Original contribution
Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects

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Abstract

Enhanced exhalation of H2O2 and TBARs have been reported in various inflammatory lung diseases. This may reflect activated phagocytes influx and free radical generation in the airways. However, to apply these compounds as markers of oxidative stress it is necessary to understand factors influencing their exhalation in healthy subjects. We investigated the concentration of H2O2 and TBARs in expired breath condensate (EBC) of 58 healthy volunteers. EBC was collected seven times every 4 h during 24 h and three times every 7 d during 2 consecutive weeks. The H2O2 exhalation revealed diurnal variation with two-peak values 0.45 ± 0.29 μM and 0.43 ± 0.22 μM at 12:00 and 24:00 h. The lowest concentrations, 0.26 ± 0.13 μM and 0.25 ± 0.26 μM, were found at 20:00 and 8:00 h. Cigarette smokers exhaled about 2.4 times more H2O2 than never smoked subjects. Moreover, in contrast to nonsmokers, cigarette smokers’ H2O2 exhalation was stable over 2 week observation. The mean H2O2 concentration estimated over the whole 2 week period was higher in subjects above 40 years regardless of smoking habit, and it positively correlated with age in never smoked subjects (p < .004). Smoking of one cigarette caused 1.8-fold rise in H2O2 exhalation (p < .01). The baseline H2O2 levels correlated with cumulative cigarette consumption (p < .05) and MEF 25% of predicted (p < .05). Neither moderate exercise nor one puff of salbutamol nor ipratropium influenced significantly the concentration of H2O2 and TBARs in EBC. Only 4 of 120 EBC specimens from never smoked subjects revealed detectable levels of TBARs. Cigarette smokers exhaled more TBARs (p < .05) than never smoked volunteers. Our results indicate that healthy never smoked subjects exhale H2O2 with diurnal variation and significant changes over 2 week observation. Cigarette smoking enhanced H2O2 generation in the airways. These results could be useful for planning studies with exhaled H2O2 as a marker of airway inflammation. Occasional detection of TBARs in EBC of never smoked persons may be a result of sufficient antioxidant activity in the airways that protects tissues from peroxidative damage.

Introduction

Measurement of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARs) in EBC is suggested to reflect free radical generation and peroxidative damage in the airways [1], [2], [3], [4]. Increased H2O2 and/or TBARs exhalation has been reported in numerous inflammatory lung disorders including bronchial asthma [1], [3], [5], [6], chronic obstructive pulmonary disease [2], [7], adult respiratory distress syndrome [8], [9], [10], pneumonia [10], and also in asymptomatic cigarette smokers [11]. Pulmonary phagocytes, type II pneumocytes, and other cells of the respiratory tract are potential sources of exhaled H2O2 [12], [13], [14], [15]. TBARs (mainly malondialdehyde) are recognized as end products of polyunsaturated fatty acid peroxidation, however, they are also formed during oxidative injury of DNA, proteins, or carbohydrates [16]. The activity of H2O2 producing cells and pathways leading to TBARs formation may change in response to many endogenic and exogenic physicochemical factors. Moreover, generation/exhalation of these compounds depends on antioxidant defense in the airways. This may explain why some healthy subjects and patients with lung inflammatory disorders did not exhale detectable amounts of H2O2 and TBARs [1], [2], [7]. Most studies on H2O2 and TBARs exhalation in patients with lung inflammatory disorders involved only single determination of these compounds [1], [2], [3], [5], [7], [11]. Little is known about variability of H2O2 and TBARs in EBC of healthy subjects.

Therefore, we investigated the circadian rhythm of H2O2 and TBARs exhalation in healthy subjects, its variability during 2 week observation and the effect of moderate exercise, acute exposure to cigarette smoke, and single dose of inhaled bronchodilators. The correlations between subjects’ age, spirometric parameters, and cigarette smoking habits were also analyzed.

Section snippets

Study populations

Fifty-eight healthy volunteers (University Hospital staff) were enrolled (18 current cigarette smokers and 40 never smoking persons) who had not suffered from any infectious diseases for at least 3 months prior to the study (Table 1). They were free of any medication and routine physical examination showed nothing abnormal.

Study design

Subjects were asked to attend the laboratory three times every 7 d during 2 consecutive weeks (visit I at 1st day, visit II at 7th day and visit III at 14th day) for EBC

Factors influencing H2O2 and TBARs levels in EBC

Neglecting of mouth washing with distilled water just before and at 7th and 14th min of EBC collection resulted in significant rise of H2O2 levels and tended to increase TBARs readings (Table 2). Wearing the nose clip while collecting EBC had no effect on both H2O2 and TBARs levels. However, the nasal airflow blockade made the whole exhaled air pass through the collecting part of the tube and undergo condensation. The yield of 20 min condensate collection was slightly higher (but not

Discussion

We found that one third of never smoked subjects and all current cigarette smokers continuously exhale detectable amounts of H2O2. Moreover, almost all never smoked healthy volunteers (35 of 40; 87.5%) had positive EBC H2O2 readings on at least one of three measurements during 2 week observation. It differs from our previous studies showing only 22% and 49% of H2O2 positive readings in healthy never smoked subjects and asymptomatic cigarette smokers, respectively [11]. In our present study we

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