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

Free Radical Biology & Medicine, Vol. 30, No. 2, pp. 178 –186, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

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Original Contribution EXHALATION OF H2O2 AND THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS) BY HEALTHY SUBJECTS DARIUSZ NOWAK,* SYLWIA KAŁUCKA,† PIOTR BIAŁASIEWICZ,*

AND

MACIEJ KRO´ L*

†

*Department of Physiology and Department of Family Medicine, Medical University of Lodz, Lodz, Poland (Received 7 June 2000; Accepted 4 October 2000)

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 ␮M and 0.43 ⫾ 0.22 ␮M at 12:00 and 24:00 h. The lowest concentrations, 0.26 ⫾ 0.13 ␮M and 0.25 ⫾ 0.26 ␮M, 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. © 2001 Elsevier Science Inc. Keywords—Hydrogen peroxide, Thiobarbituric acid reactive substances, Reactive oxygen substances, Expired breath condensate, Cigarette smoking, Free radicals

INTRODUCTION

[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 [12–15]. 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

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– 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 –10], pneumonia Address correspondence to: Dariusz Nowak, Department of Physiology, Medical University of Lodz, Mazowiecka St. 6/8, 90-131 Lodz, Poland; Tel: ⫹48 (42) 678-2661; Fax: ⫹48 (42) 678-2661; E-Mail: [email protected]. 178

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Table 1. Characteristic of the Study Subjects Parameter

Whole group

Cigarette smokers*

Nonsmokers

N Sex, M/F Age [yrs] BMI [kg/m2] FVC [% predict.] FEV1 [% predict.] FEV1/VC [%] MEF 25 [% predict.] MEF 50 [% predict.]

58 31/27 37 ⫾ 12 23.8 ⫾ 3.3 93.7 ⫾ 18.4 103.3 ⫾ 13.9 101.7 ⫾ 7.5 90.7 ⫾ 27.1 91.7 ⫾ 22.5

18 7/11 38 ⫾ 11 24.6 ⫾ 3.3 104.4 ⫾ 17.3 107.6 ⫾ 11.3 97.9 ⫾ 7.3 81.2 ⫾ 22.1 89.3 ⫾ 21.1

40 24/16 36 ⫾ 12 23.5 ⫾ 3.2 88.9 ⫾ 16.8 101.3 ⫾ 14.6 103.4 ⫾ 6.9 95.2 ⫾ 28.1 92.7 ⫾ 23.0

Spirometric parameters are expressed as % of predicted [23]. FVC ⫽ forced vital capacity; FEV1 ⫽ forced expiratory volume in 1 s; MEF 50 and MEF 25 ⫽ maximal expiratory flow at 50 and 75% of expired vital capacity; respectively, BMI ⫽ body mass index. *Present cigarette consumption and cumulative cigarette consumption was 17 ⫾ 4 cigarettes a day and 17.8 ⫾ 10.5 pack-years, respectively.

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 [1–3,5,7,11]. 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. MATERIAL AND METHODS

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 collection and pulmonary function measurement. The spirometry was performed only at visit I, before EBC, with Flowscreen (Erich Jaeger GmbH co., Marburg, Germany) equipped with software compatible to American Thoracic Society standards [17]. The EBC was always collected between 8:00 and 10:00 am. Cigarette smokers had to refrain from cigarette smoking for 12 h before the visit. If the patient failed to refrain from smoking the

visit was rescheduled within 1–3 d. Apart from determination of baseline morning H2O2 and TBARs exhalation, four experiments were performed with volunteers recruited from the whole group: 1. Determination of circadian rhythm of H2O2 and TBARs exhalation. This was performed with 12 never smoked physicians employed in our University Hospital that revealed detectable morning H2O2 concentration in EBC. They were on 24 h duty and attended the laboratory seven times that allowed us to collect EBC at 8:00, 12:00, 16:00, 20:00, 24:00, 4:00, and 8:00. 2. Effect of acute cigarette smoking on H2O2 and TBARs levels in EBC. Seventeen cigarette smokers (10 women, 7 men, present daily cigarette consumption 17 ⫾ 5, cumulative cigarette consumption 16 ⫾ 11 pack-years) who refrained from cigarette smoking for 12 h attended the laboratory at 8:00 am. EBC was collected just before and 30 min after smoking of one cigarette. Pulmonary function was measured before the first condensate collection. 3. Effect of moderate exercise on H2O2 and TBARs exhalation. This involved 12 subjects (6 cigarette smokers and 6 nonsmokers) that after 20 min of rest (at 8:00 am) started exercise test (bicycle ergometer, external workload 120 W). The performance was finished after 6 min or until the heart rate reached at least 120/min. EBC was collected just before and after the exercise. 4. Effect of inhaled single dose of salbutamol (0.1 mg) or ipratropium (0.02 mg) (MDI, Polfa SA, Poznan´, Poland) on H2O2 and TBARs exhalation was studied. Eleven subjects (5 cigarette smokers, 6 nonsmokers) or 12 subjects (6 cigarette smokers and 6 nonsmokers) were asked to attend the laboratory at 8:00 am to collect EBC just before and 30 min after inhalation of ipratropium or salbutamol, respectively.

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The EBC was collected as described previously [1,11]. Subjects were asked to breathe out spontaneously through a mouthpiece with a saliva trap connected to the tube and to breathe in with the mouthpiece removed, for 20 min. The collecting part of the tube was covered with ice and salt. Each subject wore a nose clip and rinsed their mouth with distilled water just before and at 7th and 14th min of collection to reduce the possible H2O2 and TBARs evaporation from saliva and nasal spaces [1,8, 11]. The EBC (3–5 ml) was immediately used for H2O2 and TBARs determination. In 12 never smoked persons, two consecutive collections of EBC were performed, the first one as described above and the second one without the mouth washing or wearing the nose clip.

peroxidation products that at low pH and at high temperature participate in nucleophilic addition reaction with thiobarbituric acid generating red fluorescent complex [16]. The content of TBARs in EBC was determined as previously described [1] except that results are expressed in ␮M [2] instead of nmol per sample. Briefly, 100 ␮l of the condensate was mixed with 2 ml of TBA solution (0.67 g dissolved in 100 ml of deionized water, then diluted 1:1 with glacial acetic acid), boiled for 30 min, cooled in ambient temperature and then chromogen was extracted into 2.5 ml of butanol by vigorous shaking for 1 min. Following centrifugation (10 min, 1500 ⫻ g, 25°C), TBARs were measured spectrofluorimetrically (excitation at 515 nm, emission at 546 nm) [19]. Readings were expressed in ␮M using the regression equation Y ⫽ 0.12X ⫺ 0.07 (where Y ⫽ ␮M of TBARs, X ⫽ intensity of emission expressed in arbitrary units). Tetramethoxypropane (0.01–50 ␮M) was used as an external standard and the method sensitivity was 0.05 ␮M [2]. The intra-assay variability was 2.1% and 2.9% for standard solutions of 0.1 ␮M and 0.5 ␮M tetramethoxypropane (n ⫽ 3), respectively.

Measurement of H2O2

Statistical analysis

No more than three subjects were investigated each day. The Ethics Committee of the Medical University of Lodz had approved the study and all the subjects had given informed consent before enrollment. Collection of EBC

The concentration of H2O2 in EBC was measured according to the method of Ruch et al. [18]. Briefly, 600 ␮l of EBC was mixed with 600 ␮l of HRP solution (1 U/ml) containing 100 ␮M homovanillic acid and was incubated for 60 min at 37°C. Then, the sample was mixed with 150 ␮l 0.1 M glycine-NaOH buffer (pH 12.0) with addition of 25 mM EDTA. The homovanillic acid oxidation product as a measure of the amount of H2O2 was determined spectrofluorimetrically using a Perkin Elmer Luminescence Spectrometer LS-50B (Norwalk, CT, USA). Excitation was at 312 nm and emission was measured at 420 nm. Contrary to our previous studies [1,11] (in which the results were expressed in nanomoles [nmol] of H2O2 per sample) readings were expressed in ␮mol/l (␮M) using the regression equation Y ⫽ (X ⫺ X0)0.0676, (where Y ⫽ micromoles of H2O2 per liter of EBC; X ⫽ intensity of emission expressed in arbitrary units; X0 ⫽ intensity of emission given by reference sample receiving distilled water instead of EBC). The lower limit of H2O2 detection was 0.083 ␮M [2]. The intra-assay variability was 1.3%, 1.8%, and 1.9% for standard solutions of 0.1 ␮M, 0.25 ␮M, and 0.5 ␮M H2O2 (n ⫽ 3), respectively. Measurement of TBARs TBARs are low molecular weight compounds formed via decomposition of certain primary and secondary lipid

Data are expressed as mean ⫾ SD. For readings that gave results below the method sensitivity the H2O2 and TBARs concentration in EBC was assumed 0 nM. The differences between groups were analyzed using the Mann-Whitney U test and the Wilcoxon matched pairs test. Correlation coefficients were calculated by the Pearson test. In all cases a p value of ⬍ .05 was considered significant. RESULTS

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 significantly) when subjects wore nose clip (4.1 ⫾ 0.5 ml vs. 3.9 ⫾ 0.4 ml, n ⫽ 6, NS). Therefore subjects wore nose clip and washed mouth with distilled water. Table 3 shows the effect of smoking one cigarette, moderate exercise, and inhalation of one dose of bronchodilators on H2O2 and TBARs levels in EBC of healthy subjects. Only smoking of one cigarette increased by 1.8-fold (p ⬍

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Table 2. Effect of Washing Mouth with Distilled Water and Wearing Nose Clip During EBC Collection on H2O2 and TBARs Readings Conditions of expired breath condensate collection Mouth washing Variable

With

Without

Wearing nose clip With

Without

H2O2 [␮M] 0.20 ⫾ 0.15 0.35 ⫾ 0.12* 0.13 ⫾ 0.12 0.09 ⫾ 0.14 TBARs [␮M] 0.04 ⫾ 0.05 0.07 ⫾ 0.04 ND ND Both experiments involved two different groups of six never smoked subjects. In experiment with mouth washing, subjects always wore noseclip and in experiment with wearing nose clip, subjects always washed mouth. * vs. mouth washing, p ⬍ .03. ND ⫽ not detectable (⫽ 0).

.01) the H2O2 exhalation. The levels of H2O2 before smoking correlated negatively with MEF 25% predicted (r ⫽ ⫺0.64, p .05) and positively with cumulative cigarette consumption expressed in pack-years (r ⫽ 0.59, p ⬍ .05) (Figs. 1 and 2). However, the H2O2 concentration after smoking of one cigarette as well as cigarette smoking habit-induced increment in H2O2 exhalation did not correlate with spirometric parameters and cumulative cigarette consumption (data not shown). Other factors (moderate exercise, one puff of ipratropium or salbutamol) did not change significantly the H2O2 and TBARs levels (Table 3). It should be pointed out that majority of condensate specimens (102 of 128), especially those from never smoked subjects (54 of 60) did not contain detectable amounts of TBARs. Circadian rhythm of H2O2 and TBARs exhalation Figure 3 shows results of 24 h monitoring of H2O2 in EBC collected every 4 h from 12 never smoking subjects. The mean H2O2 concentration was highest at 12:00 (0.45 ⫾ 0.29 ␮M) and at 24:00 (0.43 ⫾ 0.22 ␮M). The lowest mean H2O2 level was observed at 20:00 (0.26 ⫾ 0.13 ␮M) and at 8:00 (0.25 ⫾ 0.26 ␮M). The significant differences were noted between the mean H2O2 level at 24:00 and at 8:00 next morning (p ⬍ .01) and at 24:00

Fig. 1. Negative correlation between H2O2 levels in EBC and MEF 25% of predicted in 17 asymptomatic cigarette smokers. Spirometry and EBC collection were performed after 12 h restraining from cigarette smoking.

and at 20:00 (p ⬍ 0.05). In this experiment no sample of EBC gave positive result for TBARs. H2O2 and TBARs exhalation during 2 week observation Table 4 shows results of monitoring of H2O2 exhalation in healthy subjects over 2 weeks. The mean H2O2 concentration in EBC in the whole group (including never smoked subjects (n ⫽ 40) and asymptomatic current cigarette smokers (n ⫽18)) was 0.24 ⫾ 0.19 ␮M and 0.23 ⫾ 0.17 ␮M at the 1st and 7th d, respectively. The H2O2 exhalation by the whole group at 14th d of observation was 1.3 times lower than those at previous timepoints. Healthy never smoked subjects had almost the same mean H2O2 concentration in EBC at day 1 and 7 while H2O2 exhalation at day 14 decreased significantly by 1.6-fold (Table 4). Current cigarette smokers exhaled

Table 3. Effect of Selected Factors on H2O2 and TBARs Concentration in EBC of Healthy Subjects H2O2 [␮M] Factor Smoking of one cigarette (n ⫽ 17) Moderate exercise# (n ⫽ 12) One puff of salbutamol (n ⫽ 12) One puff of ipratropium (n ⫽ 11)

TBARs [␮M]

Before

After

Before

After

0.17 ⫾ 0.13 0.31 ⫾ 0.44 0.37 ⫾ 0.13 0.34 ⫾ 0.08

0.31 ⫾ 0.23* 0.23 ⫾ 0.22 0.36 ⫾ 0.13 0.38 ⫾ 0.24

0.01 ⫾ 0.01 0.02 ⫾ 0.05 ND ND

0.02 ⫾ 0.02 0.01 ⫾ 0.02 ND ND

* vs. value before smoking of one cigarette, p ⬍ .01. In parentheses number of subjects included in particulate trial. # The mean pulse rate and respiratory rate calculated during condensate collection before and after exercise were 72 ⫾ 9 min⫺1, 16 ⫾ 3 min⫺1 and 141 ⫾ 9 min⫺1, 27 ⫾ 8 min⫺1, respectively. ND ⫽ not detectable (⫽ 0).

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7th D

14th D

Subjects

N

Whole group Never smoked Current smokers

58 0.24 ⫾ 0.19# 0.23 ⫾ 0.17## 0.18 ⫾ 0.18 40 0.19 ⫾ 0.20# 0.18 ⫾ 0.16## 0.12 ⫾ 0.16 18 0.41 ⫾ 0.34* 0.37 ⫾ 0.19** 0.37 ⫾ 0.22**

Breath condensate was collected three times every 7 d and H2O2 measured just after collection termination. * vs. never smoked subjects, p ⬍ .003. ** vs. never smoked subjects, p ⬍ .0001. # vs value found at day 14th, p ⬍ .05. ## vs. value found at day 14th, p ⬍ .01.

Fig. 2. Positive correlation between H2O2 levels in EBC and cumulative cigarette consumption (pack-years) in 17 asymptomatic cigarette smokers. EBC was collected after 12 h restraining from cigarette smoking.

0.41 ⫾ 0.34 ␮M of H2O2 at day 1 and this did not change significantly at day 7 and 14. Thus the decrease in H2O2 exhalation observed for the whole group at 14th d was the result of H2O2 decline in EBC of never smoked subjects. Analysis of individual changes in H2O2 exhalation showed significant inverse correlation between increments of H2O2 exhalation during the 1st and the 2nd

week of observation. This was observed for the whole group as well as for never smoked subjects and current cigarette smokers (Table 5). The mean H2O2 concentration in EBC of current cigarette smokers was significantly higher at all three time-points (2.16-, 2.05-, 3.08-fold for the 1st, 7th, and 14th d, respectively) than that in EBC of never smoked subjects. In all cigarette smokers H2O2 concentration was above method sensitivity at any time-point. In never smoked subgroup 13 (4 women), 12 (4 women), and 22 (10 women) of 40 subjects had not detectable (ND) H2O2 concentrations in EBC at day 1, 7, and 14, respectively. In five never smoked persons (1 woman) we found no H2O2 readings during 2 week monitoring. On the other hand, 13 of 40 never smoked volunteers exhaled detectable amounts of H2O2 at each investigated time-point. The mean H2O2 concentration calculated for these subjects did not differ from each other and were 0.22 ⫾ 0.10 ␮M, 0.27 ⫾ 0.13 ␮M, 0.25 ⫾ 0.11 ␮M at day 1, 7, and 14, respectively. Table 6 shows the effect of gender on H2O2 exhalation by healthy subjects. Never smoked women exhaled 1.7, 1.6, and 1.3 times more H2O2 than never smoked men at day 1, 7, and 14, respectively. However, the difference at the 1st d of observation reached only the border of significance (p ⬍ .08), probably due to high within-group variability. The decrease in H2O2 exhalaTable 5. Correlations Between Individual Interweek Changes in H2O2 Exhalation in Healthy Subjects Mean increment in H2O2 exhalation [␮M] Subjects

Fig. 3. Circadian rhythm of H2O2 exhalation by 12 healthy never smoked subjects. Results represent mean H2O2 concentration in EBC specimens collected every 4 during 24 h. * vs. value at 24:00 h, p ⬍ .01; ** vs. value at 24:00 h, p ⬍ .05.

Day 7—day 1 Day 14—day 7

r

Whole group ⫺0.01 ⫾ 0.23 ⫺0.05 ⫾ 0.13* ⫺0.55 Never smoked ⫺0.02 ⫾ 0.26 ⫺0.06 ⫾ 0.14* ⫺0.53 Current smokers ⫺0.04 ⫾ 0.22 ⫺0.00 ⫾ 0.12 ⫺0.82 * vs. current smokers, p ⬍ .05.

p ⬍.0001 ⬍.0001 ⬍.0001

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Table 6. Effect of Gender on H2O2 Exhalation in Healthy Volunteers Including Never Smoked Subjects and Asymptomatic Cigarette Smokers During 2 Week Observation Concentration of exhaled H2O2 [␮M] Subjects

N

1st D

7th D

14th D

Female never smoked Male never smoked Female current smokers Male current smokers

17 23 11 7

0.25 ⫾ 0.21† 0.15 ⫾ 0.18 0.36 ⫾ 0.07** 0.33 ⫾ 0.17*

0.22 ⫾ 0.17## 0.14 ⫾ 0.14# 0.36 ⫾ 0.14** 0.33 ⫾ 0.14*

0.13 ⫾ 0.20 0.10 ⫾ 0.13 0.35 ⫾ 0.09** 0.32 ⫾ 0.13*

EBC was collected three times every 7 d. Present cigarette consumption and cumulative cigarette consumption was 18 ⫾ 3 cigarettes a day and 17.1 ⫾ 9.8 pack-years and 16 ⫾ 5 cigarettes a day and 18.7 ⫾ 11.8 pack-years for female and male current cigarette smokers, respectively. All subgroups did not differ significantly in respect of age. † vs. male never smoked subjects, p ⬍ .08. # vs. value at day 14, p ⬍ .08. ## vs. value at day 14, p ⬍ .04. * vs. male never smoked subjects, p ⬍ .02. ** vs. female never smoked subjects, p ⬍ .03.

tion at day 14, more distinct in the female subgroup, was observed. On the other hand, the H2O2 concentrations in EBC were almost equal and stable in male and female current cigarette smokers over 2 week observation. The differences between current smokers and never smoked subjects were more distinct for male volunteers (Table 6). This may result from higher H2O2 concentrations in EBC of never smoked women. Contrary to H2O2 the TBARs were detected in a few (n ⫽ 4) EBC specimens of never smoked subjects. Thus, the mean TBARs concentration observed at all three time-points was 0 ␮M. In asymptomatic current cigarette smokers the ratio of TBARs positive readings and the mean TBARs concentration were higher (p ⬍ .05) and reached 6/18 and 0.03 ⫾ 0.05 ␮M, 7/18 and 0.05 ⫾ 0.07 ␮M, 6/18 and 0.04 ⫾ 0.06 ␮M for 1st, 7th, and 14th day, respectively.

Mean H2O2 and TBARs exhalation estimated over the whole 2 week period The mean H2O2 and TBARs exhalation estimated over the whole 2 week period was calculated as a mean of three consecutive measurements performed at day 1, 7, and 14. This was 2.4-fold higher in asymptomatic cigarette smokers than in never smoked subjects (0.39 ⫾ 0.24 ␮M, n ⫽18 vs. 0.16 ⫾ 0.13 ␮M, n ⫽ 40, p ⬍ .05). Both asymptomatic current cigarette smokers and healthy never smoked subjects over 40 years exhaled more H2O2 than matched volunteers under 40 years. This was accompanied by significantly lower MEF 25 values (p ⬍ 0.05) and tendency to decrease in MEF 50 in respective subgroups over 40 years (Table 7). In addition, correlation between subjects age and mean H2O2 exhalation estimated over 2 week period in the never smoked healthy volunteers (Fig. 4) as well as in the

Table 7. Comparison of Mean H2O2 and TBARs Exhalation Extimated Over the Whole 2 Week Period, Selected Spirometric Parameters and Body Mass Index in Healthy Subjects Under and Over 40 Years Never smoked subjects Variable H2O2 [␮M] TBARs [␮M] MEF 25 [% predict.] MEF 50 [% predict.] FEV1/VC [%] FEV1 [%] BMI [kg/m2]

Current cigarette smokers

⬍ 40 years; n ⫽ 28

⬎ 40 years; n ⫽ 12

⬍ 40 years; n ⫽ 10

⬎ 40 years; n ⫽ 8

0.12 ⫾ 0.08 ND 96.9 ⫾ 27.0 97.5 ⫾ 22.6 104.7 ⫾ 6.6 101.5 ⫾ 14.5 22.6 ⫾ 3.3

0.25 ⫾ 0.17# ND 74.9 ⫾ 32.2# 81.7 ⫾ 21.8 100.4 ⫾ 7.3 100.5 ⫾ 15.9 25.4 ⫾ 2.4#

0.36 ⫾ 0.12* 0.06 ⫾ 0.10* 92.2 ⫾ 22.1 91.8 ⫾ 22.8 97.8 ⫾ 8.1* 104.9 ⫾ 9.2 23.7 ⫾ 2.3

0.42 ⫾ 0.34*# 0.02 ⫾ 0.02* 67.5 ⫾ 15.5# 86.2 ⫾ 21.5 98.2 ⫾ 7.1* 111.1 ⫾ 13.9 25.6 ⫾ 4.3#

The mean H2O2 and TBARs exhalation estimated over the whole 2 week period was calculated as a mean of three consecutive measurements performed at days 1st, 7th, and 14th. Spirometric parameters are expressed as percent of predicted [23]. The daily cigarette consumption and cumulative cigarette consumption was 18.5 ⫾ 3.3 cigarettes a day and 9.3 ⫾ 5.0 pack-years for current smokers under 40 years and 16.3 ⫾ 5.2 cigarettes a day and 21.7 ⫾ 5.9 pack-years for current smokers over 40 years. TBARs ⫽ thiobarbituric acid reactive substances; MEF 50 and 25 ⫽ maximal expiratory flow at 50% and at 75% of expired vital capacity, respectively; FEV1 ⫽ forced expiratory volume in 1 s; BMI ⫽ body mass index ND ⫽ not detectable (0 ␮M).

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Fig. 4. Positive correlation between mean H2O2 exhalation estimated over the whole 2 week period and age of healthy never smoking subjects.

subgroup under 40 years (r ⫽ 0.45, p ⬍ .02). However, no significant association between subjects age and H2O2 exhalation in current cigarette smokers was found (r ⫽ 0.28, p ⫽ .25). The mean TBARs concentration estimated over 2 week period in never smoked subjects was far below the method sensitivity and was significantly lower than that found for current cigarette smokers (ND, n ⫽ 40 vs. 0.04 ⫾ 0.06 ␮M, n ⫽ 18). This was also observed in current cigarette smokers divided in subgroups below and over 40 years (Table 7). Although the mean TBARs level in EBC was 3.2 times higher in current cigarette smokers under 40 years than that in smokers over 40 years, this difference was not significant. Contrary to H2O2 concentrations, TBARs levels did not correlate with subjects age (r ⫽ 0.28, p ⫽ .24 for the whole group of cigarette smokers; r ⫽ 0.31, p ⫽ 0.38 for cigarette smokers under 40 years; and r ⫽ 0.46, p ⫽ 0.25 for smokers over 40 years). Similarly, no correlation was found between mean H2O2 and TBARs exhalation estimated over 2 week period and clinical and physiological parameters including spirometric parameters and BMI either in asymptomatic cigarette smokers or healthy never smoked subjects (data not shown). 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 used higher volumes of EBC to avoid significant specimen dilution by reagent solutions and to increase the method sensitivity. In addition, H2O2 determination was performed just after condensate collection. Although freshly prepared aqueous H2O2 solutions stored at ⫺80°C are stable for 2 weeks [11], the presence of trace amounts of various proteins in EBC [20] may cause H2O2 decomposition and lead to increased ratio of negative results. Nevertheless, we confirmed our previous finding that asymptomatic current cigarette smokers exhaled more H2O2 than healthy never smoked subjects [11]. Moreover, this difference was observed at three time points over the 2 week observation. Apart from higher H2O2 levels cigarette smokers revealed lower variability of H2O2 exhalation than healthy never smoked subjects. Cigarette smokers have higher number of activated macrophages and polymorphonuclear leukocytes in the lower airways [14,21,22], and these cells seem to be the main source of exhaled H2O2 [4,11]. Increased activity of xanthine oxidase in lungs may also contribute to increased H2O2 levels in EBC of cigarette smokers [24]. On the other hand, alveolar phagocytes of healthy never smoked subjects are less activated and produce less H2O2 [14], and a significant part of H2O2 may originate from alveolar epithelium [12,15]. Therefore, some nonsmokers did not exhale detectable amounts of H2O2, which to some extent may contribute to high variability of H2O2 readings in this group. Unexpectedly, the mean H2O2 level in EBC fluctuated over the 2 week observation. At day 14 it differed from those observed at two first time-points (1st and 7th days). This was related to higher ratio of negative readings at this time point. We don’t know factors and mechanisms responsible for fluctuations of H2O2 exhalation in never smoked subjects. However, stimulation of H2O2 exhalation by cigarette smoking (e.g., via phagocyte recruitment) seems to abolish this phenomenon. The procedure of EBC collection (using a nose clip, expiration through the tube) and also preceding routine physical examination may stress subjects involved in the study. It cannot be excluded that these subjects were much less stressed during the third session of condensate collection (14th day) because they had time to adjust to requirements of the procedure. This may be partially responsible for decline in exhaled H2O2 in never smoked subjects at the end of the 2 week observation. Since current cigarette smokers exhale more H2O2, the influence of adaptation phenomenon on H2O2 levels in EBC was not noticeable in this group. Exhalation of H2O2 in healthy never smoked subjects

Exhaled H2O2 and TBARs

revealed also circadian rhythm with peak values at 12:00 and 24:00. Fluctuation in the number and activity of alveolar phagocytes and ability of epithelial cells to produce H2O2 may be responsible for this observation. This may also involve diurnal changes of leukocyte receptors [25] and circulating levels of adhesion molecules [26]. Another possible mechanism may consist of antioxidant enzymes activity changes (e.g., glutathione peroxidase or catalase) and/or concentration of low molecular weight antioxidants in epithelial lining fluid. Diurnal variation of glutathione content in hepatic and cardiac tissue in animals can support this hypothesis [27]. Smoking of one cigarette caused the rise in H2O2 exhalation in cigarette smokers who refrained from cigarette smoking for 12 h preceding the experiment. It may be the result of presence of free radicals in tobacco smoke, since aqueous solution of cigarette smoke is a rich source of H2O2 and superoxide anion [28,29]. Additionally, cigarette smoke may recruit and stimulate, for instance by complement activation [30] of macrophages and polymorphonuclear leukocytes to produce more reactive oxygen species, including H2O2 .The influence of cigarette smoke on H2O2 generation by type II pneumocytes cannot be excluded. However, the increase in H2O2 exhalation was not accompanied by the significant rise in TBARS levels in EBC. This may result from enhanced antioxidant defense in the airways of cigarette smokers [31,32]. It is consistent with results of our previous study showing no correlation between these variables in EBC of subjects suffering from chronic obstructive pulmonary disease (COPD) [2]. On the other hand, the baseline TBARs exhalation was higher in asymptomatic cigarette smokers, and majority of never smoked subjects did not reveal detectable amounts of TBARs in EBC. This indicates that formation of volatile TBARs in the airways of healthy subjects is sufficiently inhibited by antioxidants. Cigarette smoking causes accumulation of inflammatory cells (neutrophils and eosinophils) within the submucosa of the small airway [33]. The inflammatory infiltrate may by responsible for higher airflow obstruction at the level of the small airways and higher H2O2 exhalation in cigarette smokers. This may explain the negative correlation between MEF 25 and H2O2 concentration in EBC, and positive relationship between pack-years smoked and H2O2 exhalation in asymptomatic cigarette smokers involved in our study. In our previous studies we failed to find any relationship between H2O2 exhalation and cigarette smoking [2,11]. The differences in H2O2 determination that resulted in higher ratio of negative H2O2 readings [11], and probably high contribution of inflammatory processes to H2O2 production in airways of COPD subjects [2], may explain these discrepancies. The positive correlation between H2O2 levels in EBC and increasing age of never smoked subjects is consistent

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with recent study showing strong positive correlation between subjects age and peak release of reactive oxygen species from activated phagocytes as measured by a whole blood chemiluminescence assay [34]. The exhalation of H2O2 was higher in current smokers and never smoked subjects older than 40 years. Current cigarette smokers tend to exhale more TBARs over 40 years. This may be a result of iron accumulation in the lung [35,36] and increased ability of circulatory phagocytes to produce reactive oxygen species including H2O2 [34]. Increased content of iron in the airways of cigarette smokers [35] may augment free radicals-induced lipid peroxidation and lead to exhalation of TBARs. There is much evidence that oxidative stress related to cigarette smoking participates in the development of COPD [4]. It is interesting that H2O2 exhalation is higher over 40 years, at the time at which COPD develops and is diagnosed. Female never smoked subjects exhaled more H2O2 than male never smoked subjects. However, contrary to results of our previous study showing higher H2O2 exhalation in male cigarette smokers [11], no effect of gender was shown in this smokers group. The exhalation of H2O2 was similar in male and female cigarette smokers and this is consistent with results observed in smoking COPD patients [2]. Although analysis of free radical generation by phagocytes in the whole blood did not shown any differences between women and men [34], the stimulatory effect of progesterone on phagocyte chemiluminescence [37] may explain the influence of sex on H2O2 exhalation in healthy never smoked subjects. However, it requires further studies involving, for example, effect of progesterone on H2O2 production by airway epithelium and association between exhaled H2O2 levels and circulating progesterone. Our results, especially those showing diurnal variation and significant changes in H2O2 concentration in EBC over 2 week observation have importance for planning clinical studies on H2O2 as the marker of oxidative stress and airway inflammation in lung diseases. The EBC should be collected at the same time of day and in long-term studies all time-points should involve condensate collections from active and control groups. Moreover, the control group cannot differ from the active group in respect to age, sex, and smoking habit. In addition, subjects washing their mouth while wearing the nose clip during condensate collection is not necessary. The usage of short acting ␤2-mimetics and anticholinergics as a rescue medication (e.g., before or in the course of EBC collection) could be allowed.

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COPD— chronic obstructive pulmonary disease BMI— body mass index EBC— expired breath condensate FVC—forced vital capacity FEV1—forced expiratory volume in 1 s MEF 50 —maximal expiratory flow at 50% of expired vital capacity MEF 25—maximal expiratory flow at 75% of expired vital capacity TBA—thiobarbituric acid TBARs—thiobarbituric acid reactive substances