Exhaled Ethane Is Elevated in Cystic Fibrosis and Correlates with Carbon Monoxide Levels and Airway Obstruction PAOLO PAREDI, SERGEI A. KHARITONOV, DAVID LEAK, PALLAV L. SHAH, DEREK CRAMER, MARGARET E. HODSON, and PETER J. BARNES Department of Thoracic Medicine, National Heart and Lung Institute; Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London; and Lung Function Unit and Cystic Fibrosis Department, Royal Brompton Hospital, London, United Kingdom
Ethane is produced from lipid peroxidation and can be measured in the exhaled air. Cystic fibrosis (CF) is characterized by recurrent respiratory infections, release of reactive oxygen species by inflammatory cells, and increased oxidative stress. We measured exhaled ethane in 23 CF subjects (mean age ⫾ SEM, 21 ⫾ 4 yr; 10 male, FEV1 62 ⫾ 4%) and compared it with two other noninvasive markers of oxidative stress and inflammation, carbon monoxide (CO) and nitric oxide (NO). Exhaled ethane was collected during a flow and pressure-controlled exhalation into a reservoir discarding dead space air contaminated with ambient air. A sample (2 ml) of the expired air was analyzed by chromatography. Ethane levels were elevated in patients not on steroids (n ⫽ 13, 1.99 ⫾ 0.20 ppb) compared with steroid-treated patients (n ⫽ 10, 0.67 ⫾ 0.09 ppb, p ⬍ 0.01) and with 14 nonsmoking control (8 men, age 33 ⫾ 2.8 yr) subjects (0.82 ⫾ 0.40 ppb, p ⬍ 0.05). In patients not on steroid treatment ethane was correlated to airway obstruction as assessed by the ratio of residual volume to total lung capacity (RV/ TLC) (r ⫽ 0.66, p ⬍ 0.05) and exhaled CO (r ⫽ 0.65, p ⬍ 0.05). CO concentrations were also higher in patients not on steroid treatment (3.4 ⫾ 0.2 ppm) than in steroid-treated patients (2.6 ⫾ 0.1 ppm, p ⬍ 0.05), whereas NO concentrations were not influenced by steroid treatment (3.0 ⫾ 0.4 ppm and 2.9 ⫾ 0.2 ppm, p ⬎ 0.05) and were lower than in a control group (7.0 ⫾ 0.4 ppb, p ⬍ 0.05). Exhaled ethane is elevated in CF, reduced in steroid-treated patients and correlates with CO and RV/TLC; therefore, it may be a useful noninvasive marker of oxidative stress.
Cystic fibrosis (CF) is characterized by recurrent respiratory tract infections leading to damage of the airways, development of bronchiectasis, and progressive airflow obstruction. In CF there is an oxidant–antioxidant imbalance with damaging consequences for the bronchial epithelium (1–3). Neutrophils entering the lumen of the infected airways undergo activation and release toxic oxygen metabolites such as superoxide anions (2) and hydrogen peroxide (4) inducing oxidative stress and progression of lung disease and FEV1 decline (5). Evidence for the involvement of reactive oxygen species as mediators of tissue damage in CF has come from studies measuring the products of lipid and protein oxidation in plasma (1), and bronchoalveolar lavage (BAL) (6). These studies have shown increased plasma concentrations of lipid peroxidation products such as thiobarbituric acid, organic hydroperoxides (1, 3) and increased 9,11-linoleic acid to 9,12-linoleic acid ratio (7) in patients with CF compared with control subjects. The determination of hydrocarbons in the exhaled air has been proposed as a means to assess lipid peroxidation in vivo (8–11), and ethane has received particular attention because
(Received in original form June 28, 1999 and in revised form October 5, 1999 ) Correspondence and requests for reprints should be addressed to Professor P. J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK. E-mail:
[email protected] Am J Respir Crit Care Med Vol 161. pp 1247–1251, 2000 Internet address: www.atsjournals.org
of its easier and faster chromatographic measurement compared with other hydrocarbons (12). The first analysis of organic compounds present in the exhaled air from human subjects was performed in the 1960s (13). 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 developed a new method in which ambient air contamination is eliminated by allowing airways washout during exhalation and applied this simplified technique to the measurement of exhaled ethane in CF. Noninvasive markers of oxidative stress would be of great benefit in disease management and monitoring and in the assessment of drug efficacy. In this respect exhaled carbon monoxide (CO) and nitric oxide (NO) have already been investigated. Exhaled NO has been shown to be a marker of inflammation in asthma (14) and bronchiectasis (15). However, despite the intense chronic airway inflammation in CF, exhaled NO concentrations are lower than normal, so that this measurement is of no utility for monitoring lung inflammation in this disease. CO is a product of heme degradation by heme oxygenase (HO). The inducible form of HO (HO-1) is activated by a variety of proinflammatory cytokines and oxidants (16). Exhaled CO levels are elevated in asthma (17) and CF (18) and may reflect oxidative stress in the airways. In view of the role of oxidative stress in the pathogenesis and progression of CF, we measured exhaled ethane as a marker of lipid peroxidation, and compared it with these two noninvasive markers of oxidative stress and inflammation, NO and CO. We also studied exhaled ethane in relation to disease severity as assessed by lung function tests.
METHODS Patients Twenty-three patients with CF (10 male, age 21 ⫾ 4 yr, FEV1 62 ⫾ 4% of predicted, 10 on oral steroid treatment with prednisone 1 mg/ kg every other day), and 14 control subjects (age 33 ⫾ 3 yr, 8 male) were recruited (Table 1). All patients were stable and lung function tests did not differ between untreated and steroid treated subjects; therefore, patients in these two groups had a similar disease severity. Patients colonized with Burkholderia cepacia, methicillin-resistant Staphylococcus aureus (MRSA) or with acute chest infection or disease exacerbation were excluded from the study because of risk of chest infection. None of the patients enrolled in this study had clinical signs of pancreatic insufficiency. Patients with history of diabetes, liver disease, lung cancer, or alcohol/drug abuse were not eligible for the study (8). All patients were lifelong nonsmokers with the exception of one patient who stopped smoking 1 yr before entering the study. The smoking status of all the subjects was confirmed by nicCheck (DynaGen, Inc., Cambridge, MA), which detects nicotine and its metabolites in urine. Active and passive smokers (smoke exposure for more than 30 min/d) were excluded from the study. All subjects had at least 1 h of rest before gas measurement, in order to eliminate
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PATIENT CHARACTERISTICS Cystic Fibrosis Not Steroid-treated (n ⫽ 13)
Exhaled Ethane
Exhaled NO and CO Measurements Exhaled CO was measured by a modified electrochemical sensor with sensitivity from 1 part per million (ppm) to 500 ppm of CO, simultaneously with NO measurement by LR2000 chemiluminescence analyzer (Logan Research Ltd, Rochester, Kent, UK) in order to control exhalation parameters (resistance 3 ⫾ 0.4 mm Hg; exhalation flow 5 to 6 L/min). The subjects exhaled slowly from total lung capacity (TLC) over 10 to 15 s maintaining a constant flow. The mean of two reproducible measurements was recorded. Ambient CO was recorded before each measurement and subtracted from the mean value obtained during the maneuvers. NO was measured as described previously (20).
Lung Function Tests After the measurement of exhaled gases, all patients underwent pulmonary function tests, including spirometry and lung volumes, using a Jaeger Master Lab Compact Transfer (Erich Jaeger Ltd., Leicestershire, UK). We used the Brompton Hospital reference standards for spirometric lung function in children age 4 to 19 yr (21) and the European Community for Steel and Coal standards for adults (22).
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TABLE 1
the effect of any possible exposure to high CO concentrations during their journey to the hospital. Lung function tests were performed after exhaled breath analysis for ethane, CO, and NO.
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 (19). The time needed to wash out the dead space (t) was estimated to be 1 to 2 s (t ⫽ dead space volume/ exhalation flow, where dead space was calculated as weight [lb] ⫹ age in years, and exhalation flow was 10 to 11 L/min). A sample (2 ml) of the collected expired end-tidal air was analyzed for ethane content using gas chromatograph model Philips PU 4500, with a column Poropak Q 1–3 m ⫻ 4 mm, column temperature 60⬚ C, injector temperature 140⬚ C, detector temperature 160⬚ C, signal output to a Schimadtzu CR6A integrator. In a preliminary study we evaluated the reproducibility of this method. The difference in exhaled ethane concentrations measured during two successive collections at 5-min intervals (single session variability) was 5.4% (n ⫽ 32 subjects); between-session variability (n ⫽ 6 subjects, 1-d interval) was 6.2%. Ethane concentration was equally stable in five polyethylene and five Tedlar reservoirs for 48 h after collection (percent increase: 5 ⫾ 2% and 3 ⫾ 1% for the polyethylene and Tedlar reservoirs, respectively).
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Age, yr* Sex, M/F RV/TLC, % predicted* FEV1⫹, % predicted* Smokers Ex-smokers
20 ⫾ 4 4/9 145 ⫾ 15 69 ⫾ 4 0 1
Cystic Fibrosis Steroid-treated (n ⫽ 10)
Normals (n ⫽ 14)
22 ⫾ 6 4/6 170 ⫾ 11 64 ⫾ 5 0 0
33 ⫾ 3 8/6 95 ⫾ 9 97 ⫾ 8 0 0
Definition of abbreviation: RV/TLC ⫽ residual volume/total lung capacity. * Values are means ⫾ SEM.
RESULTS Exhaled Ethane
Ethane levels were elevated in patients not on steroids (1.99 ⫾ 0.20 parts per billion [ppb]) compared with steroid-treated patients (0.67 ⫾ 0.11 ppb, p ⬍ 0.01) and with control subjects (0.82 ⫾ 0.09 ppb, p ⬍ 0.05, Figure 1A). In patients not on steroid treatment ethane was correlated with CO concentrations (r ⫽ 0.65, p ⬍ 0.05, Figure 2) and airway obstruction as assessed by the ratio of residual volume and total lung capacity (RV/TLC) (r ⫽ 0.66, p ⬍ 0.05, Figure 1B). The level of bronchoconstriction was not significantly different in steroid-treated (RV/TLC: 170 ⫾ 11% predicted, FEV1 64 ⫾ 5%) compared with untreated patients (RV/TLC: 145 ⫾ 15% predicted, FEV1 69 ⫾ 4% predicted, p ⬎ 0.05). There was no significant correlation between exhaled ethane concentration and body weight or body surface area. Exhaled CO
Exhaled CO levels were higher in untreated patients (3.4 ⫾ 0.2 ppm) than in steroid-treated patients (2.6 ⫾ 0.1 ppm, p ⬍ 0.05) and normal subjects (2.1 ⫾ 0.1 ppm, p ⬍ 0.05, Figure 3A). There was a significant correlation between CO and RV/ TLC (r ⫽ 0.76, p ⬍ 0.65, Figure 3B).
Statistics
Exhaled NO
Comparisons between groups were made by one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. Data were expressed as means ⫾ SEM and confidence intervals of differences. Significance was defined as a p value of ⬍ 0.05.
NO concentrations were not influenced by steroid treatment (3.0 ⫾ 0.4 ppb and 2.9 ⫾ 0.2 ppb, p ⬎ 0.05) and were lower in patients with CF than in the control group (7.0 ⫾ 0.4 ppb, p ⬍ 0.05, Figure 4).
Figure 1. Concentrations of exhaled ethane in normal subjects (open squares), patients with CF not on steroid treatment (closed circles), and steroid-treated patients (open circles) (panel A). Panel B shows the correlation of exhaled ethane with residual volume/ total lung capacity (RV/TLC).
Paredi, Kharitonov, Leak, et al.: Exhaled Gases in Cystic Fibrosis
Figure 2. Correlation of exhaled ethane with exhaled CO in patients with CF not on steroid treatment (closed circles) and steroid-treated patients (open circles).
DISCUSSION We have demonstrated that patients with CF have elevated levels of exhaled ethane, which significantly correlate with CO and airway obstruction as assessed by RV/TLC. We interpret these findings as confirmation that oxidative stress and lipid peroxidation are increased in the airways of patients with CF. Oxidative stress and reactive oxygen species (ROS) have been implicated in the pathogenesis of CF (1–3). Even though hydrogen peroxide levels are not elevated in CF patients (23), other ROS such as superoxide anion (O2⭈⫺) (2), and hydroxyl radicals (2, 4), produced by activated neutrophils and macrophages (2, 24) may cause oxidation of nucleic acids, proteins, and membrane lipids (1, 3, 6). Breath hydrocarbons have been studied as a measure of lipid peroxidation (8–11). Polyunsaturated fatty acids are present in cellular and subcellular membranes and are prone to lipid peroxidation, as a result of the weak binding of the hydrogen atoms to the carbon chain. Ethane and pentane are the main hydrocarbons released during lipid peroxidation. The noninvasive nature of hydrocarbon assessment renders the hydrocarbons breath test an attractive means of assessing lipid peroxidation and oxidative stress in humans (8). Because an oxidant/antioxidant imbalance contributes to the pathogenesis of CF (1) and can lead to lung injury owing to direct oxidative damage to epithelial cells, we measured exhaled ethane as an index of lipid peroxidation and compared it
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with two other noninvasive markers of inflammation and oxidative stress, CO and NO. Exhaled ethane levels were elevated in CF patients, confirming that lipid peroxidation is increased in the airways of CF patients. It is noteworthy that exhaled ethane concentrations were correlated with CO, confirming the hypothesis that both gases reflect oxidative stress in the lung. CO is a product of heme degradation by HO. The inducible form of HO (HO-1) is activated in the alveolar macrophages of asthmatic patients compared with normal subjects (17) as a result of increased oxidative stress. Elevated levels of CO in exhaled air may reflect the degree of HO-1 induction as confirmed by higher levels of CO during allergen challenge (25). In the present study we found high levels of exhaled CO in patients with CF as previously shown (18). Heme oxygenase is present in the pulmonary vascular endothelium and alveolar macrophages (26) and can be upregulated by oxidative stress and proinflammatory cytokines (27), thus increasing the production of CO. We presume that the high concentrations of exhaled CO found in patients with CF are caused by inflammatory cytokines or ROS-induced HO-1 expression and therefore that the measurement of exhaled CO may reflect inflammation, oxidative stress, or both. NO is a gas produced by several types of pulmonary cells, including inflammatory, endothelial, and airway epithelial cells. Elevated levels of exhaled NO in asthma (14), and bronchiectasis (15) are likely to be due to the activation of the inducible form of NO synthase (iNOS) and therefore may reflect airway inflammation. Inflammation also plays an important role in CF. However, despite an increase in cytokines such as interleukin-1 (IL1), and tumor necrosis factor-alpha (TNF-␣) (28) which are known to upregulate the inducible form of NO synthase (iNOS), exhaled NO is not elevated in patients with CF. One reason for this may be the absence of detectable immunoreactivity for iNOS in lung epithelium (29). Alternatively, high concentrations of CO may inhibit iNOS activity and therefore reduce the concentrations of exhaled NO. Decreased exhaled NO concentrations may also be caused by retention and metabolism of NO within the airways as shown by an increased production of NO metabolites (nitrates and nitrites) in tracheal secretions (30). Furthermore, CF is characterized by an intense neutrophilic inflammation in the airways. Activated neutrophils decrease NO production by epithelial cells increasing the formation of peroxinitrates in a rapid interaction between NO and superoxide anions released from neutrophils (31). The finding of low concentrations of exhaled NO associ-
Figure 3. Concentrations of exhaled CO in normal subjects (open squares), patients with CF not on steroid treatment (closed circles), and steroidtreated patients (open circles) (panel A). Panel B shows the correlation of CO with RV/TLC.
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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. The profile of exhaled gases observed in this study may be of pathophysiological significance. It will be necessary to intervene with specific inhibitors of HO and lipid peroxidation or with antioxidants in order to explore this possibility further. References
Figure 4. Concentrations of exhaled NO in normal subjects (open squares), patients with CF not on steroid treatment (closed circles), and steroid-treated patients (open circles).
ated with high concentrations of exhaled ethane and CO may be of assistance in the diagnosis of the disease and may provide an insight into the pathogenesis of the disease. 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 be complementary in patients with exacerbations where 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. In patients not on steroid treatment there was a significant correlation between exhaled ethane, CO, and RV/TLC which is a sensitive test of small airway disease in CF. This observation is in keeping with the findings in smokers of a negative correlation between exhaled ethane and airway obstruction in smokers (32). The loss of this correlation in steroid-treated patients may be a result of the normalization of ethane concentrations and reduction of individual differences between patients. The level of bronchoconstriction was similar in steroid-treated and untreated patients; therefore, this may not be a contributing factor to the different concentrations of exhaled ethane in these two groups. The inverse correlation between exhaled ethane, CO, and airway obstruction indicates that patients with more severe disease have higher concentrations of exhaled ethane, possibly reflecting a more active underlying lipid peroxidation. Steroid treatment was associated with lower concentrations of exhaled ethane and CO. Steroids, in fact, by reducing inflammation, attenuating the release of oxidants by inflammatory cells (33), and suppressing proinflammatory cytokines production (34) may reduce HO-1 expression (27) 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 (29) that despite the inhibition of its activity by steroids (35) and the attenuation of oxidants release by inflammatory cells (33) exhaled NO concentrations remain unchanged. Measurement of exhaled ethane and CO may provide a means of detecting and monitoring cytokine-mediated inflammation and oxidant stress in the airways and of assessing the
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