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(Chest. 2006;130:1448-1453.)
© 2006 American College of Chest Physicians

The Effect of Challenge Method on Sensitivity and Reactivity to Adenosine 5'-Monophosphate in Subjects With Suspected Asthma*

Luis Prieto, MD, PhD; Isabel Reig, MD; Rocio Rojas, RN; Anna Ferrer, MD and Juan Domenech, MD, PhD

* From the Sección de Alergología (Drs. Prieto, Ferrer, and Domenech, and Ms. Rojas), Hospital Universitario Dr. Peset, Valencia, Spain; and Servicio de Alergología (Dr. Reig), Hospital Clínico, Madrid, Spain.

Correspondence to: Luis Prieto, MD, PhD, Sección de Alergología, Hospital Universitario Dr. Peset, C/ Gaspar Aguilar 90, 46017 Valencia, Spain; e-mail: prieto_jes{at}gva.es

Abstract

Background: The following two methods of inhalation challenge have been used to determine the airway responsiveness: the tidal-breathing method; and the dosimeter method. The objective of the study was to determine the influence of the challenge method on the response to adenosine 5'-monophosphate (AMP).

Methods: This study measured airway responsiveness to AMP by dosimeter and tidal-breathing methods in 25 subjects with suspected asthma. The two AMP challenges were conducted in random order, on separate days, at the same time of day, at intervals of 1 to 5 days. Concentration-response curves were characterized by the provocative concentration of a substance causing a 20% fall in FEV1 (PC20) and slope.

Results: The dosimeter PC20 values were significantly higher than the tidal-breathing PC20 values, with geometric mean (95% confidence interval [CI]) values of 50.35 mg/mL (95% CI, 19.50 to 129.72 mg/mL) and 28.97 mg/mL (95% CI, 11.99 to 69.98 mg/mL; p = 0.02), respectively. The mean difference in the PC20 values obtained with each method was 0.80 doubling concentrations (95% CI, 0.16 to 1.44 doubling concentrations). The mean values for the slope were 17.0%/log mg/mL (95% CI, 12.5 to 21.5 mg/mL) with the tidal breathing method and 13.8%/log mg/mL (95% CI, 9.0 to 18.7 mg/mL; p = 0.03) with the dosimeter.

Conclusions: The tidal-breathing method produces AMP PC20 values that are significantly lower than the dosimeter method and slope values that are significantly higher than the dosimeter method. These data suggest that the results obtained with each method of testing may not be comparable.

Key Words: adenosine 5'-monophosphate • airway responsiveness • asthma • challenge methods • reactivity

Although direct bronchoconstrictor agents, such as histamine or methacholine, are the most commonly used triggers to quantify bronchial hyperresponsiveness in the laboratory, a further level of complexity has been revealed by using a wide range of different indirect stimuli, including adenosine 5'-monophosphate (AMP), that cause bronchoconstriction through the secondary release of mediators from primed mast cells.12 Several studies345 suggest that the bronchial response to AMP may be a more sensitive marker of airway inflammation than histamine or methacholine, and hence more specific for asthma. Thus, the determination of airway responsiveness to AMP is increasingly used clinically and for research purposes. Guidelines for methacholine and histamine challenge testing have been published,67 but efforts to develop recommendations concerning the role of indirect airway challenges in the assessment and monitoring of airway diseases have been made only in the past few years.8

Different methods for the analysis of the concentration-response curves have been developed, but the most commonly employed is the measurement of the provocative concentration of a substance causing a 20% fall in FEV1 (PC20). However, previous studies9 have suggested a distinction between the slope of the concentration-response curve, which is referred to as reactivity, and the PC20, which is referred to as sensitivity. It was suggested that, at least in the identification of the response to direct bronchoconstrictor agents, different mechanisms may determine reactivity and sensitivity, and that both should be determined when the findings of bronchial provocation tests are being interpreted.9

At present, two methods of inhalation challenge have been used for the study of the response to histamine and methacholine. The first, introduced by Cockcroft and colleagues,10 consists of the continuous generation of aerosol and inhalation by quiet tidal breathing at spontaneous frequency for 2 min. The second, proposed by Chai and coworkers,11 consists of intermittent aerosolization with the use of a breath-activated dosimeter for five inhalations. Similar methods have been used to determine the response to inhaled AMP. Many authors have recommended the five-breath dosimeter method,12131415 but the 2 min of tidal breathing method has also been used successfully in both asthmatic and nonasthmatic subjects.16171819

Initial studies202122 concluded that the two methods give comparable results for methacholine sensitivity, and, for this reason, both methods of methacholine challenge testing are recommended by guidelines from the American Thoracic Society7 and European Respiratory Society.6 However, recent studies2324 have suggested that the tidal breathing method may produce methacholine PC20 values that are significantly lower than the dosimeter method. This is due, at least in part, to the bronchoprotective effect of the five deep inhalations, which are an integral part of the dosimeter method.25 These findings were, nevertheless, obtained in studies performed with methacholine, and it remains uncertain whether the challenge method may have some influence on the sensitivity and reactivity to AMP. Thus, the aim of this study was to determine the influence of the challenge method on the response to AMP in adults with suspected asthma.

Subjects and Methods

Subjects
Twenty-five subjects with suspected asthma were recruited from our outpatient Allergy Clinic. At the time of the study, the FEV1 was ≥ 80% predicted. All subjects were current nonsmokers, and none had a history of chronic bronchitis, emphysema, or respiratory tract infections during the 4 weeks before the study. Current smokers, pregnant women, and patients with significant renal, hepatic, or cardiovascular disease were specifically excluded from the study. The study protocol was approved by the local ethics committee, and written informed consent was obtained from all participants.

Study Design
This was an open, randomized, crossover study. The use of short-acting inhaled ß2-agonists (n = 24) was withheld for at least 6 h, of inhaled corticosteroids (n = 13) for at least 2 weeks, and of oral antihistamines (n = 10) for at least 72 h before each challenge. Long-acting inhaled ß2-agonists, theophylline, oral corticosteroids, leukotriene receptor antagonists, sodium cromoglycate, nedocromil sodium, and anticholinergic bronchodilators were not used by any subjects during the month prior to being studied. Subjects using a nasal topical corticosteroid (n = 8) kept the dose constant throughout the study.

Subjects attended the laboratory on three occasions at the same time of day. On the first day, all subjects were evaluated for suitability, and spirometry was performed. On each of the next two visits (at least 1 day apart but not > 5 days), spirometry and AMP challenges using either the tidal-breathing or dosimeter method were performed. The AMP challenges with each method were conducted on separate days with the order of challenge randomized. For each test, the baseline FEV1 was required to be within 10% of the initial baseline FEV1.

Pulmonary Function and AMP Challenge Procedures
Lung function was measured using a calibrated pneumotachograph (Jaeger MasterScope; Erich Jaeger GmbH; Würzburg, Germany) according to standardized guidelines.2627

Airway responsiveness to AMP was assessed using a standardized tidal-breathing method.10 Following baseline spirometry, FEV1 was measured after the inhalation of normal saline solution for 2 min, followed by doubling concentrations of AMP (Sigma Chemical; St Louis, MO) in a normal saline solution in concentrations ranging from 1.56 to 400 mg/mL. Aerosols were delivered with a nebulizer (model 1720; Hudson; Temecula, CA) with 2 mL of test solution in the container and a mean (± SD) delivery rate of 0.13 ± 0.02 mL/min. The nebulizer delivers particles with an aerodynamic mass median diameter of 2.2 µm. The nebulizer was connected directly to a mouthpiece, a nose clip was worn, and the aerosol was inhaled through the mouth by tidal breathing for 2 min. A single determination of FEV1 (without a full FVC maneuver) was taken 60 to 90 s after the inhalation of each concentration28 unless the forced expiratory maneuver was judged to be technically unsatisfactory. The test was interrupted when FEV1 decreased by ≥ 20% from its post-saline solution inhalation value or when the highest concentration of AMP had been administered.

The other AMP challenge procedure was performed using a jet nebulizer that was attached to a breath-activated dosimeter11 (model MB3; Mefar; Brescia, Italy) at a nebulization time of 1 s with a pause time of 6 s. The nebulizer delivers particles with an aerodynamic mass median diameter of 3.5 to 4.0 µm at an output of 11 µL per breath. The five-breath dosimeter AMP challenge was performed using the identical AMP solutions (range, 1.56 to 400.00 mg/mL), starting concentration, and FEV1 timing, as the tidal-breathing method. Only the inhalation method was different. Patients inhaled the aerosolized solutions in five inhalations from functional residual capacity with the patient’s wearing a nose clip. The test was interrupted when a fall in FEV1 of at least 20% from the post-saline solution inhalation value was recorded or the maximum concentration had been administered.

Statistical Analysis
Concentration-response curves were plotted for each challenge test as the percentage fall in FEV1 against the log AMP concentration, and were characterized by their sensitivity (PC20) and slope. AMP PC20 was calculated using an algebraic formula.29 The slope of the concentration-response curve was calculated by linear regression analysis using the method of least squares. The first point showing a measurable reduction in FEV1 (> 5%) and all subsequent points were used in the regression. At least three points were needed for the curve to be kept. The slope of the concentration-response curve, expressed as the percentage change in FEV1/log AMP concentration (in milligrams per milliliter) was thus obtained. The study had > 90% power to detect a one half concentration difference in PC20.

All PC20 values were log-transformed before analysis and presented as geometric means with 95% confidence intervals (CIs). All other numerical variables are reported as arithmetic means with 95% CI. To evaluate the normality of the distributions, the Kolmogorov-Smirnov test was used and a p value of > 0.05 was obtained. Thus, the FEV1, PC20, and slope values obtained with each method were compared by paired t tests. Differences in PC20 values were expressed in terms of doubling concentrations of AMP calculated as {Delta}log PC20/log 2. In addition, differences in PC20 and slope values between the tidal-breathing and dosimeter methods were shown graphically by plotting the difference against the mean, as recommended by Bland and Altman.30 The p values were two-sided, and values < 0.05 were considered to be statistically significant. Data were analyzed with a statistical software package (InStat for Windows, version 3.00; GraphPad Software; San Diego, CA).

Results

Twenty-five subjects were studied. The mean baseline values of FEV1 were 3.27 L (95% CI, 2.86 to 3.76 L) on the tidal-breathing study day, and 3.27 L (95% CI, 2.86 to 3.68 L; p = 0.99) on the dosimeter study day. Baseline characteristics of the patients are presented in Table 1 . The order of the challenge methods had no influence on the PC20 and slope values (p = 0.12 and 0.42, respectively).


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  		Table 1. Subject Characteristics at Baseline*

 
Technically satisfactory AMP challenges with both methods were obtained in all 25 subjects. Of these, eight subjects did not achieve a 20% fall in FEV1 with either method, five subjects achieved a ≥ 20% fall with the tidal-breathing method but not with the dosimeter method, while 12 subjects achieved a ≥ 20% fall with both methods. In the 17 subjects in whom it was possible to determine the PC20 value for at least one method, the dosimeter PC20 values were significantly higher than the tidal-breathing PC20 values (Fig 1 ), with geometric mean values of 50.35 mg/mL (95% CI, 19.50 to 129.72 mg/mL) and 28.97 mg/mL (95% CI, 11.99 to 69.98 mg/mL; p = 0.02), respectively. The AMP PC20 values obtained with the tidal-breathing and the dosimeter method are shown in Figure 2 as differences between values obtained with each method plotted against the mean according to Bland and Altman.30 The mean difference in the PC20 values obtained with each method was 0.80 doubling concentrations (95% CI, 0.16 to 1.44 doubling concentrations).


Figure 1
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  		Figure 1. Comparison of tidal-breathing PC20 and dosimeter PC20 values in 17 subjects with measurable PC20 values for at least one method. Horizontal lines = geometric means; dashed lines = the five subjects with unmeasurable PC20 by dosimeter (in these subjects, PC20 values were censored to 400 mg/mL).

 

Figure 2
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  		Figure 2. Difference between tidal-breathing and dosimeter measurements of AMP PC20 (ie, dosimeter – tidal breathing) plotted against the mean of the two values, in 17 subjects with no censored values (PC20, ≤ 400 mg/mL) for at least one method. Continuous line = the mean difference; dashed lines = ± 2 SDs for the differences. There was no indication of greater difference between the two methods when the PC20 value increased.

 
The slope could be calculated in 24 subjects because 1 subject showed concentration-response curves with only two points for the regression analysis with both the tidal-breathing and dosimeter methods. The mean values of the slope were 17.0%/log mg/mL (95% CI, 12.5 to 21.5%/log mg/mL) on the tidal-breathing study day and 13.8%/log mg/mL (95% CI, 9.0 to 18.7%/log mg/mL; p = 0.03) on the dosimeter study day (Fig 3 ). The mean difference in the slope values (Fig 4 ) obtained with each method was 3.1%/log mg/mL (95% CI, 0.2 to 6.1%/log mg/mL). In addition, there was no indication of greater variability in the difference between the two measurements as either the PC20 or slope increases (Fig 2, 4).


Figure 3
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  		Figure 3. Comparison of the slope values obtained with each method. Horizontal lines = means.

 

Figure 4
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  		Figure 4. Difference between the tidal-breathing and the dosimeter measurements of the slope of the concentration-response curve (tidal breathing – dosimeter) plotted against the mean of the two values. Continuous line = mean difference; dashed lines = ± 2 SDs for the differences. There was no indication of a greater difference between the two methods when the slope value increased.

 
Discussion

In this study, we have shown that the 2-min tidal-breathing method produced AMP PC20 values that were significantly lower and slope values that were significantly higher than the dosimeter method. These data suggest that the results obtained with each method of testing may not be comparable.

Bronchoprovocation challenges with direct and indirect bronchoconstrictor agents are commonly conducted by using one of the following two methods: the 2-min tidal-breathing method; or the five-breath dosimeter method. Previous studies232425 have demonstrated that, in subjects with suspected or diagnosed asthma, the dosimeter methacholine challenge produces significantly less response (ie, higher PC20 values) than the tidal-breathing method. This is probably due to the smaller volume of aerosolized bronchoconstrictor administered with the dosimeter compared to the tidal-breathing method, but the bronchoprotective effect of the five deep inhalations, which are an integral part of the dosimeter method, may also be important.25 To the best of our knowledge, this is the first study to demonstrate differences in sensitivity and reactivity to AMP between the two methods that are commonly used to determine the response to this indirect bronchoconstrictor agent. The results of our study clearly demonstrate that the tidal-breathing method is associated with AMP PC20 values that are significantly lower than those obtained with the dosimeter method and slope values that are significantly higher. Thus, the results obtained with each challenge method may not be comparable, and their clinical significance may be different.

In a previous study,24 our laboratory showed that the two challenge methods provide similar values for the slope of the concentration-response curve for methacholine. In contrast, the results of the present study clearly demonstrate that the challenge method has an important influence on the slope of the concentration-response curve to AMP. Although a simple explanation for these results might be a selective influence of the challenge method on the response to each bronchoconstrictor agent, there are some differences in technical factors that might be relevant. In the methacholine study,24 the test was interrupted when a fall in FEV1 of at least 40% from the post-saline solution inhalation value was recorded, whereas in the present study the test was stopped when a fall in FEV1 of ≥ 20% occurred. Obviously, more data points were included in the regression analysis for the determination of the slope in our methacholine study, and this difference makes comparison difficult.

Although this study was not designed to examine the mechanisms that induce different responses with the two methods of challenge, it has been demonstrated23 that the tidal-breathing procedure would deliver approximately twice as much aerosol to the mouthpiece as the dosimeter. Our dosimeter method uses a nebulizer with a greater output than that in the method recommended by the American Thoracic Society guidelines7 (11 vs 9 µL per breath). However, the volume of aerosolized bronchoconstrictor administered using the dosimeter was substantially lower (approximately 60%) than the volume delivered to the mouth with the tidal-breathing method. Therefore, the effect of the method of AMP challenge on the sensitivity and reactivity to this bronchoconstrictor agent might be the consequence of a greater dose of the agonist delivered to the mouth using the tidal-breathing method than using the dosimeter method. However, in a recent study25 performed with methacholine, it was suggested that the reduction of the response with the dosimeter method compared with that of the tidal-breathing method is due, at least partially, to the bronchoprotective effect of the five deep inhalations, which are an integral part of the dosimeter method. There are no published data about the effects of deep inspiration on the response to AMP. In the absence of this information, we are unable to evaluate the influence of this factor on the differences in sensitivity and reactivity to AMP between the two challenge methods.

We do not believe that differences in AMP responsiveness can be explained by measurement errors, since we used methods that have previously been shown to be reliable. First, both nebulizer output and aerosol particle size were those recommended in the guidelines.8 This is important because it has been demonstrated that nebulizer output and particle size are factors that can affect the response to the bronchoconstrictor agent. Second, the baseline FEV1 values prior to bronchial challenge in each study day were not significantly different. Thus, effects caused by differing baseline airway caliber on the subsequent determination of AMP responsiveness could be eliminated. Finally, inhalation challenges were performed randomly and were separated by 1 to 5 days. This is relevant to the evaluation of AMP responsiveness because it has been demonstrated that, at least in atopic nonasthmatic subjects, repeated inhalation challenge with AMP induces a loss of response or refractoriness to the nucleotide.31 However, the time taken for the AMP PC20 values to return to the starting baseline value ranged from 2 to 6 h, and, to our knowledge, tachyphylaxis to AMP has not been seen after 24 h. Our studies were carried out at least a day apart, and we can accept, therefore, that the observed differences in the responses to both challenges are very likely to be due to the method of AMP challenge.

Purposely, our data were obtained in a group of subjects who were evaluated for suspected asthma with baseline FEV1 values of > 80% of predicted, not only to avoid the effect of initial airway caliber on the subsequent determination of PC20 but primarily because these patients are representative of the type of patients in whom AMP challenge testing may be clinically useful. However, the results of a recently published study25 suggest that the differences in methacholine PC20 values between the two challenge methods becomes more apparent in those subjects with higher PC20 values. Our patients had mild-to-moderate sensitivity to AMP, and the conclusions of this study may not necessarily apply to markedly hyperresponsive subjects with diagnosed asthma. This needs to be examined in future studies.

In conclusion, airway responsiveness to inhaled AMP can be measured by using a simple nebulizer and tidal breathing or by a more complex dosimeter and inspiratory capacity breaths. However, the results obtained with each challenge method are not comparable. This is important for the interpretation of the results obtained with the two methods of testing that are commonly used clinically and for research purposes.

Footnotes

Abbreviations: AMP = adenosine 5'-monophosphate; CI = confidence interval; PC20 = provocative concentration of a substance causing a 20% fall in FEV1

This study is part of the NAOMI project.

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.

Received for publication February 8, 2006. Accepted for publication April 29, 2006.

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