(Chest. 2000;118:1371-1377.)
© 2000
American College of Chest Physicians
Different Response to Doubling and Fourfold Dose Increases in Methacholine Provocation Tests in Healthy Subjects*
Britt-Marie Sundblad, BSc;
Per Malmberg, MD, PhD and
Kjell Larsson, MD, PhD, FCCP
*
From the Programme for Respiratory Health and Climate, National Institute for Working Life, Stockholm, Sweden.
Correspondence to: Britt-Marie Sundblad, BSc, Program for Respiratory Health and Climate, National Institute for Working Life, S-112 79 Stockholm, Sweden; e-mail: Britt-Marie.Sundblad{at}niwl.se
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Abstract
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Rationale: In a modified methacholine provocation test
that was used to study changes in airway responsiveness to occupational
irritants or sensitizers in healthy subjects, two protocols were used:
a long protocol (doubling methacholine concentrations between dose
steps) or a short protocol (fourfold increases in concentration). This
modified methacholine provocation allows measurements of the
provocative dose causing 20% decrease in FEV1
(PD20) in a high proportion of a normal population.
Methods: The distribution of PD20 was
investigated in healthy nonatopic men without history of allergy or
asthma symptoms using the long protocol (n = 101) or the short
protocol (n = 309). In addition, 30 healthy subjects underwent
methacholine provocation tests using both protocols.
Results: PD20 was defined in 79% of subjects
with the long protocol and in 48% of subjects with the short protocol.
The provocative concentration of methacholine causing a 20% decline in
FEV1 (PC20) and PD20 were
significantly lower using the long protocol: long-protocol
PC20 (median [25th to 75th percentile]), 19.9 mg/mL (3.9
to > 32 mg/mL) compared with short-protocol PC20, > 32
mg/mL (8.7 to >32 mg/mL; p < 0.0001); long-protocol
PD20, 4.2 mg (1.6 to 20 mg) compared with short-protocol
PD20, > 13.7 (2.6 to > 13.7 mg; p = 0.006). The
differences in PD20 using short and long protocols were
confirmed in a randomized trial of 30 healthy subjects tested with both
protocols.
Conclusion: Using doubling concentrations,
PC20 and PD20 could be defined in a higher
proportion of healthy subjects than a protocol using fourfold dose
increases. Furthermore, the doubling protocol results in a
PD20 estimate that is less than half the value obtained
when using a protocol with fourfold concentrations between dose steps.
The difference remains, whether the methacholine effect is regarded as
cumulative or noncumulative. The explanation for the difference between
the protocols is unclear.
Key Words: bronchial responsiveness • dose step • methacholine provocation test
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Introduction
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Bronchial
responsiveness is often assessed by bronchial provocation tests using
direct stimuli such as methacholine or histamine. Administration of the
bronchoconstrictor is commonly conducted with a dosimeter or a tidal
breathing method. Inspiratory flow and inhaled dose of the agent are
poorly controlled in most methods that have become standard in the
clinical evaluation of asthma.1
2
The methods of Orehek et
al3
and Eiser et al,4
as distinguished from
other methods, includes control of the inspired volume and flow, and
drying of the nebulized solution, resulting in a reduction of the
pharyngeal deposition.
In all these methods, repeated deep inspirations are performed when the
agent is inhaled and/or when forced expirations are made in order to
measure FEV1. Previously it has been demonstrated
that the decrease in FEV1 caused by
administration of a bronchoconstricting agent is attenuated for up to
10 min by a deep inhalation performed before or after administration of
the agent.5
Shortening the protocol by reducing the time
interval between dose steps reduces the time interval between the deep
inhalations required to measure FEV1, which
therefore influences the outcome of the FEV1
recorded at the next dose step.
In order to study changes in bronchial responsiveness induced by
occupational irritants or sensitizers in symptom-free subjects, we have
designed a modified protocol, based on a previously described method.
In this methacholine-provocation protocol, deep inhalations are
minimized and the dose delivered to the lower airways is
controlled.6
Using this method, a high proportion of
healthy subjects reacts with a 
20% decrease in
FEV1. Furthermore, this method discriminates
between exposed and nonexposed subjects in several occupational
settings.7
8
9
10
11
12
With our method, the nebulized solution is
dried prior to inhalation, which reduces the size of the droplets and
increases the deposition of methacholine in the lower airways. Flow and
time of inhalation, and inhaled dose are controlled, and only one deep
inhalation (FEV1 measurement) is performed at
each dose step.
The duration of a standard bronchial provocation test with doubling
doses of methacholine usually is 30 to 60 min. In studies with large
numbers of subjects, a more rapid method would be useful. We therefore
modified the test for healthy nonasthmatic subjects by using fourfold
increases of the methacholine concentration rather than doubling doses.
It was, however, not clear whether or not the outcome of the bronchial
provocation test would be the same for the two provocation protocols.
Data obtained from different studies in working populations were
compiled in order to elucidate the comparability of the two protocols
(doubling and fourfold increases of the concentration).
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Materials and Methods
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Two methacholine provocation protocols were used. The standard
protocol (the long protocol) employed doubling concentrations of
methacholine, starting at 0.5 mg/mL, followed by doubling
concentrations until a fall in FEV1 of 20%
or a concentration of 64 mg/mL was reached. The short protocol was
identical, with one exception: the methacholine concentration was
increased in fourfold steps up to the highest concentration (32 mg/mL).
The bronchial provocation tests were performed as parts of a number of
studies in different working and healthy referral populations. Details
about the studies have been published previously (Table 1
). Subjects with the diagnosis of asthma or a history of other chronic
lung diseases were excluded.
Nonatopic Men
Nonatopic men (n = 410) 38 years of age (range, 19 to 78
years) with no history of asthma or other lung disease, or allergic
disease, and with negative skin-prick test result or negative
Phadiatope (Pharmacia; Uppsala, Sweden) test result participated in the
study. This group was selected to avoid possible influences of sex and
atopy on the outcome of the two protocols. The long protocol was used
in 101 men and the short protocol in 309 men.
Randomized Trial
Since the long and short protocols were studied in different
subjects, a randomized trial in 30 healthy nonsmoking volunteers (12
women) with no history of atopy or asthma (mean age, 37 years; range,
21 to 61 years) using both the long and short protocols was performed.
The two methacholine tests were performed 1 to 2 weeks apart in random
order. Characteristics of the different groups are given in Table 2
. The project was approved by the ethics committee at the Karolinska
Institute, and all subjects gave their informed consent
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Table 2.. Characteristics of the Nonatopic Male Subjects
According to Long Protocol (Doubling Doses) and Short Protocol
(Fourfold Doses) Groups*
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Spirometry
Spirometry was performed with a wedge spirometer (Vitalograph;
Buckingham, UK). The highest of three reproducible measurements (the
two highest within 5%) of FEV1 and FVC maneuvers
were selected as baseline values.
Methacholine Test
The methacholine test commenced 5 to 10 min after the
spirometry. Following inhalation of the diluent, increasing
concentrations of methacholine were inhaled at exactly 6-min intervals,
ie, 6 min from the start of one inhalation to the start of
the next inhalation, until FEV1 had decreased by
20% or the highest concentration was reached. The methacholine
concentrations were 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/mL (long
protocol) or 0.5, 2, 8, and 32 mg/mL (short protocol). The highest
concentrations of methacholine given in the two protocols were 32 mg/mL
and 64 mg/mL, which corresponds to a cumulated dose of 8.5 mg (43
µmol/L) and 25.5 mg (130 µmol/L), respectively, at a nebulizer
output of 0.4 mg/min. The inhalation flow (0.4 L/s), inhalation time (2
s) and exhalation time (2 s), volume (0.8 L), and the number of breaths
(15 breaths, guided by the pace of a metronome) were controlled during
the provocation procedure that lasted for exactly 1 min. Only one
FEV1 measurement was performed at each dose step
4 min after the start of inhalation.5
If this measurement
was technically unsatisfactory (on rare occasions), the next dose was
given only if no symptoms were reported. The subjects were instructed
not to take deep breaths and to avoid coughing during the entire
procedure, including the time between the spirometry and start of the
methacholine test.
The methacholine was continuously administered with a jet nebulizer
(Astra Meditec; Gothenburg, Sweden) in the nonatopic men group and with
a Sidestream nebulizer (Medic-Aid; Pagham, UK) in the randomized trial.
The jet nebulizer used dry compressed air (390 kPa) producing an
aerosol of 0.1 L/s. The system was supplied with additional dry air
(0.3 L/s) that was led through a drying device.6
In the
Sidestream nebulizer, all air (0.4 L/s) was led through the nebulizer.
The nebulizate was led through a metal device (3.4 L), where the
aerosol was dried for about 8 s before inhalation. The volume
output of the nebulizers was measured as weight loss after 1 min of
nebulization, and the different nebulizers were calibrated every day. A
correction was applied in order to convert volume output (including
water used to humidify air passing the nebulizer) into the amount of
methacholine that leaves the nebulizers. This correction was 0.928 for
the jet nebulizer and 0.75 for the Sidestream nebulizer (where four
times more air passes through the nebulizer). The correction factors
were calculated from measurements of concentration increase in the
nebulizer solution and from comparison of methacholine
responses.13
PC20 is the estimated provocative concentration
of methacholine causing a 20% decrease in FEV1
compared to the value measured after inhalation of the diluent. The
value was calculated by interpolation using the first
FEV1 
80% of the postdiluent value and the
preceding FEV1 on a logarithmically transformed
methacholine concentration scale. The provocative dose of methacholine
causing a 20% decline in FEV1
(PD20; the cumulated dose) was calculated in a
corresponding fashion, using logarithms of the cumulated doses rather
than concentrations of methacholine. The dose was calculated from the
corrected nebulizer output and the methacholine concentration in the
nebulizer.
A dose-response slope (DRS) was calculated as the percent change in
FEV1 as a function of the cumulated methacholine
dose and was calculated by linear regression using all measure points,
the first point representing the mean value of the preexposure and
postdiluent values. This method differs from the previously reported
methods when only the end point (ie, the last value) is
considered.14
15
16
17
18
All calculations of PC20,
PD20, and DRS were made using appropriate
algorithms in a datasheet (Excel; Microsoft; Redmond, WA). Because of
the difference in concentrations of methacholine in the two protocols,
the number of subjects who reacted with > 20% decrease in
FEV1 at a cumulated dose of 6 mg methacholine
was calculated.
Atopy was assessed by skin-prick test with a standard panel with the 10
most common allergens in Sweden (Pharmacia; Uppsala, Sweden and ALK;
Copenhagen, Denmark) or with Phadiatope (n = 228).
Statistics
Results are expressed as median (25th to 75th percentiles)
values unless otherwise stated. Comparisons were calculated by using
2 test, Mann-Whitney U test, and
Wilcoxon’s signed rank test for paired data. A p value < 0.05 was
considered significant. Data were analyzed with JMP version 3.0 (SAS
Institute; Cary, NC) and Statview version 4.02 (Abacus Concepts;
Berkeley, CA).
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Results
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Nonatopic Men
PC20 was defined in 80 of 101 subjects
(79%) who underwent the long protocol and 150 of 309 subjects (48%)
using the short protocol (p = 0.01; Fig 1
). PC20 and PD20 were
significantly lower when the long protocol was used than when the
methacholine concentration was increased in fourfold concentration
steps (p < 0.0001 and p = 0.0006, respectively; Fig 1
and Table 3 ) The DRS did not differ significantly between the two protocols
(p = 0.15). There was a slight DRS difference in the middle part of
the cumulative frequency of curve (Fig 1
, bottom,
c, and Table 3
). Fifty-nine percent of the subjects using
the long protocol and 43% using the short protocol had 20% fall
in FEV1 at cumulated methacholine dose of 6 mg
(31 µmol/L; p = 0.01; Table 3
).
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Figure 1.. Bronchial responsiveness measured with doubling
(n = 101) and fourfold (n = 309) increases of the methacholine
concentration in healthy, nonatopic men. Top,
a: PC20. Middle,
B: Cumulative PD20. Bottom,
C: Average change in FEV1 as a function of
the methacholine dose. Long protocol = 21 subjects (21%) did not
react with 20% decrease in FEV1. Short protocol = 159
subjects (52%) did not react with 20% decrease in FEV1.
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Randomized Trial
In the randomized trial, four of the subjects did not reach a 20%
decrease in FEV1 when performing the long
protocol. The corresponding number when performing the fourfold
increases in methacholine concentrations was 12 subjects (p < 0.01;
Fig 2 ,
3
).
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Figure 2.. Bronchial responsiveness measured with doubling
and fourfold increases of the methacholine concentration at two
different occasions in 30 healthy, nonatopic subjects.
Top, a: PC20.
Middle, b: Cumulative PD20.
Bottom, c: Average change in
FEV1 as a function of the methacholine dose. Long
protocol = Four subjects (13%) did not react with 20% decrease in
FEV1. Short protocol = 12 subjects (30%) did not react
with 20% decrease in FEV1.
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PC20 and PD20 were
significantly lower using the long protocol than using the short
protocol (p = 0.008 and p = 0,04, respectively; Fig 2
,
top, a, and middle, b). The
DRS differed significantly between the two protocols (p = 0.01; Fig 2
, bottom, c). The differences between the two
techniques were calculated in 26 subjects (the 4 subjects who did not
have a > 20% decrease in FEV1 in either
provocation protocol were excluded from the comparison).
Eighteen of 30 subjects (60%) reacted with a 20%
FEV1 decrease after an inhaled dose of 6 mg (31
µmol/L) methacholine when performing doubling increases. The
corresponding number using the short protocol was 7 of 30 subjects
(23%; p = 0.009; Table 3
).
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Discussion
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The present study demonstrates that the protocol using fourfold
increases in methacholine concentrations results in apparently lower
bronchial sensitivity to methacholine, compared to the standard
doubling doses protocol as assessed by PC20 or
PD20 in healthy subjects. It has been previously
shown that methacholine has a small cumulative effect.19
In the present study, the discrepancy between the two protocols remains
also if the dose is expressed as cumulated inhaled dose. The difference
between the two protocols is thus not depending on whether the
bronchial effect is regarded as cumulative or noncumulative.
The finding of the difference between the long and short protocols is
intriguing, and we have no convincing explanation. The difference
between the groups is smaller if provocative dose rather than
provocative concentration values were used, suggesting that a
cumulative degree of airway provocation is a better estimate of the
dose than the provocative concentration values. The difference between
the protocols cannot be explained by overestimation of the cumulative
dose, since the duration of the long test is longer than the fourfold
increase test. It could be assumed that the effect of the first
methacholine doses starts to decline during the provocation procedure,
but this would lead to even lower PD20 in the
fourfold protocol than when doses are doubled. When plotting
FEV1 change over log concentration of
methacholine (or cumulated dose), a curvilinear relation was found. If
the two points before and after crossing the 80% of basal
FEV1 line are wider separated on the x-axis (as
is the case for the short protocol compared to doubling dose
increases), the intersection with the 80% FEV1
line would tend to move to the left with fourfold increases
(ie, suggesting more sensitive airways, which is the
opposite to the finding of the present study). Thus, the discrepancy
between the protocols does not seem to be explained by an artifact
based on the way provocative concentration or provocative dose values
were calculated. Airway hyperresponsiveness is influenced by atopy and
airway caliber, but also by factors such as sex, age, and
smoking.20
Since different subjects underwent the twofold
and the fourfold test, it cannot be excluded that true differences in
responsiveness between the groups partly explain the findings. However,
the results in the randomized trial, in which the same subjects
underwent both protocols, contradict such an interpretation since the
differences between the protocols remained.
A deep inhalation shortly before the inhalation of methacholine
influences the airways and the FEV1 reduction
after inhalation of methacholine is attenuated, compared to the
situation in which deep inhalations before methacholine are avoided.
This effect lasts for 6 to 10 min. Repeated deep inhalations after
inhalation of methacholine also attenuate the
FEV1 response, however, less effectively than a
deep inhalation performed in relaxed airways, shortly before
administration of the methacholine.5
21
It is thus
possible that in the short protocol, the effect of the previous doses
is less and the smooth muscles are less contracted before the dose when
FEV1 decreases 20%, than in the long
protocol. We suggest that this difference may depend on the fact that
there are fewer inhaled doses to reach the same dose in the short
protocol than in the long protocol. The magnitude of such an effect is,
however, unclear, and other mechanisms yet unknown may also contribute
to the observed effects.
In conclusion, the difference between the two protocols emphasizes the
importance to strictly adhere to a given protocol, using doubling or
fourfold increasing dose steps, avoiding mixtures between the two
protocols. For results to be comparable also, the time between dose
steps must be constant and strictly adhered to. The long protocol is
the most preferable method to assess changes and differences in
bronchial responsiveness, because of smaller differences in
methacholine concentration between the different dose steps. Therefore,
the long protocol may be more useful in research. However, for
clinicians who want to discriminate between asthma and nonasthmatic
conditions, the short protocol may be equally useful. This was,
however, not examined in the present study.
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Acknowledgements
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The authors thank Charlotte Müller-Suur and
Zhiping Wang for their help with challenge tests and Lars Eklund with
technical assistance and help in data analysis.
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Footnotes
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Abbreviations: DRS = dose-response slope;
PC20 = provocative concentration of methacholine causing
a 20% decline in FEV1; PD20 = provocative
dose of methacholine causing a 20% decline in FEV1
Studies in this article were supported by the Swedish Work
Environmental Fund Grants 84–1272 and 89–1422, and the Swedish Heart
Lung Foundation.
Received for publication December 28, 1999.
Accepted for publication June 2, 2000.
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Abstract
Introduction
