Lung DOI 10.1007/s00408-014-9591-8
Exhaled Nitric Oxide Predicts Eosinophilic Airway Inflammation in COPD Kun-Ta Chou • Kang-Cheng Su • Shiang-Fen Huang • Yi-Han Hsiao • Ching-Min Tseng • Vincent Yi-Fong Su Shih-Chieh Hung • Diahn-Warng Perng
•
Received: 1 October 2013 / Accepted: 21 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Purpose Chronic obstructive pulmonary disease (COPD) with eosinophilic airway inflammation may represent a unique phenotype, possibly with shared features of COPD and asthma. The role of exhaled nitric oxide (eNO) in identifying COPD patients with sputum eosinophilia was examined in this study. Methods Ninety COPD patients without past medical history of asthma or allergic diseases were prospectively enrolled, and their eNO, lung function, and cellular profile of induced sputum were measured. Eosinophil cationic protein and IgE in sputum and venous blood also were determined. Subjects with and without sputum eosinophilia ([3 %) were compared. The role of eNO in the prediction
Kun-Ta Chou and Kang-Cheng Su are joint first authors. K.-T. Chou K.-C. Su S.-F. Huang Y.-H. Hsiao C.-M. Tseng V. Y.-F. Su D.-W. Perng (&) Department of Chest Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shi-Pai Road, Taipei 112, Taiwan e-mail: [email protected]
of sputum eosinophilia was assessed in a logistic regression model. Results Patients with sputum eosinophilia had significantly higher levels of eNO (29 vs. 18 ppb, p = 0.01) than those without. The difference in serum total IgE (168 vs. 84.9 IU/ ml, p = 0.057) and percentages of positive allergen test results (48.3 vs. 29.5 %, p = 0.082) showed a trend toward significance. The sputum eosinophil level was significantly correlated to the eNO level (r = 0.485, p \ 0.001). The eNO level at the cutoff of 23.5 ppb had the maximum sum of sensitivity (62.1 %) and specificity (70.5 %). The unadjusted and adjusted odds ratios of a higher eNO level ([23.5 ppb) in the prediction of sputum eosinophilia were 3.909 (confidence interval (CI) 1.542–9.91, p = 0.004) and 4.329 (CI 1.306–14.356, p = 0.017), respectively. Conclusions eNO is a good marker to identify COPD patients with eosinophilic airway inflammation. Keywords Chronic obstructive pulmonary disease Eosinophil Nitric oxide
Introduction K.-T. Chou Center of Sleep Medicine, Taipei Veterans General Hospital, Taipei, Taiwan K.-T. Chou S.-C. Hung Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan K.-T. Chou D.-W. Perng Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan K.-C. Su Institute of Emergency and Critical Care Medicine, National Yang-Ming University, Taipei, Taiwan
Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation associated with an enhanced chronic inflammatory response. To date, treatment is guided mostly by the severity of airflow limitation, symptom assessment, and exacerbation rate [1]. Given the complexity and heterogeneity of COPD patients, there is an urgent need to classify them into distinct phenotypes for the purpose of diagnosis, predicting treatment response, or guiding therapies. Despite this, current guidelines for COPD have not met the need for phenotype characterization very well.
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It is not easy to differentiate COPD from asthma based exclusively on clinical and physiological features [2]. The difference between COPD and asthma lies in the pathophysiology, which can reflect the characteristics of cellular inflammation in the airway [1, 3]. The presence of eosinophilic inflammation often is viewed as a distinguishing feature of asthma and is helpful in differentiating it from COPD [4]. Nonetheless, a substantial portion of asthma patients present with neutrophilic airway inflammation and COPD patients with eosinophilic inflammation [5–7]. Subjects with both asthma and COPD diagnoses were reported to have more severe respiratory discomfort, more frequent exacerbations, and an enhanced response to inhaled corticosteroids compared with those with COPD alone [8, 9]. Moreover, the recent Spanish COPD guidelines proposed mixed COPD-asthma as a distinct phenotype of COPD patients, based on the presence of asthma-like evidence, including sputum eosinophilia, an enhanced bronchodilator reversibility, elevated serum total immunoglobulin E (IgE), and a history of asthma or atopy [6, 10]. Although the cases defined by their criteria may be heterogeneous in nature, a step forward in dealing with the indistinct area amid COPD and asthma has been made. Exhaled nitric oxide (eNO) correlates with eosinophilic airway inflammation [11] and is considered a useful biomarker to guide treatment in asthma [12, 13]. For COPD, Papi et al. [14] reported that patients with partial reversibility of airflow limitation had higher levels of eNO and sputum eosinophils. A randomized, controlled trial by Siva et al. [15] showed that aggressive treatment for COPD patients designed to minimize sputum eosinophilia may decrease the frequency of hospital admissions and severe exacerbation. The current study was designed to clarify further the use of eNO to predict the presence of sputum eosinophilia in COPD patients, and this may offer an easy way to identify this subpopulation.
acute respiratory tract diseases, atopy, past medical history of asthma, or other allergic diseases, such as allergic rhinitis, allergic conjunctivitis, and allergic dermatitis, were excluded. Bronchodilator reversibility was defined as the change of FEV1 (DFEV1) or FVC (DFVC) above the prebronchodilator baseline 30 min after inhalation of 400 lg of salbutamol [16]. Severity of symptoms was evaluated with the COPD assessment test (CAT), as recommended in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2011 document [1]. Sputum Induction, Blood Sampling, and Measurement of Mediators Sputum was induced and processed as described previously [17]. The supernatant was aspirated and frozen at -80 °C before measurement. Meanwhile, we collected peripheral venous blood to measure mediators. The levels of eosinophil cationic protein (ECP, Pharmaca Diagnostics) in supernatants of sputum and serum were assayed by commercial kits per the manufacturer’s instructions [18]. Serum total immunoglobulin E (IgE) and specific IgE levels against common aeroallergens [Aspergillus fumigatus, Bermuda grass (Cynodon dactylon), cat dander, cockroach (Blattella germanica), dog dander, dust mite (Dermatophagoides pteronyssinus and Blomia tropicalis), ragweed (Ambrosia elatior), Penicillium chrysogenum, latex, cotton seed, Candida albicans] were measured (ImmunoCAP, fluoroenzyme immunoassay, Pharmaca Diagnostics) [18, 19]. Specific IgE tests were interpreted using 0.35 kU/l as a cutoff. eNO We measured eNO levels with hand-held analyzers (NIOX MINO, Aerocrine) at a constant expiratory flow of 50 ml/s. The analyzers were calibrated according to the manufacturer’s instructions. Exhaled gases were always collected prior to pulmonary function tests for fear of interference.
Materials and Methods Statistical Analyses Study Subjects The hospital’s ethics committee approved the study (VGHIRB No. 201005005IC), and signed informed consent was obtained from all subjects before the study commenced. From July 1, 2011 to June 30, 2012, subjects with a clinical diagnosis of COPD [1] were enrolled from outpatient clinics in Taipei Veterans General Hospital, a tertiary medical center and a university-affiliated teaching hospital in Taiwan, if they were older than 40 years with a smoking history C20 pack-years, had a post-bronchodilator FEV1/FVC (forced expiratory volume in 1 s/forced vital capacity) \70 %, and were treatment-naive. Subjects with
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Statistical analyses were performed utilizing SPSS software (Version 17.0; SPSS, Inc., Chicago, IL). Data were expressed as median (interquartile range) or percentage. Comparisons of continuous variables were assessed using the Mann–Whitney U test due to their nonparametric distribution. Categorical variables were compared by v2/ Fisher’s exact test. Correlation was examined with Pearson’s correlation test. To predict sputum eosinophilia ([3 %) in COPD patients [20, 21], a receiver operating characteristic (ROC) curve was performed to determine an optimal cutoff point of eNO with the maximum sum of a sensitivity and a specificity. A binary logistic regression
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model was used for multivariate adjustment. Values of two-sided p \ 0.05 were considered significant.
Table 1 Comparison of COPD patients with versus without sputum eosinophilia Sputum eosinophil count [3 %
B3 %
n
29
61
Sputum eosinophil (%)
8 (4–26)
1 (0–2)
Age (years)
79 (65.5–82.5)
77 (69.5–81)
0.635
Male (%)
93.1
93.4
1.000*
Current smoker (%)
51.7
50.8
0.936
Smoking index (pack-years)
50 (40–63)
50 (40–60)
0.876
CAT
11 (7.25–17)
10 (6–16)
0.537
Positive allergen test (%)
48.3
29.5
0.082
eNO (ppb)
29 (13–40)
18 (11–27)
0.010
FVC (L)
2.16 (1.79–2.41)
2.27 (1.93–2.87)
0.168
FVC (% predicted)
73 (63–75.5)
71 (65.5–84)
0.586
FVC reversibility (%)
12 (3.5–19)
10 (5–15.5)
0.314
FEV1 (L)
1.18 (0.85–1.42)
1.11 (0.99–1.65)
0.397
FEV1 (% predicted)
56 (43–66.5)
54 (44.5–67.5)
0.796
FEV1 reversibility (%)
11 (7–18)
10 (3–14.5)
0.223
FEV1/FVC (%)
55 (49–60.5)
54 (45.5–60.5)
0.557
Results Characteristics of Enrolled Subjects
p value
Baseline data
Ninety patients were enrolled, among which 29 (32.2 %) had sputum eosinophilia (sputum eosinophil [3 %). Patients with sputum eosinophilia had significantly higher levels of eNO (29 vs. 18 ppb, p = 0.01) compared with those without. Differences in serum total IgE (168 vs. 84.9 IU/ml, p = 0.057) and percentages of positive allergen tests (48.3 vs. 29.5 %, p = 0.082) showed a trend toward significance. There were no differences in the other clinical or demographic variables between the groups (Table 1). eNO, Sputum Eosinophils, and Serum IgE Significant correlations between levels of sputum eosinophils, eNO, and serum IgE in the COPD patients were observed (Table 2). Correlations remained evident even if the analysis was restricted to ex-smokers or current smokers, with the exception of the relationship between sputum eosinophil and serum IgE levels in ex-smokers (Table 2).
Pulmonary function
Sputum Total IgE (IU/ml)
4.31 (3.84–4.92)
4.17 (3.63–4.56)
0.374
ECP (lg/l)
126 (41.4–328.5)
141.5 (66–378.9)
0.534
Total IgE (IU/ml)
168 (46.2–701.5)
84.9 (33.5–223)
0.057
ECP (lg/l)
9.37 (4.93–19.95)
7.59 (4.67–11.78)
0.31
ROC Curve Analysis Serum
eNO at the cutoff of 23.5 ppb had the maximum sum of a sensitivity (62.1 %) and a specificity (70.5 %) in the predication of sputum eosinophilia in the subjects stratified at the eNO level of 23.5 ppb (Table 3).
Data are shown as median (interquartile range) or %
Assessment of the Role of eNO in the Prediction of Sputum Eosinophilia in a Logistic Regression Model The crude (unadjusted) odds ratio (OR) of a higher eNO level ([23.5 ppb) in the prediction of sputum eosinophilia was 3.909 (CI 1.542–9.91, p = 0.004). Serial adjustment for age, gender, smoking (smoking status and index), serum IgE, and allergen test results (Table 1) still showed the independent role of eNO in predicting sputum eosinophilia. The fully adjusted OR was 4.329 (CI 1.306–14.356, p = 0.017; Table 4).
Discussion Our results demonstrated that the eNO level is a good marker for predicting the presence of eosinophilic airway
CAT COPD assessment test; eNO exhaled nitric oxide; FEV1 forced expiratory volume in 1 s; FVC forced vital capacity; ECP eosinophil cationic protein Item with p \ 0.05 is highlighted in bold * Fisher’s exact test
inflammation in COPD patients. In addition, the role of the eNO level in COPD remained significant after adjustment for potential confounders, such as gender, atopy, or smoking status [22]. Because eNO can be measured easily and instantly, its utility in picking out COPD patients with eosinophilic airway inflammation may help to provide these patients earlier or more aggressive management. Accumulated evidence has shown the beneficial effects of steroid in COPD patients with concomitant eosinophilic airway inflammation, as evidenced by sputum eosinophilia. Brightling et al. [23] reported that there was significant
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Current smoker (n = 46)
Ex-smoker (n = 44)
r
r
p
r
p
p
Table 4 Role of eNO ([23.5 ppb) in the prediction of sputum eosinophilia in COPD patients eNO
OR
95 % CI
p value
Crude (unadjusted)
3.909
1.542–9.910
0.004
Model 1
4.727
1.715–13.025
0.003
Eos %–eNO
0.485 \0.001
0.699
\0.001
0.402
0.007
Model 2
6.234
2.010–19.339
0.002
Eos %–IgE
0.317
0.004
0.394
0.008
0.196
0.251
Model 3
4.614
1.404–15.156
0.012
IgE–eNO
0.342
0.002
0.307
0.042
0.395
0.017
Model 4
4.329
1.306–14.356
0.017
r correlation coefficient; Eos % percentage of eosinophils in sputum; eNO exhaled nitric oxide Table 3 Characteristics of COPD patients stratified by the eNO at 23.5 ppb COPD, n = 90
eNO C23.5 ppb
eNO \23.5 ppb
n
36
54
eNO (ppb)
36.5 (29–46.75)
13.5 (10–18)
p value
Age (years)
79.5 (73.3–83)
76 (65.8–81)
0.076
Male gender (%)
100
88.9
0.077*
Current smoker (%)
33.3
63
0.387
Smoking index (pack-years)
45 (30–55)
55 (45–60.3)
0.006
CAT
10 (5–20)
12 (7–16)
0.433
Positive allergen test (%)
47.2
27.8
0.059
FVC (% predicted)
79 (69.3–89.5)
81.5 (74–92)
0.301
FEV1 (% predicted)
60 (49.8–71.8)
62.5 (50.8–74.3)
0.561
FVC reversibility (%)
10 (3.3–17.8)
11.5 (5.8–17)
0.938
Pulmonary function
FEV1 reversibility (%)
9.5 (3.25–16.8)
10 (4.8–15)
0.908
FEV1/FVC (%)
54 (45. 5–62.5)
54 (46.8–59.3)
0.764
Eosinophils (%)
3.5 (0–24.5)
1 (0–3)
0.007
Total IgE (IU/ml)
4.35 (3.73–4.96)
4.13 (3.64–4.52)
0.234
ECP (lg/l)
148 (58.9–371)
138 (48.6–316)
0.462
Sputum
Serum Total IgE (IU/ml)
224.5 (53.4–658)
74.5 (33.2–156)
0.015
ECP (lg/l)
8.79 (4.97–23.8)
6.97 (4.88–11.5)
0.172
Data are shown as median (interquartile range) or % unless mentioned otherwise CAT COPD assessment test; eNO exhaled nitric oxide; FEV1 forced expiratory volume in 1 s; FVC forced vital capacity; ECP eosinophil cationic protein Items with p \ 0.05 were highlighted in bold * Fisher’s exact test
improvement in post-bronchodilator FEV1, health status (chronic respiratory disease questionnaire), and shuttle walk distance only for those COPD patients with sputum eosinophilia ([4.5 %) after prednisolone treatment. Inhaled medication containing steroid (salmeterol/fluticasone) also was reported to be able to reduce sputum
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eNO exhaled nitric oxide; OR odds ratio; CI confidence interval Model 1 adjusted for age, gender Model 2 adjusted for age, gender, current vs. ex-smoker, smoking index Model 3 adjusted for variables in Model 2 plus serum IgE Model 4 adjusted for variables in Model 3 plus allergen test results
eosinophils and increase lung function [21, 24]. In our study, the eNO level was significantly correlated to the percentage of sputum eosinophils. Taken together, eNO could be a useful biomarker to predict eosinophilic airway inflammation in COPD and could identify a subgroup of COPD patients that is likely to respond particularly well to treatment with inhaled or oral corticosteroids. Some studies have indicated COPD patients with a higher level of sputum eosinophils may have higher eNO, although a correlative relationship has not been proved [14, 25]. Due to a concern about smoking potentially interfering with eNO, the subjects in these studies were restricted to ex-smokers with COPD, which may not be representative of the whole population of COPD patients in the clinical setting. Our results revealing a significant correlation between the levels of eNO and sputum eosinophils in COPD patients, regardless of whether they were exsmokers or current smokers, may aid in broadening the usefulness of eNO in COPD. In the current study, specific serum IgEs for common antigens, rather than the skin prick test, were measured and adjusted. Although the two tests have their pros and cons in the detection of allergic diseases or atopy, the specific IgE was chosen for its convenience and objectivity. Some asthmatics may develop an irreversible airflow limitation because of long-term exposure to cigarette smoke. This could constitute a major part of the overlap between asthma and COPD. However, this may not be the case in our study, because patients with asthma or an asthma-like history were screened out. Eosinophilic airway inflammation in our enrolled COPD patients may arise due to late-onset atopy, which could be related to sensitization against environmental stimulants (allergen) or the patients’ inherited predisposition to allergy. There also is a possibility that cigarette smoking could induce or maintain eosinophilic inflammation in the airways [26–28].
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Our study has several limitations that should be mentioned. First, induced sputum cannot fully reflect the inflammatory profile in the airway as accurately as biopsy can. Despite this, induction of sputum is inexpensive, easy to perform, and less invasive in nature. Second, our enrollees were mostly male and relatively older. Hence, extrapolation of our results to younger or female populations may require further evaluation. Furthermore, the external validity of our results may be a concern since our study enrollees were all of Chinese ethnicity. In conclusion, eNO may help predict the presence of eosinophilic airway inflammation in COPD patients, thereby potentially allowing the provision of more specific and useful therapy. Acknowledgments This work was supported by research Grant CI100-39 from Yen Tjing Ling Medical Foundation. Conflict of interest conflict of interest.
All the authors declare that there is no potential
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Exhaled Nitric Oxide Predicts Eosinophilic Airway Inflammation in COPD Kun-Ta Chou • Kang-Cheng Su • Shiang-Fen Huang • Yi-Han Hsiao • Ching-Min Tseng • Vincent Yi-Fong Su Shih-Chieh Hung • Diahn-Warng Perng
•
Received: 1 October 2013 / Accepted: 21 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Purpose Chronic obstructive pulmonary disease (COPD) with eosinophilic airway inflammation may represent a unique phenotype, possibly with shared features of COPD and asthma. The role of exhaled nitric oxide (eNO) in identifying COPD patients with sputum eosinophilia was examined in this study. Methods Ninety COPD patients without past medical history of asthma or allergic diseases were prospectively enrolled, and their eNO, lung function, and cellular profile of induced sputum were measured. Eosinophil cationic protein and IgE in sputum and venous blood also were determined. Subjects with and without sputum eosinophilia ([3 %) were compared. The role of eNO in the prediction
Kun-Ta Chou and Kang-Cheng Su are joint first authors. K.-T. Chou K.-C. Su S.-F. Huang Y.-H. Hsiao C.-M. Tseng V. Y.-F. Su D.-W. Perng (&) Department of Chest Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shi-Pai Road, Taipei 112, Taiwan e-mail: [email protected]
of sputum eosinophilia was assessed in a logistic regression model. Results Patients with sputum eosinophilia had significantly higher levels of eNO (29 vs. 18 ppb, p = 0.01) than those without. The difference in serum total IgE (168 vs. 84.9 IU/ ml, p = 0.057) and percentages of positive allergen test results (48.3 vs. 29.5 %, p = 0.082) showed a trend toward significance. The sputum eosinophil level was significantly correlated to the eNO level (r = 0.485, p \ 0.001). The eNO level at the cutoff of 23.5 ppb had the maximum sum of sensitivity (62.1 %) and specificity (70.5 %). The unadjusted and adjusted odds ratios of a higher eNO level ([23.5 ppb) in the prediction of sputum eosinophilia were 3.909 (confidence interval (CI) 1.542–9.91, p = 0.004) and 4.329 (CI 1.306–14.356, p = 0.017), respectively. Conclusions eNO is a good marker to identify COPD patients with eosinophilic airway inflammation. Keywords Chronic obstructive pulmonary disease Eosinophil Nitric oxide
Introduction K.-T. Chou Center of Sleep Medicine, Taipei Veterans General Hospital, Taipei, Taiwan K.-T. Chou S.-C. Hung Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan K.-T. Chou D.-W. Perng Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan K.-C. Su Institute of Emergency and Critical Care Medicine, National Yang-Ming University, Taipei, Taiwan
Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation associated with an enhanced chronic inflammatory response. To date, treatment is guided mostly by the severity of airflow limitation, symptom assessment, and exacerbation rate [1]. Given the complexity and heterogeneity of COPD patients, there is an urgent need to classify them into distinct phenotypes for the purpose of diagnosis, predicting treatment response, or guiding therapies. Despite this, current guidelines for COPD have not met the need for phenotype characterization very well.
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It is not easy to differentiate COPD from asthma based exclusively on clinical and physiological features [2]. The difference between COPD and asthma lies in the pathophysiology, which can reflect the characteristics of cellular inflammation in the airway [1, 3]. The presence of eosinophilic inflammation often is viewed as a distinguishing feature of asthma and is helpful in differentiating it from COPD [4]. Nonetheless, a substantial portion of asthma patients present with neutrophilic airway inflammation and COPD patients with eosinophilic inflammation [5–7]. Subjects with both asthma and COPD diagnoses were reported to have more severe respiratory discomfort, more frequent exacerbations, and an enhanced response to inhaled corticosteroids compared with those with COPD alone [8, 9]. Moreover, the recent Spanish COPD guidelines proposed mixed COPD-asthma as a distinct phenotype of COPD patients, based on the presence of asthma-like evidence, including sputum eosinophilia, an enhanced bronchodilator reversibility, elevated serum total immunoglobulin E (IgE), and a history of asthma or atopy [6, 10]. Although the cases defined by their criteria may be heterogeneous in nature, a step forward in dealing with the indistinct area amid COPD and asthma has been made. Exhaled nitric oxide (eNO) correlates with eosinophilic airway inflammation [11] and is considered a useful biomarker to guide treatment in asthma [12, 13]. For COPD, Papi et al. [14] reported that patients with partial reversibility of airflow limitation had higher levels of eNO and sputum eosinophils. A randomized, controlled trial by Siva et al. [15] showed that aggressive treatment for COPD patients designed to minimize sputum eosinophilia may decrease the frequency of hospital admissions and severe exacerbation. The current study was designed to clarify further the use of eNO to predict the presence of sputum eosinophilia in COPD patients, and this may offer an easy way to identify this subpopulation.
acute respiratory tract diseases, atopy, past medical history of asthma, or other allergic diseases, such as allergic rhinitis, allergic conjunctivitis, and allergic dermatitis, were excluded. Bronchodilator reversibility was defined as the change of FEV1 (DFEV1) or FVC (DFVC) above the prebronchodilator baseline 30 min after inhalation of 400 lg of salbutamol [16]. Severity of symptoms was evaluated with the COPD assessment test (CAT), as recommended in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2011 document [1]. Sputum Induction, Blood Sampling, and Measurement of Mediators Sputum was induced and processed as described previously [17]. The supernatant was aspirated and frozen at -80 °C before measurement. Meanwhile, we collected peripheral venous blood to measure mediators. The levels of eosinophil cationic protein (ECP, Pharmaca Diagnostics) in supernatants of sputum and serum were assayed by commercial kits per the manufacturer’s instructions [18]. Serum total immunoglobulin E (IgE) and specific IgE levels against common aeroallergens [Aspergillus fumigatus, Bermuda grass (Cynodon dactylon), cat dander, cockroach (Blattella germanica), dog dander, dust mite (Dermatophagoides pteronyssinus and Blomia tropicalis), ragweed (Ambrosia elatior), Penicillium chrysogenum, latex, cotton seed, Candida albicans] were measured (ImmunoCAP, fluoroenzyme immunoassay, Pharmaca Diagnostics) [18, 19]. Specific IgE tests were interpreted using 0.35 kU/l as a cutoff. eNO We measured eNO levels with hand-held analyzers (NIOX MINO, Aerocrine) at a constant expiratory flow of 50 ml/s. The analyzers were calibrated according to the manufacturer’s instructions. Exhaled gases were always collected prior to pulmonary function tests for fear of interference.
Materials and Methods Statistical Analyses Study Subjects The hospital’s ethics committee approved the study (VGHIRB No. 201005005IC), and signed informed consent was obtained from all subjects before the study commenced. From July 1, 2011 to June 30, 2012, subjects with a clinical diagnosis of COPD [1] were enrolled from outpatient clinics in Taipei Veterans General Hospital, a tertiary medical center and a university-affiliated teaching hospital in Taiwan, if they were older than 40 years with a smoking history C20 pack-years, had a post-bronchodilator FEV1/FVC (forced expiratory volume in 1 s/forced vital capacity) \70 %, and were treatment-naive. Subjects with
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Statistical analyses were performed utilizing SPSS software (Version 17.0; SPSS, Inc., Chicago, IL). Data were expressed as median (interquartile range) or percentage. Comparisons of continuous variables were assessed using the Mann–Whitney U test due to their nonparametric distribution. Categorical variables were compared by v2/ Fisher’s exact test. Correlation was examined with Pearson’s correlation test. To predict sputum eosinophilia ([3 %) in COPD patients [20, 21], a receiver operating characteristic (ROC) curve was performed to determine an optimal cutoff point of eNO with the maximum sum of a sensitivity and a specificity. A binary logistic regression
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model was used for multivariate adjustment. Values of two-sided p \ 0.05 were considered significant.
Table 1 Comparison of COPD patients with versus without sputum eosinophilia Sputum eosinophil count [3 %
B3 %
n
29
61
Sputum eosinophil (%)
8 (4–26)
1 (0–2)
Age (years)
79 (65.5–82.5)
77 (69.5–81)
0.635
Male (%)
93.1
93.4
1.000*
Current smoker (%)
51.7
50.8
0.936
Smoking index (pack-years)
50 (40–63)
50 (40–60)
0.876
CAT
11 (7.25–17)
10 (6–16)
0.537
Positive allergen test (%)
48.3
29.5
0.082
eNO (ppb)
29 (13–40)
18 (11–27)
0.010
FVC (L)
2.16 (1.79–2.41)
2.27 (1.93–2.87)
0.168
FVC (% predicted)
73 (63–75.5)
71 (65.5–84)
0.586
FVC reversibility (%)
12 (3.5–19)
10 (5–15.5)
0.314
FEV1 (L)
1.18 (0.85–1.42)
1.11 (0.99–1.65)
0.397
FEV1 (% predicted)
56 (43–66.5)
54 (44.5–67.5)
0.796
FEV1 reversibility (%)
11 (7–18)
10 (3–14.5)
0.223
FEV1/FVC (%)
55 (49–60.5)
54 (45.5–60.5)
0.557
Results Characteristics of Enrolled Subjects
p value
Baseline data
Ninety patients were enrolled, among which 29 (32.2 %) had sputum eosinophilia (sputum eosinophil [3 %). Patients with sputum eosinophilia had significantly higher levels of eNO (29 vs. 18 ppb, p = 0.01) compared with those without. Differences in serum total IgE (168 vs. 84.9 IU/ml, p = 0.057) and percentages of positive allergen tests (48.3 vs. 29.5 %, p = 0.082) showed a trend toward significance. There were no differences in the other clinical or demographic variables between the groups (Table 1). eNO, Sputum Eosinophils, and Serum IgE Significant correlations between levels of sputum eosinophils, eNO, and serum IgE in the COPD patients were observed (Table 2). Correlations remained evident even if the analysis was restricted to ex-smokers or current smokers, with the exception of the relationship between sputum eosinophil and serum IgE levels in ex-smokers (Table 2).
Pulmonary function
Sputum Total IgE (IU/ml)
4.31 (3.84–4.92)
4.17 (3.63–4.56)
0.374
ECP (lg/l)
126 (41.4–328.5)
141.5 (66–378.9)
0.534
Total IgE (IU/ml)
168 (46.2–701.5)
84.9 (33.5–223)
0.057
ECP (lg/l)
9.37 (4.93–19.95)
7.59 (4.67–11.78)
0.31
ROC Curve Analysis Serum
eNO at the cutoff of 23.5 ppb had the maximum sum of a sensitivity (62.1 %) and a specificity (70.5 %) in the predication of sputum eosinophilia in the subjects stratified at the eNO level of 23.5 ppb (Table 3).
Data are shown as median (interquartile range) or %
Assessment of the Role of eNO in the Prediction of Sputum Eosinophilia in a Logistic Regression Model The crude (unadjusted) odds ratio (OR) of a higher eNO level ([23.5 ppb) in the prediction of sputum eosinophilia was 3.909 (CI 1.542–9.91, p = 0.004). Serial adjustment for age, gender, smoking (smoking status and index), serum IgE, and allergen test results (Table 1) still showed the independent role of eNO in predicting sputum eosinophilia. The fully adjusted OR was 4.329 (CI 1.306–14.356, p = 0.017; Table 4).
Discussion Our results demonstrated that the eNO level is a good marker for predicting the presence of eosinophilic airway
CAT COPD assessment test; eNO exhaled nitric oxide; FEV1 forced expiratory volume in 1 s; FVC forced vital capacity; ECP eosinophil cationic protein Item with p \ 0.05 is highlighted in bold * Fisher’s exact test
inflammation in COPD patients. In addition, the role of the eNO level in COPD remained significant after adjustment for potential confounders, such as gender, atopy, or smoking status [22]. Because eNO can be measured easily and instantly, its utility in picking out COPD patients with eosinophilic airway inflammation may help to provide these patients earlier or more aggressive management. Accumulated evidence has shown the beneficial effects of steroid in COPD patients with concomitant eosinophilic airway inflammation, as evidenced by sputum eosinophilia. Brightling et al. [23] reported that there was significant
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Lung Table 2 Correlations between sputum eosinophils, exhaled nitric oxide, and serum total IgE levels in COPD patients All (n = 90)
Current smoker (n = 46)
Ex-smoker (n = 44)
r
r
p
r
p
p
Table 4 Role of eNO ([23.5 ppb) in the prediction of sputum eosinophilia in COPD patients eNO
OR
95 % CI
p value
Crude (unadjusted)
3.909
1.542–9.910
0.004
Model 1
4.727
1.715–13.025
0.003
Eos %–eNO
0.485 \0.001
0.699
\0.001
0.402
0.007
Model 2
6.234
2.010–19.339
0.002
Eos %–IgE
0.317
0.004
0.394
0.008
0.196
0.251
Model 3
4.614
1.404–15.156
0.012
IgE–eNO
0.342
0.002
0.307
0.042
0.395
0.017
Model 4
4.329
1.306–14.356
0.017
r correlation coefficient; Eos % percentage of eosinophils in sputum; eNO exhaled nitric oxide Table 3 Characteristics of COPD patients stratified by the eNO at 23.5 ppb COPD, n = 90
eNO C23.5 ppb
eNO \23.5 ppb
n
36
54
eNO (ppb)
36.5 (29–46.75)
13.5 (10–18)
p value
Age (years)
79.5 (73.3–83)
76 (65.8–81)
0.076
Male gender (%)
100
88.9
0.077*
Current smoker (%)
33.3
63
0.387
Smoking index (pack-years)
45 (30–55)
55 (45–60.3)
0.006
CAT
10 (5–20)
12 (7–16)
0.433
Positive allergen test (%)
47.2
27.8
0.059
FVC (% predicted)
79 (69.3–89.5)
81.5 (74–92)
0.301
FEV1 (% predicted)
60 (49.8–71.8)
62.5 (50.8–74.3)
0.561
FVC reversibility (%)
10 (3.3–17.8)
11.5 (5.8–17)
0.938
Pulmonary function
FEV1 reversibility (%)
9.5 (3.25–16.8)
10 (4.8–15)
0.908
FEV1/FVC (%)
54 (45. 5–62.5)
54 (46.8–59.3)
0.764
Eosinophils (%)
3.5 (0–24.5)
1 (0–3)
0.007
Total IgE (IU/ml)
4.35 (3.73–4.96)
4.13 (3.64–4.52)
0.234
ECP (lg/l)
148 (58.9–371)
138 (48.6–316)
0.462
Sputum
Serum Total IgE (IU/ml)
224.5 (53.4–658)
74.5 (33.2–156)
0.015
ECP (lg/l)
8.79 (4.97–23.8)
6.97 (4.88–11.5)
0.172
Data are shown as median (interquartile range) or % unless mentioned otherwise CAT COPD assessment test; eNO exhaled nitric oxide; FEV1 forced expiratory volume in 1 s; FVC forced vital capacity; ECP eosinophil cationic protein Items with p \ 0.05 were highlighted in bold * Fisher’s exact test
improvement in post-bronchodilator FEV1, health status (chronic respiratory disease questionnaire), and shuttle walk distance only for those COPD patients with sputum eosinophilia ([4.5 %) after prednisolone treatment. Inhaled medication containing steroid (salmeterol/fluticasone) also was reported to be able to reduce sputum
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eNO exhaled nitric oxide; OR odds ratio; CI confidence interval Model 1 adjusted for age, gender Model 2 adjusted for age, gender, current vs. ex-smoker, smoking index Model 3 adjusted for variables in Model 2 plus serum IgE Model 4 adjusted for variables in Model 3 plus allergen test results
eosinophils and increase lung function [21, 24]. In our study, the eNO level was significantly correlated to the percentage of sputum eosinophils. Taken together, eNO could be a useful biomarker to predict eosinophilic airway inflammation in COPD and could identify a subgroup of COPD patients that is likely to respond particularly well to treatment with inhaled or oral corticosteroids. Some studies have indicated COPD patients with a higher level of sputum eosinophils may have higher eNO, although a correlative relationship has not been proved [14, 25]. Due to a concern about smoking potentially interfering with eNO, the subjects in these studies were restricted to ex-smokers with COPD, which may not be representative of the whole population of COPD patients in the clinical setting. Our results revealing a significant correlation between the levels of eNO and sputum eosinophils in COPD patients, regardless of whether they were exsmokers or current smokers, may aid in broadening the usefulness of eNO in COPD. In the current study, specific serum IgEs for common antigens, rather than the skin prick test, were measured and adjusted. Although the two tests have their pros and cons in the detection of allergic diseases or atopy, the specific IgE was chosen for its convenience and objectivity. Some asthmatics may develop an irreversible airflow limitation because of long-term exposure to cigarette smoke. This could constitute a major part of the overlap between asthma and COPD. However, this may not be the case in our study, because patients with asthma or an asthma-like history were screened out. Eosinophilic airway inflammation in our enrolled COPD patients may arise due to late-onset atopy, which could be related to sensitization against environmental stimulants (allergen) or the patients’ inherited predisposition to allergy. There also is a possibility that cigarette smoking could induce or maintain eosinophilic inflammation in the airways [26–28].
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Our study has several limitations that should be mentioned. First, induced sputum cannot fully reflect the inflammatory profile in the airway as accurately as biopsy can. Despite this, induction of sputum is inexpensive, easy to perform, and less invasive in nature. Second, our enrollees were mostly male and relatively older. Hence, extrapolation of our results to younger or female populations may require further evaluation. Furthermore, the external validity of our results may be a concern since our study enrollees were all of Chinese ethnicity. In conclusion, eNO may help predict the presence of eosinophilic airway inflammation in COPD patients, thereby potentially allowing the provision of more specific and useful therapy. Acknowledgments This work was supported by research Grant CI100-39 from Yen Tjing Ling Medical Foundation. Conflict of interest conflict of interest.
All the authors declare that there is no potential
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