Jia LiuBCDEF, Paul S. ThomasEFG
Faculty of Medicine, University of New South Wales and Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, NSW, Australia
Author’s address: Associate Professor Paul S. Thomas, Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, NSW 2031, Australia, e-mail: [email protected]
Source of support: Departmental sources
Most of the methods of investigating lung diseases have been invasive until the discovery that exhaled nitric oxide can be used as a surrogate marker of airway infl ammation, particularly in asthma. Exhaled nitric oxide (NO) is now established as a marker of airway infl ammation. It has been shown to correlate well with eosinophilic asthmatic airway infl ammation, and to be able to predict decline in asthma control and airway function. Altered levels of NO are also associated with other infl ammatory lung diseases. In addition, polymorphisms of the genes encoding the three nitric oxide synthases are associated with phenotypic differences associated with lung diseases. Exhaled NO is, however, non-specifi c. It is therefore of importance that collecting exhaled breath condensate (EBC) has emerged as a potential tool in the study of pulmonary diseases. The exhaled breath is collected in a cooling system which allows water vapour to condense. The EBC contains a number of mediators relating to the NO pathway, including nitrite as a metabolite of nitric oxide, nitrotyrosine, nitrosothiols plus small molecular mediators associated with oxidative stress, including hydrogen ions, and hydrogen peroxide. In addition, reports are emerging of the detection of larger molecules which not only include leukotrienes, prostaglandins, albumin and other proteins, such as cytokines, but also macromolecules, for example, DNA. EBC is becoming a technique which will allow repeated non-invasive sampling from the respiratory tract thus assisting pulmonary research and possibly the monitoring of lung diseases.
Key words: exhaled breath condensate • infl ammation • nitric oxide • airway disease
Source: Med Sci Monit, 2005; 11(8): MT53-62
Nitric oxide (NO) plays an important role in the regulation of smooth muscle tone of pulmonary blood vessels and bronchi. NO is well known for mediating vasodilation and it is involved in control of vascular tone related to cardiovascular diseases, inhibition of platelet aggregation and platelet adhesion, and is also a diffusible signaling molecule which acts as a neurotransmitter [1,2]. Although NO participates in a variety of physiological processes, excess or decreased NO production will have detrimental effects such as an abnormal response to infl ammation and injury, hypo- or hypertension [3,4]. An important role of NO in the lung is non-specifi c host defense and antimicrobial activity against various pathogens. An increase in NO may be due to activation of inducible NO synthase (iNOS) expressed by epithelial cells in response to proinfl ammatory cytokines and oxidants. Viral infection of epithelial cells also increases NO production, and the elevation in NO may limit infection by inhibition of viral replication and mediate the antiviral effect of interferon-g [5,6]. In addition, non-infectious diseases of the airway such as asthma are associated with increased iNOS expression and exhaled NO . Monitoring infl ammatory diseases in the lung can be performed by invasive methods, but newer approaches are generating insights into the applicability of non-invasive techniques. Exhaling through a cooling system generates exhaled breath condensate (EBC) as the breath is saturated with water vapour. It contains volatile and non-volatile substances including nitrite/nitrate as end products of NO metabolism, which can be used to monitor infl ammatory lung diseases.
Nitric oxide (NO) is a low molecular weight and highly reactive gas. In the atmosphere, it is a component of air pollution, but is also a free radical released by a variety of tissues by nitric oxide synthases (NOS). Since endothelium-derived relaxing factor (EDRF) and NO have been proved to have identical biological activity, stability and susceptibility to specifi c inhibitors, NO has been viewed with increasing interest [8–11].
Synthesis and release of nitric oxide
NO is enzymatically produced by NOS, then oxidised to nitrite and nitrate by several mechanisms including macrophage activation [12,13]. Mitchell et al. fi rst suggested that mammals produced oxides of nitrogen in 1916 and Furchgott et al. identifi ed EDRF in vessels with an intact endothelium in 1982 [14–18]. In 1988, Palmer and others suggested that L-arginine is the precursor for NO synthesis in vascular endothelial cells and confi rmed that NO is the intermediary of the L-arginine to nitrite and nitrate pathway [13,19].
The NOS enzyme includes three distinct isoforms representing three distinct gene products, including neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2) and endothelial NOS (ecNOS, NOS3). The three isoforms vary according to their different chromosomal locations, amino acid sequences and functions. nNOS and ecNOS are termed constitutive NOS (cNOS) which generate small amounts of NO depending on calcium/calmodulin binding . All three isoforms are found in normal lung tissue, while iNOS is up-regulated in diseases of the airway and at the alveolar level. mRNA of the ecNOS and iNOS genes are expressed in human airway epithelial cells, and have been proved to be correlated with biochemical activity [21–23]. While it is clear that NO plays an important role in the normal and the diseased lung, it is becoming apparent that NOS genotypic differences are also associated with the development of lung diseases.
Polymorphisms of nitric oxide synthases in relation to the lung
Neuronal NOS in the lung
The first NOS was purifi ed from rat cerebellum in 1990 by Bredt and Snyder and was identifi ed as a calmodulin-requiring enzyme . This was later designated as nNOS and found to be constitutively expressed in human alveolar and bronchial epithelial cells in 1994 .
The gene for nNOS in humans is located on chromosome 12q24 and polymorphisms have been linked to the diagnosis of asthma in family studies. Allelic frequencies of a polymorphism in exon 29 of the nNOS gene were reported by Grasemann et al. to be signifi cantly different between asthmatics and controls. The C®T transition which is located 276 base pairs downstream of the translation termination site has been proved to be lower in asthmatic patients than that in normal controls, which suggests that variants of the nNOS gene may contribute to asthma . Grasemann et al. also reported high AAT repeat numbers in an intronic repeat polymorphism in the nNOS gene in cystic fi brosis (CF) patients in 2000 . This was associated with a decrease of nNOS expression in airway epithelial cells in those cystic fi brosis patients who were more likely than others to have low airway NO concentrations . In addition, dinucleotide GT repeats in the proximal region of the nNOS gene, associated with progression of lung disease in patients with CF were found by Texereau et al. in 2004. These polygenic associations may be important for understanding the phenotypic disparities of patients with the same cystic fi brosis transmembrane conductance regulator (CFTR) mutations, and implicate NOS in this lung disease .
In addition to cystic fibrosis, nNOS is thought to play a role in other diseases. Airway smooth muscle hyperplasia and hypertrophy are considered to contribute to airway infl ammatory diseases. Thus, enhanced airway smooth muscle cell proliferation is thought to be associated with the chronic stages of asthma and chronic obstructive pulmonary disease (COPD). Patel et al. demonstrated increased nNOS expression in airway smooth muscle cells, which is thought to inhibit these proliferative responses .
Inducible NOS in the lung
iNOS is generally not expressed unless the cells have been induced by certain cytokines, microbes, microbial products or infl ammatory cytokines in contrast to cNOS, which suggests that NO produced by expression of iNOS acts as a major effector molecule in host defence mechanisms [15,27]. Nearly every tissue, however, in the body has the ability to express iNOS when stimulated, and it produces sustained high concentrations (μM) of NO [30–32]. Koide et al.  reported that a high level of intracellular cAMP, especially if combined with infl ammatory cytokines, increases nitric oxide production via iNOS mRNA expression in vascular smooth muscle cells, suggesting that iNOS is involved in infl ammatory reactions. Likewise, Robbins et al. have shown that TNF and IL-1 up-regulate iNOS expression in airway epithelium [34,35].
Guo et al. demonstrated that NO synthesis is due in part to the continuous expression of the iNOS isoform in airway epithelial cells of normal subjects . Abundant iNOS mRNA expression was reported in normal airway epithelial cells, while it is not detected in other resting pulmonary cells, indicating that airway epithelial cells are unique in their continuous pattern of iNOS expression. In situ analysis reveals all airway epithelial cell types express iNOS. Expression is, however, strikingly decreased by inhaled glucocorticosteroids and b-adrenergic agonists, which are commonly used medications in the treatment of infl ammatory airway diseases .
The gene encoding iNOS is located at chromosome 17q11.2–q12 and Konno et al. reported that the (CCTTT)n repeat polymorphism in the promoter of the iNOS gene is inversely associated with atopy. This polymorphism affects promoter activity and is a risk factor for the development of atopy, which seems to be an independent risk factor for asthma . The (CCTTT)n polymorphism has been proved to be associated with increased NO production in healthy children, while no data were reported on asthmatic patients . Kharitonov et al. and Hansel et al. have demonstrated that a selective inhibitor of iNOS has a rapid onset and longterm suppression of exhaled NO levels asthmatic patients. This suggests that inhibition of iNOS may have some therapeutic potential for asthma, because selective iNOS inhibitors suppress eosinophil infi ltration to the lung, decrease lung chemokine expression and inhibit allergic airway infl ammation in murine models of asthma[7,39–41]. While decreasing iNOS activity may be advantageous in some situations, a defi ciency of nitric oxide production has been found in the bronchial epithelium of some CF patients, due to a reduction in iNOS expression whether they had received glucocorticosteroids or not. Such a reduction may play a role in susceptibility to pulmonary fungal, viral and bacterial infections [27,42,43].
Endothelial NOS in the lung
ecNOS and nNOS are calcium/calmodulin-dependent enzymes, which produce low concentrations of NO (nM), but which are critical for the maintenance of endothelial function, to inhibit the adhesion of platelets and to suppress the replication of smooth-muscle cells [44,45]. The human ec- NOS gene is located at 7q35–36, and in 1994, Nadaud et al. cloned the human endothelial NO synthase gene and determined its structure, being composed of 26 exons [46,48]. ecNOS polymorphisms are associated with many different diseases, principally relating to the cardiovascular system, which has been extensively studied in this regard. ecNOS polymorphisms comprise three recognised functional allelic variants including the ecNOS4a in intron 4, T-786C, and G5557T in exon 7.
This distribution of the ecNOS intron 4 polymorphism varies greatly in the normal population among different ethic groups [49,50]. The polymorphism in intron 4, known as ecNOS4a, has four tandem 27-bp repeats, while fi ve tandem 27-bp repeats is regarded as the common, wild-type allele ecNOS 4b . Genotypic frequencies of ecNOS 4b/b, ecNOS 4b/a and ecNOS 4a/a in an Australian population were 66.7%, 32.7% and 0.7%, respectively, as reported by Wang et al. in 1996, although the frequency of ecNOS 4b/b is about 80% in Japanese and 75% in normal South Koreans [49,52–54]. The 27-bp repeat polymorphism in intron 4 was identifi ed as being associated with coronary artery disease (CAD), and myocardial infarction (MI), smoking-associated coronary heart disease and hypertension [49,55–58]. In a small study that we conducted in normal Australian subjects, 3 of total 16 were a/b (18.75%), while 13 people were type b/b (81.75%), and no a/a were detected, probably related to the small sample size (unpublished data).
Homozygous ecNOS 4a/a subjects who have the smaller allele of four repeats, had a signifi cantly lower exhaled NO level than either heterozygous ecNOS 4a/b subjects or wildtype ecNOS 4b/b subjects. Also, ecNOS 4a/a patients were more likely to have more diseased coronary vessels, which suggests an association between a reduced ability of the ecNOS4a allele to generate NO and an increased risk of CAD [49,55]. No evidence of such as association, however, was found with CAD in a hospital-based Taiwanese population . The distribution of genotype ecNOS 4b/b was signifi cantly higher in subjects with asthma than normal controls in a South Korean population, but it did not segregate with asthmatic severity . The ecNOS 4b/b genotype was reported also to be more frequent in patients with lung cancer compared to a control group . Other polymorphisms of ecNOS have been identifi ed, one being the T-786C substitution in the promoter region and another the G5557T in exon 7, also known as exon 7 Glu298Asp.
In a disease involving vascular dysfunction, both the Glu298Asp variant and the ecNOS 4a allele were reported to be signifi cantly associated with high-altitude pulmonary edema, which may underlie impaired NO synthesis in the pulmonary circulation .
Exhaled nitric oxide (eNO)
Immunostaining has shown nNOS expression in the nerves and epithelial cells of normal human airways and iNOS and ecNOS expression in airway epithelial cells [23,61]. These sources of NO are thought to be responsible for the NO detected in the breath, although the principal contributors are thought to be airway epithethial cells and perhaps pulmonary macrophages. The measurement of exhaled nitric oxide is highly reproducible in both healthy and asthmatic adults and children . Exhaled NO is increased in asthmatic patients and decreases after glucocorticosteroid therapy [63,64]. Patients with severe stable COPD have reduced levels of exhaled NO compared with normal subjects. Exhaled NO levels are only to a minor extent related to the severity of airfl ow obstruction in this disease . A number of factors may affect exhaled NO levels, including the technique used for sample collection as well as some factors within the subjects. There are two methods of collection. On-line sampling measurement requires the subject to exhale directly to the NO analyser, while off-line sampling uses a gas impermeable bag to collect the exhaled breath for later analysis. This latter method allows exhaled NO measurements in large populations, especially in remote area [66,67].
It has been demonstrated that fl ow rate is a major factor affecting the level of exhaled NO. Deykin et al. have reported that the values of exhaled NO decreased with increasing expiratory fl ow rate within the range of 50 to 500 ml/s by the off-line method and 42 to 250 ml/s by the on-line measurement [66,68]. Different inspiratory fl ow rates did not appear to infl uence exhaled NO levels . Nasal NO contamination is also a common factor confounding exhaled NO levels. NO levels in the nasal cavity have been proved to be higher than other parts of respiratory tract [70,71]. Ensuring that the subject keeps a positive intra- oral pressure during exhalation has been reported as a way of avoiding nasal NO contamination due to vellum closure .
Caffeine decreases exhaled NO levels in normal subjects, although a study of asthmatic patients showed no effect [72,73]. Alcohol also decreases exhaled NO but only in asthmatic subjects and not in normal individuals which suggests that the effect is via iNOS . Cigarette smoke is another important factor affecting exhaled nitric oxide levels. Both active and passive smoking is able to reduce exhaled NO signifi cantly in normal subjects [75,76]. Diet probably also has an effect on NO production as either inhaled or ingested L-arginine, the substrate for NOS, increased the exhaled NO level in both normal and asthmatic subjects [77,78].
Exhaled breath condensate (EBC) may be collected from breath saturated with water vapour, via a cooling system. EBC contains volatile and non-volatile substances, which can be used to monitor infl ammatory lung diseases. These include endogenous substances, such as hydrogen peroxide, thiobarbituric acid-reactive substances, isoprostanes, prostaglandins, leukotrienes and nitrite/nitrate as the end products of NO metabolism. These non-volatile mediators in exhaled breath can be used as markers of oxidative stress and infl ammation to investigate, and perhaps monitor, lung diseases . In contrast to bronchoalveolar lavage, collecting exhaled breath condensate is a totally non-invasive and a safe way to assess breath constituents and it can be repeated within a short period of time, even in asthmatic children. It does not infl uence airway function, nor induce infl ammation, unlike bronchoalveolar lavage or sputum induction . Scheideler et al.  reported that the origin of the proteins detected in breath condensate are partially from the naso-oropharyngeal tract and partially from lower regions of the airways.
Collection of exhaled breath condensate
Exhaled breath condensate collection can be performed using either commercially available equipment or simple cooling devices with glass condensing chambers whereby an inner chamber is suspended in a larger glass chamber and cooled by means of ice. Subjects are instructed to take normal tidal breaths, exhaling through a cooling system to form a condensate. In addition to simple glass collection devices, successful collection has been reported using a variety of devices, such as Tefl on-lined tubing in an ice-fi lled bucket and a commercially available condenser (Erich Jaeger GmbH, Hoechberg, Germany) .
During collection, nasal secretions, saliva and sputum may contaminate EBC. The following modifi cations may help to exclude nasal contamination: (1) inhalation and exhalation without a nose-clip; and (2) exhalation against a resistance to ensure velum closure and minimize nasal contamination . To prevent salivary contamination, subjects should rinse their mouth before collection and keep the mouth dry by periodically swallowing their saliva. Measuring amylase concentrations in samples can exclude signifi cant salivary contamination . A number of factors affect EBC volume during collection which include the temperature and humidity of exhaled air. Water loss in the expired breath is not great except under extreme conditions of exercise at altitudes when minute ventilation is very high [83,84]. We have demonstrated that EBC volume is proportioned to tidal and minute volume and also showed that deep breathing to vital capacity can give an signifi cantly larger yield of EBC than tidal breathing . Other studies have shown that controlling breathing by using visual cues can improve reproducibility of EBC volume, but nonetheless poor repeatability of infl ammatory markers may be seen .
Markers in exhaled breath condensate
Nitric oxide is difficult to measure because it is a free radical which reacts rapidly with oxygen, superoxide, water, thiols, amines, and lipids to form products with biochemical activities ranging from bronchodilation to cytotoxicity . Nitrates and nitrites are products of nitric oxide metabolism, which can be detected in EBC. Nitrite is rapidly oxidised to nitrate in aqueous solutions. Moshage et al. reported that in whole blood >95% nitrite is very rapidly oxidized to nitrate within 1 hour , thus a single measurement of plasma nitrite alone is probably meaningless.
Production of nitric oxide is generally increased in infl ammatory diseases, including asthma. Nitrite/nitrate levels in EBC are raised in asthmatic patients , which may be clinically useful in the management of infl ammatory lung diseases. Corradi et al. measured oxides of nitrogen (NOx) in EBC and showed that NOx values in smokers, asthmatic and community-acquired pneumonia patients were significantly higher than normal controls and decreased during the recovery phase of pneumonia [90,91].
We have compared total NOx levels in EBC and plasma among smoking, ex-smoking and non-smoking subjects. The total NOx concentration in samples of exhaled breath condensate and plasma was quantifi ed as nitrite by the method of nitrate reductase and the reaction of nitrite with 2,3-diaminonaphthalene (DAN) . We demonstrated that while plasma NOx concentrations in smokers are signifi - cantly higher than either non-smokers or ex-smokers, there was no correlation between plasma and EBC NOx levels . This indicates a disparity between NOx in EBC and plasma, and the complexity of NO regulation is further illustrated by the fact that exhaled NO decreases after smoking, while NOx in EBC appears to increase [75,76].
Vascular endothelial and smooth muscle cells, together with infl ammatory cells, especially eosinophils, produce superoxide anions (O2 –) and release several reactive oxygen- and nitrogen-derived species such as NO [94,95]. Superoxide anion radicals and nitric oxide react rapidly to form the peroxynitrite anion (ONOO–), a potent oxidant capable of damaging lipids and proteins in biological membranes, and which can also cause airway hyperresponsiveness and enhancement of infl ammatory cell recruitment [95–97]. Increased peroxynitrite together with elevated expression of iNOS have been observed in asthmatic patients compared with control subjects . Peroxynitrite is able to add a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine to form nitrotyrosine .
Abundant expression of iNOS with the formation of nitrotyrosine in airway epithelium and infl ammatory cells has been observed in bronchial tissue from asthmatic patients compared with little or no nitrotyrosine in normal airway epithelium. A signifi cant inverse correlation was demonstrated between the nitrotyrosine in infl ammatory and epithelial cells with pulmonary function in patients with asthma . This suggests that nitrotyrosine is a marker of oxidative stress in asthmatic airways, owing to the signifi cant correlation between nitrotyrosine in EBC and exhaled NO.
Glucocorticosteroid treatment reduces the formation of nitrotyrosine in asthmatic patients [94,95]. Elevated nitrotyrosine in EBC has been reported in patients with cystic fi - brosis and increased nitrotyrosine together with ecNOS and iNOS expression in the pulmonary lesions of human tuberculosis has been reported by Choi et al. [99,100].
Glutathione (GSH) is a low molecular weight and endogenous thiol in human lung and the reaction of nitric oxide and glutathione can form nitrosothiol, which is detectable in EBC . Corradi et al.  noted high levels of nitrosothiols in EBC of smokers and patients with severe asthma, chronic obstructive pulmonary disease (COPD) and cystic fi brosis (CF). A positive correlation between nitrosothiol values and smoking history in current smokers was also found. Nitrosothiols were not found to be elevated in mild asthmatic patients, and it was suggested that this may be helpful to classify asthmatic severity .
Other non-nitrogenous markers
Hydrogen ions, measured as the pH value of EBC, is another simple, reproducible and useful marker for airway infl ammation. Kostikas et al. reported that a signifi cantly lower EBC pH value is present in patients with COPD and bronchiectasis compared with either asthmatic patients or control subjects . Patients with moderate asthma had signifi cantly lower pH values compared with mild asthmatic patients, and EBC pH is lower in cystic fi brosis children than in healthy control subjects, in addition, the EBC pH of CF patients with an exacerbation was signifi cantly lower than that of stable patients with CF [102,103].
van Beurden et al. showed that hydrogen peroxide (H2O2) was a reproducible marker in EBC which can remain stable for a period of up to 40 days of frozen at –70°C . Hydrogen peroxide in EBC was reported to be increased during the common cold, and returned to normal within 2 weeks of recovery in otherwise healthy subjects, thus this marker appears to refl ect upper respiratory tract infl ammation . Expired breath condensate H2O2 is not elevated in patients with cystic fi brosis although others have indicated that the variation in expiratory fl ow rates may affect and limit the usefulness of exhaled hydrogen peroxide as a marker of airway infl ammation [106,107]. A recent study showed that mean concentration of H2O2 was significantly elevated in patients with COPD compared to control subjects, and H2O2 levels in patients with severe and moderate COPD were signifi cantly higher than those with mild COPD .
Many lung diseases, including asthma, COPD, cystic fi brosis and interstitial lung disease, involve chronic infl ammation and oxidative stress [109–112]. Several infl ammatory mediators such as nitrotyrosine, H2O2, leukotrienes, etc, have been identifi ed in EBC and may be used as non-invasive techniques to monitor airway infl ammation.
NO plays a major role in the regulation of the smooth muscle tone of pulmonary blood vessels and airway infl ammatory diseases not only as a marker, but it can also have anti-infl ammatory effects . The balance of NO-related products between nitrite/nitrate, nitrosothiols and nitrotyrosine refl ected by EBC may give insights to NO synthesis degradation and long term changes in NO production .
Asthma, a common chronic infl ammatory disease of airways, is characterized by reversible airway obstruction. The prevalence of asthma has become unacceptably high in recent years. It was ranked among the most common chronic conditions in the United States, with a rapid increase in prevalence from 30.7 per 1,000 persons in 1980 to 56.8 per 1,000 persons in 1995 . Similar increases of asthmatic prevalence were reported in UK, Australia and Poland [115–117]. Thus, monitoring and control of asthma is a substantial global health problem.
Exhaled nitric oxide was fi rst reported to be elevated in asthmatic patients by Alving et al. in 1993 . Kharitonov et al. demonstrated that glucocorticosteroid(GCS)-naïve asthmatic subjects had signifi cantly higher exhaled NO concentrations than asthmatic patients receiving inhaled GCS, who had similar levels of eNO to those of normal control subjects. This, and subsequent studies, have indicated that exhaled NO could be regarded as a sensitive marker of asthmatic airway infl ammation .
As nitric oxide is rapidly oxidised, metabolites of NO can be detected in EBC. Total nitrite/nitrate as the end products of nitric oxide metabolism, are elevated in mild asthmatic patients compared to normal control subjects, and these were signifi cantly affected by both glucocorticosteroid therapy and smoking habit . Furthermore, increased levels of the end product of NO degradation, NOx, correlated with the increase in iNOS protein and mRNA in human lung epithelial cells, as reported by Robbins et al. .
Nitrotyrosine in EBC was demonstrated to be elevated in mild asthmatic patients not treated by GCS compared to normal subjects and the authors speculated that nitrotyrosine in EBC may be a more sensitive marker than exhaled NO to evaluate the contribution of oxidative stress to the airway infl ammation of asthma .
Other markers of oxidative stress, including hydrogen peroxide, in EBC were reported to be correlated with airway hyper- responsiveness and sputum eosinophils, the latter being positively correlated with exhaled NO. Both H2O2 in EBC, and eNO were demonstrated to be elevated in mild asthmatic patients when compared with normal control subjects. Moreover, high levels of H2O2 in the EBC of patients with severe asthma poorly controlled on high doses of inhaled GCS were found, whereas exhaled NO was normal, which indicates that H2O2 in EBC may be more useful in monitoring control of asthmatic infl ammation .
Exogenous adenosine and adenosine monophosphate (AMP) cause similar dose-related bronchoconstriction when inhaled by asthmatic patients [120,121]. Endogenous adenosine in EBC was correlated with eNO in asthmatic patients, and was signifi cantly higher in glucocorticosteroid-naive patients than in both healthy control subjects and glucocorticosteroid- treated patients, consistent with a previous study which showed increased adenosine levels in bronchoalveolar lavage (BAL) of asthmatic patients [122,123].
A recent study showed that leukotrienes (LTs) B4, D4, and E4 were detectable in EBC, while LTC4 was undetectable, which is consistent with a rapid and complete pulmonary conversion of LTC4 to LTE4. Leukotriene concentrations in EBC increased with age in healthy subjects, and were also increased in both adults and children with asthma when compared with age matched healthy controls [124–126]. Another useful marker in EBC, which has been proved to be positively correlated with exhaled NO, is 8-isoprostane of the arachidonic acid cascade [127–129]. 8-Isoprostane concentration has been demonstrated to be elevated in asthmatic patients, while it decreased after GCS treatment [129–132].
Chronic obstructive pulmonary disease (COPD) is characterised by chronic infl ammation of the respiratory tract with a major component being oxidative stress. Since H2O2 is a marker of oxidative stress, it is not surprising that H2O2 was elevated in the EBC of both stable and COPD patients with an exacerbation when compared to normal subjects [108,133,134]. This marker was thought to be more repeatable and sensitive than 8-isoprostane in the assessment of the infl ammatory process .
Exhaled nitric oxide of current smokers with COPD was reported to be higher than that of otherwise healthy smokers, while other studies have shown that exhaled NO was only elevated in COPD during an exacerbation, and the reduction of exhaled NO may be due to the effect of smoking [112,135,136]. Nitrite/nitrate in EBC, as products of nitric oxide, have not been demonstrated to be signifi cantly different between COPD patients and normal subjects, however, nitrosothiols as other products of nitric oxide are higher in the EBC of COPD patients than those of normal subjects [90,101].
Bucchioni et al. investigated the presence of interleukin-6 (IL-6: a cytokine secreted by monocytes/macrophages, T cells, B cells and endothelial cells) in the exhaled breath condensate. The results showed that detected IL-6 levels in EBC were higher in the ex-smokers with moderate COPD than in the healthy non-smokers . Also, LTB4 was increased during an exacerbation of COPD and decreased after treatment, which may suggest that this leukotriene may take part in mediating airway infl ammatory changes .
Cystic fi brosis
Cystic fi brosis is characterized by recurrent respiratory tract infections leading to airway damage. Several studies have shown that exhaled nitric oxide levels in normal subjects did not differ from those with cystic fi brosis (CF) patients even during an exacerbation, although higher levels of NO synthase activity were demonstrated in patients with CF compared with normal subjects [139–143]. There are a number of possible explanations for this paradox, one of which is that NO is a highly reactive molecule which cannot persist as the gaseous moiety, thus, NOx in EBC as the end product of NO is presumed to refl ect total NO production [87,144]. The neutrophilic infl ammation associated with CF infl ammation may increase conversion of NO to NOx. A study in 1998 showed that NOx levels in EBC were signifi cantly higher in stable CF patients compared to normal subjects, however, NOx levels in EBC did not correlate with spirometry, which suggests that NOx in EBC may refl ect airway infl ammation rather than the end result of airway damage .
Another stable metabolite of NO, 3-nitrotyrosine was reported to be elevated in EBC of CF patients, which may indicate increased airway oxidative stress in CF patients . Nitrosothiol levels were also demonstrated to be higher in EBC of CF patients than those of normal controls, which may be partially because of the additional stimulus caused by airway acidifi cation leading to nitrosothiol formation [101,103,145]. It is suggested that metabolites of NO such as nitrite, nitrotyrosine and nitrosothiol in EBC of patients with CF may be more valuable than exhaled NO in monitoring airway infl ammation.
CF patients with an acute exacerbation had abnormally high levels of hydrogen peroxide in EBC, which decreased after treatment with antibiotics . Another marker in EBC, 8-isoprostane, was also reported higher in CF patients than that of normal subjects, which is consistent with increased 8-isoprostane levels in plasma of CF patients [146–148]. Furthermore, both LTB4 and IL-6 were demonstrated to be increased in EBC of patients with CF during acute infective exacerbations compared to those in both stable CF patients and normal subjects .
The only study as yet to assess markers in EBC of patients with community-acquired pneumonia (CAP) was reported by Corradi et al. in 2003. The results show that NOx levels in EBC were elevated in CAP patients during the acute phase and fell following recovery, being consistent with the elevated NOx level in bronchoalveolar lavage (BAL) of patients with infective pneumonia demonstrated previously [90,150].
Cigarette smoking is thought to be a risk factor of a number of pulmonary diseases. Exhaled NO concentrations have been demonstrated to be reduced in habitual cigarette smokers compared to healthy non-smokers, being consistent with decreased iNOS protein and mRNA expression in the airway epithelial cells of smokers [75,151]. Thus, some diseases related with cigarette smoking may be due to NOS dysfunction.
Acute smoking has different effects on smokers and nonsmoking healthy controls. Exhaled NO levels of the lower respiratory tract were reported to increase rapidly after actively smoking a cigarette in habitual cigarette smokers, but decreased exhaled NO was observed shortly after exposure to tobacco smoke in normal subjects in a single-blinded study [76,152,153]. In contrast, a study of current healthy smokers found a rapid and transient increase in NOx in EBC, 30 minutes after active smoking, and then a decrease of nitrite/ nitrate to baseline by 90 minutes . Kevin et al. reported that nitrites, protein concentrations and neutrophil chemotactic activities in EBC were signifi cantly higher in healthy young smokers than those of non-smokers . We have also demonstrated that NOx concentrations in the EBC of smokers are signifi cantly higher than those of non-smokers, which is consistent with elevated nitrite/nitrate levels in plasma of smokers compared with either non-smokers or ex-smokers . This indicates a disparity between EBC NOx observations and the fact that there is a fall in exhaled NO after smoking [75,76]. It is possible that smoking increases degradation of NO to NOx and that as cigarette smoke is a rich source of NO, that this contributes to the increase in NOx.
IL-6 and LTB4 have both been demonstrated to be elevated in EBC of current smokers compared with non-smokers . H2O2 levels in current smokers with COPD were signifi cantly higher than those levels seen in healthy control subjects .
Indicators of dilution of EBC are needed as references to calculate the concentrations of markers from the airway and lung, although all of the non-volatile substances in EBC are thought to be diluted to the same degree [157,158]. Effros et al. also found that concentrations of electrolytes in EBC were too low to be reliable denominators, and suggested that measurements such as conductivity should be developed and to be able to adjust for very low concentrations of markers in EBC [157,158]. Another study suggested that urea and protein in EBC may be helpful denominators, but in the past these have proved to be of limited use in bronchoalveolar lavage .
In addition to low molecular weight infl ammatory mediators, high molecular weight molecules including DNA have been detected. From these studies, mutations of the p53 gene have been detected in EBC of non-small cell lung cancer patients, which suggests a potential area of investigation . Newer and more sensitive assays may be developed to detect other markers and the combination of such markers may identify the characteristic ‘fi ngerprints’ of specifi c infl ammatory cells and their activation. Thus, the activity and number of eosinophils, neutrophils and other infl ammatory cells in the respiratory tract could be assessed indirectly .
NO is associated with a variety of physiological and pathological processes. Products of NO, as nitrite, nitrate, nitrotyrosine and nitrosothiols can be detected in EBC, which is collected by a simple, safe and non-invasive technique, are becoming useful tools for monitoring airway infl ammation. The detection of infl ammatory markers in EBC is not yet a clinical tool because few of the variables studied have been demonstrated to have clinical diagnostic or prognostic utility. Prospective studies will need to investigate any potential markers in EBC and compare them with current ‘gold standards’ for diagnosis and disease activity. Nonetheless, by virtue of its non-invasive nature, this technique holds considerable promise. Already on-line measurements of hydrogen peroxide are available and more bedside analyses are likely to become available. Sensitive assays are needed to measure markers in EBC which have low concentrations, and to explore new markers, but this technique will have far-reaching applicability in both research and possibly clinical medicine.
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