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* From the Departments of Occupational and Environmental Medicine (Drs. Olin and Torén), and Respiratory Medicine and Allergology (Dr. Bake), Sahlgrenska University Hospital, Göteborg, Sweden.
Correspondence to: Anna-Carin Olin, MD, Department of Occupational and Environmental Medicine, Sahlgrenska University Hospital, Box 414, SE 405 30 Göteborg, Sweden; e-mail anna-carin.olin{at}amm.gu.se
Abstract
Background: The measurement of fractional exhaled nitric oxide (FENO) is used as a marker of airway inflammation. The aim of this study was to establish reference values of FENO for adults.
Methods: FENO at a flow rate of 50 mL/s was analyzed in 3,376 adults using a chemiluminescence analyzer according to American Thoracic Society/European Respiratory Society guidelines. Blood samples were analyzed, and atopy was defined as the presence of specific IgE. All subjects responded to a respiratory questionnaire. Those who had never smoked (n = 1,803) were selected for this study. After the exclusion of subjects with physician-diagnosed asthma, asthma symptoms, ever wheezing, dry cough, or use of inhaled steroids, 1,131 healthy never-smokers remained, including 845 nonatopic and 286 atopic subjects.
Results: Based on multiple regression modeling, we propose the following reference equation for healthy never-smoking adults: Ln(FENO) = 0.057 + 0.013 x height (in centimeters) + 0.0088 x age (in years). The residual SD was 0.51, and the explanatory value was 9%. In a model, based on nonatopic subjects alone, the reference equation obtained was slightly different, as follows: Ln(FENO) = –0.0026 + 0.013 x height (in centimeters) + 0.010 x age (in years). The residual SD for this equation was 0.48, and the explanatory value was 11%.
Conclusions: Normal values of FENO for adults may be predicted on the basis of age and height. However, as the reference equations only account for about 9 to 11% of the variation, the most important information that could be extracted from the study is that the upper limits of FENO range from 24.0 to 54.0 parts per billion, depending on age and height.
Key Words: allergy • asthma • practice
Nitric oxide (NO) in exhaled air was first described in 1991 by Gustafsson et al1 and a few years later it was shown that asthmatics had increased levels.2 The European Respiratory Society and the American Thoracic Society have jointly published recommendations3 regarding the procedures for the measurement of the fraction of exhaled NO (FENO), and these recommendations provide a basis for comparable results and for establishing useful reference values. There are reports regarding reference values of exhaled NO in children, young adults, and healthy students and teachers.45678 However, we are not aware of any general population study among adults presenting reference values of FENO at the recommended exhalation flow rate of 50 mL/s. There is a known need for such values.39
We have recently published10 findings from a large random general population sample of adults showing that height and age, but not sex, were associated with FENO at the recommended exhalation flow rate of 50 mL/s. Furthermore, asthma symptoms, the reported use of inhaled steroids, and atopy were positively and independently associated with FENO, while there was a negative association with smoking. The aim of the present study was to establish reference values for FENO at an exhalation flow rate of 50 mL/s for healthy adult lifelong never-smokers using data from an ongoing study of a large random population in Göteborg, Sweden.
Materials and Methods
A general population sample of men and women who were 25 to 75 years old was randomly selected from the population register in Göteborg, Sweden. Between April 2001 and November 2005, FENO was investigated in 3,376 adult subjects. All subjects received a postal questionnaire and an invitation to undergo a clinical examination, as previously described.10 Those who had never smoked (n = 1,803) were selected for this study. Of these, 507 subjects reporting physician-diagnosed asthma,11 asthma symptoms, ever wheezing, dry cough, or the use of inhaled steroids were excluded. Weight data were missing in a further seven subjects, and consequently 1,131 healthy never-smokers remained for the present study.
FENO was measured with the subject in a seated position using a chemiluminescence analyzer (NIOX; Aerocrine AB; Stockholm, Sweden) during a single slow exhalation against an oral pressure of 5 cm H2O.12 Measurements were taken for 10 s aiming at an exhalation flow rate of 50 mL/s (±10%) between seconds 6 and 10 of the exhalation phase. All measurements were performed in duplicate, all within 10% deviation, and the mean concentration (in parts per billion [ppb]) was registered.3 All subjects had previously fasted for 4 h.13 A two-point calibration of the chemiluminescence analyzer was performed every 14 days with a certified NO calibration gas, according to the standards of the manufacturer.
Spirometry was performed with a dry wedge spirometer (Vitalograph; Buckingham, UK), and percentages of predicted values of lung function variables (ie, FEV1, FVC, and FEV1/FVC ratio) were calculated.14 NO measurements were obtained before spirometry. Blood samples were analyzed for specific IgE (Phadiatop; Pharmacia & Upjohn Diagnostics; Uppsala, Sweden). Class 0 was regarded as negative, and class 1 as positive (atopic).15 Subjects were weighed, and height was measured with participants wearing in light clothes and without shoes. The study was approved by the ethics committee of Göteborg University and all subjects gave their informed consent.
Statistical Analysis
Statistics were calculated using a statistical software package (SAS, version 8; SAS Institute; Cary, NC). Since the distribution of FENO was log-normally distributed, all analyses were performed on log-transformed values of FENO. In the univariate analyses, the geometric means with 95% reference intervals (RIs) were calculated. The RI was assessed as the geometric mean ± 1.96 SDs.
The reference equations were obtained from multiple linear regression modeling. From the predictors selected a priori, the variables for the final regression model were chosen using backward stepwise regression analysis. The lower and upper reference limits for the predictor were determined by calculating the 2.5th and 97.5th percentiles, which equaled the predicted value ± 1.96 x RSD (where RSD is the residual SD of the regression equation). In addition, the upper 95% cutoff limits were calculated from the regression model based on the whole population, including both atopic and nonatopic subjects.
Results
Table 1 presents basic data about the subjects. The geometric mean of FENO for the whole population was 16.6 ppb (95% RI, 5.87 to 47.14). FENO was higher among subjects with atopy, 18.8 ppb (95% RI, 6.03 to 58.74) compared to 16.0 ppb (95% RI, 5.91 to 58.76) among nonatopic subjects. FENO was higher among men (18.5 ppb; 95% RI, 16.67 to 51.13) compared to women (14.9 ppb; 95% RI, 5.36 to 41.52). However, when comparing men and women with similar height and age, FENO was similar between the sexes.
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Discussion
This study gives reference equations for predicted normal FENO for adults, based on 1,131 healthy never-smokers from a random general population sample. As previously shown, age and height are significant explanatory variables, but sex is not when height is allowed for. Our study contributes new knowledge as it is the first study that has outlined reference values according to age and height among adults.
The equations have a low explanatory value resulting in wide RIs. The equations explained only 9 to 12% of the variation, which could be compared to the prediction equations of FEV1 where height, age, and sex explain 50 to 70% of the variation. Hence, there is much remaining variation that is unexplained, and several factors may be involved in addition to methodological factors, for example, subclinical airway inflammation of various causes, nutritional history,13 and genetic factors.16
We suggest that the upper normal values of FENO in never-smoking adults, irrespective of the presence of atopy, range from 24.0 to 53.0 ppb, depending on age and height. In the same age class (35 to 44 years of age), the upper limit of FENO increased from 24.5 ppb among short subjects (ie, height < 160 cm) to 42.5 ppb in subjects 180 to 189 cm in height. A recent review9 suggested 33 ppb as a reasonable upper limit of FENO for adults, a value that is within our suggested limits.
To our knowledge, there have been only two previous studies816 of FENO aimed at establishing reference values for healthy adults. One study comprised a sample of 48 healthy subjects, but it is unclear how this sample was obtained and whether nonsymptomatic atopic subjects were included.16 Among subjects > 60 years of age, the median FENO was 36.9 ppb, and among subjects < 30 years of age it was 18.7 ppb. In an Italian study,8 204 healthy students and teachers from the medical faculty were investigated, and the 95th percentile for FENO was 19.7 ppb. Among children, there have been several studies467 presenting normal values for FENO, including one study7 that presented a regression equation. After regression modeling, the authors found that height was the only predictor to be included in their final model. The presented model was FENO = e(0.64 + height x 0.011), which is not dissimilar to our models. Baraldi et al4 established reference values for FENO from a large sample of healthy children; however, they did not present any reference equation, making comparisons with our results difficult. Buchvald et al6 investigated 405 children and found upper 95% cutoff limits ranging from 15.7 ppb at 4 years to 39.2 ppb at 14 to 17 years. These limits included both atopic and nonatopic subjects.6
We think it is of importance to have reference values for an unselected, healthy, never-smoking population. The smoking history was based on questionnaires, and we made no attempt to validate smoking information by the measurement of cotinine levels in urine or carbon monoxide levels in blood. We defined a healthy population as subjects with no history of asthma; no reporting of asthma symptoms, wheezing, or dry cough; or use of inhaled steroids. This resulted in the exclusion of 507 of 1,803 originally never-smoking selected subjects. We did not exclude those subjects reporting having "symptoms of rhinitis without having a cold after the age of 15 years." This comprised about 45% of the subjects, and they did not, on the group level, have increased FENOs. As we did include both atopic and nonatopic subjects, some subjects with allergic rhinitis were included. Also, as measurements were performed both during and outside the pollen season, some of these subjects may have had increased levels of FENO. Equation 1 shows that atopic subjects have 23% higher FENOs (e0.21 = 1.23). The inclusion of subjects reporting rhinitis might be one explanation for this, but not the only one. Also, when excluding all subjects reporting rhinitis, nonsymptomatic atopic subjects had higher FENOs when the coefficient in the reference equation was 0.20.
Whether to include nonsymptomatic subjects with atopy in a healthy reference population is not clear cut. At present, there is only limited knowledge of what it means to produce increased amounts of specific anti-IgE antibodies without having symptoms, it certainly does not imply the presence of disease, but may rather mean that these subjects are protected against symptoms by T regulatory cells or other, as-yet-unknown factors.17
Our large sample size also allowed us to present normal values (equation 3) for solely nonatopic subjects. The changes in the predicted equation and the upper cut-off limits differ only marginally compared to those obtained from the whole population (the estimate for age increased from e0.088 to e0.013). By using equation 3, we also calculated the 95% upper limits of FENO. However, these limits were only marginally different (< 0.5 ppb) from the limits already presented for the whole population and will hence not be presented.
We have also performed a separate analysis including atopy in the model (Table 2, equation 1). There is, however, no straightforward way to use atopy in regression modeling. In our study, we have defined atopy as being positive for IgE (Phadiatop; Pharmacia & Upjohn Diagnostics), but there are other several other approaches such as the presence of positive skin prick test results, self-reported manifestations, or high concentrations of total IgE. Formally, this regression equation is only applicable if there is information on specific IgE concentrations (Phadiatop; Pharmacia & Upjohn Diagnostics), but the atopy term can probably also be applied using other definitions of atopy. However, when the clinician (ie, general practitioner) initially examines a subject with suspected asthma and wants to use FENO as a diagnostic tool, information about atopy is often missing. Hence, we propose the use of reference values including both atopic and nonatopic subjects, which is given by equation 2. For atopic subjects, this implies that the cut-off limits are lower than if they were based on only atopic subjects, and that the number of atopic subjects classified as having pathologic FENOs will be increased. However, atopy is of less importance for asthma onset among adults than among children.1819
In conclusion, normal values of FENO for adults may be predicted on the basis of age and height. However, the reference equation only accounts for about 9 to 12% of the variation. The most important information that could be extracted from the study is that the upper limits of FENO range from 24.0 to 54.0 ppb, depending on age and height.
Footnotes
Abbreviations: FENO = fraction of exhaled nitric oxide; NO = nitric oxide; ppb = parts per billion; RI = reference interval
The study was supported by the Swedish Council for Work Life and Social Research (FAS), the Swedish Heart and Lung Foundation and Astra-Zeneca, Sweden.
The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
Received for publication December 5, 2006. Accepted for publication March 2, 2007.
References
scher, R, et al Effects of sex and of gene variants in constitutive nitric oxide synthases on exhaled nitric oxide. Am J Respir Crit Care Med 2003;167,1113-1116This article has been cited by other articles:
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  K. G. Lim and C. Mottram The Use of Fraction of Exhaled Nitric Oxide in Pulmonary Practice Chest, May 1, 2008; 133(5): 1232 - 1242. [Abstract] [Full Text] [PDF]
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N. M. Grob and R. A. Dweik Exhaled Nitric Oxide in Asthma: From Diagnosis, to Monitoring, to Screening: Are We There Yet? Chest, April 1, 2008; 133(4): 837 - 839. [Full Text] [PDF]
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M. Olivieri, M. Malerba, G. Talamini, and M. Corradi Reference Values for Exhaled Nitric Oxide in the General Population Chest, March 1, 2008; 133(3): 831 - 832. [Full Text] [PDF]
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A.-C. Olin, B. Bake, and K. Toren Exhaled Nitric Oxide and Gender Chest, October 1, 2007; 132(4): 1410 - 1410. [Full Text] [PDF]
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