Extended Exhaled NO Measurement Differentiates between Alveolar and Bronchial Inflammation LAURI LEHTIMÄKI, HANNU KANKAANRANTA, SEPPO SAARELAINEN, PAULA HAHTOLA, RITVA JÄRVENPÄÄ, TIMO KOIVULA, VÄINÖ TURJANMAA, and EEVA MOILANEN Immunopharmacological Research Group, University of Tampere Medical School; and Departments of Clinical Chemistry, Respiratory Medicine, Radiology, and Clinical Physiology, Tampere University Hospital, Tampere, Finland
Lower respiratory tract inflammation can be detected by measuring exhaled nitric oxide (NO) concentration at a single exhalation flow rate, but this does not differentiate between alveolar and bronchial NO production. We assessed alveolar NO concentration and bronchial NO flux with an extended method of measuring exhaled NO at several exhalation flow rates in 40 patients with asthma, 17 patients with alveolitis, and 57 healthy control subjects. Bronchial NO flux was higher in asthma (2.5 0.3 nl/s, p 0.001) than in alveolitis (0.7 0.1 nl/s) and healthy control subjects (0.7 0.1 nl/s). Alveolar NO concentration was higher in alveolitis (4.1 0.3 ppb, p 0.001) than in asthma (1.1 0.2 ppb) and healthy control subjects (1.1 0.1 ppb). In asthma, bronchial NO flux correlated with serum level of eosinophil protein X (EPX) (r 0.60, p 0.001) and bronchial hyperresponsiveness (r 0.55, p 0.001). In alveolitis, alveolar NO concentration correlated inversely with pulmonary diffusing capacity (r 0.55, p 0.022) and pulmonary restriction. Glucocorticoid treatment or allergen avoidance normalized bronchial NO flux in asthma and decreased alveolar NO concentration toward normal in alveolitis. In conclusion, extended exhaled NO measurement can be used to separately assess alveolar and bronchial inflammation and to assess disease activity/severity in asthma and alveolitis.
Nitric oxide (NO) is a gaseous signaling molecule produced by constitutive NO synthases (cNOS) in physiologic state and in much higher quantities by inducible NO synthase (iNOS) in inflammation (1). Expression of iNOS is enhanced in inflamed airways in asthma (2, 3) and in lung parenchyma in idiopathic pulmonary fibrosis (IPF) (4). Exhaled NO concentrations are increased in these diseases (5–7) and decrease toward normal simultaneously with reduction of iNOS expression during treatment with anti-inflammatory steroids (3, 7). Exhaled NO measurement is considered a promising noninvasive method to detect respiratory tract inflammation (8). Exhaled NO has usually been measured at a single exhalation flow rate. However, this method can not determine whether the enhanced NO production, and thus the inflammation, is located at conducting airways or lung parenchyma. Recently, Tsoukias and George published a mathematical model on pulmonary NO dynamics (9). They divided lung into alveolar (respiratory bronchioles and alveolar region) and bronchial compartments. According to Tsoukias and George, NO output (VNO [nl/s] exhaled NO concentration [ppb] exhalation flow rate [L/s]) during exhalation is described by the · equation VNO FNO,Br QE CAlv (equation 12 from Ref. 9), (Received in original form October 30, 2000 and in revised form March 7, 2001) Supported by grants from the National Technology Agency (Tekes), the Medical Research Fund of Tampere University Hospital, and the Tampere Tuberculosis Foundation. Correspondence and requests for reprints should be addressed to Prof. Eeva Moilanen, Medical School, FIN-33014 University of Tampere, Tampere, Finland. E-mail: [email protected]
Am J Respir Crit Care Med Vol 163. pp 1557–1561, 2001 Internet address: www.atsjournals.org
· where FNO,Br [nl/s] is bronchial NO flux, QE [L/s] is exhalation flow rate, and CAlv [ppb] is steady-state NO concentration in alveolar air. Bronchial NO flux describes the quantity of NO transferred from bronchial wall to luminal air per unit time. It depends on NO diffusivity across the bronchial wall, and on NO concentration difference between bronchial wall and luminal air that drives the diffusion. Alveolar NO concentration and bronchial NO flux can be approximated by measuring exhaled NO at several exhalation flow rates and plotting NO output against exhalation flow rate. The slope and the intercept of a linear regression line between NO output and exhalation flow rate are alveolar NO concentration and bronchial NO flux, respectively (9). Thus, increased alveolar NO concentration in alveolar inflammation should cause a steeper slope of the regression line, whereas increased bronchial NO flux in bronchial inflammation should cause higher intercept value. Our preliminary results suggest that this method could be used to differentiate between alveolar and bronchial inflammation (10). Recent guidelines by the American Thoracic Society recommend the use of a single low exhalation flow rate of 0.05 L/s in exhaled NO measurements, but acknowledge the possible advantage of using several exhalation flow rates (11). There are not yet enough data, however, to interpret the value of using several exhalation flow rates. The aim of this study was (1) to study the contributions of conducting airways and lung parenchyma to exhaled NO in alveolitis and asthma by assessing bronchial NO flux and alveolar NO concentration, (2) to study the relation of alveolar NO concentration and bronchial NO flux to disease activity/severity in patients with alveolitis and asthma, and (3) to study the effect of steroid treatment or allergen avoidance on alveolar NO concentration and bronchial NO flux in these patients.
METHODS Subjects Exhaled NO, bronchial hyperresponsiveness, and serum eosinophil protein X (EPX) levels were measured in 40 steroid-naive patients with newly diagnosed asthma (Table 1). Asthma diagnosis was based on clinical symptoms and reversible or variable airflow obstruction (10). Exhaled NO, pulmonary diffusing capacity of CO (DLCO), and lung volumes were measured in 17 patients with alveolitis [seven farmers with hypersensitivity pneumonitis (HP) (12) and 10 subjects with IPF (13)]. Six patients with HP were predisposed to moldy hay (farmer’s lung) and one to moldy wood chips. Patients with IPF had typical reticular pattern in high-resolution computed tomography (HRCT) and patients with HP had ground-glass opacity in chest radiograph or HRCT. In three patients with IPF, the histopathologic diagnosis of usual interstitial pneumonia (UIP) was obtained and in the remaining seven patients the clinical presentation was typical of UIP (Tables 1 and 2). A subset of patients was tested also after treatment. Exhaled NO was measured in 16 asthmatic patients after 4 wk of treatment with inhaled fluticasone propionate (Flixotide Diskus; GlaxoWellcome, Ware, UK, 500 g twice a day). Exhaled NO and DLCO were measured in
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TABLE 1. SUBJECT CHARACTERISTICS Group
FEV1 (% pred )
PD20FEV1 of Methacholine
VC (% pred )
DLCO (% pred )
8 M, 32 F
2.6 mg: 29 2.6 mg: 8 —
— — 76 4
— — 4.5 0.4
— — 52 3
10 M, 7 F
Definition of abbreviation: VA alveolar volume.
seven patients with alveolitis after 2 mo of allergen avoidance or drug treatment (Table 2). Exhaled NO was measured also in 57 agematched ( 5 yr) and sex-matched healthy control subjects. The study was approved by the ethical committee of Tampere University Hospital, and all patients gave written informed consent.
Exhaled NO Exhaled NO concentration was measured with a Sievers NOA 280 analyzer (Sievers Instruments, Boulder, CO). Three exhalation flow rates (100, 175, and 370 ml/s) were used with mouth pressure of 9 cm H2O, as previously described (10). Alveolar NO concentration and bronchial NO flux were assessed according to Tsoukias and George (9). Linearity between NO output and exhalation flow rate was high (r 0.98 in control subjects, 0.93 in patients with asthma, and 0.99 in patients with alveolitis). The analyzer was calibrated daily with known NO concentration (103 or 101 ppm; AGA, Lidingö, Sweden) and before every subject with NO-free air (Sievers zero-air-filter).
Serum EPX and Lung Function EPX level in venous blood serum was measured with radioimmunoassay (EPX-RIA; Pharmacia AB, Uppsala, Sweden). Bronchial responsiveness was studied by letting patients inhale increasing doses of methacholine and measuring spirometry after each dose (Vmax 20C; SensorMedics, Yorba Linda, CA) (14). Logarithm of dose–response slope (percent decline in FEV1 divided by the total dose of methacholine inhaled) was used in analysis (15). DLCO was measured by the single-breath technique (Vmax 22 or model 2200; SensorMedics) (16).
Statistics All the results were normally distributed (Kolmogorov-Smirnov test). Differences in NO parameters between the three groups were anaTABLE 2. DIAGNOSES AND TREATMENT IN PATIENTS WITH ALVEOLITIS
M F M M M M F M M
61 54 35 56 32 27 64 75 69
HP HP HP HP HP HP HP IPF IPF
F M F F
71 59 66 40
IPF IPF IPF IPF
M F F M
63 70 78 82
IPF IPF IPF IPF
Treatment at First NO Measurement
Intervention in Patients Joining 2-mo Follow-up* Allergen avoidance Pred 20 mg Pred 20 mg Allergen avoidance Allergen avoidance
lyzed using analysis of variance with Games-Howell multiple comparison post-test. The change in NO parameters during the treatment was analyzed with paired t test. Unpaired t test was used to compare NO values in control subjects with post-treatment values in patients. The relations between NO parameters and other markers were analyzed with Pearson’s correlation (17). SPSS 9.01 (SPSS Inc., Chicago, IL) software was used for analysis. A value of p 0.05 was considered significant. Results are presented as mean SEM.
RESULTS Alveolar and Bronchial NO
Patients with alveolitis had higher alveolar NO concentration (4.1 0.3 ppb, p 0.001) than patients with asthma (1.1 0.2 ppb) and healthy control subjects (1.1 0.1 ppb). Patients with asthma had higher bronchial NO flux (2.5 0.3 nl/s, p 0.001) than patients with alveolitis (0.7 0.1 nl/s) and healthy control subjects (0.7 0.1 nl/s) (Figure 1). Factors Increasing Alveolar NO Concentration in Alveolitis
Alveolar NO concentration (CAlv [ppb]) is described by the equation C Alv = V NO,Alv ⁄ DL NO
where VNO,Alv [nl/s] is NO diffusing rate from tissue to alveolar air and DLNO [nl/s/ppb] is NO diffusing capacity from alveolar space to pulmonary vessels (18). Thus, CAlv can be increased because of increased NO production in lung parenchyma causing increased NO diffusion to alveolar air (VNO,Alv), or because of decreased diffusion of NO from alveolar air to pulmonary blood caused by decreased alveolar NO diffusing capacity (DLNO). An attempt was made to estimate the contributions of increased VNO,Alv and decreased DLNO to increased alveolar NO concentration in the patients with alveolitis. As DLNO is approximately 4 DLCO (19), Equation 1 can be rearranged to give V NO,Alv = C Alv ⋅ 4 ⋅ DL CO
Pred 5 mg Pred 40 mg Pred 20 mg, Aza 150 mg Pred 20 mg UIP Pred 5 mg UIP UIP
Pred 40 mg, Aza 100 mg Pred 60 mg
Definition of abbreviations: Aza azathioprine (drug doses are given in mg/d), Pred prednisolone. * In all patients with HP joining the follow-up, the intervention included allergen avoidance, which is not shown if any drug treatment was also given.
Figure 1. Alveolar NO concentration (ppb) and bronchial NO flux (nl/s) assessed by measuring exhaled NO at several exhalation flow rates in 17 patients with alveolitis, 57 healthy control subjects, and 40 patients with steroid-naive asthma.
Lehtimäki, Kankaanranta, Saarelainen, et al.: Exhaled NO in Alveolitis and Asthma
Figure 4. Change in bronchial NO flux during 4 wk of treatment with inhaled fluticasone (500 g twice a day) in 16 patients with asthma. Figure 2. Correlation between bronchial NO flux and logarithm of dose–response slope of methacholine (A, n 37) and serum concentration of EPX (B, n 40) in patients with steroid-naive asthma.
Hence, the diffusion rate of NO to alveolar air (VNO,Alv) can be assessed on the basis of CAlv and DLCO measurements. According to these calculations, patients with alveolitis had VNO,Alv of 2.9 0.2 nl/s. Healthy control subjects and asthmatics had VNO,Alv of 1.6 0.1 and 1.7 0.3 nl/s, respectively, if their DLCO is supposed to equal age-, sex-, and height-matched reference value (20). VNO,Alv was higher in patients with alveolitis than in control subjects (p 0.001) and asthmatics (p 0.014). NO Parameters and Disease Activity/Severity
Methacholine challenge test was not carried out on three patients with asthma because of FEV1/FVC 70%. Twentynine of the remaining 37 asthmatics had bronchial hyperresponsiveness indicated by a provocative dose of methacholine causing a 20% reduction in FEV1 (PD20FEV1) less than 2.6 mg. Logarithm of dose–response slope of methacholine in the remaining 37 patients correlated with bronchial NO flux (r 0.55, p 0.001) but not with alveolar NO concentration (r 0.11, p 0.506). Serum EPX level was 37.4 3.6 g/L in the 40 asthmatics. Serum EPX level correlated with bronchial NO flux (r 0.60, p 0.001) but not with alveolar NO concentration (r 0.09, p 0.570) (Figure 2). In patients with alveolitis, DLCO percentage of predicted value (52% 3%) correlated negatively with alveolar NO concentration (r 0.55, p 0.022) but not with bronchial NO flux (r 0.15, p 0.564). Alveolar NO concentration correlated also with vital capacity (VC) (r 0.54, p 0.027) and with alveolar volume (r 0.49, p 0.047), whereas bronchial NO flux did not correlate with these factors (r 0.12, p 0.637 and r 0.22, p 0.399, respectively) (Figure 3). The Effect of Treatment on Alveolar and Bronchial NO
Steroid treatment in 16 patients with asthma decreased bronchial NO flux from the original value of 3.6 0.4 nl/s to 0.7 0.1 nl/s (p 0.001) (Figure 4). After steroid treatment, bronchial NO flux was similar to that of healthy control subjects (p 0.745). In these asthmatics, steroid treatment had no effect on alveolar NO concentration (before 1.2 0.5 ppb, and after treatment 1.5 0.3 ppb, p 0.491).
Figure 3. Correlation between alveolar NO concentration and DLCO percentage of predicted (A) and alveolar volume (B) in 17 patients with alveolitis.
In patients with alveolitis, during clinical recovery (increase in DLCO from 59% 6% to 76% 3%, p 0.006) owing to allergen avoidance or drug treatment, alveolar NO concentration decreased from 4.0 0.6 ppb to 2.4 0.5 ppb (p 0.011) (Figure 5). However, alveolar NO concentration after recovery was still higher than in control subjects (p 0.008). There was no change in bronchial NO flux (before 0.5 0.1, after 0.6 0.1 nl/s, p 0.340) during recovery in patients with alveolitis.
DISCUSSION Our results show increased alveolar NO concentration in alveolitis and increased bronchial NO flux in asthma. Markers of disease activity/severity correlated with alveolar NO concentration in alveolitis and with bronchial NO flux in asthma. Steroid treatment normalized bronchial NO flux in asthma, and allergen avoidance or drug treatment decreased alveolar NO concentration toward normal in alveolitis. Alveolar and Bronchial NO in Asthma
Exhaled NO concentration is increased in asthma (5, 6), but the anatomic site of enhanced NO production has not been clear. Our results show increased bronchial NO flux but normal alveolar NO concentration in newly diagnosed steroid-naive asthmatics, suggesting that asthmatic inflammation affects principally airways. This is in accordance with our preliminary results in a small number of patients (10) and with those of Högman and colleagues in a study published in abstract using similar methodology (21). They reported increased bronchial NO flux and normal alveolar NO concentration in asthma (1.4 0.5 nl/s and 2.2 0.5 ppb, respectively) compared with control subjects (0.3 0.1 nl/s and 1.8 0.2, ppb respectively) (21). However, their bronchial NO fluxes were slightly lower and alveolar NO concentrations higher than in our patients with asthma or control subjects.
Figure 5. Change in alveolar NO concentration (A) and DLCO percentage of predicted (B) during 2 mo of allergen avoidance or drug treatment; or both, in seven patients with alveolitis.
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Recent publications show that bronchial NO flux can be divided into bronchial wall NO concentration and bronchial NO diffusion factor, if very low exhalation flow rates are used in NO measurements (22, 23). According to results by Silkoff and coworkers, increased airway diffusing factor of NO rather than increased airway wall NO concentration causes increased bronchial NO flux in asthma (22). However, Högman and colleagues reported also increased airway wall NO concentration in addition to increased airway diffusing factor in asthma in their recent abstract (24). Increased airway wall NO concentration could be explained by the enhanced iNOS expression shown in asthmatic airways (2, 3), but the increased airway diffusion factor is more difficult to interpret in terms of histopathologic changes in airways. Unfortunately, we did not use low enough exhalation flow rates in this study to allow approximation of airway wall NO concentration and NO diffusing factor. We found normal alveolar NO concentration in asthma, although signs of alveolar inflammation have been shown in a group of patients with chronic asthma (25). The intensity and the site of asthmatic inflammation might change during the course of the disease. As our group of patients had newly diagnosed asthma, they might have only mild or negligible alveolar inflammation, explaining the normal alveolar NO concentration. However, there were a few subjects among our group of asthmatics who had slightly elevated alveolar NO concentration, although the group mean was normal. On the other hand, alveolar NO concentration might not increase significantly even if the alveolar NO production was slightly increased, as the normal alveolar-capillary membrane in asthma allows rapid diffusion of NO into pulmonary circulation where it is trapped by hemoglobin. Alveolar and Bronchial NO in Alveolitis
This is the first study to show increased alveolar NO concentration in patients with IPF or HP. Our DLCO measurements and calculations based on Equation 2 suggest that both decreased alveolar diffusing capacity of NO (DLNO 4 DLCO) and enhanced parenchymal NO production (increased VNO,Alv) contribute to the increased alveolar NO concentration in these patients. The decreased pulmonary gas diffusing capacity is typical for interstitial lung diseases (ILD), and the increased alveolar NO production is in line with earlier studies reporting enhanced iNOS expression in alveolar epithelium in IPF (4). However, direct measurements of DLNO instead of approximating it from DLCO are needed to get more reliable results on contributions of increased NO production and decreased DLNO to increased alveolar NO concentration. We found normal bronchial NO flux in alveolitis, although peribronchiolar inflammatory changes may affect the peripheral bronchioles in HP and IPF. Exhaled NO is more closely related to airway epithelial than subepithelial NO production (26), and, hence, peribronchiolar inflammatory changes might not affect bronchial NO flux as much as mucosal inflammation in airway diseases like asthma. NO Parameters and Disease Activity/Severity
Bronchial hyperresponsiveness to methacholine in asthma is associated with airway inflammation (27), and serum EPX level has been shown to correlate with disease activity (28). Both of these markers correlated with bronchial NO flux but not with alveolar NO concentration in asthma. This further supports the predominant role of conducting airways in enhanced NO production in asthma. Measurement of DLCO is considered as a sensitive method to detect ILD and has been shown to correlate with pulmo-
nary fibrosis and inflammatory cell infiltration of alveoli (29). Pulmonary restriction is also a typical finding in ILD (13). DLCO and pulmonary volumes negatively correlated with alveolar NO concentration but not with bronchial NO flux in alveolitis. There was no correlation between NO parameters and FVC or VC in asthmatics or control subjects, suggesting that alveolar NO concentration is not related to pulmonary volume as such. Thus, increased alveolar NO concentration in alveolitis seems to be an independent marker of disease severity and not due to pulmonary restriction. Correlation between NO parameters and these other markers suggests that extended exhaled NO measurement can be used to assess bronchial and alveolar inflammation and disease activity/severity in asthma and alveolitis. The Effect of Treatment on Alveolar and Bronchial NO
Inhaled steroids have been shown to reduce iNOS expression in asthmatic airways (3). This could explain the reduction in bronchial NO flux in asthmatics during fluticasone treatment in our study. Earlier results by Silkoff and colleagues also suggest that steroid treatment in asthma reduces airway wall NO production (22). However, they did not study the effect of steroid treatment on alveolar NO concentration. We found no change in alveolar NO concentration in asthma during steroid treatment, suggesting that inflammation in these patients was located in conducting airways. Allergen avoidance or drug treatment decreased alveolar NO concentration in alveolitis but had no effect on bronchial NO flux. However, alveolar NO concentration remained at higher level than in healthy control subjects. This is in concert with the finding that the improvement in clinical condition was not complete at the end of the follow-up period (DLCO remained at 76% 3%). Measurement of bronchial NO flux and alveolar NO concentration might therefore be used in assessing the effect of anti-inflammatory treatment on airway and parenchymal inflammation in these diseases. Intensity of iNOS expression in alveolar epithelium of patients with IPF is related to the histologic abnormalities found in lung specimen. Those patients with early to intermediate stage of disease with signs of active inflammation have significantly more iNOS in alveolar epithelium than those patients with end-stage disease with honeycombing and fibrosis (4). In pulmonary fibrosis associated with systemic sclerosis, patients with increased number of inflammatory cells in bronchoalveolar lavage (BAL) have significantly higher exhaled NO concentration at a single exhalation flow rate than patients with normal BAL (7). Because iNOS expression and increased exhaled NO seem to be related predominantly to active inflammatory or fibrosing process in pulmonary fibrosis, assessment of parenchymal NO production might be used to predict response to drug treatment in IPF, as patients with active disease respond more favorably to treatment (13). In conclusion, measuring exhaled NO at several exhalation flow rates to assess alveolar NO concentration and bronchial NO flux is a promising novel method to separately assess alveolar and bronchial inflammatory activity, and to study the efficacy of anti-inflammatory treatment. Alveolar and bronchial NO parameters also correlate with disease activity/severity in alveolitis and asthma. Further studies comparing alveolar and bronchial NO parameters to parenchymal and airway biopsy samples and to clinical outcome would allow more detailed evaluation of the value of this method in assessing disease activity in various lung diseases. Acknowledgment : The authors thank Paula Pirttimäki and Marja-Leena Lampén for their skillful assistance with NO measurements, methacholine challenge tests, and serum EPX measurements; and Heli Määttä for secre-
Lehtimäki, Kankaanranta, Saarelainen, et al.: Exhaled NO in Alveolitis and Asthma tarial help. The nursing team of the outpatient clinic of the Department of Respiratory Medicine, Tampere University Hospital, is acknowledged for help with patient recruitment and records.
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