(Chest. 2005;128:2387-2392.)
© 2005
American College of Chest Physicians
Exhaled Ethane*
An In Vivo Biomarker of Lipid Peroxidation in Interstitial Lung Diseases
Soichiro Kanoh, MD, PhD;
Hideo Kobayashi, MD, PhD, FCCP and
Kazuo Motoyoshi, MD, PhD
* From the Third Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
Correspondence to: Soichiro Kanoh, MD, Third Department of Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan; e-mail: kanoh{at}ndmc.ac.jp
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Abstract
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Background: Oxidative stress plays a role in the pathogenesis and progression of interstitial lung disease (ILD). Exhaled ethane is a product of lipid peroxidation that has been proposed as a biomarker of oxidative stress in vivo.
Objectives: To determine whether the exhaled ethane level is elevated in patients with ILD and to compare it with other clinical parameters.
Methods: Breath samples were collected from 34 patients with ILD, including 13 with idiopathic pulmonary fibrosis (IPF), 9 patients with cryptogenic organizing pneumonia, 6 patients with collagen vascular disease-associated interstitial pneumonia, and 6 patients with pulmonary sarcoidosis. Gas samples were obtained at hospital admission and after 3 weeks. After each expired sample was concentrated using a trap-and-purge procedure, the ethane level was analyzed by gas chromatography.
Results: Exhaled ethane levels were elevated in ILD patients (n = 34, mean ± SD, 8.5 ± 8.0 pmol/dL) compared with healthy volunteers (n = 16, 2.9 ± 1.0 pmol/dL; p < 0.001). Serial measurements revealed that increase and decrease of ethane levels were largely consistent with the clinical course. Four patients with IPF who had persistently high ethane levels died or deteriorated, whereas those with ethane levels < 5.0 pmol/dL remained stable or improved. Exhaled ethane concentrations were positively correlated with levels of lactate dehydrogenase (Spearman rank correlation coefficient [rs], 0.28, p = 0.026) and C-reactive protein (rs, 0.38, p = 0.025) and were inversely correlated with PaO2 (rs, – 0.40, p = 0.0026). Patients showing increased uptake on 67Ga scintigraphy demonstrated higher ethane levels (n = 19, 7.5 ± 5.7 pmol/dL) compared with those who did not show increased uptake on scintigraphy (n = 10, 3.0 ± 2.4 pmol/dL; p < 0.01).
Conclusions: Exhaled ethane is elevated in patients with ILD and is correlated with the clinical outcome, suggesting that it provides useful information about ongoing oxidative stress, and thereby disease activity and severity in ILD.
Key Words: exhaled ethane • idiopathic pulmonary fibrosis • interstitial lung disease • lipid peroxidation • oxidative stress
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Introduction
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Oxidative stress resulting from toxic effects of reactive oxygen species (ROS) may play a part in the pathogenesis and progression of various pulmonary disorders, especially interstitial lung disease (ILD).123 Pulmonary cells are vulnerable to ROS because in addition to their own oxygen metabolites these cells are exposed to oxidant air pollutants, reactive xenobiotic-drug metabolites, and inflammatory cells.45 Excessive production of ROS results in an imbalance between oxidants and antioxidants that leads to cell and tissue damage. One mechanism by which oxidants can cause lung injury is lipid peroxidation, ie, oxidative degradation of polyunsaturated fatty acids.6 However, monitoring in vivo lipid peroxidation is difficult due to the lack of appropriate biomarkers.
Measurement of exhaled hydrocarbons has been proposed as a means of assessing lipid peroxidation in vivo.67 Polyunsaturated fatty acids in cellular biomembranes are the main targets of free radicals, and hydrocarbons are produced through peroxidation of these fatty acids by ROS. In fact, several studies89 have shown that the exhaled ethane concentration is elevated in patients with oxidative stress-mediated pulmonary diseases such as asthma and COPD. Ethane is an end product of lipid peroxidation of (n-3) fatty acids (linolenic acid and derivatives) and is expired as a volatile gas.6 Therefore, we measured exhaled ethane levels in patients with ILD as an indicator of lipid peroxidation and compared it with other clinical parameters to determine whether measurement of ethane could be useful in clinical practice.
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Materials and Methods
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Study Population
The study population included 34 hospitalized patients with ILD. Current smokers and patients with a history of diabetes, liver disease, or lung cancer were excluded.7101112 There were 13 patients with idiopathic pulmonary fibrosis (IPF) [8 men and 5 women; mean age ± SD, 63.0 ± 8.8 years], 9 patients with cryptogenic organizing pneumonia (COP) [4 men and 5 women; mean age, 52.4 ± 14.2 years], 6 patients with collagen vascular disease-associated interstitial pneumonia (CVD-IP) [1 man and 5 women; mean age, 54.7 ± 7.0 years], and 6 patients with pulmonary sarcoidosis in radiologic stage II or III (5 men and 1 woman; mean age, 40.0 ± 17.2 years). The diagnosis of IPF was based on American Thoracic Society/European Respiratory Society criteria.1314 In all patients, a medical history was obtained and physical examinations, lung function testing, arterial blood gas analysis, and high-resolution CT were performed. Histopathologic examination was carried out after transbronchial (n = 10) or thoracoscopic lung biopsy (n = 3). The diagnosis of COP was based on the histologic features of transbronchial lung biopsy specimens and American Thoracic Society/European Respiratory Society criteria. The patients who had CVD-IP included three patients with polymyositis/dermatomyositis, two patients with rheumatoid arthritis, and one patient with primary Sjögren syndrome. The diagnosis of CVD-IP was confirmed by physical findings, serologic testing, and high-resolution CT findings compatible with ILD. Histologic evaluation of transbronchial lung biopsy specimens was undertaken only for exclusion of other specific diseases. The diagnosis of sarcoidosis was established by histologic detection of noncaseating granulomas in transbronchial lung biopsy specimens. The control subjects consisted of 16 healthy volunteers (12 men and 4 women; mean age, 28.8 ± 4.2 years).
The following clinical parameters were measured in the ILD patients: serum lactate dehydrogenase (LDH; reference range, 80 to 225 U/L), C-reactive protein (CRP; reference range, <0.3 mg/dL), PaO2, vital capacity (VC), and diffusion capacity of the lung for carbon monoxide (DLCO). 67Ga scintigraphy was performed as a marker of disease activity. These tests were done within 1 week of the exhaled ethane measurement. Informed consent was obtained from all of the subjects before breath samples were taken. The characteristics of the study population are summarized in Table 1
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Measurement of Exhaled Ethane
To avoid contamination by environmental air, the subjects quietly breathed hydrocarbon-free air through a secure facemask (connected to a two-way nonbreathing valve) for 10 min before the measurement of ethane. In a preliminary study, we employed washout periods of variable length and found that ethane concentrations in the exhaled breath decline rapidly and reached a plateau within 5 min (data not shown). In accordance with previous reports,615 a washout period of 10 min is considered to be sufficient to wash environmental ethane from the lungs.
The expired air was directed into a gas-tight 5.0-L Tedlar bag (Iuchi; Osaka, Japan). Next, 3.0 L of the collected gas was withdrawn for analysis and was concentrated using a trap-and-purge procedure, as previously described.16 In brief, the sampled gas was passed through Teflon tubing to a stainless steel column containing 3.0 g of activated coconut charcoal with a grain size of 0.4 to 0.8 mm (corresponding to a 20/40 mesh) surrounded by dry ice to ensure retention of the ethane. The charcoal in the column was heated to 240°C for 5 min to drive off the absorbed ethane, and 1 mL of the desorbed gas was transferred to an airtight syringe for analysis. Measurement of ethane was performed by using a gas chromatograph (model GC-9AM; Shimadzu; Kyoto, Japan) with 0.5 x 3-mm, 60/80 mesh column (Shincarbon S; Shinwa Kako; Kyoto, Japan) and a flame ionization detector under the following conditions: column temperature, 100 to 160°C (increased at a rate of 20°C/min); injector temperature, 100°C; detector temperature, 250°C. The gas chromatograph was calibrated using known quantities of ethane. Serial measurement of ethane levels was performed on hospital admission and after 3 weeks in all patients with ILD, except for one patient who deteriorated rapidly and died within 2 weeks.
Statistical Analysis
Data are expressed as the mean ± SD. Comparisons between two groups were performed using the nonparametric Mann-Whitney U test. Nonparametric Spearman rank correlation coefficients (rs) were calculated to assess the relationship between exhaled ethane and other clinical parameters, and p < 0.05 was considered significant.
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Results
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The exhaled ethane level in the ILD patients on hospital admission and the healthy subjects was 8.5 ± 8.0 pmol/dL and 2.9 ± 1.0 pmol/dL, respectively (Fig 1
). Ethane levels were significantly higher in the ILD patients than the healthy subjects (p < 0.001). As a provisional reference value, we set the normal range for ethane at 
4.9 pmol/dL, which was the mean value plus 2 SD in the healthy volunteers. All of the ILD patients who had ethane levels < 5.0 pmol/dL (n = 17) had a stable clinical course (eg, no worsening of dyspnea, no signs of ongoing inflammation, and maintenance of PaO2) without treatment except for two patients treated with steroids. We further examined the 17 ILD patients with elevated ethane levels (
5.0 pmol/dL). In addition, we classified the ILD patients into an IPF group and a non-IPF group (COP, CVD-IP, and sarcoidosis) and compared their clinical courses. As shown in Figure 2
, exhaled ethane levels were elevated in eight patients with IPF. Among them, four patients showed a decrease of ethane to < 5.0 pmol/dL after 3 weeks of treatment with corticosteroids and immunosuppressive agents. These patients showed no subsequent exacerbations and remained in a stable condition. In the four other patients, the ethane concentration increased further or remained high despite the similar treatment with corticosteroids and immunosuppressive agents, and they died or showed deterioration of their condition requiring long-term oxygen therapy, indicating the progression of IPF. Nine patients in the non-IPF group also had elevated ethane levels, and they were treated with (n = 2) or without (n = 5) corticosteroids alone or in combination with immunosuppressive agents (n = 2). Six patients showed improvement, and their ethane levels decreased to < 5.0 pmol/dL by 3 weeks after the first measurement. In the other three patients, ethane levels remained > 5.0 pmol/dL but decreased over time, and the patients had no exacerbations. Thus, the observed increase and decrease of ethane levels was largely consistent with the clinical status of our ILD patients. However, there was not statistically significance as to ethane levels between presteroid and poststeroid treatment (data not shown). Furthermore, none of 30 ILD patients other than 4 IPF patients with highly sustained levels of ethane were readmitted for exacerbations within at least 6 months.
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Figure 1.. Exhaled ethane levels in healthy young volunteers and patients with ILD. Horizontal bars show mean values. The dotted line indicates an exhaled ethane level of 5.0 pmol/dL.
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Figure 2.. Individual exhaled ethane concentrations in IPF and non-IPF patients with ethane levels > 5.0 pmol/dL on hospital admission and the changes seen after 3 weeks. Solid symbols represent data for patients who died (closed squares) or showed deterioration of their condition (closed circles). The dotted line indicates an exhaled ethane level of 5.0 pmol/dL.
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We then examined the relationship between ethane levels and several clinical parameters. As shown in Figure 3
, the exhaled ethane concentration of the patients with ILD was positively correlated with the level of LDH (rs, 0.28, p = 0.026) and CRP (rs, 0.38, p = 0.025) and was inversely correlated with PaO2 (rs, – 0.40, p = 0.0026) but not with VC (rs, – 0.27, p = 0.12) and DLCO (rs, – 0.14, p = 0.35). To elucidate whether exhaled ethane levels reflected disease activity, 67Ga lung scintigraphy was performed in 29 of the ILD patients. An ethane level > 5.0 pmol/dL was only observed in 1 of the 10 patients without increased gallium uptake, whereas 11 of the 19 patients with increased gallium uptake had high ethane levels (Fig 4
). Ethane levels were higher in the patients with increased gallium uptake (7.5 ± 5.7 pmol/dL), compared with those who did not have increased uptake (3.0 ± 2.4 pmol/dL, p < 0.01).
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Figure 4.. Exhaled ethane levels in ILD patients stratified according to 67Ga uptake. Horizontal bars show mean values. The dotted line indicates an exhaled ethane level of 5.0 pmol/dL.
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Discussion
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We demonstrated that the exhaled ethane levels in patients with ILD were threefold higher than those of healthy subjects. Although the control subjects in the present study were younger than ILD patients, the level of ethane in the exhaled breath is not affected by age difference.17 Therefore, the older age of the patients with ILD compared with the control group does not explain the difference in exhaled ethane levels in these two groups. Moreover, the ethane concentration was even higher in patients with active and progressive disease. Ethane is one of the best volatile markers among the numerous end products of lipid peroxidation that are derived from constant formation of ROS in the body,615 and these results are consistent with the previous notion that inflammation and oxidative stress are associated with the pathogenesis of ILD.123 Accordingly, it is possible that increased ethane observed in ILD patients may not be solely of lung origin because ethane formed elsewhere in the body may be transported to the lung for elimination. Therefore, the systemic oxidative stress may contribute the concentration of ethane in the exhaled breath, particularly among patients with systemic disease such as CVD-IP and sarcoidosis. However, respiratory symptoms were the first and main complaint in all the patients enrolled in this study, and the symptoms related other organs were so little that their performance status was not interfered. Namely, exhaled ethane in our study was largely produced by the lung.
Importantly, marked or persistent elevation of exhaled ethane was observed during exacerbations of ILD. These findings suggest the occurrence of increased and ongoing lipid peroxidation in these patients, and that ethane is not only a marker of oxidative stress/inflammation in ILD but is also a potential predictor of clinical progression. This was further confirmed by the finding that reduced and normalized ethane levels were associated with the clinical improvement of ILD. In particular, it is noteworthy that all of the IPF patients with low ethane levels on hospital admission became stable. Recently, Paredi and colleagues89 reported that exhaled ethane is elevated in untreated patients with asthma or COPD and is correlated with the severity of airway obstruction. They also found that exhaled ethane concentrations were lower in steroid-treated and stable patients than in untreated patients, indicating that the exhaled ethane level may reflect the activity and severity of oxidative stress-mediated lung diseases. Taken together, it appears that ethane in the exhaled breath is an indicator of acute inflammatory response or a predictor of acute exacerbation or deterioration in the near future.
To further assess the clinical value of measuring exhaled ethane, we analyzed the correlation between exhaled ethane levels and several clinical parameters. We found that the exhaled ethane concentration was statistically correlated with the level of LDH, CRP, and PaO2, which reflect cellular damage, the intensity of inflammation, and the severity of respiratory insufficiency, respectively. Our results implied that exhaled ethane might have a different clinical significance to conventional clinical examinations. Ethane is a detector of lipid peroxidation in vivo, and production of this alkane reflects the extent of biomembrane damage caused by ROS.17 That is, an increased ethane level is associated with cellular dysfunction or injury due to disruption of the cell membrane, which may lead to various pathophysiologic features of ILD. However, oxidative stress can have a number of consequences both locally and systemically, including the alteration of enzyme activities, promotion of neutrophil migration into the lungs, signal transduction and gene expression for proinflammatory mediators, oxidation of structural proteins, and DNA strand breakage.45181920 A complex interplay between these various biological effects may result in the progression of ILD. Therefore, measurement of exhaled ethane may provide useful information about the clinical course that is different from conventional parameters, although the exact explanation for its clinical relevance still remains uncertain. Further studies will be necessary to determine how ethane relates to other parameters and to understand its clinical implication in ILD.
Based on the result of 67Ga lung scanning, exhaled ethane concentrations were elevated in patients showing increased uptake compared to those without increased uptake. However, there was some overlap between the exhaled ethane values in patients with increased and normal gallium accumulation, indicating that exhaled ethane reflects some, but not all, of the pathology of ILD. Despite this, it is striking that 11 of the 12 patients with high ethane levels (> 5.0 pmol/dL) showed increased gallium activity. Thus, we speculate that measurement of exhaled ethane may be another means of assessing oxidative inflammation and monitoring disease activity in ILD. To avoid radiation exposure and the high cost of scintigraphy, there may be advantages in performing exhaled ethane measurement rather than radionuclide imaging. Because this breath test is completely noninvasive, it can also be repeated frequently and performed safely in patients with severe ILD.2021
In conclusion, we demonstrated that oxidative stress was increased in patients with ILD as revealed by increased levels of exhaled ethane. This noninvasive biomarker obtained by analysis of breath sample may be a promising method of monitoring disease activity and severity that can quantify inflammation and oxidative stress in ILD. Measurement of exhaled ethane is feasible, and it may become a reliable clinical tool. Further studies are necessary to evaluate possible associations between the exhaled ethane concentration and disease outcomes.
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Acknowledgements
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The authors thank all the volunteers who participated as subjects in this study. We also thank Dr. S. Suzuki for his assistance in measuring exhaled ethane and for his helpful suggestions.
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Footnotes
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Abbreviations: COP = cryptogenic organizing pneumonia; CRP = C-reactive protein; CVD-IP = collagen vascular disease associated interstitial pneumonia; DLCO = diffusion capacity of the lung for carbon monoxide; ILD = interstitial lung disease; IPF = idiopathic pulmonary fibrosis; LDH = lactate dehydrogenase; ROS = reactive oxygen species; VC = vital capacity
Received for publication June 16, 2004.
Accepted for publication May 25, 2005.
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Abstract
Introduction
