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Hydrogen Peroxide in Exhaled Breath Condensate in Patients with Asthma
CHEST
Original Research ASTHMA
Hydrogen Peroxide in Exhaled Breath Condensate in Patients with Asthma A Promising Biomarker? Yue Teng, MSc; Peili Sun, MD; Jingying Zhang, MSc; Rongbin Yu, PhD; Jianling Bai, PhD; Xin Yao, MD, PhD; Mao Huang, MD, PhD; Ian M. Adcock, PhD; and Peter J. Barnes, MD, FCCP
Background: The measurement of hydrogen peroxide (H2O2) in exhaled breath condensate (EBC) has been proposed as a noninvasive way of monitoring airway inflammation. However, results from individual studies on EBC H2O2 evaluation of asthma are conflicting. The purpose of this study was to explore whether EBC H2O2 is elevated in people with asthma and whether it reflects disease severity and disease control or responds to corticosteroid treatment. Methods: Studies were identified by searching PubMed, Embase, Cochrane Database, Cumulative Index to Nursing and Allied Health Literature (CINAHL), and www.controlled-trials.com for relevant reports published before September 2010. Observational studies comparing levels of EBC H2O2 between patients with asthma who were nonsmokers and healthy subjects were included. Data were independently extracted by two investigators and analyzed using Stata 10.0 software. Results: Eight studies (involving 728 participants) were included. EBC H2O2 concentrations were significantly higher in patients with asthma who were nonsmokers compared with healthy subjects, and higher values of EBC H2O2 were observed at each level of asthma, classified either by severity or control level, and the values were negatively correlated with FEV1. In addition, EBC H2O2 concentrations were lower in patients with asthma treated with corticosteroids than in patients with asthma not treated with corticosteroids. Conclusions: H2O2 might be a promising biomarker for guiding asthma treatment. However, further investigation is needed to establish its role. CHEST 2011; 140(1):108–116 Abbreviations: CINAHL 5 Cumulative Index to Nursing and Allied Health Literature; EBC 5 exhaled breath condensate; eNO 5 exhaled nitric oxide; FEV1 % predicted 5 percentage predicted values of FEV1; H2O2 5 hydrogen peroxide; SMD 5 standard mean difference; WMD 5 weighted mean difference
is a chronic inflammatory disorder of the Asthma airways in which many cells and cellular elements
play a role.1,2 The assessment of airway inflammation is important in the pharmacologic treatment of asthma.3 Bronchoscopy, including BAL and bronchial biopsies, which are considered the most direct methods for evaluating airway inflammation, is too invasive for use in clinical practice. Much attention has been paid recently to the use of noninvasive measurements of airway inflammation for assessing the severity and guiding the treatment of asthma; such measurement tools include induced sputum,4 exhaled nitric oxide (eNO),5-7 exhaled carbon monoxide,8 and an electronic nose.9 Among these, eNO has been the most widely studied. However, a meta-analysis involving 1,010 participants
showed that the level of eNO might not be useful as a guide in making treatment decisions in the pharmacologic treatment of asthma, since it did not reduce exacerbations or improve FEV1 measurements compared with traditional assessment methods, such as symptoms and spirometry.10 The presence of oxidative stress has long been linked to the development and severity of asthma.11 Oxidative stress, defined as an increased exposure to oxidants and/or decreased antioxidant capacities, contributes to or accelerates the pathophysiologic characteristics of airway inflammation, resulting in airway hyperresponsiveness and leading to clinical symptoms of asthma.12 Hydrogen peroxide (H2O2) is a mediator of oxidative stress and can play an important role in asthma.13 Dohlman et al14 first demonstrated
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that H2O2 within expired breath was a marker of acute airway inflammation in pediatric patients with asthma in 1993. The measurement of exhaled breath condensate (EBC) is a noninvasive method for sampling airway secretions. Several biomolecules, including H2O2, 8-isoprostane, leukotrienes, prostaglandins, adenosine, glutathione, nitrites, and nitrates have been detected in the EBC.15 The presence of 8-isoprostane, leukotriene B4, and prostaglandin E2 in EBC has been confirmed by using reverse-phase liquid chromatography and mass spectrometry.16-21 The presence of H2O2 in the EBC of patients with asthma has been the focus of much subsequent research. Indeed, EBC H2O2 levels were increased in patients with asthma compared with healthy subjects.14,22,23 However, some studies failed to replicate these results, bringing into question whether EBC H2O2 levels could be used to discriminate patients with asthma from healthy subjects.24 The primary objectives of this meta-analysis were to determine (1) whether EBC H2O2 levels are elevated in patients with asthma, (2) the relationship between EBC H2O2 levels and spirometry results, (3) whether EBC H2O2 levels can reflect clinical severity or degree of symptom control, and (4) whether EBC H2O2 levels respond to corticosteroid treatment.
Materials and Methods Studies were identified by searching the literature using PubMed (1966 to September 2010), Embase (1980 to September 2010), Cochrane Database (1972 to September 2010), Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1981 to September 2010), and www.controlled-trials.com for relevant reports, using both keywords and free text words: (“asthma” or “airway inflammation” or “bronchial constriction” or “bronchial spasm” or “bronchial hyperreactivity” or “wheeze” or “wheezing”) Manuscript received October 31, 2010; revision accepted March 1, 2011. Affiliations: From the Department of Respiratory Medicine (Mss Teng and Zhang and Drs Sun, Yao, and Huang), The First Affiliated Hospital of Nanjing Medical University, Nanjing, China; the Department of Epidemiology and Biostatistics (Drs Yu and Bai), School of Public Health, Nanjing Medical University, Nanjing, China; and the Airway Disease Section (Drs Adcock and Barnes), National Heart and Lung Institute, Imperial College, London, England. Ms Teng and Dr Sun contributed equally to this work. Funding/Support: This study was supported by the National Natural Science Foundation of China [Grants 30700342, 81070025], Jiangsu Health Promotion Project, and “the Six Great Talents” [Grant 09-B1-001]. Correspondence to: Xin Yao, MD, PhD, Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, 210029, China; e-mail: [email protected] © 2011 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-2816 www.chestpubs.org
THE BOTTOM LINE How does this work advance the field? This meta-analysis suggests a relationship between concentrations of hydrogen peroxide in exhaled breath condensate and asthma severity, asthma control status, lung function, and the response to corticosteroid treatment. It also provides evidence to support the measurement of hydrogen peroxide in exhaled breath condensate as a noninvasive biomarker to assess airway inflammation in patients with asthma.
What are the clinical implications? Measurement of hydrogen peroxide in exhaled breath condensate has potential as a biomarker for guiding asthma treatment, although further investigations with more carefully measured concentrations of hydrogen peroxide in exhaled breath condensate are needed.
and (“EBC” or “exhaled” or “expired”) and (“hydrogen peroxide” or “peroxide” or “H2O2”). There were no language restrictions. Inclusion criteria included the following: (1) observational studies, (2) studies comparing levels of EBC H2O2 between patients with asthma with an established diagnosis and healthy subjects, and (3) studies in which both patients with asthma and healthy subjects were nonsmokers and that salivary contamination had been eliminated. Exclusion criteria included the following: (1) studies with patients with asthma with diseases that could affect H2O2 levels, such as COPD,25 ARDS,26,27 and acute hypoxemic respiratory failure,28 (2) studies without healthy subjects as a control group,29,30 and (3) studies where smokers were not specifically excluded.23,31-33 Figure 1 shows the procedure and results of the search. Based on the explicit criteria, two reviewers (Y. T. and J. Z.) respectively checked the titles and abstract sections of all the articles retrieved. Full articles were searched when the information met the inclusion criteria. If there were any doubts about the titles or abstracts, we also reviewed the full articles for clarification. Any disagreements about adjudications were resolved through a third party (X. Y.). We contacted the authors for further clarification when it was not possible to resolve the disagreement. For each accepted study, the following data, when available, were extracted: the number of participants; age; gender; clinical features; treatments; method of measuring EBC H2O2; mean value, SD, SEM, and 95% CI of both patients with asthma and healthy subjects; and the percentage predicted values of FEV1 (FEV1 % predicted). Subject characteristics are given in more detail in Table 1. The SEM or 95% CI was transformed into SD, using statistical formulas. Analyses were performed using Stata software, version 10.0 (StataCorp LP; College Station, Texas). The weighted mean difference (WMD) was chosen to combine statistics; if the difference in mean value was too large and resulted in large heterogeneity, the standard mean difference (SMD) was chosen. Summary estimates and the weightings for each outcome were evaluated based on DerSimonian-Laird random effects models. Heterogeneity of study results was tested using the Mantel-Haenszel method, and results were considered heterogeneous if the P value was , .05. The I2 statistic was used to measure the extent of discrepancy among results. Data were pooled using the random effects model if there was marked heterogeneity (I2 . 50%), and the fixed effects model was used in other circumstances. Publication bias was evaluated through visual inspection of funnel plots, using the Begg test39 and the Egger asymmetry test.40 Publication bias was assumed to be present if the P value was , .05. CHEST / 140 / 1 / JULY, 2011
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Overall EBC H2O2 Levels in Patients With Asthma and Healthy Subjects Data were extracted from the eight articles regardless of the age, disease severity, and treatment measures of subjects. The overall WMD of EBC H2O2 between patients with asthma and healthy subjects was 0.49 mM (95% CI, 0.18-0.79; P 5 .002; I2 5 98.3%; random effects model). Patients with asthma showed significantly higher EBC H2O2 levels compared with healthy subjects. (Fig 3) EBC H2O2 Levels in Adult and Childhood Asthma There were five articles focused on adult asthma and two on childhood asthma. The remaining article did not mention the age of the participants, therefore, it could not be included in this subgroup analysis. The EBC H2O2 levels in adults with asthma were significantly higher than those in healthy adults (WMD, 0.58 mM; 95% CI, 0.20-0.97; P 5 .003; I2 5 99.0%; random effects model), while there was no statistical difference between children with asthma and healthy children (WMD, 0.23 mM; 95% CI, 20.02 to 0.49; P 5 .074; I2 5 31.9%; fixed effects model) (Fig 4).
Figure 1. The results of the systematic literature search. CINAHL 5 Cumulative Index to Nursing and Allied Health Literature; EBC 5 exhaled breath condensate; H2O2 5 hydrogen peroxide.
Results The systematic search (Fig 1) yielded 267 total references, of which 176 were unique. After a title review, 70 studies were excluded, yielding 106 articles. A subsequent abstract review rejected a further 80 of these references, yielding 26 candidate studies. Among the 26 studies, eight of them lacked a control group, and six of them did not provide sufficient EBC H2O2 data such as mean value, SD, SEM, or 95% CI. In addition, four of the studies did not specifically exclude smokers. We tried to contact the authors by e-mail to obtain further information. Finally, eight articles involving a total of 728 subjects14,22,24,34-38 were included in our meta-analysis. Among the eight studies, four directly provided SDs, three studies used the SEMs, and the remaining study provided 95% CIs. As the unequal variances were not mentioned, the classic t test was used to transform 95% CIs into SDs. All studies provided basic information and details of methodology related to patients and control subjects (Table 1). There was no evidence of publication bias (Begg test, P 5 .54; Egger test, P 5 .06). The funnel plot is shown in Figure 2.
EBC H2O2 Concentrations in Different Asthma Severities Asthma severity was classified according to four levels: intermittent, mild persistent, moderate persistent, and severe persistent. Because of the limited information provided by the studies, we could only separate patients with asthma into an intermittent group (four studies), a mild to moderate persistent group (five studies), and a severe persistent group (one study). The WMD of EBC H2O2 was 0.53 mM (95% CI, 0.25-0.82; P , .0001; I2 5 98.7%; random effects model) between patients with intermittent asthma and healthy subjects, and it was 0.62 mM (95% CI, 0.23-1.01; P 5 .002; I2 5 98.3%; random effects model) between patients with mild to moderate persistent asthma and healthy subjects. The values of EBC H2O2 for patients with each level of asthma classified by severity were higher than those of the healthy subjects (Fig 5). EBC H2O2 Concentrations and Asthma Control According to GINA (Global Initiative for Asthma),41 there are three categories of asthma control: controlled, partly controlled, and uncontrolled. However, we could not differentiate controlled asthma from partly controlled asthma because of the limited information provided by the articles, so we separated the patients with asthma into two groups: patients with
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111.7 ⫾ 19.4a 96 ⫾ 5c 92 ⫾ 7c 64 ⫾ 9c 95 ⫾ 3c 96.0 ⫾ 2.3b 101.8 ⫾ 1.5b 80 ⫾ 9.07a 69 ⫾ 9.87a 101 ⫾ 2.25a ... ... ... ... ... ... ... ... ... ...
19-34 30.5 ⫾ 6 29 ⫾ 5 29 ⫾ 7 30 ⫾ 4 10.7 ⫾ 3.0 10.0 ⫾ 0.4 17-56 17-56 ... ... ... ... ... 26-77 21-73 25-63 31-78 21-78 20-24
17 (7/10) 10 (NA) 10 (NA) 10 (NA) 10 (NA) 10 (NA) 15 (NA) 64 (47/17) 50 (27/23) 25 25 15 27 (NA) 43 (NA) 32 (NA) 27 (NA) 18 (12/6) 7 (5/2) 9 (6/3) 21 (8/13) 55 (31/24) 58 (47/11)
Healthy Mild intermittent asthma Mild persistent asthma Steroid-untreated asthma Steroid-treated asthma Moderate persistent asthma Steroid-untreated asthma Steroid-treated asthma Healthy Asthma
Healthy Intermittent asthma Persistent asthma Healthy Cough variant asthma Classic asthma Chronic cough Healthy Mild intermittent asthma Mild persistent asthma Moderate persistent asthma Severe persistent asthma Total asthma Healthy ... ... ... ... ... ... ... ... ... ... ...
... 50% of children had poor asthma control ... Unstable
Clinically stable for at least 4 wk Nocturnal wheezing and daily asthma symptoms ... Nocturnal wheezing and daily asthma symptoms (unstable) ... Clinically stable and no acute exacerbation for at least 4 wk
... ...
Clinical Feature
0.72 ⫾ 0.06b 0.43 ⫾ 0.08b 0.78 ⫾ 0.16b
b2-Agonist as required ICS (H) Oral steroids and ICS (H)
...
... b2-Agonist and/or theophylline bid, no systemic steroids ... ICS (⫾)
0.83-1.34c 0.46-1.02c 0.16-0.24c 2.0 ⫾ 0.22b
b2-Agonist ICS (M/H) and b2-agonist ... ICS (M)/oral steroids/ leukotriene antagonists ... No systematic steroids
2.2 ⫾ 0.31b 0.66 ⫾ 0.05a 1.14 ⫾ 0.36a 0.23 ⫾ 0.03a 0.83 ⫾ 0.34a 0.89 ⫾ 0.36a 0.24 ⫾ 0.22a 0.29 ⫾ 0.7a 1.85 ⫾ 0.10b 2.26 ⫾ 0.25b 1.65 ⫾ 0.13b 1.89 ⫾ 0.40b 1.89 ⫾ 0.07b 0.59 ⫾ 0.03b
0.40-0.87c 0.41-0.64c
0.024 ⫾ 0.016a 0.23-0.32c
0.27 ⫾ 0.04b 0.127 ⫾ 0.083a
b2-Agonist ICS (M/H) and b2-agonist
... b2-Agonist as required, steroids naive within 3 mo ... b2-Agonist
0.54 ⫾ 0.56 0.25 ⫾ 0.27a
Oral steroids/ICS/b2-agonist ... a
H2O2 (mM)
Treatment
Fluorometer
Spectrophotometer, 450 nm
Perkin Elmer UltravioletVIS Spectrometer L 10, 450 nm Spectrophotometer, 450 nm
Spectrophotometer (model 46; Lomo; St. Petersburg, Russia), 450 nm Spectrophotometer (Uvicon 940; Kontron Instruments; Zurich, Switzerland), 450 nm
Fluorometer (LS-50; Perkin-Elmer Corp; Norwalk, Connecticut) Spectrophotometer (AR 8003; Labtech Int Ltd; Uckfield, England), 450 nm
Measurement
F 5 female; H2O2 5 hydrogen peroxide; ICS 5 inhaled corticosteroids; ICS (H) 5 high dose of inhaled corticosteroids; ICS (M) 5 medium dose of inhaled corticosteroids; M 5 male; NA 5 not available; FEV1 % predicted 5 percentage predicted values of FEV1. amean ⫾ SD. bmean ⫾ SEM. c95% CI.
Ueno et al38/2008
Al Obaidi37/2007
Al-Obaidy and Al-Samarai36/2007
Robroeks et al24/2007
Loukides et al35/2002
97 ⫾ 3b 82 ⫾ 19.6a
31 ⫾ 1.8 18-62
35 (18/17) 70 (20/50)
86 ⫾ 2.4b 76 ⫾ 5b 64 ⫾ 5b
28 ⫾ 1.3 34 ⫾ 1.9 37 ⫾ 3
Healthy Steroid-untreated asthma
Emelyanov et al34/2001
... ...
7-18 ...
72 (42/30) 30 (14/16) 14 (5/9)
FEV1 % predicted
Steroid-untreated asthma Steroid-treated asthma
Age, y
Horváth et al22/1998
25 (NA) 11 (NA)
No. (M/F)
Asthma Healthy
14
Subject Group
Dohlman et al /1993
Study/Year
Table 1—Study Characteristics
Figure 2. The Begg funnel plot. WMD 5 weighted mean difference.
controlled and partly controlled asthma (defined as clinically stable for at least 4 weeks and/or use of b2-agonist as required; two studies) and patients with uncontrolled asthma (defined as nocturnal wheezing, daily asthma symptoms, and use of b2-agonist as required; two studies). The other studies lacked information on asthma control and were, therefore, excluded. The difference in mean value was very large in the uncontrolled asthma group, which resulted in a large heterogeneity, thus the SMD was chosen to combine statistics. Patients with controlled and partly controlled asthma showed higher levels of EBC H2O2 than healthy subjects, with an SMD of 1.06 (95% CI, 0.44-1.69; P 5 .001; I2 5 65.6%; random effects model). Much higher values of EBC H2O2 were observed in patients with uncontrolled asthma, with an SMD of 1.37 (95% CI, 0.93-1.80; P , .0001; I2 5 0%; fixed effects model). There was no significant difference between patients with controlled and partly controlled asthma vs patients with uncontrolled asthma; however, a trend of increasing EBC H2O2 levels was shown (Fig 6).
Figure 3. Overall EBC H2O2 levels in patients with asthma. See Figure 1 legend for expansion of the abbreviations.
Figure 4. EBC H2O2 levels in adults and children with asthma. See Figure 1 legend for expansion of the abbreviations.
EBC H2O2 Levels in Patients With SteroidUntreated and Steroid-Treated Asthma The studies were divided into two groups according to corticosteroid treatment, namely a steroiduntreated group (defined as not receiving regular inhaled or systemic corticosteroids and/or receiving b2-agonists as a relief medication on demand; five studies) and a steroid-treated group (defined as current use of inhaled or systemic corticosteroids regardless of use of b2-agonists or leukotriene receptor antagonists; two studies). Compared with healthy subjects, increased levels of EBC H2O2 were found in both the steroid-untreated group (WMD, 0.49 mM; 95% CI, 0.17-0.82; P 5 .003; I2 5 97.9%; random effects model) and the steroid-treated group (WMD, 0.37 mM; 95% CI, 0.19-0.56; P , .0001; I2 5 64.9%; random effects model). Patients with steroid-untreated asthma
Figure 5. EBC H2O2 concentrations in patients with different asthma severities. See Figure 1 legend for expansion of the abbreviations.
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formation. The combined r9 between the EBC H2O2 and the FEV1 % predicted was 20.14, which indicated that there was a significant negative correlation between the EBC H2O2 and the FEV1 % predicted (Fig 8).
Discussion
Figure 6. EBC H2O2 concentrations and levels of asthma control. See Figure 1 legend for expansion of the abbreviations.
showed significantly higher EBC H2O2 levels than patients with steroid-treated asthma (WMD, 0.19 mM; 95% CI, 0.05-0.34; P 5 .009; I2 5 0%; fixed effects model) (Fig 7). Correlation Coefficient Between EBC H2O2 Levels and the FEV1 % predicted The correlation coefficient between the EBC H2O2 value and the FEV1 % predicted value was reported in three out of eight studies. Fisher z9 transformation42 was used to transform r to z, which is considered as the normal distribution. The z value was 20.79 (95% CI, 2 1.13 to 20.44; P 5 .007; I2 5 79.7%; random effects model). Then the combined r9 was derived from z by using the reverse Fisher z9 trans-
Figure 7. EBC H2O2 levels in patients with steroid-untreated and steroid-treated asthma. See Figure 1 legend for expansion of the abbreviations. www.chestpubs.org
Based on eight studies involving 500 patients with asthma and 228 healthy subjects, higher values of EBC H2O2 were observed at each level of asthma, classified according to either severity or degree of control. EBC H2O2 levels were negatively correlated with the FEV1 % predicted in patients with asthma. Furthermore, the average EBC H2O2 levels showed a trend toward disease severity and the control level, although this did not reach statistical significance in our meta-regression analysis. In addition, EBC H2O2 was sensitive to corticosteroid treatment. H2O2 is one form of reactive oxygen species, which are derived from spontaneous or enzyme-catalyzed dismutation of superoxide anions.13 H2O2 contributes to oxidative stress in the airways and might cause the activation of various intracellular signaling pathways, leading to secretion of a variety of proinflammatory cytokines and chemokines.43 In addition, the predominant cells producing H2O2 are activated inflammatory cells such as eosinophils and neutrophils,35 suggesting that H2O2 might reflect the influx of inflammatory granulocytes and, therefore, disease severity. Our meta-analysis demonstrated that adults with asthma had higher EBC H2O2 levels than healthy subjects, indicative of an enhancement of oxidative stress in these patients, although no statistical significance was found between children with asthma and the control group. This might be the result of
Figure 8. Correlation coefficient (r) between EBC H2O2 and the percentage predicted values of FEV1. r9 5 20.14 (using the reverse Fisher z9 transformation); z 5 the value of r after using Fisher z9 transformation. See Figure 1 legend for expansion of other abbreviations. CHEST / 140 / 1 / JULY, 2011
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the lower levels of EBC H2O2 in children,44 which reduced the accuracy of detection, or may reflect the recent genome-wide association study data indicating differences between childhood onset of asthma and adult asthma.45 Corticosteroid treatment and the expiratory flow rate46 may also have an impact on the results. Since there were only two samples (89 children with asthma and 61 healthy children) included in the childhood group, further investigations are required to address whether there is an impact of age on EBC H2O2 levels. In our meta-analysis, patients with intermittent, mild to moderate, and severe persistent asthma expressed significantly higher levels of EBC H2O2 than the healthy subjects, although there was no statistical significance on severity using meta-regression analysis (P 5 .649) (Fig 9). Furthermore, levels of EBC H2O2 also showed a significant negative correlation with FEV1 % predicted. In addition, in patients with intermittent asthma, whose FEV1 % predicted values were normal, the values of H2O2 were still higher than in healthy subjects, suggesting the detection of H2O2 might be more sensitive than traditional spirometry in the less severe population, although further studies are needed to confirm this. In our meta-analysis, EBC H2O2 levels in patients with steroid-treated asthma were significantly lower than in patients with steroid-untreated asthma, and the patients with controlled and partly controlled asthma had lower average levels of EBC H2O2 than patients with uncontrolled asthma. Therefore, the level of EBC H2O2 might be a promising biomarker, with the potential of guiding asthma therapy. However, it should be pointed out that the studies to date
reporting the effects of steroids and asthma control status on EBC H2O2 level are mainly cross-sectional in design. Therefore, more randomized controlled trials are needed to confirm the influence of steroids and the relationship between EBC H2O2 level and asthma severity and control. There are some limitations to EBC H2O2 measurements. The most frequently used methods for measuring H2O2 in the EBC are the spectrophotometric and fluorimetric methods. However, the reproducibility of EBC H2O2 has not been provided in most studies, and the stated values vary between reports; some showed a satisfactory agreement,47,48 while others have a rather high coefficient of variation, especially with fluorimetric methods.46,49 Since the method of quantifying H2O2 can have an impact on the concentrations of EBC H2O2, we compared these two methods (spectrophotometric method, six studies; fluorimetric method, two studies). No statistical differences were observed in the WMD of participants, irrespective of whether the studies using the fluorimetric method were excluded (WMD, 0.39 mM; 95% CI, 0.12-0.65; I2 5 97.3%) or not (WMD, 0.49 mM; 95% CI, 0.18-0.79; I2 5 98.3%). Thus, we did not exclude these studies despite the methodologic differences in H2O2 detection. Cigarette smoke contains organic radicals, which can react with molecular oxygen in a redox-dependent manner to form superoxide anions, hydroxyl radicals, and H2O2.50 Previous studies have found that smoking increases EBC H2O2 concentrations, both in patients with asthma and in healthy subjects,33,51,52 so that smoking status must be taken into consideration when using EBC H2O2 levels to assess airway inflammation.
Figure 9. EBC H2O2 WMD (95% CI) between patients with different types of asthma and healthy subjects. The arrowheads indicate the mean of the WMD between patients with specific types of asthma and healthy subjects. The vertical lines indicate the 95% CI of the WMD between patients with specific types of asthma and healthy subjects. See Figure 1 and 2 legends for expansion of abbreviations. 114
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The relative lack of studies based on normal populations and the relatively high variability of EBC H2O2 levels in healthy individuals52-54 are the critical limitations for the clinical application of EBC H2O2. Further studies on normal populations are required to establish reference values using standardized methods of measurement.
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Conclusions H2O2 in the EBC is elevated in adult patients who have asthma and are nonsmokers compared with healthy adult subjects. EBC H 2O 2 levels demonstrated trends toward correlations with disease severity and the degree of asthma control and had a negative correlation with FEV1 % predicted. In addition, EBC H2O2 is sensitive to corticosteroid treatment. EBC H2O2 might, therefore, be a promising biomarker in guiding asthma treatment, although further large-scale studies are needed. Acknowledgments Author contributions: Ms Teng: read and met the International Committee of Medical Journal Editors (ICMJE) criteria for authorship, designed this study, extracted data, performed the analysis, wrote the first draft of the manuscript, and read and approved the final manuscript. Dr Sun: read and met the ICMJE criteria for authorship, designed this study, and read and approved the final manuscript. Ms Zhang: read and met the ICMJE criteria for authorship, extracted data, and read and approved the final manuscript. Dr Yu: read and met the ICMJE criteria for authorship, performed the analysis, and read and approved the final manuscript. Dr Bai: read and met the ICMJE criteria for authorship, performed the analysis, and read and approved the final manuscript. Dr Yao: read and met the ICMJE criteria for authorship, designed this study, extracted data, performed the analysis, critically revised the manuscript, and read and approved the final manuscript. Dr Huang: read and met the ICMJE criteria for authorship, designed this study, and read and approved the final manuscript. Dr Adcock: read and met the ICMJE criteria for authorship, critically revised the manuscript, and read and approved the final manuscript. Dr Barnes: read and met the ICMJE criteria for authorship, critically revised the manuscript, and read and approved the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors had no role in the design and conduct of the study, in the data extraction, analysis, or interpretation of the data, or in the preparation, review, or approval of the manuscript.
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tion and asthma severity. Am J Respir Crit Care Med. 2000; 161(1):9-16. Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med. 2000;161(1):64-72. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet. 1994;343(8890):133-135. Malerba M, Ragnoli B, Radaeli A, Tantucci C. Usefulness of exhaled nitric oxide and sputum eosinophils in the longterm control of eosinophilic asthma. Chest. 2008;134(4): 733-739. Montuschi P, Mondino C, Koch P, Ciabattoni G, Barnes PJ, Baviera G. Effects of montelukast treatment and withdrawal on fractional exhaled nitric oxide and lung function in children with asthma. Chest. 2007;132(6):1876-1881. Zhang J, Yao X, Yu R, et al. Exhaled carbon monoxide in asthmatics: a meta-analysis. Respir Res. 2010;11:50. Montuschi P, Santonico M, Mondino C, et al. Diagnostic performance of an electronic nose, fractional exhaled nitric oxide, and lung function testing in asthma. Chest. 2010;137(4): 790-796. Petsky HL, Cates CJ, Li A, Kynaston JA, Turner C, Chang AB. Tailored interventions based on exhaled nitric oxide versus clinical symptoms for asthma in children and adults. Cochrane Database Syst Rev. 2009;(4):CD006340. Rahman I, Morrison D, Donaldson K, MacNee W. Systemic oxidative stress in asthma, COPD, and smokers. Am J Respir Crit Care Med. 1996;154(4 Pt 1):1055-1060. Sugiura H, Ichinose M. Oxidative and nitrative stress in bronchial asthma. Antioxid Redox Signal. 2008;10(4):785-797. Comhair SA, Erzurum SC. Redox control of asthma: molecular mechanisms and therapeutic opportunities [published correction appears in Antioxid Redox Signal. 2010;12(2):321]. Antioxid Redox Signal. 2010;12(1):93-124. Dohlman AW, Black HR, Royall JA. Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma. Am Rev Respir Dis. 1993;148(4 Pt 1): 955-960. Montuschi P. Analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications. Ther Adv Respir Dis. 2007;1(1):5-23. Carpenter CT, Price PV, Christman BW. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest. 1998;114(6):1653-1659. Montuschi P, Ragazzoni E, Valente S, et al. Validation of 8-isoprostane and prostaglandin E(2) measurements in exhaled breath condensate. Inflamm Res. 2003;52(12):502-507. Montuschi P, Ragazzoni E, Valente S, et al. Validation of leukotriene B4 measurements in exhaled breath condensate. Inflamm Res. 2003;52(2):69-73. Montuschi P, Martello S, Felli M, Mondino C, Barnes PJ, Chiarotti M. Liquid chromatography/mass spectrometry analysis of exhaled leukotriene B4 in asthmatic children. Respir Res. 2005;6:119. Montuschi P, Martello S, Felli M, Mondino C, Chiarotti M. Ion trap liquid chromatography/tandem mass spectrometry analysis of leukotriene B4 in exhaled breath condensate. Rapid Commun Mass Spectrom. 2004;18(22):2723-2729. Montuschi P. LC/MS/MS analysis of leukotriene B4 and other eicosanoids in exhaled breath condensate for assessing lung inflammation. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877(13):1272-1280. Horváth I, Donnelly LE, Kiss A, et al. Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma. Am J Respir Crit Care Med. 1998;158(4):1042-1046. CHEST / 140 / 1 / JULY, 2011
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23. Antczak A, Nowak D, Shariati B, Król M, Piasecka G, Kurmanowska Z. Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients. Eur Respir J. 1997;10(6):1235-1241. 24. Robroeks CM, van de Kant KD, Jöbsis Q, et al. Exhaled nitric oxide and biomarkers in exhaled breath condensate indicate the presence, severity and control of childhood asthma. Clin Exp Allergy. 2007;37(9):1303-1311. 25. Fireman E, Shtark M, Priel IE, et al. Hydrogen peroxide in exhaled breath condensate (EBC) vs eosinophil count in induced sputum (IS) in parenchymal vs airways lung diseases. Inflammation. 2007;30(1-2):44-51. 26. Kietzmann D, Kahl R, Müller M, Burchardi H, Kettler D. Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS. Intensive Care Med. 1993;19(2):78-81. 27. Baldwin SR, Simon RH, Grum CM, Ketai LH, Boxer LA, Devall LJ. Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet. 1986;1(8471): 11-14. 28. Sznajder JI, Fraiman A, Hall JB, et al. Increased hydrogen peroxide in the expired breath of patients with acute hypoxemic respiratory failure. Chest. 1989;96(3):606-612. 29. Al Obaidi AH, Al Samarai AM. Biochemical markers as a response guide for steroid therapy in asthma. J Asthma. 2008; 45(5):425-428. 30. Robroeks CM, van de Kant KD, van Vliet D, et al. Comparison of the anti-inflammatory effects of extra-fine hydrofluoroalkanebeclomethasone vs fluticasone dry powder inhaler on exhaled inflammatory markers in childhood asthma. Ann Allergy Asthma Immunol. 2008;100(6):601-607. 31. Antczak A, Nowak D, Bialasiewicz P, Kasielski M. Hydrogen peroxide in expired air condensate correlates positively with early steps of peripheral neutrophil activation in asthmatic patients. Arch Immunol Ther Exp (Warsz). 1999;47(2):119-126. 32. Svensson S, Olin AC, Lärstad M, Ljungkvist G, Torén K. Determination of hydrogen peroxide in exhaled breath condensate by flow injection analysis with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2004; 809(2):199-203. 33. Horváth I, Donnelly LE, Kiss A, Balint B, Kharitonov SA, Barnes PJ. Exhaled nitric oxide and hydrogen peroxide concentrations in asthmatic smokers. Respiration. 2004;71(5): 463-468. 34. Emelyanov A, Fedoseev G, Abulimity A, et al. Elevated concentrations of exhaled hydrogen peroxide in asthmatic patients. Chest. 2001;120(4):1136-1139. 35. Loukides S, Bouros D, Papatheodorou G, Panagou P, Siafakas NM. The relationships among hydrogen peroxide in expired breath condensate, airway inflammation, and asthma severity. Chest. 2002;121(2):338-346. 36. Al-Obaidy AH, Al-Samarai AG. Exhaled breath condensate pH and hydrogen peroxide as non-invasive markers for asthma. Saudi Med J. 2007;28(12):1860-1863. 37. Al Obaidi AH. Expired breath condensate hydrogen peroxide concentration and pH for screening cough variant
38. 39. 40. 41.
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53. 54.
asthma among chronic cough. Ann Thorac Med. 2007;2(1): 18-22. Ueno T, Kataoka M, Hirano A, et al. Inflammatory markers in exhaled breath condensate from patients with asthma. Respirology. 2008;13(5):654-663. Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics. 1994;50(4): 1088-1101. Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997;315(7109):629-634. Global Initiative for Asthma. GINA report, global strategy for asthma management and prevention. Global Initiative for Asthma Web site. www.ginasthma.com. Updated December 2010. Accessed April 28, 2011. Fisher R. Statistical Methods for Research Workers. Newdelhi, India; Genesis Publishing Pvt Ltd; 2006. Rahman I, Yang SR, Biswas SK. Current concepts of redox signaling in the lungs. Antioxid Redox Signal. 2006;8(3-4): 681-689. Jöbsis Q, Raatgeep HC, Schellekens SL, Hop WC, Hermans PW, de Jongste JC. Hydrogen peroxide in exhaled air of healthy children: Reference values. Eur Respir J. 1998;12(2):483-485. Moffatt MF, Gut IG, Demenais F, et al; GABRIEL Consortium. A large-scale, consortium-based genomewide association study of asthma. N Engl J Med. 2010;363(13):1211-1221. Schleiss MB, Holz O, Behnke M, Richter K, Magnussen H, Jörres RA. The concentration of hydrogen peroxide in exhaled air depends on expiratory flow rate. Eur Respir J. 2000;16(6): 1115-1118. Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S. Oxidative stress in expired breath condensate of patients with COPD. Chest. 2003;124(4):1373-1380. Ho LP, Faccenda J, Innes JA, Greening AP. Expired hydrogen peroxide in breath condensate of cystic fibrosis patients. Eur Respir J. 1999;13(1):103-106. van Beurden WJ, Dekhuijzen PN, Harff GA, Smeenk FW. Variability of exhaled hydrogen peroxide in stable COPD patients and matched healthy controls. Respiration. 2002; 69(3):211-216. Nakayama T, Church DF, Pryor WA. Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts. Free Radic Biol Med. 1989;7(1):9-15. Barreto M, Villa MP, Corradi M, et al. Non-invasive assessment of airway inflammation in ship-engine workers. Int J Immunopathol Pharmacol. 2006;19(3):601-608. Guatura SB, Martinez JA, Santos Bueno PC, Santos ML. Increased exhalation of hydrogen peroxide in healthy subjects following cigarette consumption. Sao Paulo Med J. 2000;118(4):93-98. Nowak D, Antczak A, Krol M, et al. Increased content of hydrogen peroxide in the expired breath of cigarette smokers. Eur Respir J. 1996;9(4):652-657. Zappacosta B, Persichilli S, Mormile F, et al. A fast chemiluminescent method for H(2)O(2) measurement in exhaled breath condensate. Clin Chim Acta. 2001;310(2):187-191.
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Original Research ASTHMA
Hydrogen Peroxide in Exhaled Breath Condensate in Patients with Asthma A Promising Biomarker? Yue Teng, MSc; Peili Sun, MD; Jingying Zhang, MSc; Rongbin Yu, PhD; Jianling Bai, PhD; Xin Yao, MD, PhD; Mao Huang, MD, PhD; Ian M. Adcock, PhD; and Peter J. Barnes, MD, FCCP
Background: The measurement of hydrogen peroxide (H2O2) in exhaled breath condensate (EBC) has been proposed as a noninvasive way of monitoring airway inflammation. However, results from individual studies on EBC H2O2 evaluation of asthma are conflicting. The purpose of this study was to explore whether EBC H2O2 is elevated in people with asthma and whether it reflects disease severity and disease control or responds to corticosteroid treatment. Methods: Studies were identified by searching PubMed, Embase, Cochrane Database, Cumulative Index to Nursing and Allied Health Literature (CINAHL), and www.controlled-trials.com for relevant reports published before September 2010. Observational studies comparing levels of EBC H2O2 between patients with asthma who were nonsmokers and healthy subjects were included. Data were independently extracted by two investigators and analyzed using Stata 10.0 software. Results: Eight studies (involving 728 participants) were included. EBC H2O2 concentrations were significantly higher in patients with asthma who were nonsmokers compared with healthy subjects, and higher values of EBC H2O2 were observed at each level of asthma, classified either by severity or control level, and the values were negatively correlated with FEV1. In addition, EBC H2O2 concentrations were lower in patients with asthma treated with corticosteroids than in patients with asthma not treated with corticosteroids. Conclusions: H2O2 might be a promising biomarker for guiding asthma treatment. However, further investigation is needed to establish its role. CHEST 2011; 140(1):108–116 Abbreviations: CINAHL 5 Cumulative Index to Nursing and Allied Health Literature; EBC 5 exhaled breath condensate; eNO 5 exhaled nitric oxide; FEV1 % predicted 5 percentage predicted values of FEV1; H2O2 5 hydrogen peroxide; SMD 5 standard mean difference; WMD 5 weighted mean difference
is a chronic inflammatory disorder of the Asthma airways in which many cells and cellular elements
play a role.1,2 The assessment of airway inflammation is important in the pharmacologic treatment of asthma.3 Bronchoscopy, including BAL and bronchial biopsies, which are considered the most direct methods for evaluating airway inflammation, is too invasive for use in clinical practice. Much attention has been paid recently to the use of noninvasive measurements of airway inflammation for assessing the severity and guiding the treatment of asthma; such measurement tools include induced sputum,4 exhaled nitric oxide (eNO),5-7 exhaled carbon monoxide,8 and an electronic nose.9 Among these, eNO has been the most widely studied. However, a meta-analysis involving 1,010 participants
showed that the level of eNO might not be useful as a guide in making treatment decisions in the pharmacologic treatment of asthma, since it did not reduce exacerbations or improve FEV1 measurements compared with traditional assessment methods, such as symptoms and spirometry.10 The presence of oxidative stress has long been linked to the development and severity of asthma.11 Oxidative stress, defined as an increased exposure to oxidants and/or decreased antioxidant capacities, contributes to or accelerates the pathophysiologic characteristics of airway inflammation, resulting in airway hyperresponsiveness and leading to clinical symptoms of asthma.12 Hydrogen peroxide (H2O2) is a mediator of oxidative stress and can play an important role in asthma.13 Dohlman et al14 first demonstrated
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that H2O2 within expired breath was a marker of acute airway inflammation in pediatric patients with asthma in 1993. The measurement of exhaled breath condensate (EBC) is a noninvasive method for sampling airway secretions. Several biomolecules, including H2O2, 8-isoprostane, leukotrienes, prostaglandins, adenosine, glutathione, nitrites, and nitrates have been detected in the EBC.15 The presence of 8-isoprostane, leukotriene B4, and prostaglandin E2 in EBC has been confirmed by using reverse-phase liquid chromatography and mass spectrometry.16-21 The presence of H2O2 in the EBC of patients with asthma has been the focus of much subsequent research. Indeed, EBC H2O2 levels were increased in patients with asthma compared with healthy subjects.14,22,23 However, some studies failed to replicate these results, bringing into question whether EBC H2O2 levels could be used to discriminate patients with asthma from healthy subjects.24 The primary objectives of this meta-analysis were to determine (1) whether EBC H2O2 levels are elevated in patients with asthma, (2) the relationship between EBC H2O2 levels and spirometry results, (3) whether EBC H2O2 levels can reflect clinical severity or degree of symptom control, and (4) whether EBC H2O2 levels respond to corticosteroid treatment.
Materials and Methods Studies were identified by searching the literature using PubMed (1966 to September 2010), Embase (1980 to September 2010), Cochrane Database (1972 to September 2010), Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1981 to September 2010), and www.controlled-trials.com for relevant reports, using both keywords and free text words: (“asthma” or “airway inflammation” or “bronchial constriction” or “bronchial spasm” or “bronchial hyperreactivity” or “wheeze” or “wheezing”) Manuscript received October 31, 2010; revision accepted March 1, 2011. Affiliations: From the Department of Respiratory Medicine (Mss Teng and Zhang and Drs Sun, Yao, and Huang), The First Affiliated Hospital of Nanjing Medical University, Nanjing, China; the Department of Epidemiology and Biostatistics (Drs Yu and Bai), School of Public Health, Nanjing Medical University, Nanjing, China; and the Airway Disease Section (Drs Adcock and Barnes), National Heart and Lung Institute, Imperial College, London, England. Ms Teng and Dr Sun contributed equally to this work. Funding/Support: This study was supported by the National Natural Science Foundation of China [Grants 30700342, 81070025], Jiangsu Health Promotion Project, and “the Six Great Talents” [Grant 09-B1-001]. Correspondence to: Xin Yao, MD, PhD, Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing, 210029, China; e-mail: [email protected] © 2011 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-2816 www.chestpubs.org
THE BOTTOM LINE How does this work advance the field? This meta-analysis suggests a relationship between concentrations of hydrogen peroxide in exhaled breath condensate and asthma severity, asthma control status, lung function, and the response to corticosteroid treatment. It also provides evidence to support the measurement of hydrogen peroxide in exhaled breath condensate as a noninvasive biomarker to assess airway inflammation in patients with asthma.
What are the clinical implications? Measurement of hydrogen peroxide in exhaled breath condensate has potential as a biomarker for guiding asthma treatment, although further investigations with more carefully measured concentrations of hydrogen peroxide in exhaled breath condensate are needed.
and (“EBC” or “exhaled” or “expired”) and (“hydrogen peroxide” or “peroxide” or “H2O2”). There were no language restrictions. Inclusion criteria included the following: (1) observational studies, (2) studies comparing levels of EBC H2O2 between patients with asthma with an established diagnosis and healthy subjects, and (3) studies in which both patients with asthma and healthy subjects were nonsmokers and that salivary contamination had been eliminated. Exclusion criteria included the following: (1) studies with patients with asthma with diseases that could affect H2O2 levels, such as COPD,25 ARDS,26,27 and acute hypoxemic respiratory failure,28 (2) studies without healthy subjects as a control group,29,30 and (3) studies where smokers were not specifically excluded.23,31-33 Figure 1 shows the procedure and results of the search. Based on the explicit criteria, two reviewers (Y. T. and J. Z.) respectively checked the titles and abstract sections of all the articles retrieved. Full articles were searched when the information met the inclusion criteria. If there were any doubts about the titles or abstracts, we also reviewed the full articles for clarification. Any disagreements about adjudications were resolved through a third party (X. Y.). We contacted the authors for further clarification when it was not possible to resolve the disagreement. For each accepted study, the following data, when available, were extracted: the number of participants; age; gender; clinical features; treatments; method of measuring EBC H2O2; mean value, SD, SEM, and 95% CI of both patients with asthma and healthy subjects; and the percentage predicted values of FEV1 (FEV1 % predicted). Subject characteristics are given in more detail in Table 1. The SEM or 95% CI was transformed into SD, using statistical formulas. Analyses were performed using Stata software, version 10.0 (StataCorp LP; College Station, Texas). The weighted mean difference (WMD) was chosen to combine statistics; if the difference in mean value was too large and resulted in large heterogeneity, the standard mean difference (SMD) was chosen. Summary estimates and the weightings for each outcome were evaluated based on DerSimonian-Laird random effects models. Heterogeneity of study results was tested using the Mantel-Haenszel method, and results were considered heterogeneous if the P value was , .05. The I2 statistic was used to measure the extent of discrepancy among results. Data were pooled using the random effects model if there was marked heterogeneity (I2 . 50%), and the fixed effects model was used in other circumstances. Publication bias was evaluated through visual inspection of funnel plots, using the Begg test39 and the Egger asymmetry test.40 Publication bias was assumed to be present if the P value was , .05. CHEST / 140 / 1 / JULY, 2011
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Overall EBC H2O2 Levels in Patients With Asthma and Healthy Subjects Data were extracted from the eight articles regardless of the age, disease severity, and treatment measures of subjects. The overall WMD of EBC H2O2 between patients with asthma and healthy subjects was 0.49 mM (95% CI, 0.18-0.79; P 5 .002; I2 5 98.3%; random effects model). Patients with asthma showed significantly higher EBC H2O2 levels compared with healthy subjects. (Fig 3) EBC H2O2 Levels in Adult and Childhood Asthma There were five articles focused on adult asthma and two on childhood asthma. The remaining article did not mention the age of the participants, therefore, it could not be included in this subgroup analysis. The EBC H2O2 levels in adults with asthma were significantly higher than those in healthy adults (WMD, 0.58 mM; 95% CI, 0.20-0.97; P 5 .003; I2 5 99.0%; random effects model), while there was no statistical difference between children with asthma and healthy children (WMD, 0.23 mM; 95% CI, 20.02 to 0.49; P 5 .074; I2 5 31.9%; fixed effects model) (Fig 4).
Figure 1. The results of the systematic literature search. CINAHL 5 Cumulative Index to Nursing and Allied Health Literature; EBC 5 exhaled breath condensate; H2O2 5 hydrogen peroxide.
Results The systematic search (Fig 1) yielded 267 total references, of which 176 were unique. After a title review, 70 studies were excluded, yielding 106 articles. A subsequent abstract review rejected a further 80 of these references, yielding 26 candidate studies. Among the 26 studies, eight of them lacked a control group, and six of them did not provide sufficient EBC H2O2 data such as mean value, SD, SEM, or 95% CI. In addition, four of the studies did not specifically exclude smokers. We tried to contact the authors by e-mail to obtain further information. Finally, eight articles involving a total of 728 subjects14,22,24,34-38 were included in our meta-analysis. Among the eight studies, four directly provided SDs, three studies used the SEMs, and the remaining study provided 95% CIs. As the unequal variances were not mentioned, the classic t test was used to transform 95% CIs into SDs. All studies provided basic information and details of methodology related to patients and control subjects (Table 1). There was no evidence of publication bias (Begg test, P 5 .54; Egger test, P 5 .06). The funnel plot is shown in Figure 2.
EBC H2O2 Concentrations in Different Asthma Severities Asthma severity was classified according to four levels: intermittent, mild persistent, moderate persistent, and severe persistent. Because of the limited information provided by the studies, we could only separate patients with asthma into an intermittent group (four studies), a mild to moderate persistent group (five studies), and a severe persistent group (one study). The WMD of EBC H2O2 was 0.53 mM (95% CI, 0.25-0.82; P , .0001; I2 5 98.7%; random effects model) between patients with intermittent asthma and healthy subjects, and it was 0.62 mM (95% CI, 0.23-1.01; P 5 .002; I2 5 98.3%; random effects model) between patients with mild to moderate persistent asthma and healthy subjects. The values of EBC H2O2 for patients with each level of asthma classified by severity were higher than those of the healthy subjects (Fig 5). EBC H2O2 Concentrations and Asthma Control According to GINA (Global Initiative for Asthma),41 there are three categories of asthma control: controlled, partly controlled, and uncontrolled. However, we could not differentiate controlled asthma from partly controlled asthma because of the limited information provided by the articles, so we separated the patients with asthma into two groups: patients with
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111.7 ⫾ 19.4a 96 ⫾ 5c 92 ⫾ 7c 64 ⫾ 9c 95 ⫾ 3c 96.0 ⫾ 2.3b 101.8 ⫾ 1.5b 80 ⫾ 9.07a 69 ⫾ 9.87a 101 ⫾ 2.25a ... ... ... ... ... ... ... ... ... ...
19-34 30.5 ⫾ 6 29 ⫾ 5 29 ⫾ 7 30 ⫾ 4 10.7 ⫾ 3.0 10.0 ⫾ 0.4 17-56 17-56 ... ... ... ... ... 26-77 21-73 25-63 31-78 21-78 20-24
17 (7/10) 10 (NA) 10 (NA) 10 (NA) 10 (NA) 10 (NA) 15 (NA) 64 (47/17) 50 (27/23) 25 25 15 27 (NA) 43 (NA) 32 (NA) 27 (NA) 18 (12/6) 7 (5/2) 9 (6/3) 21 (8/13) 55 (31/24) 58 (47/11)
Healthy Mild intermittent asthma Mild persistent asthma Steroid-untreated asthma Steroid-treated asthma Moderate persistent asthma Steroid-untreated asthma Steroid-treated asthma Healthy Asthma
Healthy Intermittent asthma Persistent asthma Healthy Cough variant asthma Classic asthma Chronic cough Healthy Mild intermittent asthma Mild persistent asthma Moderate persistent asthma Severe persistent asthma Total asthma Healthy ... ... ... ... ... ... ... ... ... ... ...
... 50% of children had poor asthma control ... Unstable
Clinically stable for at least 4 wk Nocturnal wheezing and daily asthma symptoms ... Nocturnal wheezing and daily asthma symptoms (unstable) ... Clinically stable and no acute exacerbation for at least 4 wk
... ...
Clinical Feature
0.72 ⫾ 0.06b 0.43 ⫾ 0.08b 0.78 ⫾ 0.16b
b2-Agonist as required ICS (H) Oral steroids and ICS (H)
...
... b2-Agonist and/or theophylline bid, no systemic steroids ... ICS (⫾)
0.83-1.34c 0.46-1.02c 0.16-0.24c 2.0 ⫾ 0.22b
b2-Agonist ICS (M/H) and b2-agonist ... ICS (M)/oral steroids/ leukotriene antagonists ... No systematic steroids
2.2 ⫾ 0.31b 0.66 ⫾ 0.05a 1.14 ⫾ 0.36a 0.23 ⫾ 0.03a 0.83 ⫾ 0.34a 0.89 ⫾ 0.36a 0.24 ⫾ 0.22a 0.29 ⫾ 0.7a 1.85 ⫾ 0.10b 2.26 ⫾ 0.25b 1.65 ⫾ 0.13b 1.89 ⫾ 0.40b 1.89 ⫾ 0.07b 0.59 ⫾ 0.03b
0.40-0.87c 0.41-0.64c
0.024 ⫾ 0.016a 0.23-0.32c
0.27 ⫾ 0.04b 0.127 ⫾ 0.083a
b2-Agonist ICS (M/H) and b2-agonist
... b2-Agonist as required, steroids naive within 3 mo ... b2-Agonist
0.54 ⫾ 0.56 0.25 ⫾ 0.27a
Oral steroids/ICS/b2-agonist ... a
H2O2 (mM)
Treatment
Fluorometer
Spectrophotometer, 450 nm
Perkin Elmer UltravioletVIS Spectrometer L 10, 450 nm Spectrophotometer, 450 nm
Spectrophotometer (model 46; Lomo; St. Petersburg, Russia), 450 nm Spectrophotometer (Uvicon 940; Kontron Instruments; Zurich, Switzerland), 450 nm
Fluorometer (LS-50; Perkin-Elmer Corp; Norwalk, Connecticut) Spectrophotometer (AR 8003; Labtech Int Ltd; Uckfield, England), 450 nm
Measurement
F 5 female; H2O2 5 hydrogen peroxide; ICS 5 inhaled corticosteroids; ICS (H) 5 high dose of inhaled corticosteroids; ICS (M) 5 medium dose of inhaled corticosteroids; M 5 male; NA 5 not available; FEV1 % predicted 5 percentage predicted values of FEV1. amean ⫾ SD. bmean ⫾ SEM. c95% CI.
Ueno et al38/2008
Al Obaidi37/2007
Al-Obaidy and Al-Samarai36/2007
Robroeks et al24/2007
Loukides et al35/2002
97 ⫾ 3b 82 ⫾ 19.6a
31 ⫾ 1.8 18-62
35 (18/17) 70 (20/50)
86 ⫾ 2.4b 76 ⫾ 5b 64 ⫾ 5b
28 ⫾ 1.3 34 ⫾ 1.9 37 ⫾ 3
Healthy Steroid-untreated asthma
Emelyanov et al34/2001
... ...
7-18 ...
72 (42/30) 30 (14/16) 14 (5/9)
FEV1 % predicted
Steroid-untreated asthma Steroid-treated asthma
Age, y
Horváth et al22/1998
25 (NA) 11 (NA)
No. (M/F)
Asthma Healthy
14
Subject Group
Dohlman et al /1993
Study/Year
Table 1—Study Characteristics
Figure 2. The Begg funnel plot. WMD 5 weighted mean difference.
controlled and partly controlled asthma (defined as clinically stable for at least 4 weeks and/or use of b2-agonist as required; two studies) and patients with uncontrolled asthma (defined as nocturnal wheezing, daily asthma symptoms, and use of b2-agonist as required; two studies). The other studies lacked information on asthma control and were, therefore, excluded. The difference in mean value was very large in the uncontrolled asthma group, which resulted in a large heterogeneity, thus the SMD was chosen to combine statistics. Patients with controlled and partly controlled asthma showed higher levels of EBC H2O2 than healthy subjects, with an SMD of 1.06 (95% CI, 0.44-1.69; P 5 .001; I2 5 65.6%; random effects model). Much higher values of EBC H2O2 were observed in patients with uncontrolled asthma, with an SMD of 1.37 (95% CI, 0.93-1.80; P , .0001; I2 5 0%; fixed effects model). There was no significant difference between patients with controlled and partly controlled asthma vs patients with uncontrolled asthma; however, a trend of increasing EBC H2O2 levels was shown (Fig 6).
Figure 3. Overall EBC H2O2 levels in patients with asthma. See Figure 1 legend for expansion of the abbreviations.
Figure 4. EBC H2O2 levels in adults and children with asthma. See Figure 1 legend for expansion of the abbreviations.
EBC H2O2 Levels in Patients With SteroidUntreated and Steroid-Treated Asthma The studies were divided into two groups according to corticosteroid treatment, namely a steroiduntreated group (defined as not receiving regular inhaled or systemic corticosteroids and/or receiving b2-agonists as a relief medication on demand; five studies) and a steroid-treated group (defined as current use of inhaled or systemic corticosteroids regardless of use of b2-agonists or leukotriene receptor antagonists; two studies). Compared with healthy subjects, increased levels of EBC H2O2 were found in both the steroid-untreated group (WMD, 0.49 mM; 95% CI, 0.17-0.82; P 5 .003; I2 5 97.9%; random effects model) and the steroid-treated group (WMD, 0.37 mM; 95% CI, 0.19-0.56; P , .0001; I2 5 64.9%; random effects model). Patients with steroid-untreated asthma
Figure 5. EBC H2O2 concentrations in patients with different asthma severities. See Figure 1 legend for expansion of the abbreviations.
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formation. The combined r9 between the EBC H2O2 and the FEV1 % predicted was 20.14, which indicated that there was a significant negative correlation between the EBC H2O2 and the FEV1 % predicted (Fig 8).
Discussion
Figure 6. EBC H2O2 concentrations and levels of asthma control. See Figure 1 legend for expansion of the abbreviations.
showed significantly higher EBC H2O2 levels than patients with steroid-treated asthma (WMD, 0.19 mM; 95% CI, 0.05-0.34; P 5 .009; I2 5 0%; fixed effects model) (Fig 7). Correlation Coefficient Between EBC H2O2 Levels and the FEV1 % predicted The correlation coefficient between the EBC H2O2 value and the FEV1 % predicted value was reported in three out of eight studies. Fisher z9 transformation42 was used to transform r to z, which is considered as the normal distribution. The z value was 20.79 (95% CI, 2 1.13 to 20.44; P 5 .007; I2 5 79.7%; random effects model). Then the combined r9 was derived from z by using the reverse Fisher z9 trans-
Figure 7. EBC H2O2 levels in patients with steroid-untreated and steroid-treated asthma. See Figure 1 legend for expansion of the abbreviations. www.chestpubs.org
Based on eight studies involving 500 patients with asthma and 228 healthy subjects, higher values of EBC H2O2 were observed at each level of asthma, classified according to either severity or degree of control. EBC H2O2 levels were negatively correlated with the FEV1 % predicted in patients with asthma. Furthermore, the average EBC H2O2 levels showed a trend toward disease severity and the control level, although this did not reach statistical significance in our meta-regression analysis. In addition, EBC H2O2 was sensitive to corticosteroid treatment. H2O2 is one form of reactive oxygen species, which are derived from spontaneous or enzyme-catalyzed dismutation of superoxide anions.13 H2O2 contributes to oxidative stress in the airways and might cause the activation of various intracellular signaling pathways, leading to secretion of a variety of proinflammatory cytokines and chemokines.43 In addition, the predominant cells producing H2O2 are activated inflammatory cells such as eosinophils and neutrophils,35 suggesting that H2O2 might reflect the influx of inflammatory granulocytes and, therefore, disease severity. Our meta-analysis demonstrated that adults with asthma had higher EBC H2O2 levels than healthy subjects, indicative of an enhancement of oxidative stress in these patients, although no statistical significance was found between children with asthma and the control group. This might be the result of
Figure 8. Correlation coefficient (r) between EBC H2O2 and the percentage predicted values of FEV1. r9 5 20.14 (using the reverse Fisher z9 transformation); z 5 the value of r after using Fisher z9 transformation. See Figure 1 legend for expansion of other abbreviations. CHEST / 140 / 1 / JULY, 2011
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the lower levels of EBC H2O2 in children,44 which reduced the accuracy of detection, or may reflect the recent genome-wide association study data indicating differences between childhood onset of asthma and adult asthma.45 Corticosteroid treatment and the expiratory flow rate46 may also have an impact on the results. Since there were only two samples (89 children with asthma and 61 healthy children) included in the childhood group, further investigations are required to address whether there is an impact of age on EBC H2O2 levels. In our meta-analysis, patients with intermittent, mild to moderate, and severe persistent asthma expressed significantly higher levels of EBC H2O2 than the healthy subjects, although there was no statistical significance on severity using meta-regression analysis (P 5 .649) (Fig 9). Furthermore, levels of EBC H2O2 also showed a significant negative correlation with FEV1 % predicted. In addition, in patients with intermittent asthma, whose FEV1 % predicted values were normal, the values of H2O2 were still higher than in healthy subjects, suggesting the detection of H2O2 might be more sensitive than traditional spirometry in the less severe population, although further studies are needed to confirm this. In our meta-analysis, EBC H2O2 levels in patients with steroid-treated asthma were significantly lower than in patients with steroid-untreated asthma, and the patients with controlled and partly controlled asthma had lower average levels of EBC H2O2 than patients with uncontrolled asthma. Therefore, the level of EBC H2O2 might be a promising biomarker, with the potential of guiding asthma therapy. However, it should be pointed out that the studies to date
reporting the effects of steroids and asthma control status on EBC H2O2 level are mainly cross-sectional in design. Therefore, more randomized controlled trials are needed to confirm the influence of steroids and the relationship between EBC H2O2 level and asthma severity and control. There are some limitations to EBC H2O2 measurements. The most frequently used methods for measuring H2O2 in the EBC are the spectrophotometric and fluorimetric methods. However, the reproducibility of EBC H2O2 has not been provided in most studies, and the stated values vary between reports; some showed a satisfactory agreement,47,48 while others have a rather high coefficient of variation, especially with fluorimetric methods.46,49 Since the method of quantifying H2O2 can have an impact on the concentrations of EBC H2O2, we compared these two methods (spectrophotometric method, six studies; fluorimetric method, two studies). No statistical differences were observed in the WMD of participants, irrespective of whether the studies using the fluorimetric method were excluded (WMD, 0.39 mM; 95% CI, 0.12-0.65; I2 5 97.3%) or not (WMD, 0.49 mM; 95% CI, 0.18-0.79; I2 5 98.3%). Thus, we did not exclude these studies despite the methodologic differences in H2O2 detection. Cigarette smoke contains organic radicals, which can react with molecular oxygen in a redox-dependent manner to form superoxide anions, hydroxyl radicals, and H2O2.50 Previous studies have found that smoking increases EBC H2O2 concentrations, both in patients with asthma and in healthy subjects,33,51,52 so that smoking status must be taken into consideration when using EBC H2O2 levels to assess airway inflammation.
Figure 9. EBC H2O2 WMD (95% CI) between patients with different types of asthma and healthy subjects. The arrowheads indicate the mean of the WMD between patients with specific types of asthma and healthy subjects. The vertical lines indicate the 95% CI of the WMD between patients with specific types of asthma and healthy subjects. See Figure 1 and 2 legends for expansion of abbreviations. 114
Original Research
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The relative lack of studies based on normal populations and the relatively high variability of EBC H2O2 levels in healthy individuals52-54 are the critical limitations for the clinical application of EBC H2O2. Further studies on normal populations are required to establish reference values using standardized methods of measurement.
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Conclusions H2O2 in the EBC is elevated in adult patients who have asthma and are nonsmokers compared with healthy adult subjects. EBC H 2O 2 levels demonstrated trends toward correlations with disease severity and the degree of asthma control and had a negative correlation with FEV1 % predicted. In addition, EBC H2O2 is sensitive to corticosteroid treatment. EBC H2O2 might, therefore, be a promising biomarker in guiding asthma treatment, although further large-scale studies are needed. Acknowledgments Author contributions: Ms Teng: read and met the International Committee of Medical Journal Editors (ICMJE) criteria for authorship, designed this study, extracted data, performed the analysis, wrote the first draft of the manuscript, and read and approved the final manuscript. Dr Sun: read and met the ICMJE criteria for authorship, designed this study, and read and approved the final manuscript. Ms Zhang: read and met the ICMJE criteria for authorship, extracted data, and read and approved the final manuscript. Dr Yu: read and met the ICMJE criteria for authorship, performed the analysis, and read and approved the final manuscript. Dr Bai: read and met the ICMJE criteria for authorship, performed the analysis, and read and approved the final manuscript. Dr Yao: read and met the ICMJE criteria for authorship, designed this study, extracted data, performed the analysis, critically revised the manuscript, and read and approved the final manuscript. Dr Huang: read and met the ICMJE criteria for authorship, designed this study, and read and approved the final manuscript. Dr Adcock: read and met the ICMJE criteria for authorship, critically revised the manuscript, and read and approved the final manuscript. Dr Barnes: read and met the ICMJE criteria for authorship, critically revised the manuscript, and read and approved the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors had no role in the design and conduct of the study, in the data extraction, analysis, or interpretation of the data, or in the preparation, review, or approval of the manuscript.
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