INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Endogenous nitric oxide (NO) is detectable in the exhaled air of normal subjects (1) and this may play an important role in the regulation of respiratory function (2, 3). Levels of exhaled nitric oxide (eNO) are elevated in patients with asthma (4, 5). Patients with asthma who are treated with inhaled steroids have levels of eNO similar to those of patients without (6) and the levels rise as the dose of inhaled steroid is reduced (7). eNO levels are also lower in cigarette smokers (8).

Sensitization to indoor allergen is a major risk factor for asthma worldwide (9). Although there is much evidence linking indoor allergen exposure with allergic sensitization (10), there is less information on the effect of exposure to indoor allergens in established asthma. Peat and co-workers have compared airway reactivity in mite-sensitized children with asthma who are living in geographic areas with differing levels of mite allergens, and found that those in areas with higher exposure had more severe airway reactivity (11).

Studies of children have suggested that levels of eNO are higher in patients with atopic asthma compared with levels in patients with nonatopic asthma and atopic patients without asthma (12), but this study did not measure domestic allergen exposure in the subjects.

The aim of this study was to identify whether patients with allergic asthma who are not taking inhaled steroids and are exposed to high levels of allergen to which they are sensitized have more severe allergic inflammation of the airways, as measured by eNO, than do patients with asthma who are not exposed to high levels of allergen. In addition, we investigated the correlation of eNO with bronchial hyperresponsiveness, and total and specific IgEs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Adult patients, between 18 and 60 yr of age, were eligible for screening if they met the following criteria: (1) a physician diagnosis of asthma, (2) asthma symptoms in the previous 12 mo, (3) receiving treatment with short-acting bronchodilators only, (4) had no change made to their asthma therapy for the previous 3 mo and (5) had been free of respiratory tract infections for the previous 4 wk, and (6) were nonsmokers. Before entry subjects received a full explanation of the study and signed a consent form. The study protocol was approved by the local ethics committee.

Study Design

Subjects meeting the preceding criteria were invited to attend for a single morning visit. The recruiting physician took a full medical history and performed a physical examination, in order to exclude subjects with respiratory disease other than asthma. Measurements of exhaled nitric oxide (eNO) were made, followed by methacholine challenge. Only those with symptomatic bronchial hyperresponsiveness (BHR) who were reactive to methacholine (PD₂₀ [provocative dose of methacholine causing a 20% drop in FEV₁] < 4 mg) were asked to proceed by (1) giving serum for total and specific IgE measurements, (2) undergoing skin prick testing to common inhalant allergens, and (3) allowing a home visit for collection of dust samples to measure allergen exposure.

To avoid potential confounding by pollen, we designed the timing of the study to be outside the grass pollen season in our area.

In addition, we measured eNO in a group of patients with nonatopic asthma, only half of whom were exposed to high levels of indoor allergen at home, and in groups of nonatopic nonasthmatic and atopic nonasthmatic subjects about whom we had no information on allergen exposure.

Nitric Oxide Measurement

All subjects had eNO measured at the screening visit, using a chemiluminesence analyzer (LR 2000; Logan Research, Rochester, UK) sensitive to NO from 1 to 5,000 parts per billion (ppb) and with a resolution of 1 ppb designed for online recording (13). The response time (10-90%) was < 0.6 s. The analyser also measured CO₂ (resolution of 0.1% CO₂; response time, 200 ms), with sample pressure and volume in real time. The sampling rate of the analyzer was 250 ml/min for all measurements. The analyser was calibrated daily using a certified NO mixture (114 ppb) in nitrogen (BOC Special Gases, Guilford, UK).

eNO was measured during slow exhalation (6 L/min) from total lung capacity to residual volume via a wide-bore Teflon tube, bypassing the analyzer and thus with minimal resistance to flow; NO was continuously sampled from a side arm. The NO reading was taken when levels had plateaued and the CO₂ concentration exceeded 5%.

During measurement of eNO, subjects wore a nose clip and pressure during expiration was kept constant (3 ± 0.4 mm Hg) by using a visual display of expiratory flow measured by the pressure and volume sensors in the analyzer. The mean value of two readings with < 5% variability was taken.

Spirometry

Forced expiratory volume in 1 s (FEV₁) was measured in all subjects, using a dry bellows spirometer (Vitalograph, Buckingham, UK). The best of three technically acceptable blows, reproducible to within 0.1 L, was recorded. Results were expressed as a percentage of the predicted value, using derived equations (14). All subjects achieved FEV₁ values greater than 50% predicted and so proceeded to methacholine challenge testing.

Methacholine Challenge Testing

The challenge was performed using a modified Yan technique (15). The dosimeter (Southampton University Medical Physics, Southampton, UK) and nebulizer were calibrated to deliver a dose of 10 µl per actuation. Three inhalations of normal saline were administered first and a post-saline FEV₁ was measured after 1 min, the higher of two measurements reproducible to within 0.1 L of each other being the value recorded. Sequential doubling doses of methacholine chloride, starting with 0.0156 mg, were then administered and the FEV₁ was measured 1 min after each dose. The challenge was stopped when the fall from post-saline FEV₁ was  20% or when the cumulative dose had reached 4 mg of methacholine. The PD₂₀ was calculated for all subjects with a positive challenge test (using linear interpolation between log doses); those who had a fall of < 20% FEV₁ were not suitable for the study.

Skin Prick Testing

Skin testing was performed using the prick method. The solutions used were positive control (histamine dihydrochloride, 10 mg/ml), negative control, Dermatophagoides pteronyssinus, cat, dog, and pollen (Bayer, Elkhart, IN) and one drop of each was applied to the appropriately marked test site on the volar aspect of the forearm. The lancet was then pressed through the drop at 90° to the skin surface, and the drops were carefully blotted off using a tissue. Wheal size was read at 15 min and was recorded as the mean of the longest and the midpoint orthogonal diameters. A mean wheal diameter of  3 mm greater than the negative control was considered positive.

Total and Specific IgE

Serum was collected and stored at 20° C while awaiting analysis. Total IgE and specific IgE to D. pteronyssinus, cat, and dog were measured using the Pharmacia (Uppsala, Sweden) CAP system.

Allergen Exposure

Dust samples were collected from a 1-m² area of the living room carpet (lrc), sofa (s), bedroom carpet (brc), and mattress (m) of each home by vacuuming for 2 min using a Medivac dust sampler (airflow, 45 L/s; Medivac, Wilmslow, Cheshire, UK) onto a 5-mm vinyl filter (Plastok Associates, Wirral, UK). Dust samples were transferred into preweighed petri dishes and stored at 4° C until extraction. A 100-mg aliquot of the dust was then extracted by rotation with 2 ml of borate-buffered saline with 0.1% Tween 20, pH 8.0, at room temperature for 2 h before being centrifuged for 20 min at 2,500 rpm at 4° C. The supernatant was stored at 20° C until analyzed for allergen content.

Allergen Assays

All samples were assayed for content of major D. pteronyssinus allergen Der p 1 and for major cat allergen Fel d 1 using monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) and for major dog allergen Can f 1 using a monoclonal/polyclonal antibody-based ELISA as previously described (16). The standard used to establish the control curve for the Der p 1 assay (UVA 93/02) was considered to contain 2,500 ng of Der p 1 per milliliter (relative to WHO/IUIS D. pteronyssinus standard NIBSC 82/518, which has been estimated to contain 12.5 µg of Der p 1 per ampoule). The UVA 91/01 standard used for Fel d 1 contained 2 U of Fel d 1 per milliliter (relative to the CBER Cat E5 standard containing 9.7 U/ml; 1 unit = 4 µg of protein). For Can f 1, UVA 94/02 (10,000 IU of Can f 1 per milliliter) was substandardized against the WHO/IUIS International Reference Preparation of dog hair and dander (NIBSC 84/685), which contains 100,000 IU of Can f 1 per milliliter. One international unit is approximately 1 ng of Can f 1 protein, and this value was used to calculate the results. The results were expressed as micrograms of allergen per gram of fine dust and the limit of detection for Der p 1 was 0.05 µg/g, 0.08 µg/g for Fel d 1, and 0.2 µg/g for Can f 1.

Statistical Analysis

Statistical analysis was performed using SPSS for Windows (version 6.1.3; SPSS, Chicago, IL). Statistical significance was set at the conventional 5% level. Results of BR, specific and total IgE, eNO, and allergen exposures were log-normally distributed and therefore are reported as geometric means (GMs) with 95% confidence intervals (CIs). Univariate and multiple linear regression analysis and independent sample t tests were used for the analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subject Demographics

Fifty-three subjects were screened for the study, of which 10 (3 of whom were male) did not show nonspecific BHR to methacholine (PD₂₀ > 4 mg of methacholine).

Of the 43 subjects with symptomatic BHR, of whom 27 were female, the mean age was 32.2 yr (95% CI 29-35.4), and the mean FEV₁ was 93.6% predicted (95% CI 89.9-96.8). The mean level of eNO was 13.5 ppb (95% CI 11.1-16.4). Of the 43 patients reactive to methacholine, 5 declined to provide dust samples for allergen analysis or moved house before dust samples could be collected. Six subjects declined skin prick testing but all provided a serum sample for total and specific IgE measurement. As a result, a full data set was available for 38 subjects.

Relationship between eNO, BHR, and IgE

There was a significant linear correlation between eNO and total IgE (r = 0.44, p = 0.003; Figure 1). In the multiple linear regression analysis the independent correlates of eNO were mite-specific IgE and BHR (p = 0.0002 and p = 0.02, respectively, adjusted r² = 0.35).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Correlation between exhaled nitric oxide (eNO) and total serum IgE; natural log values given. p = 0.003, r = 0.44.

Sensitization

As some subjects refused skin prick testing, subjects were considered to be sensitized if either the skin test was positive or the specific IgE was raised. More than 95% of the subjects were atopic, with more than 75% showing IgE-mediated hypersensitivity to dust mite; the most common pattern of sensitization was to mite, cat, and dog (44%). Sixty-four percent of subjects were sensitized to cat allergen, with 60% being sensitized to dog allergen.

Indoor Allergen Exposure

Of the 38 homes sampled, Der p 1 exceeded the lower limit of detection in at least one reservoir in all but one home. Der p 1 levels (GM and 95% CI values) were as follows: lrc, 1.21 µg/g (0.61-2.39); s, 0.82 µg/g (0.41-1.65); brc, 0.57 µg/g (0.28-1.17); and m, 1.02 µg/g (0.5-2.29). In 27 homes at least 1 reservoir contained Der p 1 levels greater than the proposed significant level of 2 µg/g (19, 20).

Levels of Fel d 1 were approximately 50-fold higher in homes with cats than in homes without cats, e.g., lrc 84.7 (33.0- 217.6) versus 1.22 (0.72-2.1). In homes with dogs the levels of Can f 1 were 50- to 200-fold higher than in homes without dogs, e.g., lrc 362.1 (107.6-1,218.7) versus 1.38 (0.78-2.43).

Sensitization and Exposure

Subjects were classified as exposed or not exposed according to previously proposed significant levels (those whose homes contained in any reservoir > 2 µg/g Der p 1, > 8 µg/g Fel d 1, and > 10 µg/g Can f 1 were considered to be exposed to a significant level of that allergen) (21). Subjects exposed to lower levels in all reservoirs were considered not to be exposed to significant levels of that allergen. All cat owners were exposed to Fel d 1 levels > 8 µg/g fine dust in at least one reservoir, as were three subjects who did not own a cat. All dog owners were exposed to Can f 1 levels > 10 µg/g in at least one dust reservoir, as were four subjects who did not keep a dog.

The subjects were divided into two groups: those sensitized and exposed to significant levels of the relevant indoor allergen ("sensitized and exposed" group) and those sensitized but not exposed to significant levels of the relevant allergen ("sensitized and not exposed" group). Of the 38 subjects, 26 were both sensitized and exposed to one or more allergen (10 to mite only, 2 to cat only, 2 to dog only, 7 to mite and cat, 4 to mite and dog, 1 to cat and dog) and the remaining 12 were sensitized, but not exposed. The levels of eNO, bronchial hyperresponsiveness, and total serum IgE for each group are illustrated in Table 1.

eNO was significantly higher in those subjects in the sensitized and exposed group than in the sensitized and not exposed group (GM and 95% CI: 17.69 [14.1-22.2] versus 9.09 [6.5-12.7], p = 0.001, Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Exhaled nitric oxide (ppb) in sensitized and exposed versus sensitized and not exposed. Horizontal bars denote geometric mean values.

For those subjects with nonatopic asthma (n = 22), there was no difference in eNO in those exposed to high levels of allergen and those exposed to low levels (GM and 95% CI: 8.96 [5.53-14.51] versus 7.50 [4.71-11.74], p = 0.81). For the nonatopic nonasthmatic subjects (n = 16) and the atopic nonasthmatic subjects (n = 36) levels tended to be lower (GM and 95% CI: 3.8 [2.68-5.49] and 6.24 [5.23-7.44]).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is the first study to show a quantitative relationship between natural exposure to indoor allergen and exhaled nitric oxide, which may be an indication of allergic airway inflammation, in patients with atopic asthma. In this population of steroid-naive patients with asthma, we have found that the levels of exhaled nitric oxide were significantly higher in patients both sensitized and exposed to indoor allergen than in those who were sensitized, but not exposed. Because of the effects of cigarette smoke and inhaled steroids on exhaled NO, we studied only nonsmoking patients with asthma with symptomatic bronchial hyperresponsiveness who were using only short-acting ₂ agonists.

The distribution of indoor allergen within the homes of the patients with asthma was similar to those we found in previous studies in our area (24, 25), and we have again found levels of pet allergen greater than the proposed significant levels in some homes that did not currently contain a pet. This suggests that, for the further assessment of personal allergen exposure, a history alone is inadequate and there is a need to sample the individual's home. The exposure to a relevant allergen also needs to be set in the context of an individual patient's sensitization, i.e., exposure and sensitization to a specific allergen in an individual patient. Allergen exposure in a nonsensitized subject and absence of exposure in a sensitized subject both seem unlikely to be clinically relevant. This raises the question of what represents a significant exposure. Levels of allergen in household reservoirs that result in significant exposure have been suggested as Group 1 mite allergen  2 µg/g, Fel d 1  8 µg/g, and Can f 1  10 µg/g in previous studies (21), and in this study we have used these suggested levels to divide the population into those exposed and those not exposed.

The levels of eNO we have recorded are similar to those in other studies of patients with mild asthma (12). In this group of patients with multiple sensitizations, it is difficult to elucidate the relative contribution of the sensitizations and exposures to the three different allergens to asthma severity.

Laboratory studies in steroid-naive patients with asthma who underwent allergen challenge have shown that those who experienced a late response to the challenge, as measured by a fall in FEV₁, had an associated elevation in the levels of exhaled nitric oxide that was not seen after a control challenge with methacholine (26). This established a relationship between allergen-induced inflammation and eNO. The current study investigated the effects of chronic exposure to allergen in the home, which is more clinically relevant, as many asthmatic subjects are exposed to high levels of allergen to which they are sensitized. Laboratory allergen challenge and domestic exposure differ significantly both in terms of allergen dose and also in terms of the size of the allergen-carrying particles. Nonetheless, the results of the current study are in agreement with those obtained under experimental conditions.

Many studies of allergen avoidance have used BHR as the indicator of response to allergen avoidance; however, a significant improvement in BHR is usually seen only after 6 mo of treatment (27). eNO may prove to be a more sensitive, reproducible, and less invasive index, but this preliminary finding needs to be confirmed in further studies.

In summary, this is the first clinical study to show that natural exposure to high levels of allergen in patient's homes is associated with high levels of eNO in sensitized patients with asthma. The allergen exposure may be driving the inflammatory process in the airways and contributing to more severe asthma. Once a subject has become sensitized to an allergen, subsequent exposure of the airways to levels of allergen found in the domestic environment may cause cytokine-mediated inflammation and increased eNO production. These findings imply that the optimal treatment for sensitized patients with asthma should be an integrated approach including domestic environmental control as well as the appropriate medication.