Determinants of exhaled nitric oxide levels in healthy, nonsmoking African American adults

Determinants of exhaled nitric oxide levels in healthy, nonsmoking African American adults Marc C. Levesque, MD, PhD,a David W. Hauswirth, MD,c Sabrena Mervin-Blake, MS,a Carolyn A. Fernandez, BS,a Kistie B. Patch, MS,a Katie M. Alexander, BS,a Sallie Allgood, MS,a Patrice D. McNair, MS,a Andrew S. Allen, PhD,d and John S. Sundy, MD, PhDa,b Durham, NC Background: Asthma is a significant cause of morbidity and mortality for African Americans. Fraction of exhaled nitric oxide (FeNO) levels are increased in patients with asthma, and airway levels of nitric oxide metabolites regulate airway inflammation and airway diameter. More needs to be known about the factors that regulate FeNO. There is a need for FeNO reference values for African Americans. Objective: We sought to establish reference values and identify factors associated with FeNO levels in healthy African American adults. Methods: FeNO levels were measured in 895 healthy, nonsmoking African Americans between the ages of 18 and 40 years. FeNO measurements were repeated in 84 subjects. Factors potentially associated with FeNO were measured, including blood pressure, height, weight, and serum total IgE, eosinophil cationic protein, C-reactive protein, and nitrate levels. Data on respiratory symptoms, including upper respiratory tract infection (URI) symptoms, were collected. Univariate and multivariate analyses of the relationship between these variables and FeNO levels were performed. Results: In healthy, nonsmoking African Americans FeNO levels were stable during repeated measurements (intraclass correlation coefficient, 0.81). Sex (P < .0001), serum total IgE levels (P < .0001), and current URI symptoms (P 5 .0002) contributed significantly to FeNO variability but together accounted for less than 50% of the variation in FeNO levels. Conclusion: The high correlation between repeated measurements of FeNO and the low correlation coefficients of known factors associated with FeNO suggest that other factors might contribute substantially to variability of FeNO levels in African Americans. (J Allergy Clin Immunol 2008;121:396-402.) Key words: African American, human subjects, nitric oxide, respiratory function tests, IgE, respiratory tract infections From athe Department of Medicine, Division of Rheumatology and Immunology; bthe Division of Pulmonary, Allergy and Critical Care Medicine; cthe Department of Pediatrics, Division of Allergy and Immunology; and dthe Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham. Supported by the Sandler Program for Asthma Research; ES011185 from the National Institute of Environmental Health Sciences; and MO1-RR-30 from the National Center for Research Resources, Clinical Research Centers Program, National Institutes of Health. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication July 30, 2007; revised September 7, 2007; accepted for publication September 11, 2007. Available online November 26, 2007. Reprint requests: Marc C. Levesque, MD, PhD, Duke University Medical Center, Box 3266, Durham, NC 27710. E-mail: [email protected]. 0091-6749/$34.00 Ó 2008 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.09.031

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Abbreviations used ATS: American Thoracic Society BMI: Body mass index BP: Blood pressure CRP: C-reactive protein ECP: Eosinophil cationic protein FeNO: Fraction of exhaled nitric oxide NO: Nitric oxide NOA: Nitric oxide analyzer NOS2: Inducible or type II nitric oxide synthase URI: Upper respiratory tract infection

Asthma is a significant cause of morbidity and mortality for African Americans, and asthma is increasing in prevalence.1,2 Fraction of exhaled nitric oxide (FeNO) levels are increased in asthma and associated with airway inflammation and upregulation of inducible nitric oxide synthase (NOS2) in respiratory epithelial cells.3-6 Atopy without asthma is associated with increased FeNO levels,5,6 which is consistent with the presence of lower airway inflammation in nonasthmatic atopic subjects.7,8 Therapy-mediated decreases in FeNO levels in asthmatic subjects are associated with improved control of asthma symptoms.9,10 Studies of asthmatic subjects reveal a dynamic relationship between NO synthase enzyme activity, FeNO levels, and airway lining fluid nitric oxide (NO) metabolites that regulate airway tone and inflammation.11-13 Therefore defining the factors and understanding the mechanisms that regulate FeNO levels in normal and diseased airways are essential to understanding the role of NO in the pathogenesis of asthma and allergen-mediated airway disease. There is a significant need for reference values for FeNO in different populations.14,15 In patients with asthma and atopy, serum total and allergenspecific IgE levels, serum eosinophil cationic protein (ECP) levels, and peripheral blood eosinophils are all associated with increased FeNO levels.16-19 In healthy adult subjects male sex, height, upper respiratory tract infection (URI), and nitrate intake have been associated with increased FeNO levels,20-27 whereas cigarette smoking28 and hypertension29 have been associated with low FeNO levels. There are conflicting reports about whether age is associated with differences in FeNO levels in adults,20,27 whereas in children there is more consensus that increasing age is associated with higher FeNO levels.6,18 Recent studies identified a direct relationship between serum total IgE and FeNO levels in healthy white subjects.27,30 To our knowledge, large studies of FeNO levels in healthy African American subjects have not been reported. Given the high prevalence and significant morbidity associated with

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asthma in African Americans1,2 compared with that in white subjects, there is a significant need to establish normative FeNO levels and to determine the relationship between basal FeNO production, IgE production, and the development and severity of asthma in African Americans. Therefore we collected and analyzed demographic, physiologic, and serum data from 895 healthy, nonsmoking, young (age range, 18-40 years) African American adults. Our analysis indicated that FeNO levels were stable during repeated measurements. Serum total IgE levels, sex, and URI symptoms contributed significantly to the variability in FeNO levels in healthy subjects, but together these factors accounted for less than 50% of the variation in FeNO levels. This suggests that unknown factors might contribute substantially to the variability of FeNO levels in healthy African Americans.

METHODS Subject recruitment and enrollment Healthy subjects between the ages of 18 and 40 years were recruited from students and employees at 4 local university campuses. The study was originally powered to target an enrollment of 1000 African American subjects (994 were actually enrolled, and 99 were excluded because of smoking; n 5 895). Informed consent was obtained as part of a protocol approved by the Duke University Institutional Review Board. After obtaining informed consent, subjects were asked to provide their birth dates and declare that they were healthy (ie, no chronic illnesses or chronic use of any medication except oral contraceptives); that they had no history of asthma, allergic rhinitis, hay fever, or atopic dermatitis; that they were nonsmokers; and that they were of African ancestry. Ethnicity was based on a self-declared description of ethnicity and determined by using a questionnaire based on that developed by the US Census bureau (http://www.census.gov/ dmd/www/2000quest.html). The ethnicity questionnaire asked subjects to declare whether they were Hispanic (Spanish, Latino, or Hispanic) or non-Hispanic and whether they were African American, Asian, white, Native American, Pacific Islander, or some other race or ethnicity. Only non-Hispanic African American subjects were enrolled in the study. Blood samples for preparation of serum were obtained. Height, weight, and body mass index (BMI; in kilograms per square meter) were determined. Blood pressure (BP) measurements were performed by using an Intellisense HEM-907 Digital Blood Pressure Monitor (Omron Healthcare, Vernon Hills, Ill). Four BP measurements with an appropriately sized cuff were obtained 1 minute apart, with the subject resting for at least 5 minutes before the first measurement. Hypertension was defined as a systolic BP of 140 mm Hg or greater, a diastolic BP of 90 mm Hg or greater, or both. FeNO levels were measured, and URI and American Thoracic Society (ATS) questionnaires were administered, as described below. BP determinations and questionnaire data were not collected from all subjects in the cohort (n 5 491 and n 5 457, respectively) because the questionnaires and BP instrument were not available at the time of FeNO measurements for some subjects.

Measurement of FeNO levels Online FeNO levels were measured in triplicate and averaged with a Sievers 280i Nitric Oxide Analyzer (NOA; GE Analytical Instruments, Boulder, Colo), according to the manufacturer’s instructions. Triplicate FeNO measurements had to have less than 10% variance to be included in the average. The NOA was calibrated each day before use. FeNO levels were measured according to ATS recommendations31 at a flow rate of 50 mL/s and against enough resistance to maintain an oropharyngeal pressure of at least 5 cm of H2O. Ambient air NO was excluded by inclusion of an activated charcoal and potassium hydroxide filter unit attached to the air intake of the NOA flowmeter.

URI and ATS questionnaires A validated URI questionnaire32,33 was administered to subjects at the time of enrollment and measurement of FeNO levels. Self-report of a cold with or without a runny nose, which is highly correlated with a clinical diagnosis of URI,32 was used to dichotimize subjects into groups with and without URI symptoms. A modified ATS questionnaire34 was also administered at the time of enrollment. The modified questionnaire collected information on smoking, cough, phlegm production, wheezing, dyspnea, and asthma history. The questionnaire also collected information about rhinitis, eczema, and other allergy symptoms. Answers from the questionnaire were used to validate subject self-report of the absence of asthma, eczema, and allergic rhinitis symptoms.

Serum ECP, C-reactive protein, total IgE, and nitrate measurements Serum ECP and C-reactive protein (CRP) levels were determined by means of ELISA with kits from MBL International (Woburn, Mass) and Alpha Diagnostic (San Antonio, Tex), respectively, according to the manufacturers’ instructions. The CRP ELISA is a high-sensitivity ELISA and results in CRP measurements comparable with those determined by means of nephelometry (http://www.4adi.com/kits/hormones/1000CRP.html). Total serum IgE levels were measured by using the Pharmacia CAP System (IgE FEIA; Pharmacia Diagnostics, Uppsala, Sweden). Serum nitrate levels were determined with a Sievers 280i NOA, according to the manufacturer’s instructions.

Determination of subject smoking status by using serum cotinine levels Serum cotinine levels were determined by using an ELISA-based assay from OraSure Technologies (Bethlehem, Pa). Serum cotinine levels were used to separate subjects into nonsmokers (<25 ng/mL, n 5 895) and smokers (25 ng/mL, n 5 99). Smokers were excluded from analyses.

Statistical analyses The JMP statistical program (version 5.0.1a; SAS Institute, Cary, NC) was used for all statistical analyses. Continuous variables (FeNO levels, age, BP, height, weight, BMI, total IgE levels, ECP levels, CRP levels, and nitrate levels) were not normally distributed (Shapiro-Wilk W test) and were log normalized before statistical analysis. The intraclass correlation coefficient was used for comparison of repeated FeNO measurements.5,35 The baseline or initial FeNO measurement was used for all other univariate and multivariate analyses. The geometric mean and SD were used for summary statistics. For univariate analyses of dichotomous variables (sex, hypertension, health history questions, and URI symptoms), Student t tests were used for comparisons of FeNO levels. The Pearson product-moment correlation coefficient was determined for linear regression of FeNO levels and age, BP, height, weight, BMI, and total IgE, ECP, CRP, and nitrate levels. Multiple linear regression was performed with variables that had an r2 value of 0.01 or greater and a P value of .1 or less in univariate analyses. All variables meeting these criteria were entered into the multiple linear regression model of FeNO. Variables were added to the FeNO model with forward stepping, and variables with P values of less than .05 were retained in the FeNO model. There were no corrections for multiple testing, and the reported r2 values were unadjusted.

RESULTS Subject characteristics and reproducibility of FeNO measurements The characteristics of the 895 subjects enrolled in the study are listed in Table I. FeNO measurements were repeated for 84 of these subjects between 5 and 850 days apart (median, 143 days

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TABLE I. Patient characteristics and univariate analyses of factors associated with FeNO levels in healthy, nonsmoking African American adults Factor

Mean 6 SD*

Ageà (y) Sex BP MAP (mm Hg) Systolic (mm Hg) Diastolic (mm Hg) Hypertension# Height (cm) Weight (kg) BMI (kg/m2) URI symptoms All** Current Past week Health history   Asthma Rhinitis Eczema Serum Total IgE (kU/L) ECP (ng/mL) CRP (ng/mL) Nitrate (mmol/L)

22 6 4.5 —

P valuey

N

.0764 <.0001{

876k Male 5 271,{ female 5 587

0.0030 0.0135 <0.0001 0.0031 0.0551 0.0075 0.0003

.2271 .0099 .9748 .2151 <.0001 .0096 .6108

491 491 491 Yes 5 26, no 5 465 889 888 888

— — —

0.0188 0.0308 0.0028

.0033 .0002 .2574

Yes 5 95, no 5 362 Yes 5 44, no 5 405 Yes 5 66, no 5 389

— — —

0.0108 0.0012 0.0046

.0278 .4647 .1564

Yes 5 37, no 5 410 Yes 5 86, no 5 354 Yes 5 24, no 5 419

0.1109 0.0170 0.0013 0.0083

<.0001 .0001 .2868 .0499

84 6 9.1 117 6 11.8 68 6 9.9 — 168 6 9.3 77 6 22 27 6 6.9

59 16 0.12 31

r2

6 6 6 6

187 25 0.41 16

0.0036§ 0.0689

871 881 890 464

MAP, Mean arterial pressure. *Geometric mean 6 SD for continuous variables.  P values for continuous variable comparisons were determined by using linear regression and ANOVA. P values for dichotomous variable comparisons were determined by using the Student t test. P values were not corrected for multiple testing. àAll continuous variables were log normalized before analysis. §Unadjusted correlation coefficients (Pearson r2 for continuous variables and coefficient of determination for dichotomous variables). kThere were 895 African American nonsmokers in the study. Complete demographic and serum data on all subjects were not available, and as such, totals for each group of subjects might not equal 895. {P values in boldface are less than .05. #Hypertension is defined as a systolic BP of 140 mm Hg or greater, a diastolic BP of 90 mm Hg or greater, or both. **All URI symptoms included self-report of cold with or without a runny nose on the day of sample collection (current) or within the past week.   Subject self-report of history of or prior diagnosis of asthma, eczema, and/or rhinitis.

apart). There was an excellent correlation between the first and second FeNO measurements for each subject (intraclass correlation coefficient, 0.81; Fig 1).

Factors associated with variability of FeNO levels We used univariate analyses to determine the relationship of FeNO levels to the factors listed in Table I. There were significant differences in FeNO levels between subjects grouped on the basis of sex and URI symptoms and significant correlations between FeNO levels and height, weight, systolic BP, and serum levels of total IgE, ECP, and nitrate (Table I). Male subjects had significantly higher FeNO levels than female subjects (geometric mean 6 SD, 27 6 26 vs 18 6 18 ppb, respectively; 95% CI, 25–29 vs 17–19 ppb, respectively; P < .0001; Table I and Fig E1 in the Online Repository at www.jaci online.org). Height, weight, and systolic BP were all significantly higher in male subjects compared with values in female subjects in our cohort (data not shown). These 3 sex-related factors (height, weight, and systolic BP) directly correlated with FeNO levels (Table I). Other factors, such as BMI, diastolic BP, and hypertension, which were not significantly different between male

and female subjects in our cohort (data not shown), were not associated with differences in FeNO levels. Taken together with the multiple linear regression analysis in Table II (see below), this suggested that sex was likely to be the major factor associated with FeNO levels among this group of variables. Despite selecting subjects who reported the absence of asthma, allergic rhinitis, and/or eczema, a number of subjects reported respiratory and skin symptoms on questionnaires. This is in keeping with other epidemiologic studies in which self-reported symptoms consistent with asthma or allergic rhinitis exceed the reported prevalence of diagnosed asthma and allergic rhinitis.36 Symptoms consistent with asthma, allergic rhinitis, and/or eczema were reported by 8.3%, 19.5%, and 5.4% of subjects, respectively (Table I). Asthma symptoms, but not allergic rhinitis or eczema symptoms, were associated with higher FeNO levels (asthma symptoms vs no asthma symptoms: geometric mean 6 SD, 26 6 33 vs 20 6 20 ppb, respectively; 95% CI, 20–34 vs 19–21 ppb, respectively; P 5 .0278; Table I and see Fig E2, A, in the Online Repository at www.jacionline.org). We determined whether several self-reported factors related to URIs were associated with increased FeNO levels. We found that a subject’s self-report of a cold with or without a runny nose on the day that samples were collected was associated with increased

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FIG 1. Reproducibility of FeNO measurements. FeNO levels were measured, and measurements were repeated 5 to 850 days later (median, 143 days) on 84 subjects (convenience sample). A, Plot of initial FeNO levels versus repeat FeNO levels (r2 5 0.69, Pearson correlation coefficient). B, Plot of difference of repeated FeNO measurements versus mean of repeated measurements (intraclass correlation coefficient [rI], 0.81). The dashed lines represent ideal repeated measurements. ENO, Exhaled nitric oxide.

FeNO levels (self-report of cold symptoms vs no cold symptoms: geometric mean 6 SD, 29 6 24 vs 19 6 20 ppb, respectively; 95% CI, 25–35 vs 18–21 ppb, respectively; P 5.0002; Table I and Fig E2, B, in the Online Repository at www.jacionline.org). Past cold symptoms within the week before sample collection were not associated with increased FeNO levels. The use of the Jackson cold scoring system did not improve our ability to discriminate differences in FeNO levels compared with a self-report of a cold with or without a runny nose on the day of sample collection. For example, no subjects in our cohort had the most restrictive definition of a cold, as defined by Jackson et al (Jackson cold scores, >13),32 and although we found that there was a significantly direct correlation between a subject’s Jackson cold score and FeNO levels, the correlation was relatively weak (r2 5 0.0233, P 5 .0011). We also determined the relationship of serum factors to FeNO levels. In agreement with prior studies,16-19,27 we found that there was a direct correlation of serum total IgE and ECP levels with FeNO levels in our cohort (Table I and Fig E3, A and B, in the Online Repository at www.jacionline.org). We also found a weak direct correlation between serum nitrate levels and FeNO levels (r2 5 0.0083, P 5 .0499; Table I and Fig E3, C).

Multiple linear regression analysis of factors associated with FeNO levels We performed a multiple linear regression analysis of FeNO levels by using variables that had an r2 value of 0.01 or greater and a P value of .1 or less in univariate analyses (Table I). Sex, current URI symptoms, and serum total IgE and ECP levels were significantly associated with FeNO levels in healthy nonsmoking African American subjects in a multiple linear regression model (Table II). In this analysis total IgE levels and sex accounted for most of the variability in FeNO levels. A multiple linear regression analysis of variables in Table II that were measured in all

TABLE II. Multiple linear regression analysis of factors associated with FeNO levels in 429 healthy, nonsmoking African American adults* Factor

Effect

Estimate

r2

P value

Total IgE  Sex Current URI symptoms§ ECP

Direct Male > female Cold > well

0.1654 0.0876 0.0913

0.1481 0.2107à 0.2457

<.0001 <.0001 <.0001

Direct

0.0863

0.2565

.0132

*P < .0001 (ANOVA) for overall multiple linear regression model.  All continuous variables were log normalized before analysis. àCumulative r2 values; that is, r2 values include effects of factor and factors in rows above it in the table. §Current URI symptoms included self-report of cold with or without a runny nose on the day of sample collection.

subjects (as opposed to the subset of 429 subjects in Table II) and that had an r2 value of 0.01 or greater and a P value of .1 or less in univariate analyses (sex and serum total IgE and ECP levels, Table I) indicated that only sex and serum total IgE levels were significantly associated with FeNO levels in our cohort (data not shown).

DISCUSSION We believe that our study represents the largest analysis to date of factors associated with FeNO levels in healthy African Americans. We found significant associations of sex, serum total IgE and ECP levels, and URI symptoms with FeNO levels (Tables I and II). The modest associations of these factors, which together accounted for less than 50% of the variability in FeNO levels, suggest that other unknown factors might contribute substantially to the variability of FeNO levels. Therefore we believe the variation in FeNO levels in African Americans is likely caused by a

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complex interaction of genetic, biologic, and environmental factors. Importantly, our study defined normative values for FeNO in African Americans, and such normative values are important when FeNO measurement is used in the clinical management of asthma.10 The results of our analysis of FeNO levels in healthy African Americans are similar to published findings in white cohorts.27,30 The FeNO studies in white subjects and our study reached nearly identical conclusions on the effect of atopy on FeNO levels and the modest effect of known factors on the variability of FeNO levels. For example, all 3 studies found comparable low correlations for positive allergen skin test responses or serum total IgE levels and FeNO levels. In contrast to the study by Olin et al,27 Travers et al30 concluded that sex and not sex-associated factors, such as height and weight, was more likely to be associated with FeNO level variation. In our multivariate analysis we found that sex, rather than height, better explained variation in FeNO levels. In addition to the difference in the relationship of sex and FeNO levels, there were other differences between our study and the published FeNO studies in white adults.27,30 First, our study was performed on a younger cohort. We only enrolled younger subjects to diminish the effects of age-related respiratory ailments, such as chronic obstructive pulmonary disease on FeNO levels. Second, our study did not collect spirometric data. However, the FeNO studies in white subjects suggested that spirometric measurements were not correlated with FeNO levels. Finally, in contrast to the FeNO studies in white subjects, our study included analysis of serum ECP levels, included collection of URI questionnaire information, included repeated FeNO measurements, and rigorously excluded smokers based on serum cotinine measurements. Although direct comparisons of FeNO levels between our study and the FeNO studies in white subjects27,30 are difficult because of methodological differences, it appears that FeNO levels are higher in our African American cohort. Travers et al30 reported a geometric mean FeNO level of 17.9 ppb in their healthy smoking and nonsmoking subjects, and we found a geometric mean of 19.7 ppb in a comparable grouping of our subjects. Likewise, Olin et al27 found a median FeNO level of approximately 17.0 ppb in nonsmokers in their cohort, and we found a median of 19.9 ppb for a similar subset of our cohort. The association of FeNO with serum total IgE levels (Tables I and II and Fig E3, A) and the known changes in FeNO levels associated with inhaled steroid therapy10,37 suggest that the association of FeNO and total IgE levels might be due to IgE-mediated allergic airways disease. However, this association appears modest, and an alternative hypothesis might provide a better explanation for the association of FeNO and serum total IgE levels. In this regard it has been suggested that the association of atopy and FeNO levels might be due to shared genetic determinants.38 Based on this, we propose an alternative hypothesis for the association of FeNO and serum total IgE levels; we believe that FeNO levels and associated NO metabolites might instead regulate IgE levels. In support of this hypothesis, T-cell responses (especially TH2 responses) are essential to the production of IgE, and NO regulates T-cell responses.39-41 The hygiene hypothesis42 proposes that early-life exposure to microbes and microbial products, such as LPS, promotes TH1 responses,43 and early exposure to LPS is associated with protection from later development of asthma and atopy.44 NO production is typically augmented by LPS and by LPS-induced cytokines (TNF-a, IFN-g, and

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IL-1b)45; as such, NO can promote TH1 responses. This might be particularly relevant to African populations, who have higher levels of serum total IgE than white populations and are dependant on TH2-mediated IgE responses for resistance to parasites.46,47 To our knowledge, our study is the first to use practical information on URI symptoms to understand what limitations URIs might impose on the interpretation of FeNO results. We found that simply asking subjects whether they had a cold identified subjects who had significantly higher FeNO levels than those seen in subjects without cold symptoms. One potential concern with interpretation of the cold symptom results is the possibility that the reported cold symptoms represent atopyrelated allergic rhinitis symptoms rather than URI symptoms. We believe that the symptoms are not atopy related for the following reasons. First, we analyzed FeNO levels from 5 subjects with cold symptoms on their first visit who had repeat FeNO measurements on a different day without cold symptoms. Four of these 5 subjects had lower FeNO levels on the day without cold symptoms (P 5 .0625, Wilcoxon signed rank test, 1-tailed). Second, we found no correlation between cold symptoms and serum total IgE levels (P 5.5486), whereas in contrast, a subject self-report of current or past rhinitis was significantly associated with higher serum total IgE levels (P 5 .0406). Finally, we found that cold symptoms were significantly associated with higher CRP levels (P 5 .0086), but CRP levels were not significantly associated with FeNO levels (Table I). Therefore we believe that a subject’s self-report of a cold is typically not due to allergy-mediated symptoms. The association of higher FeNO levels with male sex remains unexplained but is consistent with prior results.20,30 Several published studies have focused on the effects of estrogen on NOS2 and endothelial or type III NO synthase expression and NO production, suggesting that FeNO levels might be lower in women because of increased estrogen levels.48-52 Estrogen suppresses NOS2 expression in endothelial cells48,49 and reduces inflammation in NO-dependent models of inflammation and ischemic injury.50,51 A study of the human airway cell line A549 and of human primary epithelial cells indicated that an estrogen receptor agonist blocked cytokine-stimulated NOS2 expression.52 The specific effect of estrogen on FeNO levels remains unclear, but these studies suggest that estrogen might decrease FeNO levels through reduced respiratory epithelial expression of NOS2. The weak but significant correlation between systolic BP and FeNO levels is not consistent with a prior report of an association of increased BP with lower FeNO levels.29 As discussed earlier, we believe that the weak association of systolic BP with FeNO levels is likely due to the association of sex with higher systolic BP and FeNO levels. We do not believe that vascular NO or endothelial or type III NO synthase contributes substantially to FeNO levels under normal circumstances and in nondiseased states. Instead, the association of serum nitrate levels with FeNO levels supports the hypothesis that gastrointestinal NO contributes to FeNO levels. Prior studies found that ingested nitrate was excreted in the saliva and converted by oral bacteria to nitrite by bacterial nitrate reductase.24 Once swallowed, nitrite reacts with H1 ions in the stomach to produce NO, which serves as an important antimicrobial and vasodilator that protects the stomach from ulceration.53,54 Some stomach NO is released in exhaled air, and therefore some fraction of serum nitrate is subsequently released in exhaled air as NO.24 Serum and salivary nitrate levels are

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significantly associated with the amount of ingested nitrate,24,55 although the effect of serum nitrate levels on the variability of FeNO levels appears small (Tables I and II and Fig E3). The relatively low correlation coefficients for association of sex, current URI symptoms, total IgE levels, and ECP levels with FeNO levels (Tables I and II) and the high correlation between repeated FeNO measurements suggest that genetic differences might be associated with FeNO levels. A Norwegian study of young adult twins found that genetic effects accounted for 60% of the variation in FeNO levels.38 Genetic studies of asthmatic patients indicate an association of FeNO levels with the neuronal isoform of NO synthase.56 In addition, studies in African populations of NOS2 promoter polymorphisms indicated associations of these polymorphisms with protection from malaria and with increased serum nitrite and nitrate levels.57,58 In summary, we examined the association of demographic, physiologic, and serum factors with FeNO levels and established FeNO reference values in a large cohort of young, healthy, nonsmoking African American adults. FeNO levels were associated with increased serum total IgE levels, and FeNO levels were higher in men compared with those in women. We believe that normative values for FeNO based on sex are important. Our data on the relationship of serum nitrate levels and URI symptoms support avoidance of measuring FeNO levels after consumption of high-nitrate meals and when cold symptoms are present on the day of the examination. Finally, the excellent correlation between repeated measurements of FeNO and the low correlation coefficients of demographic and serum factors associated with FeNO provide a rationale for genetic studies of FeNO variability. We thank Edna Scarlett and Catherine Foss for their assistance during subject enrollment and sample collection. We also thank Clayton Thomas and Charles Tao for assistance with computer data entry. Finally, we appreciate the use of J. Brice Weinberg’s NO analyzer and Wesley Burks’ CAP-FEIA instrument for measurement of FeNO and total IgE levels, respectively.

9.

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15. 16.

17. 18.

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20. 21.

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Clinical implications: Sex, increased serum total IgE levels, and current URI symptoms should be considered when evaluating FeNO levels in adults. Furthermore, these studies provide FeNO reference values for healthy African American adults.

23. 24. 25.

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FIG E1. Comparison of FeNO levels in healthy, nonsmoking African American men and women. Box plot comparison of FeNO levels from male (n 5 271) and female (n 5 587) subjects are shown. For comparison of male and female subjects, FeNO levels were log normalized and compared by using an unpaired Student t test (P < .0001). The lower and upper limits of the rectangular box plots represent the 25th and 75th percentiles, respectively, for the data in each set. The upper and lower limits of the whisker plots represent the upper and lower quartiles plus and minus 1.5 times the interquartile range, respectively, in each dataset. The horizontal line in the middle of each box plot represents the median for that dataset, and the solid circle represents the geometric mean for each dataset.

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FIG E2. Comparisons of FeNO levels in healthy, nonsmoking African American adults based on subject selfreports of upper respiratory tract symptoms. Box plot comparisons of FeNO levels from subjects based on (A) current URI symptoms (report of a cold with or without a runny nose on the day of sample collection; r2 5 0.0308; n 5 44 [Cold Sxs] and n 5 405 [No Cold Sxs]; P 5 .0002 for comparison of subjects with and without current URI symptoms) and based on (B) self-report of prior asthma diagnosis (r2 5 0.0108; n 5 37 [Asthma] and n 5 410 [No Asthma]; P 5 .0278 for comparison of subjects with and without a prior asthma diagnosis) are shown. FeNO levels from each group of subjects were log normalized and compared by using unpaired Student t tests. The lower and upper limits of the rectangular box plots represent the 25th and 75th percentiles, respectively, for the data in each set. The upper and lower limits of the whisker plots represent the upper and lower quartiles plus and minus 1.5 times the interquartile range, respectively, in each dataset. The horizontal line in the middle of each box plot represents the median for that dataset, and the filled circle represents the geometric mean for each dataset.

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FIG E3. Linear regression analyses of FeNO and serum IgE, ECP, and nitrate levels in healthy, nonsmoking African American adults. Plots of FeNO and serum levels of total IgE (A), ECP (B), and nitrate (C) are shown. FeNO, total IgE, ECP, and nitrate levels were log normalized before analysis. Linear regression and the Pearson product-moment correlation coefficient were used to examine the relationship between FeNO levels and serum levels of total IgE (Fig E3, A; r2 5 0.1109, P < .0001, n 5 871), ECP (Fig E3, B; r2 5 0.0170, P 5 .0001, n 5 881), and nitrate (Fig E3, C; r2 5 0.0083, P 5 .0449, n 5 464). The line in each plot was derived from a linear regression analysis of the points in each plot.