers for detection of human liver diseases: preliminary study

Chronic liver disease is initially occult, has multiple aetiologies, involves complex diagnostic questions, and requires follow-up because progression is likely. Blood tests and biopsies are generally used, but have disadvantages. We have developed a new test for liver disease based on abnormal concentrations of metabolic products detected in exhaled breath. This test can be used, in conjunction with other clinically accepted diagnostic protocols, to detect and classify chronic liver diseases. Samples of breath collected from spontaneously breathing human subjects (86 patients presenting with 13 liver diseases and 109 subjects with normal liver function) were concentrated cryogenically and analysed by wide-bore capillary gas chromatography using various detectors. The concentrations of various molecules in exhaled breath were examined for potential use as biomarkers of liver function. Subjects with chronic liver diseases could be differentiated from those with normal liver function by comparing levels of breath carbonyl sulphide, carbon disulphide and isoprene; these differences were confirmed and correlated by comparing the levels with standard clinical blood markers of liver damage. The presence of chronic liver failure can thus be detected with sensitivity and specificity by quantifying sulphur-containing compounds arising from the abnormal metabolism associated with liver disease. The breath test we have developed appears to distinguish between hepatocellular and biliary tract aetiologies, and allows staging for severity. This approach may provide the clinician with a simple, non-invasive technique for use in the screening of large populations and follow-up for patients with chronic liver disease.     

Biomarkers of some pulmonary diseases in exhaled breath

Analysis of various biomarkers in exhaled breath allows completely non-invasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchiectasis and interstitial lung diseases. The technique is simple to perform, may be repeated frequently, and can be applied to children, including neonates, and patients with severe disease in whom more invasive procedures are not possible. Several volatile chemicals can be measured in the breath (nitric oxide, carbon monoxide, ammonia), and many non-volatile molecules (mediators, oxidation and nitration products, proteins) may be measured in exhaled breath condensate. Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in differential diagnosis of airway disease and in the monitoring of therapy. Most progress has been made with exhaled nitric oxide (NO), which is increased in atopic asthma, is correlated with other inflammatory indices and is reduced by treatment with corticosteroids and antileukotrienes, but not (β₂-agonists. In contrast, exhaled NO is normal in COPD, reduced in CF and diagnostically low in primary ciliary dyskinesia. Exhaled carbon monoxide (CO) is increased in asthma, COPD and CF. Increased concentrations of 8-isoprostane, hydrogen peroxide, nitrite and 3-nitrotyrosine are found in exhaled breath condensate in inflammatory lung diseases. Furthermore, increased levels of lipid mediators are found in these diseases, with a differential pattern depending on the nature of the disease process. In the future it is likely that smaller and more sensitive analysers will extend the discriminatory value of exhaled breath analysis and that these techniques may be available to diagnose and monitor respiratory diseases in the general practice and home setting.     

Effect of alcoholic cirrhosis on ethane and pentane levels in breath

Lipid peroxidation can be monitored by measuring one or several highly volatile alkanes in exhaled air. The concentrations of ethane and pentane were determined in breath samples from patients with alcoholic and non-alcoholic cirrhosis as well as from healthy subjects. The greatest increase of exhaled pentane was found in 17 patients with alcoholic cirrhosis (2.85 +/- 2.37 pmol/ml) in comparison with 10 patients with non-alcoholic cirrhosis (0.71 +/- 0.33 pmol/ml) and 10 control subjects (0.59 +/- 0.41 pmol/ml). On the contrary, no significant difference was detected as far as exhaled ethane is concerned. These data suggest that: a) gas-chromatographic determination of exhaled pentane may play a significant role in detecting alcohol-induced liver disease; b) hepatic injury may be mediated by lipid peroxidation in these patients.     

Nitric oxide in the breath of bottlenose dolphins: Effects of breath hold duration,feeding, and lung disease

Breath analysis, including measurement of nitric oxide (NO), is a noninvasive diagnostic tool that may help evaluate cetacean health. This is the first report on the effects of breath hold duration, feeding, and lung disease on NO in dolphin exhaled breath. Three healthy dolphins were trained to hold their breath for 30, 60, 90, and 120 s and then exhale into an underwater funnel. Exhaled NO values from 157 breath samples were compared among three healthy dolphins by breath hold time and after fasting and feeding. Exhaled NO values were also measured in two dolphins with pulmonary disease. NO in dolphin breath was higher compared to ambient air; healthy dolphins had higher NO concentrations in their breath after feeding compared to after overnight fasting; and there were no significant differences in exhaled NO levels by breath hold duration. A dolphin with Mycoplasma‐associated pneumonia and chronic gastrointestinal disease had higher postprandial exhaled NO levels compared to healthy controls. This study demonstrates, contrary to previous publications, that dolphins exhale NO. Given the high standard deviations present in exhaled breath NO values, future studies are needed to further standardize collection methods or identify more reliable samples (e.g., blood).     

Abnormal exhaled ethane concentrations in scleroderma.

Scleroderma (systemic sclerosis) is a chronic multisystem autoimmune disease in which oxidative stress is suspected to play a role in the pathophysiology. Therefore, it was postulated that patients with scleroderma would have abnormally high breath ethane concentrations, which is a volatile product of free-radical-mediated lipid peroxidation, compared with a group of controls. There was a significant difference (p<0.05) between the mean exhaled ethane concentration of 5.27 pmol ml(-1) CO(2) (SEM=0.76) in the scleroderma patients (n=36) versus the mean exhaled concentration of 2.72 pmol ml(-1) CO(2) (SEM=0.71) in a group of healthy controls (n=21). Within the scleroderma group, those subjects taking a calcium channel blocker had lower ethane concentrations compared with patients who were not taking these drugs (p=0.05). There was a significant inverse association between lung diffusion capacity for carbon monoxide (per cent of predicted) and ethane concentration (b=-2.8, p=0.026, CI=-5.2 to -0.35). These data support the presence of increased oxidative stress among patients with scleroderma that is detected by measuring breath ethane concentrations.     

Influenza virus in human exhaled breath: an observational study

Background

Recent studies suggest that humans exhale fine particles during tidal breathing but little is known of their composition, particularly during infection.

Methodology/Principal Findings

We conducted a study of influenza infected patients to characterize influenza virus and particle concentrations in their exhaled breath. Patients presenting with influenza-like-illness, confirmed influenza A or B virus by rapid test, and onset within 3 days were recruited at three clinics in Hong Kong, China. We collected exhaled breath from each subject onto Teflon filters and measured exhaled particle concentrations using an optical particle counter. Filters were analyzed for influenza A and B viruses by quantitative polymerase chain reaction (qPCR). Twelve out of thirteen rapid test positive patients provided exhaled breath filter samples (7 subjects infected with influenza B virus and 5 subjects infected with influenza A virus). We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects–three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. Exhaled influenza virus RNA generation rates ranged from <3.2 to 20 influenza virus RNA particles per minute. Over 87% of particles exhaled were under 1 µm in diameter.

Conclusions

These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.     

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.     

Biomarkers 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.     

Exhaled volatile organic compounds for phenotyping chronic obstructive pulmonary disease: a cross-sectional study

Background

Non-invasive phenotyping of chronic respiratory diseases would be highly beneficial in the personalised medicine of the future. Volatile organic compounds can be measured in the exhaled breath and may be produced or altered by disease processes. We investigated whether distinct patterns of these compounds were present in chronic obstructive pulmonary disease (COPD) and clinically relevant disease phenotypes.

Methods

Breath samples from 39 COPD subjects and 32 healthy controls were collected and analysed using gas chromatography time-of-flight mass spectrometry. Subjects with COPD also underwent sputum induction. Discriminatory compounds were identified by univariate logistic regression followed by multivariate analysis: 1. principal component analysis; 2. multivariate logistic regression; 3. receiver operating characteristic (ROC) analysis.

Results

Comparing COPD versus healthy controls, principal component analysis clustered the 20 best-discriminating compounds into four components explaining 71% of the variance. Multivariate logistic regression constructed an optimised model using two components with an accuracy of 69%. The model had 85% sensitivity, 50% specificity and ROC area under the curve of 0.74. Analysis of COPD subgroups showed the method could classify COPD subjects with far greater accuracy. Models were constructed which classified subjects with ≥2% sputum eosinophilia with ROC area under the curve of 0.94 and those having frequent exacerbations 0.95. Potential biomarkers correlated to clinical variables were identified in each subgroup.

Conclusion

The exhaled breath volatile organic compound profile discriminated between COPD and healthy controls and identified clinically relevant COPD subgroups. If these findings are validated in prospective cohorts, they may have diagnostic and management value in this disease.     

Metallic elements in exhaled breath condensate and serum of patients with exacerbation of chronic obstructive pulmonary disease

Biomarkers in exacerbated chronic obstructive pulmonary disease may be useful in aiding diagnosis, defining specific phenotypes of disease, monitoring the disease and evaluating the effects of drugs. The aim of this study was the characterization of metallic elements in exhaled breath condensate and serum as novel biomarkers of exposure and susceptibility in exacerbated chronic obstructive pulmonary disease using reference analytical techniques. C-Reactive protein and procalcitonin were assessed as previously validated diagnostic and prognostic biomarkers which have been associated with disease exacerbation, thus useful as a basis of comparison with metal levels. Exhaled breath condensate and serum were obtained in 28 patients at the beginning of an episode of disease exacerbation and when they recovered. Trace elements and toxic metals were measured by inductively coupled plasma-mass spectrometry. Serum biomarkers were measured by immunoassay. Exhaled manganese and magnesium levels were influenced by exacerbation of chronic obstructive pulmonary disease, an increase in their concentrations--respectively by 20 and 50%--being observed at exacerbation in comparison with values obtained at recovery; serum elemental composition was not modified by exacerbation; serum levels of C-reactive protein and procalcitonin at exacerbation were higher than values at recovery. In outpatients who experienced a mild-moderate chronic obstructive pulmonary disease exacerbation, manganese and magnesium levels in exhaled breath condensate are elevated at admission in comparison with values at recovery, whereas no other changes were observed in metallic elements at both the pulmonary and systemic level.     

呼吸中的疾病诊断标志物及检测技术

人类呼出气体中的各种化合物能提供各种疾病和健康状况的重要信息。近年来,由于红外、电化学、化学发光等新技术的重大突破和质谱仪的使用,使得在极低浓度下精确测量呼出的挥发性有机化合物(VOCs)和气溶胶颗粒成为可能,呼吸检测领域因而取得了重大进展:,呼吸检测因其可以作为一种实时、快速和无创的方法来评估和监测各种疾病与健康状况信息,在科学研究、临床运用中引起了广泛关注。本综述主要概述呼出气体成分分析方法及在疾病诊断中的研究与应用情况,旨在为将来疾病的实时、快速和无创诊断提供一种新的策略.     

Molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 μL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses.Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 μL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5-1.0 μm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.     

Non-invasive diagnosis of liver diseases by breath analysis using an optimized ion–molecule reaction-mass spectrometry approach: a pilot study

Breath composition is altered in liver diseases. We tested if ion–molecule-reaction mass spectrometry (IMR-MS) combined with a new statistical modality improves the diagnostic accuracy of breath analysis in liver diseases. We analysed 114 molecules in the breath of 126 individuals (healthy controls, and patients with non-alcoholic and alcoholic fatty liver disease and liver cirrhosis) by IMR-MS. Characteristic exhalation patterns were identified for each group. Combining two to seven molecules in the new stacked feature ranking model reached a diagnostic accuracy (area under the curve) for individual liver diseases between 0.88 and 0.97. IMR-MS followed by sophisticated statistical analysis is a promising tool for liver diagnostics by breath analysis.     

Abnormal exhaled ethane concentrations in scleroderma

Abstract

Scleroderma (systemic sclerosis) is a chronic multisystem autoimmune disease in which oxidative stress is suspected to play a role in the pathophysiology. Therefore, it was postulated that patients with scleroderma would have abnormally high breath ethane concentrations, which is a volatile product of free-radical-mediated lipid peroxidation, compared with a group of controls. There was a significant difference (p<0.05) between the mean exhaled ethane concentration of 5.27 pmol ml^–1 CO₂ (SEM=0.76) in the scleroderma patients (n=36) versus the mean exhaled concentration of 2.72 pmol ml^?1 CO₂ (SEM=0.71) in a group of healthy controls (n=21). Within the scleroderma group, those subjects taking a calcium channel blocker had lower ethane concentrations compared with patients who were not taking these drugs (p=0.05). There was a significant inverse association between lung diffusion capacity for carbon monoxide (per cent of predicted) and ethane concentration (b=?2.8, p=0.026, CI=?5.2 to ?0.35). These data support the presence of increased oxidative stress among patients with scleroderma that is detected by measuring breath ethane concentrations.     

Exhaled breath condensate as matrix for toluene detection: A preliminary study

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 µg l^-1 (range 0.15-0.55 µg l^-1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.     

Exhaled Breath Analysis Using Electronic Nose in Cystic Fibrosis and Primary Ciliary Dyskinesia Patients with Chronic Pulmonary Infections

The current diagnostic work-up and monitoring of pulmonary infections may be perceived as invasive, is time consuming and expensive. In this explorative study, we investigated whether or not a non-invasive exhaled breath analysis using an electronic nose would discriminate between cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) with or without various well characterized chronic pulmonary infections. We recruited 64 patients with CF and 21 with PCD based on known chronic infection status. 21 healthy volunteers served as controls. An electronic nose was employed to analyze exhaled breath samples. Principal component reduction and discriminant analysis were used to construct internally cross-validated receiver operator characteristic (ROC) curves. Breath profiles of CF and PCD patients differed significantly from healthy controls p = 0.001 and p = 0.005, respectively. Profiles of CF patients having a chronic P. aeruginosa infection differed significantly from to non-chronically infected CF patients p = 0.044. We confirmed the previously established discriminative power of exhaled breath analysis in separation between healthy subjects and patients with CF or PCD. Furthermore, this method significantly discriminates CF patients suffering from a chronic pulmonary P. aeruginosa (PA) infection from CF patients without a chronic pulmonary infection. Further studies are needed for verification and to investigate the role of electronic nose technology in the very early diagnostic workup of pulmonary infections before the establishment of a chronic infection.     

Exhaled breath condensate as matrix for toluene detection: A preliminary study

Abstract

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 µg l^?1 (range 0.15–0.55 µg l^?1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.     

Fractionated breath condensate sampling: H2O2 concentrations of the alveolar fraction may be related to asthma control in children

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Low levels of nitric oxide and carbon monoxide in alpha 1-antitrypsin deficiency.

Quantitations of exhaled nitric oxide (NO) and carbon monoxide (CO) have been proposed as noninvasive markers of airway inflammation. We hypothesized that exhaled CO is increased in individuals with alpha(1)-antitrypsin (AT) deficiency, who have lung inflammation and injury related to oxidative and proteolytic processes. Nineteen individuals with alpha(1)-AT deficiency, 22 healthy controls, and 12 patients with non-alpha(1)-AT-deficient chronic obstructive pulmonary disease (COPD) had NO, CO, CO(2), and O(2) measured in exhaled breath. Individuals with alpha(1)-AT deficiency had lower levels of NO and CO than control or COPD individuals. Alpha(1)-AT-deficient and COPD patients had lower exhaled CO(2) than controls, although only alpha(1)-AT-deficient patients had higher exhaled O(2) than healthy controls. NO was correlated inversely with exhaled O(2) and directly with exhaled CO(2), supporting a role for NO in regulation of gas exchange. Exhaled gases were not significantly related to corticosteroid use or lung function. Demonstration of lower than normal CO and NO levels may be useful as an additional noninvasive method to evaluate alpha(1)-AT deficiency in individuals with a severe, early onset of obstructive lung disease.     

Non-invasive biomarkers of oxidative stress: reproducibility and methodological issues

Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs 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 to examine the affected compartments, to the patient's discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H2O2, F2-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.