Levels of nitric oxide oxidation products are increased in the epithelial lining fluid of children with persistent asthma

Article Outline

• Abstract
• Methods
  • Sample
  • Procedures
  • Statistical analysis
• Results
  • NO oxidation products in the proximal and distal airway lavage fluid
  • Relationship of NO oxidation products to FENO values and other clinical features in asthmatic children
• Discussion
• Acknowledgment
• Fig E1. 
• Table E1. 
• Table E2. 
• Table E3. 
• Table E4. 
• Table E5. 
• Table E6. 
• Table E7. 
• Table E8. 
• References
• Copyright

Background

Children with severe allergic asthma have persistent airway inflammation and oxidant stress.

Objectives

We hypothesized that children with severe allergic asthma would have increased concentrations of the nitric oxide (NO) oxidation products nitrite, nitrate, and nitrotyrosine in the proximal and distal airway epithelial lining fluid (ELF). We further hypothesized that NO oxidation products would be associated with higher exhaled NO values (fraction of exhaled nitric oxide [F_ENO]), greater allergic sensitization, and lower pulmonary function.

Methods

Bronchoalveolar lavage fluid was obtained from 15 children with mild-to-moderate asthma, 30 children with severe allergic asthma, 5 nonasthmatic children, and 20 nonsmoking adults. The bronchoalveolar lavage fluid was divided into proximal and distal portions and nitrite, nitrate, and nitrotyrosine values were quantified.

Results

Children with mild-to-moderate and severe allergic asthma had increased concentrations of nitrite (adult control subjects, 15 ± 3 μmol/L; pediatric control subjects, 23 ± 4 μmol/L; subjects with mild-to-moderate asthma, 56 ± 26 μmol/L; subjects with severe asthma, 74 ± 18 μmol/L), nitrate (37 ± 13 vs 145 ± 38 vs 711 ± 155 vs 870 ± 168 μmol/L, respectively) and nitrotyrosine (2 ± 1 vs 3 ± 1 vs 9 ± 3 vs 10 ± 4 μmol/L, respectively) in the proximal ELF. Similar results were seen in the distal ELF, although the concentrations were significantly lower (P < .05 for each). Although univariate analyses revealed no associations between NO oxidation products and clinical features, multivariate analyses revealed F_ENO values to be a significant predictor of NO oxidation in asthmatic children.

Conclusions

NO oxidation products are increased in the ELF of asthmatic children. The relationship between F_ENO values and airway nitrosative stress is complicated and requires further study.

Key words: Asthma, children, nitric oxide, nitrogen oxides, nitrosation, nitrosative stress, reactive nitrogen species

Abbreviations used: BAL, Bronchoalveolar lavage, ELF, Epithelial lining fluid, F_ENO, Fraction of exhaled nitric oxide, ICS, Inhaled corticosteroid, NO, Nitric oxide, NOS, Nitric oxide synthase

Severe allergic asthma in school-aged children is a complex disorder characterized by persistent airway inflammation, ongoing symptoms, and increased exhaled nitric oxide (NO; fraction of exhaled nitric oxide [F_ENO]) concentrations despite treatment with high doses of inhaled and oral corticosteroids.¹, ² Although airway NO is essential for epithelial signaling and host defense, excessive NO production results in NO oxidation and potential toxicity.³ This process of excessive NO oxidation is commonly referred to as “nitrosative stress”⁴ and ultimately promotes protein nitration, resulting in structural and functional protein alterations that might enhance the inflammatory response.⁵ Thus excessive airway NO concentrations in children with severe allergic asthma might contribute to an ongoing cycle of airway destruction with airway injury.⁶

In the human airway, the most readily detectable NO oxidation products include nitrite (NO₂⁻) and nitrate (NO₃⁻), which can be derived from NO through a series of reactions involving superoxide anion (O·₂⁻) and oxygen (Fig 1). Nitrotyrosine is also easily measured in airway samples and reflects the overall degree of protein nitration.⁷ Indeed, previous studies have noted increased nitrite, nitrate, and nitrotyrosine concentrations in the exhaled breath condensate of asthmatic children⁸, ⁹, ¹⁰ and in the epithelial lining fluid (ELF) of adults with mild-to-moderate and severe asthma.¹¹, ¹² However, no study to date has examined NO oxidation products in the ELF of asthmatic children. Because children with severe asthma have profound airway oxidant stress,¹³ the purpose of this study was to quantify NO oxidation products in the ELF of children with mild-to-moderate and severe allergic asthma. The secondary purpose of this study was to determine the association between increased ELF NO oxidation products and clinical features of asthma severity in children. We hypothesized that children with severe allergic asthma would have increased concentrations of the NO oxidation products nitrite, nitrate, and nitrotyrosine in the proximal and distal airway ELF. We further hypothesized that these increased NO oxidation products would be associated with increased F_ENO values, greater allergic sensitization, and lower pulmonary function.

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• Fig 1. 

  Diagram of NO metabolite formation in the airways.

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Methods 

Sample 

Children 5 to 17 years of age with symptomatic asthma attending an asthma clinic at Emory University were invited to participate in this study. Asthmatic children met published criteria for persistent asthma¹⁴ and had a history of at least a 12% change in FEV₁ after albuterol administration.¹⁵ Severe asthma was diagnosed according to criteria developed by the National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program,¹ which were adapted from the American Thoracic Society's Consensus Panel Report (see Table E1 in this article's Online Repository at www.jacionline.org).¹⁶ Thresholds for high-dose inhaled corticosteroids (ICSs) were defined as 440 μg or greater of fluticasone equivalent per day for children less than 12 years of age and 880 μg or greater for children 12 to 17 years of age.¹⁴ Children with severe allergic asthma were treated with a stable dose of ICSs or oral corticosteroids for at least 6 months before recruitment. Adherence to ICS therapy was monitored by an analysis of prescription refills. Informed consent was obtained from all caregivers. Children also provided verbal and written assent.

Children who fit the criteria for severe allergic asthma underwent flexible bronchoscopy with bronchoalveolar lavage (BAL) as indicated for persistent asthma symptoms despite appropriate treatment with high-dose inhaled and systemic corticosteroids.¹⁷ Children with mild-to-moderate asthma underwent bronchoscopy for suspected foreign body aspiration, recurrent pneumonia, persistent cough, and suspected congenital anomalies. Control subjects for this study included children with psychogenic (habit) cough or vocal cord dysfunction undergoing bronchoscopy for definitive diagnosis and healthy, nonsmoking adult volunteers. Control subjects were nonsmokers with no family history of asthma and a negative bronchodilator response.

Procedures 

Spirometry was performed before and after 2 inhalations of albuterol sulfate (90 μg per inhalation) with a portable spirometer (KoKo Legend; Ferraris, Louisville, Colo). The results fulfilled American Thoracic Society criteria for reproducibility¹⁸ and were interpreted according to reference standards.¹⁹ Atopic sensitization was assessed by means of skin prick testing with a standard kit (Multi-Test II; Lincoln Diagnostics, Decatur, Ill) containing tree pollen, grass pollen, ragweed pollen, weed pollen, dog hair, cat epithelium, Alternaria species, Cladosporium species, Aspergillus species, Dermatophagoides pteronyssinus, Dermatophagoides farinae, cockroach, normal saline, and histamine extracts (Greer Laboratories, Lenoir, NC). The application site was examined 15 minutes after application and considered positive if both a wheal of 3 mm in diameter or larger and erythema of 10 mm in diameter or larger were present.

On the day of bronchoscopy, participants submitted F_ENO samples and underwent venipuncture. F_ENO was collected with a reservoir bag within 1 hour before bronchoscopy. For this procedure, subjects took 2 tidal breaths of NO-free air through a scrubbing filter, followed by a 6-second exhalation at a fixed flow rate of 0.35 L/s.²⁰ The first 150 mL exhaled was discarded. Subjects repeated this procedure 3 times. The resulting samples were analyzed offline by means of chemiluminescence (Sievers NOATM 280-I; Ionic Instruments, Boulder, Colo) within 1 hour of collection. The data were averaged to reflect mean F_ENO values. Serum immunoglobulin (IgE) concentrations and plasma urea value were determined after venipuncture.

Bronchoscopy in pediatric participants was performed by pediatric pulmonologists using a laryngeal mask airway. BAL fluid was collected from the right middle lobe with three 1 mL/kg (50 mL maximum) saline lavages flushed through the suction channel of a flexible bronchoscope (Olympus BF-3C160 [3.7 mm] or BF-P160 [4.9 mm]; Olympus America, Inc, Melville, NY). Bronchoscopy was performed in adults with a flexible bronchoscope (Olympus BF-1T20D) passed transnasally into the right middle lobe. Three 50-mL saline aliquots were instilled and immediately aspirated. The first lavage sample from all participants was reserved for evaluation of proximal airway constituents.²¹ The second and third lavage samples were pooled for distal airway constituent analysis. In children the BAL return volume was divided between the research and clinical laboratories.

BAL fluid was centrifuged at 1200 rpm within 1 hour of collection for 7 minutes at 4°C to separate the supernatant and cellular fractions. Given the limited number of cells present in the proximal airway lavage sample, the cell pellets from the proximal and distal airway lavage samples were pooled and resuspended in 10 mL of Dulbecco modified Eagle medium with 10% FCS for cell counting. Total cell counts were performed manually with a hemocytometer, and cellular differentials were determined from 300 consecutive cells after Wright staining.

The protein content of the BAL fluid supernatant was assessed by using a Coomassie (Bradford) protein assay (Pierce Biotechnology, Rockford, Ill) read at an absorbance of 595 nm with a detection limit 1 μg/mL. Urea nitrogen levels were measured in plasma and BAL supernatants by using a quantitative colorimetric assay (Pointe Scientific, Canton, Mich) with a sensitivity of 0.05 to 150 mg/dL. The dilution of the proximal and distal BAL fluid was calculated as follows: [Urea]plasma/[Urea]BAL.²²

Nitrite and total nitrite plus nitrate concentrations were determined from the BAL supernatant by using a colorimetric assay (Cayman Chemical, Ann Arbor, Mich) analyzed at 540 nmol/L with a lower detection limit of 0.1 μmol/L. All samples were analyzed in duplicate. For this assay, nitrate was converted to nitrite with nitrite reductase, followed by the addition of Griess reagent. Nitrate concentrations were determined by subtracting the concentration of nitrite from total nitrite plus nitrate concentrations. Samples were analyzed immediately after thawing to minimize false nitrate and nitrate readings during the assay. The background nitrite and nitrate content in the saline lavage fluid was predetermined and subtracted from the final concentration values.

Nitrotyrosine concentrations were determined spectrophotometrically by using a microplate sandwich ELISA (Oxis International, Foster City, Calif) with a sensitivity of 2.0 nmol/L and interassay precision of 11%. Samples were analyzed in duplicate and corrected for the background levels of nitrotyrosine in the saline lavage fluid. Absorbance was measured at 450 nm.

Statistical analysis 

Data were analyzed with SPSS software (Version 15; SPSS, Inc, Chicago, Ill). Nitrite, nitrate, and nitrotyrosine concentrations from the proximal and distal airway lavage fluid were adjusted according to the urea dilution²² and were logarithmically transformed. Nitrotyrosine concentrations were further adjusted for the total protein content of the BAL supernatant. Differences between groups and post-hoc tests were assessed by using Kruskal-Wallis tests and Mann-Whitney U tests, respectively. Pearson correlations were used to examine associations between NO oxidation products and clinical features. To evaluate factors that might affect NO oxidation in the ELF of asthmatic children, multivariate backward elimination linear regression was performed with total nitrite plus nitrate concentrations in the proximal and distal ELF as dependent variables and age, sex, ethnicity, ICS dose, FEV₁, FEV₁ bronchodilator reversibility, serum IgE level, history of hospitalization, F_ENO value, and the percentage of airway eosinophils and neutrophils as predictors. Multicolinearity between predictors was assessed with tolerance statistics. Entry and removal probabilities were set at .05 and .10, respectively. Significance was defined as a 2-tailed α value of .05 or less for all tests.

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Results 

Initially, 49 asthmatic children (severe asthma, n = 32), 7 pediatric control subjects, and 20 healthy adult control subjects were recruited for this study. However, 6 children, including 2 pediatric control subjects, 2 subjects with mild-to-moderate asthma, and 2 subjects with severe asthma, were infected with Streptococcus pneumoniae, Haemophilus influenzae, and/or Moraxella catarrhalis and were excluded from data analysis because of potential denitrification.²³ The features of the excluded children appear in the Tables E2 and E3 (available in this article's Online Repository at www.jacionline.org). Thus the final sample included in data analysis contained 30 children with severe allergic asthma, 15 children with mild-to-moderate asthma, 5 pediatric control subjects, and 20 adult control subjects.

Because bronchoscopy was performed only for clinical indications, all of the asthmatic subjects were symptomatic. None of the children with mild-to-moderate asthma had evidence of airway infection or chronic aspiration syndromes. The features of the final sample are presented in Table I. Whereas all (100%) children with severe asthma had allergic sensitization, allergic sensitization was present in only half (53%) of the children with mild-to-moderate asthma (see Table E4 in this article's Online Repository at www.jacionline.org). Children with severe allergic asthma were also treated with higher doses of ICSs but had significantly lower baseline pulmonary function and increased bronchodilator reversibility. Although F_ENO values were increased in both groups of asthmatic subjects, there were no differences in F_ENO values between children with mild-to-moderate and severe allergic asthma (Table I).

Table I. Features of the sample

	Adult control subjects (n = 20)	Pediatric control subjects (n = 5)	Subjects with mild-to-moderate asthma (n = 15)	Subjects with severe asthma (n = 30)
Age (y)	39 ± 10	11 ± 4∗	10 ± 4∗	10 ± 4∗
Male sex	8 (40)	3 (60)	10 (67)	15 (50)
White	8 (40)	4 (80)	14 (93)∗	9 (30)†‡
African American	11 (55)	1 (20)	1 (7)∗	20 (67)†‡
ICS dose (μg of fluticasone/d)	0	0	262 ± 189∗†	917 ± 236∗†‡
Asthma medications
Budesonide	0	0	3 (20)	7 (23)
Fluticasone	0	0	1 (7)	1 (3)
Fluticasone/salmeterol	0	0	8 (53)∗†	22 (73)∗†‡
Montelukast	0	0	10 (67)∗†	28 (93)∗†‡
Prednisone	0	0	0	11 (37)∗†‡
Emergency department visit (previous year)	0	0	3 (20)	28 (93)∗†‡
Hospitalization (previous year)	0	0	1 (7)	26 (87)∗†‡
Intensive care unit admission (ever)	0	0	0	14 (47)∗†‡
Intubation (ever)	0	0	0	6 (20)∗†‡
FVC (% predicted)	98 ± 16	102 ± 18	102 ± 15	87 ± 19∗†‡
FEV1 (% predicted)	103 ± 16	101 ± 15	100 ± 15	73 ± 20∗†‡
FEV1/FVC ratio	0.86 ± 0.07	0.89 ± 0.03	0.87 ± 0.06	0.74 ± 0.12∗†‡
FEF25-75 (% predicted)	121 ± 32	92 ± 16∗	94 ± 23∗	51 ± 25∗†‡
FEV1 bronchodilator reversibility (%)§	3 ± 6	6 ± 5	9 ± 11	23 ± 17
FENO (offline, ppb)	5 ± 3	7 ± 4	11 ± 12∗	13 ± 10∗†
Increased baseline FENO (>10 ppb)	4 (20)	2 (40)	3 (20)	20 (67)∗†‡
Reported allergies	Not assessed	2 (40)	9 (60)	25 (83)
Reported atopic dermatitis	Not assessed	0	5 (33)	21 (70)
No. of skin prick responses	Not assessed	0	2 ± 2	5 ± 3†‡
Serum IgE (kU/L)	100 ± 194	80 ± 64	94 ± 139	487 ± 730∗†‡

Data represent means ± SDs or frequencies (percentages).

FVC, Forced vital capacity; FEF_25-75, forced expiratory flow.

The characteristics of the BAL fluid are presented in Table E5 (available in this article's Online Repository at www.jacionline.org). Although larger lavage volumes were used for adult control subjects, the percentage of BAL return was similar between adult and pediatric control subjects (proximal lavage, 23% vs 27%; distal lavage, 49% vs 35%; adult vs pediatric control subjects). However, the BAL samples from adult control subjects were characterized by higher total cell counts (adult control subjects, 7.81 ± 3.61 × 10⁶; pediatric control subjects, 3.53 ± 2.32 × 10⁶; subjects with mild-to-moderate asthma, 3.81 ± 3.06 × 10⁶; subjects with severe asthma, 3.32 ± 2.02 × 10⁶; P < .01). Whereas subjects with severe asthma had the highest percentage of BAL eosinophils (adult control subjects, 0.4% ± 0.5%; pediatric control subjects, 0.3% ± 0.5%; subjects with mild-to-moderate asthma, 0.7% ± 0.7%; subjects with severe asthma, 1.9% ± 3.2%; P = .03), subjects with mild-to-moderate and severe asthma had higher percentages of neutrophils compared with both groups of control subjects (adult control subjects, 3.5% ± 3.2%; pediatric control subjects, 3.2% ± 1.4%; subjects with mild-to-moderate asthma, 5.3% ± 3.9%; subjects with severe asthma, 5.2% ± 3.2%; P = .04).

NO oxidation products in the proximal and distal airway lavage fluid 

Compared with control subjects, children with mild-to-moderate and severe allergic asthma had significantly higher concentrations of nitrite, nitrate, and nitrotyrosine in the ELF (Fig 2). However, no significant differences in concentrations of NO oxidation products were observed between children with mild-to-moderate and severe allergic asthma. In each group nitrate was the most abundant NO oxidation product measured, with concentrations nearly 10-fold higher than those of nitrite. Furthermore, nitrite, nitrate, and nitrotyrosine concentrations were also consistently higher in the proximal versus the distal airway ELF (Fig 2). Similar increases in concentrations of NO oxidation products were also apparent in the raw BAL samples without adjustment for the urea dilution (see Fig E1 in this article's Online Repository at www.jacionline.org). Analysis of the entire sample (all asthmatic and control subjects) revealed strong correlations between proximal and distal airway ELF concentrations of total nitrite plus nitrate (r = 0.76, P < .01), nitrite (r = 0.50, P < .01), nitrate (r = 0.76, P < .01), and nitrotyrosine (r = 0.34, P = .02). When this analysis was restricted only to asthmatic children, similar correlations between the proximal and distal ELF NO oxidation products were observed (total nitrite plus nitrate: r = 0.44, P ≤ 001; nitrite: r = 0.58, P < .01; nitrate: r = 0.31, P = .05; nitrotyrosine: r = 0.35, P = 0.05). Within the proximal and distal airway ELF, high agreement was further observed between the measured concentrations of total nitrite plus nitrate and nitrotyrosine (Fig 3).

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• Fig 2. 

  Total nitrite plus nitrate (A), nitrite (B), nitrate (C), and nitrotyrosine (D) concentrations (in micromoles per liter) in the proximal (black bars) and distal (gray bars) airway ELF. Data represent means ± SEMs with adult control subjects (AC), pediatric control subjects (PC), subjects with mild-to-moderate asthma (MA), and subjects with severe asthma (SA). ^aP < .05 versus adult control subjects. ^bP < .05 versus pediatric control subjects.

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• Fig 3. 

  Scatterplot depicting the relationship between total nitrite plus nitrate and nitrotyrosine concentrations (in micromoles per liter) in the proximal (black circles) and distal (gray circles) airway ELF. Data were logarithmically transformed.

Relationship of NO oxidation products to F_ENO values and other clinical features in asthmatic children 

Correlational analysis was first performed between NO oxidation products and clinical features of asthma severity, including F_ENO, serum IgE levels, the number of skin prick test responses, FEV₁, FEV₁ bronchodilator reversibility, and the percentage of BAL eosinophils and neutrophils, to determine the clinical implications of increased ELF oxidation products in children with mild-to-moderate and severe asthma. This analysis was restricted to children with mild-to-moderate and severe asthma and did not include control subjects. No significant correlations were observed between NO oxidation products and any of the clinical features measured, including F_ENO values (see Table E6 in this article's Online Repository at www.jacionline.org). However, F_ENO values were significantly associated with the percentage of BAL eosinophils (r = 0.35, P = .04) and serum IgE levels (r = 0.30, P = .02).

To further evaluate factors that might affect NO oxidation in the ELF of asthmatic children, multivariate backward elimination linear regression was performed by using total nitrite plus nitrate concentrations in the proximal and distal ELF as the dependent variables and age, sex, ethnicity, ICS dose, FEV₁, FEV₁ bronchodilator reversibility, serum IgE levels, history of hospitalization, F_ENO values, and the percentage of airway eosinophils and neutrophils as predictors. Control data were excluded. In the proximal ELF age (P < .01), sex (P = .02), and F_ENO value (P = .06) were significant predictors of nitrite plus nitrate concentrations (final model: R² = 0.49, P = .01; see Table E7 in this article's Online Repository at www.jacionline.org). Likewise, sex (P = .05) and F_ENO values (P = .05) were significant predictors of total nitrite plus nitrate concentrations in the distal ELF of children with mild-to-moderate and severe asthma (final model: R² = 0.25, P = .05; see Table E8 in this article's Online Repository at www.jacionline.org). In both the proximal and distal airway ELF, the relationship between F_ENO values and NO oxidation was negative, such that higher F_ENO values were associated with lower NO oxidation product formation.

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Discussion 

To our knowledge, this is the first study to directly measure NO oxidation products in the ELF of children with persistent asthma. Compared with control subjects, children with mild-to-moderate and severe allergic asthma had increased concentrations of nitrite, nitrate, and nitrotyrosine in the ELF that were consistently higher in the proximal versus the distal airways. Contrary to our hypothesis, we did not detect significant differences in concentrations of NO oxidation products between children with mild-to-moderate and severe asthma. Furthermore, no associations between NO oxidation products and clinical features, such as F_ENO values, were detected by using univariate analyses. However, with multivariate modeling to control for the potential confounding effects of ICSs and atopy on NO synthesis, F_ENO values were identified as a modest predictor of NO oxidation product formation. Although the clinical relevance of this finding is yet unclear, these data highlight the complexity of NO biology in children with asthma and suggest that the relationship between F_ENO values and NO oxidation is not directly proportional. Thus in children with severe asthma, lower F_ENO values might not necessarily indicate the absence of airway inflammation but instead might reflect decreased NO bioavailability from increased NO oxidation.

Airway NO biochemistry is complex, and the exact contribution of NO to the pathogenesis of asthma is not fully understood. NO is produced by nitric oxide synthases (NOSs) in a variety of cell types and serves as an important signaling molecule both within and outside of the cell.³ NO production is also vital to the epithelial antiviral and immune defenses of the airways.²⁴ Although the generation of NO oxidation products from NO is important for transcription factor activation and the regulation of airway inflammation,³ excessive airway NO production from altered NOS isoforms or lack of endogenous NOS inhibition can lead to the oxidation of NO and potential nitrogen oxide toxicity.²⁵ The resulting nitrosative stress might ultimately contribute to protein dysfunction and airway cellular destruction.²⁶ Our findings of increased nitrite, nitrate, and nitrotyrosine concentrations in the ELF of asthmatic children confirm that nitrosative stress is a distinguishing feature of the asthmatic airway. However, the underlying mechanisms responsible for this finding are unclear and warrant further study.

Although this is the first study to directly measure NO oxidation products in the ELF of children with mild-to-moderate and severe allergic asthma, our findings support previously reported observations in exhaled breath condensate. In these previous studies baseline concentrations of nitrite, nitrate, and nitrotyrosine were significantly higher in the exhaled breath condensate of asthmatic children.⁸, ¹⁰, ²⁷ Whereas others have shown reductions in nitrite and nitrate concentrations after 8 weeks of ICS therapy,²⁸ we observed NO oxidation in the ELF of children with mild-to-moderate and severe allergic asthma despite ICS treatment. This observation is intriguing and might reflect decreased sensitivity to ICSs in this population. Alternatively, NO oxidation products in the ELF might reflect complex biochemical abnormalities that are distinct from other types of airway inflammation and are not necessarily influenced by ICS treatment.²⁹

Although there is increasing evidence of distal airway inflammation in human³⁰ and experimental³¹ models of asthma, our results show that airway nitrosative stress is consistently higher in the proximal versus the distal airways. For this study, we performed sequential BAL of the right middle lobe to separate proximal and distal airway constituents.²¹ Because this method of lavage was adapted for children to account for different body weights, our data might not accurately reflect nitrosative stress in the bronchial versus alveolar airspace. Thus our distal airway samples might have contained a pooling of bronchial and alveolar NO oxidation products. However, our findings are similar to those of others showing increased inflammation in the bronchial versus alveolar space in asthmatic adults³² and lend support to the more proximal involvement of the airways in asthmatic children.

Our data do not show clear linear associations between ELF NO oxidation products and clinical features of asthma in children, which might be a function of our limited sample size or our patient selection. In addition, it is possible that our measurements of F_ENO values and concentrations of NO oxidation products were confounded by ICSs and atopy. In steroid-naive asthmatic subjects F_ENO values decrease in a dose-dependent manner after the initiation of ICSs.³³ Allergic sensitization is also associated with increased F_ENO values independent of asthma,³⁴ a finding that might be attributable to a late-phase influx of eosinophils. In the present study all of the children with severe asthma were treated with ICSs and had objective evidence of aeroallergen sensitization. Furthermore, 80% (n = 12) of the children with mild-to-moderate asthma were taking daily ICSs, and 53% (n = 8) had positive skin prick test responses. Whereas NO metabolites were not associated with any clinical features, like others,³⁵ we did observe an association between F_ENO values and airway eosinophils. This finding might explain the utility of F_ENO values in guiding ICS reduction and evaluating asthma control.³⁶ Because there might also be neutrophilic or other patterns of airway inflammation in children with severe asthma, our findings also might reflect the marked heterogeneity of this group of patients. Alternatively, the differences in F_ENO values among asthmatic subjects might be due to airway pH³⁷ or altered S-nitrosothiol metabolism³⁸ and not NO oxidation.

This study had a number of limitations. Because bronchoscopy cannot be ethically performed in healthy children, our pediatric control group was limited to nonasthmatic children with significant respiratory symptoms. The inclusion of these children might have resulted in inadvertent selection of a group of children with significant nitrosative stress. It is also possible that some of our subjects with mild-to-moderate asthma were undertreated. Thus the concentrations of NO oxidation products measured in our group of children with mild-to-moderate asthma might not be reflective of the larger population and might have been reduced with more aggressive ICS treatment.

In summary, we have demonstrated significant increases in the formation of NO oxidation products in the proximal and distal airway ELF of children with persistent asthma. Contrary to our hypothesis, concentrations of NO oxidation products did not differ between children with mild-to-moderate and those with severe allergic asthma. Although these data highlight the magnitude of oxidant stress that is present in the airways of children with symptomatic asthma, the relationship of this nitrosative stress to asthma severity is yet unclear. Additional studies are warranted to determine the clinical utility of measuring NO oxidation products in asthmatic children, particularly given the marked heterogeneity of the disease. It might be that targeted interventions to reduce nitrosative stress are indicated in children with significant nitrosative stress, despite ICS treatment.

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The Severe Asthma Research Program is a multicenter asthma research group funded by the National Heart, Lung, and Blood Institute and consists of the following contributors (∗Steering Committee members): Brigham and Women's Hospital, Boston, Mass—Elliot Israel,∗ Bruce D. Levy, and Gautham Marigowda; Cleveland Clinic, Cleveland, Ohio—Serpil C. Erzurum,∗ Raed A. Dweik, Suzy A. A. Comhair, Abigail R. Lara, Sumita Khatri, Marcelle Baaklini, Daniel Laskowski, and Jacqueline Sharp; Emory University, Atlanta, Ga—W. Gerald Teague,∗ and Anne M. Fitzpatrick; Imperial College School of Medicine, London, United Kingdom—Kian F. Chung,∗ Mark Hew, and Sally Meah; National Jewish Medical and Research Center, Denver—Colo—Sally E. Wenzel∗; University of Pittsburgh, Pittsburgh, Pa—William J. Calhoun,∗ and Bill T. Ameredes; University of Virginia, Charlottesville, Va—Benjamin Gaston∗; University of Wisconsin, Madison, Wis—William W. Busse,∗ Nizar Jarjour, and Cheri Swenson; Wake Forest University, Winston-Salem, NC—Eugene R. Bleecker,∗ Deborah Meyers, Wendy Moore, Stephen Peters, Annette Hastie, and Gregory Hawkins; Washington University in St Louis, St Louis, Mo—Mario Castro,∗ Leonard Bacharier, Iftikhar Hussain, and Jaime Tarsi; Data Coordinating Center, Denver, Colo—Douglas Curran-Everett∗; and National Heart, Lung, and Blood Institute, Bethesda, Md—Patricia Noel.

We thank Dr Benjamin Gaston and Dr Serpil Erzurum for their thoughtful assistance and careful review of this manuscript. We also thank Eric Hunter for his assistance with subject recruitment and Meredith Brown for her assistance with sample preparation and laboratory analyses.

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Fig E1. 

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• Total nitrite plus nitrate (A), nitrite (B), nitrate (C), and nitrotyrosine (D) concentrations (in micromoles per liter) in the proximal (black bars) and distal (gray bars) in the raw BAL supernatant. Data were not adjusted for the urea dilution. Data represent means ± SEMs with adult control subjects (AC), pediatric control subjects (PC), subjects with mild-to-moderate asthma (MA), and subjects with severe asthma (SA). ^aP < .05 versus adult control subjects. ^bP < .05 versus pediatric control subjects.

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Table E1. 

Criteria for severe asthma in children, as defined by the National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program

Major criteria (must have ≥1)

Daily high-dose ICSs (≥6 continuous months)

Children <12 y: ≥440 μg of fluticasone equivalent/d

Children ≥12 y: ≥880 μg of fluticasone equivalent/d

Daily oral corticosteroid use (≥6 continuous months)

Minor criteria (must have ≥2)

Treatment with a daily controller medication in addition to ICSs

Daily short-acting bronchodilator use (≥5 of 7 d)

Airway obstruction with FEV1 < 80% of predicted value at baseline

One or more emergency department visits in the previous 12 mo

Three or more oral corticosteroid bursts in the previous 12 mo

History of worsening symptoms with a reduction in corticosteroid dose

History of intubation

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Table E2. 

Characteristics of pediatric subjects excluded from data analysis

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Table E3. 

Biomarkers of nitrosative stress in children excluded from data analysis

Data are expressed as micromoles per liter per milliliter of ELF and represent means ± SDs.

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Table E4. 

Skin prick test responses to aeroallergens in children with mild-to-moderate and severe allergic asthma

	Mild-to-moderate asthma (n = 15)	Severe asthma (n = 30)
House dust mites
Dermatophagoides pteronyssinus	4 (27)	17 (57)
Dermatophagoides farinae	4 (27)	20 (67)∗
Mold species
Alternaria	4 (27)	17 (57)
Cladosporium	3 (20)	12 (40)
Aspergillus	3 (20)	12 (40)
Weeds
Weed mix	0	9 (30)∗
Ragweed	1 (7)	11 (37)
Animal dander
Dogs	3 (20)	6 (20)
Cats	3 (20)	17 (57)∗
Cockroach	1 (7)	12 (40)∗
Tree mix	3 (20)	9 (30)
Grass mix	3 (20)	5 (17)

Data represent frequencies (percentages).

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Table E5. 

Characteristics of the BAL fluid from control subjects and children with asthma

	Adult control subjects (n = 20)	Pediatric control subjects (n = 5)	Subjects with mild-to-moderate asthma (n = 15)	Subjects with severe asthma (n = 30)
BAL fluid recovery (% of volume instilled)
Proximal airway lavage	23 ± 9	27 ± 9	38 ± 11∗	37 ± 8∗
Distal airway lavage	49 ± 13	35 ± 6	42 ± 16	38 ± 14
Protein (μg/dL)
Proximal airway lavage	81 ± 73	227 ± 190∗	194 ± 159∗	238 ± 221∗
Distal airway lavage	118 ± 79	223 ± 142∗	241 ± 113∗	219 ± 166∗
Urea (mg/dL)
Proximal airway lavage	0.36 ± 0.30	0.34 ± 0.24	0.27 ± 0.24	0.27 ± 0.44
Distal airway lavage	0.50 ± 0.28	0.44 ± 0.63	0.43 ± 0.53	0.40 ± 0.51
Total leukocyte cell count (× 106)‡	7.81 ± 3.61	3.53 ± 2.32∗	3.81 ± 3.06∗	3.32 ± 2.02∗
Cellular differential (%)‡
Macrophages/monocytes	90.1 ± 4.3	90.2 ± 7.2	89.4 ± 7.5	88.0 ± 4.8
Neutrophils	3.5 ± 3.2	3.8 ± 1.4	5.3 ± 3.9∗†	5.2 ± 3.2∗†
Eosinophils	0.4 ± 0.5	0.3 ± 0.5	0.7 ± 0.7	1.9 ± 3.2∗†
Lymphocytes	4.9 ± 2.6	3.9 ± 2.8	3.5 ± 2.9	4.7 ± 3.0

Data represent means ± SDs.

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Table E6. 

Correlations between NO oxidation products (in micromoles per liter) and clinical features in asthmatic children∗

Proximal airway lavage	Total nitrite plus nitrate	Nitrite (NO2−)	Nitrate (NO3−)	Nitrotyrosine
FENO†	−0.12 (.53)	0.31 (.11)	−0.15 (.43)	−0.35 (.11)
Serum IgE†	−0.18 (.31)	−0.09 (.63)	−0.21 (.22)	0.09 (.66)
No. of skin prick test responses	−0.05 (.83)	0.33 (.11)	−0.07 (.74)	−0.25 (.29)
FEV1 (% predicted)	−0.06 (.73)	0.03 (.88)	−0.03 (.88)	−0.08 (.70)
FEV1/FVC (ratio)	−0.08 (.65)	−0.29 (.11)	0.02 (.90)	−0.05 (.80)
FEV1 bronchodilator reversibility (%)	0.80 (.70)	−0.14 (.49)	0.02 (.92)	0.21 (.38)
BAL eosinophils (%)†	0.02 (.91)	−0.18 (.32)	0.02 (.92)	−0.17 (.41)
BAL neutrophils (%)†	0.17 (.35)	0.33 (.06)	0.12 (.49)	−0.06 (.76)
Distal airway lavage
FENO†	−0.21 (.27)	0.31 (.10)	−0.30 (.11)	−0.00 (.99)
Serum IgE†	0.30 (.06)	0.06 (.73)	0.29 (.07)	0.10 (.60)
No. of skin prick test responses	0.02 (.91)	0.32 (.11)	−0.02 (.92)	0.09 (.70)
FEV1 (% predicted)	−0.28 (.09)	0.14 (.40)	−0.27 (.10)	−0.05 (.78)
FEV1/FVC (ratio)	−0.09 (.59)	−0.11 (.52)	−0.07 (.66)	−0.25 (.19)
FEV1 bronchodilator reversibility (%)	0.03 (.89)	−0.13 (.48)	0.04 (.83)	0.10 (.64)
BAL eosinophils (%)†	0.05 (.77)	−0.20 (.22)	0.03 (.88)	−0.22 (.25)
BAL neutrophils (%)†	0.07 (.66)	−0.06 (.70)	0.07 (.65)	−0.11 (.59)

Data shown represent the Pearson correlation coefficient (r) and the P value (in parentheses). NO oxidation products were logarithmically transformed due to a nonnormal distribution before analysis.

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Table E7. 

Results of backward linear regression of selected clinical features on the concentration of total nitrite plus nitrate in the proximal ELF

Model	Regression coefficient	SE	t	P value	95% CI
Included variables∗
Constant	8.82	0.68	13.05	<.001	7.39 to 10.26
Age	−0.15	0.04	−3.34	<.01	−0.24 to −0.05
Sex	0.71	0.28	2.54	.02	0.18 to 1.31
FENO†	−0.45	0.22	−2.00	.06	−0.93 to 0.03
Excluded variables
Ethnicity	0.02		0.11	.91
ICS dose	0.01		0.06	.95
History of hospitalization	−0.05		−0.29	.78
FEV1 (% predicted)	−0.20		−1.05	.31
FEV1 bronchodilator reversibility (%)	0.03		0.13	.90
Serum IgE (kU/L)†	−0.16		−0.85	.41
BAL eosinophils (%)†	−0.09		−0.45	.66
BAL neutrophils (%)†	0.09		0.49	.63

Total nitrite plus nitrate data were logarithmically transformed. Data are from children with mild-to-moderate and severe allergic asthma. Adult and pediatric control subjects were excluded.

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Table E8. 

Results of backward linear regression of selected clinical features on the concentration of total nitrite plus nitrate in the distal ELF

Model	Regression coefficient	SE	t	P value	95% CI
Included variables∗
Constant	7.27	0.78	9.39	<.001	5.66 to 8.89
Sex	0.89	0.42	2.09	.05	0.00 to 1.77
FENO†	−0.74	0.36	−2.07	.05	−1.48 to 0.01
Excluded variables
Age	−0.31		−1.69	.11
Ethnicity	0.14		0.69	.50
ICS dose	−0.18		−0.91	.38
History of hospitalization	−0.04		−0.18	.86
FEV1 (% predicted)	−0.18		−0.88	.39
FEV1 bronchodilator reversibility (%)	−0.11		−0.49	.63
Serum IgE (kU/L)†	0.09		0.46	.65
BAL eosinophils (%)†	0.08		0.35	.73
BAL neutrophils (%)†	−0.04		−0.17	.86

Total nitrite plus nitrate data were logarithmically transformed. Data are from children with mild-to-moderate and severe allergic asthma. Adult and pediatric control subjects were excluded.

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