Maximal Response Plateau to Adenosine 5′-Monophosphate in Asthma

. . CHEST --

Original Research ASTHMA

--

Maximal Response Plateau to Adenosine 5'-Monophosphate in Asthma* Relationship With the Response to Methacholine, Exhaled Nitric Oxide, and Exhaled Breath Condensate pH Luis Prieto, PhD; Saioa Esnal, MD; Victoria Lopez, MD; Desire Barato, MD; Rocio Rojas, RN; and julio Marin, PhD

Background: No information is available on the plateau in response to adenosine 5'-monophosphate (AMP). The aims of the present study were (1) to determine whether plateau can be detected with AMP and the relation with the plateau in response to methacholine, and (2) to identify the relation between the plateau and indirect markers of airway inflammation, sucb as exhaled nitric oxide (ENO) and exhaled breath condensate (EBC) pH. Methods: Airway responsiveness to high concentrations of methacholine and AMP, ENO levels, and EBC pH values were obtained in 31 subjects with well-controlled asthma. Concentrationresponse curves were characterized by their concentration of agonist that produces a decrease in FEV 1 of 20% and, if possible, by the level of plateau. Results: Although the prevalence ofplateau with methacholine (48%) and AMP (58%) was similar, the two challenges did not identify plateau in exactly the same individuals. In 14 subjects who showed plateau with both bronchoconstrictor agents, the mean plateau level for methacholine was 26.0% (95% confidence interval [Cll, 21.3 to 30.8), compared with 16.5% (95% CI, 12.2 to 20.8; P < 0.0001) for AMP. Both ENO and EBC pH values were similarin subjects with plateau and in those without plateau. Conclusions: In well-eontrolled asthmatics, the plateau in response to AMP can be identified at a milder degree of obstruction than the plateau in response to methacholine, but the two agonists are not identifying the same airway abnormalities. Furthermore, if ENO and EBC pH are markers of inflammation, the determination of the presence or level of plateau is not a reliable method to identifY airway inflammation in asthma. (CHEST 2009; 135:1521-1526) Key words: adenosine 5' -monophosphate; airway responsiveness; asthma; exhaled breath condensate; methacholine; nitric oxide; pH; plateau Abbreviations: AMP = adenosine 5' -monophosphate; CI = confidence interval; EBC = exhaled breath condensate; ENO = exhaled nitric oxide; ICS = inhaled corticosteroid; PC zo = provocative concentration of a substance causing a 20% decrease in FEV 1; ppb = parts per billion

Clinically and for research purposes, airway responsiveness is measured by bronchial challenge, usually with methacholine or histamine.': how'From the Asociacion Valenciana de Investigaciones Clinicas (Drs. Prieto, Lopez, and Barato, and Ms. Rojas), Valencia, Spain; Servicio de Alergologia(Dr. Esnal), Hospital Santiago Apostol,Vitoria, Spain; and Universidad de Valencia (Dr. Marin), Valencia, Spain. The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Manuscript received October 3, 2008; revision accepted January 9,2009. www.chestjournal.org

ever, adenosine 5'-monophosphate (AMP) has been introduced more recently as a bronchoconstrictive stimulus. 2 - 4 The most commonly employed method for the analysis of the concentration-response curve Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjoumal. orgisite/miscireprints.xhtml). Correspondence to: Luis Prieto, PhD, Seccion de Alergologia, Hospital Universitario Dr Peset, Gaspar Aguilar 90, 46017 Valencia, Spain; e-mail: [email protected] DOl: lO.1378/chest.08-2392 CHEST /135/6/ JUNE, 2009

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is the measurement of the sensitivity, which is identified by the provocative concentration of a substance causing a 20% decrease in FEV1 (PC 20) or the provocative dose of a substance causing a 20% decrease in FEVI' An increased sensitivity is often regarded as being synonymous with the term hyperresponsiveness. However, some investigations5--7 have focused on the importance of characterizing the entire dose-response curve not only by sensitivity, but also by the maximal airway narrowing response value (plateau). Evidence indicates that the mechanisms that modulate sensitivity and the plateau level are at least partially different.v" All these investigations were performed, however, with inhaled methacholine (a direct bronchoconstrictor agent), but no information is available on the maximal response plateau obtained with AMP (an indirect bronchoconstrictor). Increased concentration of exhaled nitric oxide (ENO)lO.ll and acidification of exhaled breath condensate (EBC)12.13 have been demonstrated in asthma. In addition, both ENO and EBC pH are correlated with the number of eosinophils in the lower respiratory tract 13.14 and, therefore, these parameters have been proposed as markers of airway inflammation and disease severity in asthma. 15,16 Previous studies demonstrated that ENO levels were significantly related with the PC 20 AMP, but not with the PC 20 for methacholine.V!" No information is available, however, on the relationship between the maximal response plateau level and either ENO or EBC pH values. Therefore, the present study was designed with two objectives: (1) to determine whether plateau can be identified on concentration-response curves obtained with AMP in subjects with well-controlled asthma and the relation with the plateau in response to methacholine; and (2) to explore the relationship between the maximal response plateau to each bronchoconstrictor agent and some indexes related to airway inflammation, such as ENO and EBC pH values.

SUBJECTS AND METHODS

had FEV] at baseline> 80% of predicted. The study protocol was approved by the local ethics committee, and informed consent was obtained from all participants. Study Design Patients attended the laboratory on 3 days, at the same time each day. Short-acting inhaled 132-agonists were withheld for at least 6 h before each challenge, ICS (n = 12) for at least 12 h, and oral antihistamines for at least 72 h. No other asthma medications were used. On the first day, all the patients were evaluated for suitability and spirometry was performed. On each of the next 2 visits (at least 1 day but not> 5 days apart), spirometry and concentration-response studies with either methacholine or AMP were performed. Methacholine and AMP challenges were conducted on separate days with the order of challenge randomized. ENO was measured at the second visit, and EBC pH was obtained at the third visit. These determinations were performed before the spirometry and challenge test. Inhalation Challenge Tests Lung function was measured using a calibrated pneumotachograph (Jaeger MasterScope; Erich Jaeger GmbH; Wiirzburg, Germany) according to standardized guidelmes.!? Inhalation provocation tests were performed using a modification of the dosimeter method,20.21 as previously reported. 22.2.1 Methacholine and AMP (Sigma Chemical; St. Louis, MO) were dissolved freshly in 0.9% saline solution to produce doubling concentration ranges of 0.19 to 100 mglmL (1 to 511 mmol/L) for methacholine and 6.25 to 3,200 mglmL (18 to 9,216 rnmol/L) for AMP. Subjects inhaled the aerosolized methacholine and AMP solutions (Mefar MB3; Brescia, Italy) in five inspiratory capacity inhalations. The nebulizer output was 10 ILL per breath. The test was interrupted when a 40% decrease in FEV l from the postsaline solution administration value was recorded or when the highest concentration was administered. END Measurement Technique ENO measurements were performed according to a standardized method.s- using a chemiluminescence analyzer (NiOx; Aerocrine; Solna, Sweden). The detailed procedure has been described elsewhere.2.5.26 ERC Collection and pH Measurement EBC was collected using the RTube collection system (Respiratory Research, Inc; Charlottesville, VA), as previously reported.e? EBC pH was measured immediately using a micro pH probe (Metrohm AG; Herisau, Switzerland), connected to a calibrated pH meter (model pH 900), after deaeration with argon for 8 minp·2H

Subjects

Statistical Analysis

Thirty-one adult subjects, nonsmokers and with a previous history of asthma, were enrolled in the study. Subjects with stable asthma requiring inhaled corticosteroids (lCSs) at low doses (budesonide, 100 to 400 ILg or equivalent daily) or inhaled short-acting 132-agonists on demand only to maintain asthma control were recruited. Most patients selected (n = 21) had pollen-induced asthma and were tested out of the pollen season, during a period of absence of symptoms. In the 3 months before the study, patients had asthma symptoms no more than once a month and did not wake at night because of asthma. All patients

The sample size requirement of this study was calculated considering a difference of 7% between the level of plateau obtained with methacholine and AMP as clinically relevant. Based on a calculated SD of 6% for the level of plateau with methacholine from previous studies,22.23 it was estimated that 15 patients with plateau and 15 without plateau to each agonist would be required to provide 90% power at the two-sided 0.05 level. Concentration-response curves were characterized by their PC 20 and, if possible, maximal response plateau level. A plateau

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Original Research

Table I-Subject Characteristics* Characteristics

40 P <0.0001

Data

Patients, No. Age, yr Gender Male Female Ex-smokers Duration of asthma, yr Skin test result positive rcs use Yes No FEV), % predicted FEV/FVC,%

31 44 (40-49) 15 16 7 18.1 (14.7-21.8) 25 12 19 101.4 (95.6-107.2) 74.7 (72.3-77.2)

*Data are presented as No. or mean (95% CI).

35 ~ ~

30

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15 10 5 0

AMP

(n = 14)

response was considered to be present when, for three or more of the highest concentrations of agonist, the FEV 1 did not change by > 5%. The level of the maximal FEV) response was obtained by averaging the data points on the plateau.w In those subjects (n = 9) who showed FEV) falls < 20% after the highest AMP concentration, the PC 20 value was censored to 3,200 mgtmL. Numerical variables were compared with t tests, and categorical variables were analyzed with the Fisher exact test. P values < 0.05 were considered statistically significant.

RESULTS

Table 1 shows the baseline characteristics of the 31 patients studied. Mean baseline FEVI values were not Significantly different before the two different provocation tests (p = 0.26) [Table 2].

Response to Methacholine and AMP Methacholine and AMP PC 20 values are shown in Table 2. A maximal response plateau was detected in a similar proportion of patients with each bronchoconstrictor agent (48% for methacholine and 58% for AMP (p = 0.61); Table 2). However, the two challenges did not identify plateau in exactly the same individuals. The presence of plateau to either methacholine or AMP was detected in 19 patients; 14 of

Table 2-Prechallenge FEV1 and Ainvay Responsiveness to Methacholine and AMP* Variables

Methacholine

Prechallenge FEV), L 3.01 (2.72-3.31) PC 20 , t mglmL 6.4 (3.3-12.1) Plateau, yes/no 15/16 Level of plateau.t % FEV 1 26.0 (21.3-30.8)

AMP 2.99 (2.69-3.29) 301.3 (148.9-609.5) 18/13 16.5 (12.2-20.8)

*Values are given as the mean (95% Cl), unless otherwise indicated. [Values are given as the geometric mean (95% CI). [In subjects who showed plateau with both bronchoconstrictor agents. www.chestjournaJ.org

Methacholine (n

= 14)

FIGURE 1. Individual values for level of plateau with methacholine and with AMP in 14 subjects who showed plateau with both bronchoconstrictor agents. Horizontal lines are means.

them showed plateau to both agonists. Among the 18 subjects with plateau to AMP, 4 had FEV j falls > 40% with methacholine without evidence of plateau; in these 4 subjects, the level of plateau with AMP was 35%, 20%, 23% and 13%, respectively. Only 1 of the 15 subjects with plateau to methacholine showed FEV I fall > 40% with AMP without evidence of plateau (the level of plateau with methacholine in this subject was 38%). In the group of 14 subjects who showed plateau with both bronchoconstrictor agents, the mean plateau level for methacholine was 26.0% (95% confidence interval [CI], 21.3 to 30.8%), compared with 16.5% (95% CI, 12.2 to 20.8%; P < 0.0001) for AMP (Fig 1).

ENG Levels and ERC pH Values in Subjects With and Without Plateau The mean ENO value for the group with plateau to AMP was 48.9 parts per billion (ppb) [95% CI, 20.6 to 77.2 ppb], compared with 54.6 ppb (95% CI, 38.3 to 70.9 ppb) [Fig 2] for the group without plateau (p = 0.71). The mean EBC pH value for subjects with plateau to AMP was 7.57 (95% CI, 7.09 to 7.86), compared with 7.38 (95% CI, 6.87 to 7.89) for subjects without plateau (p = 0.76) [Fig 3]. Differences in either ENO or EBC pH values between subjects with and without plateau in response to methacholine were also not statistically significant (data not shown).

Correlations There was a significant correlation between methacholine and AMP PC 2 0 values (r = 0.78; CHEST /135/6 / JUNE, 2009

1523

200

P

•

=0.71

DISCUSSION

175 _150

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25

AMP Plateau

AMP No Plateau

FIGURE 2. Individual values for ENO in subjects with and without plateau in response to AMP. Horizontal lines = means.

p < 0.001), but the correlation between the maximal response plateau level for methacholine and AMP did not reach statistical significance (r = 0.50; P = 0.07). No significant correlation was found between methacholine PC20 values and either ENO (r = - 0.19; P = 0.31) or EBC pH (r = 0.12; P = 0.49). A weak, although significant, correlation was found between AMP PC 20 and ENO values (r = - 0.38; P = 0.03), but no correlation was found between PC 20 AMP and EBC pH values (r = 0.12; P = 0.51). The correlation between the level of plateau for methacholine or AMP and either ENO or EBC pH values was also not Significant.

P = 0.76

8.5

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AMP No Plateau

FIGURE 3. Individual values for EBC pH in subjects with plateau and in those without plateau in response to AMP. Horizontal lines = means.

1524

The results of the present study demonstrate that a maximal response plateau on the concentrationresponse curves of subjects with well-controlled asthma can be identified not only with methacholine, but also with an indirect bronchoconstrictor such as AMP. Interestingly, the plateau in response to AMP can be identified at a milder degree of obstruction than the plateau in response to methacholine. However, the two bronchoconstrictor agents do not identifY plateau in exactly the same patients, and there is no correlation between the level of plateau obtained with methacholine and with AMP. Moreover, ENO and EBC pH values are similar in subjects with and without plateau. These findings suggest that, at least in subjects with well-controlled asthma, methacholine and AMP challenges are identifying different abnormalities of the airways. Furthermore, if ENO and EBC pH values reflect the presence of airway inflammation, our results suggest that the identification of the presence or level of plateau is not a reliable method to identify airway inflammation in asthma. To the best of our knowledge, this is the first study designed to investigate the prevalence and level of plateau to AMP, compared with methacholine, in subjects with asthma. In our study, plateau in response to AMP was identified at a milder degree of obstruction than plateau in response to methacholine. This appears to indicate that AMP is more convenient than methacholine to identify plateau because this agonist allows the identification of plateau with lower risk of excessive bronchoconstriction than methacholine. However, our results also demonstrate that the levels of plateau obtained with methacholine and AMP are not significantly related, and the two bronchoconstrictor agents do not identify plateau in the same individuals. These results are consistent with the suggestion that the response to each agonist may be a consequence of different structural changes in the lower airways,30-33 and they suggest that the determination of the presence and level of plateau with AMP might not be reliable to identify the plateau in response to methacholine. However, to obtain the required number of asthmatic subjects with plateau, we purposely selected subjects with a high degree of asthma control or patients with pollen-induced asthma not exposed to sensitizing allergens. Although the effect of these two factors on the plateau in response to AMP has not been studied, it has been demonstrated that both treatment with ICS34 and allergen avoidance3.5 have a greater influence on AMP than on methacholine sensitivity. Therefore, it seems reasonably to speculate that differences in the level of Original Research

plateau between the two bronchoconstrictor agents might be the consequence, at least in part, of the clinical characteristics of the subjects selected. Additionally, this apparent decreased prevalence and level of plateau with AMP compared with methacholine raises a question regarding dose equivalence. It has been demonstrated that inhaled AMP is, on average, 17 times less potent than methacholine in constricting the airways of asthmatic patients. 31,36 On a molar basis, however, the doses of AMP delivered to the mouth of each patient were I8-fold greater than the corresponding doses of methacholine. Therefore, it is unlikely that differences in the prevalence and level of plateau between the concentration-response curves obtained with each agonist may be the consequence of differences in the dose of the bronchoconstrictor administered. When this study was designed, our original expectations were (I) a better correlation between inflammatory markers (ENO or EBC pH) and AMP responsiveness than between these markers and methacholine responsiveness, and (2) a higher concentration of inflammatory markers in subjects without plateau than in those with plateau. These initial expectations were only partially confirmed in our study. In keeping with previous reports,l7·18 we have detected a significant correlation between ENO levels and AMP sensitivity, but not between ENO values and methacholine sensitivity. If ENO levels reflect the presence of airway inflammation, our findings support the hypothesis that sensitivity to AMP may be a more sensitive marker for assessing airway inflammation in asthma than sensitivity to methacholine. By contrast, no significant correlation was found between the level of plateau with either methacholine or AMP and ENO levels. In addition, differences in ENO values between subjects with and without plateau were not statistically significant. These findings suggest that, at least for AMP, the PC 20 value is a better indicator of airway inflammation than the plateau level, and the identification of plateau does not provide relevant additional information on the degree of airway inflammation to that obtained with the PC 20 . To the best of our knowledge, only one study has determined the relationship between the level of plateau and inflammatory changes in the lower airways of subjects with asthma. In a study performed on bronchial biopsies from subjects with atopic asthma, Moller et al37 demonstrated a positive correlation between the number of activated eosinophils (EG2+) in the lamina propria and the level of plateau. Because ENO has been proposed as a marker of eosinophilic airway inflammation.l" our results appear to contradict those obtained by Moller et al,37 However, the correlation between ENO and direct measures of airway inflamwww.chestjournal.org

mation have been of relatively small magnitude, 14 and therefore the precise mechanismts) that link(s) nitric oxide with eosinophilic airway inflammation, and whether elevated ENO concentrations are caused by enhanced activity of eosinophils or by enhanced diffusion through the airway wall due to structural damage, remain to be elucidated. However, no correlation was found between EBC pH value and airway responsiveness (sensitivity and plateau) to either methacholine or AMP. Moreover, EBC pH values were similar in subjects with plateau and in those without plateau in response to either methacholine or AMP. Because EBC pH has been purposed as a procedure for the evaluation of the inflammatory process.P-':' our results might be interpreted as an additional argument for the absence of relationship between airway inflammation and responsiveness. However, it must be acknowledged that the interpretation of EBC pH is controversial due to technical factors,38,39 and there is a debate as to whether orally collected EBC pH assays reflect acidification of the lower airways.s? In conclusion, inhaled AMP can be used to identifY plateau in subjects with well-controlled asthma, but the presence of plateau in response to AMP is not necessarily associated with plateau in response to methacholine. Furthermore, there is no correlation between the level of plateau and some purposed markers of airway inflammation, such as ENO and EBC pH. These results suggest that information provided by the plateau in response to methacholine and AMP is not interchangeable, and the determination of the presence or level of plateau is not a reliable method to identify airway inflammation in asthma,

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Original Research