Exhaled Biomarkers

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.