Use of Exhaled Nitric Oxide Measurement to Identify a Reactive, at-Risk Phenotype among Patients with Asthma

Abstract

Section:

Rationale: Exhaled nitric oxide (Fe_NO) is a biomarker of airway inflammation in mild to moderate asthma. However, whether Fe_NO levels are informative regarding airway inflammation in patients with severe asthma, who are refractory to conventional treatment, is unknown. Here, we hypothesized that classification of severe asthma based on airway inflammation as defined by Fe_NO levels would identify a more reactive, at-risk asthma phenotype.

Methods: Fe_NO and major features of asthma, including airway inflammation, airflow limitation, hyperinflation, hyperresponsiveness, and atopy, were determined in 446 individuals with various degrees of asthma severity (175 severe, 271 nonsevere) and 49 healthy subjects enrolled in the Severe Asthma Research Program.

Measurements and Main Results: Fe_NO levels were similar among patients with severe and nonsevere asthma. The proportion of individuals with high Fe_NO levels (>35 ppb) was the same (40%) among groups despite greater corticosteroid therapy in severe asthma. All patients with asthma and high Fe_NO had more airway reactivity (maximal reversal in response to bronchodilator administration and by methacholine challenge), more evidence of allergic airway inflammation (sputum eosinophils), more evidence of atopy (positive skin tests, higher serum IgE and blood eosinophils), and more hyperinflation, but decreased awareness of their symptoms. High Fe_NO identified those patients with severe asthma characterized by the greatest airflow obstruction and hyperinflation and most frequent use of emergency care.

Conclusions: Grouping of asthma by Fe_NO provides an independent classification of asthma severity, and among patients with severe asthma identifies the most reactive and worrisome asthma phenotype.

Keywords:

nitric oxide; severe asthma; phenotype; airway reactivity; exhaled breath

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

Exhaled nitric oxide (Fe_NO) is a biomarker of airway inflammation in mild to moderate asthma. However, whether Fe_NO levels are informative regarding airway inflammation in patients with severe asthma, who are refractory to conventional treatment, is unknown.

What This Study Adds to the Field

Here, we demonstrate that grouping of asthma by Fe_NO provides an independent classification of asthma severity, and among patients with severe asthma identifies the most reactive and worrisome asthma phenotype.

Despite progress that has been made in the understanding and treatment of mild and moderate asthma, severe asthma is poorly understood, refractory to established treatments, and accounts for a high proportion of the adverse financial impact, morbidity, and mortality of asthma in the United States (1–4). The underlying reasons why certain individuals with asthma have severe, refractory disease are poorly defined. Although sputum eosinophils have been shown to predict acute exacerbations in asthma (5, 6), sputum induction is not easy to do or widely available. Thus, there is a need for a noninvasive, easy-to-perform test to monitor patients with severe asthma and predict acute and often life-threatening asthma exacerbations, and thus allow for determination of whether or not therapy is adequate (1–4). As a free radical that reacts with oxidants and antioxidants, nitric oxide (NO) in exhaled breath (Fe_NO) reflects the redox state of the airway and has been proposed as a marker of airway inflammation and guide for antiinflammatory therapy in asthma (7). High levels of Fe_NO are well documented in nonsevere asthma (8–21) and decrease in response to treatment with corticosteroids (22–27). However, measures of Fe_NO in 50 patients with severe asthma in the European multicenter study of chronic severe asthma suggest that Fe_NO levels of patients with severe asthma, who are refractory to conventional treatments, may not be suppressed by corticosteroids (28). Although the mean Fe_NO levels of patients with severe asthma were similar to those of patients with nonsevere asthma, 22 (44%) of the subjects with severe asthma who were receiving high-dose oral corticosteroids had threefold higher Fe_NO than those receiving inhaled corticosteroids, which suggested that a substantial subpopulation of patients with severe asthma had persistent airway inflammation and possible relative corticosteroid resistance.

In this study, we hypothesized that classification of severe asthma based on airway inflammation as defined by Fe_NO levels would identify a more severe asthma phenotype. The present study was designed to assess alterations of Fe_NO in patients with severe asthma as compared with patients with nonsevere asthma and healthy control subjects, and the relationship between Fe_NO and asthma severity, airflow limitation, hyperinflation, hyperresponsiveness, and atopy. Although the average Fe_NO levels in severe and nonsevere asthma were previously reported to be similar (29), when asthma was classified on the basis of Fe_NO levels, a distinct asthma phenotype emerged. In general, patients with asthma and high Fe_NO levels tended to be younger and diagnosed with asthma at a younger age. They were more likely to be atopic and to have evidence of airway inflammation. Furthermore, patients with severe asthma and high Fe_NO levels had the greatest airway reactivity, the most hyperinflation, and the least awareness of their asthma symptoms. The findings provide evidence that Fe_NO levels are informative for classification of severe asthma phenotypes and allow identification of a particularly worrisome subgroup of patients with severe asthma. Some of the results of these studies have been previously reported in the form of an abstract (30).

METHODS

Section:

Detailed methods and statistical analyses are provided in the online supplement. A brief description is provided here.

Subject Enrollment and Characterization

All subjects were recruited by centers participating in the Severe Asthma Research Program (SARP) and gave written informed consent by signing a consent document approved by the institutional review board at the enrolling center and the SARP Data Safety and Monitoring Board (DSMB). All subjects were screened by history, physical examination, spirometry (before and after two puffs of inhaled albuterol), methacholine provocation, and allergy prick skin testing to a standard panel of aeroallergens. Subjects were nonsmokers, and classified as healthy control subjects if they were free of respiratory symptoms, had normal baseline spirometry, a negative methacholine challenge test, and nitric oxide level less than 50 ppb. Asthma was defined according to the National Asthma Education and Prevention Program guidelines, which include episodic respiratory symptoms, reversible airflow obstruction (documentation of variability of FEV₁ and/or FVC by 12% and 200 cm³ either spontaneously or after two puffs of inhaled albuterol), and/or a positive methacholine challenge test (4). Severe asthma was based on the definition used by the proceedings of the American Thoracic Society Workshop on Refractory Asthma (2).

Lung Function

Spirometry was performed with an automated spirometer, consistent with American Thoracic Society (ATS) standards (31). Plethysmographic lung volumes, including total lung capacity (TLC) and residual volume (RV), were measured in 62 subjects with severe asthma and 53 subjects with nonsevere asthma, using methods conforming to ATS guidelines (32), and recorded as the percentage of predicted values obtained with the equations of Stocks and Quanjer (33), with adjustments for African Americans per ATS recommendations (34).

Atopy

Allergy skin testing was done once on each subject during the study. Skin prick testing to 14 common allergens was performed at all SARP sites with the Multi-Test II (Lincoln Diagnostics, Inc., Decatur, IL). Blood was collected for measurement of total serum IgE and a complete blood count.

Exhaled NO (Fe_NO)

All SARP centers performed online and/or offline NO measurements according to the standards published by the ATS (35). Online Fe_NO values were used in all data analyses in this article. NO levels were measured online by chemiluminescence at a constant expiratory flow (50 ml/s) in all participating centers. The analyzers were calibrated in accordance with the manufacturer's instructions. Because spirometry can affect the Fe_NO levels, exhaled gases were collected before spirometry, if completed on the same day. On the basis of data suggesting poor asthma control when Fe_NO is more than 35 ppb (7), we evaluated clinical characteristics of asthma populations in subgroups of high (>35 ppb) and low (<35 ppb) NO. The rationale for selecting 35 ppb as a cutoff point for high and low NO was based on the published literature (7) and analysis of the data collected in this study. In addition to the published literature, Figure 1 provides the rationale for selecting 35 ppb as a cutoff point for high and low Fe_NO that is the basis for all data analyses in this study. Relevant variables (as outlined in Table E1 in the online supplement) in the database were analyzed on the basis of receiver operator characteristic (ROC) curves with Fe_NO as a continuous variable. The cutoff point for each variable was determined on the basis of these ROC curves. Figure 1 represents the frequency distribution of all these cutoff points. The median of all cutoff points for the variables (both categorical and continuous) that showed a significant relationship with Fe_NO was 37 ppb. This provided support for the validity of our selection of 35 ppb as the cutoff point between high and low Fe_NO.

Figure 1. Relevant variables (as outlined in Table E1 in the online supplement) in the database were analyzed on the basis of receiver operator characteristic (ROC) curves with Fe_NO as a continuous variable. The cutoff point for each variable was determined on the basis of these ROC curves. Shown here is the frequency distribution of all these cutoff points. The median of all cutoff points for the variables (both categorical and continuous) that showed a significant relationship with Fe_NO was 37 ppb. This provided support for the validity of our selection of 35 ppb as the cutoff point between high and low Fe_NO.

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Total NO Reaction Products

NO reaction products (NOx) in serum samples were measured by an amperometric NO sensor in combination with acidified iodide for the detection of NO derived from total nitrite and nitrate after cadmium/copper-mediated reduction of nitrate to nitrite (ISO-NOP, Nitralyzer II; World Precision Instruments, Sarasota, FL) (36).

Statistical Analyses

Categorical data were summarized as frequencies, and statistical comparisons for categorical variables were performed using Fisher's exact test. Subgroup comparisons within NO level or asthma severity were performed using appropriate contrasts from a logistic regression model including NO level, asthma severity, and their interaction as independent variables. Continuous variables were summarized using the sample size, mean, and standard deviation, and alternatively using the median and interquartile range for variables with skewed distributions. Associations between NO levels and other variables were assessed by linear regression for Fe_NO as a continuous variable and multiple logistic regression for Fe_NO (high or low) as categorical variables. Multiple logistic regression modeling is described in more detail in Results. All tests and model fitting were performed with the JMP statistical program, version 5.0 (SAS Institute Inc., Cary, NC) and R version 2.4.1 (www.R-project.org) (37). Models for Fe_NO as a continuous outcome in a linear regression model and as a dichotomous outcome classified as high or low in a logistic regression model were created. For multivariate analyses and modeling, parsimonious selection of independent variables was performed to avoid confounding that would render the estimated associations with the outcome as noninterpretable or misleading. Similarly, a logistic regression model for which the Fe_NO outcome would be classified as high or low had to be parsimonious to be mathematically stable.

RESULTS

Section:

Characterization of Study Population

Online Fe_NO levels were measured in 495 individuals enrolled in the Severe Asthma Research Program (SARP). Baseline characteristics are shown in Table 1. On average, healthy control subjects and patients with nonsevere asthma were younger than patients with severe asthma (P < 0.05) (Table 1). As expected, lung functions were lower in patients with severe asthma than in patients with nonsevere asthma or healthy control subjects (Table 1). The detailed clinical description of individuals in the SARP data set was previously published (29). The SARP population included in the current study does not overlap with the SARP subpopulation of children with offline NO values published previously (38).

TABLE 1. **DEMOGRAPHICS, PULMONARY FUNCTION, EXHALED NITRIC OXIDE LEVELS, AND CORTICOSTEROIDS USAGE FOR ALL SUBJECTS**
	Control Subjects (n = 49)	n	Nonsevere Asthma (n = 271)	n	Severe Asthma (n = 175)	n
Mean age, yr^*	32 ± 11	49	34 ± 12	271	41 ± 13	175
Baseline %FEV₁^*	101 ± 15	49	83 ± 16	271	58 ± 20	175
Maximal %FEV₁^*	108 ± 15	37	93 ± 15	256	76 ± 20	168
% FVC^*	103 ± 11	49	94 ± 14	271	80 ± 19	176
% FEV₁/FVC^*, ppb	97 ± 7	49	88 ± 12	271	77 ± 14	175
Fe_NO, ppb	17 ± 9	49	43 ± 42	271	42 ± 41	175
Median, IQR^*	14 (11–19)	49	27 (17–55)	271	27 (17–52)	175
Sex (male), no.	13	49	86	271	65	176
Race, C/AA/AI/A/NH/O/U/R/MR	40/5/0/2/0/0/0/0/2	49	172/80/0/4/0/8/1/0/6	271	116/44/0/7/0/2/0/0/6	175
Corticosteroids
Inhaled, %^*	0	49	64%	271	100%	175
Oral, %^*	0	49	3%	271	44%	175
Injected, %	0	49	0%	271	3%	175
Serum IgE levels^*	58 ± 87	45	267 ± 380	239	318 ± 730	147
Median, IQR^*	32 (10–60)	45	145 (60–330)	239	124 (40–320)	147
BAL eosinophils, %^*	0.2 ± 0.7	21	1.1 ± 0.4	73	1.9 ± 0.5	49
Median, IQR^*	0 (0–0.4)	21	0.3 (0–1.2)	73	0.5 (0–1.5)	49
Blood eosinophils, %^*	2.3 ± 1	45	4.1 ± 3	252	4.1 ± 5	168
Median, IQR^*	2 (1–2.85)	45	3.7 (2–5)	252	3 (1.5–5)	168

Definition of abbreviations: A = Asian; AA =African American; AI = American Indian or Alaska native; BAL = bronchoalveolar lavage; C = Caucasian; Fe_NO = exhaled nitric oxide level; IQR = interquartile range; MR = multiple races; n = number of individuals with available data; NH = Native Hawaiian; O = other; R = refused; U = uncertain.

^* Fisher's, analysis of variance, or Kruskal-Wallis P < 0.05 among three groups.

NO in Asthma

NO levels were higher in patients with asthma as compared with control subjects, but there was no significant difference in average Fe_NO between severe and nonsevere asthma (Fe_NO [ppb]: control, 17 ± 9; nonsevere, 43 ± 42; severe, 42 ± 41; P = 0.01) (Table 1). The proportion of individuals with high Fe_NO was the same in severe and nonsevere asthma (nonsevere, 109/271 [40%]; severe, 70/175 [40%]).

The High-NO Phenotype in Asthma

There were equal proportions of patients with severe and nonsevere asthma in the low- and high-Fe_NO groups. In general, patients with asthma and high Fe_NO demonstrated several distinct characteristics when compared with patients with asthma and low Fe_NO. Demographically, patients with asthma and high Fe_NO were younger (age, yr [mean ± SD]: low Fe_NO, 38 ± 12; high Fe_NO, 36 ± 13; P = 0.03) and diagnosed with asthma at a younger age (age, yr [mean ± SD]: low Fe_NO, 16 ± 13; high Fe_NO, 14 ± 14; P = 0.05) and less likely to be female (female [% of population]: low Fe_NO, 70%; high Fe_NO, 60%; P = 0.02).

On pulmonary function testing, the high- and low-Fe_NO groups had similar baseline FEV₁ and FVC, but the FEV₁/FVC ratio (% predicted) was lower in high Fe_NO, indicating increased airflow limitation in this group. The high-Fe_NO group also had more airway reactivity as shown by greater FEV₁ reversibility after maximal bronchodilation and lower PC₂₀ (provocative concentration of methacholine causing a 20% fall in FEV₁). They had more hyperinflation with a higher total lung capacity (TLC), a higher residual lung volume (RV), and a higher RV/TLC ratio (Table 2).

TABLE 2. **PULMONARY FUNCTION BY EXHALED NITRIC OXIDE**
Characteristic	Low Fe_NO (≤35 ppb)	n	High Fe_NO (>35 ppb)	n	P Value
Baseline FVC, % predicted	85 ± 18	267	87 ± 19	179	0.20
Maximal FVC, % of predicted	93 ± 16	253	100 ± 15	170	<0.001
Baseline FEV₁, % predicted	74 ± 20	267	73 ± 23	179	0.80
Maximal FEV₁, % of predicted	85 ± 20	253	90 ± 18	170	0.005
FEV₁/FVC ratio, % of predicted	86 ± 14	267	81 ± 14	179	<0.001
Maximal FEV₁ reversal, %	14 ± 16	253	20 ± 17	170
Median (IQR)^*	10 (5–18)	253	16 (8–26)	170	<0.001
PC₂₀	4.3 ± 6	203	1.7 ± 3	123
Median (IQR)^*	1.8 (0.5–4.9)	203	0.7 (0.3–1.6)	123	<0.001
TLC, % predicted	106 ± 12	88	115 ± 14	43	0.002
FRC, % predicted	101 ± 24	84	119 ± 30	40	<0.001
FRC/TLC, % predicted	95 ± 18	84	103 ± 16	40	0.008
RV, % predicted	124 ± 42	88	153 ± 57	43	0.003
RV/TLC, % predicted	111 ± 30	88	126 ± 40	42	0.03

Definition of abbreviations: IQR = interquartile range; PC₂₀ = provocative concentration of methacholine causing a 20% fall in FEV₁; RV = reserve volume; TLC = total lung capacity.

^* Wilcoxon rank sum P values reported rather than t test.

High-Fe_NO patients with asthma, whether severe or nonsevere, were more likely to be atopic as shown by more positive skin tests (number of positive skin tests [mean ± SD]: low Fe_NO, 3.4 ± 3; high Fe_NO, 4.2 ± 3; P = 0.004), higher serum IgE level (serum IgE [mean ± SD]: low Fe_NO, 219 ± 366; high Fe_NO, 340 ± 402; P = 0.0001), and higher blood eosinophils (% blood eosinophils [mean ± SD]: low Fe_NO, 3.4 ± 3.7; high Fe_NO, 5.1 ± 3.9; P = 0.0001). They also had more eosinophils in sputum (% sputum eosinophils [mean ± SD]: low Fe_NO, 3 ± 7; high Fe_NO, 13 ± 23; P = 0.0001), suggesting more evidence of allergic airway inflammation. Interestingly, patients with asthma and high Fe_NO levels were less likely to have seen a physician in the last 12 months (%: low Fe_NO, 72%; high Fe_NO, 63%; P = 0.04), but more likely to have been in the emergency room (%: low Fe_NO, 66%; high Fe_NO, 73%; P = 0.05) over the same time period, or admitted to the intensive care unit in the past (%: low Fe_NO, 16%; high Fe_NO, 25%; P = 0.02).

Patients with asthma and low NO levels were more likely to be overweight (body mass index, kg/m² [mean ± SD]: low Fe_NO, 31 ± 9; high Fe_NO, 28 ± 8; P = 0.002), have systemic hypertension (%: low Fe_NO, 16%; high Fe_NO, 8%; P = 0.05), and be on treatment for diabetes (%: low Fe_NO, 40%; high Fe_NO, 11%; P = 0.01).

Characterizing the High-Fe_NO Phenotype in Severe Asthma

In patients with severe asthma, high Fe_NO levels identified a phenotype that appeared to be the most severe of all groups, including low-Fe_NO severe asthma, and high- or low-Fe_NO nonsevere asthma groups. Individuals with severe asthma and high Fe_NO levels tended to share several characteristics. They had the greatest airway reactivity of any group defined as the magnitude of FEV₁ reversal after maximal bronchodilation and by PC₂₀. They had the greatest degree of airflow limitation and the most hyperinflation (Table 3). They also had high numbers of eosinophils in the sputum (Table 4). Emergency room use and intensive care unit admissions were greatest in this group (Table 3). In contrast to Fe_NO, NO metabolites (NOx) in serum were higher in all patients with severe asthma as a group in comparison with patients with nonsevere asthma (NOx, μM: nonsevere, 36 ± 23; severe, 42 ± 24; P = 0.0009) and were unrelated to Fe_NO levels (R = 0.002; P = 0.5). Serum NOx was not related to clinical characteristics such as lung function or atopy (all P > 0.2).

TABLE 3. **PULMONARY FUNCTION AND EXHALED NITRIC OXIDE LEVEL BY SEVERITY**
Characteristic	Severe Asthma, Low Fe_NO	n	Nonsevere Asthma, Low Fe_NO	n	Severe Asthma, High Fe_NO	n	Nonsevere Asthma, High Fe_NO	n	Low Fe_NO: Severe vs. Nonsevere Asthma	High Fe_NO: Severe vs. Nonsevere Asthma	Severe Asthma: Low vs. High Fe_NO	Nonsevere Asthma: Low vs. High Fe_NO
Baseline FVC, % predicted	75 ± 18	105	92 ± 15	162	75 ± 21	70	95 ± 14	109	<0.001	<0.001	0.97	0.13
Maximal FVC, % predicted	88 ± 17	101	97 ± 14	153	95 ± 18	67	103 ± 12	103	<0.001	<0.001	0.004	<0.001
Baseline FEV₁, % predicted	60 ± 19	105	83 ± 16	162	56 ± 22	70	83 ± 17	109	<0.001	<0.001	0.24	0.76
Maximal FEV₁, % predicted	74 ± 20	101	91 ± 15	153	80 ± 19	67	96 ± 15	103	<0.001	<0.001	0.009	0.04
FEV₁/FVC ratio, % predicted	79 ± 15	105	90 ± 11	162	74 ± 14	70	86 ± 12	109	<0.001	<0.001	0.011	0.03
Maximal FEV₁ reversal, %	18 ± 23	101	11 ± 9	153	23 ± 19	67	17 ± 15	103
Median (IQR)^*	14 (6–22)	101	8 (5–14)	153	21 (9–29)	67	13 (7–22)	103	0.002	0.01	0.005	<0.001
PC₂₀	3.9 ± 6	53	4.4 ± 6	149	1.5 ± 3	27	1.7 ± 3	96
Median (IQR)^*	1 (0.2–4.5)	53	2 (0.6–5)	149	0.6 (0.2–1.7)	27	0.7 (0.3–1.6)	96	0.10	0.40	0.01	<0.001
TLC, % predicted	107 ± 13	41	104 ± 12	31	117 ± 17	21	112 ± 10	22	0.55	0.28	0.006	0.05
FRC, % predicted	103 ± 27	38	96 ± 21	31	124 ± 34	18	115 ± 26	22	0.40	0.36	0.005	0.01
FRC/TLC, % predicted	96 ± 21	38	92 ± 14	31	104 ± 16	18	102 ± 17	22	0.43	0.84	0.10	0.03
RV, % predicted	143 ± 44	41	109 ± 34	31	176 ± 58	21	131 ± 47	22	0.004	0.001	0.005	0.11
RV/TLC, % predicted	128 ± 30	41	99 ± 21	31	141 ± 41	20	111 ± 33	22	<0.001	0.002	0.12	0.21
ER in past 12 mo	38%	105	12%	161	53%	70	18%	109	<0.001	<0.001	0.05	0.14
Ever had an ICU admission due to asthma	34%	105	5%	162	44%	70	12%	108	<0.001	<0.001	0.26	0.04
BMI	32 ± 8	103	30 ± 9	162	30 ± 8	66	28 ± 7	109	0.08	0.13	0.08	0.016

Definition of abbreviations: BMI = body mass index; ER = emergency room; ICU = intensive care unit; IQR = interquartile range; RV = reserve volume; TLC = total lung capacity.

High Fe_NO defined as >35 ppb, and low Fe_NO as ≤35 ppb.

^* Wilcoxon rank sum P values reported rather than those based on contrasts from analysis of variance.

TABLE 4. **INFLAMMATORY CELLS IN BLOOD, BRONCHOALVEOLAR LAVAGE, AND SPUTUM BY SEVERITY AND EXHALED NITRIC OXIDE LEVELS**
Characteristic	Severe Asthma, Low Fe_NO	n	Nonsevere Asthma, Low Fe_NO	n	Severe Asthma, High Fe_NO	n	Nonsevere Asthma, High Fe_NO	n	Low Fe_NO: Severe vs. Nonsevere Asthma	High Fe_NO: Severe vs. Nonsevere Asthma	Severe Asthma: Low vs. High Fe_NO	Nonsevere Asthma: Low vs. High Fe_NO
Inflammatory cells in blood
Total WBC	7.8 ± 3	103	6.8 ± 2	153	8 ± 2	64	6.5 ± 2	104	0.001	<0.001	0.76	0.19
Monocytes, %	5.7 ± 2	103	6.4 ± 2	152	6.0 ± 3	63	6.8 ± 2	104	0.23	0.83	0.06	0.16
Neutrophils, %	62 ± 12	103	58 ± 10	152	63 ± 15	64	55 ± 10	104	0.27	0.46	0.81	0.63
Lymphocytes, %	28 ± 11	103	32 ± 9	152	25 ± 10	64	33 ± 9	104	0.16	<0.001	0.27	0.11
Eosinophils, %	3.8 ± 5.2	103	3.1 ± 2.1	152	4.7 ± 3.9	64	5.3 ± 3.9	104	0.20	0.27	0.13	<0.001
Basophils, %	0.4 ± 0.5	103	0.6 ± 0.5	144	0.5 ± 0.5	64	0.5 ± 0.6	104	0.005	0.36	0.34	0.60
Inflammatory cells in BAL
BAL total cells	8 ± 6	27	9 ± 8	48	4.8 ± 3	22	8.9 ± 6	27	0.55	0.02	0.09	0.95
BAL macrophages, %	91 ± 10	27	90 ± 16	47	82 ± 24	22	81 ± 26	26	0.74	0.85	0.10	0.07
BAL neutrophils, %	3.0 ± 4	27	2.1 ± 4	47	2.6 ± 4	22	3.2 ± 7	26	0.42	0.62	0.72	0.34
BAL lymphocytes, %	3.8 ± 3	27	5.3 ± 6	47	8.9 ± 13	22	6.4 ± 6	26	0.40	0.23	0.02	0.56
BAL eosinophils, %	1.9 ± 6	27	0.8 ± 2.2	47	1.8 ± 3	22	1.5 ± 2.4	26	0.18	0.77	0.93	0.39
Inflammatory cells in sputum
Total cells, millions	4.1 ± 7	61	3.0 ± 4	117	3.3 ± 5	34	2.4 ± 3	80	0.24	0.98	0.52	0.76
Total WBCs, millions	3.1 ± 6	61	2.1 ± 4	117	2.5 ± 5	34	2.4 ± 9	79	0.27	0.92	0.61	0.76
Viability of WBCs, %	61 ± 24	62	63 ± 21	117	58 ± 23	34	62 ± 22	79	0.48	0.30	0.53	0.83
Bronchial epithelial cells, %	4.8 ± 6	62	2.9 ± 5	117	2.4 ± 3	34	3.7 ± 4	79	0.02	0.21	0.03	0.30
Sputum macrophages, %	52 ± 25	56	56 ± 26	99	39 ± 32	28	63 ± 26	66	0.38	<0.001	0.05	0.11
Sputum lymphocytes, %	4.2 ± 5	56	2.5 ± 3	99	3.1 ± 6	28	2.7 ± 2	66	0.008	0.64	0.20	0.82
Sputum neutrophils, %	40 ± 25	56	39 ± 26	99	32 ± 30	28	27 ± 23	66	0.79	0.36	0.23	0.006
Sputum eosinophils, %	3 ± 5	56	2.2 ± 4	99	25 ± 33	28	7 ± 13	66	0.38	<0.001	<0.001	0.02

Definition of abbreviation: BAL = bronchoalveolar lavage; WBCs = white blood cells.

Characterizing the High-Fe_NO Phenotype in Nonsevere Asthma

In patients with nonsevere asthma, high Fe_NO similarly identified a more severe subgroup. In fact, the patients with nonsevere asthma and high Fe_NO shared more similarities with patients with severe asthma and high Fe_NO than with patients with nonsevere asthma and low Fe_NO. For instance, the nonsevere group with high Fe_NO had more airway reactivity defined by the magnitude of FEV₁ reversal after maximal bronchodilation and by PC₂₀, and significantly more airflow limitation and hyperinflation than patients with nonsevere asthma and low Fe_NO levels. They also had more eosinophilic inflammation (Table 4) and more intensive care unit (although not emergency room) admissions. These individuals were the thinnest among all groups (Table 3).

Fe_NO and Lung Volumes

TLC increased linearly with increased air trapping as measured by elevated ratio of RV to TLC. In addition, there was an independent additive increase in TLC in subjects with higher Fe_NO (P = 0.0005 for Fe_NO effect, P < 0.0001 for RV/TLC effect; analysis of covariance). There was no effect of the designated severe or nonsevere asthma grouping (P > 0.9) on TLC independent of air trapping and Fe_NO effects within each of the severity groups. This indicates that air trapping and Fe_NO are independent determinants for lung hyperinflation in asthma.

Fe_NO and Use of Corticosteroids and Other Medications

The greater reactivity in the high-Fe_NO asthma subgroups suggested that these patients had greater airway inflammation and/or less antiinflammatory therapy. All patients with severe asthma in this study were by definition receiving some form of corticosteroids (2). There was no difference in the use of inhaled corticosteroid or leukotriene modifiers among patients with asthma and high or low Fe_NO, but more patients in the high-Fe_NO group were taking oral corticosteroids (Table 5). Individuals with high Fe_NO were more likely to be taking theophylline (Table 5). When corticosteroid use was further analyzed by asthma severity in addition to Fe_NO levels, again there was no significant difference in inhaled corticosteroid use between the high- and low-Fe_NO groups regardless of severity. The group with high Fe_NO and severe asthma had the highest proportion of oral corticosteroid use (percent oral corticosteroid use: severe asthma–low Fe_NO, 37%; nonsevere asthma–low Fe_NO, 1%; severe asthma–high Fe_NO, 56%; nonsevere asthma–high Fe_NO, 5%; P = 0.01). Whereas only a small number of individuals were taking theophylline, patients with severe asthma and high Fe_NO levels were much more likely to be taking daily theophylline than members of any of the other groups (percent theophylline use: severe asthma–low Fe_NO, 13%; nonsevere asthma–low Fe_NO, 1%; severe asthma–high Fe_NO, 29%; nonsevere asthma–high Fe_NO, 3%; P = 0.01). Thus, the finding of high Fe_NO in the severe and nonsevere asthma subgroups was likely not due to less corticosteroid therapy than in the low-Fe_NO subgroups. Multivariate analyses and modeling for determinants of Fe_NO did not indicate an influence of corticosteroid use on Fe_NO levels (Table 6).

TABLE 5. **MEDICATION USE BY EXHALED NITRIC OXIDE LEVELS**
Characteristic	Low Fe_NO (≤35 ppb)	n	High Fe_NO (>35 ppb)	n	P Value^*
Inhaled corticosteroids	28%	267	28%	179	0.9
Oral corticosteroids	15%	267	25%	179	0.01
Injectable corticosteroids	2.2%	267	3.9%	179	0.3
Inhaled corticosteroids and β-agonist	57%	267	50%	179	0.1
Total β-agonists	91%	267	91%	179	0.9
Total long-acting β-agonist	65%	267	59%	179	0.2
Total inhaled corticosteroids	73%	267	70%	179	0.5
Total other corticosteroids	16%	267	25%	179	0.02
Leukotriene modifiers	29%	267	31%	179	0.6
Theophylline	6%	267	13%	179	0.01

^* Fisher's exact test P values.

TABLE 6. **RESULTS OF MULTIVARIATE LOGISTIC REGRESSION ANALYSIS WITH HIGH EXHALED NITRIC OXIDE (>35 ppb) AS THE OUTCOME**
	All Asthma (n = 335)			Nonsevere Asthma (n = 210)
Characteristic	Odds Ratio (95% CI)	P Value	Odds Ratio (95% CI)	P Value	Odds Ratio (95% CI)	P Value
Sex	0.99 (0.55–1.61)	0.96	1.04 (0.50–2.05)	0.90	1.11 (0.40–2.70)	0.82
Age	0.99 (0.97–1.01)	0.57	0.97 (0.95–1.00)	0.058	1.02 (0.99–1.05)	0.22
BMI	0.99 (0.95–1.02)	0.40	1.02 (0.98–1.07)	0.36	0.94 (0.89–1.00)	0.06
Activity score	1.42 (1.10–1.80)	0.005	1.47 (1.08–2.03)	0.02	1.52 (0.89–2.31)	0.08
Wheezing	1.14 (0.92–1.38)	0.17	1.17 (0.89–1.50)	0.24	1.19 (0.80–1.68)	0.33
Log maximal FEV₁ reversal	1.19 (1.01–1.41)	0.03	1.22 (0.99–1.51)	0.06	1.29 (0.91–1.73)	0.1
Log IgE	1.88 (1.19–3.01)	0.007	2.71 (1.38–5.36)	0.004	1.52 (0.74–3.20)	0.25
Log percent eosinophils in the blood	1.35 (1.11–1.62)	0.002	1.15 (1.14–1.93)	0.003	1.31 (0.97–1.78)	0.07
Total ICS	0.74 (0.40–1.39)	0.34	0.68 (0.34–1.40)	0.30	3.14 (0.07–95.54)	0.53
Total other CS	1.34 (0.66–3.20)	0.41	2.05 (0.18–22.45)	0.55	1.76 (0.68–5.15)	0.23
Theophylline use	2.92 (1.23–7.54)	0.02	9.0 (0.71–102.62)	0.08	2.9 (1.13–9.30)	0.04
Seen a doctor in the last 12 mo	0.58 (0.31–1.09)	0.09	0.75 (0.35–1.61)	0.46	0.5 (0.12–2.89)	0.38
Visited ER in the last 12 mo	2.33 (1.20–4.69)	0.01	2.65 (0.87–7.90)	0.09	2.5 (1.08–7.69)	0.05

Definition of abbreviations: BMI = body mass index; CI = confidence interval; CS = corticosteroids; ER = emergency room; ICS = inhaled corticosteroids.

n = number of individuals who had a complete set of all variables to run the model.

DISCUSSION

Section:

This study provides evidence that subclassification by Fe_NO defines asthma phenotypes independent of current definitions for asthma severity. Patients with asthma who have high Fe_NO levels share several characteristics regardless of their asthma severity as it is currently defined. Patients with asthma and high Fe_NO are younger and diagnosed with asthma at a younger age. They are atopic and have more eosinophilic airway inflammation, more airway reactivity, more airflow limitation, and more hyperinflation. The fact that patients with high Fe_NO were more likely to have gone to an emergency room or admitted to an ICU over the past 12 months also suggests that they may be less aware of early symptoms of their disease. Within the severe asthma group of subjects, high Fe_NO identifies a severe asthma phenotype that has the greatest eosinophilic airway inflammation, the most severe airflow limitation, and uses emergent care most often.

NO is produced by nitric oxide synthases (NOS), including constitutive (neuronal, or type 1, and endothelial, or type 3) and inducible (type 2) enzymes, all isoforms of which are present in the lung (39, 40). Abnormalities in NOS1 and NOS2 genotype and expression are associated with asthma and increased NOS activity is associated with increased Fe_NO levels (41–43). Fe_NO represents a balance between NO production and consumption (10). In particular, NO is rapidly consumed by reaction with superoxide. There is direct evidence that more severe obstruction in asthma is associated with increased spontaneous and stimulus-induced generation of superoxide by inflammatory cells in the airway (44).

The independent association of elevated Fe_NO with increased TLC is a novel finding, and suggests that there is an inflammatory component affecting lung mechanics that is separate from the air-trapping mechanism. Increased TLC has been associated with acute asthma exacerbation and with poorly controlled chronic asthma, and many of these subjects exhibit a decrease in TLC after therapy with bronchodilators and corticosteroids (45–47). Further studies are needed to determine the nature of the interaction between NO and TLC in asthma, but the current study shows that Fe_NO is associated with hyperinflation in asthma. Furthermore, Fe_NO in severe asthma might reflect airway-remodeling processes (48, 49). Because many of the variables that are related to NO are also related to severity, relationships between variables and Fe_NO were also evaluated within severity group by multivariate analyses. Multivariate analyses and modeling confirmed most of the associations suggested by the univariate analyses and revealed new findings as well. For instance, the relationship between Fe_NO and markers of atopy and eosinophilic inflammation was confirmed in nonsevere asthma, which suggests a strong dependence of Fe_NO on these variables. However, the multivariate significance of factors that influence Fe_NO in all asthma was driven primarily by the nonsevere asthma group. Most features did not significantly influence Fe_NO in severe asthma. This suggests that features other than those evaluated in this study may be determinants of high Fe_NO levels in severe asthma.

There are several possible explanations for the presence of high Fe_NO in patients with severe asthma. Because the Fe_NO levels of patients with nonsevere asthma decrease in response to corticosteroid therapy (22–27), the greater corticosteroid use in the severe asthma group would be expected to result in low levels of Fe_NO. In this context, one possible explanation for the high Fe_NO may be inadequate corticosteroid therapy. However, high Fe_NO levels in patients with severe asthma on high-dose oral or injectable corticosteroids are difficult to explain on this basis. Noncompliance with therapy is possible, but this explanation needs to invoke that patients with severe asthma with high Fe_NO are less compliant than patients with severe asthma with low Fe_NO, even though they report similar corticosteroid use. Importantly, high Fe_NO was not related to corticosteroid therapy in any analysis. Furthermore, the current ATS workshop criteria are meant to distinguish patients with severe asthma, but not to define those with nonsevere asthma. This may partially explain why the patients with high Fe_NO have similar characteristics in both patient groups. Taken together with previous studies (1, 3, 23, 28, 50–54), the high-Fe_NO severe asthma group may have relative resistance to corticosteroid therapy. In support of this, individuals with severe asthma and high Fe_NO are more likely to be taking theophylline. Given the current clinical practice of reserving theophylline use in asthma for individuals not responding to traditional therapies, greater use of daily theophylline in the high-Fe_NO group is another indirect marker of more difficult to treat, less corticosteroid-responsive asthma.

Analyses of the characteristics of individuals with low Fe_NO and severe asthma in this study also provide new information about asthma phenotypes. Fe_NO levels reflect a balance between the rates of NO production and its consumption, which is largely related to oxidant–NO reactions (10, 55, 56). Thus, low levels of Fe_NO in asthma may be related to less NO synthesis or greater oxidative consumption. Mechanisms that affect NO production include factors that modify NOS enzyme activity or expression, alter nonenzymatic release of NO from storage pools, or change the denitrifying organisms that colonize the upper airway (57–61). However, the end products of NO consumption are greater in severe asthma than in nonsevere asthma, which suggests that total NO production is greater in severe asthma but may not be reflected by levels in the exhaled breath because of greater oxidative consumption. In support of this concept, features of metabolic syndrome, which is characterized by oxidative stress and abnormalities of NO metabolism, were observed in the low-Fe_NO asthma group, that is, higher body mass index, hypertension, and diabetes.

Strengths of this study include the large cohort, the well-characterized population, and the prospective and standardized method of data collection. The main imitation of this study is also the fact that it is a cohort and not a randomized controlled trial. Fe_NO levels were analyzed in a cross-sectional fashion and not based on or before and after an intervention. Certain variables such as compliance with therapy could not be completely accounted for and verified.

Clinical asthma phenotypes have been recognized for some time (51, 62), but quantitative biomarkers have not been previously identified in severe asthma (51). This has limited the clear discrimination and understanding of severe asthma. Detailed and quantitative phenotypes will further our understanding of the pathobiology and genetics that contribute to severe asthma genesis (51). Although the current definition of asthma severity is useful for clinical research, it is cumbersome to use and impractical for the busy office setting. The availability of an easy-to-measure, noninvasive marker would greatly simplify and improve severe asthma management (63). The current findings suggest that evaluation of multiple quantitative biologic markers, such as Fe_NO, circulating NO reaction products, and sputum eosinophils, may provide a cumulative index for definition of asthma severity in the future. Here, Fe_NO is identified as a biomarker that distinguishes a group of patients with severe airflow obstruction, hyperreactivity, hyperinflation, and persistent airway inflammation. Although the retrospective nature of our analysis has precluded us from determining whether Fe_NO could predict future risk of exacerbations in asthma, its correlation with ER visits and hospital and ICU admission suggests a great potential for Fe_NO in identifying those patients with the most severe disease in clinical practice. Prospective studies would be helpful in confirming this fact that is suggested by our findings and to ascertain the determinants of the high-Fe_NO phenotype in patients with severe asthma, who are refractory to therapy.

The authors thank A. Janocha for the performance of NOx analyses.

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Correspondence and requests for reprints should be addressed to Raed A. Dweik, M.D., Department of Pulmonary, Allergy, and Critical Care Medicine/Respiratory Institute, Desk A-90, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: [email protected]

American Journal of Respiratory and Critical Care Medicine

Abstract

Abstract

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