Introduction
Chronic obstructive pulmonary disease (COPD) is a complex polyclinic lung disease characterized by an abnormal inflammatory and a progressive condition with declining lung function.1 Although airway inflammation in COPD is generally considered to be caused by type 1 immune response, type 2 airway inflammation can also occur in some COPD patients during stable or exacerbation state.2 Some studies have found that some COPD patients have gene expression of type 2 inflammation in the airway, and these patients have a good response to corticosteroid.3 In recent years, there have been a large number of studies on the role of type 2 biomarkers in COPD, including eosinophils, IgE and FeNO50.4 Nitric oxide (NO) is biosynthesized from L-arginine and oxygen by the enzyme NO synthase (NOS) endogenously, fractional concentration of exhaled nitric oxide at a flow rate of 50mL/s (FeNO50) is a known marker of airway inflammation. As a noninvasive, convenient and highly reproducible method for assessing airway inflammation, FeNO measurement has been used to evaluate type 2 inflammation of asthma and guide anti-inflammatory treatment.5,6 However, the role of FeNO50 in COPD remains controversial.7–10
According to the latest technical standard published by the European Respiratory Society (ERS) in 2017, FeNO50 (usually abbreviated as FeNO, representing the exhaled NO value at the flow rate of 50mL/s) mainly reflects large airway inflammation from bronchi to respiratory bronchioles, but cannot reflect small airway inflammation.11 COPD is a respiratory disease characterized by chronic inflammation of the small airway. As a marker of peripheral/small airway inflammation, more and more studies began to pay attention to the clinical value of CaNO (concentration of alveolar NO) in COPD patients. Several studies reported that CaNO was increased in COPD patients and was correlated with a single nitrogen washout curve (dN2) and diffusion capacity for carbon monoxide (DLCO).12–17 Nonetheless, some researchers found that there was no difference in CaNO between COPD patients and healthy population.18 In addition, whether CaNO could guide the treatment of corticosteroid in COPD patients needs further study.
The estimation of CaNO requires three exhalations at three different flow rates that follow the conventional linear regression model. The procedure is complex, especially for AECOPD patients, it is challenging to complete.16 Therefore, it is necessary to have a simpler and more convenient index than CaNO, but it can also reflect peripheral/small airway inflammation. In 2014, Peter J Barnes found that FeNO200 (fractional concentration of exhaled nitric oxide at a flow rate of 200 mL/s) is proportional to CaNO. By measuring the exhaled NO at the flow rates of 50 mL/s and 200 mL/s, the inflammation of central and peripheral airways can be distinguished.19 Therefore, FeNO200 (fractional concentration of exhaled NO at a flow rate of 200 mL/s) was used as it maximizes the alveolar fraction of eNO in hereditary hemorrhagic telangiectasia, hepatopulmonary syndrome, and liver cirrhosis.20–22 However, the role of FeNO200 in patients with COPD has not been studied.
Our prospective study measured pulmonary function parameters and exhaled nitric oxide (FeNO50, FeNO200, and CaNO) in COPD patients with different stages and controls. For AECOPD patients, we compared these parameters before and after corticosteroid treatment. The purpose of this study was to evaluate a change in peripheral small airway/alveolar sites of NO detected by FeNO200 and CaNO in COPD. Besides, whether FeNO200 or CaNO could be used as an evaluation tool for corticosteroids therapy in AECOPD patients was further explored.
Materials and Methods
Study Subjects
Stable COPD patients and AECOPD patients were diagnosed based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2017 guidelines. Healthy subjects (HS) with normal lung function as controls were included in the study. All participants were restricted to adults 60 years and older. The physician prescribed the therapy of AECOPD. Patients enrolled at the Department of Respiratory Medicine at the Wuxi People’s Hospital from November 2017 to December 2019.
For all participants with a history of asthma or other respiratory diseases were excluded from the study. The main exclusion criteria were treated with systemic corticosteroids or antibiotics 4 weeks before admission. The hospital Ethics Committee approved the study. We fully abide by the guidelines in the Helsinki declaration and written informed consent was obtained from all participants enrolled in the study.
Study Design
Lung function, exhaled nitric oxide, the white blood cell count (WBC), and blood C-reactive protein (CRP) concentration were measured among the controls and COPD patients. For AECOPD patients, methylprednisolone 40mg/day, intravenous administration for 1 week was given. Moreover, before discharged from the hospital, lung function and exhaled nitric oxide would be measured again in AECOPD patients.
Exhaled NO Measurement
Exhaled NO was measured using the Nano Coulomb Breath Analyzer (Sunvou-CA2122, Wuxi, China), in line with the American Thoracic Society/European Respiratory Society (ATS/ERS) recommendations.23 One hour before the test, eating, smoking, drinking, strenuous exercise or pulmonary function test were prohibited.
Ten healthy controls, ten stable COPD patients, and ten AECOPD patients participated in the preliminary clinical trial. They were required to exhale via a mouthpiece at multiple flow rates: 100, 200, 350mL/s. At each flow rate, the mean value was calculated. FeNO50 was measured at the single flow rate of 50mL/s. CaNO was estimated with a mathematical approach based on a two-compartment linear model published by Tsoukias et al.24 However, the success rate of measurement at a flow rate of 350 mL/s was only 60% for AECOPD patients (Table 1).
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Table 1 The Success Rate of Exhaled Nitric Oxide Measurement at Different Expiratory Flow Rates
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A simplified estimation method of alveolar nitric oxide was developed to improve the success rate of measurement. Subjects were informed about inhaling NO-free air and exhaling via a mouthpiece at two constant flow rates: 50, 200mL/s. FeNO50 and FeNO200 were recorded. CaNO was calculated based on the linear model published by ERS:
FeNO = CaNO + JawNO/VE
FeNO (fractional concentration of exhaled NO) is recorded in ppb (1ppb = 1×10-9mol/L). The exhalation flow rate is given as a subscript in mL/s. A flow rate of 50 mL/s is written FeNO50 and a low rate of 200 mL/s is written FeNO200. FeNO is a flow rate dependent index. The smaller the flow rate is, the higher the value is, the better it can reflect NO in a large airway; the higher the flow rate, the smaller the value, the better it can reflect NO in a small airway. JawNO, the total flux of NO in the conducting airway compartment (nl/s), is not affected by the flow rate and only reflects the inflammation of the central/large airway. CaNO, the concentration of alveolar NO, is not affected by the flow rate and only reflects the inflammation of peripheral/small airway.
Statistical Analysis
SPSS software vsrsion22.0 was used to process data. All continuous variables were checked for normal distribution by Kolmogorov–Smirnov normality test. Normally distributed variables are expressed as mean ± standard deviation, and skewed variables are expressed as the median (interquartile range, IQR). The significance of the difference between the two groups was assessed with a two-tailed Manne–Whitney test (t-test). The differences between multiple groups were evaluated with the nonparametric Kruskal–Wallis test (ANOVA) with Dunn post-test. The chi-square test was used to compare categorical variables between the two groups. Statistical significance was considered to exist when P<0.05.
Results
Subject Characteristics
A total of 98 COPD patients (59 stable COPD patients, 39 AECOPD patients) and 73 healthy controls were enrolled in this study. The characteristics of the study patients are shown in Table 2. There were no statistical differences in terms of age, blood eosinophil percentage, and eosinophil counts. Elevated CRP concentration was seen in COPD patients. Pulmonary function parameters were significantly higher in healthy controls than in COPD patients. In the stable COPD and AECOPD patients, lung function parameters and maintenance therapy were similar. There was no significant difference in the distribution of disease severity between the COPD and AECOPD patients.
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Table 2 The Clinical Characteristics of the Patients and Control
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Increased Exhaled Nitric Oxide in Different Stages of COPD
FeNO200 elevated in patients with stable COPD [median: 11.0 ppb, IQR: (9.0, 15.0) ppb; P<0.05] and AECOPD patients [meidan: 11.0 ppb, IQR: (10.0, 14.0) ppb; P<0.05] compared with healthy controls [median: 9.0 ppb, IQR: (6.0, 11.0) ppb]. FeNO50 increased in patients with stable COPD [median: 22.0 ppb, IQR: (17.0, 30.0) ppb; P<0.05] and exacerbated COPD patients [median: 21.0 ppb, IQR: (18.0, 28.0) ppb; P<0.05] compared with healthy controls [median: 20.0 ppb, IQR: (15.0, 25.0) ppb]. However, there was no significant difference in FeNO50 and FeNO200 between stable and exacerbated COPD patients. CaNO demonstrated no significant difference among control subjects [median: 4.4 ppb, IQR: (1.5, 6.7) ppb], stable [median: 5.9 ppb, IQR: (3.5, 8.2) ppb] and exacerbated COPD patients [median: 5.4 ppb, IQR: (2.6, 8.5) ppb] (Figure 1).
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Figure 1 Exhaled nitric oxide in healthy controls, stable and exacerbated COPD patients. FeNO50 increased in stable and exacerbated COPD patients (A). FeNO200 increased in stable and exacerbated COPD patients (B). There was no significant difference of CaNO in healthy controls, stable and exacerbated COPD patients (C).
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There was a positive correlation between FeNO50 and CRP (r=0.384, p<0.05) in stable COPD patients. FeNO200 was associated with CRP (r=0.335, p<0.05) as well. CaNO did not correlate with CRP (p>0.05). Forty-seven percent stable COPD patients and 64% AECOPD patients with blood eosinophil counts ≥100cells/ul. No correlation was found between exhaled nitric oxide (FeNO50, FeNO200, CaNO) and eosinophils (P>0.05). In AECOPD patients, there was no correlation between exhaled nitric oxide and eosinophils and CRP (p>0.05).
Effect of Corticosteroids on Exhaled Nitric Oxide in AECOPD Patients
Exhaled NO was repeatedly measured in 39 patients with AECOPD before discharge from the hospital following adequate corticosteroid treatment. During hospitalization, all patients were treated continuously with systemic corticosteroids. There were significant improvements in FeNO50, lung function (FEV 1%, PEF, FEF25, FEF50, FEF75) (Table 3).
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Table 3 Changes in Exhaled NO and Pulmonary Function in AECOPD Patients After Treatment
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FeNO200 >10ppb Predicted a Good Corticosteroid Response in AECOPD
We grouped all patients according to the cut-off point of FeNO50, FeNO200 and CaNO. According to the clinical guideline published by ATS in 2011, FeNO50 values greater than 25ppb indicate eosinophilic inflammation and the likelihood of corticosteroid responsiveness. No guidelines have published cut-off points for FeNO200 and CaNO. Based on the literature, the normal values of FeNO200 and CaNO in healthy people do not exceed 7.4 ppb and 4.7 ppb, so we use 10ppb and 5ppb as the cut-off point.20,25,26
There were 11 patients with initial FeNO50> 25 ppb, FeNO50 decreased significantly from 51.7±22.7 ppb to 30.3±9.0 ppb (p<0.05) after a week of corticosteroid treatment (Figure 2A). For 28 patients with initial FeNO50 ≤ 25 ppb, FeNO50 did not change significantly (19.0±3.2 vs 20.0±9.3 ppb; p=0.764) (Figure 2B). There were 23 patients with initial FeNO200> 10 ppb, FeNO200 decreased from 16.1±7.8 to 12.2±5.2 after treatment (p<0.05) (Figure 2C). And for 16 patients with initial FeNO200 ≤ 10 ppb, there was no significant change after treatment (8.7±1.4 vs 10.3±4.3ppb; p=0.112) (Figure 2D). There were 18 patients with initial CaNO >5 ppb and 21 patients ≤5 ppb, and no significant change was found in neither group (10.3±5.9 vs 7.4 ±5.0 ppb, p=0.181; 2.6±1.5 vs 4.5±5.7 ppb, p=0.148; Figure 2E and F).
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Figure 2 Changes in FeNO50, FeNO200 and CaNO in different groups. FeNO50 in patients with initial FeNO50> 25 ppb decreased after treatment (A), FeNO50 in patients with initial FeNO50 ≤ 25 ppb did not change after treatment (B), FeNO200 in patients with initial FeNO200> 10 ppb decreased after treatment (C), FeNO200 in patients with initial FeNO200 ≤ 10 ppb did not change after treatment (D), CaNO in patients with initial CaNO > 5 ppb did not change after treatment (E), CaNO in patients with initial CaNO ≤ 5 ppb did not change after treatment (F).
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The patients in the high FeNO50 group (FeNO50> 25 ppb) had greater improvement in FeNO50 and CAT than the low FeNO50 group (FeNO50≤ 25 ppb) (Table 4). Similar results were found between the high FeNO200 group (FeNO200 > 10 ppb) and low FeNO200 group (FeNO200 ≤ 10 ppb) (Table 5). No improvement was observed in both high and low CaNO groups (Table 6).
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Table 4 The Difference from Baseline in FeNO50, CAT and Pulmonary Function Test
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Table 5 The Difference from Baseline in FeNO200, CAT and Pulmonary Function Test
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Table 6 The Difference from Baseline in CaNO, CAT and Pulmonary Function Test
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No Relationship Between COPD Severity and Exhaled Nitric Oxide
According to the GOLD criteria, four patients were categorized as GOLD stage1 (FEV1% pred≥80%), 32 as GOLD stage 2 (80% > FEV1% pred ≥50%), 41 as GOLD stage 3 (50% > FEV1% pred ≥30%) and 21 as stage 4 (FEV1% pred <30%).
The increase of CaNO from GOLD1-2 to GOLD3, GOLD 3 to GOLD 4 was insignificant, but there was a significant increase from GOLD 1–2 to GOLD 4 (Figure 3C). And no difference was found between different GOLD stages in FeNO50 and FeNO200 (Figure 3A and B).
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Figure 3 FeNO50, FeNO200 and CaNO in healthy controls and COPD patients of different severity according to the classification of the Global Initiative for Chronic Obstructive Lung Disease (GOLD). FeNO50 was not correlated with different GOLD stages (A). FeNO200 was not correlated with GOLD stages (B). CaNO was elevated at stage of GOLD4 (C).
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No correlations were found between FeNO50, FeNO200, CaNO and FEV1 (p>0.05).
Discussion
Invasive sampling, such as lung biopsy or bronchoalveolar lavage (BAL), poses a risk to COPD patients, making it difficult to assess small airway and alveoli inflammation directly. Exhaled NO has been used as a noninvasive biomarker of airway inflammation since it was discovered. According to ERS guideline, FENO50 = CANO+JawNO/50 at the flow rate of 50mL/s and FENO200 = CANO+JawNO/200 at the flow rate of 200mL/s. JawNO is the NO flux in the large airway, which reflects the NO production and inflammation in the large airway, and is not affected by the flow rate; similarly, CANO is the alveolar NO concentration, which completely reflects the small airway inflammation and is not affected by the flow rate. Therefore, FENO50 reflects more JawNO, that is, NO concentration in large airway, while FeNO200 reflects more CANO, that is, NO concentration in small airway. We observed that FeNO200 elevated in both stable and acute exacerbated COPD patients. Further subgroup analysis implied that FeNO200 in AECOPD patients with greater than 10ppb decreased after systemic corticosteroid therapy. However, no similar changes in CaNO were found. As we know, this is the first report of assessing the role of FeNO200 in COPD and may have important clinical significance.
We discovered that FeNO200 in COPD and AECOPD patients were higher than those in the healthy control group, reflecting the peripheral airway inflammation in COPD patients. Also, our research showed that FeNO200 was correlated with CRP in stable COPD patients, which was consistent with some findings in CaNO.15,16 Nevertheless, the same changes of CaNO were not found in this study. Our results are in line with a few studies, suggesting that CaNO did not differentiate healthy controls from COPD patients.18,27 However, several studies have found that the CaNO of COPD and AECOPD patients were higher than those of healthy controls.15,16 The inconsistency may be due to the use of different types of NO analyzers in these studies. CaNO varies with different flow rates, velocities, and various calculation models. Low, medium and high exhalation flow rates are needed for CaNO calculation, but this method is challenging to apply in COPD patients.11 Our pre-test showed that nitric oxide measurement only had a 70% success rate at 300 mL/s expiratory flow rate. Thus, FeNO200 can reflect peripheral airway inflammation more directly and accurately. Compared with the complex operation and calculation of CaNO, FeNO200 is an effective and simple method to evaluate small airway inflammation, especially in AECOPD patients. A larger sample size is needed for further research.
The peripheral airways NO measured by FeNO200 and the simplified CaNO were not elevated as we expected in AECOPD compared with the stable condition. Zsófia Lázár also reported the same results in CaNO.16 The airway production of NO would change along with the expression of endothelial, neuronal, and inducible isoforms of NO synthase (eNOS, nNOS, and iNOS) in the peripheral lung tissue of COPD patients. iNOS is believed to play a critical role in the inflammatory response. iNOS is increased by inflammatory mediators and can generate tremendous amounts of NO.28 On the one hand, the activity of iNOS in AECOPD patients is enhanced by airway inflammation.29 On the other hand, hypoxia can induce damage to pulmonary capillaries endothelial cells and decrease eNOS activity.30 The NO concentration produced by eNOS decreased when it diffused from alveolar capillaries to alveoli. Therefore, influenced by multiple factors, the level of NO did not change significantly in AECOPD patients. Further research is needed to understand the mechanism better.
Our findings showed that FeNO50 of AECOPD and stable COPD patients were higher than that of healthy controls, which agreed with previous studies. Zhiyu Lu reported that only a mild elevation of FeNO50 levels patients with stable COPD.31 However, no significant difference was found in FeNO50 between stable and exacerbated COPD patients. The reason may be consistent with FeNO200 as described above, and large airway NO could be suppressed by ICS.31 Besides, the bacterial infection is an important cause of exacerbation of COPD. There was evidence showed that bacterial infection decreased FeNO50 levels.32 NO levels at lower expiratory flow rates mainly indicate the bronchial inflammation; therefore, FeNO50 cannot exactly reflect peripheral airway inflammation in COPD patients.
For AECOPD patients, there was no significant difference between FeNO200 and CaNO after systemic corticosteroid treatment. This result may be attributed to the low initial exhaled NO values in some patients, and these patients had an inadequate response to corticosteroid treatment. By classifying patients based on the cut-off point of FeNO50> 25, We identified that 31% of patients with higher FeNO50 levels had a significant FeNO50 decrease and improved CAT after corticosteroid treatment, suggesting an excellent response to corticosteroid. This finding agreed with ATS’s guideline in 2011 that FeNO50 greater than 25ppb indicates eosinophilic inflammation and high FeNO50 in COPD patients predict a good corticosteroid response.18,33
Although there are no guidelines for FeNO200 and CaNO, based on existing clinical studies, the FeNO200 and CaNO values of healthy people are less than 10 ppb and 5 ppb, respectively.18,20,35–40 Our research showed a significant decrease in CAT and FeNO200 in patients with FeNO200 greater than 10 ppb after corticosteroid therapy. Patients with FeNO200 below 10 ppb did not change after treatment, whereas CaNO did not change after 1 week’s corticosteroid treatment neither in the high CaNO value group nor in the low CaNO value group, which is similar to the previous study.16 The results implied that AECOPD patients with FeNO200 > 10 ppb would benefit from corticosteroid.
GOLD recommended that COPD patients with circulating eosinophils >100 cells/µL can benefit from corticosteroid application. For patients with eosinophils <100 cells/µL, corticosteroid treatment is restrained due to poor response and the increased risk of pneumonia.41 In our study, 64% of patients had eosinophil counts greater than 100 cells/µL, similar to previous results.42 There were 59% AECOPD patients with FeNO200 >10 ppb. However, only 31% of patients with AECOPD had FeNO50 greater than 25 ppb. Therefore, FeNO50 cannot truly reflect COPD’s inflammatory state, and only measuring FeNO50 may miss some patients who would benefit from corticosteroid application. Compared with FeNO50, FeNO200 may be a better indicator of corticosteroid therapy in COPD patients.
Conclusion
This study proved that FeNO200 was a simpler and more patient-friendly method to directly measure the NO levels of peripheral airway/alveoli in COPD patients. In AECOPD patients with FeNO200 > 10 ppb presented a better response to corticosteroid treatment. Moreover, it is consistent with the guidance of peripheral blood eosinophils counts. Therefore, FeNO200 can be a type 2 inflammation biomarker and a useful tool for corticosteroid treatment in COPD.
Ethics Approval and Consent to Participate
The study was approved by the ethical review board of Wuxi People’s Hospital Affiliated to Nanjing Medical University. The ethics approval number was KS000024.
Acknowledgments
The study was supported by the project of the project of Jiangsu commission of Health [BJ15009 and BJ17009] and the Natural Science Foundation of Wuxi [JZYX01].
Disclosure
The authors declare that they have no conflicts of interest in this work.
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