Exhaled nitric oxide from lung periphery is increased in COPD

Subjects

_MEFeNO was measured in 81 subjects, comprising 18 nonsmokers, 16 smokers, 14 with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 0 (at risk), seven with stage 1 (mild), 11 with stage 2 (moderate), eight with stage 3 (severe) and seven with stage 4 (very severe) COPD, according to the GOLD classification (table 1⇓).

Study design

All subjects underwent _MEFeNO measurements between 09:00–10:00 h, consisting of two exhalations at each flow, which is sufficient to obtain reproducible NO results 1. In addition, the reproducibility and diurnal variation of _MEFeNO measurements were examined in 36 subjects (nine nonsmokers, 12 smokers-GOLD₀, 10 with GOLD stage 1–2 and five with GOLD stage 3–4) by measuring their _MEFeNO t.i.d. between 09:00–10:00, 12:00–13:00 and 15:00–16:00 h. Day-to-day variation, as well as the diurnal variation, of _MEFeNO was assessed in eight COPD patients (GOLD₂) when their _MEFeNO was measured between 09:00–10:00, 12:00–13:00 and 15:00–16:00 h on two consecutive days. _MEFeNO levels and forced expiratory volume in one second (FEV₁) were also measured before and 45 min after ipratropium bromide (40 µg) given via a spacer in randomly allocated patients to investigate any potential effect of air calibre changes on C_alv,NO, J_aw,NO and D_aw,NO. All patients refrained from using bronchodilators before the measurements. The effects of smoking and current treatment with ICS were also assessed by comparing values in current versus ex-smokers and patients who were treated with and without inhaled steroids. The effect of ambient NO on C_alv,NO was studied in five COPD patients (GOLD_0–1) whose _MEFeNO measurements were made after either one inhalation of NO-free air from the analyser (standard operating procedure for the single exhalation eNO and _MEFeNO measurements in the present authors' laboratory) versus five consecutive breaths of NO-free air made in two sessions separated by 3 h with recording of ambient NO.

None of the subjects studied had recent (4 weeks prior the study) upper respiratory tract infections, chest infection or COPD exacerbations. All of the subjects were advised not to consume any nitrite-enriched food (i.e. spinach) before the study visits. The study was approved by the Ethics Committee of the Royal Brompton Hospital and Harefield NHS Trust (London, UK) and all the patients gave written informed consent.

Lung function

Measurements of FEV₁ and FEV₁/forced vital capacity (FVC) were made with a dry spirometer (Vitalograph-S; Vitalograph Ltd, Buckingham, UK) which met American Thoracic Society (ATS) standards. Lung volumes and carbon monoxide gas transfer were measured with a Jaeger Master Lab Compact Transfer (Erich Jaeger Ltd, Hoechberg, UK), as described previously 20.

_MEFeNO measurements

Standardised single eNO (expiratory flow 50 mL·s⁻¹) was measured by a chemiluminescence analyser (NIOX®; Aerocrine AB, Stockholm, Sweden), as described previously 1. _MEFeNO was measured at expiratory rates 10, 100, 200 and 260 mL·s⁻¹ by applying resistors of 10, 100, 200 and 300 cm H₂O mL·s^-1 to create and maintain the target flow rates. _MEFeNO was measured using a vital capacity manoeuvre performed in duplicate 1 to collect plateau NO concentrations. Mean eNO at each expiratory flow was calculated by the analyser during an NO plateau of ≥3 s with NO variability within 10% of the plateau or ±1 ppb 1. The patients inhaled NO-free air from the analyser and then exhaled against different linear resistors. The exhalation time was 20 s for 10 mL·s⁻¹, 10 s for 50 and 100 mL·s⁻¹, and 6 s for 200 and 260 mL·s⁻¹. The second manoeuvre (to estimate the effect of ambient NO on C_alv,NO) involved five breaths of NO-free air from the analyser followed by a standard vital capacity manoeuvre. J_aw,NO, C_alv,NO, and D_aw,NO were calculated as previously described (Appendix 1) 18, 24.

Statistical methods

The correlation between C_alv,NO and FEV₁ and other parameters was determined using the Spearman rank correlation test. Intra-class correlation coefficient (ICC; table 2⇓) was used for each flow separately for all the subjects. Nonparametric tests were applied as the distribution of these variables was not known and there were insufficient data for normal distribution analysis. Data were expressed as mean±sd and/or ±sem, or mean (95% confidence intervals (CI)). ANOVA was performed using the Kruskall-Wallis test for multiple comparisons. For comparison of two groups, the Mann-Whitney U-test after Bonferroni correction was used. Reproducibility was assessed by the pooled sd and Bland-Altman test. A value of p<0.05 was considered statistically significant.

_MEFeNO: repeatability, reproducibility and diurnal variation

The mean pooled sd₁ of all measurements was 0.32±0.60 ppb (table 2⇓) and _MEFeNO measurements were highly reproducible (fig 1⇓). Pooled sd analysis of all the subjects at the flows 10, 50, 100, 200 and 260 mL·s⁻¹ was the highest at the lowest exhalation flow of 10 mL·s⁻¹ (table 2⇓). C_alv,NO and other measurements (data not shown) made at different visits and time points were highly reproducible and there was no significant day-to-day or diurnal variation (fig. 2⇓).

Effect of ambient NO and NO-free air inhalation on _MEFeNO

There was no effect of ambient NO on C_alv,NO, J_aw,NO or D_aw,NO (data not shown). There was no difference in C_alv,NO measured after one (2.0±1.13 ppb; 95% CI 0.63–3.43 ppb; ambient NO 72±27 ppm; 95% CI 38–106 ppm) versus five consecutive breaths (2.0±1.45 ppb; 95% CI 0.20–3.80 ppb; ambient NO 64±29 ppm; 95% CI 28–100 ppm) of NO-free air.

Effect of bronchodilator

There was no effect of the iptratropium bromide on the C_alv,NO, J_aw,NO, D_aw,NO and patient's lung function (fig. 2⇓).

Effect of smoking and ICS on C_alv,NO, J_aw,NO and D_aw,NO

There was no difference in either C_alv,NO or D_aw,NO between the current and ex-smokers COPD patients (3.2±0.2 versus 3.4±0.1 ppb and 18.1±1.4 versus 13.5±0.9 pL·ppb⁻¹s⁻¹, respectively; fig. 3⇓). Current COPD smokers, however, had lower J_aw,NO (497±88.1 pL·s⁻¹) than ex-smokers (711±81, pL·s⁻¹; p<0.01). Although there was a trend towards a lower J_aw,NO in COPD patients taking ICS, this was not significant. ICS did not have any effect on C_alv,NO (no ICS 3.4±0.1 ppb versus treatment with ICS 3.3±0.2; p>0.05), nor on D_aw,NO (no ICS 14±0.1 versus treatment with ICS 17±1.4 pL ppb⁻¹s⁻¹, p>0.05; fig. 3⇓).

COPD severity and C_alv,NO, J_aw,NO and D_aw,NO

There was a significant increase in C_alv,NO in COPD patients (fig. 4a⇓; table 3⇓), and significant negative correlation between C_alv,NO and FEV₁ (r = −0.6, p<0.0001), in COPD patients and normal smokers (fig. 5⇓). The levels of J_aw,NO (fig. 4b⇓; table 3⇓) were lower in smokers-GOLD₀ and COPD GOLD_3–4 than in healthy nonsmoking control subjects. The levels of D_aw,NO (fig. 4c⇓; table 3⇓) were increased in COPD patients compared with smokers and healthy controls. A weak negative correlation (r = −0.4, p = 0.006) was found between the D_aw,NO and FEV₁ across all COPD and smoking subjects. No correlation was found between J_aw,NO and FEV₁ or between C_alv,NO, J_aw,NO and D_aw,NO and alveolar volume, transfer of the lung for carbon monoxide or mean forced expiratory flow during the 25% of the FVC (data not shown).

Reproducibility and variability

Identifying the variability of this novel technique is important for the potential clinical utility of the _MEFeNO parameters to document longitudinal changes in COPD. The high level of reproducibility of _MEFeNO measurements (ICC 0.96) was similar to the high reproducibility of the ATS-standardised eNO measurements (ICC 0.99) 1. The variability of _MEFeNO was not greater in more severe COPD patients, despite the fact that some individuals found it difficult to complete the high exhalation flow manoeuvre (260 mL·s⁻¹) before the required 6 s exhalation time. The most difficult flow to maintain was 10 mL·s⁻¹ (pooled sd₁ 0.8±1.4 ppb), because of the long duration of exhalation (20 s), but this was unrelated to the degree of airway obstruction. The lack of diurnal variability of the _MEFeNO in COPD was expected considering the nature of the disease and previously published data demonstrating no diurnal variation of C_alv,NO and J_aw,NO in either asthmatics or patients with fibrosing alveolitis 13.

Effect of ambient NO

Ambient NO (0–138 ppm) did not influence C_alv,NO (the lowest amongst the other NO parameters measured by _MEFeNO), and a single inhalation versus five inhalations of NO-free air had the same effect on C_alv,NO. This gives further support for the development of portable NO analysers capable of the _MEFeNO measurements, so that C_alv,NO might be monitored by COPD patients at home in the future.

Effect of bronchodilators

Although no changes were seen in C_alv,NO and other NO parameters or FEV₁ after inhalation of ipratropium bromide that may reduce air trapping, it still too early to say to what extent C_alv,NO is related to small airway obstruction and/or to small airway inflammation. Perhaps, both of these factors contribute to elevated levels of C_alv,NO in COPD, but their contribution may only be determined once effective anti-inflammatory treatments are developed.

Effect of smoking

There was no difference in C_alv,NO between current smokers and ex-smokers, although J_aw,NO was reduced, thus, confirming previous studies 8, 13 which show a reduced eNO using the single exhalation technique. An acute and transient (1–5 min) increase in J_aw,NO has been previously reported after smoking a cigarette 13. This is most likely to be due to release of inhaled NO from haemoglobin and/or nitrosothiols. The reduction in J_aw,NO and eNO using the single exhalation technique 8 is likely to be due to down-regulation of both eNOS 11 and iNOS in lung epithelial cells of large airways 12.

In contrast, the increase in C_alv,NO in COPD patients (that was almost twice as high as in smokers) is mainly from the peripheral lung and is most likely derived from iNOS in macrophages and alveolar walls that are not directly affected by smoking 6. The present authors speculate that the elevated C_alv,NO in COPD and its relation to the disease severity may be a manifestation of peripheral lung inflammation involving alveolar walls and small airways. Smoking may trigger this inflammatory cascade, but does not have a direct effect on the source of C_alv,NO.

Effect of corticosteroids

The role of ICS in COPD is under scrutiny, as there is little evidence of their effect on various inflammatory markers in COPD. The current study has shown no effect of ICS on C_alv,NO or D_aw,NO and a rather small reduction in J_aw,NO. This is markedly different from asthma, where D_aw,NO and J_aw,NO, but not C_alv,NO are significantly reduced by ICS 15, 22, 25. A short course of oral steroids, but not the inhaled steroids, was able to reduce C_alv,NO in patients with moderate asthma 26 suggesting that inhaled steroids may not reach inflammation in peripheral airways. This indicates an important advantage of _MEFeNO measurements in COPD for monitoring the inflammatory process that is clearly different from asthma.

COPD severity

The progression of COPD from GOLD stage 0 to GOLD stage 4 is most strongly associated with thickening of the wall of small airways by a repair or remodelling process 4, and with the intensity of the inflammation response in the walls of these airways. The present authors speculate that the severity-related increase of the C_alv,NO in COPD may reflect this mechanism of disease progression. Low J_aw,NO and high C_alv,NO may have different pathophysiological effects and roles, and, therefore, may be pharmacologically corrected using different drugs, including iNOS inhibitors or NOS donors 7. The present study has shown a significant correlation between C_alv,NO and both FEV₁ and FEV₁/FVC ratio, which was not seen in either healthy subjects 27 or in mild asthmatics 15, 26, suggesting that C_alv,NO in COPD patients may reflect peripheral inflammation and remodelling resulting in increased peripheral resistance. C_alv,NO might be an early and simple marker to diagnose early stages of peripheral inflammation in COPD. Therefore, even COPD and some asthmatic patients may have a similar degree of fixed airway obstruction. The nature of the airway and lung parenchyma inflammation in these diseases is different 28 and elevated C_alv,NO may reflect the predominant small airway inflammation in COPD.

The elevated levels of C_alv,NO in the present study were similar to the levels reported by Hogman et al. 17, and may indicate accumulation of NO and NO-related species in the periphery of the lungs, as high iNOS expression has been reported in macrophages 5, alveolar walls, small airway epithelium and vascular smooth muscles of COPD patients 5, 6. Significantly higher numbers of iNOS-positive cells in alveolar walls in more severe COPD patients 29 may explain the high levels of C_alv,NO in GOLD_3–4. Interestingly, the C_alv,NO was most elevated in patients with GOLD_1–2 and GOLD_3–4, but there was no significant difference between these two groups. Although the number of patients in the current study with stage GOLD₄ is small because of the difficulty in performing the manoeuvre, this may reflect the fact that patients with severe emphysema show a lower percentage of iNOS-positive alveolar macrophages than patients with milder disease 30. In fact, some of the stage GOLD₂ patients had C_alv,NO levels similar to those of more severe patients.

Thickening and fibrosis of the airway walls and increased production of mucus might be expected to decrease D_aw,NO in COPD patients by increasing the diffusion distance for NO. However, the current authors found higher D_aw,NO in COPD patients compared with smokers-GOLD₀ and healthy nonsmokers. The inflammation may increase the surface area of airways producing NO by stimulating iNOS and increasing the D_aw,NO. Consumption of NO is also a possibility; in vivo NO reacts with several substrates like oxygen, protein thiols (glutathione) and superoxide. In COPD there are an increased number of neutrophils producing toxic radicals, including superoxide, which can react quickly with NO to form peroxynitrite, nitrite and nitrate.

Advantages and limitations

A major advantage of this noninvasive and relatively simple approach of eNO analysis is to monitor eNO, as a marker of inflammation, derived from the lung periphery and to assess the main site of inflammation in COPD. There are, however, potential sources of error in the measurements or interpretation of _MEFeNO values that need to be considered. The structural changes in COPD comprise mucoid impaction and atelectasis, often coexisting bronchiectasis, bronchial dilatation and bronchial wall thickening. Bronchoconstriction, small airways obstruction, air trapping, airway hypersecretion and accumulation of inflammatory mucous exudates in the lumen of small airway may result in an overestimation of C_alv,NO production and bronchial wall thickening and airway hypersecretion may distort D_aw,NO values. Presently, the weakness of current mathematical models, which are used to calculate the NO parameters is that these structural abnormalities are not yet integrated into the analysis. Another factor which may affect _MEFeNO in COPD patients of different severity is the surface area of the lung which participates in the exchange process. However, no significant correlation was found between NO exchange parameters and alveolar volume in COPD patients, nor in normal subjects. Finally, the current model that was used in the current authors' calculations assumes that NO is nonreactive. It may be speculated that increased production of superoxide in the airway by neutrophils lowers the NO concentration which may be left unaccounted for in a two-compartment model.

Conclusion

The present authors conclude that measurements of exhaled nitric oxide at multiple expired flows to determine alveolar nitric oxide concentration reflect inflammation in the peripheral lung of patients with chronic obstructive pulmonary disease; alveolar nitric oxide concentration is not affected by inhaled corticosteroid therapy or smoking and, thus, may provide valuable additional information for assessing the inflammatory process and its response to different therapies.