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Am. J. Respir. Crit. Care Med., Volume 158, Number 4, October 1998, 1042-1046

Combined Use of Exhaled Hydrogen Peroxide and Nitric Oxide in Monitoring Asthma

ILDIKÓ HORVÁTH, LOUISE E. DONNELLY, ANDRÁS KISS, SERGEI A. KHARITONOV, SAM LIM, K. FAN CHUNG, and PETER J. BARNES

Department of Thoracic Medicine, National Heart and Lung Institute at Imperial College School of Medicine, and Royal Brompton Hospital, London, United Kingdom

	ABSTRACT

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Oxidative stress contributes to airway inflammation and exhaled hydrogen peroxide (H₂O₂) and nitric oxide (NO) are elevated in asthmatic patients. We determined the concentrations of expired H₂O₂ and NO in 116 asthmatic (72 stable steroid-naive, 30 stable steroid-treated, and 14 severe steroid-treated unstable patients) and in 35 healthy subjects, and studied the relation between exhaled H₂O₂, NO, FEV₁, airway responsiveness, and eosinophils in induced sputum. Both exhaled H₂O₂ and NO levels were elevated in steroid-naive asthmatic patients compared with normal subjects (0.72 ± 0.06 versus 0.27 ± 0.04 µM and 29 ± 1.9 versus 6.5 ± 0.32 ppb, respectively; p < 0.001) and were reduced in stable steroid-treated patients (0.43 ± 0.08 µM, p < 0.05, and 9.9 ± 0.97 ppb, p < 0.001). In unstable steroid-treated asthmatics, however, H₂O₂ levels were increased, but exhaled NO levels were low (0.78 ± 0.16 µM and 6.7 ± 1.0 ppb, respectively). There was a correlation between expired H₂O₂, sputum eosinophils and airway hyperresponsiveness (methacholine PC₂₀). Exhaled NO also correlated with sputum eosinophils, but not with airway hyperresponsiveness. Our findings indicate that measurement of expired H₂O₂ and NO in asthmatic patients provides complementary data for monitoring of disease activity.

	INTRODUCTION

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Oxidative stress, defined as an increased exposure to oxidants and/or decreased antioxidant capacities, is implicated in airway inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma (1- 4). Inflammatory cells, especially eosinophils which produce more superoxide anions (O₂·) than neutrophils or macrophages, release several reactive oxygen- and nitrogen-derived species such as nitric oxide (NO). O₂· and NO combine to form peroxynitrite (ONOO), which is highly reactive and may damage airway epithelium (5). NO has several biological activities in the airways and rapidly reacts to generate peroxynitrite anions (9, 10). O₂ is rapidly metabolized to form hydrogen peroxide (H₂O₂), which diffuses into the airway lining fluid and may evaporate into exhaled air.

H₂O₂ in exhaled breath condensate is increased in COPD, adult respiratory distress syndrome, cigarette smokers, and asthma (11), and may be used as a noninvasive marker of oxidative stress. However, the effect of corticosteroids on production of reactive oxygen species (ROS) or H₂O₂ is not certain. Exhaled NO is another noninvasive marker of airway inflammation, but could be dose-dependently reduced following corticosteroid treatment (17), and may be of limited value in assessment of asthmatic inflammation in patients with asthma treated with steroids. Therefore, we have evaluated the utility these two noninvasive markers of oxidative stress and airway inflammation in asthma of different severity and have compared them with the eosinophils in induced sputum and airway hyperresponsiveness.

	METHODS

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Subjects

Four groups of nonsmoking subjects were studied (Table 1). None of the 35 nonsmoking nonatopic normal subjects had a history of respiratory or cardiovascular disease or was receiving any long-term medication; 72 steroid-naive atopic asthmatic patients were receiving ₂-agonists only as required; 30 stable steroid-treated atopic asthmatic patients (without asthma symptoms for > 4 wk prior to study with no history of upper respiratory infection in the last month) were treated with inhaled corticosteroids; 14 steroid-treated patients (3 nonatopic) were unstable (peak flow variability > 20%, nocturnal wheezing and daily asthma symptoms) despite treatment with inhaled and oral steroids. Atopic status was assessed by positive skin prick tests (> 3 mm). The protocol was approved by the ethics committee of the Royal Brompton Hospital, and informed consent from each subject was obtained.

View this table: [in this window] [in a new window]   TABLE 1 SUBJECT CHARACTERISTICS AND EXHALED H2O2 AND NO LEVELS*

Study Design

Subjects' details were obtained and then baseline spirometry and exhaled NO were measured, followed by collection of expired breath condensate. In some patients, this was followed by methacholine challenge and/or sputum induction.

Expired Breath Condensate and H₂O₂ Measurement

Expired breath condensate was collected by using a glass condensing device, with an inner glass chamber which contained ice and was suspended in a larger chamber (Figure 1). Condensate was formed on the outside surface of the inside glass that was separated from ambient air. After rinsing their mouth, subjects breathed tidally through a mouthpiece connected to the inlet for 15 min while wearing a nose-clip. The mouthpiece was also used as a saliva trap. Approximately 1 ml of breath condensate was collected and stored at 70° C. H₂O₂ was measured using a colorimetric assay as described previously (20). Briefly, 100 µl of condensate was mixed with 100 µl of 420 µM 3'3'5'5'-tetramethylbenzidine in 0.42 M 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 stopped by the addition of 10 µl of 2 N sulfuric acid. The product was measured spectrophotometrically (Model AR 8003; Labtech Int. Ltd., Uckfield, UK) at 450 nm. A standard curve of H₂O₂ was performed for each assay with a detection limit of 0.1 µM. In 21 normal subjects the variation between the H₂O₂ values on separate days was minor (7.6%).

View larger version (22K): [in this window] [in a new window]   Figure 1.   Schematic representation of the apparatus for collection of exhaled condensate.

Exhaled NO Measurements

Exhaled NO was measured by a chemiluminescence analyzer (Model LR2000; Logan Research, Rochester, UK) as previously described (21, 22). The analyzer is sensitive to NO from 1 (ppb, by volume) to 5,000 ppb, and with a resolution of 0.3 ppb. In addition to NO, the analyzer also measures CO₂ (resolution 0.1% CO₂; response time, 200 ms) and sample pressure and volume in real time. The analyzer was calibrated using certified NO mixtures (90 ppb and 436 ppb) in nitrogen (BOC Special Gases, Guildford, UK). Measurements of exhaled NO were made by slow exhalation (5-6 L/min) from total lung capacity for 15 to 20 s against a mild resistance to exclude nasal contamination. The value corresponding to the plateau of the end- exhaled CO₂ reading (5% CO₂) was taken as representative of an alveolar sample. In these measurements the pressure during expiration was kept constant (3 ± 0.4 mm Hg) by using a visual display of expiratory flow measured by pressure and volume sensors in the analyzer.

Sputum Induction

Subjects (n = 32) were instructed to wash their mouth thoroughly with water and then to inhale 3.5% saline nebulized via an ultrasonic nebulizer (De Vilbiss 99; De Vilbiss, Heston, UK). The sputum samples were kept at 4° C for no longer than 2 h prior to processing. The volume of the sample was recorded and the sample was diluted with 2 ml of Hanks' balanced salt solution containing 1% dithiothreitol (Sigma Chemicals, Poole, UK). Total cell counts were done on a hemocytometer using Kimura stain, and slides were made with a cytospin (Shandon, Runcorn, UK) and stained with May-Grunwald-Giemsa stain for differential cell counts.

Pulmonary Function and Methacholine Provocation Test

Forced expiratory volume (FEV₁) was measured by a dry spirometer (Vitalograph, Buckingham, UK). Airway responsiveness was assessed by methacholine challenge. After an initial 0.9% NaCl inhalation patients were exposed to doubling concentrations of methacholine delivered as five breaths from a dosimeter (Dosimeter MB3; Mefar, Bovezzo, Italy) at 3-min intervals until FEV₁ fell by > 20% from the postsaline value. FEV₁ was measured 2 min after each inhalation. The concentration of histamine giving a 20% fall in FEV₁ (PC₂₀) was determined by linear interpolation from the log₁₀ concentration-response curve.

Statistical Analysis

Data are given as means ± SEM. Comparisons of H₂O₂ and NO concentrations between groups were made by Kruskal-Wallis test and subsequent Dunn's multiple comparison test. A two tailed p < 0.05 was considered significant. Spearman correlation tests were performed to detect a correlation between variables.

	RESULTS

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Normal subjects had the lowest H₂O₂ levels and it was below the limit of detection in 10 healthy subjects (Table 1). In steroid-naive asthmatic patients, concentrations of exhaled H₂O₂ and NO were significantly elevated, whereas those well-controlled on inhaled steroid treatment exhibited significantly lower levels of H₂O₂ and NO in their exhaled air, which were similar to normal values. Unstable asthmatic patients had elevated H₂O₂ levels in expired condensate, whereas their exhaled NO was similar to levels in normal subjects (Figure 2). There was a negative correlation between H₂O₂ and PC₂₀ methacholine (Figure 3A), and positive correlation between eosinophil count in induced sputum and H₂O₂ (Figure 3B) and NO levels (Figure 3C). However, there was no correlation between the concentrations of exhaled NO and PC₂₀ values in these patients. The relationship between H₂O₂, NO, and FEV₁ was assessed in all asthmatic subjects and no correlation was found.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Individual data for H2O2 concentrations in exhaled condensate (A) and for NO in exhaled air (B).

View larger version (13K): [in this window] [in a new window]   Figure 3.   (A) Relation between PC20 methacholine and exhaled H2O2. (B) Eosinophil count in induced sputum and exhaled H2O2. (C ) Eosinophil count in induced sputum and exhaled NO.

	DISCUSSION

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We have found that levels of exhaled H₂O₂ and NO were elevated in mild asthmatic patients compared with normal control subjects and were related to the eosinophil differential counts in induced sputum. Exhaled H₂O₂ levels were also related to airway responsiveness. Expired H₂O₂ and NO levels were normal in well-controlled asthma patients treated with inhaled corticosteroids, suggesting that these exhaled markers reflect airway inflammation in asthma. However, in unstable severe patients, the levels of exhaled H₂O₂ were elevated, while exhaled NO levels were normal.

The concentrations of exhaled H₂O₂ measured in normal subjects were similar to those reported in previous studies (11, 13). In asthmatic patients, the mean level of exhaled H₂O₂ (0.72 µM) is also comparable to levels obtained previously from stable asthmatic children (0.8 µM) (14). However, higher levels have been reported in patients with an exacerbation of asthma (1.5 µM) (15).

Increased oxidative stress is implicated in asthma (7) and this may be reflected by expired H₂O₂. An increased concentration of exhaled H₂O₂ may represent an increased production of oxidants and/or a reduced free radical scavenging capacity in asthmatic airways. Oxygen radicals may contribute to epithelial shedding, release of inflammatory mediators, and development of airway hyperresponsiveness. Thus, a close relationship between O₂· produced by circulating neutrophils and the degree of airway hyperresponsiveness has been shown in asthmatic patients (23). Furthermore, peroxynitrite formed by a reaction between O₂· and NO causes airway hyperresponsiveness in animals (24). Indeed, we found a significant correlation between exhaled H₂O₂ concentrations and the degree of airway hyperresponsiveness to methacholine in our patients.

Activation of inflammatory cells, and particularly eosinophils, is a key feature of asthmatic inflammation (4, 7), and may be reflected by eosinophils in induced sputum (18, 25). In the present study, we found a significant correlation between exhaled H₂O₂ and NO and eosinophil counts in induced sputum. Because eosinophil differential counts in sputum have been shown to relate to the intensity of airway inflammation in asthma measured in bronchial biopsies and to the severity of the disease (25, 26), this relationship supports the use of expired H₂O₂ and NO in assessing airway inflammation in asthmatic patients.

However, measurement of exhaled H₂O₂ and NO may give different information about the inflammatory process. Although almost all of the steroid-naive asthmatic patients had elevated levels of exhaled NO, only approximately 70% of them had increased levels of exhaled H₂O₂, suggesting that in some patients there is no overproduction of oxidants. This finding implies that measurement of exhaled NO is a more sensitive technique in diagnosing asthma, but that not all of these patients have enhanced oxidative stress. Some of these patients did not need to use a bronchodilator for several months before the study, although they were diagnosed as asthmatic and had airway hyperresponsiveness. In patients well-controlled with inhaled steroids, both exhaled NO and H₂O₂ showed a similar pattern. Corticosteroids inhibit the oxidative burst of leukocytes and also inhibit expression of inducible nitric oxide synthase resulting in a reduction of exhaled H₂O₂ and NO. Steroid treatment also inhibits eosinophil recruitment to the airways, therefore it may influence exhaled mediator levels by preventing the influx of H₂O₂ and NO producing cells. The differences in exhaled H₂O₂ levels between the inhaled steroid-naive and the steroid-treated patients imply differences in oxidative stress between these two groups. However, in severe unstable asthmatics, exhaled H₂O₂ levels were elevated, despite the use of high doses of inhaled and of oral steroids, whereas concentrations of exhaled NO were almost normal. Thus, the patients with severe asthma who are still symptomatic presumably have ongoing inflammation as reflected by sputum eosinophil despite steroid therapy, whereas exhaled NO is normalized since iNOS induction sensitive to steroids, while oxidative stress is still present because of the residual inflammation.

In summary, our study shows that the levels of exhaled H₂O₂ and NO are elevated in asthmatic patients and this is related to other indices of airway inflammation. The fact that expired H₂O₂ is elevated in patients with severe asthma not well controlled on high doses of inhaled steroids, whereas exhaled NO is normal, indicates that this measurement may be more useful in monitoring control of asthmatic inflammation.

	Footnotes

Correspondence and requests for reprints should be addressed to Professor P. J. Barnes, M.A., D.M., D.Sc., F.R.C.P., Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.

(Received in original form October 28, 1997 and in revised form May 6, 1998).

Dr. Horváth was supported by the European Respiratory Society and the Hungarian National Scientific Research Foundation (OTKA-F017050), Dr. Donnelly by the National Asthma Campaign (UK), and Dr. Kharitonov by the British Lung Foundation.

Acknowledgments: The authors thank Julaiha Mohamed and Susan Woollett for their excellent technical help.

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				I Horvath, S Loukides, T Wodehouse, E Csiszer, P J Cole, S A Kharitonov, and P J Barnes Comparison of exhaled and nasal nitric oxide and exhaled carbon monoxide levels in bronchiectatic patients with and without primary ciliary dyskinesia Thorax, January 1, 2003; 58(1): 68 - 72. [Abstract] [Full Text] [PDF]

				R. P. Bowler and J. D. Crapo Oxidative Stress in Airways: Is There a Role for Extracellular Superoxide Dismutase? Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): S38 - 43. [Abstract] [Full Text] [PDF]

				E. Huszar, G. Vass, E. Vizi, Zs. Csoma, E. Barat, Gy. Molnar Vilagos, I. Herjavecz, and I. Horvath Adenosine in exhaled breath condensate in healthy volunteers and in patients with asthma Eur. Respir. J., December 1, 2002; 20(6): 1393 - 1398. [Abstract] [Full Text] [PDF]

				R. Pawliczak, X.-L. Huang, U. B. Nanavaty, M. Lawrence, P. Madara, and J. H. Shelhamer Oxidative Stress Induces Arachidonate Release from Human Lung Cells through the Epithelial Growth Factor Receptor Pathway Am. J. Respir. Cell Mol. Biol., December 1, 2002; 27(6): 722 - 731. [Abstract] [Full Text] [PDF]

				A. Emelyanov, G. Fedoseev, O. Krasnoschekova, A. Abulimity, T. Trendeleva, and P.J. Barnes Treatment of asthma with lipid extract of New Zealand green-lipped mussel: a randomised clinical trial Eur. Respir. J., September 1, 2002; 20(3): 596 - 600. [Abstract] [Full Text] [PDF]

				K. Kostikas, G. Papatheodorou, K. Ganas, K. Psathakis, P. Panagou, and S. Loukides pH in Expired Breath Condensate of Patients with Inflammatory Airway Diseases Am. J. Respir. Crit. Care Med., May 15, 2002; 165(10): 1364 - 1370. [Abstract] [Full Text] [PDF]

				A. Schwingshackl, R. Moqbel, and M. Duszyk Nitric oxide activates ATP-dependent K+ channels in human eosinophils J. Leukoc. Biol., May 1, 2002; 71(5): 807 - 812. [Abstract] [Full Text] [PDF]

				T J Warke, P S Fitch, V Brown, R Taylor, J D M Lyons, M Ennis, and M D Shields Exhaled nitric oxide correlates with airway eosinophils in childhood asthma Thorax, May 1, 2002; 57(5): 383 - 387. [Abstract] [Full Text] [PDF]

				S. Loukides, D. Bouros, G. Papatheodorou, P. Panagou, and N. M. Siafakas The Relationships Among Hydrogen Peroxide in Expired Breath Condensate, Airway Inflammation, and Asthma Severity Chest, February 1, 2002; 121(2): 338 - 346. [Abstract] [Full Text] [PDF]

				A. Emelyanov, G. Fedoseev, A. Abulimity, K. Rudinski, A. Fedoulov, A. Karabanov, and P. J. Barnes Elevated Concentrations of Exhaled Hydrogen Peroxide in Asthmatic Patients Chest, October 1, 2001; 120(4): 1136 - 1139. [Abstract] [Full Text] [PDF]

				S. A. KHARITONOV and P. J. BARNES Exhaled Markers of Pulmonary Disease Am. J. Respir. Crit. Care Med., June 1, 2001; 163(7): 1693 - 1722. [Full Text] [PDF]

				S. LIM, D. GRONEBERG, A. FISCHER, T. OATES, G. CARAMORI, W. MATTOS, I. ADCOCK, P. J. BARNES, and K. F. CHUNG Expression of Heme Oxygenase Isoenzymes 1 and 2 in Normal and Asthmatic Airways . Effect of Inhaled Corticosteroids Am. J. Respir. Crit. Care Med., November 1, 2000; 162(5): 1912 - 1918. [Abstract] [Full Text]

				L. M. van den TOORN, J.-B. PRINS, S. E. OVERBEEK, H. C. HOOGSTEDEN, and J. C. de JONGSTE Adolescents in Clinical Remission of Atopic Asthma Have Elevated Exhaled Nitric Oxide Levels and Bronchial Hyperresponsiveness Am. J. Respir. Crit. Care Med., September 1, 2000; 162(3): 953 - 957. [Abstract] [Full Text]

				G. L. LARSEN and P. G. HOLT The Concept of Airway Inflammation Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): S2 - 6. [Full Text] [PDF]

				J. C. de JONGSTE and K. ALVING Gas Analysis Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): S23 - 27. [Full Text] [PDF]

				M. Silvestri, D. Spallarossa, E. Battistini, V. Brusasco, and G. A Rossi Dissociation between exhaled nitric oxide and hyperresponsiveness in children with mild intermittent asthma Thorax, June 1, 2000; 55(6): 484 - 488. [Abstract] [Full Text]

				J. A. NIGHTINGALE, D. F. ROGERS, K. FAN CHUNG, and P. J. BARNES No Effect of Inhaled Budesonide on the Response to Inhaled Ozone in Normal Subjects Am. J. Respir. Crit. Care Med., February 1, 2000; 161(2): 479 - 486. [Abstract] [Full Text]