Abstract
8-Isoprostane is a potential in vivo marker for oxidant burden, but its usefulness in induced sputum of smokers and chronic obstructive pulmonary disease (COPD) has not been investigated.
The current study investigated 58 subjects comprising 11 never-smokers, 11 ex-smokers, 13 healthy current smokers and 23 COPD with stage 0–III disease (according to the Global Initiative for Chronic Obstructive Lung Disease criteria). 8-Isoprostane was determined from induced sputum by enzyme immunoassay.
Sputum 8-isoprostane levels were similar in the never-smokers and ex-smokers, but were elevated in the healthy smokers compared with nonsmokers, and in those with stage I–III COPD. Sputum 8-isoprostane levels could not differentiate nonsymptomatic smokers from those with Stage 0 COPD. There was a correlation between sputum 8-isoprostane level and lung function parameters (forced expiratory volume in one second/forced vital capacity and sputum neutrophils.
In conclusion, sputum 8-isoprostane levels correlate with the severity of chronic obstructive pulmonary disease. However, they do not appear to differentiate healthy smokers from those who are at risk of developing chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease stage 0).
Chronic obstructive pulmonary disease (COPD) is generally diagnosed when lung function parameters have become significantly reduced and a major part of the lung has been damaged. The pathogenesis of COPD has been strongly associated with reactive oxygen species 1–4, although it is not known how oxidative/nitrosative stress predicts the disease progression. Several oxidant markers and “footprints” of oxidative/nitrosative damage have been detected in COPD lung tissue, sputum, exhaled air and exhaled breath condensate 3–6, but it has not been unequivocally resolved whether these biomarkers can be used in the early assessment of cigarette-smoke-related lung diseases, their progression or whether they relate to smoking alone.
8-Epi-prostaglandinF2α (8-isoprostane) has been suggested to be the most reliable approach to monitor oxidative stress in vivo 7, 8. Isoprostanes are formed by free-radical-catalysed lipid peroxidation of arachidonic acid and cell membrane phospholipids. Isoprostanes can also be released into the circulation, secretions and urine where levels have been found to be stable and reproducible in many experimental approaches 8, 9. Isoprostanes also have potent biological actions and therefore they may significantly contribute to the progression of oxidant-mediated lung diseases, such as COPD.
Several studies have shown elevated 8-isoprostane in the exhaled breath condensate of COPD patients 10–13. There are, however, a number of uncertainties with respect to the usefulness and standardisation of exhaled breath condensate 14–17. One recent study also failed to detect 8-isoprostane in exhaled breath condensate in the majority of cigarette smokers 18; the reasons suggested were the high dilution of all biological constituents in the breath condensate and the low sensitivity of the 8-isoprostane enzyme immunoassay (EIA) method. Induced sputum is a standardised method that reliably reflects local airway inflammation. 8-Isoprostane has not been previously investigated using the induced sputum of smokers.
The present study was undertaken to obtain a more accurate insight into the significance of the local oxidant burden in the airways of healthy smokers and smokers who are evidently at risk of developing COPD (stage 0 COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria) 19 by analysing the concentrations of 8-isoprostane from induced sputum specimens. The study included age-matched subjects, never-smokers, ex-smokers, healthy smokers, smokers with stage 0 COPD (symptoms with normal lung function parameters, GOLD criteria 19), and those with stable COPD (stage I–III).
SUBJECTS AND METHODS
Subjects
A total of 58 subjects were included in the present study, comprising 11 never-smokers, 11 ex-smokers (who had stopped smoking ≥20 yrs before the study with a smoking history of <15 pack-yrs), 13 healthy nonsymptomatic smokers, nine smokers with symptoms (St George’s questionnaire for symptoms such as cough and sputum production) whose lung function parameters were normal (stage 0 COPD) 19, and 14 COPD patients with stage I–III disease. The diagnosis of COPD was based on the GOLD criteria with post-bronchodilator forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) <70% with the post-bronchodilator effect <12%. Atopy and allergies were excluded. Healthy controls and stage 0 COPD subjects had not been treated with any anti-inflammatory medication for 2 months, but two individuals had been prescribed a short-acting bronchodilator. The medications for stage I–III included inhaled short-acting bronchodilators in 100% of cases, long-acting bronchodilators in 79% and inhaled steroids in 64%. None of the subjects were allowed to smoke during the 12 h prior to specimen collection and none had suffered from any viral infection for 2 months. Each subject underwent spirometry with the bronchodilator test and an assessment of total lung capacity and diffusion capacity 20.
This study was approved by the Ethics Committee of Helsinki University Hospital with written consent obtained from every subject. The study is registered by the hospital (www.hus.fi/clinicaltrials).
Sputum processing
Sputum was induced, as described by the European Respiratory Society Task Force, with 4.5% physiological saline solution 21, and samples processed as previously described 22. Briefly, expectorated samples were processed with four volumes of dithioerythritol (DTE; Sigma, Munich, Germany). Suspensions were filtered through 70-μm nylon gauze and centrifuged at 400×g at 4°C for 10 min. In preliminary studies, the sputum was also collected by the same protocol in PBS without DTE to test the possible effect of DTE on the 8-isoprostane EIA analysis. After centrifugation, the pellet was resuspended, and the viabilities and absolute numbers of cells were calculated by the trypan blue exclusion test. All samples in DTE or PBS were immediately frozen at -80°C. Cytospins were prepared and stained using the May–Grunwald–Giemsa method for cell differential counts. The cytospins were frozen at -20°C.
Analyses
Free 8-isoprostane was analysed by EIA according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA). The values were expressed as pg·mL-1. The EIA method is highly specific for 8-isoprostane and has been used previously for the assessment of 8-isoprostane from exhaled breath condensate 10, 23, bronchoalveolar lavage 24, plasma 25 and induced sputum 26. The method has been found to show strong association with the results obtained with gas chromatography-mass spectroscopy (GC-MS) 26. To test the analysis further, sputum samples were analysed at three different dilutions, giving good reproducibility (% coefficient of variation 10.5, intra-class correlation coefficient 0.87). When one individual sputum specimen (divided originally in small aliquots) was run in 10 separate assays the reproducibility was good; the values of this specimen ranged 29.7–37.1 pg·mL-1 (sd 3.8). The effect of DTE had been tested in the preliminary studies by adding the corresponding DTE concentration as in the sputum specimens to the standards; these determinations showed very consistent results (r = 0.99). In addition, eight inductions had been processed with PBS without DTE. These specimens were further divided into two parts and treated with DTE or PBS, and analysed for 8-isoprostane. The results with or without DTE were also very similar (r = 0.75). The dilutions were made in the buffer provided by the manufacturer.
Statistics
Data are presented as mean±sem or median (range) for not normally distributed data. Data for all groups were analysed by the Kruskall–Wallis test and differences between individual variables from two groups were analysed by the Mann–Whitney U-test. Correlations between variables were sought using the Spearman rank correlation test. A p-value <0.05 was considered significant. For values below the detection limit in 8-isoprostane analysis, random number interpolation was used; this was only needed for five samples in the healthy control group.
RESULTS
Patient characteristics are shown in table 1⇓. As expected, current smokers had higher numbers of neutrophils in the induced sputum than nonsmokers.
Patient characteristics
The percentage and number of sputum neutrophils were very similar in never-smokers and ex-smokers (who had stopped smoking ≥20 yrs ago). The percentage of sputum neutrophils tended to be higher in those smokers who had symptoms but normal lung function parameters (stage 0 COPD) compared with nonsymptomatic smokers (p = 0.06; table 1⇑). However, there was no difference in the total number of neutrophils between these two groups (p = 0.23).
The levels of 8-isoprostane in the induced sputum were higher in healthy smokers (median 108.4 pg·mL-1) than in nonsmokers (median 15.3 pg·mL-1; p = 0.005), but did not differ between healthy smokers and stage 0 COPD (median 66.6 pg·mL-1; fig. 1a⇓). The levels of 8-isoprostane did not differ between never-smokers and ex-smokers. The levels significantly increased in COPD (median 202.2 pg·mL-1, stage I–III; p<0.0001 and p = 0.02 compared with nonsmokers and healthy smokers, respectively). There was a significant correlation between the 8-isoprostane levels and lung function parameters (8-isoprostane versus FEV1/FVC r = -0.66, p<0.0001, versus FEV1 r = -0.48, p = 0.006; fig. 1b⇓) and between the 8-isoprostane level and smoking history evaluated from smoking pack-yrs (r = 0.56, p = 0.001; fig. 1c⇓). The 8-isoprostane level in the sputum significantly correlated with sputum neutrophils (total neutrophils r = 0.37, p = 0.02; fig. 1d⇓). Three subjects (33%) with stage 0 COPD had stopped smoking ≥1 yr ago (mean time from quitting 2 yrs). However, there was no difference between these ex-smokers and current smokers in stage 0 COPD group in any of the parameters measured. In patients with stage I COPD or worse, six patients (43%) had stopped smoking (mean time from quitting 3.6 yrs). In this group, current smokers tended to have higher sputum 8-isoprotane levels than ex-smokers but the difference was not significant (p = 0.09). No significant differences could be found between current smokers and ex-smokers in these COPD patients. When all the subjects were divided into two groups, current smokers and nonsmokers, then the level of 8-isoprostane in sputum was significantly higher in current smokers (p = 0.001). In the COPD group, 64% of the patients used inhaled steroids. However, there were no significant differences between 8-isoprostane levels in patients with inhaled steroids or without them.
a) 8-isoprostane (8-IP) levels (pg·mL-1) in sputum samples. HC: healthy controls; HS: healthy smokers; COPD: chronic obstructive pulmonary disease. #: p<0.0001; ¶: p = 0.02; +: p = 0.005. Horizontal bars indicate median values. p-Values between all groups were calculated with Kruskall–Wallis test and between two groups with Mann–Whitney U-test. Spearman rank correlations between sputum 8-IP and b) forced expiratory volume in one second (FEV1)/forced vital capacity (FVC; = -0.66, p = 0.0001), c) pack-yrs (r = 0.56, p = 0.001), and d) total number of sputum neutrophils (r = 0.37, p = 0.02).
DISCUSSION
The main aim of the present study was to assess 8-isoprostane in induced sputum specimens, since these are probably the most sensitive noninvasive way of assessing oxidative stress in the airways. As far as the current authors are aware, there are no studies on the levels of 8-isoprostane in the induced sputum of COPD. The levels of 8-isoprostane were already significantly elevated in healthy smokers and consistently increased in COPD, and a significant correlation with the lung function parameters was noted.
The EIA method used appeared to be reproducible. Several earlier studies have also assessed 8-isoprostane in the exhaled breath condensate and bronchoalveolar lavage 10, 23, 24. The current results are also in line with a recent study where this same method was used to examine the induced sputum of asthma and found to have highly significant correlation (r>0.9) with GC-MS and 100% spiking recovery 26.
Given that 8-isoprostane was also related to smoking without COPD, the possibility that sputum 8-isoprostane levels may differentiate healthy smokers from those who are at risk of developng COPD was investigated. Therefore, healthy smokers and those who were exhibiting symptoms, i.e. stage 0 COPD, were included in the study. The levels of 8-isoprostane were significantly elevated in smokers but there was no difference in the sputum 8-isoprostane level between nonsymptomatic smokers and stage 0 COPD. None of the smokers had smoked for 12 h. 8-Isoprostane was, however, significantly higher in the sputum of COPD (stage≥1) when compared with stage 0. These results clearly suggest that 8-isoprostane is not a reliable marker in differentiating healthy smokers from those who are probably at risk of developing COPD. It also needs to be emphasised that stage 0 COPD does not necessarily lead to the development of COPD 27–30. Overall, 8-isoprostane is already elevated in the sputum of smokers but it still remains unclear whether this or other markers of oxidative stress are sensitive enough in finding those smokers who really are at risk for COPD development.
In conclusion, 8-isoprostane levels are clearly increased in the induced sputum of smokers and especially in moderate-to-severe chronic obstructive pulmonary disease, but do not appear to be very sensitive in differentiating healthy smokers from those who probably have a risk of developing chronic obstructive pulmonary disease.
Acknowledgments
The authors would like to thank M. Luukkonen and T. Marjomaa for their excellent technical assistance and T. Rehn for assistance with subject recruitment.
- Received February 14, 2006.
- Accepted September 19, 2006.
- © ERS Journals Ltd
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