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(Chest. 2005;128:3585-3589.)
© 2005 American College of Chest Physicians

The Physical and Biological Doses of Methacholine Are Different for Mefar MB3 and Jaeger APS Sidestream Nebulizers*

Georg Praml, PhD; Eva Scharrer, MD; Dorothea de la Motte, PhD; Dennis Nowak, MD; Gerhard Scheuch, PhD; Knut Sommerer, PhD and Katja Radon, PhD

* From the Institute of Occupational and Environmental Medicine (Drs. Praml, Scharrer, de la Motte, and Nowak, and Radon), Ludwig-Maximilians-University, Munich; and Inamed GmbH (Drs. Scheuch and Sommerer), Institute for Aerosol Medicine, Gauting, Germany.

Correspondence to: Georg Praml, PhD, Institute of Occupational and Environmental Medicine, Ziemssenstr. 1, 80336 Munich, Germany; e-mail: Georg.Praml{at}med.uni-muenchen.de


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Within a study on respiratory symptoms in rural areas, we used the European Community Respiratory Health Survey methacholine challenge protocol. For quicker and more reliable handling, we had to change the nebulizer in the bronchial challenge system from Mefar model MB3 (Bovezzo, Italy) to Jaeger APS Sidestream (similar to Mefar; Würzburg, Germany). Therefore, we compared the physical properties of the two systems, adapted the challenge protocol, and compared the results of both systems in subjects with and without airway hyperresponsiveness to methacholine.

Method: The physical properties of both systems were characterized by the residual method indicating a similar particle size distribution and an average output of 6 µL/s for Mefar MB3 and 1.25 µL/s for APS Sidestream. In the comparison study, 34 subjects were included. Airway responsiveness was quantified by provocative dose of methacholine causing a 20% fall in FEV1.

Results: A significant difference was found between the two challenge systems (p =0.004, McNemar test). Nine subjects reached a 20% drop in FEV1 with the APS Sidestream only. The FEV1 dropped by > 20% using either system in eight subjects. In 17 subjects, none of the two systems caused a 20% decrease in FEV1.

Conclusion: Even if the physical dose is determined with elaborate methods, the biological dose may vary between two nebulizer systems, causing incomparable outcomes for subjects tested with different systems.

Key Words: bronchial hyperresponsiveness • Lower Saxony Lung Study • methacholine • residual method


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Lower Saxony Lung Study (Niedersächsische Lungenstudie) is a cross-sectional survey assessing the respiratory health of young adults in a rural area with intensive livestock farming in northwest Germany. Methods used in this study are mainly taken from the European Community Respiratory Health Survey (ECRHS).12 The baseline survey of this study was done in the city of Hamburg in 1991/1992 within a random sample of young adults aged 20 to 44 years.1 In order to compare the results of this urban population to the rural study population taking part in the Lower Saxony Lung Study, similar methods were used in our survey.

Within the ECRHS, a standardized protocol for measurements of bronchial responsiveness to methacholine was developed.3 In the ECRHS, methacholine chloride was administered through an aerosol dosimeter (model MB3; Mefar; Bovezzo, Italy). However, during the study it found that the aerosol output of these nebulizers may vary largely.45

Since the start of the ECRHS in 1991, several new nebulizers have been developed, among them the APS Sidestream (Jaeger; Würzburg, Germany). Advantages of the APS Sidestream nebulizer include the use of a filter absorbing the methacholine in the exhalate, which reduces unknown extrainhalation of methacholine, and undesirable exposure to the technician handling the system. Furthermore, control of the dosimeter is an integral part of the lung function measurement software. To achieve comparable inhalation of methacholine for both protocols, we adapted the ECRHS protocol to the APS Sidestream device. Therefore, we compared output and particle size of methacholine droplets of the Mefar MB3 and the APS Sidestream dosimeters. Furthermore, we tested both systems in a sample of adult subjects with various degrees of bronchial hyperresponsiveness.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Physical Measurements
All physical measurements were performed in a specialized laboratory for aerosol medicine (Inamed GmbH, Institute for Aerosol Medicine; Gauting, Germany).

Particle Size: The particle size in terms of mass median aerodynamic diameter (MMAD) was determined with the residual method6: a 99mTc-tagged NaCl solution of known concentration was nebulized with the dosimeter and passed into a 300-L glass container that was continuously flushed with dry air (24 L/min, relative humidity < 10%), thus drying up the aerosol droplets. The particles of the solid residual were then size classified with an eight-stage cascade impactor (Andersen Mark II; Thermo Electron Corp; Minneapolis, MN). MMAD and geometric SD of the original droplets were obtained by measuring the count rates of the deposits on the respective impactor stages with a scintillation counter, taking into account the aerodynamic diameter and the density of the dry residuals as well as the NaCl concentration.

Three different Mefar MB3 nebulizers were each filled as prescribed by the manufacturer with 3 mL of 99mTc-tagged 5% NaCl solution and activated 30 times for 1 s (ECRHS setting; total nebulizing time, 30 s). Filling volume for the APS Sidestream was 1.03 mL. Since APS Sidestream software allows for precise timing of the nebulizing period, three cycles of 0.2 s, 0.4 s, and 1 s, respectively, were performed with a total nebulizing time of 30 s each. All of the cycles were followed by a 5-min impactor sampling period and were done twice.

Output: The same 99mTc-labeled 5% NaCl solution was nebulized and deposited directly on a filter (B50TE; Pall Corporation; East Hills, NY) without any further treatment; the output was then calculated from the results of a scintillation counter. Three Mefar MB3 nebulizers were filled with 3 mL of solution; the dosimeter was activated 10 times for 1 s. The APS Sidestream nebulizer was equally filled with 1.03 mL of solution; output was measured at 0.2-s, 0.4-s, and 1-s nebulizing periods (total nebulizing time, 10 s each). All of the tests were done twice.

Biological Measurements
Subjects: A convenience sample of 34 subjects took part in the comparison study. Mean age was 35 ± 9 years (± SD); 12 of the subjects were male. The mean baseline FEV1 was 108.4 ± 13.6% of predicted7; eight subjects reported current wheeze. The study was approved by the Ethics Committee of the Faculty of Medicine at the Ludwig-Maximilians-University, Munich, Germany.

Baseline Lung Function Measurements: Prior to each methacholine challenge, lung function measurements including body plethysmography and spirometry were performed in all subjects according to American Thoracic Society criteria.8 All lung function measurements were done by two well-trained and experienced lung function technicians. For each subject, both challenges were done by the same technician.

Methacholine Testing: Methacholine challenge was done according to the protocol of the ECRHS.3 In short, baseline FEV1 was measured in the body plethysmograph according to American Thoracic Society recommendations.8 Participants were asked to avoid smoking for 1 h, refrain from using ß2-agonists or anticholinergic inhalers for 4 h, and not to use oral medications (ß2-agonists, theophylline, or antimuscarinics) for 8 h before each test.

Subjects who reported symptoms suggestive of asthma or chronic bronchitis or with a baseline FEV1 < 90% of predicted received doubling doses of methacholine (long protocol). For participants without such symptoms, fourfold increments of methacholine were administered until the FEV1 had dropped by > 10% from the control value. After that, doubling doses were used. The challenge was terminated if the FEV1 had dropped by > 20% from the control value or the maximum cumulative dose (1.2 mg) had been reached.

Prior to methacholine challenge, saline solution diluent (0.9%) was administered on both challenge days. The maximum post-saline solution FEV1 recorded 2 min after inhalation of the solution was used as the control value. In both methacholine challenge procedures, the subjects were asked to inhale deep and slowly from residual lung volume to near total lung capacity. FEV1 maneuvers were performed 2 min after inhalation and repeated until FEV1 maneuvers were technically acceptable.9 The participants were challenged twice on two different days within an 11-day interval using the Mefar MB3 and the APS Sidestream in random order. Both measurements were done at the same time of day.

Mefar MB3 Protocol
The challenge protocol was taken from the ECRHS manual. However, the first dose level had been omitted in order to shorten the duration of the procedure in the field study. Within the study, the Mefar MB3 dosimeter of the ECRHS center in Erfurt was used. The methacholine challenge doses for the short and long protocols are given in Table 1 . In addition to the dose assumed in the ECRHS protocol, the effective dose was calculated based on the mean output of the dosimeter calculated from the physical measurements. After expiration, subjects placed their lips around the mouthpiece, inspired to near total lung capacity, held their breath for at least 3 s, and exhaled into the challenge chamber. As in the ECRHS, the aerosol was delivered over a period of 1 s for each inhalation. If the FEV1 did not drop to 20% below the control FEV1, the next dose was administered immediately.


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  		Table 1.. The Mefar MB3 Challenge Protocol*

 
APS Sidestream Protocol
The challenging protocol was based on the mean output of 1.27 µL/s obtained from the physical measurements at a nebulization period of 0.4 s. The duration of aerosol delivery as well as the number of inhalations and the methacholine concentration in each challenge step are given in Table 2 . The duration of aerosol delivery was chosen in a narrow range from 0.3 to 0.6 s. Subjects placed their lips around the mouthpiece during the whole time at each challenge level. When inhalation became steady, subjects were asked to exhale and inspire slowly to near total lung capacity, to hold their breath, and to exhale into the mouthpiece. The APS Sidestream was triggered 0.1 s after start of inspiration. If the FEV1 did not drop to 20% below the control FEV1, the next dose was administered immediately.


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  		Table 2.. The APS Sidestream Challenge Protocol

 
Statistical Analysis
The provocative dose of methacholine causing a 20% drop in FEV1 (PD20) was calculated according to Sterk et al.10 The overall percentage agreement was calculated in relation to a PD20 cut point of 1.2 mg. In order to assess the level of agreement, the McNemar test was used.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Physical Measurements
The results of the physical measurements are given in Table 3 . The mean MMAD for the three Mefar MB3 nebulizers tested was 5.4 µm. The mean MMAD for the APS Sidestream nebulizer was 5.1 µm, slightly increasing over the nebulizing period. On average, the fine particle fraction (< 5 µm) was 46.3% for Mefar MB3 nebulizers and 49.7% for the APS Sidestream nebulizer. As particle size and size distribution of both systems were quite similar, it was concluded that an APS Sidestream protocol may legitimately be adapted to the ECRHS Mefar MB3 protocol by taking only the aerosol output into account.


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  		Table 3.. Results of the Physical Measurements

 
The mean Mefar MB3 output totaled 0.3 mg of NaCl/s (6 µL/s; relative SD, 6%). Mean APS Sidestream output was 0.06 mg NaCl/s (1.25 µL/s; relative SD, 4.9%). Fine particle mass output (< 5 µm) was, on average, 0.14 mg/s for Mefar MB3 and 0.03 mg/s for APS Sidestream.

Consequently, the APS Sidestream protocol was reliably adaptable to the ECRHS Mefar MB3 protocol assuming an output of 1.27 µL/s, provided the nebulization periods remain fairly close to 0.4 s. Additionally, the 6 µL/s Mefar MB3 output turned out to be substantially lower than the 10 µL/s output assumed during the ECRHS survey. Therefore, the 6 µL/s output found in the laboratory was used to establish the corresponding bronchial challenge steps for the biological measurements (Tables 1 , 2).

Biological Measurements
Technically satisfactory methacholine challenges with both methods were obtained in all 34 subjects. All subjects had baseline FEV1 > 80% predicted in both tests. The mean ± SD variation in baseline FEV1 between the two challenge days was 2.0 ± 1.7%. The maximum difference between the two baseline measurements was 8.5%. On average, the measurements were done within 5 days.

Using the McNemar test, the two methods resulted in significantly different results (p = 0.004): nine subjects reached a 20% drop in FEV1 with APS Sidestream only, and eight subjects did so with both systems (Table 4 ). Half of the subjects did not reach a 20% drop in FEV1 in either protocol. Thus, the overall agreement was 74%.


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  		Table 4.. Agreement in PD20 Between Mefar MB3 and APS Sidestream Protocols*

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
With this study, we aimed to adapt an APS Sidestream scheme for bronchial challenge with methacholine to the Mefar MB3 protocol used in the ECHRS. Therefore, we used a two-step approach: elaborate physical measurements preceded a comparison study in volunteers.

In order to adapt the Mefar MB3 protocol to the APS Sidestream protocol, the physical properties of the two nebulizing systems had to be characterized in detail. This can only be done in a specialized laboratory for aerosol medicine, as the aerosol particles behave highly dynamically. Particle size and MMAD may change rapidly because of evaporation losses.6 Therefore, determining aerosol output by measuring weight loss—as often recommended by manufacturers—is inadequate.

Our study has shown that in spite of identical particle size and physical output, the dose producing a 20% fall in FEV1 differed significantly between the two nebulizer systems. Basically, a larger number of PD20-positive subjects was found with the APS Sidestream system.

In general, the intraindividual variation of methacholine challenge performed within a couple of days is low.1112 Likewise, a high level of agreement was shown for the Mefar MB3 dosimeter protocol using different methods and different challenge agents. Siersted and colleagues13 compared the ECRHS Mefar MB3 dosimeter protocol to the 2-min tidal breathing method, and found an overall agreement of 89% between the two methods and therefore much higher than in our study. Toelle et al14 compared the Mefar MB3 dosimeter to the protocol by Yan et al,15 and also showed an agreement of 89% between the two tests.

In these studies,1415 the variation between the two methods seemed to be at random, while in our study all subjects who were defined as bronchial hyperresponsive by the Mefar MB3 method were also bronchial hyperresponsive using the APS Sidestream method. Therefore, our result might indicate a systematic underestimation of the biological dose using the APS Sidestream protocol. One has to consider that not only particle size, fine particle mass, and nebulizer output determine the effective dose. In addition, the nebulization period and the triggering time might influence the amount of aerosols that is deposited in the lungs of the subjects as well as the site of aerosol deposition.

We did not control for caffeine consumption on the two challenge days.16 Likewise, we did not assess use of longer-acting bronchodilating agents prior to testing. However, due to the small intraindividual differences in baseline FEV1 between the two challenge days, we do not think that our results were largely affected. For the physical measurements, pure 5% NaCl solution was used, while the bronchial challenge was done with methacholine diluted in 0.9% NaCl solution. It would be desirable to use tagged methacholine solution for the physical measurements; however, this is unfortunately not feasible with the sophisticated method used to determine the output of the two nebulizers. Different filling volumes were used for the physical measurements in the APS Sidestream (1.03 mL) and Mefar MB3 (3 mL) nebulizers. This was done because these volumes are usually applied in the challenge protocols. The same filling volumes were used in our biological measurements because our physical measurements have shown that higher filling volumes, especially in the APS Sidestream system, might slightly increase the output (data not shown).

In conclusion, even if the physical dose of the nebulizer systems is determined with elaborate methods, the biological dose may vary largely. Therefore, in clinical practice results of bronchial challenge tests in a patient using an Mefar MB3 may not be comparable to results using the APS Sidestream.


   Acknowledgements
 
We thank Martina Dutschke, Julia Post, and Alexandra Koenig for field work, and the subjects for participation in the study. We are grateful to Dr. Rob van Strien for helpful comments on a draft of this article.


   Footnotes
 
Abbreviations: ECRHS = European Community Respiratory Health Survey; MMAD = mass median aerodynamic diameter; PD20 = provocative dose of methacholine causing a 20% drop in FEV1

The study was funded by the Lower Saxony Ministry for Social Affairs and the European Union.

Received for publication December 21, 2004. Accepted for publication June 1, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  10. Sterk, PJ, Fabbri, LM, Quanjer, PH, et al Airway responsiveness: standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults; report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal Official Statement of the European Respiratory Society. Eur Respir J Suppl 1993;16,53-83[Medline]
  11. Juniper, EF, Frith, PA, Dunnett, C, et al Reproducibility and comparison of responses to inhaled histamine and methacholine. Thorax 1978;33,705-710[Abstract/Free Full Text]
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  16. Bara, AI, Barley, EA Caffeine for asthma. Cochrane Database Syst Rev 2001; CD001112




This Article
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