The measurement of the exhaled nitric oxide fraction (FE_NO) has been proposed as a useful marker of airway inflammation.1 FE_NO levels are elevated in inflammatory lung diseases, such as asthma.1-3 Establishing reference ranges in healthy subjects would be useful for the interpretation of FE_NO measurements. Although the measurement procedures have been standardized, the normal upper limits of FE_NO levels have not been specified.1

Previous studies have demonstrated that there are several determinants of FE_NO, such as age, gender, atopy, smoking status, and diet.1, 4-17 It has been reported that the FE_NO levels in Asian children are significantly higher than those in Caucasian children.15-17 However, in healthy adults, there have been a few studies of the reference ranges for FE_NO in Caucasians.4-6 A community study in other regions may reveal any possible ethnic differences in the FE_NO levels.

In the present study, the reference ranges for FE_NO in healthy Japanese adults were estimated using two different statistical methods recommended by International Federation of Clinical Chemistry and Laboratory Medicine,18 and the results were compared with those of Caucasians. Furthermore, factors influencing the FE_NO levels were also investigated.

A general population sample of people aged 18 years or older was randomly selected from the population register in Wakayama, Fukuoka, Kanagawa, and Tokyo, Japan. All participants were interviewed by physicians and a total of 240 healthy adults were recruited. The study was approved by the local ethics committee and informed consent was obtained from each subject. To avoid the influence of the pollen season, the enrollment was performed from May to July 2009. The study subjects had neither history of atopic rhinitis, atopic dermatitis, food allergy, nor history of asthma, or other lung diseases. We did not include those subjects who reported having symptoms of either asthma or rhinitis based on guidelines.19, 20 Subjects were not included if they were current smokers or ex-smokers with more than 20 pack-years, had had an airway infection or were taking any form of corticosteroids in the 4 weeks preceding the study. Baseline demographics of the subjects are presented in Table 1.

This was a multi-center cross-sectional study. The subjects attended the outpatient clinic on one occasion for physical examination and FE_NO measurements.

FE_NO was measured by an online electrochemical nitric oxide analyzer (NIOX MINO; Aerocrine AB, Solna, Sweden). This nitric oxide analyzer has been approved by the U.S. Food and Drug Administration for clinical use. Measurements of FE_NO were performed asking the subjects to empty their lungs and then to inhale to total lung capacity through the mouthpiece and finally exhale into device at a constant flow rate of 50 mL/s; the software within the device automatically checks that the breathing manoeuvre is performed according to American Thoracic Society/European Respiratory Society guidelines.1 The calibration of the analyzer is automatically performed by the software. The sensor on the device was changed periodically according to the manufacture's guidance. Repeated exhalations were performed to obtain two acceptable measurements that agreed within 10% deviation, and the average of these two values was registered. All subjects were fasted for one hour before the FE_NO measurements.

The reference ranges for FE_NO were estimated using two different statistical methods recommended by International Federation of Clinical Chemistry and Laboratory Medicine: 1) 95% prediction interval (2.5 to 97.5 percentiles) of FE_NO values; 2) Since normality assumptions were met for the logarithm of FE_NO, the logarithm-transformed value was used to calculate the mean ± two standard deviations (SD) and then back-transformed values were estimated.18

The factors influencing the FE_NO values were examined in a regression tree-based model with the logarithm-transformed FE_NO as the response variable in relation to the different explanatory variables, age, gender, height, and past smoking history. Comparisons of mean FE_NO values between the groups (gender and smoking status) were performed by unpaired t tests. Pearson's correlation coefficients were calculated to determine the correlation between the FE_NO values and continuous data (age and height). All data were expressed as mean ± SD and significance was defined as a p value of less than 0.05.

The reproducibility of FE_NO measurements was expressed as the limit of agreement (intraclass correlation coefficient = 0.97). The mean FE_NO was 16.9 ppb (parts per billion) with a 95% prediction interval of 6.5 to 35.0 ppb. Normality assumptions were met for the logarithm-transformed FE_NO (Kolmogorov-Smirnov test, p = 0.43) and back-transformed values were used in the subsequent analysis. The geometric mean FE_NO for all subjects was 15.4 ppb with a mean ± 2 SD of 6.5 to 36.8 ppb in healthy Japanese adults (Table 2).

The mean FE_NO was 1.13 times higher in males, 16.5 ppb, compared to the 14.6 ppb in females (p <0.05), and that was 1.04 times higher in ex-smokers, 15.9 ppb, compared to the 15.3 ppb in nonsmokers (p = 0.62). However, the mean ± 2 SDs of FE_NO values were similar in each group although it was not statistically analyzed (Table 2). The distribution of the FE_NO levels in all subjects, males, and females, and the upper limits of FE_NO in each group are shown in Figure 1. In a linear regression analysis, no correlations were found between FE_NO and the values of age (r = 0.12, p = 0.07) or height (r = -0.02, p = 0.82). The regression tree analysis showed that age, gender, height, and past smoking history were not significant factors influencing the FE_NO levels.

The present study is one of the largest that investigated the FE_NO levels in a community sample of adults. We have shown that the reference range for FE_NO was approximately 6.5 to 36.0 ppb in healthy Japanese adults.

For healthy Caucasian adults, a few reports have shown the reference ranges for FE_NO measurements according to the current guidelines.4-6, 8 Travers et al. recruited 193 normal subjects categorized as follows: no physician diagnosis of lung disease, no symptoms of lung disease, no inhaled medication, and no allergic rhinitis.5 The criteria were similar to ours although 19 current smokers were included in the study population. The geometric mean FE_NO was 17.9 ppb with a 90% confidence interval of 7.8 to 41.1 ppb.5 In another study comprising 30 healthy non-atopic subjects, the mean FE_NO was 16.3 ppb with SD of 8.4 ppb in adults.7 On the basis of this report, a recent review suggested 33.1 ppb (mean + 2 SD) as the upper limit of FE_NO for adults.8 Olin et al. recruited healthy adults from the general population, and 1,131 never smokers, including 845 non-atopic and 286 atopic subjects, were selected for the study.6 Subjects with physician-diagnosed asthma or asthma symptoms, and those using inhaled steroids were excluded. Using a reference equation based on multiple regression modeling, they proposed upper limits of FE_NO ranging from 24.0 ppb to 54.0 ppb depending on age and height.6 This upper limit of FE_NO seems definitely higher than those reported in previous studies and ours. In their study, the subjects having symptoms of rhinitis were not excluded, and this population comprised about 45% of the study subjects. The high prevalence of subjects with nasal symptoms may explain the increased FE_NO levels. In the present study, we estimated the reference ranges for FE_NO using two different statistical methods recommended by the guidelines,18 and this study demonstrated approximately 36.0 ppb as a reasonable upper limit of FE_NO for healthy Japanese adults, a value that is within previously suggested limits for Caucasians.

It has been shown that the mean FE_NO levels in healthy Asian children are significantly higher than those in healthy Caucasian children.15-17 Several factors, such as dietary differences, environmental differences, and genetic variation have been proposed to explain the ethnic differences.15-17 However, these studies included less than 70 subjects who were ethnic minorities, and the reference ranges for FE_NO in each ethnic group were not estimated. In addition, FE_NO was measured according to the current guidelines in only one of these studies.17 Further study with large samples using standardized methods will be necessary to clarify ethnic differences in the FE_NO levels of children.

The normal range for FE_NO is influenced by patient factors. Previous studies have shown that there are several determinants of FE_NO levels, such as age, gender, height, atopy, smoking, and diet.1, 4-17 The finding of a higher FE_NO in subjects with atopy has been reported previously,5, 9-11 while chronically reduced levels of FE_NO have been demonstrated in current smokers.5, 9, 12 Therefore, current smokers and subjects having a history of atopic disease were not included in this study. Furthermore, the FE_NO level has been found to be elevated after the intake of a nitrate-rich meal.13, 14 Thus, the subjects were fasted for one hour before the FE_NO measurements. In the present study, the mean FE_NO level for males was significantly higher than that for females, which is consistent with previous reports.4, 5, 7, 15 However, the upper limits of FE_NO were similar in each gender group as shown in Figure 1. In addition, age, gender, height, and past smoking history were not significant predictors for FE_NO. Although the association between FE_NO and age, gender, height is still controversial in adults,1, 4-9 the present results suggested that these demographics are not critical factors in establishing the normal range for FE_NO.

In the present study, subjects were recruited based on interview. Although the subjects with a history of atopic disease were carefully excluded according to the guidelines,19, 20 we made no attempt to validate information by skin prick testing. However, a detailed interview is a basic approach to investigate individual demographics, and it will not always be practical for clinicians to perform skin prick testing before FE_NO measurements. In clinical practice, the proposed reference data would be useful for interpretation of the FE_NO measurements as a reasonably approximate value. Additionally, the reference ranges for FE_NO in Caucasians adults were analyzed by different measurement system, chemiluminescene analyzer.5-7 However, it has been shown that FE_NO values measured by the electrochemical analyzer are reproducible, reliable, and in agreement with the chemiluminescene analyzer.21-24

In summary, the reference ranges for FE_NO in healthy Japanese adults were similar to those of Caucasians. It seems reasonable that the upper limits of FE_NO for healthy adults should be set at approximately 36.0 ppb irrespective of ethnic differences.

ACKNOWLEDGEMENTS

We thank Mr. Brent Bell for reading of the manuscript, and also thank Mr. Satoru Fukinbara for helping in statistical analysis.

This work was supported by grant H20-meneki-ippan-012 from the Ministry of Health, Labour and Welfare, Japan.

REFERENCES

1
American Thoracic Society; European Respiratory Society. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide. Am J Respir Crit Care Med 2005; 171: 912-30.

2
Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6: 1368-70.
 

3
Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133-5.
 

4
Olivieri M, Talamini G, Corradi M et al. Reference values for exhaled nitric oxide (REVENO) study. Respir Res 2006; 7: 94.

5
Travers J, Marsh S, Aldington S et al. Reference ranges for exhaled nitric oxide derived from a random community survey of adults. Am J Respir Crit Care Med 2007; 176: 238-42.
 

6
Olin AC, Bake B, Toren K. Fraction of exhaled nitric oxide at 50 mL/s: reference values for adult lifelong never smokers. Chest 2007; 131: 1852-6.

7
Kharitonov SA, Gonio F, Kelly C, Meah S, Barnes PJ. Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children. Eur Respir J 2003; 21: 433-8.
 

8
Taylor DR, Pijnenburg MW, Smith AD, De Jongste JC. Exhaled nitric oxide measurements: clinical application and interpretation. Thorax 2006; 61: 817-27.
 

9
Olin AC, Rosengren A, Thelle DS, Lissner L, Bake B, Toren K. Height, age, and atopy are associated with fraction of exhaled nitric oxide in a large adult general population sample. Chest 2006; 130: 1319-25.

10
Gratziou C, Lignos M, Dassiou M, Roussos C. Influence of atopy on exhaled nitric oxide in patients with stable asthma and rhinitis. Eur Respir J 1999; 14: 897-901.
 

11
Salome CM, Roberts AM, Brown NJ, Dermand J, Markes GB, Woolcock AJ. Exhaled nitric oxide measurements in a population sample of young adults. Am J Respir Crit Care Med 1999; 159: 911-6.
 

12
Persson MG, Zetterstrom O, Agrenius V, Ihre E, Gustafsson LE. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 1994; 343: 133-5.

13
Zetterquist W, Pedroletti C, Lundberg JO, Alving K. Salivary contribution to exhaled nitric oxide. Eur Respir J 1999; 13: 327-33.
 

14
Olin AC, Aldenbratt A, Ekman A et al. Increased nitric oxide in exhaled air after intake of a nitrate-rich meal. Respir Med 2001; 95: 153-8.
 

15
Wong GWK, Liu EKH, Leung TF et al. High levels and gender difference of exhaled nitric oxide in Chinese schoolchildren. Clin Exp Allergy 2005; 35: 889-93.
 

16
Kovesi T, Kulka R, Dales R. Exhaled nitric oxide concentration is affected by age, height, and race in healthy 9-to 12-year old children. Chest 2008; 133: 169-75.
 

17
Linn WS, Rappaport EB, Berhane KT, Bastain TM, Avol E, Gilliland FD. Exhaled nitric oxide in a population-based study of Southern California schoolchildren. Respir Res 2009; 10: 28.

18
Solberg HE. The International Federation of Clinical Chemistry recommendation on estimation of reference intervals. Clin Chem Lab Med 2004; 42: 710-4.
 

19
National Heart, Lung, and Blood Institute/World Health Organization. Global Initiative for Asthma, Global Strategy for Asthma Management and Prevention, Updated 2006. Bethesda: NIH Publication, 2006.

20
Bousquet J, Khaltaev N, Cruz AA et al, and World Health Organization; GA(2)LEN; AllerGen. Allergic Rhinitis and its Impact on Asthma(ARIA)2008 update in collaboration with the World Health Organization, GA(2)LEN and AllerGen. Allergy 2008; 63 (Suppl 86): 8-160.

21
Alving K, Janson C, Nordvall L. Performance of a new hand-held device for exhaled nitric oxide measurement in adults and children. Respir Res 2006; 7: 67.
 

22
Menzies D, Nair A, Lipworth BJ. Portable exhaled nitric oxide measurement: Comparison with the "Gold standard" technique. Chest 2007; 131: 410-4.
 

23
Pizzimenti S, Bugiani M, Piccioni P et al. Exhaled nitric oxide measurements: Correction equation to compare hand-held device to stationary analyzer. Respir Med 2008; 102: 1272-5.

24
Boot JD, de Ridder L, de Kam ML, Calderon C, Mascelli MA, Diamant Z. Comparison of exhaled nitric oxide measurements between NIOX MINO electrochemical and Ecomedics chemiluminescene analyzer. Respir Med 2008; 102: 1667-71.