Introduction

Asthma and chronic obstructive pulmonary disease (COPD) are chronic respiratory diseases which involves small airway inflammation and dysfunction,1–3 while Asthma-COPD Overlap (ACO) is characterized by the concurrent presence of features of both asthma and COPD.4–6 FENO is a non-invasive, portable and convenient method, which has been extensively utilized in the diagnosis and therapeutic evaluation of asthma7–9 and may be a potential biomarker for assessing ICS response in COPD.10,11 Exhaled nitric oxide measured at varying expiratory flow rates is acknowledged to reflect the inflammation status within airways of different diameters.12

FENO50 is one of the most widely used biomarkers for airway inflammation, predominantly reflecting the inflammation status of large central airways but is insensitive to the changes of inflammation in peripheral airways. In recent years, CANO has garnered increased interest as a biomarker for peripheral airway inflammation, offering unique advantages over FENO50 in the assessment of pulmonary function.13,14 Nevertheless, to obtain the data needed to compute CANO values patients are required to perform multiple exhalations at different flow rates, which can be challenging for some patients. The study by Fan et al had pointed out that FENO200 was associated with small airway function in COPD and might be an alternative to CANO.15 Despite these findings, the comparative efficacy between CANO and FENO200 remains not fully elucidated, and the clinical relevance of FENO200 and CANO in ACO has been rarely reported. Hence, this study is an extension of the former studies and the primary objective of the present study is to investigate the differential clinical significance of FENO200 and CANO in the context of asthma, COPD and ACO.

Methods Patients and Study Design

This was a retrospective study conducted in Shenzhen Hospital, Southern Medical University. Data of outpatients diagnosed with asthma, COPD or ACO who presented to the respiratory department between January 2019 and January 2023 were collected. Only cases with available data for pulmonary function and exhaled nitric oxide were included. COPD was diagnosed based on the presence of non-fully reversible airflow limitation (FEV1/FVC<0.7 post-bronchodilation) according to the GOLD criteria.16 Asthma diagnoses were established following the GINA guidelines.17 Patients fulfilling the diagnostic criteria for COPD and exhibited reversible airflow limitation (post-bronchodilation increase in FEV1≥12% and 200 mL from baseline) were diagnosed with ACO.17 All participants were in a stable condition. The primary exclusion criteria encompassed the absence of pulmonary function data, coexistence of severe cardiovascular disease or other underlying pulmonary diseases such as lung cancer, bronchiectasis, tuberculosis, and interstitial lung disease. The flowchart of patient selection is depicted in Figure 1. A total of 178 patients were finally included, with 82 patients in asthma group, 47 patients in COPD group and 49 patients in ACO group. The study was in compliance with the Helsinki Declaration and approved by Medical Ethics Committee of Shenzhen Hospital of Southern Medical University (Ethical approval number: NYSZYYEC20240013). Informed consent was waived by the ethics committee since only pseudonymous data were analyzed and published.

Figure 1 Flow chart showing the enrollment of patients with asthma, COPD and ACO.

Spirometry and Bronchodilator Test

Spirometry and bronchodilator test was performed using the Jaeger Masterscope System (Jaeger, Wuerzburg, Germany). The procedures were in accordance with the recommended guidelines by ATS/ERS.18 In spirometry, subjects were instructed to perform a complete and forceful inhalation of total lung capacity (TLC) followed by a maximal exhalation of residual volume (RV). This maneuver was executed a minimum of three times to ensure reliable measurements. The best of the three spirometry values was used for analysis. For the bronchodilator test, subjects should abstain from short-acting inhaled drugs for 4 hours and from long-acting β-agonist bronchodilators or aminophylline for 12 hours prior to the test. After the baseline spirometry was performed, subjects inhaled salbutamol in four separate doses of 100 mcg. Post-bronchodilator spirometry was performed after 15 minutes and changes in forced expiratory volume in 1 second (FEV1) were measured.18,19

Measurement of Exhaled Nitric Oxide

Measurement of exhaled nitric oxide was conducted based on the Sunvou-CA2122 analyzer (Sunvou, Wuxi, China). The operation process complied with the ATS/ERS standards.20 Participants were instructed to inhale for 2–3 seconds to TLC and then exhaled immediately at a certain flow rate. The fractional concentrations of exhaled nitric oxide at flow rates of 50 mL/s and 200 mL/s were denoted as FENO50 and FENO200, respectively. CANO was calculated by a two-compartment model proposed by Tsoukias NM and George SC.12,21

Statistical Analysis

Statistical analyses were performed using IBM SPSS software, version 27.0 (SPSS, Chicago, USA). Categorical data were presented as n(%) and continuous data as mean ± standard deviation (SD) or median (interquartile range) (IQR) based on the distribution normality. Z-scores of spirometry parameters were calculated based on the GLI references.22 For categorical data, differences between groups were compared using chi-square. For continuous data, differences between groups were compared using ANOVA for normally distributed variables and Kruskal–Wallis for non-normally distributed variables. Changes of pulmonary function in the bronchodilator test were assessed by paired t-test or Wilcoxon signed-rank test. The association between clinical parameters and exhaled nitric oxide levels was evaluated by Spearman’s rank correlation analysis. The discriminant canonical analysis was performed using the Canonical Discriminant Function tool of SPSS. Statistical significance was defined as a p-value <0.05.

Results Comparison of General Information and Pulmonary Function

The comparative analysis of demographic data and pulmonary function parameters among the asthma, COPD and ACO groups is detailed in Table 1. Significant intergroup differences were noted in sex distribution, age, FVC, FEV1, FEV1/FVC, maximum mid-expiratory flow (MMEF) and maximum expiratory flow at 25% of vital capacity (MEF25). The asthma group demonstrated a significantly lower proportion of male subjects and younger age compared to the COPD and ACO groups. There are no significant differences observed in height, weight or BMI among the three groups. The ACO group exhibited significantly impaired pulmonary function, including FVC, FEV1, FEV1/FVC, MMEF and MEF25, compared to the asthma and COPD groups. A graphical representation of FENO50, FENO200, and CANO comparisons is provided in Figure 2. The values of FENO200 in asthma, COPD and ACO groups were 11.0(7.0–22.3), 8.0(6.0–11.0) and 9.0(6.5–19.5) ppb, respectively. The values of CANO in asthma, COPD and ACO groups were 2.5(0.5–6.2), 2.8(1.8–5.0) and 3.8(1.8–8.8) ppb, respectively. The asthma group showed the highest FENO50 and FENO200 values, while the ACO group had the highest CANO values. However, the differences in CANO do not reach the level of significance.

Table 1 General Information and Pulmonary Function Parameters Among Asthma, COPD and ACO Groups

Figure 2 Values of FENO50, FENO200 and CANO among asthma, COPD and ACO groups showing median and interquartile range (IQR).

Correlation Between Exhaled Nitric Oxide and Clinical Parameters

Correlation between exhaled nitric oxide and clinical parameters for each group is delineated in Tables 2–4. In the asthma group, FENO50 exhibited significant or near-significant negative correlations with pulmonary function parameters such including FEV1/FVC (ρ = −0.241, P < 0.05), MMEF (ρ = −0.239, P < 0.05), MEF50 (ρ = −0.029, P < 0.05) and MEF25 (ρ = −0.215, P = 0.054). FENO200 is significantly correlated with FEV1/FVC (ρ = −0.240, P < 0.05), MMEF (ρ = −0.219, P < 0.05) and MEF50 (ρ = −0.234, P < 0.05). No significant correlation was observed between CANO and pulmonary function parameters. Within the COPD group, FENO50 was significantly correlated only with MEF50 (ρ = −0.296, P < 0.05), while both FENO200 and CANO showed significant or near-significant negative correlation with pulmonary function parameters including FVC, FEV1, PEF, MMEF, MEF75, MEF50. In the ACO group, neither FENO50 nor FENO200 demonstrated significant correlations with pulmonary function parameters, while CANO significantly correlated with FEV1(ρ = −0.309, P < 0.05), PEF (ρ = −0.373, P < 0.05), MMEF (ρ = −0.349, P < 0.05) and MEF50 (ρ = −0.315, P < 0.05). Additionally, across all three groups, FENO200 exhibited significant positive correlations with both FENO50 and CANO (P < 0.01).

Table 2 Correlation Coefficients Between FENO50, FENO200, CANO and Clinical Parameters in Asthma Group

Table 3 Correlation Coefficients Between FENO50, FENO200, CANO and Clinical Parameters in COPD Group

Table 4 Correlation Coefficients Between FENO50, FENO200, CANO and Clinical Parameters in ACO Group

Correlation Between Bronchodilator Reversibility and Exhaled Nitric Oxide in COPD and ACO

The bronchodilator reversibility of FEV1 and FVC for COPD and ACO patients is presented in Table 5. Increases in FEV1 were 4.82 ± 4.57% for the COPD group and 17.86(12.41–25.21)% for the ACO group. Corresponding increases in FVC were 2.58 ± 5.29% and 13.96(8.77–21.06)%, respectively. Significant changes were observed between pre- and post-bronchodilator FEV1 and FVC in both COPD and ACO groups. In addition, Spearman correlation analysis was conducted to explore the potential associations between bronchodilator reversibility and exhaled nitric oxide including FENO50, FENO200 and CANO. In COPD group, changes in FEV1(ΔFEV1) was found to be significantly correlated with FENO50(ρ = −0.320, P < 0.05) and FENO200(ρ = −0.302, P < 0.05). In the ACO group, ΔFEV1 was significantly correlated only with CANO (ρ = 0.286, P < 0.05). The results of the correlation analysis is presented in Figure 3.

Table 5 Changes of FEV1 and FVC in the Bronchodilation Test of COPD and ACO Patients

Figure 3 Correlation between ΔFEV1 and exhaled nitric oxide in COPD and ACO. (A) Correlation between ΔFEV1 (L) and FENO200 in COPD; (B) Correlation between ΔFEV1 (%pred) and CANO in ACO.

Discriminant Canonical Analysis

Clinical parameters including gender, age, height, weight, FVC, FEV1, PEF, MMEF, MEF75, MEF50, MEF25, FENO50, FENO200 and CANO were included into the linear discriminant analysis (LDA). The LDA based on all 14 parameters from which by stepwise exclusion 4 remained in the model is graphically represented in Figure 4. The overall prediction accuracy was 70.2%. Based on Wilks’ lambda (Λ) test statistics four parameters including gender (Λ = 0.457), age (Λ = 0.567), MEF75 (Λ = 0.456) and FENO50 (Λ = 0.466) discriminated between the three groups of asthma, COPD and ACO.

Figure 4 Linear discriminant analysis based on 14 parameters from which by stepwise exclusion 4 remained in the model (gender, age, MEF75, FENO50), differentiating between asthma, COPD and ACO.

Discussion

To the best of our knowledge, this is the first report concerning the differences between FENO200 and CANO. Both FENO200 and CANO have the potential to serve as biomarkers for peripheral airway inflammation.23,24 In our study, FENO200 was observed to be positively correlated with CANO across all the groups. However, it was noteworthy that the correlation analyses yielded results that were not entirely congruent. FENO200 demonstrated a predominant association with pulmonary function in asthma and COPD, whereas CANO showed a stronger association with pulmonary function in COPD and ACO. Our findings suggest that FENO200 and CANO may be differentially selected for the assessment of distinct pulmonary diseases.

FENO is recognized as a biomarker for type-2 airway inflammation, which is mainly presented in asthma and a subset of COPD patients, playing an important role in both stable condition and exacerbation.25–27 Previous researches have suggested that patients with asthma or ACO exhibited significantly higher FENO levels than COPD,28,29 which is consistent with our study. FENO50 and FENO200 were found to be highest in the asthma group, followed by the ACO group, and lowest in the COPD group (Table 1). Notably, the highest level of CANO was observed in the ACO group. Moreover, the ACO group also presented with significantly impaired pulmonary function compared to the other two groups. A possible explanation is that ACO exhibits the characteristics of both asthma and COPD, the chronic inflammatory response and oxidative stress in the distal airways may be more pronounced,30,31 which warrant further investigation.30

According to the European Respiratory Society (ERS) recommendations, unlike FENO50, there are no universally accepted reference values for CANO and FENO200.12 Similar to our findings, Sy Duong-Quy et al reported the highest CANO levels and the poorest pulmonary function in patients with ACO instead of asthma and COPD,28 but the values of CANO in all three groups (4±2ppb, 3±2ppb and 6±3ppb for asthma, COPD and ACO group, respectively) were slightly higher than in our study (2.5(0.5–6.2)ppb, 2.8(1.8–5.0)ppb and 3.8(1.8–8.8)ppb for asthma, COPD and ACO group, respectively). The discrepancies observed in CANO values may be attributed to several factors, including disease severity, model of nitric oxide analyzer, expiratory flow rates and computational methods for CANO measurement.32 Furthermore, the literature on reference values for FENO200 is scarce. Fan et al previously reported a FENO200 value of 11.0(9.0–15.0)ppb in stable COPD patients,15 while a study on cough variant asthma reported a value of 17.0(10.0–24.0)ppb.33 There is no study regarding FENO200 values in ACO patients. Our study provides novel reference values for FENO200 and CANO in asthma, COPD, and especially ACO.

The American Thoracic Society (ATS) has endorsed the use of FENO50 in the diagnosis and management of asthma.26 Elevated FENO50 levels indicate a poor prognosis for asthma and are correlated with dysfunction of small airways,34 aligning with our study’s findings (Table 2). Besides, a similar correlation was observed in FENO200. Our study revealed that FENO200 was inversely associated with pulmonary function parameters including FEV1/FVC, MMEF and MEF50. Conversely, no significant correlation was observed between CANO and pulmonary function parameters. Whether CANO is associated with impairment of pulmonary function in asthma remains a contentious issue.35 While a study on refractory asthma in pediatric populations reported a negative association between CANO and MMEF, in another study on adult asthma, no significant correlation was observed between CANO and either MMEF or MEF50.36,37 The observed differences were likely attributable to the patient characteristics, disease severity and the treatment modalities utilized. Additionally, a recent study by Bai et al had shown that FENO200 exhibited a more robust correlation with small airway function and was potentially more useful than CANO in diagnosing cough variant asthma.33 Our results suggest that FENO200 may offer advantages over CANO for the assessment of pulmonary function in asthma.

It is believed that airway inflammation in COPD is primarily driven by type-1 immune response.25 However, type-2 inflammatory immunity is also present in 15–37% of COPD patients, indicating its role in COPD pathogenesis.38,39 Compared with asthma, influenced by factors including smoking habits, phenotypic heterogeneity and disease severity, the role of FENO in COPD has not been well defined.40 Previous study suggested that elevated FENO levels is associated with increased symptoms, impaired pulmonary function and frequent exacerbations in COPD.34 In our study, both FENO200 and CANO demonstrated significant inverse correlation with pulmonary function parameters, which indicates that FENO200 and CANO are efficient in reflecting the inflammatory levels and pulmonary function in COPD.

ACO is characterized by an incompletely reversible airway obstruction accompanied by symptoms or signs of increased reversibility of obstruction.32 Former studies have shown that ACO patients are more symptomatic and exhibit higher frequency of exacerbations and a worse quality of life.41,42 Without appropriate treatment, patients with ACO may have worse prognosis than those with asthma of COPD alone. A study by Deng et al on ACO had found that there was no significant correlation between FENO50 and pulmonary function parameters including FEV1 and FEV1/FVC, which was in agreement with our study.43 As mentioned in the introduction, there have been very few studies on the clinical significance of FENO200 and CANO in patients with ACO. In the current study, we observed that pulmonary function parameters such as FEV1, PEF, MMEF and MEF50 were only correlated with CANO but not FENO200. Since this is the first report comparing FENO200 and CANO, further in-depth studies are required to understand the mechanisms behind these discrepancies.

In COPD and ACO groups, we further investigated the correlation between bronchodilator reversibility and exhaled nitric oxide. It is well established in the literature that exhaled nitric oxide is associated with eosinophilic airway inflammation and the response to glucocorticoids in asthma and ACO.44,45 Our study showed that ACO patients with elevated CANO levels exhibited better bronchodilator reversibility. In contrast, we found that elevated FENO200 levels was associated with worse bronchodilator reversibility in COPD. It had been previously reported in a few studies that a minor degree of airway obstruction in COPD could result in a reduced ΔFEV1% during bronchodilator test and an increase in FENO values.23,46–48 Given that the majority of COPD patients enrolled in our study were mainly classified within GOLD I–II stages, this might provide a plausible explanation for the results.

Notably, the canonical discriminant analysis revealed that age exerted the most discriminative power, succeeded by FENO50, gender and MEF75. FENO200 and CANO were excluded from the final model. This implies that, although FENO200 and CANO demonstrate significant correlation with spirometry parameters as well as bronchodilator reversibility, FENO50 may have a better discriminatory ability in distinguishing asthma, COPD and ACO.

Some limitations need to be noted regarding the present study. First, as this was a retrospective study, the differences in the inherent disease characteristics might have influenced the results. Second, since previous study has indicated that plethysmography measurements such as sRawtot and sRaweff provide additional value in the evaluation of peripheral airway dysfunction,49 the plethysmography measurements should be incorporated in the future studies. Finally, the underlying mechanisms that contribute to the observed correlation between FENO200 and small airways necessitate further exploration and elucidation.

Conclusion

In asthma, COPD and ACO, FENO200 has demonstrated a robust correlation with CANO. Elevated FENO200 levels are predominantly indicative of pulmonary function impairment in asthma and COPD, whereas elevated CANO levels are mainly correlated with pulmonary function impairment in COPD and ACO. Compared with FENO200 and CANO, FENO50 may have a better discriminatory ability in distinguishing asthma, COPD and ACO.

Ethical Approval

This retrospective cohort study was approved by the Medical Ethics Committee of Shenzhen Hospital of Southern Medical University (Ethical approval number: NYSZYYEC20240013).

Acknowledgments

The authors thank all the staff of the pulmonary function department at Shenzhen Hospital, Southern Medical University for their valuable contribution to patient care.

Funding

This work was supported by Medical and Health Research Project of Bao’an District (2024JD051).

Disclosure

The authors report no conflicts of interest in this work.

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