European Respiratory Society
Exhaled Biomarkers

Exhaled air contains numerous substances, often in extremely low concentrations. The development of sensitive detection techniques has made it possible to examine the composition of exhaled air in relation to a variety of airway diseases and other disorders. In this book, an overview of current cutting-edge breath analysis techniques and their clinical applications is provided for the clinician. The various contributions give a fascinating perspective of a future where new, highly sensitive methodologies will enable clinicians to diagnose and monitor a wide variety of diseases merely by taking the patient's breath.

  • European Respiratory Society Monographs
  1. Page v
  2. Page vi
  3. Page vii
  4. Page 1
    Correspondence: K. Alving, Dept of Women's and Children's Health, Uppsala University Hospital, SE-751 85, Uppsala, Sweden. Email:

    Nitric oxide (NO) in orally exhaled air mainly originates from the respiratory epithelium. NO is produced by inducible NO synthase (iNOS), which is regulated by signal transducer and activator of transcription (STAT)-1 under the influence of homeostatic interferon-γ. In patients with asthma, iNOS expression is upregulated by interleukin (IL)-4 and IL-13 via the activation of STAT-6 in the bronchial epithelium. Thus, exhaled NO primarily signals local T-helper cell type 2-driven inflammation in the bronchial mucosa. With these characteristics, exhaled NO will be a suitable marker for predicting the response to inhaled corticosteroids, and to monitor the anti-inflammatory effect.

    The methodology for measuring exhaled NO has been standardised based on international consensus. The determinants of exhaled NO levels are fairly well characterised, with the most important being cigarette smoking, nitrate intake, air pollution, allergen sensitisation and exposure, along with height, sex and age. A future development may be the estimation of peripheral airway inflammation by measuring exhaled NO at multiple exhalation flow rates.

  5. Page 32
    Correspondence: D.E. Shaw, Respiratory Biomedical Research Unit, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK. Email:

    Exhaled nitric oxide fraction (FeNO) is an easy to measure, noninvasive surrogate measure of airway inflammation which provides an immediate result and is particularly well suited to repeated measurement in the clinic, especially for young children. FeNO is increased in asthma compared with normal controls and is reduced after taking inhaled corticosteroids and other anti-inflammatory treatment. However, measurements of FeNO have a complex relationship with differential sputum eosinophil counts, and are affected by several confounding factors; therefore its role is not yet clear in the management of asthma. FeNO measurements may provide information on pathological processes, and response to treatment, within the distal lung.

  6. Page 45
    Correspondence: M.W.H. Pijnenburg, Dept of Paediatrics/Paediatric Respiratory Medicine, Erasmus MC - Sophia Children's Hospital, PO Box 2060, 3000 CB, Rotterdam, The Netherlands, Email:

    Although the use of exhaled nitric oxide (exhaled nitric oxide fraction; FeNO) in the management of asthma is well accepted nowadays, the use of FeNO in respiratory disorders other than asthma is much more controversial.

    In chronic obstructive pulmonary disease (COPD), FeNO may be of help in discriminating COPD from asthma and, to some extent, in predicting the response to steroids. In this regard, measurements of alveolar nitric oxide seem more promising; however, data on the value of these measurements in COPD are very limited.

    During upper and lower respiratory tract infections, FeNO values are elevated, both in healthy subjects and in asthmatics. However, despite the massive inflammation in the airways of patients with cystic fibrosis, FeNO measurements have not been clinically helpful in cystic fibrosis. In primary ciliary dyskinesia, FeNO measurements again are not helpful, but nasal nitric oxide is extremely low, and may become the screening tool of choice.

    While serial FeNO measurements may be able to predict the early development of bronchiolitis obliterans or pulmonary infections after lung transplantation, its value as a screening tool seems to be limited due to its low sensitivity. Finally, FeNO measurements seem valuable in the monitoring of occupational respiratory disease and in the monitoring of the effects of air pollution on the respiratory system.

  7. Page 56
    Correspondence: P. Latzin, Division of Respiratory Medicine, University Hospital of Bern, 3010 Bern, Switzerland, Email:

    Exhaled nitric oxide (NO) is increasingly used for the diagnosis and monitoring of asthma. The standard single-breath technique cannot be applied in infants. Thus, three different methods have been developed: the tidal-breathing online method, the tidal-breathing off-line (or bag) method, and the single-breath method.

    While it is not clear whether NO actually reflects airway inflammation in infants, the ability of NO measurements to differentiate between health and disease has been demonstrated for various disease groups. Comparable to older subjects, NO is elevated in atopic infants with and without wheezing episodes. Viral-triggered wheeze is less clear, partly due to overlapping phenotype definitions. In high-risk infants, NO may help to predict occurrence of respiratory symptoms.

    The role of NO metabolism during normal and abnormal lung development seems important during infancy as evidence in animal models suggests. Results on NO in infants with bronchopulmonary dysplasia are less clear. However, NO seems to be useful to monitor environmental impact, e.g. tobacco smoke, particularly during rapid lung growth.

    Taken together, NO measurements in infants are promising, especially as noninvasive estimates of environmental exposures, as predictors of subsequent respiratory diseases and in epidemiological studies to assess influences of maturational and inflammatory processes early in life.

  8. Page 71
    Correspondence: J.O. Lundberg, Dept of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, Email:

    Nitric oxide (NO) is continuously generated in the human nasal airways and this NO can be readily measured with noninvasive techniques. The main source of nasal NO seems to be the paranasal sinus epithelium where an inducible NO synthase is constantly expressed. In this chapter, we will discuss the proposed role of NO in regulation of nasal and pulmonary physiology as well as the usefulness of nasal NO measurements in diagnosis and monitoring of upper airway disorders.

  9. Page 82
    Correspondence: S.W. Ryter, 75 Francis St, Boston, MA 02115, USA, Email:

    Carbon monoxide (CO) is detectable in the exhaled breath. Exhaled CO (eCO) levels represent the sum of the endogenous production of the gas and airway contamination from environmental exposure. CO is easily detectable in breath with portable electrochemical methods in the parts per million ranges. Smoking and ambient CO have a significant impact on baseline eCO levels. In the absence of high background exposure, eCO may correlate with cellular haem oxygenase-derived CO production. Haem oxygenase-1 enzyme activity is highly induced upon oxidative stress in inflammatory and resident lung cells and, in turn, eCO levels may reflect inflammatory responses in human disease. Although eCO is suggested as a potential biomarker in pulmonary and systemic inflammatory diseases, further studies are necessary to identify its role in human diagnostics.

  10. Page 96
    Correspondence: A. Amann, University-Clinic for Anaesthesia, Anichstr. 35, A-6020 Innsbruck, Austria, Email:

    Recommended standardised procedures have been developed for measurement of exhaled lower respiratory nitric oxide (NO) and nasal NO. It would be desirable to develop similar guidelines for the sampling of exhaled breath related to other compounds. For such systemic volatile organic compounds (VOCs), CO2-controlled sampling is recommended to assure reliable and consistent sample quality for within- and between-subject comparisons.

    There are two basic approaches for analysing VOCs in breath: real-time analysis and off-line laboratory analysis, each with its particular advantages. Real-time analysis of exhaled breath is most promising for reactive compounds and for compounds that change rapidly as a function of external influence. Off-line laboratory analysis of exhaled breath generally employs some form of pre-concentration of analytes followed by a separation step using high-resolution gas chromatography-mass spectrometry (GCMS)-based detection. GCMS gives the most detailed and specific results for identifying the VOCs contained in breath, but the processes of sample storage, pre-concentration, injection and chromatographic separation may limit the detection of reactive or thermally labile metabolites.

    This chapter discusses the state-of-the-art analyses of exhaled breath for clinical/medical applications, presents current concerns about methods, implementation for different instruments and techniques, and provides specific guidance for standardisation to introduce noninvasive breath-based technology into clinical practice.

  11. Page 115
    Correspondence: C. Di Natale, Dept of Electronic Engineering, University of Rome Tor Vergata, Via del Politecnico 1, 00133 Rome, Italy, Email:

    It is current opinion that the composition of breath contains information which enables the detection of a number of different diseases. Even if there is no clear explanation about the connection between diseases and volatile compounds, several studies involving instruments, such as gas chromatography and electronic nose, have provided evidence that in the case of certain diseases it is possible to correctly identify the breath of patients with respect to the breath of healthy individuals. Most of these studies are focused on lung cancer.

    Correct interpretation of the results from these studies requires medical and instrumental consideration of the problem. The medical approach has to consider the bio-chemical processes that connect the disease to the production of small volatile molecules which can be found in breath. These molecules are revealed by instrumental analyses. Following this, volatile organic compounds are detected by the interaction between the molecules in the exhaled breath and the sensors. This interaction is a transduction operation ruled by three fundamental instrumental characteristics: working principle, selectivity and sensitivity. In this chapter the complete interpretation procedure is viewed as a short review of the medical interpretation of the volatile organic compounds in exhaled breath and the related diseases.

  12. Page 130
    Correspondence: P.J. Mazzone, Respiratory Institute, The Cleveland Clinic, 9500 Euclid Avenue, A90, Cleveland, OH 44195, USA, Email:

    Inflammatory and metabolic changes associated with lung diseases may lead to differences in the production and processing of volatile organic compounds within the body. These differences might be expressed in the exhaled breath. If these differences are robust and unique, and a sensing technology is able to accurately and consistently detect them, then the sensing technology could be developed into a useful clinical test. Such a test could be capable of diagnosing a disease, or identifying the nature and activity of the disease.

    Exhaled volatile organic compound biomarkers have been sought for a variety of lung diseases. There is evidence to support the development of clinical tests based on the detection of volatile exhaled breath biomarkers for asthma, chronic obstructive pulmonary disease, granulomatous lung diseases, interstitial lung diseases, lung cancer, and lung transplant rejection. The evidence is most robust for lung cancer detection, but has shown promise in each of these areas. Methodological and technological advancements are occurring which will allow us to recognise the full potential of this line of investigation.

  13. Page 140
    Correspondence: M. Corradi, Dept of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Via Gramsci 14, 43100 Parma, Italy, Email:

    Volatile organic compounds (VOCs), are mainly blood-borne and are exhaled via the blood–breath interface in the lungs. VOCs diffuse across the pulmonary alveolar membrane from the compartment with the higher vapour pressure to the lower; thus, exhaled VOC analysis is suitable for the study of nonrespiratory diseases.

    VOC levels seem to be modified in some nonrespiratory diseases, such as breast cancer, heart transplant rejection, diabetes, heart failure and pre-eclampsia. In some diseases, the difference in the pattern of exhaled VOCs between patients and controls is marked. For instance, breath VOCs distinguished between females with breast cancer and healthy volunteers with a sensitivity of 94.1% and a specificity of 73.8%. Exhaled VOCs showed significant differences in patients with type-2 diabetes and healthy control subjects with a sensitivity and specificity of 90% and 92%, respectively.

    Changes in exhaled VOC levels in nonrespiratory diseases may have different reasons, such as the increased production of reactive oxygen species or a change in absorption, distribution, biotransformation and excretion rate of endogenous or exogenous VOCs.

    Exhaled VOC analysis may become an attractive option for large-scale testing of at-risk populations; however, the establishment of exhaled VOCs as metabolic markers requires additional confirmatory investigations.

  14. Page 152
    Correspondence: E. Dompeling, Dept of Paediatric Pulmonology, MUMC, PO Box 5800, 6202 AZ Maastricht, Netherlands, Email:

    The diagnosis and management of patients with chronic lung disease may benefit from noninvasive assessments of airway inflammation. The measurement of inflammatory biomarkers in condensate of exhaled breath (EBC) is an innovative, noninvasive technique. To collect EBC, exhaled breath is cooled in a condenser system. In 2005, the task force of the American Thoracic Society/European Respiratory Society formulated recommendations on the standardisation of EBC measurements.

    It is evident from studies that the use of different condensers will contribute to the variability of condensate volume and biomarker levels, which can be explained by variations in the condensation surface, cooling temperature, coating of the condenser tube, sampling time, breath recirculation and collection vial. Open issues include the ideal coatings for markers other than proteins and eicosanoids, and the influence of cleaning procedures, breathing patterns, ambient air pollution and the dilution factor on biomarkers in EBC.

    The use of a highly efficient standard condenser for both children and adults, with breath recirculation and with the possibility to sample with different coatings (depending on the biomarker of interest) will be helpful for the further evaluation of the full potential of EBC measurements, and will facilitate standardisation within and between different centres.

  15. Page 162
    Correspondence: S. Loukides, Smolika 2 16673, Athens, Greece, Email:

    The lung is susceptible to oxidative injury in the form of several reactive oxygen species, including hydrogen peroxide (H2O2). The main cellular sources for H2O2 are neutrophils, eosinophils, alveolar macrophages, epithelial cells and endothelial cells.

    H2O2 has been extensively studied in exhaled breath condensate (EBC) incorporating various techniques, including the onsite measurement with an automated amperometric biosensor.

    EBC H2O2 has been measured in various diseases, mainly in asthma and chronic obstructive pulmonary disease, with most of the clinical studies reporting higher levels compared to normal subjects. Associations of EBC H2O2 levels with parameters obtained from other biological fluids, such as bronchoalveolar lavage and induced sputum have been reported. These findings are interpreted in the context of increased oxidative stress in various lung inflammatory diseases. Results related to treatment intervention lack validity, since they are mainly designed in an uncontrolled manner.

    In conclusion, the usefulness of H2O2 in clinical research and practice is limited, despite the fact that it offers the possibility to be measured in the field, mainly due to methodological issues.

  16. Page 173
    Correspondence: I. Horvath, Dept of Pulmonology, Semmelweis University, Dios arok 1/C Budapest, 1125, Hungary, Email:

    Exhaled breath condensate (EBC) pH is the most well-standardised EBC biomarker and provides surrogate information on acid-base balance in the airway surface lining fluid. Standardised modes for EBC pH determination have been published and a large number of data are available on factors that influence EBC pH values. Acidification of EBC has been reported in several inflammatory airway diseases including asthma, chronic obstructive airway disease, cystic fibrosis and post-transplant bronchiolitis obliterans syndrome. Airway acidification has important pathophysiological effects and influences the bioavailability of inhaled drugs. Even if airway pH cannot be precisely estimated from EBC pH values, the potential of EBC pH as a biomarker of different airway diseases has been suggested. The validity and clinical usefulness of this measurement is still under investigation.

  17. Page 183
    Correspondence: Z. Lázár, Dept of Pulmonology, Semmelweis University, 1/C Diósarok Str., H -1125, Budapest, Hungary, Email:

    An overwhelming body of in vitro and in vivo evidence underscores the importance of purinergic signalling pathways in the regulation of crucial airway mechanisms, such as mucociliary clearance, airway inflammation and smooth muscle reactivity. Purinergic mediators have been measured in airway samples from patients with asthma, cystic fibrosis and chronic obstructive pulmonary disease. Adenosine triphosphate (ATP) and its metabolites, especially adenosine, have also been detected in exhaled breath condensate (EBC) to aid diagnosis and follow-up of patients who present with airway pathology. EBC is an ideal biological fluid for measuring purinergic molecules, as factors confounding mediator concentrations in other airway samples do not influence measurements in the condensate fluid. Recently, the simultaneous measurements of purines and a dilution marker have been validated to overcome the varying dilution of airway droplets in EBC. The use of these mediators as disease biomarkers, however, needs further evaluation.

    This chapter discusses the physiological and pathophysiological effects of ATP and adenosine in the airways, and the methodological issues, together with the most recent findings of their measurements in EBC.

  18. Page 196
    Correspondence: P. Montuschi, Dept of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart Largo F. Vito, 1 00168 Rome, Italy, Email:

    Eicosanoids, including leukotrienes (LTs), prostaglandins (PGs) and thromboxane A2 (TxA2), are lipid mediators involved in the pathophysiology of airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease. Isoprostanes or isoeicosanoids, PG-like compounds primarily produced by peroxidation of arachidonic acid induced by reactive oxygen species, are considered among the best biomarkers of oxidative stress and lipid peroxidation. Using immunoassays, several eicosanoids and 8-isoprostane have been detected in exhaled breath condensate (EBC) and found to be selectively increased in patients with different lung diseases. LTB4, 8-isoprostane and PGE2 in EBC has been confirmed by mass spectrometry.

    Measurements of LTs, 8-isoprostane and prostanoids in EBC may provide insights into the pathophysiology of lung diseases. Selective eicosanoid profiles in EBC may prove to be important for differentiating between inflammatory lung diseases. This approach is potentially useful for quantifying lung inflammation and monitoring pharmacological therapy. EBC analysis of eicosanoids and isoprostanes might clarify the effects of drugs on lung inflammation and oxidative stress. However, due to the lack of a standardised procedure for EBC analysis of eicosanoids and current methodological limitations, comparisons of results from different laboratories are difficult.

    Important methodological issues need to be addressed before analysis of eicosanoids in EBC can be considered in the clinical setting. Mass spectrometry techniques are being applied to the measurement of eicosanoids in EBC making it possible to obtain accurate quantitative assessment of eicosanoid concentrations in EBC.

  19. Page 207
    Correspondence: L.E. Donnelly, Airway Disease, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, UK, Email:

    Exhaled breath condensate (EBC) is a simple, safe and noninvasive method of sampling the lower airways. To this end, measurement of inflammatory markers within these samples could be of benefit in monitoring disease severity, exacerbations and responses to treatment.

    Measurement of metabolites of nitric oxide (NO,) such as nitrite and nitrate, have been measured extensively in EBC from subjects with a variety of pulmonary diseases including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Although there remain methodological issues regarding the source of nitrite and nitrate in EBC, due to acute cigarette smoking and bacterial contamination of the oral cavity, these can be overcome by smoking cessation prior to sample collection and the use of an antibacterial mouthwash. Using these techniques, levels of NO-related compounds have been reported to be elevated in many inflammatory lung conditions, including asthma and COPD.

    Measurements of nitrotyrosine and S-nitrosothiols in EBC are also possible and more recently, techniques have become available to measure peroxynitrite in EBC. However, more studies are required to validate these measurements. Data currently available indicate that levels of these compounds are elevated in a number of respiratory conditions and that these levels can be altered by treatments such as glucocorticosteroids. Therefore, assessment of nitrosative stress in the airways using measurement of NO-related compounds may be of benefit for monitoring novel therapies that target these pathways directly.

  20. Page 217
    Correspondence: H. Wirtz, Dept of Respiratory Medicine, University of Leipzig, Liebigstrasse 20, 04103 Leipzig, Germany, Email:

    Exhaled breath condensate (EBC) has been used to measure various proteins. These proteins may be related to total protein, which does not appear to vary very much in most diseases. The proteins measured are mostly smaller proteins, such as cytokines and hormones, but also structural proteins, such as cytokeratins.

    Asthma and, to a lesser extent, chronic obstructive pulmonary disease are diseases that have been investigated most intensively with EBC measurements. Most often, the aim of the investigation is the estimation and differentiation of the inflammation seen in these diseases. In other diseases, such as lung cancer, the aim is to detect the disease using highly specific markers. Some progress has been made with a small number of factors and with newer techniques involving more markers, such as fluorescent bead-based immunoassays. The very low concentrations of these markers are still a challenge for the sensitivity of the tests involved, and the methodology of sampling and preparing. The chapter will outline and review protein markers that have been used and diseases in which they have been investigated.

  21. Page 231
    Correspondence: E. Baraldi, Dept of Paediatrics, Allergy and Respiratory Medicine Unit, Via Giustiniani 3, 35128 Padova, Italy, Email:

    The metabolomic approach is defined as the analysis and interpretation of the global metabolic data expressing the response of living systems to genetic modification, pathophysiological stimuli and environmental influences. Metabolomics is a comprehensive approach, which enables the characterisation of the “metabolic fingerprint” of a sample. This approach promises to enable the detection of states of disease, the stratification of patients based on biochemical profiles and the monitoring of disease progression. Metabolomic analysis may also be able to orient the choice of therapy, identify responders and predict toxicity, paving the way to a customised therapy.

    In the field of respiratory medicine, a promising application of metabolomics is in the analysis of exhaled breath condensate, a biofluid collected noninvasively which reflects the composition of airway lining fluid.

    In patients with respiratory disease, metabolomic analysis of exhaled breath condensate might have a role in the characterisation of diseases subphenotypes and in the prediction of response to specific treatments.

  22. Page 237
    Correspondence: P.J. Barnes, Airway Disease Section, National Heart and Lung Institute, Dovehouse St, London, SW3 6LY, UK, Email:

    There have been major developments in measuring biomarkers in breath in the last decade. Breath analysis is now becoming feasible in the clinic and exhaled nitric oxide (NO) fraction is already widely used in the clinic for diagnosis and monitoring of asthma. However, it has so far proved difficult to demonstrate any clinical benefit in improving asthma control and studies in more selected patient groups are now needed. Partitioning of exhaled NO into airway and peripheral fractions may be more useful for monitoring chronic obstructive pulmonary disease and severe asthma in the future. The development of cheap and sensitive NO detectors will make it possible for patients to undertake monitoring at home.

    Exhaled breath condensate has the potential to measure semi-volatile lipid mediators and pH, and there are novel electrochemical assays that may allow on-line detection. However, a major problem limiting the usefulness of this technique is variable dilution with water vapour. Many volatile organic compounds have been detected in the breath and the pattern of molecules can now be characterised with various electronic nose devices and by mass spectrometry profiles. Use of smaller and more sensitive mass spectrometry approaches allows the identification of the discriminatory chemicals so that more selective detectors and small portable devices may be developed in the future. Breathomics may become a reality in the clinic, not only for the diagnosis and monitoring of pulmonary diseases, but also for systemic diseases.

  23. Page 247
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