- Email: [email protected]
Oxidative Stress in Expired Breath Condensate of Patients With COPD
Oxidative Stress in Expired Breath Condensate of Patients With COPD* Konstantinos Kostikas, MD; Georgios Papatheodorou, PhD; Konstantinos Psathakis, MD; Panos Panagou, MD; and Stelios Loukides, MD
Objective: To evaluate the levels of hydrogen peroxide (H2O2) and 8-isoprostane in the expired breath condensate (EBC) of patients with COPD, and to assess the relationship between the above markers of oxidative stress and parameters expressing inflammatory process and disease severity. Setting: Inpatient respiratory unit and outpatient clinic in tertiary care hospital. Design: Cross-sectional study. Patients: Thirty stable COPD patients (all smokers) with disease severity ranging from mild to severe. Ten subjects who were smokers with stage 0 disease (ie, at risk for COPD; mean [ⴞ SD] FEV1, 88 ⴞ 5% predicted) were studied as a control group. Methods: H2O2 and 8-isoprostane levels were measured in EBC, and the values were correlated with variables expressing COPD severity (ie, FEV1 percent predicted, dyspnea severity score (ie, Medical Research Council scale) and airway inflammation (ie, differential cell counts from induced sputum). Results: The mean concentration of H2O2 was significantly elevated in COPD patients compared to control subjects (mean, 0.66 mol/L [95% confidence interval (CI), 0.54 to 0.68 mol/L) vs 0.31 mol/L [95% CI, 0.26 to 0.35 mol/L], respectively; p < 0.0001). The difference was primarily due to the elevation of H2O2 in patients with severe and moderate COPD, whose expired breath H2O2 levels were significantly higher than those of patients with mild disease (mean, 0.96 mol/L [95% CI, 0.79 to 1.13 mol/L], 0.68 mol/L [95% CI, 0.55 to 0.81 mol/L], and 0.33 mol/L [95% CI, 0.24 to 0.43 mol/L], respectively, p < 0.0001). The mean concentration of 8-isoprostane was significantly elevated in patients with COPD compared to that of the control group (47 pg/mL [95% CI, 41 to 53 pg/mL] vs 29 pg/mL [95% CI, 25 to 33 pg/mL], respectively; p < 0.0001) but did not differ significantly among the different stages of the disease (p ⴝ 0.43). Repeatability and stability data within measurements showed that H2O2 has a better repeatability and stability than 8-isoprostane. Furthermore, we observed significant correlations of H2O2 with FEV1, neutrophil count, and dyspnea score. Those correlations existed only in patients with moderate and severe disease. No correlations were found between levels of 8-isoprostane and the above parameters. Conclusions: We conclude that levels of H2O2 and 8-isoprostane are elevated in the EBC of patients with COPD, but that H2O2 seems to be a more repeatable and a more sensitive index of the inflammatory process and the severity of the disease. (CHEST 2003; 124:1373–1380) Key words: COPD; expired breath condensate; inflammation; oxidative stress; severity Abbreviations: CI ⫽ confidence interval; EBC ⫽ expired breath condensate; GOLD ⫽ Global Initiative for Chronic Obstructive Lung Disease; H2O2 ⫽ hydrogen peroxide; ICS ⫽ inhaled corticosteroid; MRC ⫽ Medical Research Council; ROS ⫽ reactive oxygen species
is considerable evidence that oxidative T here stress is increased in patients with COPD and that reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) contribute to the pathophysiology *From the Pneumonology and Clinical Research Department, Athens Army General Hospital, Athens, Greece. Manuscript received November 7, 2002; revision accepted May 5, 2003. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Stelios Loukides, MD, Smolika 2 16673, Voula Athens, Greece; e-mail: [email protected] www.chestjournal.org
of this disease entity.1 Oxidative stress also leads to the formation of isoprostanes by the direct oxidation of arachidonic acid.2 ROS may contribute to the pathophysiology of COPD in several ways, including the damage of serum protease inhibitors, the potentiation of elastase activity, and increased mucus secretion. In addition, they activate the transcription factor NFB, which orchestrates the transcription of many inflammatory genes, including interleukin-8, nitric oxide synthase, and inducible cyclooxygenase.3 ROS are present in cigarette smoke and are produced endogenously by activated inflammatory CHEST / 124 / 4 / OCTOBER, 2003
1373
cells, including neutrophils and alveolar macrophages. The increased production of endogenous ROS is demonstrated by the increased levels of H2O24 and 8-isoprostane5 in the expired breath condensate (EBC) of patients with COPD. However, a number of important issues regarding the value of assessing H2O2 and 8-isoprostane levels in patients with COPD remain unclear, the main one being whether the oxidative stress seen in the EBC of stable COPD patients may express the presence of underlying airway inflammation or predict the severity of the disease. Our primary aim was to evaluate the concentrations of H2O2 and 8-isoprostane in the EBC of COPD patients, to investigate the reproducibility and stability of these measurements, and to see whether there is an association between the levels of those two markers in EBC and airway inflammation or disease severity. Furthermore, we investigated which inflammatory cells are the main sources of H2O2 and 8-isoprostane, and whether these cells differ regarding the classification of severity or the use of inhaled corticosteroids (ICSs). As indexes of airway inflammation in sputum, we studied validated variables such as differential cell counts. Disease severity was assessed according to the results of pulmonary function tests and dyspnea according to a severity score (ie, the Medical Research Council [MRC] scale). Finally, the classification of the COPD patients was made according to the recent Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.6 To account for the possible confounding effects of therapy with ICSs, further analysis was conducted after subdividing the patients on this basis.
Organization Workshop on the Global Initiative for COPD.6 Thirty nonatopic, stable patients with COPD who were smokers (mean smoking, 53 pack-years [SD, 13]; range, 41 to 71 pack-years; all men; mean age, 58 years [SD, 9]; age range, 42 to 76 years; mean FEV1, 57% predicted [SD, 24]; FEV1 range, 21 to 88% predicted) with disease severity ranging from mild to severe (mild COPD, 10 patients [FEV1 83% predicted; SD, 2% predicted; FEV1 range, 80 to 88% predicted]; moderate COPD, 10 patients [FEV1, 60% predicted; SD, 7% predicted; FEV1 range, 50 to 69% predicted]; and severe COPD, 10 patients [FEV1, 27% predicted; SD, 3% predicted; FEV1 range, 21 to 30% predicted]) were studied. Ten healthy, nonatopic subjects who smoked and fulfilled the criteria of COPD stage 0 at risk, according to the GOLD guidelines6 (ie, mean age of all current smokers, 55 years; SD, 9 years; age range, 43 to 77 years; mean FEV1, 88 years; SD, 5 years; FEV1 range, 81 to 99% predicted), served as a control group. Patients were included in the study only if they were clinically stable and had no evidence of an acute exacerbation for at least 4 weeks prior to the study. None of our patients had reversibility with inhaled salbutamol therapy of ⬎ 12% of the predicted FEV1. Fifteen patients were receiving therapy with ICSs (fluticasone propionate, 500 to 1,000 g daily). Six patients were receiving theophylline, 300 mg twice daily. None was receiving any other anti-inflammatory treatment, including therapy with leukotriene antagonist agents, inhaled or oral mucolytic agents, or long-term oxygen therapy. Nineteen patients were receiving long-acting 2-agonists twice daily, and 11 patients occasionally were receiving short-acting 2-agonists or anticholinergics as relief medication. Control subjects had a negative history of allergy (ie, negative responses to skin prick tests for common allergens), normal spirometry results, normal FEV1/FVC ratios, no history of any disease, and were not receiving medical treatment for any reason. They were all male smokers as were the patients (mean smoking history, 49 pack-years; SD, 12 pack-years; range, 38 to 67 pack-years). All patients had normal bronchial reactivity with a provocative dose of histamine causing a 20% fall in FEV1 of ⬎ 0.800 mg (mean, 1.45 mg; range, 0.920 to 1.85 mg). The Scientific Ethics Committee of our hospital approved the study protocol, and all participants gave informed written consent. Assessment of Disease Severity
Materials and Methods Subjects Subject characteristics are summarized in Table diagnosis of COPD and the classification of the severity in our patients was established according National Heart, Lung, and Blood Institute/World
1. The disease to the Health
All the subjects were instructed to define the severity of their daily breathlessness by using the MRC questionnaire6 (scale of 0 of 4 as follows: 0, breathlessness only with strenuous exercise; 1, shortness of breath when hurrying on the level or walking up a slight hill; 2, walk slower than people of the same age on the level because of breathlessness or have to stop for breath when walking on their own pace on the level; 3, stop for breath after walking
Table 1—Subject Characteristics*
Characteristics Age, yr FEV1, % predicted FEV1/FVC ratio, % Smoking habit, pack-yr Dyspnea score Neutrophils, %
Control Subjects (n ⫽ 10) 55 (9) (43–77) 88 (5) (81–99) 86 (7) (81–95) 49 (12) (58–67) 0 38 (11) (32–47)
COPD Patients All (n ⫽ 30)
Mild (n ⫽ 10)
58 (9) (42–76) 57 (24) (21–88) 51 (11) (41–74) 53 (13) (40–71) 2.2 (1) (1–4) 56 (13) (31–70)
60 (5) (53–76) 84 (2) (80–88) 55 (14) (45–74) 53 (16) (40–70) 1.4 (0.7) (1–3) 39 (5) (31–46)
Moderate (n ⫽ 10) 59.5 (9) (42–71) 60 (7) (50–69) 54 (10) (44–64) 56 (12) (42–71) 1.9 (0.6) (1–3) 63 (6.5) (50–70)
Severe (n ⫽ 10) 58 (9) (47–70) 27 (3) (21–30) 44 (9) (41–59) 50 (11) (47–64) 3.4 (0.5) (3–4) 66 (2) (63–70)
*Values given as mean (SD) [ranges]. 1374
Clinical Investigations
about 100 yards or after a few minutes on the level; 4, too breathless to leave the house or breathless when dressing or undressing). Each patient was asked to quantify the impact of dyspnea on his daily health status. The questionnaires were started from the last level, and if a positive answer was given, no further levels were evaluated. Lung Function FEV1 and FVC were measured with a dry spirometer (model VEP2, Vica-test; Mijnhardt; Rotterdam, Holland) using the American Thoracic Society criteria for the standardization of spirometry.7 The best value of three maneuvers was expressed as the percentage of the predicted value. FEV1/FVC ratio was measured in all persons in order to confirm the presence or absence of airway obstruction in patients and in control subjects, respectively.6 Airway responsiveness was measured by histamine provocation challenge in control subjects, using the guidelines provided by the American Thoracic Society.8 According to these criteria, patients were asked not to smoke for 6 h before undergoing the challenge. Histamine challenge in healthy subjects was performed on a separate day in order to avoid any effect of the challenge occurring on the inflammatory cells and oxidative stress values.
8-Isoprostane Measurements: 8-Isoprostane concentration was determined by a competitive enzyme immunoassay kit (Cayman Chemical; Ann Arbor, MI), as previously described.5 The detection limit of the assay was 4 pg/mL. Sputum Induction and Processing Sputum was induced by the inhalation of an aerosol 3.5% hypertonic saline solution that was generated by an ultrasonic nebulizer (model 2696; DeVilbiss; Somerset PA), with modifications to improve its safety.12 At least 2 mL sputum was collected into a sterile container. The sample from the first cough was discarded, as it was heavily contaminated with squamous epithelial cells.13 The sample was determined to be adequate if the number of squamous epithelial cells was ⬍ 30% of the total number of inflammatory cells.14 Cytospin slides were prepared and stained with May-Gru¨ nwald-Giemsa stain. The person who did the differential cell counts was not aware of the clinical and functional status of the patients or of the EBC measurements. Two slides were used for counting, and at least 300 inflammatory cells were counted for each slide. The inflammatory cells in the sputum samples are shown as the percentage of total nonsquamous cells. Sputum measurements were performed on the same day for all the patients. Statistical Analysis
Collection and Measurements of EBC Collection of EBC: The collection of EBC was performed as previously described.9 Approximately 2 mL EBC was collected. The repeatability of H2O2 and 8-isoprostane measurements and the stability of the frozen samples were estimated as previously described10 in 5 control subjects and 12 patients with COPD (4 persons from each subgroup). In order to assess the repeatability of the measurements, EBC was collected on 2 consecutive days under the same conditions. To assess the stability of the frozen condensate samples, 4 mL EBC was collected. The abovementioned concentration was divided into 1-mL aliquots, in which H2O2 and 8-isoprostane concentrations were determined after 2 days, 1 week, 2 weeks, and 3 weeks of storage (the maximum time between collection and measurement in any sample). All condensate samples were tested for salivary contamination by the determination of amylase activity. Briefly, amylase activity was carried out spectrophotometrically (kinetic method) using a commercial reagent kit (model 981362; KONE Instruments; Espoo, Finland). In this procedure, the ␣-amylase of the sample and the enzyme ␣-glycosidase hydrolyze the substrate p-nitrophenyl-␣-D-maltoheptaoside to glucose and p-nitrophenol. The liberation of p-nitrophenol was observed at 405 nm (37°C) for 2 min. Two samples were spiked with saliva to ensure that this could be detected by our method. In all the samples tested for saliva, no amylase was detected, suggesting that there was no contamination of breath condensate. The samples that were spiked with saliva showed levels of salivary amylase of ⬎ 5,000 IU. In all study subjects, EBC was collected first, followed by the sputum-induction procedure. Subjects had been asked not to smoke for 2 h prior to the collection of EBC and sputum. In order to ensure that, all subjects stayed in our laboratory for 2 h before the study, under the supervision of one of the investigators. H2O2 Measurements: H2O2 concentration was determined by an enzymatic assay using horseradish peroxidase (Sigma Chemical; St Louis, MO), as previously described.11 The detection limit of the assay was 0.1 mol/L. www.chestjournal.org
Data concerning subject characteristics are expressed as the mean (SD) with ranges. Data concerning the comparisons among the various parameters in the study groups are given as the mean with 95% confidence intervals (CIs) for the differences. The data were examined for normal distribution, and when it was not normally distributed the nonparametric Mann-Whitney test was used for statistical comparisons. For normally distributed data, the paired t test was used for statistical comparisons between the two groups. The normality of distribution was tested with a Shapiro-Wilks test. Parameters from the four groups of patients with COPD were compared using the one-way analysis of variance with an appropriate post hoc test (ie, Bonferroni test) for multiple comparisons. The Pearson correlation coefficient was used to investigate the relations among the parameters. A p value of ⬍ 0.05 was considered to be significant.
Results Repeated measurements on 2 consecutive days revealed a mean within-subject difference of 0.08 mol/L (SD, 0.05 mol/L) for H2O2 (p ⫽ 0.26), and 13 pg/mL (SD, 4 pg/mL) for 8-isoprostane (p ⫽ 0.04). When we checked the stability of H2O2 in the frozen samples, we noticed no significant differences among the four measurements (after 2 days, 0.49 mol/L [SD, 0.11 mol/L]; after 1 week, 0.51 mol/L [SD, 0.1 mol/L]; after 2 weeks, 0.52 mol/L [SD, 0.09 mol/L]; and after 3 weeks, 0.47 mol/L [SD, 0.14 mol/L]; p ⫽ 0.43). Regarding the stability of 8-isoprostane in frozen samples, we noticed marginal differences among the four measurements, which were not significant (after 2 days, 33 pg/mL [SD 11 pg/mL]; after 1 week, 38 pg/mL [SD 10.3 pg/mL]; after 2 weeks, 31 pg/mL [SD 8 pg/mL]; after 3 weeks, 39 pg/mL [SD 7.3 pg/mL]; p ⫽ 0.07). The mean concentration of H2O2 was significantly CHEST / 124 / 4 / OCTOBER, 2003
1375
elevated in patients with COPD compared to the control subjects (0.66 mol/L [95% CI, 0.54 to 0.68 mol/L] vs 0.31 mol/L [95% CI, 0.26 to 0.35 mol/L]; p ⬍ 0.0001) [Fig 1, top, A]. The difference was primarily due to the elevation of H2O2 levels in patients with severe and moderate COPD, whose H2O2 levels in EBC were significantly
higher than those of patients with mild disease (0.96 mol/L [95% CI, 0.79 to 1.13 mol/L], 0.68 mol/L [95% CI, 0.55 to 0.81 mol/L], and 0.33 mol/L [95% CI, 0.24 to 0.43 mol/L] respectively; p ⬍ 0.0001) [Fig 1, top, A]. The mean concentration of 8-isoprostane was significantly elevated in patients with COPD compared to that of the control group (47 pg/mL [95% CI, 41 to 53 pg/mL] vs 29 pg/mL [95% CI, 25 to 33 pg/mL], respectively; p ⬍ 0.0001) [Fig 1, bottom, B] but did not differ significantly among the different stages of the disease (patients with mild disease, 42 pg/mL [95% CI, 36 to 49 pg/mL]; patients with moderate disease, 48 pg/mL [95% CI, 33 to 63 pg/mL]; and patients with severe disease, 51 pg/mL [95% CI, 41 to 61 pg/mL]; p ⫽ 0.43) [Fig 1, bottom, B]. Subanalysis for the Effects of ICSs COPD patients receiving ICS therapy had significantly higher H2O2 values compared with steroid-naive patients (0.8 mol/L [95% CI, 0.61 to 0.94 mol/L] vs 0.52 mol/L [95% CI, 0.39 to 0.64 mol/L], respectively; p ⬍ 0.0001) [Fig 1, top, A]. This difference may be attributed to the fact that most of the COPD patients who were receiving ICS therapy had severe disease. The mean concentration of 8-isoprostane did not differ significantly between patients receiving ICS therapy compared with steroid-naive patients (46 pg/mL [95% CI, 38 to 54 pg/mL] vs 48 pg/mL [95% CI, 39 to 57.5 pg/mL], respectively; p ⫽ 0.63) [Fig 1, bottom, B]. Airway Inflammatory Cells
Figure 1. Top, A: H2O2 concentration in the EBC of control subjects (10 subjects) and patients with COPD (total, 30 patients; mild disease, 10 patients; moderate disease, 10 patients; severe disease, 10 patients; ICS-treated patients, 15 patients; and steroid-naive patients, 15 patients). Patients with COPD have significantly higher values compared to control subjects (p ⬍ 0.0001). Patients with severe disease have significantly higher values than those with mild and moderate disease (p ⬍ 0.0001). Patients treated with ICSs have significantly higher values compared with steroid-naive patients (p ⬍ 0.0001). Each symbol represents one individual. Horizontal bars represent mean values. Bottom, B: 8-isoprostane concentration in the EBC of control subjects (total, 10 subjects) and of patients with COPD (total, 30 patients; mild disease, 10 patients; moderate disease, 10 patients; severe disease, 10 patients; ICS-treated, 15 patients; and steroid-naive patients, 15 patients). Patients with COPD had significantly higher values compared to control subjects (p ⬍ 0.0001). No significant differences were observed among patients with mild, moderate, and severe disease (p ⫽ 0.43). Patients treated with ICSs had similar values compared with steroid-naive patients (p ⫽ 0.63). Each symbol represents one individual. Horizontal bars represent mean values. ICS(⫹) ⫽ ICS-treated; ICS(-) ⫽ steroid-naive. 1376
COPD patients had higher levels of sputum neutrophilia than did control subjects (56% [95% CI, 51 to 61%] and 528 ⫻ 103 cells/g [95% CI, 488 to 534 ⫻ 103 cells/g] vs 38% [95% CI, 32 to 40%] and 315 ⫻ 103 cells/g [95% CI, 288 –334 ⫻ 10 cells/g], respectively; p ⬍ 0.0001) [Fig 2, Table 1]. The difference was primarily due to the elevation of neutrophils in patients with moderate and severe disease (mild disease, 39% [95% CI, 36 to 43%] and 315 ⫻ 103 cells/g [95% CI, 288 to 334 ⫻ 103 cells/g]; moderate disease, 63% [95% CI, 58 to 67.5%] and 604 ⫻ 103 cells/g [95% CI, 548 to 635 ⫻ 103 cells/g]; and severe disease, 67% [95% CI, 64 to 68%] and 667 ⫻ 103 cells/g [95% CI, 603 to 688 ⫻ 103 cells/g]; p ⬍ 0.002) [Fig 2, Table 1]. The percentage and number of macrophages was significantly higher in control subjects than in COPD patients (62% [95% CI, 54 to 66%] and 578 ⫻ 103 cells/g [95% CI, 509 to 601 ⫻ 103 cells/g] vs 42% [95% CI, 38 to 48%] and 349 ⫻ 103 cells/g [95% CI, 308 to 421 ⫻ 103 cells/g], respectively; p ⬍ 0.0001) [Fig 2] and was significantly lower in patients with severe disease than in Clinical Investigations
Discussion In this prospective, cross-sectional study we have found that oxidative stress, as assessed by the levels of H2O2 and 8-isoprostane, is elevated in the EBC of patients with COPD. The measurement of H2O2 levels seems to be a more repeatable and more sensitive index of the inflammatory process and disease severity compared with that of 8-isoprostane levels. The increased levels of H2O2 and 8-isoprostane observed in this study are similar to those previously reported.4,5 It has been shown that the concentration of 8-isoprostane is increased in patients with COPD, irrespective of smoking status and lung function impairment.5 Moreover, the levels of H2O2 in the EBC of stable patients with COPD have been found to be higher than those of healthy subjects and lower than those of unstable patients with COPD.4 In addition, no correlation of either H2O2 or 8-isoprostane and lung function impairment has been reported. An important question arising from these studies was the identification of the cellular sources of these biomarkers of oxidative stress and the quantification of their possible role in predicting the severity of the disease. Another issue was the absence of data regarding the repeatability and stability of H2O2 and 8-isoprostane measurements in EBC. In order to identify the relationship between the measurements and disease severity, we believe that the critical point is to classify patients according to widely accepted guidelines, such as those proposed by GOLD,6 and simultaneously to avoid using terms like unstable, since they do not provide precise characteristics of the disease and, additionally, depend on multiple factors that might affect our measurements. Our results showed that evaluating the H2O2 concentration may provide useful information about the severity of the disease. On the other hand, we found no equivalent data regarding the levels of 8-isoprostane. One plausible explanation for these observations might depend on the underlying antioxidant defense mechanisms. An increased concentration of biomarkers of oxidative stress may repre-
Figure 2. Percentage of differential cell counts in the induced sputum of patients with COPD and in that of control subjects. * ⫽ statistically significantly higher percentage of neutrophils in the induced sputum of patients with COPD compared to control subjects (p ⬍ 0.0001); ** ⫽ statistically significant lower percentage of macrophages in the induced sputum of patients with COPD compared to control subjects (p ⬍ 0.0001); *** ⫽ statistically significant higher percentage of neutrophils in the induced sputum of patients with severe disease compared to those with mild and moderate disease (p ⬍ 0.002).
those with mild and moderate disease (severe disease, 32% [95% CI, 31 to 34%]; 301 ⫻ 103 cells/g [95% CI, 275 to 342 ⫻ 103 cells/g]; mild disease, 36% [95% CI, 31– 41%]; 357 ⫻ 103/g [95% CI, 312–384 ⫻ 103 cells/g]; moderate disease, 60% [95% CI, 47 to 63%]; 530 ⫻ 103 cells/g [95% CI, 421 to 542 ⫻ 103 cells/g]; p ⬍ 0.05) [Fig 2]. No significant differences were observed between mean neutrophil levels in steroid-naive and steroid-treated patients (54 ⫾ 13% [95% CI, 47 to 62%] vs 58 ⫾ 13 [95% CI, 51 to 61%], respectively; p ⫽ 0.6). Correlations Among H2O2 and 8-Isoprostane Concentrations, Inflammatory Cells in Induced Sputum, Lung Function, and Dyspnea Severity Scale in Patients With COPD Major correlations are presented in Tables 2, 3, and 4, and in Figure 3.
Table 2—Correlations of H2O2 in the Four Groups of Patients With COPD* All
Mild
Moderate
Severe
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score 8-Isoprostane
⫺0.83 0.83 0.68 0.01
⬍ 0.0001 ⬍ 0.0001 ⬍ 0.0001 0.46
⫺0.46 0.44 0.57 0.02
0.76 0.19 0.07 0.67
⫺0.76 0.67 0.51 0.2
0.009 0.02 0.11 0.15
⫺0.04 0.76 0.42 0.17
0.89 0.001 0.22 0.5
*Correlations were conducted using the Pearson correlation coefficient. www.chestjournal.org
CHEST / 124 / 4 / OCTOBER, 2003
1377
Table 3—Correlations of 8-Isoprostane in the Four Groups of Patients With COPD* All
Mild
Moderate
Severe
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score
⫺0.23 0.2 0.21
0.22 0.28 0.25
⫺0.26 0.37 0.56
0.44 0.29 0.08
⫺0.06 0.27 0.32
0.84 0.43 0.35
⫺0.33 0.23 0.26
0.3 0.51 0.45
*Correlations were conducted using the Pearson correlation coefficient.
sent an increased production of ROS or a reduced number of free radicals scavenging capacity in the airways of COPD subjects, or even a combination of these two parameters. Long-term antioxidant treatment attenuates H2O2 formation in the airways of COPD subjects but has no significant effect on the levels of lipid peroxidation products.15,16 It has been shown17,18 that dietary antioxidant supplies may scavenge the ROS, but they fail to reduce lipid peroxidation. There is some evidence that the deficiency of antioxidant defenses is related to the degree of airway obstruction in patients with COPD,19 while no data exist to support a significant correlation between lipid peroxidation and lung function impairment. Since the classification of the disease by GOLD is based on FEV1 values, and taking all the above points under consideration, we believe that there may well be close relationships among H2O2 concentration, lung function, and antioxidant mechanisms. In contrast, no respective data exist for the products of lipid peroxidation. Based on the above hypothesis and on part of our data, we may assume that, as the severity of the disease progresses, there is also a progressive course of H2O2 concentration, antioxidant status, and changes in lung function, with the last two decreasing and the first one increasing or just overcoming the defenses. The difference we have observed in the data for 8-isoprostane might be explained by the fact that lipid peroxidation is the result of a complicated metabolic pathway, which starts from free radicals that trigger lipid peroxidation chain reactions.20 In this metabolic pathway, the antioxidant defenses do not play an equally impor-
tant role, and the concentration of 8-isoprostane eventually might reach a point at which the underlying triggering ROS-mediated mechanism has little, if any, effect that leads to a further increase of its end-products such as 8-isoprostane. This is partially supported by the absence of significant correlation between the levels of H2O2 and 8-isoprostane that we observed in this study. Our data suggest that H2O2 is a more repeatable and more stable product than 8-isoprostane. Since 8-isoprostane is an end-product of a metabolic pathway that is chemically stable, one might expect that its measurements would be more stable and repeatable. However, its overall production is based on a complicated metabolic pathway, in which many factors are involved that might significantly affect its measurement. Those factors may be independent of the inflammatory process of COPD. The strong repeatability and stability of the H2O2 measurements might be explained by the fact that H2O2 production mainly depends on the underlying inflammatory cells (primarily neutrophils) and the effect of the antioxidant defenses. There is incoming evidence from the literature that these two factors remain unchanged through the course of COPD in stable patients.21,22 Most observations dealing with inflammatory cells in patients with COPD have focused on neutrophils, since they undoubtedly have an impact on the inflammatory process and may alter the oxidantantioxidant balance.21–23 Similar results were observed in our study, using a noninvasive and widely accepted method, such as sputum induction. Increased numbers of neutrophils appear to be pro-
Table 4 —Correlations of H2O2 and 8-Isoprostane in Steroid-Naive and Steroid-Treated Patients With COPD* H2O2 Steroid-Naive
8-Isoprostane Steroid-Treated
Steroid-Naive
Steroid-Treated
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score 8-Isoprostane
⫺0.82 0.88 0.37 0.08
0.0001 ⬍ 0.0001 0.14 0.75
⫺0.8 0.74 0.79 0.3
0.0004 0.001 0.0004 0.1
⫺0.007 0.3 0.17
0.7 0.2 0.51
⫺0.06 0.11 0.25
0.63 0.27 0.31
*Correlations were conducted using the Pearson correlation coefficient. 1378
Clinical Investigations
Figure 3. Correlations between H2O2 concentration in the EBC of patients with COPD and (top, A) the percentage of neutrophils in induced sputum (r ⫽ 0.83; p ⬍ 0.0001), (middle, B) dyspnea MRC scale (r ⫽ 0.68; p ⬍ 0.0001), and (bottom, C) the FEV1 percent predicted (r ⫽ ⫺0.83; p ⬍ 0.0001). f ⫽ individual data points.
ducing increased amounts of ROS in the lung of COPD patients.2 Examining all the above points together (ie, the high percentage of neutrophils in the sputum of COPD patients), the strong correlation of neutrophil levels with H2O2 concentration that we have observed in this study, and the lack of a positive correlation between macrophage levels and H2O2 concentration, we may come to the plauwww.chestjournal.org
sible conclusion that neutrophils are the main source of H2O2 production in patients with COPD. We were not able to ascertain the cellular source of 8-isoprostane using the data of this study. The source of the increased ROS production in patients with COPD might derive from structural cells of the airways.24 However, in our study there are no data to support this. Another important issue deriving from the data presented is the difference between the control group and the groups of patients with different stages of COPD. It is well-known that smoking contributes to the burden of oxidants and free radicals in the lower airways.25 Our results showed that in patients with COPD with similar smoking habits the differences observed in oxidative stress values are related mainly to the underlying inflammatory process. Similar results have been presented in previous studies,26 from which we were not able to come to any conclusions since they did not provide cellular data. Another main advantage of our study was the fact that as a control group we used patients who were at risk for COPD (ie, stage 0) and had a similar smoking history. A possible explanation might be that in subjects who are at risk to develop COPD, the underlying oxidative stress is mainly related to smoking but when the disease appears the inflammatory process surpasses the smoking contribution. This idea is partially supported by the absence of significant correlation between H2O2 and neutrophil levels in both the control group and the group of patients with mild COPD in whom the inflammatory process seems to be limited. The interindividual differences in the correlations data within groups might well be explained by the limited range of FEV1 and dyspnea severity score values, particularly in patients with severe disease. This is partially confirmed by the significant correlations we observed in patients with moderate disease, who presented a wide range of FEV1 values and symptom scores. The present cross-sectional study cannot demonstrate a causal relationship between treatment with ICSs and the concentrations of 8-isoprostane and H2O2 in EBC. In fact, patients receiving ICS therapy had higher values compared to steroid-naive patients. This might be due to the fact that the patients treated with ICSs in our study had more severe forms of COPD. The ineffectiveness of ICS therapy on H2O2 concentration might be explained by the fact that steroids fail to inhibit the production of H2O2 from polymorphonuclear leukocytes,27 probably because of their inability to inhibit the underlying neutrophilic inflammation.28 In this study, we were not able to clarify the role of treatment with ICSs on the levels of 8-isoprostane, since there are currently CHEST / 124 / 4 / OCTOBER, 2003
1379
no sufficient data regarding the cellular source of 8-isoprostane. The presence of significant correlations between H2O2 and study variables, irrespective of ICS usage, and simultaneously the absence of similar correlation data for 8-isoprostane supports the recommendations6 that treatment with ICSs does not significantly change the inflammatory, functional, and clinical course of the disease. In conclusion, studying those two different components of oxidative stress, we report that both H2O2 and 8-isoprostane levels are increased in stable patients with COPD. The measurement of H2O2 is a more repeatable and more sensitive marker of the underlying inflammatory process and the severity of the disease than are the end-products of lipid peroxidation, as reflected by the levels of 8-isoprostane. The role of lipid peroxidation in the inflammatory burden of COPD still requires further study to be clarified. References 1 Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154:813– 816 2 Gutteridge GM. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41:1819 –1828 3 Rahman I, MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 1998; 53:601– 612 4 Dekhuijzen RPN, Aben KKH, Dekker I, et al. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154:813– 816 5 Montuschi P, Collins JV, Ciabattoni G, et al. Exhaled 8isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 2000; 162:1175–1177 6 Global Initiative for Chronic Obstructive Pulmonary Disease. NHLBI/WHO workshop report. Bethesda, MD: National Institutes of Health, April 2001; NIH Publication No. 2701 7 American Thoracic Society. Standardization of spirometry: 1994 update. Am J Respir Crit Care Med 1995; 152:1107– 1136 8 American Thoracic Society. Guidelines for methacholine and exercise challenge testing 1999. Am J Respir Crit Care Med 2000; 161:309 –329 9 Ganas K, Loukides S, Papatheodorou G, et al. Total nitrite/ nitrate in expired breath condensate of patients with asthma. Respir Med 2001; 95:649 – 654 10 Jobsis Q, Raatgeep HC, Schellekens SL, et al. Hydrogen peroxide in exhaled air of healthy children: reference values. Eur Respir J 1998; 12:483– 485 11 Gallati H, Pracht I. Horseradish peroxidase: kinetic studies and optimization of peroxidase activity determination using the substrates H2O2 and 3, 3⬘, 5, 5⬘-tetramethylbenzidine. J Clin Chem Clin Biochem 1985; 23:453– 460
1380
12 Pizzichini MM, Pizzichini E, Clelland L, et al. Sputum in severe exacerbations of asthma: kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med 1997; 155:1501–1508 13 Keatings VM, Jatakanon A, Worsdell YM, et al. Effect of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997; 155:542–548 14 Keatings VM, O’Connor BJ, Wright LG, et al. Late response to allergen is associated with increased concentrations on TNF-a and interleukin-5 in induced sputum. J Allergy Clin Immunol 1997; 99:693– 698 15 Kasielski M, Nowak D. Long-term administration of Nacetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease. Respir Med 2001; 95:448 – 456 16 Gillissen A, Jaworska M, Orth M, et al. Nacystelyn, a novel lysine salt of N-acetylcysteine, to augment cellular antioxidant defense in vitro. Respir Med 1997; 91:159 –168 17 Andeson R, Theron AJ, Ras JG. Ascorbic acid neutralizes reactive oxidants released by hyperactive phagocytes from cigarette smokers. Lung 1988; 166:149 –159 18 Habib MP, Tank LJ, Lane LC, et al. Effect of vitamin E on exhaled ethane in cigarette smokers. Chest 1999; 115:684 – 690 19 Taylor JC, Madison R, Kosinka D. Is antioxidant deficiency related to chronic obstructive pulmonary disease? Am Rev Respir Dis 1986; 134:285–289 20 Montuschi P. Isoprostanes and other exhaled markers in respiratory diseases. Eur Respir Rev 1999; 68:249 –253 21 Rahman I, MacNee W. Oxidant/antioxidant imbalance in smokers and in chronic obstructive pulmonary disease. Thorax 1996; 51:348 –350 22 Peleman RA, Rytila PH, Kips JC, et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839 – 843 23 Selby C, Drost E, Lannan S, et al. Neutrophil retention in the lungs of patients with chronic obstructive pulmonary diseases. Am Rev Respir Dis 1991; 143:1359 –1364 24 Rahman I. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Mol Biol 2003; 36:95–109 25 Rahman I, MacNee W. Role of oxidants/antioxidants in smoking induced lung diseases. Free Radic Biol Med 1996; 21:669 – 681 26 Nowak D, Kasielski M, Pietras T, et al. Cigarette smoking does not increase hydrogen peroxide levels in expired breath condensate of patients with stable COPD. Monaldi Arch Chest Dis 1998; 53:268 –273 27 McLeish KR, Miller FN, Stelzer GT, et al. Mechanism by which methylprednisolone inhibits acute immune complex induced changes in vascular permeability. Inflammation 1986; 10:321–332 28 Culpitt SV, Maziak W, Loukides S, et al. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:1635–1639
Clinical Investigations
Objective: To evaluate the levels of hydrogen peroxide (H2O2) and 8-isoprostane in the expired breath condensate (EBC) of patients with COPD, and to assess the relationship between the above markers of oxidative stress and parameters expressing inflammatory process and disease severity. Setting: Inpatient respiratory unit and outpatient clinic in tertiary care hospital. Design: Cross-sectional study. Patients: Thirty stable COPD patients (all smokers) with disease severity ranging from mild to severe. Ten subjects who were smokers with stage 0 disease (ie, at risk for COPD; mean [ⴞ SD] FEV1, 88 ⴞ 5% predicted) were studied as a control group. Methods: H2O2 and 8-isoprostane levels were measured in EBC, and the values were correlated with variables expressing COPD severity (ie, FEV1 percent predicted, dyspnea severity score (ie, Medical Research Council scale) and airway inflammation (ie, differential cell counts from induced sputum). Results: The mean concentration of H2O2 was significantly elevated in COPD patients compared to control subjects (mean, 0.66 mol/L [95% confidence interval (CI), 0.54 to 0.68 mol/L) vs 0.31 mol/L [95% CI, 0.26 to 0.35 mol/L], respectively; p < 0.0001). The difference was primarily due to the elevation of H2O2 in patients with severe and moderate COPD, whose expired breath H2O2 levels were significantly higher than those of patients with mild disease (mean, 0.96 mol/L [95% CI, 0.79 to 1.13 mol/L], 0.68 mol/L [95% CI, 0.55 to 0.81 mol/L], and 0.33 mol/L [95% CI, 0.24 to 0.43 mol/L], respectively, p < 0.0001). The mean concentration of 8-isoprostane was significantly elevated in patients with COPD compared to that of the control group (47 pg/mL [95% CI, 41 to 53 pg/mL] vs 29 pg/mL [95% CI, 25 to 33 pg/mL], respectively; p < 0.0001) but did not differ significantly among the different stages of the disease (p ⴝ 0.43). Repeatability and stability data within measurements showed that H2O2 has a better repeatability and stability than 8-isoprostane. Furthermore, we observed significant correlations of H2O2 with FEV1, neutrophil count, and dyspnea score. Those correlations existed only in patients with moderate and severe disease. No correlations were found between levels of 8-isoprostane and the above parameters. Conclusions: We conclude that levels of H2O2 and 8-isoprostane are elevated in the EBC of patients with COPD, but that H2O2 seems to be a more repeatable and a more sensitive index of the inflammatory process and the severity of the disease. (CHEST 2003; 124:1373–1380) Key words: COPD; expired breath condensate; inflammation; oxidative stress; severity Abbreviations: CI ⫽ confidence interval; EBC ⫽ expired breath condensate; GOLD ⫽ Global Initiative for Chronic Obstructive Lung Disease; H2O2 ⫽ hydrogen peroxide; ICS ⫽ inhaled corticosteroid; MRC ⫽ Medical Research Council; ROS ⫽ reactive oxygen species
is considerable evidence that oxidative T here stress is increased in patients with COPD and that reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) contribute to the pathophysiology *From the Pneumonology and Clinical Research Department, Athens Army General Hospital, Athens, Greece. Manuscript received November 7, 2002; revision accepted May 5, 2003. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Stelios Loukides, MD, Smolika 2 16673, Voula Athens, Greece; e-mail: [email protected] www.chestjournal.org
of this disease entity.1 Oxidative stress also leads to the formation of isoprostanes by the direct oxidation of arachidonic acid.2 ROS may contribute to the pathophysiology of COPD in several ways, including the damage of serum protease inhibitors, the potentiation of elastase activity, and increased mucus secretion. In addition, they activate the transcription factor NFB, which orchestrates the transcription of many inflammatory genes, including interleukin-8, nitric oxide synthase, and inducible cyclooxygenase.3 ROS are present in cigarette smoke and are produced endogenously by activated inflammatory CHEST / 124 / 4 / OCTOBER, 2003
1373
cells, including neutrophils and alveolar macrophages. The increased production of endogenous ROS is demonstrated by the increased levels of H2O24 and 8-isoprostane5 in the expired breath condensate (EBC) of patients with COPD. However, a number of important issues regarding the value of assessing H2O2 and 8-isoprostane levels in patients with COPD remain unclear, the main one being whether the oxidative stress seen in the EBC of stable COPD patients may express the presence of underlying airway inflammation or predict the severity of the disease. Our primary aim was to evaluate the concentrations of H2O2 and 8-isoprostane in the EBC of COPD patients, to investigate the reproducibility and stability of these measurements, and to see whether there is an association between the levels of those two markers in EBC and airway inflammation or disease severity. Furthermore, we investigated which inflammatory cells are the main sources of H2O2 and 8-isoprostane, and whether these cells differ regarding the classification of severity or the use of inhaled corticosteroids (ICSs). As indexes of airway inflammation in sputum, we studied validated variables such as differential cell counts. Disease severity was assessed according to the results of pulmonary function tests and dyspnea according to a severity score (ie, the Medical Research Council [MRC] scale). Finally, the classification of the COPD patients was made according to the recent Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.6 To account for the possible confounding effects of therapy with ICSs, further analysis was conducted after subdividing the patients on this basis.
Organization Workshop on the Global Initiative for COPD.6 Thirty nonatopic, stable patients with COPD who were smokers (mean smoking, 53 pack-years [SD, 13]; range, 41 to 71 pack-years; all men; mean age, 58 years [SD, 9]; age range, 42 to 76 years; mean FEV1, 57% predicted [SD, 24]; FEV1 range, 21 to 88% predicted) with disease severity ranging from mild to severe (mild COPD, 10 patients [FEV1 83% predicted; SD, 2% predicted; FEV1 range, 80 to 88% predicted]; moderate COPD, 10 patients [FEV1, 60% predicted; SD, 7% predicted; FEV1 range, 50 to 69% predicted]; and severe COPD, 10 patients [FEV1, 27% predicted; SD, 3% predicted; FEV1 range, 21 to 30% predicted]) were studied. Ten healthy, nonatopic subjects who smoked and fulfilled the criteria of COPD stage 0 at risk, according to the GOLD guidelines6 (ie, mean age of all current smokers, 55 years; SD, 9 years; age range, 43 to 77 years; mean FEV1, 88 years; SD, 5 years; FEV1 range, 81 to 99% predicted), served as a control group. Patients were included in the study only if they were clinically stable and had no evidence of an acute exacerbation for at least 4 weeks prior to the study. None of our patients had reversibility with inhaled salbutamol therapy of ⬎ 12% of the predicted FEV1. Fifteen patients were receiving therapy with ICSs (fluticasone propionate, 500 to 1,000 g daily). Six patients were receiving theophylline, 300 mg twice daily. None was receiving any other anti-inflammatory treatment, including therapy with leukotriene antagonist agents, inhaled or oral mucolytic agents, or long-term oxygen therapy. Nineteen patients were receiving long-acting 2-agonists twice daily, and 11 patients occasionally were receiving short-acting 2-agonists or anticholinergics as relief medication. Control subjects had a negative history of allergy (ie, negative responses to skin prick tests for common allergens), normal spirometry results, normal FEV1/FVC ratios, no history of any disease, and were not receiving medical treatment for any reason. They were all male smokers as were the patients (mean smoking history, 49 pack-years; SD, 12 pack-years; range, 38 to 67 pack-years). All patients had normal bronchial reactivity with a provocative dose of histamine causing a 20% fall in FEV1 of ⬎ 0.800 mg (mean, 1.45 mg; range, 0.920 to 1.85 mg). The Scientific Ethics Committee of our hospital approved the study protocol, and all participants gave informed written consent. Assessment of Disease Severity
Materials and Methods Subjects Subject characteristics are summarized in Table diagnosis of COPD and the classification of the severity in our patients was established according National Heart, Lung, and Blood Institute/World
1. The disease to the Health
All the subjects were instructed to define the severity of their daily breathlessness by using the MRC questionnaire6 (scale of 0 of 4 as follows: 0, breathlessness only with strenuous exercise; 1, shortness of breath when hurrying on the level or walking up a slight hill; 2, walk slower than people of the same age on the level because of breathlessness or have to stop for breath when walking on their own pace on the level; 3, stop for breath after walking
Table 1—Subject Characteristics*
Characteristics Age, yr FEV1, % predicted FEV1/FVC ratio, % Smoking habit, pack-yr Dyspnea score Neutrophils, %
Control Subjects (n ⫽ 10) 55 (9) (43–77) 88 (5) (81–99) 86 (7) (81–95) 49 (12) (58–67) 0 38 (11) (32–47)
COPD Patients All (n ⫽ 30)
Mild (n ⫽ 10)
58 (9) (42–76) 57 (24) (21–88) 51 (11) (41–74) 53 (13) (40–71) 2.2 (1) (1–4) 56 (13) (31–70)
60 (5) (53–76) 84 (2) (80–88) 55 (14) (45–74) 53 (16) (40–70) 1.4 (0.7) (1–3) 39 (5) (31–46)
Moderate (n ⫽ 10) 59.5 (9) (42–71) 60 (7) (50–69) 54 (10) (44–64) 56 (12) (42–71) 1.9 (0.6) (1–3) 63 (6.5) (50–70)
Severe (n ⫽ 10) 58 (9) (47–70) 27 (3) (21–30) 44 (9) (41–59) 50 (11) (47–64) 3.4 (0.5) (3–4) 66 (2) (63–70)
*Values given as mean (SD) [ranges]. 1374
Clinical Investigations
about 100 yards or after a few minutes on the level; 4, too breathless to leave the house or breathless when dressing or undressing). Each patient was asked to quantify the impact of dyspnea on his daily health status. The questionnaires were started from the last level, and if a positive answer was given, no further levels were evaluated. Lung Function FEV1 and FVC were measured with a dry spirometer (model VEP2, Vica-test; Mijnhardt; Rotterdam, Holland) using the American Thoracic Society criteria for the standardization of spirometry.7 The best value of three maneuvers was expressed as the percentage of the predicted value. FEV1/FVC ratio was measured in all persons in order to confirm the presence or absence of airway obstruction in patients and in control subjects, respectively.6 Airway responsiveness was measured by histamine provocation challenge in control subjects, using the guidelines provided by the American Thoracic Society.8 According to these criteria, patients were asked not to smoke for 6 h before undergoing the challenge. Histamine challenge in healthy subjects was performed on a separate day in order to avoid any effect of the challenge occurring on the inflammatory cells and oxidative stress values.
8-Isoprostane Measurements: 8-Isoprostane concentration was determined by a competitive enzyme immunoassay kit (Cayman Chemical; Ann Arbor, MI), as previously described.5 The detection limit of the assay was 4 pg/mL. Sputum Induction and Processing Sputum was induced by the inhalation of an aerosol 3.5% hypertonic saline solution that was generated by an ultrasonic nebulizer (model 2696; DeVilbiss; Somerset PA), with modifications to improve its safety.12 At least 2 mL sputum was collected into a sterile container. The sample from the first cough was discarded, as it was heavily contaminated with squamous epithelial cells.13 The sample was determined to be adequate if the number of squamous epithelial cells was ⬍ 30% of the total number of inflammatory cells.14 Cytospin slides were prepared and stained with May-Gru¨ nwald-Giemsa stain. The person who did the differential cell counts was not aware of the clinical and functional status of the patients or of the EBC measurements. Two slides were used for counting, and at least 300 inflammatory cells were counted for each slide. The inflammatory cells in the sputum samples are shown as the percentage of total nonsquamous cells. Sputum measurements were performed on the same day for all the patients. Statistical Analysis
Collection and Measurements of EBC Collection of EBC: The collection of EBC was performed as previously described.9 Approximately 2 mL EBC was collected. The repeatability of H2O2 and 8-isoprostane measurements and the stability of the frozen samples were estimated as previously described10 in 5 control subjects and 12 patients with COPD (4 persons from each subgroup). In order to assess the repeatability of the measurements, EBC was collected on 2 consecutive days under the same conditions. To assess the stability of the frozen condensate samples, 4 mL EBC was collected. The abovementioned concentration was divided into 1-mL aliquots, in which H2O2 and 8-isoprostane concentrations were determined after 2 days, 1 week, 2 weeks, and 3 weeks of storage (the maximum time between collection and measurement in any sample). All condensate samples were tested for salivary contamination by the determination of amylase activity. Briefly, amylase activity was carried out spectrophotometrically (kinetic method) using a commercial reagent kit (model 981362; KONE Instruments; Espoo, Finland). In this procedure, the ␣-amylase of the sample and the enzyme ␣-glycosidase hydrolyze the substrate p-nitrophenyl-␣-D-maltoheptaoside to glucose and p-nitrophenol. The liberation of p-nitrophenol was observed at 405 nm (37°C) for 2 min. Two samples were spiked with saliva to ensure that this could be detected by our method. In all the samples tested for saliva, no amylase was detected, suggesting that there was no contamination of breath condensate. The samples that were spiked with saliva showed levels of salivary amylase of ⬎ 5,000 IU. In all study subjects, EBC was collected first, followed by the sputum-induction procedure. Subjects had been asked not to smoke for 2 h prior to the collection of EBC and sputum. In order to ensure that, all subjects stayed in our laboratory for 2 h before the study, under the supervision of one of the investigators. H2O2 Measurements: H2O2 concentration was determined by an enzymatic assay using horseradish peroxidase (Sigma Chemical; St Louis, MO), as previously described.11 The detection limit of the assay was 0.1 mol/L. www.chestjournal.org
Data concerning subject characteristics are expressed as the mean (SD) with ranges. Data concerning the comparisons among the various parameters in the study groups are given as the mean with 95% confidence intervals (CIs) for the differences. The data were examined for normal distribution, and when it was not normally distributed the nonparametric Mann-Whitney test was used for statistical comparisons. For normally distributed data, the paired t test was used for statistical comparisons between the two groups. The normality of distribution was tested with a Shapiro-Wilks test. Parameters from the four groups of patients with COPD were compared using the one-way analysis of variance with an appropriate post hoc test (ie, Bonferroni test) for multiple comparisons. The Pearson correlation coefficient was used to investigate the relations among the parameters. A p value of ⬍ 0.05 was considered to be significant.
Results Repeated measurements on 2 consecutive days revealed a mean within-subject difference of 0.08 mol/L (SD, 0.05 mol/L) for H2O2 (p ⫽ 0.26), and 13 pg/mL (SD, 4 pg/mL) for 8-isoprostane (p ⫽ 0.04). When we checked the stability of H2O2 in the frozen samples, we noticed no significant differences among the four measurements (after 2 days, 0.49 mol/L [SD, 0.11 mol/L]; after 1 week, 0.51 mol/L [SD, 0.1 mol/L]; after 2 weeks, 0.52 mol/L [SD, 0.09 mol/L]; and after 3 weeks, 0.47 mol/L [SD, 0.14 mol/L]; p ⫽ 0.43). Regarding the stability of 8-isoprostane in frozen samples, we noticed marginal differences among the four measurements, which were not significant (after 2 days, 33 pg/mL [SD 11 pg/mL]; after 1 week, 38 pg/mL [SD 10.3 pg/mL]; after 2 weeks, 31 pg/mL [SD 8 pg/mL]; after 3 weeks, 39 pg/mL [SD 7.3 pg/mL]; p ⫽ 0.07). The mean concentration of H2O2 was significantly CHEST / 124 / 4 / OCTOBER, 2003
1375
elevated in patients with COPD compared to the control subjects (0.66 mol/L [95% CI, 0.54 to 0.68 mol/L] vs 0.31 mol/L [95% CI, 0.26 to 0.35 mol/L]; p ⬍ 0.0001) [Fig 1, top, A]. The difference was primarily due to the elevation of H2O2 levels in patients with severe and moderate COPD, whose H2O2 levels in EBC were significantly
higher than those of patients with mild disease (0.96 mol/L [95% CI, 0.79 to 1.13 mol/L], 0.68 mol/L [95% CI, 0.55 to 0.81 mol/L], and 0.33 mol/L [95% CI, 0.24 to 0.43 mol/L] respectively; p ⬍ 0.0001) [Fig 1, top, A]. The mean concentration of 8-isoprostane was significantly elevated in patients with COPD compared to that of the control group (47 pg/mL [95% CI, 41 to 53 pg/mL] vs 29 pg/mL [95% CI, 25 to 33 pg/mL], respectively; p ⬍ 0.0001) [Fig 1, bottom, B] but did not differ significantly among the different stages of the disease (patients with mild disease, 42 pg/mL [95% CI, 36 to 49 pg/mL]; patients with moderate disease, 48 pg/mL [95% CI, 33 to 63 pg/mL]; and patients with severe disease, 51 pg/mL [95% CI, 41 to 61 pg/mL]; p ⫽ 0.43) [Fig 1, bottom, B]. Subanalysis for the Effects of ICSs COPD patients receiving ICS therapy had significantly higher H2O2 values compared with steroid-naive patients (0.8 mol/L [95% CI, 0.61 to 0.94 mol/L] vs 0.52 mol/L [95% CI, 0.39 to 0.64 mol/L], respectively; p ⬍ 0.0001) [Fig 1, top, A]. This difference may be attributed to the fact that most of the COPD patients who were receiving ICS therapy had severe disease. The mean concentration of 8-isoprostane did not differ significantly between patients receiving ICS therapy compared with steroid-naive patients (46 pg/mL [95% CI, 38 to 54 pg/mL] vs 48 pg/mL [95% CI, 39 to 57.5 pg/mL], respectively; p ⫽ 0.63) [Fig 1, bottom, B]. Airway Inflammatory Cells
Figure 1. Top, A: H2O2 concentration in the EBC of control subjects (10 subjects) and patients with COPD (total, 30 patients; mild disease, 10 patients; moderate disease, 10 patients; severe disease, 10 patients; ICS-treated patients, 15 patients; and steroid-naive patients, 15 patients). Patients with COPD have significantly higher values compared to control subjects (p ⬍ 0.0001). Patients with severe disease have significantly higher values than those with mild and moderate disease (p ⬍ 0.0001). Patients treated with ICSs have significantly higher values compared with steroid-naive patients (p ⬍ 0.0001). Each symbol represents one individual. Horizontal bars represent mean values. Bottom, B: 8-isoprostane concentration in the EBC of control subjects (total, 10 subjects) and of patients with COPD (total, 30 patients; mild disease, 10 patients; moderate disease, 10 patients; severe disease, 10 patients; ICS-treated, 15 patients; and steroid-naive patients, 15 patients). Patients with COPD had significantly higher values compared to control subjects (p ⬍ 0.0001). No significant differences were observed among patients with mild, moderate, and severe disease (p ⫽ 0.43). Patients treated with ICSs had similar values compared with steroid-naive patients (p ⫽ 0.63). Each symbol represents one individual. Horizontal bars represent mean values. ICS(⫹) ⫽ ICS-treated; ICS(-) ⫽ steroid-naive. 1376
COPD patients had higher levels of sputum neutrophilia than did control subjects (56% [95% CI, 51 to 61%] and 528 ⫻ 103 cells/g [95% CI, 488 to 534 ⫻ 103 cells/g] vs 38% [95% CI, 32 to 40%] and 315 ⫻ 103 cells/g [95% CI, 288 –334 ⫻ 10 cells/g], respectively; p ⬍ 0.0001) [Fig 2, Table 1]. The difference was primarily due to the elevation of neutrophils in patients with moderate and severe disease (mild disease, 39% [95% CI, 36 to 43%] and 315 ⫻ 103 cells/g [95% CI, 288 to 334 ⫻ 103 cells/g]; moderate disease, 63% [95% CI, 58 to 67.5%] and 604 ⫻ 103 cells/g [95% CI, 548 to 635 ⫻ 103 cells/g]; and severe disease, 67% [95% CI, 64 to 68%] and 667 ⫻ 103 cells/g [95% CI, 603 to 688 ⫻ 103 cells/g]; p ⬍ 0.002) [Fig 2, Table 1]. The percentage and number of macrophages was significantly higher in control subjects than in COPD patients (62% [95% CI, 54 to 66%] and 578 ⫻ 103 cells/g [95% CI, 509 to 601 ⫻ 103 cells/g] vs 42% [95% CI, 38 to 48%] and 349 ⫻ 103 cells/g [95% CI, 308 to 421 ⫻ 103 cells/g], respectively; p ⬍ 0.0001) [Fig 2] and was significantly lower in patients with severe disease than in Clinical Investigations
Discussion In this prospective, cross-sectional study we have found that oxidative stress, as assessed by the levels of H2O2 and 8-isoprostane, is elevated in the EBC of patients with COPD. The measurement of H2O2 levels seems to be a more repeatable and more sensitive index of the inflammatory process and disease severity compared with that of 8-isoprostane levels. The increased levels of H2O2 and 8-isoprostane observed in this study are similar to those previously reported.4,5 It has been shown that the concentration of 8-isoprostane is increased in patients with COPD, irrespective of smoking status and lung function impairment.5 Moreover, the levels of H2O2 in the EBC of stable patients with COPD have been found to be higher than those of healthy subjects and lower than those of unstable patients with COPD.4 In addition, no correlation of either H2O2 or 8-isoprostane and lung function impairment has been reported. An important question arising from these studies was the identification of the cellular sources of these biomarkers of oxidative stress and the quantification of their possible role in predicting the severity of the disease. Another issue was the absence of data regarding the repeatability and stability of H2O2 and 8-isoprostane measurements in EBC. In order to identify the relationship between the measurements and disease severity, we believe that the critical point is to classify patients according to widely accepted guidelines, such as those proposed by GOLD,6 and simultaneously to avoid using terms like unstable, since they do not provide precise characteristics of the disease and, additionally, depend on multiple factors that might affect our measurements. Our results showed that evaluating the H2O2 concentration may provide useful information about the severity of the disease. On the other hand, we found no equivalent data regarding the levels of 8-isoprostane. One plausible explanation for these observations might depend on the underlying antioxidant defense mechanisms. An increased concentration of biomarkers of oxidative stress may repre-
Figure 2. Percentage of differential cell counts in the induced sputum of patients with COPD and in that of control subjects. * ⫽ statistically significantly higher percentage of neutrophils in the induced sputum of patients with COPD compared to control subjects (p ⬍ 0.0001); ** ⫽ statistically significant lower percentage of macrophages in the induced sputum of patients with COPD compared to control subjects (p ⬍ 0.0001); *** ⫽ statistically significant higher percentage of neutrophils in the induced sputum of patients with severe disease compared to those with mild and moderate disease (p ⬍ 0.002).
those with mild and moderate disease (severe disease, 32% [95% CI, 31 to 34%]; 301 ⫻ 103 cells/g [95% CI, 275 to 342 ⫻ 103 cells/g]; mild disease, 36% [95% CI, 31– 41%]; 357 ⫻ 103/g [95% CI, 312–384 ⫻ 103 cells/g]; moderate disease, 60% [95% CI, 47 to 63%]; 530 ⫻ 103 cells/g [95% CI, 421 to 542 ⫻ 103 cells/g]; p ⬍ 0.05) [Fig 2]. No significant differences were observed between mean neutrophil levels in steroid-naive and steroid-treated patients (54 ⫾ 13% [95% CI, 47 to 62%] vs 58 ⫾ 13 [95% CI, 51 to 61%], respectively; p ⫽ 0.6). Correlations Among H2O2 and 8-Isoprostane Concentrations, Inflammatory Cells in Induced Sputum, Lung Function, and Dyspnea Severity Scale in Patients With COPD Major correlations are presented in Tables 2, 3, and 4, and in Figure 3.
Table 2—Correlations of H2O2 in the Four Groups of Patients With COPD* All
Mild
Moderate
Severe
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score 8-Isoprostane
⫺0.83 0.83 0.68 0.01
⬍ 0.0001 ⬍ 0.0001 ⬍ 0.0001 0.46
⫺0.46 0.44 0.57 0.02
0.76 0.19 0.07 0.67
⫺0.76 0.67 0.51 0.2
0.009 0.02 0.11 0.15
⫺0.04 0.76 0.42 0.17
0.89 0.001 0.22 0.5
*Correlations were conducted using the Pearson correlation coefficient. www.chestjournal.org
CHEST / 124 / 4 / OCTOBER, 2003
1377
Table 3—Correlations of 8-Isoprostane in the Four Groups of Patients With COPD* All
Mild
Moderate
Severe
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score
⫺0.23 0.2 0.21
0.22 0.28 0.25
⫺0.26 0.37 0.56
0.44 0.29 0.08
⫺0.06 0.27 0.32
0.84 0.43 0.35
⫺0.33 0.23 0.26
0.3 0.51 0.45
*Correlations were conducted using the Pearson correlation coefficient.
sent an increased production of ROS or a reduced number of free radicals scavenging capacity in the airways of COPD subjects, or even a combination of these two parameters. Long-term antioxidant treatment attenuates H2O2 formation in the airways of COPD subjects but has no significant effect on the levels of lipid peroxidation products.15,16 It has been shown17,18 that dietary antioxidant supplies may scavenge the ROS, but they fail to reduce lipid peroxidation. There is some evidence that the deficiency of antioxidant defenses is related to the degree of airway obstruction in patients with COPD,19 while no data exist to support a significant correlation between lipid peroxidation and lung function impairment. Since the classification of the disease by GOLD is based on FEV1 values, and taking all the above points under consideration, we believe that there may well be close relationships among H2O2 concentration, lung function, and antioxidant mechanisms. In contrast, no respective data exist for the products of lipid peroxidation. Based on the above hypothesis and on part of our data, we may assume that, as the severity of the disease progresses, there is also a progressive course of H2O2 concentration, antioxidant status, and changes in lung function, with the last two decreasing and the first one increasing or just overcoming the defenses. The difference we have observed in the data for 8-isoprostane might be explained by the fact that lipid peroxidation is the result of a complicated metabolic pathway, which starts from free radicals that trigger lipid peroxidation chain reactions.20 In this metabolic pathway, the antioxidant defenses do not play an equally impor-
tant role, and the concentration of 8-isoprostane eventually might reach a point at which the underlying triggering ROS-mediated mechanism has little, if any, effect that leads to a further increase of its end-products such as 8-isoprostane. This is partially supported by the absence of significant correlation between the levels of H2O2 and 8-isoprostane that we observed in this study. Our data suggest that H2O2 is a more repeatable and more stable product than 8-isoprostane. Since 8-isoprostane is an end-product of a metabolic pathway that is chemically stable, one might expect that its measurements would be more stable and repeatable. However, its overall production is based on a complicated metabolic pathway, in which many factors are involved that might significantly affect its measurement. Those factors may be independent of the inflammatory process of COPD. The strong repeatability and stability of the H2O2 measurements might be explained by the fact that H2O2 production mainly depends on the underlying inflammatory cells (primarily neutrophils) and the effect of the antioxidant defenses. There is incoming evidence from the literature that these two factors remain unchanged through the course of COPD in stable patients.21,22 Most observations dealing with inflammatory cells in patients with COPD have focused on neutrophils, since they undoubtedly have an impact on the inflammatory process and may alter the oxidantantioxidant balance.21–23 Similar results were observed in our study, using a noninvasive and widely accepted method, such as sputum induction. Increased numbers of neutrophils appear to be pro-
Table 4 —Correlations of H2O2 and 8-Isoprostane in Steroid-Naive and Steroid-Treated Patients With COPD* H2O2 Steroid-Naive
8-Isoprostane Steroid-Treated
Steroid-Naive
Steroid-Treated
Variables
r Value
p Value
r Value
p Value
r Value
p Value
r Value
p Value
FEV1 Neutrophils Dyspnea score 8-Isoprostane
⫺0.82 0.88 0.37 0.08
0.0001 ⬍ 0.0001 0.14 0.75
⫺0.8 0.74 0.79 0.3
0.0004 0.001 0.0004 0.1
⫺0.007 0.3 0.17
0.7 0.2 0.51
⫺0.06 0.11 0.25
0.63 0.27 0.31
*Correlations were conducted using the Pearson correlation coefficient. 1378
Clinical Investigations
Figure 3. Correlations between H2O2 concentration in the EBC of patients with COPD and (top, A) the percentage of neutrophils in induced sputum (r ⫽ 0.83; p ⬍ 0.0001), (middle, B) dyspnea MRC scale (r ⫽ 0.68; p ⬍ 0.0001), and (bottom, C) the FEV1 percent predicted (r ⫽ ⫺0.83; p ⬍ 0.0001). f ⫽ individual data points.
ducing increased amounts of ROS in the lung of COPD patients.2 Examining all the above points together (ie, the high percentage of neutrophils in the sputum of COPD patients), the strong correlation of neutrophil levels with H2O2 concentration that we have observed in this study, and the lack of a positive correlation between macrophage levels and H2O2 concentration, we may come to the plauwww.chestjournal.org
sible conclusion that neutrophils are the main source of H2O2 production in patients with COPD. We were not able to ascertain the cellular source of 8-isoprostane using the data of this study. The source of the increased ROS production in patients with COPD might derive from structural cells of the airways.24 However, in our study there are no data to support this. Another important issue deriving from the data presented is the difference between the control group and the groups of patients with different stages of COPD. It is well-known that smoking contributes to the burden of oxidants and free radicals in the lower airways.25 Our results showed that in patients with COPD with similar smoking habits the differences observed in oxidative stress values are related mainly to the underlying inflammatory process. Similar results have been presented in previous studies,26 from which we were not able to come to any conclusions since they did not provide cellular data. Another main advantage of our study was the fact that as a control group we used patients who were at risk for COPD (ie, stage 0) and had a similar smoking history. A possible explanation might be that in subjects who are at risk to develop COPD, the underlying oxidative stress is mainly related to smoking but when the disease appears the inflammatory process surpasses the smoking contribution. This idea is partially supported by the absence of significant correlation between H2O2 and neutrophil levels in both the control group and the group of patients with mild COPD in whom the inflammatory process seems to be limited. The interindividual differences in the correlations data within groups might well be explained by the limited range of FEV1 and dyspnea severity score values, particularly in patients with severe disease. This is partially confirmed by the significant correlations we observed in patients with moderate disease, who presented a wide range of FEV1 values and symptom scores. The present cross-sectional study cannot demonstrate a causal relationship between treatment with ICSs and the concentrations of 8-isoprostane and H2O2 in EBC. In fact, patients receiving ICS therapy had higher values compared to steroid-naive patients. This might be due to the fact that the patients treated with ICSs in our study had more severe forms of COPD. The ineffectiveness of ICS therapy on H2O2 concentration might be explained by the fact that steroids fail to inhibit the production of H2O2 from polymorphonuclear leukocytes,27 probably because of their inability to inhibit the underlying neutrophilic inflammation.28 In this study, we were not able to clarify the role of treatment with ICSs on the levels of 8-isoprostane, since there are currently CHEST / 124 / 4 / OCTOBER, 2003
1379
no sufficient data regarding the cellular source of 8-isoprostane. The presence of significant correlations between H2O2 and study variables, irrespective of ICS usage, and simultaneously the absence of similar correlation data for 8-isoprostane supports the recommendations6 that treatment with ICSs does not significantly change the inflammatory, functional, and clinical course of the disease. In conclusion, studying those two different components of oxidative stress, we report that both H2O2 and 8-isoprostane levels are increased in stable patients with COPD. The measurement of H2O2 is a more repeatable and more sensitive marker of the underlying inflammatory process and the severity of the disease than are the end-products of lipid peroxidation, as reflected by the levels of 8-isoprostane. The role of lipid peroxidation in the inflammatory burden of COPD still requires further study to be clarified. References 1 Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154:813– 816 2 Gutteridge GM. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41:1819 –1828 3 Rahman I, MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 1998; 53:601– 612 4 Dekhuijzen RPN, Aben KKH, Dekker I, et al. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154:813– 816 5 Montuschi P, Collins JV, Ciabattoni G, et al. Exhaled 8isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 2000; 162:1175–1177 6 Global Initiative for Chronic Obstructive Pulmonary Disease. NHLBI/WHO workshop report. Bethesda, MD: National Institutes of Health, April 2001; NIH Publication No. 2701 7 American Thoracic Society. Standardization of spirometry: 1994 update. Am J Respir Crit Care Med 1995; 152:1107– 1136 8 American Thoracic Society. Guidelines for methacholine and exercise challenge testing 1999. Am J Respir Crit Care Med 2000; 161:309 –329 9 Ganas K, Loukides S, Papatheodorou G, et al. Total nitrite/ nitrate in expired breath condensate of patients with asthma. Respir Med 2001; 95:649 – 654 10 Jobsis Q, Raatgeep HC, Schellekens SL, et al. Hydrogen peroxide in exhaled air of healthy children: reference values. Eur Respir J 1998; 12:483– 485 11 Gallati H, Pracht I. Horseradish peroxidase: kinetic studies and optimization of peroxidase activity determination using the substrates H2O2 and 3, 3⬘, 5, 5⬘-tetramethylbenzidine. J Clin Chem Clin Biochem 1985; 23:453– 460
1380
12 Pizzichini MM, Pizzichini E, Clelland L, et al. Sputum in severe exacerbations of asthma: kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med 1997; 155:1501–1508 13 Keatings VM, Jatakanon A, Worsdell YM, et al. Effect of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997; 155:542–548 14 Keatings VM, O’Connor BJ, Wright LG, et al. Late response to allergen is associated with increased concentrations on TNF-a and interleukin-5 in induced sputum. J Allergy Clin Immunol 1997; 99:693– 698 15 Kasielski M, Nowak D. Long-term administration of Nacetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease. Respir Med 2001; 95:448 – 456 16 Gillissen A, Jaworska M, Orth M, et al. Nacystelyn, a novel lysine salt of N-acetylcysteine, to augment cellular antioxidant defense in vitro. Respir Med 1997; 91:159 –168 17 Andeson R, Theron AJ, Ras JG. Ascorbic acid neutralizes reactive oxidants released by hyperactive phagocytes from cigarette smokers. Lung 1988; 166:149 –159 18 Habib MP, Tank LJ, Lane LC, et al. Effect of vitamin E on exhaled ethane in cigarette smokers. Chest 1999; 115:684 – 690 19 Taylor JC, Madison R, Kosinka D. Is antioxidant deficiency related to chronic obstructive pulmonary disease? Am Rev Respir Dis 1986; 134:285–289 20 Montuschi P. Isoprostanes and other exhaled markers in respiratory diseases. Eur Respir Rev 1999; 68:249 –253 21 Rahman I, MacNee W. Oxidant/antioxidant imbalance in smokers and in chronic obstructive pulmonary disease. Thorax 1996; 51:348 –350 22 Peleman RA, Rytila PH, Kips JC, et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839 – 843 23 Selby C, Drost E, Lannan S, et al. Neutrophil retention in the lungs of patients with chronic obstructive pulmonary diseases. Am Rev Respir Dis 1991; 143:1359 –1364 24 Rahman I. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Mol Biol 2003; 36:95–109 25 Rahman I, MacNee W. Role of oxidants/antioxidants in smoking induced lung diseases. Free Radic Biol Med 1996; 21:669 – 681 26 Nowak D, Kasielski M, Pietras T, et al. Cigarette smoking does not increase hydrogen peroxide levels in expired breath condensate of patients with stable COPD. Monaldi Arch Chest Dis 1998; 53:268 –273 27 McLeish KR, Miller FN, Stelzer GT, et al. Mechanism by which methylprednisolone inhibits acute immune complex induced changes in vascular permeability. Inflammation 1986; 10:321–332 28 Culpitt SV, Maziak W, Loukides S, et al. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:1635–1639
Clinical Investigations