Abstract
Chronic inflammation is a critical feature of chronic obstructive pulmonary disease, cystic fibrosis, and asthma. This inflammation is associated with the increased production of reactive oxygen species or oxidative stress in the lungs. Oxidative stress may have several adverse effects and may amplify the inflammatory process; however, monitoring oxidative stress is difficult and may not be reflected by changes in blood markers. We have therefore developed several noninvasive markers in the exhaled breath that may indicate oxidative stress in the lungs, and we studied these in relationship to the severity of chronic inflammatory lung diseases. We analyzed the exhaled breath for the content of nitric oxide as a marker of inflammation, carbon monoxide as a marker of oxidative stress, and ethane, which is one of the end products of lipid peroxidation. In addition, we measured the concentration of markers of oxidative stress such as isoprostanes in exhaled breath condensate. Our results confirm that there are increased inflammation, oxidative stress, and lipid peroxidation in lung disease, as shown by elevated levels of nitric oxide, carbon monoxide, and ethane, respectively. The finding of lower levels of these gases in patients on steroid treatment and of higher levels in those with more severe lung disease, as assessed by lung function tests and clinical symptoms, reinforces the hypothesis that the noninvasive measurement of exhaled gases maybe useful in monitoring the underlying pathologic pathways of lung disease. Longitudinal studies are required to assess the clinical usefulness of these measurements in the monitoring of chronic inflammatory lung disease.
Oxidative stress is implicated in the pathogenesis and progression of asthma (1), chronic obstructive respiratory disease (COPD) (2), and cystic fibrosis (CF) (3). Reactive oxygen species are unstable compounds with unpaired electrons, capable of initiating oxidation. Several of the inflammatory cells that participate in the inflammatory response, such as macrophages, neutrophils, and eosinophils, release increased amounts of reactive oxygen species exceeding the already reduced tissue antioxidant defenses of patients with asthma and patients with COPD.
Oxidative stress may have several adverse effects and may amplify the inflammatory process; however, monitoring oxidative stress is difficult and may not be reflected by changes in blood markers. Therefore, we studied exhaled nitric oxide (NO) and carbon monoxide (CO) as markers of inflammation and oxidative stress and ethane as a marker of lipid peroxidation (Figure 1)
Figure 1. Schematic representation of the pathways for the synthesis of nitric oxide (NO), carbon monoxide (CO), and ethane.
[More] [Minimize]The development of noninvasive methods of assessment of inflammation and oxidative stress may provide a means of monitoring disease progress and response to therapy.
CO is produced endogenously from the stress protein heme oxygenase (HO)-1, which is induced by oxidants, inflammatory cytokines, and other forms of cellular stress in a variety of cell types. HO-1 plays an important role in the response to oxidative stress (4). HO-1 converts heme and hemin to biliverdin with the formation of CO. Biliverdin is rapidly converted to bilirubin, which is a potent antioxidant. CO may also be produced by the activity of HO-2, a constitutive enzyme highly expressed in the brain and testes.
The precise mechanisms for antioxidant protection are not fully understood, but both the degradation of heme with removal of iron and induction of ferritin and the generation of bilirubin (an antioxidant) may be involved. There is evidence that the deleterious effects of reactive oxygen species, such as superoxide and hydrogen peroxide, are dependent on the presence of iron. The intracellular pool of free iron can react with both hydrogen peroxide and superoxide, giving rise to the hydrogen oxide radical via the Fenton reaction. The free iron that is not metabolized intracellularly sequestered in cells as ferritin. Thus, ferritin serves as a reservoir to restrict iron from participating in the Fenton reaction. It has been shown that free iron released from heme by HO may induce ferritin synthesis, and heme-induced HO-1 protein also activates ferritin via mRNA expression. Furthermore, the metabolite of heme degradation, bilirubin, is itself an effective antioxidant of peroxynitrite-mediated protein oxidation and may be even more effective than vitamin E in preventing lipid peroxidation. Moderate overexpression of HO-1 improves the resistance of cells to oxygen toxicity.
Exhaled NO can be measured in the airways and has been suggested as a marker of airway inflammation. The inducible enzyme responsible for the synthesis of NO (iNOS) can be activated by inflammatory cytokines, whereas HO-1 is induced both by cytokines and oxidants; therefore, exhaled NO may reflect inflammation, whereas CO may be a marker of inflammation and oxidative stress (Figure 2)
Figure 2. Levels of exhaled carbon monoxide (CO) in normal subjects (open squares) and patients not on steroid treatment (closed circles) and patients on steroid treatment (open circles) for asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF).
[More] [Minimize]In most studies, CO is measured electrochemically. The sensors used are selective and provide reproducible results. CO can also be measured by laser spectrophotometer and near-infrared CO analyzers. Gas chromatography is a reference method for CO measurements, but its use is limited to specialized laboratories.
The exhalation maneuver is a single exhalation at a constant flow rate (5–6 L/minute) after inspiration to total lung capacity from functional residual capacity. The measurement is completely noninvasive and reproducible. It is well tolerated by patients with poor lung volumes and by children.
There is an increase in exhaled CO in patients with asthma (6) (Figure 2), and we have demonstrated that this is associated with HO-1 expression in macrophages in induced sputum (7). There is a further increase of CO during allergen challenge, whereas its levels are not modified by methacoline-induced bronchocostriction (8), confirming that exhaled CO is a marker of oxidative stress in the airways and is not influenced by bronchocostriction per se.
The difference in exhaled CO between normal subjects and subjects with asthma, however, is much less than in exhaled NO, and the effect of inhaled steroids on exhaled CO in patients with mild asthma, as it has been reported recently, is negligible (4). Both HO-1 and HO-2 are extensively distributed in airways of normal subjects and subjects with asthma. The increased levels in stable asthma are likely to be due to preferential increase of HO-1 expression, which is seen in alveolar macrophages in induced sputum of patients with asthma (7). There is also an increase in the concentration of bilirubin in induced sputum, indicating increased HO-1 activity. Further evidence that exhaled CO increases may reflect HO activity is the demonstration that inhaled hemin, which is a substrate for HO, results in a significant increase in exhaled CO concentration in normal subjects and subjects with asthma (7). Increased levels of exhaled CO are seen in acute exacerbations of asthma and are reduced after treatment with oral corticosteroids (9).
Smoking causes an acute increase in exhaled breath CO, making this measurement less useful in this group of patients. We found high levels of exhaled CO in ex-smoker COPD patients (10) (Figure 2). Exhaled CO levels are further increased during acute exacerbations of COPD, with a decline after recovery.
Heme oxygenase is present in the pulmonary vascular endothelium and alveolar macrophages and can be upregulated by oxidative stress and proinflammatory cytokines, thus increasing the production of CO. We presume that the high levels of exhaled CO found in COPD patients are due to inflammatory cytokines or reactive oxygen species–induced HO-1 expression and therefore that the measurement of exhaled CO may reflect inflammation, oxidative stress, or both.
Exhaled CO levels were markedly elevated in patients with stable CF (11) and increased further during exacerbations and reduced with antibacterial treatment (Figure 2) (12). This suggests that exhaled CO is not only a marker of oxidative stress/inflammation in CF but is also a marker of disease severity. This is further confirmed by the finding of lower CO levels in patients receiving oral corticosteroid treatment (11). In fact, by reducing airway inflammation and the release of oxidants by inflammatory cells, steroids may attenuate HO-1 expression and the synthesis of CO. We have shown that patients homozygous for the CF transmembrane regulator ΔF508 mutation have higher exhaled CO levels than do heterozygous patients (11). Considering the growing interest in gene therapy in CF, further studies are needed to investigate the role of CO levels in the assessment of effective therapeutic gene delivery or to confirm the diagnosis in patients with borderline sweat tests where more extensive genetic analysis is not available.
The determination of hydrocarbons in the exhaled air has been proposed as a means to assess lipid peroxidation in vivo (13). Ethane and pentane are hydrocarbons released during lipid peroxidation in biologic tissues. Ethane specifically results from the effects of free radicals on the omega-3 fatty acids such as 9,12,15-linolenic acid, whereas pentane derives from the peroxidation of n-6 polyunsaturated acids such as 9,12,15-linoleic and arachidonic acid.
Ethane has received particular attention because of its easier and faster chromatographic measurement compared with other hydrocarbons. The first analyses of organic compounds present in the exhaled air from human subjects was performed in the 1960s (14). Since then, the research in this area has progressed slowly because of the technical and practical problems, such as the influence of ambient hydrocarbons on exhaled breath levels of these gases.
We modified a previously developed technique for single breath analysis of exhaled hydrocarbons (15) by allowing airways dead space washout during exhalation, eliminating ambient contamination of the exhaled breath.
Exhaled air was collected during a flow- and pressure-controlled exhalation into a reservoir discarding dead space air contaminated with ambient air, as previously described (16). A sample (2 ml) of the collected expired air was analyzed for ethane content using gas chromatography.
We found elevated levels of exhaled ethane in patients with asthma compared with normal subjects (17) (Figure 3)
Figure 3. Levels of exhaled ethane in normal subjects (open squares), patients with asthma not on steroid treatment (closed circles), and patients with asthma on steroid treatment (open circles).
[More] [Minimize]Patients with COPD have elevated levels of exhaled CO and ethane, and the latter correlates with disease severity as assessed by FEV1 (Figure 4)
Figure 4. (A) Levels of exhaled ethane in normal subjects (open squares), patients with chronic obstructive pulmonary disease not on steroid treatment (closed circles) and on steroid treatment (open circles). (B) The correlation of exhaled ethane with FEV1.
[More] [Minimize]Pentane and isoprene are increased in normal smokers. Although vitamin E given for 3 weeks failed to reduce exhaled ethane in cigarette smokers, those whose ethane values fell the most tended to have better preserved lung function (20).
Increased levels of volatile organic compounds in exhaled breath could be used as biochemical markers of exposure to cigarette smoke and oxidative damage caused by smoking.
Patients with CF have elevated levels of exhaled ethane, which is significantly correlated with exhaled CO and airway obstruction (21), supporting the view that oxidative stress and lipid peroxidation are increased in the airways of patients with CF.
Steroid treatment was associated with lower levels of exhaled ethane and CO. Steroids, in fact, by reducing inflammation, attenuating the release of oxidants by inflammatory cells, and suppressing proinflammatory cytokines production may reduce HO-1 expression and lipid peroxidation and, therefore, the synthesis of CO and ethane. This finding indicates that ethane and CO are better markers of steroid activity than NO, which is not affected by steroid treatment. One explanation for this may be that the expression of iNOS is so reduced in patients with CF (22) that despite the inhibition of its activity by steroids and the attenuation of oxidants release by inflammatory cells, exhaled NO levels remain unchanged.
The measurement of NO has been suggested as a noninvasive tool to assess inflammation; however, because of the interactions of this gas with the synthesis of CO, which is a marker of oxidative stress, the measurement of exhaled NO may also reflect oxidative stress in the airways.
NO is a gas produced by several types of pulmonary cells, including inflammatory cells and endothelial and airway epithelial cells. NO is the oxidation product of the terminal guanidine group of l-arginine. The reaction is sterospecific and catalyzed by a number of isoforms of NOS. Three different NOS isoforms have been identified. Two are constitutively expressed in the epithelium, one termed “eNOS” is found in the endothelium, cardiac myocytes, platelets, hippocampus, whereas the second termed “nNOS” is found in neural tissue and in the skeletal muscles. Conversely, the iNOS is not expressed in most tissues but is induced by bacterial lypopolysaccharide or cytokines.
Besides being a reactive free radical, NO is also a highly diffusible gas. The rapid reaction of NO with metalloproteins (e.g., hemoglobin, guanyl cyclase), superoxide as well as thiol groups (e.g., cysteine) makes direct measurements in biologic tissue difficult. Its poor solubility in aqueous solutions and stability in the gas phase at low concentrations means that NO produced in superficial structures of hollow organs will tend to diffuse and be detectable in gas collected from the lumen.
Expiratory flow, soft palate closure, and dead space air may all influence exhaled NO levels. Therefore, exhaled NO is usually determined during single-breath exhalations against a resistance to prevent contamination with nasal NO or using reservoir collection with discarding of the dead space. The levels of exhaled NO are measured by chemiluminescence. This method is reproducible and has been standardized.
Elevated levels of exhaled NO in asthma (23), after allergen challenge (8) and in interstitial lung disease (24), are likely to be due to the activation of the iNOS (25) and therefore may reflect airway inflammation. In COPD, the measurement of exhaled NO as a noninvasive marker of inflammation has previously been investigated (10, 26). We found elevated NO levels in COPD patients, which were not influenced by steroid treatment (10). This is consistent with the finding that inflammation in COPD is not suppressed by inhaled or oral corticosteroids, even at high doses (27). This lack of effect may be due to the fact that corticosteroids prolong the survival of neutrophils (28).
It is interesting to note that despite an increase in cytokines such as interleukin-1β and tumor necrosis factor-α (29), which are known to upregulate the iNOS, exhaled NO is not elevated in patients with CF (11). One reason for this may be the absence of detectable immunoreactivity for iNOS in lung epithelium (30). Decreased exhaled NO concentrations may also be due to retention and metabolism of NO within the airways, as shown by an increased production of NO metabolites (nitrates and nitrites) in tracheal secretions (31). Furthermore, CF is characterized by an intense neutrophilic inflammation in the airways. Activated neutrophils decrease NO production by epithelial cells, increasing the formation of peroxynitrates in a rapid interaction between NO and superoxide anions released from neutrophils (32).
Exhaled breath condensate is collected by cooling or freezing exhaled air and is totally noninvasive. Potentially, condensate measurements reflect different markers and molecules derived from the mouth (oral cavity and oropharynx), tracheobronchial system, and alveoli, and their proportional contribution has not yet been sufficiently studied. It is assumed that airway surface liquid becomes aerosolized during turbulent airflow so that the content of the condensate reflects the composition of airway surface liquid, although large molecules may not aerosolize as well as small soluble molecules.
Several nonvolatile compounds have now been detected in breath condensate. Hydrogen peroxide (18), leukotrienes (33), thiobarbituric acid–reactive substances (34), and isoprostanes (35) have all been shown to be elevated in asthma, COPD, and CF and have been suggested as possible markers of inflammation and oxidative stress (36). In summary, the concentration of hydrogen peroxide is related to the number of sputum eosinophils and airway responsiveness in asthma. In patients with mild asthma, levels of leukotriene E4, C4, and D4 in exhaled condensate are increased during the late asthmatic response to allergen challenge and are elevated significantly in patients with moderate or severe asthma (33). The concentrations of 8-isoprostanes have also been shown to be doubled in patients with asthma compared with normal subjects and increased by three times in those with severe asthma (35).
The reaction of NO and superoxide anions (O2−) in the airway results in the formation of peroxynitrite, a highly reactive oxidant species. Peroxynitrite reacts with tyrosine residues in proteins to form the stable product nitrotyrosine. The analysis of exhaled breath condensate of patients with asthma has shown elevated levels of 3-nitrotyrosine (33), confirming that this group of patients have increased oxidative stress in the airways.
The research in the field of exhaled breath condensate has shown promising results; however, these findings still need to be confirmed and validated. Further studies are necessary to standardize the method of sample collection and analysis.
Accurate assessment of airway inflammation and oxidative stress, its severity, and its location within the lung is important for the clinical management of lung disease.
Inflammation and oxidative stress are a complex reaction to stimuli such as infection, trauma, or exposure to exogenous toxins or irritants. The assessment and the measurement of the intensity of inflammation and oxidative stress would allow us to tailor the therapy according to the specific need of the patients.
Considering the complexity of inflammation and oxidative stress and the interaction of one with the other, it is unlikely that a single molecule measured in the exhaled breath, or in other biological fluids, may provide a complete picture. Therefore, several markers have been studied, each of them reflecting different pathways in the inflammatory process (Figure 1).
Our results confirm that there is increased inflammation (NO), oxidative stress (CO), and lipid peroxidation (ethane) in patients with asthma, COPD, and CF. Patients on steroid treatment had lower levels of exhaled gases, confirming that these noninvasive measurements maybe a useful tool to monitor disease activity. Steroid treatment was associated with lower levels of exhaled NO confirming the data of previous publications (37). Steroids, in fact, by reducing inflammation, attenuating the release of oxidants by inflammatory cells (38), and suppressing proinflammatory cytokine production (39) may reduce iNOS (40) expression and therefore the synthesis of NO. It is noteworthy that patients with CF have low levels of NO and that patients on steroid treatment do not have lower levels of exhaled NO compared with untreated patients. This is probably due to the reduced iNOS activity in CF despite inflammation in the airways and the release of compounds known to activate iNOS such as interleukin-1β and tumor necrosis factor-α (41), which are known to upregulate iNOS. This makes the measurement of NO not suitable for the evaluation of airway inflammation in CF; however, the finding of high levels of CO and ethane in patients with CF and their reduction in steroid-treated patients suggest the simultaneous measurements of NO, CO, and ethane maybe a new tool to assess disease progression; furthermore, the finding of high ethane and CO levels together with low NO in the exhaled breath of a patient with lung disease could aid in the diagnosis of CF.
Exhaled ethane was also reduced in steroid-treated patients with asthma, COPD, and CF, showing that steroid treatment can effectively attenuate lipid peroxidation, as already shown in previous studies (42–44). However, 8-isoprostane, another marker of lipid peroxidation, shows resistance to steroid treatment. This may be due to the fact that 8-isoprostane may also have an enzymatic synthesis by cyclooxygenase-1 and cyclooxygenase-2 besides deriving from the free radical peroxidation of the arachidonic acid; therefore, the final levels of 8-isoprostanes may reflect a more complex phenomenon rather than the lipid peroxidation per se.
In patients not on steroid treatment, there was a significant correlation between exhaled ethane, CO, residual volume to total lung capacity ratio, and FEV1 in patients with CF and COPD. This observation is in keeping with the findings in smokers of a negative correlation between exhaled ethane and airway obstruction (20).
The inverse correlation between exhaled ethane, CO, and airway obstruction indicates that patients with more severe disease have higher levels of exhaled ethane, possibly reflecting a more active underlying lipid peroxidation.
Exhaled ethane is a marker of lipid peroxidation, and therefore, it reflects the damage of cell membranes caused by reactive oxygen species. On the other hand, exhaled CO is an indirect measurement of oxidative stress mediated by HO activity. The measurement of exhaled ethane and CO may complement each other in patients with COPD exacerbations in which the intensity of the oxidative stress and the actual cell damage may provide different values. The combined use of exhaled ethane and CO as noninvasive markers of oxidative stress is particularly appealing if one considers that decreasing the inflammatory response may prevent structural damage to the airways.
Even though monitoring airway inflammation and oxidative stress by measuring exhaled gases is appealing and promising, further studies are required to clarify the following points:
Standardization. The measurement of exhaled NO has been standardized and guidelines for its measurement have been defined in two International Taskforce meetings (45, 46); similar standardization methods are now needed for exhaled ethane and CO.
Clinical implications. Whether repeated measurements of exhaled markers will help in the clinical management of lung diseases needs to be determined by longitudinal studies relating exhaled markers to other measurements of asthma control.
Cost. There is a need for cheaper analyzers. The systems of detection used for the analysis of exhaled NO and ethane are expensive. In addition, although the measurement of exhaled NO is simple and requires only little practice by the operator, the measurement of exhaled ethane requires the expertise of a gas cromatograph technician and is time consuming.
Effect of antioxidants. Studying the effect of antioxidants on the levels of the compounds reflecting oxidative stress in the exhaled breath may offer an insight into the pathogenesis of lung disease as well as providing a clinical tool in the assessment of drug efficacy.
The histologic examination of the airway wall has revealed increased vascularity (47) and bronchial blood flow (48) in asthma. These changes contribute to airway remodeling and are correlated with the severity of the disease (49). Bronchial blood flow (50) and exhaled breath temperature (51) may reflect the final common pathways of inflammation. The measurement of these parameters maybe another noninvasive way to assess the level of inflammation in the airways as suggested by the correlation of exhaled NO and exhaled breath temperature (52, 53).
Numerous new compounds can be measured in exhaled breath condensate such as hydrogen peroxide, eicosanoids, product of lipid peroxidation, 8-isoprostane, 3-nitrotyrosine, nitrosothiols; however, the clinical usefulness of these measurements and their reproducibility are still to be confirmed.
The measurement of exhaled gases may provide a means of detecting and monitoring cytokine-mediated inflammation and oxidant stress in the airways and of assessing the efficacy of treatment. Because the measurement of exhaled gases is simple and noninvasive, it can be repeated and may be applied to children and to patients with severe disease. Because the techniques are noninvasive, it is possible to make repeated measurements without disturbing the system, in contrast to the invasive procedures currently used.
Longitudinal studies are required to investigate the usefulness of these measurements in a clinical setting; furthermore, less sophisticated and cheaper devices are required to allow the day to day use of these techniques in the clinical management of inflammatory lung diseases.
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