INTRODUCTION

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Oxidative stress is thought to be associated with the pathogenesis and progression of asthma (1, 2). Reactive oxygen species (ROS) 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 ROS (1) exceeding the already reduced tissue antioxidant defenses of asthmatic patients (3).

One mechanism by which oxidants may cause lung injury is through lipid peroxidation. ROS, such as superoxide anion (O₂^.), and hydrogen peroxide (H₂O₂) released by activated immune and inflammatory cells can induce the lipid peroxidation of polyunsaturated membrane fatty acids (4), impair membrane function and inactivate membrane-bound receptors and enzymes, increase tissue permeability (5), and therefore promote airflow limitation. That lipid peroxidation is increased in asthmatic patients is confirmed by the finding of increased concentrations of 8-isoprostane, a product of oxidation of arachidonic acid, in exhaled air condensate (6).

The measurement of exhaled hydrocarbons has been proposed as a means to assess lipid peroxidation in vivo in several studies (4, 7, 8). In this group of exhaled gases, ethane has received more attention because of its easier and faster chromatographic measurement compared with other hydrocarbons (9). The exhaled air of human subjects was first analyzed for the presence of hydrocarbons in the 1960s (10). Since then, the research in this area has progressed slowly because of 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 (9) by allowing airways dead space washout during exhalation, eliminating ambient contamination of the exhaled breath. We applied this simplified technique to the measurement of exhaled ethane in asthmatic patients.

Noninvasive markers of inflammation and oxidative stress would be of great benefit in disease management and monitoring and in the assessment of drug efficacy. In this respect other exhaled gases have already been investigated. In asthma high concentrations of exhaled nitric oxide (NO) (11) may reflect the release of several mediators, including cytokines and ROS, that can activate inducible NO synthase (iNOS), the enzyme that catalyzes the production of NO.

In view of the role of oxidative stress, lipid peroxidation, and inflammation in the pathogenesis of asthma, we measured exhaled ethane as a marker of lipid peroxidation, and compared it with NO, a noninvasive volatile marker of inflammation.

	METHODS

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Patients

Twenty-six patients (15 male, age 38 ± 8 yr, FEV₁ 60 ± 4% of predicted, 14 on steroid treatment, nine of whom had severe persistent asthma and five moderate persistent asthma), and 14 control subjects (age 33 ± 3 yr, eight male) were recruited from our outpatient clinic (Table 1). Review of medical records confirmed that the diagnosis of asthma was established in each patient according to American Thoracic Society criteria (12). Patients with acute chest infection, upper respiratory tract infection, or disease exacerbation during the month before enrollment were excluded from the study. None of the patients was on a vitamin supplement diet. Patients with history of diabetes, liver disease, lung cancer, or alcohol/drug abuse were not eligible for the study. All patients were lifelong nonsmokers; 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 ¹/₂ h/d) were excluded from the study. All subjects had at least 1 h of rest before gas measurement, in order to eliminate the effect of any possible exposure to high ambient gas concentrations during their journey to the hospital. Lung function tests were performed after exhaled breath analysis for ethane and NO.

Exhaled Ethane

Exhaled breath was collected into a collapsible reservoir during a single exhalation from TLC to residual volume (RV) at a constant flow (10 to 11 L/min) over 20 to 30 s against a mild resistance (13). During exhalation the air coming from the dead space, contaminated with nasal and ambient ethane, was discarded in the atmosphere by a three-way valve. The time needed to wash out the dead space (t) is estimated to be 1 to 2 s (t = dead space volume/exhalation flow where dead space is calculated as weight [lb] + age in years, and exhalation flow was 10 to 11 L/min).

Once the exhaled air was collected, the reservoir was immediately sealed and the sample was kept at room temperature and analyzed for ethane content within 48 h. Two samples of exhaled breath and one of ambient were collected for each patient. The concentration of ambient ethane was subtracted from the final exhaled ethane concentration to reduce exhaled breath contamination. The mean of two samples (with a variability lower than 3%) was recorded.

A sample (2 ml) of the collected expired air was analyzed for ethane content using a gas chromatograph (model PU 4500; Phillips), with a column Poropak Q 1 to 3 m × 4 mm, column temperature 60° C, injector temperature of 140° C, detector temperature of 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), whereas 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).

Exhaled NO Measurements

Exhaled NO was measured using a modified chemiluminescence analyzer (model LR2000; Logan Research, Rochester, UK), sensitive to NO from 1 to 5,000 parts per billion (ppb) (by volume), and with a resolution of 0.3 ppb, which was designed for online recording of exhaled NO concentration. The analyzer was calibrated using certified NO mixtures (90 ppb and 436 ppb) in nitrogen (BOC Special Gases; Guildford, UK). Measurements of exhaled NO were made by slow exhalation (5 to 6 L/min) from TLC for 20 to 30 s against a resistance (3 ± 0.4 mm Hg).

Lung Function Tests

After the measurement of exhaled gases, all patients underwent lung function testing, including spirometry and lung volumes, using a Jaeger Master Lab Compact Transfer (Erich Jaeger Ltd., Leicestershire, UK).

Statistics

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 less than 0.05.

	RESULTS

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Exhaled Ethane

Ethane levels were elevated in patients who were not receiving steroids (2.06 ± 0.30 ppb) compared with steroid-treated patients (0.79 ± 0.10 ppb, p < 0.01) and with control subjects (0.88 ± 0.09 ppb, p < 0.05, Figure 1A). When patients were divided into two groups according to their FEV₁ we found that among patients not undergoing steroid treatment, those with more severe disease (FEV₁ < 60% predicted, Group A) had higher concentrations of ethane (2.86 ± 0.37 ppb) compared with untreated patients with FEV₁ > 60% (Group B) (1.26 ± 0.12 ppb, Figure 1B). In both groups the concentrations of exhaled ethane were reduced in steroid-treated patients (0.59 ± 0.05 ppb and 0.94 ± 0.16 ppb for patients with FEV₁ < 60% and > 60%, respectively); furthermore, patients in Group A had higher ethane concentrations compared with those in Group B irrespective of steroid treatment (1.76 ± 0.31 ppb and 0.92 ± 0.30 ppb, respectively, p < 0.05). In patients not receiving steroid treatment, ethane was correlated with NO (r = 0.55, p < 0.05, Figure 2) and air trapping, as assessed by the ratio of RV/TLC (r = 0.60, p < 0.05). Exhaled ethane was not significantly correlated with FEV₁ even though there was a tendency for higher concentrations in patients with more severe bronchoconstriction (r = 0.47, p > 0.05). There was no correlation with any other lung function tests.

View larger version (10K): [in this window] [in a new window]   Figure 1.   (A) Concentrations of exhaled ethane in normal subjects (open squares), patients with asthma not receiving steroid treatment (closed circles), and steroid-treated asthmatic patients (open circles). (B) Exhaled ethane concentrations in asthmatic patients divided according to their FEV1 and steroid treatment.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Correlation of exhaled ethane with exhaled NO in patients with asthma not receiving steroid treatment (closed circles) and steroid-treated (open circles).

Exhaled NO

NO concentrations were higher in steroid-naïve (14.7 ± 1.7 ppb) compared with steroid-treated patients (8.6 ± 0.5 ppb, p < 0.05) and with the control group (6.7 ± 0.5 ppb, p < 0.05, Figure 3A). There was a tendency for higher concentrations of exhaled NO in untreated patients in Group A (15.5 ± 3.3 ppb) compared with Group B (14.0 ± 1.7 ppb) but this was not significant (Figure 3B). In group B untreated patients (14.0 ± 1.7 ppb) had higher concentrations of exhaled NO compared with steroid-treated patients (9.7 ± 0.6 ppb, p < 0.05); such difference was also significant in Group A where exhaled NO was reduced in treated (7.9 ± 0.7 ppb) compared with untreated patients (15.5 ± 3.3 ppb, p < 0.05).

View larger version (10K): [in this window] [in a new window]   Figure 3.   (A) Concentrations of exhaled NO in normal subjects (open squares), patients with asthma not receiving steroid treatment (closed circles) and steroid-treated patients (open circles). (B) Exhlaed NO in asthmatic patients divided according to their FEV1 and steroid treatment.

	DISCUSSION

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We have demonstrated that patients with asthma have elevated levels of exhaled ethane, which is significantly correlated with NO and air trapping as measured by RV/TLC. We also found that the concentrations of ethane and NO are reduced in steroid-treated patients and that patients with more severe bronchoconstriction have higher concentrations of exhaled ethane. We interpret these findings as confirmation that oxidative stress and lipid peroxidation are increased in the airways of asthmatic patients.

Asthma is defined by reversible airflow obstruction, airway hyperresponsiveness, and chronic inflammation characterized by an influx and activation of inflammatory cells, generation of inflammatory mediators, and epithelial cell shedding (12, 14). ROS, such as superoxide anions (O₂·) and hydrogen peroxide (H₂O₂) are produced by activated neutrophils, macrophages, and eosinophils and may lead to oxidation of nucleic acids, proteins, and membrane lipids (15). There is evidence for an increased lipid peroxidation in asthma (6, 16) and breath hydrocarbons have been studied as a measure of its activity in several studies (4, 7, 8). Polyunsaturated fatty acids are found in the cellular and subcellular membranes and are prone to lipid peroxidation as a result of the weak binding of hydrogen atoms to the carbon chain. 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 been used as a noninvasive marker of lipid peroxidation since the 1960s (10) and has been confirmed as a potential marker of lipid peroxidation in vivo (4). We favor ethane over pentane as a measure of lipid peroxidation because of the more rapid metabolism of pentane and its difficult chromatographic separation from isoprene, another product of lipid peroxidation.

Because an oxidant/antioxidant imbalance contributes to the severity of asthma and bronchial hyperreactivity (17) and because ROS can result in lung injury owing to direct oxidative damage of epithelial cells, we measured exhaled ethane as an index of lipid peroxidation and compared it with NO as a noninvasive marker of inflammation and oxidative stress. We found elevated levels of exhaled ethane in patients with asthma compared with normal subjects. These results indicate that there is increased lipid peroxidation in these patients, confirming previous studies showing elevated concentrations of other markers of lipid peroxidation such as thiobarbituric acid-reactive products (16) and of 8-isoprostane (6) in exhaled breath condensate. Although this is the first study in which exhaled ethane has been measured in stable asthmatic patients, Olopade and coworkers (18) have investigated the concentrations of pentane as a marker of lipid peroxidation during asthma exacerbations. In contrast with our results, the concentrations of pentane were elevated only during acute asthma exacerbations and were normal in stable patients. A similar difference between ethane and pentane has also been shown in multiple sclerosis (19) and alcoholic cirrhosis (20) and may be a result of the more rapid metabolism of pentane compared with ethane (21).

In steroid-naïve patients we found a negative correlation between exhaled ethane and air trapping, as assessed by RV/ TLC. The loss of this correlation in steroid-treated patients is likely to be due to the normalization of ethane concentrations and reduction of individual differences between patients. Evaluation of physiologic and pathologic studies suggests that the distal lung units, including the small airways (< 2 mm) and lung parenchyma, participate in the pathogenesis of asthma (22). However, the challenge lies in improving the ability to assess their structure and function noninvasively. We hypothesize that the correlation between exhaled ethane and RV/TLC may be due to obstruction of small airways. This may be the only pulmonary abnormality detectable in asthmatics during asymptomatic periods (23) and is related to bronchial hyperreactivity (24). Exhaled ethane measurement therefore may be a new tool to investigate small airway disease in asthma. There was no correlation between exhaled ethane and FEV₁, suggesting that the concentrations of this gas do not reflect obstruction of large airways. Our data indicate that in more severe patients lipid peroxidation is greater. This is further confirmed by the finding of higher exhaled ethane and of a tendency for higher concentrations of exhaled NO in subjects with FEV₁ lower than 60% of the predicted value (Group A) irrespective of steroid treatment, indicating exhaled ethane as a possible predictor of disease severity. Further studies are necessary to evaluate a possible association between the concentration of exhaled ethane, pathologic findings, and disease progression.

The fact that concentrations of exhaled ethane were not increased in the inhaled steroid-treated group of patients suggests that the increased concentrations in untreated patients are likely to be derived from the respiratory tract; however, it is possible that exhaled ethane may not be solely of lung origin but may be transported to the lung for elimination. Ethane is produced in other organs such as the intestine, brain, kidney, liver, heart, diaphragm, and testis (25); therefore, the systemic oxidative stress that characterizes asthma (2) may contribute to the final concentration of ethane in the exhaled breath.

There was a positive correlation between exhaled ethane and NO. We speculate that patients with more active inflammation in the airways are more likely to have an increased release of ROS and cytokines by inflammatory cells (26) and therefore induction of lipid peroxidation and inflammation with the resulting increased concentrations of exhaled ethane and NO. However, this correlation was weak and more studies, possibly including the use of NO synthase inhibitors, are necessary to confirm this finding.

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 (11) are likely to be due to the activation of the inducible form of NO synthase (iNOS) (27) and therefore may reflect airway inflammation.

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 NO is an indirect measurement of oxidative stress mediated by iNOS activity. The measurement of exhaled ethane and NO 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 NO as noninvasive markers of oxidative stress is particularly appealing as decreasing the inflammatory response may prevent structural damage to the airways and disease progression (28).

Steroid treatment was associated with lower concentrations of exhaled NO, confirming the data of previous publications (29). Steroids, in fact, by reducing inflammation, attenuating the release of oxidants by inflammatory cells (30), and suppressing proinflammatory cytokine production (31) may reduce iNOS (32) expression and therefore the synthesis of NO. Exhaled ethane was also reduced in steroid-treated patients, showing that steroid treatment can effectively attenuate lipid peroxidation, as already shown in previous studies (33); however, there was an overlap between the values of exhaled ethane in steroid-treated and untreated subjects, indicating that the measurement of exhaled ethane is not a predictor of steroid treatment by itself. 8-isoprostane, another marker of lipid peroxidation, shows resistance to steroid treatment (6), possibly because 8-isoprostane may also have an enzymatic synthesis by cyclooxygenase (COX-1 and COX-2) besides deriving from the free radical peroxidation of the arachidonic acid; therefore the final concentrations of 8-isoprostanes may reflect a more complex phenomenon rather than lipid peroxidation per se.

Measurement of exhaled ethane may be another means of detecting and monitoring cytokine- and oxidant-mediated inflammation and of assessing anti-inflammatory treatments. Further studies are necessary to investigate the correlation of exhaled ethane with other markers of lipid peroxidation and the clinical utility of exhaled ethane measurement in the follow-up of patients with asthma.