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(Chest. 2005;127:125-130.)
© 2005 American College of Chest Physicians

Phenol-Containing Saline Solution as a Diluent for Adenosine 5'-Monophosphate in Bronchial Challenge Testing*

Luis Prieto, PhD; Carlos Badiola, MD; Julio Cortijo, PhD; Carmen Pérez-Francés, MD; Valentina Gutiérrez, PhD and Amparo Lanuza, PhD

* From the Sección de Alergología, Hospital Universitario Dr Peset (Drs. Prieto, Pérez-Francés, Gutiérrez, and Lanuza), Valencia, Spain; Fundación de Investigación del Hospital General Universitario (Dr. Cortijo), Valencia; and GlaxoSmithKline (Dr. Badiola), Madrid, Spain.

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


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objectives: To investigate the effect of dissolving adenosine 5'-monophosphate (AMP) with phenol-containing saline solution on the stability and the bronchoconstrictive properties of this indirect agonist.

Methods: Eleven subjects with asthma well controlled with short-acting inhaled ß2-agonists as required or with inhaled corticosteroids were studied. Bronchial challenge tests with AMP dissolved with either normal saline solution or saline solution containing 0.4% phenol were performed on separate days. Furthermore, to assess the potential influence of the phenol-containing saline solution on the stability of the bronchoconstrictor agent, AMP solutions in concentrations of 40 µg/mL and 400 µg/mL were prepared in saline solution and phenol-containing saline solution and, after 30 min, the AMP levels were determined by high-performance liquid chromatography (HPLC) assay.

Results: The geometric mean AMP provocative concentration causing a 20% fall in FEV1 (PC20) was 13.49 mg/mL (95% confidence interval [CI], 6.76 to 26.91) for the saline solution method, and AMP PC20 for the saline solution with phenol method was 8.91 mg/mL (95% CI, 3.39 to 23.44) [p = 0.18]. No significant differences were found between the concentrations of AMP made in saline solution compared to those made in phenol-containing saline solution measured by HPLC.

Conclusion: These observations indicate that normal saline solution with or without phenol can be used as the diluent for AMP. However, since a potential risk with AMP of industrial sources is the bacterial contamination, adding a preservative such as phenol to a saline solution diluent might be recommended.

Key Words: adenosine 5'-monophosphate • airway responsiveness • asthma • high-performance liquid chromatography


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bronchial hyperresponsiveness and airway inflammation are characteristic features of asthma. Methacholine and histamine are the most frequently used spasmogens for the investigation of bronchial responsiveness. These two substances cause bronchoconstriction by a direct effect on airway smooth muscle cells. In contrast, inhaled adenosine 5'-monophosphate (AMP) acts indirectly, causing "primed" mast cell degranulation and the release of histamine and other mediators with subsequent smooth muscle contraction.1 Thus, AMP may provide additional information about airway inflammation and cellular activation state in asthma.2 Bronchial responsiveness to inhaled AMP may also better reflect acute changes in airway inflammation induced by allergen avoidance34 or by treatment with inhaled steroids.5 Therefore, 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 recently.8

The abnormal airway responsiveness in asthmatic patients can be studied by constructing concentration-response curves to pharmacologic bronchoconstrictors. Several methods for the analysis of the concentration-response curves have been developed, but the most commonly employed is the measurement of the concentration or dose that produces a predetermined response, such as provocative concentration causing a 20% fall in FEV1 (PC20) or a 35% reduction in specific airway conductance. However, previous studies9 have suggested a distinction between the slope of the concentration-response curve, referred to as reactivity, and the PC20 or the 35% reduction in specific airway conductance, referred to as sensitivity. It was suggested9 that different mechanisms may determine reactivity and sensitivity, and that both should be determined when bronchial provocation tests are being interpreted.

Food and Drug Administration (FDA)-approved methacholine (Provocholine; Methapharm; Brantford, ON, Canada) is available in prepackaged, sealed 100-mg vials, but FDA-approved AMP is not available. Therefore, the sodium salt of AMP from standard commercial sources is the agent of choice for challenge testing. This is preferential to adenosine because it is more soluble in aqueous solution. Sterile, normal saline solution (0.9% sodium chloride) may be used for the dilution of methacholine67 and AMP.8 However, because the package insert for Provocholine specifies the use of normal saline solution containing 0.4% phenol as the diluent, this phenol-containing saline solution is also widely used.

The potential benefit of adding phenol is reducing the potential for bacterial contamination. When Provocholine is used, there is no evidence that adding a preservative such as phenol to sterile saline solution diluent is necessary.7 On the contrary, because of its industrial origin and absence of certification of sterility, it might be preferable to dissolve AMP in saline solution containing phenol. However, it remains uncertain whether the use of saline solution with phenol to dissolve AMP may affect its stability or its capacity to induce bronchoconstriction. The purpose of this study was to investigate the effect of dissolving AMP with phenol-containing saline solution on the stability and the bronchoconstrictive properties of this indirect agent.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Patients 18 to 65 years old with a history of asthma and who had been treated with short-acting inhaled ß2-agonists as required or with inhaled corticosteroids (beclomethasone, 200 to 1,000 µg or equivalent daily) were enrolled in the study. Asthma was diagnosed by the presence of symptoms of wheeze, breathlessness, or cough plus methacholine airway hyperresponsiveness with a methacholine PC20 of < 8 mg/mL if the FEV1/FVC was ≥ 70%, or an improvement of the FEV1 from predicted of ≥ 15% after 200 µg inhaled albuterol if the FEV1/FVC was < 70%. Subjects were clinically stable at the time of testing, and FEV1 at baseline had to be ≥ 80% of predicted. All patients were nonsmokers, and none had history of chronic bronchitis, emphysema, or respiratory tract infections during the 4 weeks before the study. Current smokers, pregnant women, patients with only seasonal symptoms and skin sensitization to pollen allergens, and patients with significant renal, hepatic, or cardiovascular disease were specifically excluded. The study protocol was approved by the local ethics committee, and written informed consent was obtained from all participants.

Study Design
Subjects attended the laboratory on 3 days, at the same time of day (± 2 h). Short-acting inhaled ß2-agonists were withheld for at least 6 h, inhaled corticosteroids (if used) for 12 to 15 h, and oral antihistamines 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 during the whole study period. Inhaled (n = 7) or nasal topical (n = 5) corticosteroids were continued in the same dose. On the first day, all subjects were evaluated for suitability and spirometry was performed. On each of the next two visits (at least 2 days but not > 7 days apart), spirometry and AMP challenges with either normal saline solution or normal saline solution with phenol as the diluent were performed. The AMP challenges with each diluent were conducted on separate days with the order of challenge randomized. For each test, the baseline FEV1 was required to be ≥ 80% of predicted,10 and within 10% of the initial baseline FEV1.

Pulmonary Function
Lung function (flow-volume curves) was measured using a calibrated pneumotachograph (Jaeger MasterScope; Erich Jaeger GmbH; Würzburg, Germany) according to standardized guidelines.11 Baseline FEV1 and FVC levels were measured until three reproducible recordings differing by < 5% were obtained. Maneuvers were accepted as technically satisfactory if the back-extrapolated volume was < 150 mL or 5% of FVC, and if the expiratory time was at least 6 s. The highest values were used for analyses. Reference values were those of the European Community for Coal and Steel.10

AMP Challenge
Airway responsiveness to AMP was assessed using a standardized dosimetric method, as described in detail previously.1213 AMP (Pro.Bio.Sint; Varese, Italy) was dissolved freshly in 0.9% saline solution with or without 0.4% phenol to produce a doubling concentration range of 0.31 to 320 mg/mL. Each solution was administered from a jet nebulizer attached to a breath-activated dosimeter (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 10 µL per breath. Patients inhaled the aerosolized AMP solutions in five inhalations from functional residual capacity to total lung capacity through a mouthpiece with the patient’s nose clipped. Single measurements of FEV1 were made 60 to 90 s after the inhalation of each concentration, unless the forced expiratory maneuver was judged to be technically unsatisfactory. The test was interrupted when a fall in FEV1 of at least 20% from the post-saline solution value was recorded or the maximum concentration had been administered.

High-Performance Liquid Chromatography Analysis
Chromatographic analysis was performed with a high-performance liquid chromatography (HPLC) system (Perkin Elmer; Norwalk, CT) equipped with an injector (Rheodyne model 7125; Rheodyne; Rohnert Park, CA) with 20-µL sample loop, a precolumn (2 µm Kromasil C18, model TR-C160K; Tecknokroma; Barcelona, Spain) connected to an analytical 5-µm column (250 x 4.6 mm inner diameter) [Supelcosil LC-18-DB; Supelco; Bellefonte, PA] operating at 22 ± 2°C, and a diode-array ultraviolet detector (model 235C; Perkin Elmer; Norwalk, CT) set at 255 nm. AMP was eluted isocratically using a mobile phase consisting of potassium phosphate buffer 0.1 mol/L (Scharlau Chemie SA; Barcelona, Spain) adjusted to pH 6.0 and passing through the column at a flow rate of 1.0 mL/min. The retention time was 10 min. Known amounts of AMP solutions in saline solution were used to obtain calibration plots by analyzing samples spiked with 10 different concentrations (from 2.5 to 1,600.0 µg/mL of AMP; three samples for each concentration in 3 separate days). Calibration graphs were constructed by unweighted linear regression analysis of the peak area of AMP vs the theoretical AMP concentration. Unknown concentrations of AMP were calculated by inverse prediction for each sample by reference to the calibration graph. Reproducibility and accuracy of AMP measurements were determined (coefficient of variation = [SD ÷ measured concentration] x 100; accuracy = [measured concentration – theoretical concentration] ÷ theoretical concentration x 100). All samples showed one chromatographic peak at the expected retention time. A significant linear relationship (p < 0.0001) was found between concentrations of AMP and the area measured in the chromatographic peak. Analytical performance study showed the coefficient of variation in the range of 2.9 to 8.8%, and the accuracy in the range 0.4 to 11.5%. To study the potential influence of the phenol-containing saline solution on the stability of AMP, two concentration levels of AMP were selected (40 and 400 µg/mL). The corresponding solutions were prepared in saline solution and phenol (0.4%)-containing saline solution and, after 30 min, the AMP levels were determined by HPLC.

Statistical Analysis
Concentration-response curves were plotted for each challenge test as percentage fall in FEV1 against the log AMP concentration, and were characterized by their PC20 and slope. The AMP PC20 was calculated using an algebraic formula,14 whereas 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 and all subsequent points were used in the regression.1516 A graphic illustration of concentration-response curves obtained with normal saline solution or phenolated saline solution as the diluent for AMP in a single patient is presented in Figure 1 . An AMP PC20 value could not be calculated in one subject (FEV1 fall < 20% after the highest concentration). Thus, there were evaluable data on 11 subjects for FEV1 and slope, and there were evaluable data on 10 subjects for the PC20. All PC20 values were log transformed before analysis and presented as geometric means with 95% confidence intervals (CIs). The concentrations of AMP measured by HPLC are presented as mean ± SEM. All other numerical variables are reported as arithmetic means with 95% CI. Differences in concentrations of AMP measured by HPLC, FEV1, slope, and PC20 values were assessed by paired t tests. The study had > 80% power with an {alpha} error set at 0.05 (two tailed) to detect a 1 doubling concentration difference in PC20. All data were analyzed with standard statistical software (InStat for Windows, version 3.00; GraphPad Software; San Diego, CA); p values are two sided, and values < 0.05 were considered statistically significant.



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  		Figure 1. Graphic illustration of concentration-response curves obtained with normal saline solution (open circles) or phenolated-saline solution (filled circles) as the diluent for AMP in a representative patient. The PC20 and slope are illustrated.

 

   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Patient demographic data are shown in Table 1 . Eleven patients (7 men and 4 women) with a mean age of 35 years (95% CI, 26 to 45) were enrolled, and all completed the study with no adverse events. Seven patients were receiving inhaled corticosteroids, which remained unchanged throughout the study. The mean FEV1 and FEV1/FVC values were 99.7% predicted (95% CI, 91.8 to 107.6) and 76.3% predicted (95% CI, 72.0 to 80.6), respectively.


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  		Table 1. Patient Demographic Details*

 
The mean baseline values of FEV1 were 3.49 L (95% CI, 2.72 to 4.26) on the saline solution study day, and 3.57 L (95% CI, 2.80 to 4.29) [p = 0.42] on the saline solution with phenol study day. There was no difference in PC20 values for the two methods (p = 0.18). The geometric mean PC20 values were 13.49 mg/mL (95% CI, 6.76 to 26.91) for the saline solution method, and 8.91 mg/mL (95% CI, 3.39 to 23.44) for the saline solution with phenol method. The mean difference in the PC20 values obtained with each method was 0.59 doubling concentrations (95% CI, – 0.33 to 1.52). The individual data points are shown in Figure 2 . The mean values of the slope were 12.51%/log mg/mL (95% CI, 9.00 to 16.02) on the saline solution study day, and 13.74%/log mg/mL (95% CI, 9.08 to 18.39) [p = 0.24] on the saline solution with phenol study day (Fig 2).



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  		Figure 2. Individual values for AMP PC20 (top, A) and slope (bottom, B) for normal saline solution and saline solution containing phenol; horizontal lines are geometric means for PC20 and arithmetic means for slope.

 
In the HPLC studies, the values determined for a nominal concentration of AMP of 40 µg/mL were 36.4 ± 0.9 µg/mL and 37.0 ± 0.9 µg/mL in saline solution and phenolated saline solution, respectively. When AMP was prepared at 400 µg/mL, the obtained values were 399.9 ± 9.0 µg/mL and 414.1 ± 9.6 µg/mL in saline solution and phenolated saline solution, respectively (n = 6 for each group; p > 0.05). Therefore, no significant differences were found between the concentrations of AMP made in saline solution compared to those made in phenol-containing saline solution measured by HPLC after 30 min of preparation (Fig 3 ).



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  		Figure 3. AMP values determined by HPLC for nominal concentrations of 40 µg/mL and 400 µg/mL in saline solution or saline solution containing phenol after 30 min of preparation. There was no significant difference in the AMP concentrations detected with both diluents.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings clearly demonstrate that normal saline solution with or without 0.4% phenol can be used as the diluent for AMP without affecting the stability of this compound or the results of the bronchial challenge test. Airway hyperresponsiveness and inflammation are thought to be important hallmarks of asthma. AMP, when used as a bronchoconstrictor stimulus, acts by releasing inflammatory mediators from primed mast cells,1 and there now seems to be sufficient evidence to suggest that the severity of the airway response to inhaled AMP reflects the involvement of inflammatory cells and their mediators in the airways.2 Thus, in addition to demonstrating the capacity of the airways to narrow in response to the endogenous release of inflammatory mediators, challenges with AMP, when followed over time, may be useful in evaluating both the short-term and long-term effects of medications used in the treatment of airway inflammation. Since AMP is more soluble in aqueous solution, allowing higher concentrations of this agonist to be delivered by aerosolization, it has replaced adenosine as the most frequently used purine nucleoside bronchoprovoking agent. At this time, no FDA-approved preparation of AMP is available; therefore, the sodium salt of AMP, commercially available as a dry crystalline powder, is the agent most commonly used for challenge testing. Although phosphate-buffered saline solution has been used for the dilution of AMP in a few studies,1718 normal saline solution (0.9% sodium chloride) is the diluent most commonly used.23451920 For this reason, recent guidelines recommend the use of normal saline solution as the diluent for AMP.8

Our results indicate that phenol-containing saline solution may also be used for the dilution of AMP. A potential risk with AMP of industrial origin is the bacterial contamination. In order to minimize this possibility, single-use ampoules should preferably be used, and the most concentrated solution must be filtered through a 0.22-µm filter and transferred into a sterile vial. Furthermore, adding a preservative such as phenol to sterile saline solution diluent might be recommendable. Therefore, of the two diluents tested here, we prefer the phenol-containing saline solution.

The results were obtained after carefully addressing such aspects of methodology as study design and challenge methods. First, 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 PC20 and slope of the concentration-response curve could be eliminated. Second, inhaled or nasal topical corticosteroids were continued unchanged during the study, and none of the patients were being treated with other medications that could have affected the response to AMP. Third, inhalation challenges were performed randomly and were carried out at the same time of the day, thus ruling out a possible influence of circadian variations on airway responsiveness. We can accept, therefore, that observed differences in the response to both challenges are very likely to be due to the degree of measurement imprecision and cannot be explained by different conditions of airway challenge. Finally, our study was adequately powered at 80% to detect a 1 doubling concentration difference in AMP PC20 at the 5% level. However, a 0.5 doubling concentration difference might be considered relevant in some studies, and a larger population needs to be studied in order to completely ensure equivalence of the two different diluents.

When normal saline solution is used as the diluent, AMP solutions ≥ 3.125 mg/mL remain stable for up to 25 weeks at 4°C.8 Our results demonstrate that when phenol-containing saline solution is used as the diluent, AMP solutions remain stable for up to 30 min at room temperature. However, we are not aware of published information on the stability of AMP in normal saline solution with phenol solution for days or weeks of conservation; such studies are needed. Therefore, in the absence of this information, we recommend that, when phenol-containing saline solution is used as the diluent, AMP solutions should be prepared freshly before each inhalation challenge.

In summary, AMP can be dissolved either in normal saline solution or phenol-containing saline solution without adversely affecting its short-term stability and its capacity to induce bronchoconstriction. However, because normal saline solution does not have the antiseptic capacity of saline solution with phenol, we recommend that AMP solutions should be prepared with phenol-containing saline solution rather than normal saline solution.


   Acknowledgements
 
The authors are grateful to Rocio Rojas and Bertha Camps for preparing the dilutions of AMP and assisting with the pulmonary function testing, and Pedro Santamaría for HPLC analysis. The authors thank Professor Esteban Morcillo for critically reviewing the manuscript.


   Footnotes
 
Abbreviations: AMP = adenosine 5'-monophosphate; CI = confidence interval; FDA = Food and Drug Administration; HPLC = high-performance liquid chromatography; PC20 = provocative concentration causing a 20% fall in FEV1

This study is part of the NAOMI Project (publication No. 14) and was supported by a grant from GlaxoSmithKline S. A. of Spain.

Received for publication February 5, 2004. Accepted for publication August 17, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Polosa, R, Ng, WH, Crimi, N, et al (1995) Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 151,624-629[Abstract]
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  7. American Thoracic Society.. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med 2000;161,309-329[Free Full Text]
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  12. Prieto, L, Gutiérrez, V, Torres, V, et al Effect of salmeterol on seasonal changes in airway responsiveness and exhaled nitric oxide in pollen-sensitive asthmatic subjects. Chest 2002;122,798-805[Abstract/Free Full Text]
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  16. Prieto, L, Gutiérrez, V, Morales, C Maximal response plateau to methacholine as a reliable index for reducing inhaled budesonide in moderate asthma. Eur Respir J 1999;13,1236-1244[Abstract]
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  20. Ketchell, RI, Jensen, MW, Spina, D, et al Dose-related effects of formoterol on airway responsiveness to adenosine 5'-monophosphate and histamine. Eur Respir J 2002;19,611-616[Abstract/Free Full Text]




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