Hydrogen peroxide in exhaled breath condensate: A clinical study.

OBJECTIVES: To study the ongoing inflammatory process of lung in healthy individuals with risk factors and comparing with that of a known diseased condition. To study the inflammatory response to treatment.
BACKGROUND: Morbidity and mortality of respiratory diseases are raising in trend due to increased smokers, urbanization and air pollution, the diagnosis of these conditions during early stage and management can improve patient's lifestyle and morbidity.
MATERIALS AND METHODS: One hundred subjects were studied from July 2010 to September 2010; the level of hydrogen peroxide concentration in exhaled breath condensate was measured using Ecocheck.
RESULTS: Of the 100 subjects studied, 23 were healthy individuals with risk factors (smoking, exposure to air pollution, and urbanization); the values of hydrogen peroxide in smokers were 200-2220 nmol/l and in non-smokers 340-760 nmol/l. In people residing in rural areas values were 20-140 nmol/l in non-smokers and 180 nmol/l in smokers. In chronic obstructive pulmonary disease cases, during acute exacerbations values were 540-3040 nmol/l and 240-480 nmol/l following treatment. In acute exacerbations of bronchial asthma, values were 400-1140 nmol/l and 100-320 nmol/l following treatment. In cases of bronchiectasis, values were 300-340 nmol/l and 200-280 nmol/l following treatment. In diagnosed pneumonia cases values were 1060-11800 nmol/l and 540-700 nmol/l following treatment. In interstitial lung diseases, values ranged from 220-720 nmol/l and 210-510 nmol/l following treatment.
CONCLUSION: Exhaled breath condensate provides a non-invasive means of sampling the lower respiratory tract. Collection of exhaled breath condensate might be useful to detect the oxidative destruction of the lung as well as early inflammation of the airways in a healthy individual with risk factors and comparing the inflammatory response to treatment.

INTRODUCTION

Airway inflammation plays an important role in various respiratory lung diseases, including recurrent wheezing, asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Several attempts have been made therefore to detect and monitor inflammatory changes and mediators using non-invasive methods. Analysis of exhaled breath condensate (EBC), a novel and a non-invasive method for studying the composition of airway lining fluid, has the potential for assessing airway inflammation.[1] Analysis of EBC is also useful for assessing the response to treatment.[2] This study helps to validate the analysis of EBC by measuring hydrogen peroxide (H2O2) concentration in healthy non-smokers, smokers, diseased, and also comparing the response to treatment. Inflammatory cells release H2O2, which can be detected in EBC. Elevated levels of H2O2 have been found in a number of respiratory disorders, thus H2O2 is considered to be a possible biomarker of airway inflammation.

MATERIALS AND METHODS

In this hospital-based study, conducted between July 2010 and September 2010, 100 randomly selected subjects were analyzed with EBC H2O2. Sputum positive tuberculosis patients, pregnant women, children less than 12 yrs and immunocompromised patients were excluded from the study. EBC was collected and analyzed using Ecocheck-Ecosreen (Jaeger, Hoechberg, Germany) device in all 100 subjects. The subjects were instructed to clean the oral cavity with water and then breathe through a mouth piece and a 2 way non-breathing valve, which also serve as a saliva trap. They were asked to breath at a normal frequency and tidal volume wearing a nose clip for a period of 15 min. About 1-3 ml of EBC was collected at –2 to –4°C [Figure 1]. The collected EBC was diluted with equal quantity of dilution buffer. The diluted sample was analyzed in a measuring chamber containing biosensors.[3] The results were analyzed statistically using t test. The amount of condensate generated per exhalation varies among individuals. Minute ventilation remains the major determinant of the amount of condensate over time. The concentration of hydrogen peroxide in exhaled air depends on expiratory flow rate.[4]

Figure 1

Schematic representation of a collection apparatus

RESULTS

Of the 100 cases studied, 23 were healthy individuals with risk factors, like smoking, exposure to air pollution and urbanization. The values of H2O2 in smokers were 200-2220 nmol/l and in non-smokers values were 340-760 nmol/l [Table 1, Figure 2]. In 10 smokers the standard deviation was 643.135 and in 13 non-smokers standard deviation was 217.279I with significant P value of 0.045 (P<0.05) [Table 2]. In people residing in rural areas values were from 20-140 nmol/l in non-smokers and 180 nmol/l in smokers. H2O2 concentrations were correlated with pack years. In majority of subjects, as the pack years increased, the H2O2 levels were also found to be increased. However in some of the subjects varied H2O2 levels were observed irrespective of pack years. For instance, one subject with 8 pack years had the H2O2 level as high as 2220 nmol/l, whereas in two subjects with 15 pack years, the values were 180 and 340 nmol/l [Table 3].

Table 1

H2O2 concentration in cases of healthy subjects

Figure 2

Analysis in healthy subjects

Table 2

Statistical analysis of H2O2 concentration in healthy subjects

Table 3

Values of H2O2 in relation to pack years

In patients who are known COPD presented with acute exacerbations (as per anthonisen criteria and GOLD criteria), the predicted FEV1% varied from 32-65% (48±16%) with H2O2 levels of 540-3040 nmol/l. These patients were treated with bronchodilators and corticosteroids as per treatment protocol. Following treatment, the predicted FEV1% varied from 35-71%(53±18%) and the concentrations of H2O2 were reduced to 240-480 nmol/l [Table 4, Figure 3]. Before treatment standard deviation was 770.076 and following treatment standard deviation was 94.571 with P value of 0.022 [Table 5].

Table 4

H2O2 concentration and predicted FEV1% in COPD

Figure 3

Analysis report in COPD

Table 5

Statistical analysis of H2O2 concentration in COPD

In cases of acute exacerbations of bronchial asthma, the values of H2O2 were 400-1140 nmol/l and following treatment the values were reduced to 100-320 nmol/l [Table 6, Figures 4 and 5] with a significant P value of 0.002 [Table 7]. In these patients predicted FEV1% varied from 18-62% (40±22%) and following treatment the predicted FEV1% drastically improved to 68-89% (78±10).

Table 6

H2O2 concentration and predicted FEV1% in bronchial asthma

Figure 4

Analysis report in bronchial asthma

Figure 5

Analysis report in bronchial asthma

Table 7

Statistical analysis of H2O2 concentration in asthma

In other conditions like bronchiectasis, values of H2O2 were 300-340 nmol/l and 200-280 nmol/l [Table 8], in pneumonia 1060-11800 nmol/l and 540-700 nmol/l [Table 9], and in patients with interstitial lung diseases 220-720 nmol/l and 210-510 nmol/l [Table 10] before and after treatment, respectively. The P values of the above three conditions could not be calculated as the sample size was small. Spirometry was also performed in all these patients but the lung function tests could not be correlated with H2O2 in all these patients as the sample size was small.

Table 8

H2O2 concentration in Bronchiectasis

Table 9

H2O2 concentration in Pneumonia

Table 10

H2O2 concentration In interstitial lung disease

DISCUSSION

A variety of inflammatory markers present in EBC have been investigated as possible biomarkers of disease activity.[5] EBC contains aerosolized airway epithelial lining fluid particles and volatile compounds. There is increasing evidence that exhaled markers may reflect biochemical changes in airway lining fluid.[6] Table 11 shows the various markers in exhaled breath. H2O2 was one of the most commonly studied markers in EBC.[78] Lung is constantly exposed to oxygen, so highly susceptible to oxidative stress in the form of reactive oxygen species (super oxide ion, hydroxyl radical, and hydrogen peroxide). These reactive oxygen species produced by active inflammatory cells like neutrophils, macrophages, activated eosinophils, epithelial cells, and endothelial cells.[9] Thus, measurement of concentration of reactive oxygen species in exhaled breath condensate can reflect the underlying inflammation. In the present study, H2O2 measurements were evaluated and analyzed.

Table 11

Contents of exhaled breath condensate

Exhaled breath condensate was measured in cigarette smokers versus healthy control subjects.[10] Cigarette smokers had a 5-fold higher mean expired breath H2O2 level than non-smokers.[1112] In another study that attempted to correlate exhaled breath H2O2 with H2O2 generated from the alveolar lining fluid, exhaled H2O2 was 5 × 104 times lower than H2O2 produced in the alveolar lining fluid. This difference was attributed to the presence of antioxidants in the lining fluid of the lower respiratory tract. The above study showed that level of H2O2 in exhaled breath condensate of smokers is increased half an hour after combustion of one cigarette. In the present study, the levels of H2O2 were elevated in healthy smokers and also in healthy non-smokers who are residing in urban area compared to those of rural area. These elevated levels can be attributed to constant exposure for vehicle and industrial pollution. The H2O2 values in healthy individuals with risk factors are more than 180 nmol/l, whereas the healthy individuals residing in rural areas with minimal risk factors had values of H2O2 varied from 20-140 nmol/l. Hence, the level of H2O2 below 200 nmol/l can be considered as normal reference value as per our study and needs further studies to support our observation in India.

Dekhuijzen and coworkers demonstrated increased H2O2 in exhaled breath condensate of patients with stable COPD relative to healthy controls with a further increase noted during an acute exacerbation.[13-16] The effect of corticosteroids on the level of hydrogen peroxide studied by van Beurden et al.[17] Levels of H2O2 also correlated with eosinophils differential counts in induced sputum. In the present study, H2O2 was increased in all stable COPD patients with further increase during acute exacerbations with reduced predicted FEV1%. Lower the value of predicted FEV1%, higher the elevated H2O2 concentration. These patients with exacerbations after treatment with bronchodilators, corticosteroids (both inhalational and parenteral) showed reduction in H2O2 levels with the improvement in predicted FEV1%.

Oxidative stress plays an important pathogenetic role in many inflammatory diseases including asthma. Emelyanov et al. studied the correlation between asthma, concentration of H2O2 and FEV1. They concluded that exhaled H2O2 may be useful to assess the degree of airway inflammation and oxidative stress in asthmatic patients and significant negative correlation among exhaled H2O2 and FEV1.[18] In the present study, the H2O2 levels were elevated during acute exacerbation with decreased predicted FEV1% and reduced H2O2 levels with significant improvement in predicted FEV1% in all cases following treatment.

Bronchiectasis, a suppurative lung disease, is characterized by significant pulmonary oxidant stress that can be measured using exhaled breath H2O2. In a study by Loukides and coworkers,[2] patients with bronchiectasis displayed exhaled H2O2 levels higher than normal controls, and a negative correlation between the H2O2 levels and FEV1 was documented. In the studied cases of bronchiectasis, H2O2 was raised significantly with reduction in the levels following treatment.

In the pilot study by Mikuls et al., patients with rheumatoid arthritis with interstitial lung diseases had increased levels of exhaled H2O2 compared with controls, suggesting that EBC H2O2 is a potentially useful biomarker.[19] In the present study, in patients with interstitial lung disease, pneumonia H2O2 estimated by EBC were found to be raised and showed decreased values following treatment.

The measurement of the H2O2 marker in exhaled breath condensate can be used routinely for i) early prediction of the ongoing inflammatory process in healthy individuals who are exposed to risk factors, and for educating them in future, ii) early tool of assessing exacerbation of the lung condition and to reduce the morbidity, iii) as a marker in assessing the inflammatory response to treatment. Hence, the detection of H2O2 in EBC can be used for routine clinical practice and research activities.

This type of facility is a rare modality available in India, as per our knowledge. The drawbacks of this facility are that the establishment of the unit is quite expensive and sensors need to be changed for every new patient, which are to be imported and of high cost.

CONCLUSION

Oxidative stress is implicated in various lung diseases. Its assessment with non-invasive technique is of great value. Collection of exhaled breath condensate is a non-invasive method. Measurement of H2O2 in EBC sample can be used as a method of measuring oxidative destruction in the lung and inflammation of the airways. Even in healthy individuals with risk factors, elevated H2O2 levels in EBC is a general marker for airway inflammation and can be used as an early predictor of the ongoing inflammatory process. This measurement can be carried out easily and its application in inflammatory airway diseases has been extensively studied. Most of the clinical studies reported higher levels of H2O2 in healthy individuals with risk factors and diseased conditions compared to normal subjects without risk factors and also the levels of H2O2 decreased following treatment.