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 .