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Elevated Concentrations of Exhaled Hydrogen Peroxide in Asthmatic Patients^*

^* From the Hospital Therapeutic Clinic (Drs. Emelyanov, Fedoseev, Abulimity, Rudinski, Fedoulov, and Karabanov), Pavlov Medical University, St. Petersburg, Russia; and the Department of Thoracic Medicine (Dr. Barnes), National Heart and Lung Institute, London, UK.

Correspondence to: Alexander Emelyanov, MD, Hospital Therapeutic Clinic, Pavlov Medical University, 6/8 L. Tolstogo St, St. Petersburg 197089, Russia; e-mail: emelav{at}netscape.net

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Background: Airway inflammation is important in the development and progression of asthma. Activation of inflammatory cells induces a respiratory burst resulting in the production of reactive oxygen species, such as H₂O₂. The aim of this study was to measure the concentration of H₂O₂ in exhaled breath condensate and its correlation with airway obstruction, airway hyperresponsiveness, and concentration of eosinophil cationic protein (ECP) in serum in 70 steroid-naive, atopic patients with unstable asthma (20 men; age range, 18 to 62 years) and 17 normal subjects (7 men; age range, 19 to 34 years).

Methods: Exhaled H₂O₂ was measured using a colorimetric assay, and the concentration of ECP in serum was measured using radioimmunoassay. Airway hyperresponsiveness was expressed as the provocative concentration of inhaled histamine causing a 20% fall in FEV₁ (PC₂₀).

Results: In patients with asthma, the mean H₂O₂ concentration was significantly elevated compared to values in normal subjects: 0.127 ± 0.083 mol/L vs 0.024 ± 0.016 mol/L (p < 0.001). There was a significant correlation among H₂O₂ concentration, FEV₁, PC₂₀, and ECP in serum.

Conclusion: We conclude that exhaled H₂O₂ is significantly elevated in asthmatic patients. This is correlated with disease severity and indirect markers of airway inflammation. Measurement of exhaled H₂O₂ may be useful to assess airway inflammation and oxidative stress in asthmatic patients.

Key Words: asthma • expired breath condensate • hydrogen peroxide

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Bronchial asthma is a chronic inflammatory disease of airways.¹ Inflammatory cells (eosinophils, neutrophils, macrophages) release superoxide anion, which then undergoes spontaneous or enzyme-catalyzed dismutation to form H₂O₂. H₂O₂ is a highly reactive oxygen species involved in cellular injury via further reactions leading to hydroxyl radical and lipid peroxidation products.² Increased production of free radicals occurs in airway inflammation, and H₂O₂ is detectable in exhaled air. Increased levels of H₂O₂ have been reported in expired breath condensate in cigarette smokers³ ; and in patients with COPD, particularly during exacerbations⁴ ; ARDS^{5 6} ; bronchiectasis⁷ ; and asthma.^{8 9 10 11} However, the relationship among exhaled H₂O₂, airway obstruction, airway hyperresponsiveness, and markers of airway inflammation in asthmatic patients is not yet certain. Therefore, we have evaluated the concentration of H₂O₂ in patients with steroid-naive asthma of differing disease severity and have compared it with FEV₁, airway hyperresponsiveness to histamine, and levels of eosinophil cationic protein (ECP) in serum.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Patients
Seventy patients (20 men and 50 women) with unstable, steroid-naive, atopic asthma (age range, 18 to 62 years; median age, 33 years) were examined at the outpatient department of the Hospital Therapeutic Clinic of Pavlov Medical University, St. Petersburg, Russia. They had nocturnal wheezing and daily asthma symptoms. Asthma was diagnosed according to American Thoracic Society criteria.¹² Diagnosis was based on clinical history, reversibility of FEV₁ > 15%, and diurnal variability of peak expiratory flow rate > 20%. Atopic status was assessed by positive skin-prick test result (> 3 mm) to common inhalant allergens. The mean duration of asthma was 10.3 ± 8.4 years (mean ± SD), and mean FEV₁ at the time of the study was 82 ± 19.6% predicted. All patients were nonsmokers. Ex-smokers and patients with active allergic rhinitis or upper respiratory tract infections during or within 4 weeks of the study were excluded. Patients were receiving short-acting, inhaled ß₂-agonists only as required. No patients had received systemic steroids within 3 months of assessment. Seventeen nonatopic, nonsmoking, normal subjects (7 men and 10 women) aged 19 to 34 years (median age, 30 years) served as a control group. They had no history of respiratory or cardiovascular disease and were not receiving any long-term medications. Their FEV₁ was 111.7 ± 19.4% predicted. The study was approved by the local ethics committee. The participants were informed in writing, and their written consent was obtained.

Lung Function Tests
FEV₁ was measured by dry spirometry (Vitalograph; Buckingham, UK). Airway hyperresponsiveness was assessed by histamine challenge (MasterScope automatic spirometer; Jaeger GmbH; Wurzburg, Germany). After an initial 0.9% sodium chloride inhalation, patients were exposed to doubling concentrations of histamine delivered as five breaths from a dosimeter (Dosimeter APS pro; Jaeger GmbH). FEV₁ was measured 2 min after each inhalation. Airway hyperresponsiveness was expressed as provocative concentration of inhaled histamine causing a 20% fall in FEV₁ (PC₂₀). PC₂₀ was determined by linear interpolation from the log₁₀ concentration-response curve. Bronchial challenge was performed in 28 asthmatic patients with FEV₁ > 70% predicted.

Expired Breath Condensate and H₂O₂ Measurement
Expired breath condensate was collected by using a glass condensing device that was placed in a large chamber with ice. After rinsing their mouth, subjects breathe tidally with normal frequency through a mouthpiece for 20 min while wearing a nose clip. The mouthpiece was also used as a saliva trap. The volume of condensate was 2 to 4 mL.

H₂O₂ assay was carried out immediately after collecting the condensate. H₂O₂ was measured by using a colorimetric assay as described previously.¹³ Briefly, 100 µL of condensate was mixed with 100 µL of 3,3',5,5' tetramethylbenzidine in 0.42 mol/L citrate buffer, pH 3.8, and 10 µL of horseradish peroxidase (52.5 U/mL). The samples were incubated at room temperature for 20 min, and the reaction was stopped by addition of 10 µL 18 N sulfuric acid. The reaction product was measured spectrophotometrically (model 46; Lomo; St. Petersburg, Russia) at 450 nm. A standard curve of H₂O₂ was performed for each assay. The variation between the H₂O₂ values on separate days in 10 normal subjects was minor (3.6%).

ECP Measurement
The concentration of ECP in serum was measured by using radioimmunoassay in duplicate (Pharmacia and Upjohn Diagnostics AB; Uppsala, Sweden). Blood samples were obtained at 9 AM to 10 AM after an overnight fast (Vacutainer; Becton-Dickenson; Meylan Cedex, France). Clotting time was 60 ± 10 min. After centrifugation, serum was frozen and stored at - 20°C within 14 days until the assay.

Statistics
Student’s unpaired two-tailed t test, Pearson correlation (r), and Spearman coefficient (rs) were used for statistical methods (Statistica for Windows 5; StatSoft; Tulsa, OK). Statistical significance was assumed at p < 0.05, and the data are expressed as mean ± SD.

	Results

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The concentrations of H₂O₂ in expired breath condensate and ECP in serum in asthmatic patients and normal subjects are shown in Table 1 . The concentrations of exhaled H₂O₂ and ECP in serum were significantly elevated in asthmatic patients compared to normal subjects. There was a negative correlation between expired H₂O₂ concentration and FEV₁ (r = - 0.45; n = 70; p < 0.001; Fig 1 ) and between H₂O₂ and PC₂₀ (rs = - 0.40; n = 28; p < 0.05; Fig 2 ), and a positive correlation between expired H₂O₂ concentration and serum ECP (r = 0.37; n = 70; p < 0.01; Fig 3 ). However, there was no correlation among the concentration ECP in serum, FEV₁ (r = - 0.07; n = 70; p > 0.05), and PC₂₀ (r = - 0.055; n = 28; p > 0.05).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Correlation between concentration of exhaled H2O2 and FEV1 in asthmatic patients.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 2.. Correlation between concentration of exhaled H2O2 and airway hyperresponsiveness to histamine (log PC20 is shown) in asthmatic patients.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 3.. Correlation between concentration of exhaled H2O2 and serum ECP in asthmatic patients.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

Exhaled H₂O₂ levels have previously been related to the eosinophil differential counts in induced sputum and activity of peripheral neutrophils in asthmatic patients.^{11 14} Therefore, elevated concentrations of H₂O₂ may result from an enhanced number and activity of inflammatory cells in the airways. Expired H₂O₂ levels were elevated even in patients with mild asthma with FEV₁ > 80% predicted and may reflect airway inflammation even in patients with mild asthma.

Increased oxidative stress is implicated in asthma and other respiratory diseases.^{15 16} H₂O₂ is one of the most stable of the reactive oxygen metabolites. Due to lack of charge, it may easily penetrate cellular membranes and may generate hydroxyl radical in presence of iron cations (Fenton reaction). H₂O₂ and hydroxyl radical are able to react with membrane and lipid components of bronchial lining fluid and cause their peroxidation.² Concentrations of H₂O₂ in expired breath condensate are raised in patients with inflammatory diseases of the airways, such as asthma, COPD, and bronchiectasis.^{4 5 6 7} Elevated exhaled H₂O₂ levels are associated with concentration of thiobarbituric acid-reactive products in expired breath condensate, nitric oxide in exhaled air, airway obstruction, and airway hyperresponsiveness to methacholine in asthmatic patients.^{9 11} This is consistent with several studies^{17 18} in animals and human airways in vitro in which reactive oxygen species could lead to airway inflammation, airway hyperresponsiveness, and subsequent bronchoconstriction.

In the present study, we found a significant negative correlation among exhaled H₂O₂, FEV₁ (percent predicted), and PC₂₀ in our asthmatic patients. The changes of these parameters may reflect ongoing airway inflammation. Airway hyperresponsiveness was inversely related to the average number of total leukocytes, especially mast cells, activated eosinophils, and CD8+ and CD45RO+ cells in bronchial biopsy specimens from asthmatic patients.^{19 20} We suggest that the increased intensity of inflammation accompanied by an elevated level of expired H₂O₂ can be followed by airway hyperresponsiveness and airway obstruction.

Activation of inflammatory cells, and particularly eosinophils, is the prominent feature of airway inflammation in patients with asthma.¹ Eosinophils release several mediators, including H₂O₂ and ECP, that may amplify the inflammatory process in the airways. ECP is suggested to be an indirect marker of airway inflammation.²¹ We found an increased level of ECP in serum in our patients. ECP may diffuse from inflammatory cells in the airways to the blood compartment. Several studies support this hypothesis.^{22 23 24 25} There was a correlation between enhanced activity of eosinophils in bronchial mucosa and serum levels of ECP.²² Suppression of eosinophilic inflammation by inhaled corticosteroids reduces the concentrations of ECP in BAL and in serum.^{23 24 25} Another explanation may be that eosinophils are activated both locally in the lungs and in the blood.²⁶ We found a significant correlation between exhaled H₂O₂ and ECP in serum. Perhaps, this may reflect activation of eosinophils in the asthmatic airways. This relation supports the use of expired H₂O₂ as a surrogate marker of airway inflammation in asthmatic patients.

However, there was no correlation among ECP, FEV₁, and PC₂₀ in our patients. This finding implies that expired H₂O₂ levels may better reflect airway hyperresponsiveness and airway obstruction than serum ECP. In conclusion, our study shows that the concentrations of exhaled H₂O₂ are elevated in steroid-naive, atopic patients with unstable asthma. This is associated with airway obstruction and the indirect markers of airway inflammation, PC₂₀, and serum ECP. Exhaled H₂O₂ may be useful to assess the degree of airway inflammation and oxidative stress in asthmatic patients.

	Footnotes

 
Abbreviations: ECP = eosinophil cationic protein; PC₂₀ = provocative concentration of inhaled histamine causing a 20% fall in FEV₁

Supported in part by a research grant from MacLab (Melbourne, Australia).

Received for publication December 4, 2000. Accepted for publication April 25, 2001.

	References

TOP Abstract Introduction Materials and Methods Results Discussion References

 

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