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Differential Flow Analysis of Exhaled Nitric Oxide in Patients With Asthma of Differing Severity^*

^* From the Section of Airway Diseases, National Heart and Lung Institute, Imperial College, London, UK.

Correspondence to: Sergei A. Kharitonov, MD, PhD, Section of Airway Disease, National Heart and Lung Institute, Imperial College, Dovehouse St, London, SW3 6LY, UK; e-mail: s.kharitonov{at}imperial.ac.uk

Abstract

Background: The majority of asthmatic patients achieve control of their illness; others do not. It is therefore crucial to validate/develop strategies that help the clinician monitor the disease, improving the response to treatment.

Methods: We have quantified the inflammation in central and peripheral airways by measuring exhaled nitric oxide (NO) at multiple exhalation flows in 56 asthmatics at different levels of severity (mild, n = 10; moderate stable, n = 17; moderate during exacerbation, n = 11; severe, n = 18, 7 of whom were receiving oral corticosteroids) and 18 healthy control subjects. The reproducibility of the measurement was also assessed.

Results: Bronchial NO (JNO) in patients with mild asthma (2,363 ± 330 pL/s) [mean ± SD] was higher than in patients with moderate stable asthma (1,300 ± 59 pL/s, p < 0.0005), in patients with severe asthma receiving inhaled corticosteroids (ICS) [1,015 ± 67 pL/s, p < 0.0005], and healthy control subjects (721 ± 22 pL/s, p < 0.0001). There were no differences between JNO in patients with mild asthma compared to patients with severe asthma receiving ICS and oral corticosteroids (2,225 ± 246 pL/s). Patients with exacerbations showed a higher JNO (3,475 ± 368.9 pL/s, p < 0.05) compared to the other groups. Alveolar NO was higher in patients with severe asthma receiving oral corticosteroids (3.0 ± 0.1 parts per billion [ppb], p < 0.0001) than in the other groups but was not significantly higher than in patients with moderate asthma during exacerbation (2.8 ± 0.3 ppb). No differences were seen in NO diffusion levels between the different asthma groups. All the measurements were highly reproducible and free of day-to-day and diurnal variations.

Conclusions: Differential flow analysis of exhaled NO provides additional information about the site of inflammation in asthma and may be useful in assessing the response of peripheral inflammation to therapy.

Key Words: asthma • different flow rates • exhaled nitric oxide • inflammation • small airways

Asthma is a complex, chronic inflammatory lung disease that is characterized by a specific pattern of airway inflammation airway smooth-muscle hypertrophy and hyperplasia and production of mucus. The pathophysiology of asthma has traditionally been attributed to an inflammatory process that occurs predominantly in the large airways.

Pathology studies in patients who have died of asthma reveal extensive involvement of the peripheral airways with inflammatory and structural changes, with luminal occlusion with viscid mucus plugs. Transbronchial biopsies have confirmed peripheral airway involvement in severe asthma including the alveoli.¹ On the basis of the physiologic and pathologic evidence, small or peripheral airways and lung parenchyma are clearly implicated in the pathogenesis of asthma, and this aspect seems to be an important feature of patients with asthma and has important implications for drug delivery.²³

Several tests have been proposed to measure the level of peripheral airway inflammation.⁴⁵⁶⁷⁸ Airway inflammation can be measured noninvasively by measuring the sputum eosinophil count and single-flow exhaled nitric oxide (NO) concentration, although these tests are limited to sampling the proximal airways. Two compartment models of pulmonary NO production have been described previously,⁹ and studies have shown that alveolar NO (CALV) is elevated in conditions associated with distal lung inflammation such as pulmonary fibrosis¹⁰ and COPD.¹¹ CALV has been shown to be related to BAL eosinophil cationic protein levels in children¹² and to BAL eosinophil counts in adults.¹³ Unlike bronchial NO (JNO), CALV production is not reduced by inhaled corticosteroids (ICS) in patients with asthma,¹⁴ suggesting that it may be derived from a site not accessed by the inhaled drug. In this study, we test whether it is possible to measure CALV concentration in patients with asthma, evaluating the reproducibility/repeatability and the effects of treatment relating to disease severity.

Materials and Methods

Subjects
Seventy-four subjects participated in the study. Patients attended outpatient clinics at the Royal Brompton Hospital, and healthy subjects were recruited from staff members and by advertisement. The characteristics of the patients are shown in Table 1 . Eighteen nonsmoking subjects had neither atopy nor history of asthma or other disease and had normal lung function.

All of the asthmatics had symptoms consistent with asthma and the following objective measures of airway responsiveness and/or documented airway obstruction and a positive skin-prick test result to at least two common allergens (cat dander, house dust mite, grass pollen, Aspergillus fumigatus). Current smokers and patients with a smoking history of > 5 pack-years were excluded from participation.

None of the patients were receiving food supplements containing L-arginine, and none were on a nitrate-rich or nitrate-restricted diet that might influence exhaled NO levels. The study was approved by the Ethics Committee of the Royal Brompton Hospital and Harefield NHS Trust, and all participants gave written informed consent

Study Design
All subjects underwent measurements of NO at multiple exhalation flows (MEFeNO) between 9:00 and 10:00 AM, consisting of two exhalations at each flow, sufficient to obtain reproducible NO results.¹⁶ In addition, the reproducibility and diurnal variation of the measurements were examined in 26 subjects (10 healthy control, 6 mild asthma, 5 moderate asthma, 4 asthma with exacerbation, 1 severe asthma) by measuring their MEFeNO between 9:00 and 10:00 AM, 12:00 noon and 1:00 PM, and 3:00 and 4:00 PM on 2 consecutive days. The effect of ambient NO on MEFeNO parameters was studied in 32 subjects (12 healthy control, 7 mild asthma, 6 moderate asthma, 5 asthma with exacerbation, 2 severe asthma) whose MEFeNO measurements were made after either one inhalation of NO-free air from the analyzer vs five consecutive breaths of NO-free air made in three sessions in 2 consecutive days with recording of ambient NO. MEFeNO levels were also measured in eight randomly allocated asthmatic patients before and 30 min after administration of 200 µg of albuterol by metered-dose inhaler to investigate any effect of air caliber changes on CALV and JNO. All patients refrained from using bronchodilators before the measurements.

Exhaled NO Measurements
Exhaled NO concentration was measured by a chemiluminescence analyzer (NIOX; Aerocrine AB; Stockholm, Sweden) at expiratory rates of 10, 50, 100, 200, and 260 mL/s by applying resistors of 10, 50, 100, 200, and 300 cm H₂O mL/s to maintain the target flow rates. The patients were comfortably seated, inhaled NO-free air from a reservoir, and then exhaled against different linear resistors. The collection only started when dead space time was subtracted from start of exhalation. The analyzer was calibrated with a known NO concentration (200 ppm). The exhalation time was 20 s for 10 mL/s, 10 s for 50 mL/s and 100 mL/s, and 6 s for 200 mL/s and 260 mL/s. The minimum waiting time between measurements was 20 s to allow the patient to rest.

JNO, CALV, and diffusion of NO (DNO) were calculated by nonlinear regression according to the equation of George et al.⁹ The slope and the intercept of a regression line between NO output and exhalation flow rate are CALV and JNO, respectively. DNO was calculated from the following equation:

where CEXH = exhaled NO concentration. Any exhalation that did not meet American Thoracic Society requirements was not accepted by the NIOX system, and the subjects were asked to perform a new exhalation maneuver.

Lung Function
Measurements of FEV₁ and FEV₁/FVC were made with a dry spirometer (Vitalograph-S; Vitalograph; Buckingham, UK), and the best value of three maneuvers was expressed as an absolute value (in liters) and as a percentage of the predicted value. Lung volumes and carbon monoxide gas transfer were measured with a Jaeger Master Lab Compact Transfer (Erich Jaeger; Hoechberg, Germany).

Methacholine Challenge
Methacholine inhalation challenge was performed according to the European Respiratory Society guidelines.¹⁷ Doubling increasing concentrations of methacholine from 0.03 to 32 mg/mL were delivered by a dosimeter (output, 9 µL per puff; MB3; Mefar; Brescia, Italy) and inhaled. Inhalations were interrupted when FEV₁ decreased by 20% from its post-saline solution value. The provocative dose of methacholine provoking a 20% decrease in FEV₁ was determined by linear interpolation of the last two experimental points.

Reversibility Testing
Testing was based on a complete FVC maneuver according to the European Respiratory Society guidelines¹⁸ and repeated three times. For each subject, a second FVC maneuver was performed, similar to the first maneuver, 20 min after inhaling 200 µg of salbutamol by metered-dose inhaler. Result of reversibility testing with salbutamol were considered positive (subject with reversible airway obstruction) when an increase in FEV₁ 12% over baseline was found.

Symptom Scores
Daytime and nighttime score were recorded on the same day as each patient performed the visit, on a 3-point scale, as follows: daytime symptoms (0 = none, no symptoms; 1 = mild, few symptoms, not troublesome; 2 = moderate, symptoms troublesome; and 3 = severe, not able to perform normal activities); and nighttime symptoms (0 = none, slept well throughout the night; 1 = mild, slept well, woken early or once by asthma; 2 = moderate, woken two or three times by asthma; and 3 = severe, bad night, kept awake most of night).

Statistical Methods
Data are expressed as mean ± SD. Nonparametric tests were applied because the distribution of these variables was not known and there were insufficient data for normal distribution analysis. For comparisons between two groups, the Mann-Whitney U test was used. Reproducibility of the measurements was assessed by means of the intraclass correlation coefficient (ICC) [expressed on a 0 to 1 scale, where 0 represents no agreement and 1 represents perfect agreement], which considers the contribution of variance between repeated measurements to the total variance and by Bland-Altman analysis. Repeatability was calculated by intrasubject SD. The pooled SD was calculated in the group of subjects who underwent the measurements three times daily on 2 consecutive days as the final measure of repeatability.

A Student paired t test was used to compare NO values at different time points in 1 day and in 2 consecutive days and to compare NO values measured after one breaths vs five breaths. The correlation between MEFeNO parameters and FEV₁ and other parameters was determined using a Spearman rank correlation test; p < 0.05 was considered statistically significant.

Results

MEFeNO:
Repeatability, Reproducibility, and Diurnal Variation

MEFeNO and NO measurements were highly reproducible (Table 2 , Fig 1, 2 ). CALV, JNO, and DNO obtained at different time points within the same day were highly reproducible (Table 3 ), and there was no significant day-to-day variation (Table 4 ).

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Bland-Altman analysis for the repeatability of fractional exhaled NO (FeNO) values measured at 10 mL/s (top left, a), 50 mL/s (top right, b), 100 mL/s (center left, c), 200 mL/s (center right, d), and 300 mL/s (bottom, e) flow rates.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Bland-Altman analysis for the repeatability of JNO (top left, a), CALV (top right, b), and DNO (bottom, C) in the studied groups.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Bland-Altman analysis for the repeatability of JNO (top left, a), CALV (top right, b), and DNO (bottom, C) measured after one breath vs five consecutive breaths.

Asthma Severity and CALV, JNO, or DNO
JNO, CALV, and DNO levels are displayed in Table 6 and Figure 4 .

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Airway wall NO concentration (JNO; top, a), CALV concentration (bottom left, b), and DNO (bottom right, c) in healthy control subjects, and in patients with mild, moderate, and severe asthma. CS = corticosteroids.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Correlation between daytime symptoms score and JNO (top left, a) and CALV (top right, b), and correlation between nighttime symptoms score and JNO (bottom left, c) and CALV (bottom right, d) in patients with severe asthma.

DNO:
All the groups had higher DNO pL/s^–1 ppb^–1 than control subjects (p < 0.05), but there were no differences in DNO levels between different asthma groups (Fig 4, bottom right, c). No correlation was found between DNO and lung function parameters.

Discussion

In this study, we have determined flow-independent NO exchange parameters characteristic of NO exchange dynamics in the lungs in patients with asthma at different levels of severity. We have also shown that the measurements are highly reproducible, free of diurnal variation, and can be applied in asthmatic patients.

We have confirmed that JNO concentration is increased in asthma, but the anatomic site of enhanced NO production is not yet certain. The increased JNO flux in patients with asthma most probably results from increased NO synthesis in bronchial epithelial cells, as a result of increased inducible NO synthase (iNOS, NOS2) expression in response to inflammatory mediators released locally in the airway wall.¹⁹²⁰²¹ Treatment with corticosteroids results in a reduction in exhaled NO levels due to the reducing effects of corticosteroids on the underlying airways inflammation, thus resulting in suppression of iNOS induction. In patients with mild asthma who were not treated with inhaled steroids, we found an increase in JNO, demonstrating the presence of an ongoing active inflammatory process, which presumably reflects the increased expression of iNOS in conducting airways.

JNO in patients with moderately stable, well-controlled, severe asthma receiving ICS was lower compared to the other asthma groups but higher than in control subjects despite treatment with ICS. In this respect, our results differ from those of Lehtimaki et al,¹⁰ who reported that steroid treatment reduces JNO values to those seen in normal subjects. However, their patients had newly diagnosed asthma, so this may have had greater potential for reversibility.

In our patients with moderate stable asthma, JNO remained elevated despite apparent clinical control. This might reflect the fact that airway wall inflammation is not completely suppressed, possibly because the inhaled drug does not reach all the sites of inflammation due to uneven deposition of the drug.

Patients with moderate asthma that was not adequately controlled during exacerbation had increased JNO compared to patients with moderate stable asthma, indicating that increased JNO may reflect not an adequate suppression of inflammation and the need to increase the dose of ICS. During an exacerbation, there was a further increase in JNO compared to other groups, again reflecting an increase in airway inflammation.²² Asthma exacerbations are acute worsening of the disease with duration of 3 days and a need of an increase in treatment. An important cause is suboptimum controller therapy and breakthrough airway inflammation.²³ Under these circumstances, the level of JNO might become a particularly useful marker directed at normalizing the inflammation.

In patients with severe asthma, JNO was elevated compared to healthy control subjects despite treatment with a high dose of inhaled steroids. This probably reflects reduced responsiveness to corticosteroid therapy, which may be an important feature of severe persistent asthma.²⁴ In patients with severe asthma receiving oral corticosteroid treatment, JNO remained elevated, suggesting a failure to respond to the therapy. Complete corticosteroid resistance is very rare; much more common is a reduced responsiveness to corticosteroids, described as corticosteroid-dependent asthma, for which large inhaled or oral doses are needed to control asthma adequately.

We found normal values of CALV in patients with moderate stable asthma, and in patients with severe asthma receiving ICS, suggesting that asthmatic inflammation principally affects the airways rather than the lung periphery, in agreement with the findings of Lehtimaki et al¹⁰ and Hogman et al.²⁵ However, CALV values in patients with mild persistent asthma were higher than in these studies, but this is probably because our patients had chronic rather than newly diagnosed asthma.

We have shown also a significant increase in CALV values in patients during exacerbations and in severe asthma on treatment with ICS and oral corticosteroids, confirming the findings of van Veen et al²⁶ and suggesting that the intensity and the site of inflammation might change during the course of the disease. This is remarkable because the systemic treatment would reduce the peripheral inflammation, whereas it appears that there is extensive peripheral airway disease that is not fully controlled by such a high level of antiinflammatory treatment. Evidence from autopsy studies and transbronchial biopsies¹²⁷ suggests that the pathologic changes in asthma occur in both the large and small airways, and small airway inflammation been suggested to contribute to instability of the disease, therapy resistance, and excessive airway narrowing. The evidence that patients receiving continuous oral corticosteroids had more peripheral inflammation than patients with severe asthma receiving inhaled steroids alone suggests that peripheral inflammation seems to be an important determinant of patients of "steroid-insensitive" asthma and the requirement of additional or alternative antiinflammatory treatment.

NO diffuses from the sites of production in the airway wall into the lumen but also diffuses across the airway wall and may be taken up and removed by the bronchial circulation. DNO represents the ability of the airway barrier to transfer NO and depends on the transfer coefficient, which describes the quantity of NO diffusing from airway wall to lumen per unit time per unit surface area.²⁸ The increased DNO in asthma could be due to either an increase in the transfer coefficient due to increased bronchial blood flow or an increase in the lumenal surface area over which diffusion is occurring. The analysis of Tsoukias and George²⁹ indicates that there are two factors affecting the transfer coefficient: airway wall thickness, which varies inversely, and the rate of NO catabolism, which varies directly with DNO. Because airway wall thickness is significantly increased in asthma, it seems likely that the increased DNO observed in asthmatics was explained by an increase in surface area, rather an increase in the transfer coefficient.

Our results showed also an increased of the DNO in asthmatic subjects independently of the use of corticosteroids. These findings are in good agreement with previously published data by Silkoff et al,³⁰ despite using a different breathing maneuver and analytic technique to estimate the flow-independent NO exchange parameters. More recently, Hogman et al²⁵ also demonstrated that DNO is increased 1.5-fold in a group of atopic asthmatic subjects.

Since corticosteroids decrease induction of iNOS and the concentration of NO depends on the rate of NO production per unit of tissue volume, it is likely that the decrease in JNO is caused by a corticosteroid-induced decrease in NO production by iNOS. The lack of steroid effect on DNO implies that airway iNOS activity was located within the area of NO production by constitutive NO synthase isoforms, which are not affected by steroids.³⁰ DNO may reflect changes in the lung that impact function, which are not affected by steroid therapy, and thus may provide clinical information not available from exhaled NO concentration alone.

The correlation of the flow-independent parameters with standard spirometry is of particular interest to the potential clinical application and interpretation of NO parameters. We did not find any correlation in asthma patients between NO parameters and respiratory flows, in agreement with the findings of Lehtimaki et al.¹⁰ These results suggest that NO parameters are not markers of airflow limitation in asthma. By contrast, Silkoff et al³⁰ reported that values of JNO and DNO after steroid therapy in asthmatic subjects were all significantly correlated with FEV₁/FVC ratio. These differences may be due to differences in study design and the technique used to estimate the flow-independent NO parameters. Nevertheless, future studies will need to continue to explore the relationship between NO parameters and lung function in asthmatic patients of differing severity and with different therapies.

JNO and CALV correlate with the symptom score in patients with severe asthma. The relationship of symptoms to inflammatory activity in patients with severe asthma is unclear, but its importance lies in the choice of treatment response to symptoms. JNO and CALV combined with the symptom score may be a useful tool for monitoring asthma control in patients with severe asthma for whom changes in lung function may have limited sensitivity.

The present study may have some limitations due to the fact that the calculation of CALV is based on a mathematical model. In five patients, negative CALV values were calculated, while the measurements were technically well performed. We would have needed additional measurements at higher flows, although this maybe difficult to achieve in patients with flow limitation.

In conclusion, the multiple expiratory flow technique was easy to perform, noninvasive, and able to estimate airway and the peripheral inflammation. It provides important information and might have implications for the assessment and treatment of patients with asthma.

Acknowledgements

The authors thank Professor K. F. Chung for allowing them to recruit some of his patients for the study, and Björn Jonsson for statistical advices.

Footnotes

Abbreviations: CALV = alveolar nitric oxide; CI = confidence interval; DNO = diffusion of nitric oxide; ICC = intraclass correlation coefficient; ICS = inhaled corticosteroids; iNOS = inducible nitric oxide synthase; JNO = bronchial nitric oxide; MEFeNO = nitric oxide at multiple exhalation flows; NO = nitric oxide; ppb = parts per billion

This study was technically supported by Aerocrine AB (Sweden).

Professor Barnes and Drs. Ito and Brindicci have no financial or other potential conflicts of interest to disclose. Dr. Kharitonov has a consultancy agreement with Aerocrine AB (Sweden).

Received for publication October 16, 2006. Accepted for publication December 30, 2006.

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This Article Abstract Full Text (PDF) Free Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Brindicci, C. Articles by Kharitonov, S. A. Search for Related Content PubMed PubMed Citation Articles by Brindicci, C. Articles by Kharitonov, S. A.