At age 32, Study members were questioned about current and previous asthma, as well as symptoms of wheezing, cough, episodic shortness of breath, hay fever and rhinitis. Current asthma was defined as reported diagnosed asthma with symptoms in the last 12 months. Current wheezing was recorded as any wheeze in the last 12 months but excluding subjects with only one or two episodes each lasting less than 1 hour. Asthma treatment was any inhaled bronchodilator, corticosteroid or cromoglycate medication. Current smoking was defined as smoking tobacco cigarettes daily for at least one month during the previous 12 months, or smoking cannabis 6 or more times during the previous 12 months. Current smokers were further subdivided into two groups: those who did and did not smoke on the study day. Ex-smokers were defined as having discontinued for at least 12 months.

Height and weight in light clothing without shoes were measured to calculate body mass index (BMI) in kg/m². F_ENO was then measured on-line using a Logan LR 2000 series chemiluminescence analyser (Logan Research Ltd., Rochester, England) in accordance with ATS/ERS guidelines at a flow rate of 50 mL/sec. [22] Exhaled nitric oxide in parts per billion (ppb) was recorded continuously throughout expiration. Individual results were read at the first nitric oxide plateau and the mean of two acceptable tests was recorded. A third was obtained only where one or both of the first two were considered to be technically unsatisfactory. The NO recording was determined for each test by two observers on a separate occasion. The first 44 Study members were tested using a flow rate of 250 mL/second and results were adjusted to 50 mL/second using a previously validated formula [23].

F_ENO measurements were obtained immediately prior to carrying out spirometry. Skin prick testing included house dust mite (D. pteronyssinus), grass, cat, dog, horse, cockroach, wool, Aspergillus fumigatus, alternaria, penicillium, and cladosporium. A weal diameter 3 mm or greater than the saline control was considered positive. Atopy was defined as a positive response to one or more allergens. A blood sample was obtained for eosinophil count and total serum immunoglobulin E (IgE).

The F_ENO values obtained from the Study population are shown in Table ​Table1.1. Data relating to smoking status are shown in Table ​Table2.2. F_ENO was on average 25% higher in males than females (males: 15.3 ppb [95% C.I.:14.3–16.3] versus females: 11.6 ppb [95% C.I.: 11.0–12.4]; p < 0.0001; Table ​Table1).1). Unadjusted analyses revealed that, for all Study members, there were also significant differences in F_ENO in relation to height, FEV₁, FEV₁ % predicted and FVC (Table ​(Table3).3). However, none of these factors remained significant after stratifying by sex. In contrast, current smoking (on the day of testing), atopy, log IgE, history of rhinitis, and current asthma and the use of inhaled corticosteroids remained significant in both males and females.

In the adjusted regression analyses, significant predictors of F_ENO were sex, body mass index (BMI), current smoking (on the day of testing), atopy, current asthma, and the interaction between sex and smoking. Current wheeze was not a significant factor. This was perhaps because of the significant confounding relationship between current smoking (resulting in reduced F_ENO) and wheeze (p = 0003; model 4; see Additional File 1). Details of all the models examined are provided in Additional File 1.

After controlling for all of the significant factors affecting F_ENO, the sex-related differences in F_ENO remained significant (p < 0.001). The factors which significantly affected F_ENO were different for males and females. For males, current smoking (all), current asthma, and atopy (any positive SPT ≥ 3 mm over the negative control) were significant independent predictors of F_ENO. For females, while current smoking (all), current asthma, and atopy (any positive SPT ≥ 3 mm over the negative control) were significant independent predictors of F_ENO, those females who were current smokers and also had asthma had an additional increase in their F_ENO (F_ENO increased by 131%; p = 0.001; Table ​Table44).

Based on these results, the equations for predicting F_ENO in our study cohort were:

For males: log F_ENO = 1.1932 - 0.3496*current smoking (smoked day of testing) - 0.0940*current smoking (not smoked day of testing) + 0.16511*atopy + 0.0973*asthma

(R² = 0.3434)

For females: log F_ENO = 1.0533 - 0.2407*current smoking (smoked day of testing) - 0.1160*current smoking (not smoked on day of testing) + 0.0388*asthma + 0.1355*atopy + 0.0531*current smoking (smoked day of testing)*asthma + 0.3630*current smoking (not smoked day of testing)*asthma

(R² = 0.2760)

where, for the terms current smoking, atopy and asthma, yes = 1, and no = 0.

Using these equations, the predicted values and ranges (95% C.I.) for clinically important populations are presented in Table ​Table5.5. For comparison, the actual values obtained from each subgroup of the study population are also presented.

A review of the literature provides somewhat conflicting data regarding this issue. It is important to take account of the different methodologies used for F_ENO measurements when making comparisons between studies, particularly with regard to expiratory flow rates. However, within studies, significant differences between males and females will still be valid, and the balance of evidence suggests that sex-related differences in F_ENO are indeed important. In early investigations, both Jilma et al. [25] and Tsang et al. [26] reported sex-related differences in F_ENO whose magnitude (50% and 53% higher in males compared to females, respectively) was comparable to the present result. More recently, Olivieri et al. have reported higher levels in males, with an upper limit of normal of 28.8 ppb, compared to 21.5 ppb for females [27]. Travers et al. [19] reported that the mean F_ENO in males was 23% higher than in females (95% C.I. 7–43; p = 0.004, n = 191). In that study, the significance of the difference persisted even after controlling for height. In the study by Berry et al. a similar highly significant difference between males and females was recorded [28]. However, in the largest study to date to focus on factors affecting F_ENO, comprising 2,200 subjects, Olin et al. has presented contrasting results [18]. Although there was a male-female F_ENO difference in non-smokers amounting to 19%, this was not statistically significant in a multiple linear regression analysis in which adjustments for all other factors were included [18]. The reasons why the difference failed to reach statistical significance are unclear.

After adjusting for sex, we found that other anthropometric factors such as height and lung function were no longer significant factors affecting F_ENO. Previously it has been argued that sex-related differences in F_ENO result from differences in the surface area of airway epithelium, the major source of exhaled NO, and for which height is an important anthropometric correlate. Thus our results are perhaps surprising. However, given that plasma levels of nitrate, a product of NO metabolism, are similarly different between the sexes [25,29], it seems unlikely that NO production in the airways is solely a reflection of differences in airway size, but rather reflects sex-related differences in endogenous NO production. This is consistent with the results of a twin study, which showed that genetic rather than environmental factors are more important in determining F_ENO [30].

Our findings raise the question as to whether guidelines for the interpretation of F_ENO should be stratified by sex, and that reference ranges for males and females should be different. In the paper by Olivieri et al. the authors propose that reference ranges should be stratified for sex [27]. Travers et al. [19] advocate reference ranges based on sex, smoking status and atopy, but not age or height. We concur with this view, and the reference ranges contained in Table ​Table55 of the present paper are based on this approach. In the study by Olin et al. [18], similar to conventional pulmonary function tests, both age and height but not sex, were deemed to be significant, although reference values as such were not provided. All studies concur that smoking and atopy are important considerations, and both are included in the reference values given here and by Travers et al. [19].

In fact, interpreting F_ENO levels in clinical practice is even more complex. Reference values which take into account background characteristics such as sex, atopy and smoking may indeed be useful in guiding the diagnosis of airways-related symptoms. In asymptomatic individuals, it is still possible that increased F_ENO reflects subclinical airway inflammation [7,28], but this interpretation is less likely if appropriate reference values which take factors such as sex into account have been used in the first place. The interpretation of F_ENO levels in the context of ongoing management of diagnosed asthma is far from clear. Despite optimal anti-inflammatory treatment, F_ENO levels may remain resolutely high [31], and it is generally agreed that normalizing F_ENO in relation to reference values for a healthy population is not a desirable therapeutic aim [8]. This point is perhaps reflected in the results obtained in the present study, which showed that in non-smoking, atopic, male asthmatics, who were all clinically stable, the upper limit of the 95% confidence interval was 38.8 ppb, considerably higher than the levels obtained in non-smoking, atopic, male, non-asthmatics (28.2 ppb).

One of the weaknesses of our study is that the F_ENO measurements were obtained in individuals who were all aged 32 years. Thus it was not possible to explore the influence of age as a factor in the regression analyses or to conclude whether reference values ought to include it as a factor. Previous studies have reported that F_ENO rises with increasing age in children [15,16,32-34]. In adults, Olin et al. [18] have reported that an effect of age also occurs: F_ENO was shown to increase over the age range 35 to 65 years, with the magnitude of effect similar to that of atopy. In contrast, in the study by Travers et al. no significant relationship was noted over a similar age range, but numbers were much smaller [19]. In children, it is suggested that the changes with age are attributable to increasing airway NO flux, probably reflecting larger airway surface area with growth [34]. If at all, any increase in F_ENO with age in adults is likely to be due to non-anthropometric factors, and if the results from Olin et al. are repeatable, this may be an important consideration.

F_ENO fraction of nitric oxide in expired air

FEV₁ forced expiratory volume in one second

FVC forced vital capacity

NO nitric oxide

mL/sec milliliters per second

ppb parts per billion

Piush Mandhane conducted statistical analysis of the results and contributed to writing the manuscript

Justina M Greene conducted statistical analysis of the results

Robert J. Hancox directed and supervised the conduct of the study and data collection

Christene R McLachlan provided technical assistance in the collection of study data

Avis J Williamson provided technical assistance in the collection of study data

Sue Filsell provided technical assistance in the collection of study data

Jan O Cowan provided technical assistance in the collection of study data

Andrew D Smith provided technical assistance in the collection of study data

Malcolm R Sears provided general study oversight and contributed to writing the manuscript