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Allergology international vol.56 no.

Allergology International. 2007;56:37-43DOI: 10.2332! Awarded Article, Annual Meeting of JSA The Relationship between Exhaled
Nitric Oxide Measured with an Off-line
Method and Airway Reversible
Obstruction in Japanese Adults with
Asthma
Takahiro Tsuburai1, Naomi Tsurikisawa1, Masami Taniguchi1, Sonoko Morita1, Emiko Ono1,
Chiyako Oshikata1, Mamoru Ohtomo1, Yuji Maeda1, Kunihiko Ikehara2 and Kazuo Akiyama1
ABSTRACT
Background: Exhaled nitric oxide (eNO) is a useful marker of eosinophilic airway inflammation in asthma pa-
tients. There is no study to show the relationship between the eNO measured by using an off-line method and
the degree of reversibility of airflow limitation in Japanese asthma patients. We sought to investigate the rela-
tionship between the eNO level measured by using an off-line method and the degree of reversibility of bron-
chial constriction in Japanese asthma patients.
Methods: The study population comprised 97 asthma patients in our outpatient clinic with some patients in
both groups who received inhaled corticosteroid treatment. We measured eNO levels, forced expiratory volume
in one second (FEV1) before and after treatment, reversible airway obstruction (∆FEV1) after inhalation of bron-
chodilator, and other parameters.
Results: eNO was significantly correlated with peripheral blood eosinophil counts in asthma patients (in
steroid-naïve asthma patients, r = 0.544, p < 0.0001; in asthma patients treated with inhaled corticosteroid, r =
0.463, p = 0.026), and subjects with severe eosinophilia in sputum showed high levels of eNO (mild eosino-
philia versus severe, p = 0.0152). Among patients with obstructive impairment, eNO levels were correlated with
∆FEV1 regardless of whether patients received (r = 0.527, p = 0.0435) or did not receive (r = 0.64, p = 0.0056)
inhaled corticosteroid. In subjects with normal pulmonary function, there was no significant relationship be-
tween eNO and ∆FEV1 with or without inhaled corticosteroid.
Conclusions: In patients with obstructive impairment, eNO reflects the degree of reversible airflow limitation.
In subjects with normal pulmonary function, eNO may facilitate the diagnosis and management of asthma,
rather than indicate reversible bronchial obstruction. eNO measurement by off-line methods is applicable as a
potential tool for the diagnosis of asthma and management of asthma patients.
KEY WORDS
airflow reversible obstruction, asthma, exhaled nitric oxide, off-line method
inflammation. Inhaled corticosteroid (ICS), the main- stay of asthma treatment, is effective because it pre- Bronchial asthma is caused by eosinophilic bronchial vents this inflammatory process. Therefore, quantifi- 1Clinical Research Center for Allergy and Rheumatology, National gawa 228−8522, Japan.
Hospital Organization, Sagamihara National Hospital and 2Ikehara Email: t tuburai@Sagamihara hosp.gr.jp Clinic, Kanagawa, Japan.
Received 17 February 2006. Accepted for publication 30 August Correspondence: Takahiro Tsuburai, Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sa- !2007 Japanese Society of Allergology gamihara National Hospital, Sakuradai 18 1, Sagamihara, Kana- Allergology International Vol 56, No1, 2007 www.jsaweb.jp! Tsuburai T et al.
cation of airway inflammation may provide additional information for both diagnosis and management ofbronchial asthma. However the current asthma man- The study population was recruited from adult outpa- Asthma) relies on monitoring respiratory function tients with bronchial asthma (n = 97) at the Clinic of and symptoms.1 Three studies have demonstrated Allergy and Respiratory Medicine in the Sagamihara that adding alternative monitoring markers such as National Hospital from June 2003 until February airway hyperresponsiveness, eosinophilia in induced 2005. All patients gave full informed consent to par- sputum, and exhaled nitric oxide (eNO) to current ticipate in the study. Each subject underwent a stan- guidelines on dose adjustment of ICS leads to im- dard clinical assessment, which included a history proved outcomes.2-4 and physical examination, laboratory tests (including According to recent studies, eNO is a useful eosinophil counts in peripheral blood), and chest ra- diograph. The diagnosis of asthma was based on ap- asthma.5,6 The increased levels of eNO are due to ac- plication of the GINA guidelines by an experienced tivation of NO synthase in airway epithelial and in- respiratory physician blinded to the result of eNO flammatory cells.7-9 Measurement of eNO is simple, measurement.1 The clinical severity of asthma (Step) noninvasive, and repeatable. eNO levels are higher in was classified according to these guidelines.1 Atopy asthma patients than in healthy subjects,10 and eNO was indicated by a positive skin test to mites or levels fall after treatment with corticosterids.11 In housedust, or serum IgE >250 IU!ml. Exclusion crite- steroid-naïve asthma patients, eNO levels correlate ria included current smokers or ex-smokers of with peripheral eosinophils in blood, eosinophils in greater than 20 packs per year, rescue use of oral cor- induced sputum, airway reversible limitation, or bron- ticosteroids within the preceding four weeks, preg- chial hyperresponsiveness,12-16 so that eNO is useful nancy, and any other respiratory disease. The study in the diagnosis of asthma patients.17,18 In asthma pa- protocol (no. 14, 2003) was approved by the ethical tients treated with ICS, eNO levels correlate with pe- committee at our hospital .
ripheral eosinophils in the blood, but are not corre-lated with eosinophils in induced sputum or bronchial hyperresponsiveness.19 However, eNO levels corre- Exhaled air was collected according to the recom- late with the following markers of disease control: mendation of ATS!ERS by using bag collection kits asthma symptoms within the prior 2 weeks, disease (Sievers Instrument, Boulder, Colo).5,6 Briefly, sub- score, and reversibility of airflow obstruction of bron- jects inhaled deep breaths of room air through the chial asthma.20 Furthermore, eNO is useful in the NO scavenging filter and exhaled through a mouth- clinical management4 of asthma patients treated with piece with a flow rate of 70 ml per second against an ICS. These findings suggest that eNO is not only use- expiratory resistance of 10 cm H2O and, 5 seconds ful in steroid-naïve asthma patients, but also useful as later, exhaled air was collected into the 1.5 liter Mylar a marker of disease control in asthma patients treated bag provided in the kit. The NO concentration in the collected exhaled air was measured within 12 hours.
Despite the utility of the eNO measurement, NO The air was drawn out of the balloons at 200 ml per analyzers are too expensive for widespread use by minute into an NO chemiluminescence analyzer general practitioners. In comparison, the off-line (bag (NOA model 280A, Sievers Instrument) with a re- collection) method of eNO measurement may be sponse time of 200 milliseconds. The normal range of more useful.5,6,21 In contrast to on-line techniques, eNO was determined in 14 healthy non-atopic, non- off-line collection methods offer the following bene- smoking volunteers. Their mean value was 21.1 ppb; fits: (1) the potential for expirate collection at sites re- the 95%CI was 15.0 27.1 ppb. Regarding this range, mote from the analyzer; (2) more efficient use of the we considered basal concentrations analyzer because exhaled gas can be collected and cutoff values.
stored for a while from several patients or severalplaces and analyzed in a batch, possibly with notable cost savings. Recent studies have demonstrated that After the eNO samples were collected, all subjects eNO measurement in the off-line method has sensi- underwent spirometry with an electric spirometer tivity and specificity in which is comparable to the on- (Minato Autospiro AS-302, Japan). The forced expira- line method,22-25 especially in asthmatic children.
tory volume in 1 second (FEV1) was expressed as However in Japan, there have been few studies on the percentage of forced vital capacity (FVC). The revers- use of eNO in asthma patients using the off-line ible change of FEV1 was expressed before and 30 method. Therefore, we sought to investigate the rela- minutes after inhaling 200 μg salbutamol. The degree tionship between eNO levels measured by using an of reversible airflow limitation was defined as ∆FEV1, off-line method and the level of bronchial reversibility which is equal to (FEV1post-bronchodilator − FEV1baseline) in Japanese asthma patients.
× 100!FEV1baseline (%).
Allergology International Vol 56, No1, 2007 www.jsaweb.jp! eNO Measurement and Airway Reversibility ween eNO and FEV1/ enced investigator (A.S.) who was blinded to the clinical data of the patients. The level of eosinophiliawas scored as: mild, no or few eosinophils through- out the entire smear; moderate, several eosinophils in each high-power (×400) microscopic field; severe, nu- merous eosinophils in each high-power (×400) micro- scopic field.
STATISTICAL ANALYSIS
Correlations were determined using the Spearman
rank correlation. A paired-sample t test was used to analyze eNO measurements and FEV1, ∆FEV1 or pe- ripheral eosinophil percentage in blood; p < 0.05 was considered significant. Analysis of variance was used to compare eNO measures between groups accord- ing to the level of eosinophilia in the sputum.
We classified the 97 asthma patients into two groups in regard to their therapeutic backgrounds: (A) 35 asthma patients treated with ICS (ICS(+)) and (B) 62 stable asthma patients treated without ICS (ICS(−))(Table 1).
There was no relationship between eNO levels and either FEV1 (L) or FEV1!FVC (%) among the patients EOSINOPHILIA IN SPUTUM
in either groups (Fig. 1A, B). The peripheral eosino- Sputum was collected and analyzed using the Han- phil percentage in blood was correlated with the eNO sel's modified method (Eosinostein, Torii Pharma- levels in each group (steroid-naïve asthma patients, ceutical Co., Tokyo, Japan).26 Briefly, each sputum r = 0.544, p < 0.0001, Fig. 2A; asthma patients with sample was smeared on a glass slide and stained for ICS, r = 0.463, p = 0.026, Fig. 2B).
45 seconds. All samples were analyzed by an experi- Sputum was collected from 7 of the 35 subjects Allergology International Vol 56, No1, 2007 www.jsaweb.jp! Tsuburai T et al.
Peripheral eosinophils in Peripheral eosinophils in results of the respiratory function tests: (1) asthmapatients with airflow limitation in the respiratory func- tion test (FEV1!FVC < 70%) and (2) asthma patientswith normal function (FEV1!FVC " 70%).
Among the patients with obstructive impairment, eNO levels were correlated with ∆FEV1 in the ICS (−)subjects (group A, r = 0.64, p = 0.0056, Fig. 4A). In ad- dition, eNO levels were also correlated with ∆FEV1 in the ICS (+) subjects (group B, r = 0.527, p = 0.0435, Fig. 4B). For subjects with normal pulmonary func- tion, there was no significant relationship betweeneNO levels and ∆FEV1 in either the ICS (−) (Fig. 5A)or the ICS (+) (Fig. 5B) subjects. The eNO levels of 24 out of 36 ICS (−) subjects or the eNO levels of 10out of 17 ICS (+) subjects were higher than 27.2 ppb despite the low levels of ∆FEV1.
Eosinophil level in sputum In asthma patients with airflow limitation who were treated either with or without ICS, eNO levels weresignificantly associated with ∆FEV asthma patients with normal respiratory function who were treated either with or without ICS, there was nosignificant relationship between eNO and ∆FEV1. Inmild asthma conditions, there is eosinophilic inflam- (20%) in the ICS (+) group and from 39 of the 62 sub- mation of bronchial mucosa, even if the patient's res- jects (62.9%) in the ICS (−) group. The eNO levels of piratory function is normal. This might explain why the subjects with severe eosinophilia in the sputum there was no significant relationship between eNO were significantly (p = 0.0152) higher than those of and ∆FEV1 in asthma patients with currently normal patients with no or mild eosinophilia in the sputum respiratory function. Furthermore this suggests that the biomarker reflecting airway inflammation can be To investigate whether the level of airway eosino- useful for diagnosis and disease control in asthma pa- philic inflammation influences the degree of obstruc- tive impairment, we classified each of the ICS (+) and eNO levels and the percentage of eosinophils in ICS (−) groups into two subgroups according to the the peripheral blood are significantly correlated, and Allergology International Vol 56, No1, 2007 www.jsaweb.jp! eNO Measurement and Airway Reversibility ween eNO and ∆FEV1 ( subjects with severe eosinophilia in sputum show vide the subjects into two groups for analysis. Recent high levels of eNO. The relationship between eNO studies have shown that eNO levels correlate with levels and the percentage of eosinophils in peripheral eosinophils in induced sputum in steroid-naïve pa- blood in our study is compatible to a recent study.12 tients, but not in asthma patients treated with The correlation between eNO levels and eosinophilia ICS.12,19 Our sputum results are mainly reflected in in sputum is not clearly demonstrated in our study. In the steroid-naïve subjects. These findings suggest our study, samples were collected from only 46 sub- that eNO is a useful marker of eosinophilic airway in- jects (7 of 35 subjects in the ICS (+) group or from 39 flammation in patients without ICS treatment, and of 62 subjects in the ICS (−) group), because the sam- eNO partially reflects eosinophilic inflammation in ples were collected from spontaneously expectorated those with ICS treatment.
sputum. The number of samples was too small to di- In asthma patients with airflow limitation who were Allergology International Vol 56, No1, 2007 www.jsaweb.jp! Tsuburai T et al.
treated either with or without ICS, eNO levels were significantly associated with ∆FEV1. The data fromthe asthma patients with airflow limitation and those Pulmonary function tests were performed by Mr.
treated with ICS suggest that eNO levels may be use- Masayuki Kimura, Ms. Yumiko Takeuchi, and Ms.
ful for detecting deterioration of asthma control, and Masayo Morie. Eosinophil sputum counts were per- the findings from the asthma patients treated without formed by Ms. Akemi Saito. The authors are in- ICS indicate that eNO may be useful for diagnosing debted to Ms. Mayumi Sato and Ms. Misuzu Matsu- asthma in the patients with reversible airway obstruc- moto for secretarial assistance. This study was sup- tion. In a recent study, Sippel et al. noted that eNO ported by a grant from the Japanese Society of Aller- levels correlate with reversibility of airflow obstruc- gology. This study received an article award at the tion in asthma patients treated with or without ICS 2004 meeting of the Japanese Society of Allergology.
and that elevated eNO levels may reflect the pres-ence of inflammation treatable with anti-inflammatory drugs.20 Our current findings support their conclu- 1. National Institutes of Health, National Heart, Lung, and
Blood Institute. Global Strategy for Asthma Management In asthma patients with normal respiratory function and Prevention: Global Initiative For Asthma 2002. NIH who were treated either with or without ICS, there Publication No 02-3659. Bethesda: National Institute ofHealth, 2002.
was no significant relationship between eNO and 2. Sont JK, Willems LN, Bel EH, van Krieken JH, Vanden-
∆FEV1. In this group, eNO levels in some subjects broucke JP, Sterk PJ. Clinical control and histopathologic were high despite low levels of ∆FEV1. In such sub- outcome of asthma when using airway hyperresponsive- jects (who have ‘cough variant' asthma), the diagno- ness as an additional guide to long-term treatment. The sis of asthma is often difficult because lung function AMPUL Study Group. Am. J. Respir. Crit. Care Med. 1999; tests such as spirometry or the reversibility of airway obstruction have a low sensitivity for diagnosing 3. Green RH, Brightling CE, McKenna S et al. Asthma exac-
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mendations for standardized procedures for the on-lineand off-line measurement of exhaled lower respiratory ni- Previous studies have shown that eNO is useful in di- tric oxide and nasal nitric oxide in adults and children- agnosing mild asthma,17,18 and our results are com- 1999. This official statement of the American Thoracic So- patible with the findings in these studies. In addition, ciety was adopted by the ATS Board of Directors, July our findings for asthma patients treated without ICS 1999. Am. J. Respir. Crit. Care Med. 1999;160:2104-2117.
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