MSc, PT M S Werkman

PhD, MSc, PT H J Hulzebos
email: h.hulzebos@umcutrecht.nl

MD H G M Arets

PhD, PT, PCS J Van Der Net

PhD, MSc, PT, PCS P J M Helders

PhD, MSc T Takken

Is static hyperinflation a limiting factor during exercise in CF? IS STATIC HYPERINFLATION A LIMITING FACTOR DURING EXERCISE IN ADOLESCENTS WITH CYSTIC FIBROSIS?

Keywords: work of breathing, ventilatory limitation, cardiopulmonary exercise testing work of breathing, ventilatory limitation, cardiopulmonary exercise testing work of breathing, ventilatory limitation, cardiopulmonary exercise testing

Is static hyperinflation a limiting factor during exercise in CF?

Summary

Increased work of breathing is considered to be a limiting factor in patients with Cystic Fibrosis (CF) during exercise. We hypothesized that adolescents with CF and with static hyperinflation are more prone to a ventilatorily limited exercise capacity than non-static hyperinflated adolescents with CF.

Exercise data of 119 adolescents with CF [range 12-18 yrs.], stratified for static hyperinflation, defined as ratio of residual volume to total lung capacity (RV/TLC) > 30%, were obtained during a progressive bicycle ergometer test with gas analysis and analyzed for ventilatory limitation.

Static hyperinflation showed a significant, though weak association (Φ 0.38; p < .001) with a ventilatorily limited exercise capacity (Breathing Reserve Index at maximal effort > 0.70; FEV 1 < 80% predicted and reduced exercise capacity, defined as VO 2peak < 85% predicted).

Analysis of association for increasing degrees of hyperinflation showed an increase to Φ .49 (p < .001) for RV/TLC > 50%. In adolescents with static hyperinflation, peak work rate (W peak ; 3.1±0.7 Watt/kg (75.1±17.3 % of predicted), peak oxygen uptake (VO 2peak /kg (ml/min/kg); 39.2±9.2 ml/min/kg (91.0±20.3 % of predicted), peak heart rate (HR peak ; 176±19 beats/min) were significantly (p < .05) decreased when compared with non-static hyperinflated adolescents (W peak 3.5 ± 0.5 Watt/kg (81.4±10.0 % of predicted)); VO 2peak /kg (ml/min/kg); 43.1±7.5 ml/min/kg (98.0±15.1 % of predicted) and HR peak 185±14 beats/min).

Additionally, no difference was found in the degree of association of FEV 1 (%) and RV/TLC (%) with VO 2peak /kg pred and W peak /kg Pred , but we found the RV/TLC (%) to be a slightly stronger predictor of VO 2peak /kg pred and W peak /kg Pred than FEV 1 (%).

These results indicate that the presence of static hyperinflation in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise and that static

Is static hyperinflation a limiting factor during exercise in CF?

Summary

Increased work of breathing is considered to be a limiting factor in patients with Cystic Fibrosis (CF) performing aerobic exercise. We hypothesized that adolescents with CF and with static hyperinflation are more prone to a ventilatorily limited exercise capacity than nonstatic hyperinflated adolescents with CF.

Exercise data of 119 adolescents with CF [range 12-18 yrs.], stratified for static hyperinflation, defined as ratio of residual volume to total lung capacity (RV/TLC) > 30%, were obtained during a progressive bicycle ergometer test with gas analysis and analyzed for ventilatory limitation.

Static hyperinflation showed a significant, though weak association (Φ 0.38; p < .001) with a ventilatorily limited exercise capacity (Breathing Reserve Index at maximal effort > 0.70; FEV 1 < 80% predicted and reduced exercise capacity, defined as VO 2peak < 85% predicted).

Analysis of association for increasing degrees of hyperinflation showed an increase to Φ .49 (p < .001) for RV/TLC > 50%. In adolescents with static hyperinflation, peak work rate (W peak ; 3.1±0.7 Watt/kg (75.1±17.3 % of predicted), peak oxygen uptake (VO 2peak /kg (ml/min/kg); 39.2±9.2 ml/min/kg (91.0±20.3 % of predicted), peak heart rate (HR peak ; 176±19 beats/min) were significantly (p < .05) decreased when compared with non-static hyperinflated adolescents (W peak 3.5 ± 0.5 Watt/kg (81.4±10.0 % of predicted)); VO 2peak /kg (ml/min/kg); 43.1±7.5 ml/min/kg (98.0±15.1 % of predicted) and HR peak 185±14 beats/min).

Additionally, no difference was found in the degree of association of FEV 1 (%) and RV/TLC (%) with VO 2peak /kg pred and W peak /kg Pred , but we found the RV/TLC (%) to be a slightly stronger predictor of VO 2peak /kg pred and W peak /kg Pred than FEV 1 (%).

These results indicate that the presence of static hyperinflation in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise and that static

Introduction

Limitation of exercise capacity in adolescents with cystic fibrosis (CF) has a multi-factorial cause. Reduced lung function and muscle mass are known to be most important factors leading to a limited exercise capacity. 1,2 A decreased muscle mass reduces skeletal muscle function, including respiratory muscle strength, in adults with CF. 3 Moreover, in children with CF a decreased skeletal muscle strength 4,5 and endurance 6 have been reported, even when corrected for a decreased lean body mass or lung function. [4][5][6] This points to a possible intrinsic abnormality in muscle oxygen uptake in patients with CF, however, currently there is no firm evidence available. [7][8][9][10][11] Due to continuous airflow obstruction, as reflected by a decreased forced expiratory volume in one second (FEV 1 ), and dynamic hyperinflation, as reflected by a decreasing inspiratory capacity (IC) during exercise 12 , children with CF develop a rapid breathing pattern during exercise with a concomitant increase in the work of breathing (WOB) [13][14][15] and oxygen cost. 13 A decreased inspiratory muscle function (strength and endurance) that has been observed in patients with CF will lead to a faster inspiratory muscle fatigue during exercise, which contributes to the reduced exercise capacity. 16 17 It seems that there is an interrelationship between lung function, muscle mass, energy expenditure, (respiratory) muscle function and exercise capacity in patients with CF. 18 The objective of the current study was to investigate whether static hyperinflation makes adolescents with CF more prone to a ventilatory limitation during exercise. We hypothesized that adolescents with static hyperinflation are more prone to a ventilatorily limited exercise capacity than non-static hyperinflated adolescents with CF. Furthermore, we questioned if the amount of static hyperinflation (RV/TLC (%)) is a stronger predictor of exercise capacity than the degree of airflow obstruction FEV 1 (%pred).

Materials and Methods

Subjects

Adolescents with CF (n = 119) of the Cystic Fibrosis Center of the University Children's Hospital and Medical Center Utrecht, the Netherlands, were measured for body weight, height, lung function and exercise capacity as part of routine assessment measures at the annual medical check-up. All measurements were part of usual care, according to the policy of the medical ethical committee of the University Medical Center Utrecht, ethical approval and informed consent were not obliged. Data of the initial test of each participant (between 1998 and 2006) were selected for this study. Participants were stratified into static hyperinflated and non-static hyperinflated. Conform previous literature about children with asthma, we defined a ratio of residual volume to total lung capacity (RV/TLC), after using a bronchodilator, higher than 30% as moderate to severe hyperinflation. 19 Analysis for increasing degrees of static hyperinflation was performed by including only patients with a certain level of static hyperinflation in the analysis (RV/TLC > 30%, > 35%, > 40%, >45% and >50%).

The definition used for determination of a ventilatory limitation during Cardio Pulmonary

Exercise Test (CPET) was previously used by Sexauer et al, including: [1] Breathing Reserve Index at maximal effort > 0.70 (Calculated as peak minute ventilation (VE peak ) divided by maximal voluntary ventilation (MVV) were MVV is calculated as 35 x FEV 1 ), [2] a FEV 1 < 80% predicted and [3] a reduced exercise capacity, defined as VO 2peak < 85% predicted. 20

Spirometry

Spirometry and body plethysmography were performed before and after bronchodilation with salbutamol (800 ug), using a pneumotach system and a volume-constant plethysmograph (Master Lab system, E. Jaeger, Würzburg, Germany). Lung function measurements included total lung capacity (TLC), residual volume (RV) and forced expiratory volume in 1 second (FEV 1 ). The results were compared with predicted values for healthy subjects matched for age, body height, and gender. 21

Cardiopulmonary exercise test (CPET)

Exercise capacity was assessed using a progressive cardiopulmonary exercise test (CPET).

CPET, after brochodilation with salbutamol, performed on an electronically braked cycle ergometer (Jaeger physis; Carefusion, Houten, The Netherlands). The seat height was adjusted to the participant's comfort and leg length. Participants rested until all measured variables were stable. Cycling started at a workload of 0 W; the workload was incremented with 15 W/min until the patient stopped due to volitional exhaustion. The workload which could be overcome for the last 30 seconds prior to exhaustion was considered to be the W peak .

Determination if a participants' effort was maximal was based on subjective and objective criteria. Subjective criteria are described as "unsteady biking", "sweating", "facial flushing" and "clear unwillingness to continue despite encouragement". Objective criteria were: [1] peak heart rate (HR) > 95% HR predicted (210-age) and [2] respiratory exchange ratio (RER) > 1.00. Based on previous literature, we defined that a participant had to meet the subjective criteria and at least 1 out of two objective criteria for the test to be considered of maximal effort and character. 22 Participants breathed through a mask that was connected to a calibrated metabolic cart (Oxycon pro, Carefusion, Houten, The Netherlands). Expired gas was passed through a flow meter, oxygen analyzer, and a carbon dioxide analyzer. The flow meter and gas analyzer were connected to a computer, which calculated breath-by-breath minute ventilation (VE), oxygen uptake (VO 2 ), carbon dioxide production (VCO 2 ) and respiratory exchange ratio (RER) from conventional equations. Relative peak oxygen uptake (VO 2peak /kg)

Reference values

Reference values for VO 2peak and W peak from healthy children and adolescents were obtained from previously studied Dutch children and adolescents. 23,24 Statistical analysis Data were expressed as mean ± SD. Data were analyzed using SPSS 15.0 for Windows and tested for normality with the Kolmogorov-Smirnov Test. An alpha value of 0.05 was considered as statistically significant. Possible differences between groups in the CFpopulation were analyzed using one-way ANOVA when normally distributed and with the Mann-Whitney U-Test when not normally distributed. Dichotomy variables were tested for association using the phi coefficient (Φ), and tested for significance using Chi-square test.

Prognostic value of FEV 1 and RV/TLC for exercise capacity was analyzed using standard multiple regression analysis.

Results

Study Group Demographics

After determination of maximal effort and screening for completeness of data set, in total 119 adolescents, 50 females and 69 males were included.

Mean age was 13.8 years ± 1.7 (range 12-18 years), with a mean FEV 1 of 82.9% ± 20.9 (% predicted). FEV 1 and anthropometric values did not differ according to gender (Table 1). All measurements were obtained after bronchodilator.

<Please, insert TABLE 1 about here>

Static hyperinflation versus non-static hyperinflation

Overall, 54 (40.3%) patients were non-static hyperinflated (20♀; 34♂; RV/TLC (%) 23.4±3.4; FEV 1 (%pred) 97.0±13.6), whereas 65 (48.5%) patients were identified as static hyperinflated (30♀; 35♂; RV/TLC (%) 40.1±9.0; FEV 1 (%pred) 71.4±18.7). Peak exercise parameters and ventilatory parameters in static hyperinflated and non-static hyperinflated adolescents with CF are shown in Table 2. VO 2peak /kg, W peak /kg, VE peak and HR peak were all significantly lower in static hyperinflated patients compared to the non-hyperinflated patients (p < 0.05). Corrected for age and gender, the differences in VO 2peak /kg Pred (98.0±15.1 % in non-static hyperinflated and 91.0±20.3 % in static hyperinflated patients; p < .05), and in W peak /kg Pred (75.1±17.3 (%) in static hyperinflated and 81.4±10.0 (%) in non-static hyperinflated patients; p < .05) remained (Table 2).

<Please, insert

Ventilatory versus Non-ventilatory limitation

Twenty (29.4%; 7♀; 13♂) out of the 65 patients with static hyperinflation were ventilatorily limited during exercise while only 1 (1♂) of the 54 patients without static hyperinflation was ventilatorily limited during exercise. Phi coefficient (Φ) between ventilatory limitation and static hyperinflation was 0.38 (p < 0.001; see Table 3). Furthermore, for increasing degrees of hyperinflation, Phi coefficient increased to .52 (p < .001) in the RV/TLC range 30% -50%.

(Table 3) <Please, insert TABLE 3

about here>

Lung function and exercise capacity

As presented in Figure 1, baseline FEV 1 (%pred) after bronchodilator, showed a fair degree of association with VO 2peak /kg Pred and W peak /kg Pred (r = .44 and r = .46 respectively (both p < .001), where RV/TLC, after bronchodilator, showed more variable degrees of association (r = -.43 and r = -.47 respectively (both p < .001) after bronchodilator). There was a strong association between FEV 1 (%pred) and RV/TLC after bronchodilator (r = -.84; p < .001).

Multiple linear regression showed that, compared to FEV1 (%pred), RV/TLC (%) was a somewhat stronger predictor for W peak /kg Pred (FEV1 (%pred) B .

Discussion

We hypothesized that adolescents with CF with static hyperinflation are more prone for a ventilatorily limited aerobic exercise capacity compared with non-static hyperinflated adolescents. We found a significant, but weak association (Φ 0.38; p < 0.001) between static hyperinflation (RV/TLC > 30%) and ventilatory limitation at peak exercise. This indicates that the presence of static hyperinflation in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise, which is in line with previous research. 20 Sexauer et al found an odds ratio of 0.96 (p = 0.76) for the RV/TLC ratio at rest as a weak non-significant predictor for ventilatory limitation in adults with CF. 20 Confirmative results were found in a study among adult COPD patients, were the change in inspiratory capacity (IC) during exercise, reflecting dynamic hyperinflation, has been shown to be superior to static hyperinflation (resting IC) in estimating exercise tolerance. 25 Moreover, after analysis of the association between the degrees of static hyperinflation and a ventilatorily limited exercise capacity, the correlation coefficient slightly increased from .38 to .49 for RV/TLC > 30% and > 50%, respectively. Additionally, we found no difference in the degree of association of baseline FEV 1 (%pred) and RV/TLC (%), after bronchodilator, with VO 2peak /kg Pred. and W peak /kg Pred , however, RV/TLC (%) was a slightly stronger predictor of VO 2peak /kg Pred. and W peak /kg Pred than FEV 1 (%pred).

A point of discussion in the present study is the cut-off point in breathing reserve used to determine ventilatory limitation. Prioux et al suggested a ventilatory reserve at peak exercise of 20% (MVV-VE / MVV x 100%), with a corresponding breathing reserve 0.8, in 11-yearold children, which increased to 30% (corresponding breathing reserve 0.7) at the age of 16 years. The mean age of our patients is 14 years, which could have influenced the prevalence of ventilatory limitation as we have used breathing reserve > 0.7 as cut-off point. Furthermore, due to the narrow age range of our population, the present results could not be extrapolated to patients that are younger or older.

Based on the results we conclude that the presence of static hyperinflation after bronchodilator (RV/TLC > 30%) in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise and that static hyperinflation, as reflected by RV/TLC (%), is only a slightly stronger predictor of W peak /kg Pred and VO 2peak /kg Pred than the FEV 1 (%pred), which is only reflecting the degree of airflow obstruction. The decreased exercise capacity in static hyperinflated adolescents could be explained by faster termination of peak exercise due to preliminary inspiratory muscle fatigue. The preliminary inspiratory muscle fatigue could be induced by the development of dynamic hyperinflation. 14,15,24 , which increases work [12][13][14][15] and oxygen cost of breathing 13 and causing intrapulmonary gas trapping and ventilation/perfusion mismatching 27 , which make a patient with CF more susceptible to ventilatory limitation during exercise. 15 Moreover, the greater fatigability of the inspiratory muscles could hypothetically induce a reflex vasoconstriction in the peripheral locomotor muscles and thereby compromises blood flow to the exercising limbs. 17 3 Moreover, in children with CF a decreased skeletal muscle strength 4, 5 and endurance 6 have been reported, even when corrected for a decreased lean body mass or lung function. [4][5][6] This points to a possible intrinsic abnormality in muscle oxygen uptake in patients with CF, however, currently there is no firm evidence available. [7][8][9][10][11] Due to continuous airflow obstruction, as reflected by a decreased forced expiratory volume in one second (FEV 1 ), and dynamic hyperinflation, as reflected by a decreasing inspiratory capacity (IC) during exercise 12 , children with CF develop a rapid breathing pattern during exercise with a concomitant increase in the work of breathing (WOB) [13][14][15] and oxygen cost. 13 A decreased inspiratory muscle function (strength and endurance) that has been observed in patients with CF will lead to a faster inspiratory muscle fatigue during exercise, which contributes to the reduced exercise capacity. 16 17 It seems that there is an interrelationship between lung function, muscle mass, energy expenditure, (respiratory) muscle function and exercise capacity in patients with CF. 18 The objective of the current study was to investigate whether static hyperinflation makes adolescents with CF more prone to a ventilatory limitation during exercise. We hypothesized that adolescents with static hyperinflation are more prone to a ventilatorily limited exercise capacity than non-static hyperinflated adolescents with CF. Furthermore, we questioned if the amount of static hyperinflation (RV/TLC (%)) is a stronger predictor of exercise capacity than the degree of airflow obstruction FEV 1 (%pred).

Materials and Methods

Subjects

Adolescents with CF (n = 119) of the Cystic Fibrosis Center of the University Children's Hospital and Medical Center Utrecht, the Netherlands, were measured for body weight, height, lung function and exercise capacity as part of routine assessment measures at the annual medical check-up. All measurements were part of usual care, according to the policy of the medical ethical committee of the University Medical Center Utrecht, ethical approval and informed consent were not obliged. Data of the initial test of each participant (between 1998 and 2006) were selected for this study. Participants were stratified into static hyperinflated and non-static hyperinflated. Conform previous literature about children with asthma, we defined a ratio of residual volume to total lung capacity (RV/TLC), after using a bronchodilator, higher than 30% as moderate to severe hyperinflation. 19 Analysis for increasing degrees of static hyperinflation was performed by including only patients with a certain level of static hyperinflation in the analysis (RV/TLC > 30%, > 35%, > 40%, >45% and >50%).

The definition used for determination of a ventilatory limitation during Cardio Pulmonary

Exercise Test (CPET) was previously used by Sexauer et al, including: [1] Breathing Reserve Index at maximal effort > 0.70 (Calculated as peak minute ventilation (VE peak ) divided by maximal voluntary ventilation (MVV) were MVV is calculated as 35 x FEV 1 ), [2] a FEV 1 < 80% predicted and [3] a reduced exercise capacity, defined as VO 2peak < 85% predicted. 20

Spirometry

Spirometry and body plethysmography were performed before and after bronchodilation with salbutamol (800 ug), using a pneumotach system and a volume-constant plethysmograph (Master Lab system, E. Jaeger, Würzburg, Germany). Lung function measurements included total lung capacity (TLC), residual volume (RV) and forced expiratory volume in 1 second (FEV 1 ). The results were compared with predicted values for healthy subjects matched for age, body height, and gender. 21

Cardiopulmonary exercise test (CPET)

Exercise capacity was assessed using a progressive cardiopulmonary exercise test (CPET).

CPET, after brochodilation with salbutamol, performed on an electronically braked cycle ergometer (Jaeger physis; Carefusion, Houten, The Netherlands). The seat height was adjusted to the participant's comfort and leg length. Participants rested until all measured variables were stable. Cycling started at a workload of 0 W; the workload was incremented with 15 W/min until the patient stopped due to volitional exhaustion. The workload which could be overcome for the last 30 seconds prior to exhaustion was considered to be the W peak .

Determination if a participants' effort was maximal was based on subjective and objective criteria. Subjective criteria are described as "unsteady biking", "sweating", "facial flushing" and "clear unwillingness to continue despite encouragement". Objective criteria were: [1] peak heart rate (HR) > 95% HR predicted (210-age) and [2] respiratory exchange ratio (RER) > 1.00. Based on previous literature, we defined that a participant had to meet the subjective criteria and at least 1 out of two objective criteria for the test to be considered of maximal effort and character. 22 Participants breathed through a mask that was connected to a calibrated metabolic cart (Oxycon pro, Carefusion, Houten, The Netherlands). Expired gas was passed through a flow meter, oxygen analyzer, and a carbon dioxide analyzer. The flow meter and gas analyzer were connected to a computer, which calculated breath-by-breath minute ventilation (VE), oxygen uptake (VO 2 ), carbon dioxide production (VCO 2 ) and respiratory exchange ratio (RER) from conventional equations. Relative peak oxygen uptake (VO 2peak /kg)

Reference values

Reference values for VO 2peak and W peak from healthy children and adolescents were obtained from previously studied Dutch children and adolescents. 23,24 Statistical analysis Data were expressed as mean ± SD. Data were analyzed using SPSS 15.0 for Windows and tested for normality with the Kolmogorov-Smirnov Test. An alpha value of 0.05 was considered as statistically significant. Possible differences between groups in the CFpopulation were analyzed using one-way ANOVA when normally distributed and with the Mann-Whitney U-Test when not normally distributed. Dichotomy variables were tested for association using the phi coefficient (Φ), and tested for significance using Chi-square test.

Prognostic value of FEV 1 and RV/TLC for exercise capacity was analyzed using standard multiple regression analysis.

Results

Study Group Demographics

After determination of maximal effort and screening for completeness of data set, in total 119 adolescents, 50 females and 69 males were included.

Mean age was 13.8 years ± 1.7 (range 12-18 years), with a mean FEV 1 of 82.9% ± 20.9 (% predicted). FEV 1 and anthropometric values did not differ according to gender (Table 1). All measurements were obtained after bronchodilator.

<Please, insert TABLE 1 about here>

Static hyperinflation versus non-static hyperinflation

Overall, 54 (40.3%) patients were non-static hyperinflated (20♀; 34♂; RV/TLC (%) 23.4±3.4; FEV 1 (%pred) 97.0±13.6), whereas 65 (48.5%) patients were identified as static hyperinflated (30♀; 35♂; RV/TLC (%) 40.1±9.0; FEV 1 (%pred) 71.4±18.7). Peak exercise parameters and ventilatory parameters in static hyperinflated and non-static hyperinflated adolescents with CF are shown in Table 2. VO 2peak /kg, W peak /kg, VE peak and HR peak were all significantly lower in static hyperinflated patients compared to the non-hyperinflated patients (p < 0.05). Corrected for age and gender, the differences in VO 2peak /kg Pred (98.0±15.1 % in non-static hyperinflated and 91.0±20.3 % in static hyperinflated patients; p < .05), and in W peak /kg Pred (75.1±17.3 (%) in static hyperinflated and 81.4±10.0 (%) in non-static hyperinflated patients; p < .05) remained (Table 2). <Please, insert

Ventilatory versus Non-ventilatory limitation

Twenty (29.4%; 7♀; 13♂) out of the 65 patients with static hyperinflation were ventilatorily limited during exercise while only 1 (1♂) of the 54 patients without static hyperinflation was ventilatorily limited during exercise. Phi coefficient (Φ) between ventilatory limitation and static hyperinflation was 0.38 (p < 0.001; see Table 3). Furthermore, for increasing degrees of hyperinflation, Phi coefficient increased to .52 (p < .001) in the RV/TLC range 30% -50%.

(Table 3) <Please, insert TABLE 3

about here>

Lung function and exercise capacity

As presented in Figure 1, baseline FEV 1 (%pred) after bronchodilator, showed a fair degree of association with VO 2peak /kg Pred and W peak /kg Pred (r = .44 and r = .46 respectively (both p < .001), where RV/TLC, after bronchodilator, showed more variable degrees of association (r = -.43 and r = -.47 respectively (both p < .001) after bronchodilator). There was a strong association between FEV 1 (%pred) and RV/TLC after bronchodilator (r = -.84; p < .001).

Multiple linear regression showed that, compared to FEV1 (%pred), RV/TLC (%) was a somewhat stronger predictor for W peak /kg Pred (FEV1 (%pred) B .

Discussion

We hypothesized that adolescents with CF with static hyperinflation are more prone for a ventilatorily limited aerobic exercise capacity compared with non-static hyperinflated adolescents. We found a significant, but weak association (Φ 0.38; p < 0.001) between static hyperinflation (RV/TLC > 30%) and ventilatory limitation at peak exercise. This indicates that the presence of static hyperinflation in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise, which is in line with previous research. 20 Sexauer et al found an odds ratio of 0.96 (p = 0.76) for the RV/TLC ratio at rest as a weak non-significant predictor for ventilatory limitation in adults with CF. 20 Confirmative results were found in a study among adult COPD patients, were the change in inspiratory capacity (IC) during exercise, reflecting dynamic hyperinflation, has been shown to be superior to static hyperinflation (resting IC) in estimating exercise tolerance. 25 Moreover, after analysis of the association between the degrees of static hyperinflation and a ventilatorily limited exercise capacity, the correlation coefficient slightly increased from .38 to .49 for RV/TLC > 30% and > 50%, respectively. Additionally, we found no difference in the degree of association of baseline FEV 1 (%pred) and RV/TLC (%), after bronchodilator, with VO 2peak /kg Pred. and W peak /kg Pred , however, RV/TLC (%) was a slightly stronger predictor of VO 2peak /kg Pred. and W peak /kg Pred than FEV 1 (%pred).

A point of discussion in the present study is the cut-off point in breathing reserve used to determine ventilatory limitation. Prioux et al suggested a ventilatory reserve at peak exercise of 20% (MVV-VE / MVV x 100%), with a corresponding breathing reserve 0.8, in 11-yearold children, which increased to 30% (corresponding breathing reserve 0.7) at the age of 16 years. The mean age of our patients is 14 years, which could have influenced the prevalence of ventilatory limitation as we have used breathing reserve > 0.7 as cut-off point. Furthermore, due to the narrow age range of our population, the present results could not be extrapolated to patients that are younger or older.

Based on the results we conclude that the presence of static hyperinflation after bronchodilator (RV/TLC > 30%) in adolescents with CF by itself does not strongly influence ventilatory constraints during exercise and that static hyperinflation, as reflected by RV/TLC (%), is only a slightly stronger predictor of W peak /kg Pred and VO 2peak /kg Pred than the FEV 1 (%pred), which is only reflecting the degree of airflow obstruction. The decreased exercise capacity in static hyperinflated adolescents could be explained by faster termination of peak exercise due to preliminary inspiratory muscle fatigue. The preliminary inspiratory muscle fatigue could be induced by the development of dynamic hyperinflation. 14,15,24 , which increases work [12][13][14][15] and oxygen cost of breathing 13 and causing intrapulmonary gas trapping and ventilation/perfusion mismatching 27 , which make a patient with CF more susceptible to ventilatory limitation during exercise. 15 Moreover, the greater fatigability of the inspiratory muscles could hypothetically induce a reflex vasoconstriction in the peripheral locomotor muscles and thereby compromises blood flow to the exercising limbs. 17

  dividing VO 2peak by total body mass. Heart rate was monitored continuously by a 3-lead electrocardiogram (Hewlett-Packard, Amstelveen, Netherlands).

  161 and ß .227 (p=.135)); RV/TLC (%) B -.371 and ß -.273 (p = .073)) and VO 2peak /kg Pred (RV/TLC (%) B -.343 and ß -.203 (p = .188); FEV 1 (%pred) B .239 and ß .272 (p = .078)). <Please, insert FIG.1 about here>

26

 26

  Furthermore, the increase in work of breathing in patients with CF as a possible factor in ventilatorily limited exercise capacity could theoretically be elicited by the development of dynamic hyperinflation during exercise, instead of the presence of static hyperinflation at rest. Beside the FEV 1 and possible dynamic hyperinflation, other factors, such as nutritional status, muscle mass, respiratory and peripheral muscle strength and habitual daily physical activity are also important predictors of exercise capacity.1, 2, 4,[START_REF] Lands | The effects of nutritional status and hyperinflation on respiratory muscle strength in children and young adults[END_REF] After all we suggest that, in future research, beside standard anthropometric and lung function measures, dynamic hyperinflation and flow-volume curves during exercise should be measured to facilitate a better understanding of the role of increased work of breathing as possible limiting factor in the exercise capacity of patients with CF.

Fig. 1 .

 1 Figure Captions

Introduction

  Limitation of exercise capacity in adolescents with cystic fibrosis (CF) has a multi-factorial cause. Reduced lung function and muscle mass are known to be most important factors leading to a limited exercise capacity.1, 2 A decreased muscle mass reduces skeletal muscle function, including respiratory muscle strength, in adults with CF.

  Is static hyperinflation a limiting factor during exercise in CF? 4 was calculated by dividing VO 2peak by total body mass. Heart rate was monitored continuously by a 3-lead electrocardiogram (Hewlett-Packard, Amstelveen, Netherlands).

  161 and ß .227 (p=.135)); RV/TLC (%) B -.371 and ß -.273 (p = .073)) and VO 2peak /kg Pred (RV/TLC (%) B -.343 and ß -.203 (p = .188); FEV 1 (%pred) B .239 and ß .272 (p = .078)). <Please, insert FIG.1 about here>

26

 26

  Furthermore, the increase in work of breathing in patients with CF as a possible factor in ventilatorily limited exercise capacity could theoretically be elicited by the development of dynamic hyperinflation during exercise, instead of the presence of static hyperinflation at rest. Beside the FEV 1 and possible dynamic hyperinflation, other factors, such as nutritional status, muscle mass, respiratory and peripheral muscle strength and habitual daily physical activity are also important predictors of exercise capacity.1, 2, 4,[START_REF] Lands | The effects of nutritional status and hyperinflation on respiratory muscle strength in children and young adults[END_REF] After all we suggest that, in future research, beside standard anthropometric and lung function measures, dynamic hyperinflation and flow-volume curves during exercise should be measured to facilitate a better understanding of the role of increased work of breathing as possible limiting factor in the exercise capacity of patients with CF.

Fig. 1 .

 1 Figure Captions

TABLE 2

 2

	about here>

TABLE 2

 2

	about here>

Table 1 .

 1 Demographic characteristics Significant difference between gender groups (p < 0.05): Non parametric tested with Mann-Whitney U-test d Significant difference between gender groups (p < 0.05) Values are presented as means ± SD. Abbreviations: BM=body mass; BMI=body mass index; RV/TLC=ratio residual volume/total lung capacity; FEV 1 =forced expiration volume in one second; HR = heart rate; RER = respiratory exchange ratio (VCO 2 /VO 2 ); VO 2 peak = peak oxygen uptake; W peak = peak work rate;

		Female (n=50)	Male (n=69)	Total (n=119)
	Age (yrs)	13.7±1.5	13.8±1.8	13.8±1.7
	Heigth (cm)	156.6±7.7	159.1±11.4	158.1±10.1
	BM (kg)	43.8±8.1	44.3±10.7	44.1±9.6
	BMI (kg/m 2 )	17.8±2.1	17.2±2.0	17.4±2.1
	RV/TLC (%) (before Ventolin)	36.3±9.8	34.4±12.8	35.2±11.6
	RV/TLC (%) (after Ventolin)	33.2±9.6	32.1±11.7	32.5±10.9
	FEV 1 (%Pred.) (before Ventolin)	80.2±19.2	76.8±22.1	78.2±20.9
	FEV 1 (%Pred.) (after Ventolin)	84.8±20.1	81.5±21.5	82.9±20.9
	HR rest (beats/min)	102.1±20.6	100.2±14.5	101.0±17.3
	RER rest (VCO 2 /VO 2 )	0.92±0.09	0.91±0.08	0.91±0.08
	VO 2peak (L/min)	1.6±0.4 c	1.9±0.5	1.8±0.5
	VO 2 peak (ml/min/kg) (%Pred.)	36.5±6.3 a	44.2±8.8	41.0±8.7
		(94.5±16.7%)	(94.0±19.6%)	(94.2±18.4%)
	W peak (Watt)	133.3±27.4 c	152.7±48.0	144.6±41.6
	W peak (Watt/ kg) (%Pred.)	3.1±0.5 a	3.5±0.7	3.3±0.7
		(79.4±12.3%)	(76.9±16.3%)	(77.9±14.8%)
	HR peak (beats/min)	180.1±15.1	179.7±19.0	179.9±17.4
	RER peak (VCO 2 /VO 2 )	1.18±0.09 b	1.13±0.08	1.15±0.09
	VE peak (L/min)	62.2±15.6 d	70.4±22.7	67.0±20.3

a Significant difference between gender groups (p < 0.001) b Significant difference between gender groups (p < 0.01) c

Table 2 .

 2 Exercise capacity in static hyperinflated and non-static hyperinflated patients with CF Values are presented as means ± SD. Abbreviations: HR peak = peak heart rate; RER peak = respiratory exchange ratio at peak exercise; VE peak = peak minute ventilation; VO 2 peak /kg = peak oxygen uptake per kilogram body mass; W peak /kg = peak work rate per kilogram body mass; a p < 0.05 Non parametric tested with Mann-Whitney U-test, b p < 0.05.

		Static hyperinflation (N = 65)	Non-static hyperinflation (N = 54)
	HR peak (beats/min)	176±19	185±14 a
	RER peak (VCO 2 /VO 2 )	1.14±0.09	1.17±0.08
	VE peak (L/min)	59.5±17.2	76.0±20.2 b
	VO 2peak /kg (ml/min/kg)	39.2±9.2	43.1±7.5 b
	VO 2peak /kg Pred (%)	91.0±20.3	98.0±15.1 b
	W peak /kg (Watt/kg)	3.1±0.7	3.5±0.5 b
	W peak /kg Pred (%)	75.1±17.3	81.4±10.0 b

Table 3 .

 3 Correlation between static hyperinflation and ventilatory limitation for different degrees of hyperinflationValues are Phi correlation coefficients with p-value. Abbreviations: RV/TLC = residual volume / total lung capacity.

		Phi coefficient p-value
	RV/TLC > 30%	.38	<.001
	RV/TLC > 35%	.52	<.001
	RV/TLC > 40%	.50	<.001
	RV/TLC > 45%	.50	<.001
	RV/TLC > 50%	.49	<.001

John Wiley & Sons, Inc.Pediatric Pulmonology

Acknowledgments

This study was funded by an unconditional research grant (DO-IT) from the Committee on Physiotherapy Research of the Royal Dutch Society for Physiotherapy (Wetenschappelijk College Fysiotherapie, Koninklijk Nederlands Genootschap voor Fysiotherapie (KNGF)). All measurements used in data analysis are part of standard routine care at the Cystic Fibrosis Center of the University Children's Hospital and Medical Center Utrecht, Utrecht, the Netherlands. Acknowledgments This study was funded by an unconditional research grant (DO-IT) from the Committee on Physiotherapy Research of the Royal Dutch Society for Physiotherapy (Wetenschappelijk College Fysiotherapie, Koninklijk Nederlands Genootschap voor Fysiotherapie (KNGF)). All measurements used in data analysis are part of standard routine care at the Cystic Fibrosis Center of the University Children's Hospital and Medical Center Utrecht, Utrecht, the Netherlands.


MD Marije Koopman

; Hein

MD, PhD J L Brackel

MD, PhD Anja A P H Vaessen- 3 Verberne

PhD Wim C Hop

MD, PhD Cornelis K Van Der Ent

H G M Arets

N J Van Den Berg

P L P Brand

E J Duiverman

H J Hendriks

J W C M Heynens

J C Van Nierop

M Nuysink

Ms Marije Koopman

MD Marije Koopman

Ms Marije Koopman

Evaluation of interrupter resistance in methacholine challenge testing in children

Keywords: Bronchial hyperresponsiveness, bronchial provocation test, forced expiratory volume in 1 second, interrupter resistance, methacholine challenge test, sensitivity, specificity Bronchial hyperresponsiveness, bronchial provocation test, forced expiratory 55 volume in 1 second, interrupter resistance, methacholine challenge test, sensitivity, 56 specificity Bronchial hyperresponsiveness, bronchial provocation test, forced expiratory 55 volume in 1 second, interrupter resistance, methacholine challenge test, sensitivity, 56 specificity

Bronchial hyperresponsiveness is a key feature of asthma and is assessed using bronchial 36 provocation tests. The primary outcome in such tests (a 20% decrease in forced expiratory 37 volume in 1 second (FEV 1 )) is difficult to measure in young patients. This study evaluated the 38 sensitivity and specificity of the interrupter resistance (R int ) technique, which does not require 39 active patient participation, by comparing it to the primary outcome measure. 40

Methacholine challenge tests were performed in children with a history of moderate 41 asthma and bronchial hyperresponsiveness. Mean and individual changes in R int and FEV 1 42 were studied. A receiver operating characteristic (ROC) curve was used to describe sensitivity 43 and specificity of R int . 44 Seventy-three children (median age: 9.2 yrs; range: 6.3-13.4 yrs) participated. There 45 was a significant (p< 0.01) increase in mean R int with increasing methacholine doses. 46 However, individual changes of R int showed large fluctuations. There was great overlap in 47 change of R int between children who did and did not reach the FEV 1 endpoint. A ROC-curve 48 showed an area under the curve of 0.65. 49

Because of low sensitivity and specificity, the use of R int to diagnose bronchial 50 hyperresponsiveness in individual patients seems limited.

INTRODUCTION 59

According to GINA guidelines bronchial hyperresponsiveness (BHR) is a key feature of 60 asthma (1). To objectify BHR, forced flow volume manoeuvres are required to measure the 61 forced expiratory volume in one second (FEV 1 ), as primary outcome measure in bronchial 62 challenge testing (2). Because of the difficulty of obtaining forced lung function 63 measurements in early childhood the diagnosis of asthma in this age group is challenging and 64 complicated. Several studies have advocated measurement of the interrupter resistance (R int ) 65 as an alternative to quantify airway obstruction in preschool children and in children with 66 developmental delay (3;4). The interrupter technique is non-invasive and can be obtained 67 during tidal breathing (5). R int can be measured in children from two years of age as it 68 requires minimal cooperation (6). The R int technique has been compared to body 69 plethysmography [START_REF] Klug | Measurement of lung function in awake 2-4-year-old asthmatic 304 children during methacholine challenge and acute asthma: a comparison of the 305 impulse oscillation technique, the interrupter technique, and transcutaneous 306 measurement of oxygen versus whole-body plethysmography[END_REF] and transcutaneous partial pressure of oxygen measurements (7-10) 70 during methacholine challenge tests, but most studies made no comparison with the outcome 71 measure FEV 1 . The one study that compared R int with FEV 1 during methacholine challenge 72 testing reported a mean increase in R int with increasing doses of methacholine [START_REF] Bisgaard | Lung function measurement in awake young children[END_REF]. However, 73 this study did not present the changes in R int for individual patients nor how these related to 74 individual changes in FEV 1 . The ability of R int to detect methacholine induced 75 bronchoconstriction in individual patients, as measured by a 20% decrease in FEV 1 , has never 76 been studied. 77

The latest ATS/ERS statement on pulmonary function testing in preschool children 78 recommended the need to determine the role of R int in bronchial challenge tests (5). The 79 current study evaluated the sensitivity and specificity of R int to detect airflow obstruction 80 during methacholine challenge tests in asthmatic children by comparing measurements with 81 FEV 1 as the gold standard. 82 Maximal expiratory flow-volume measurements were performed with standardized 115 equipment, using reference values of Stanojevic (16). 116 117 Analysis 118

Changes in FEV 1 and R int were analyzed as percentages change from baseline. Change in R int 119 was also assessed as change in percentage predicted and change in z-score, computed from 120 the latest R int reference values (15). Wilcoxon Signed Ranks test was used to investigate 121 whether there was a significant increase in mean change from baseline of R int at each dose 122 step. To quantify the variability of FEV 1 and R int within individuals between the various 123 dosesteps, coefficients of variation (CV) were calculated (see online supplement for 124 calculation). The resulting individual paired CV's of FEV 1 and R int were compared using 125 Wilcoxon's test. Comparison of mean CV's between centers was done using the Wallis test. The association between changes in FEV 1 and changes in R int at PD 20 was 127 analysed using Spearman's correlation (r S ). At each separate dose step Mann Whitney tests 128 were computed to compare the difference in changes from baseline between subjects who did 129 and who did not reach the PD 20 endpoint at the dose step. Cox regression, with change from 130 baseline of R int as time-dependent variable, was used to analyse whether these changes were 131 predictive for having reached the PD 20 endpoint at the subsequent dose steps. The sensitivity 132 At the first COMBO-study visit 73 children with moderate asthma (60 % boys) with a median 143 age of 9.2 years (range: 6.3 -13.4 years) performed both R int and FEV 1 measurements during 144 methacholine challenge. Baseline characteristics are shown in Table 1. Fifty-two children 145 reached the PD 20 endpoint and were defined as responders (Table 2). 146

There was a significant increase from baseline of R int starting at the sixth methacholine 147 dose step and all dose steps thereafter (all p<0.01). From the first dose step mean FEV 1 was 148 significantly different from baseline value (all p< 0.01), except for dose step 2. Visual 149 inspection of individual R int profiles during the challenge test showed large fluctuations with 150 rises and falls at subsequent dose steps. In contrast, much smoother profiles with gradual falls 151 were found for FEV 1 (see E-figure 1 in the online supplement). To express the variability in 152 individual curves, the CV of measured values was calculated per patient. For R int the mean 153 CV of all patients was 10.6% (10 th -90 th percentile: 5.5% -16.3%). The mean CV for FEV 1 154 was significantly smaller: 2.9% (1.7% -4.3%, p<0.001). Further analysis of CV R int showed 155 that there was no correlation with age (r S = -0.13, p = 0.26). Also centres did not significantly 156 differ from each other (Kruskal-Wallis test: p=0.17). 157

Analysing the 52 children who reached the PD 20 endpoint, there was no significant 158 correlation between changes from baseline of FEV 1 and changes from baseline of R int at the 159 last dose step at which the PD 20 endpoint was reached (r S =0.11, p = 0.44). 160 Figure 1 demonstrates the mean changes from baseline of FEV 1 and R int at each dose 161 step, while at each dose step patients are grouped according to whether or not the PD 20 162 endpoint was reached at the dose step. At none of the dose steps there was a significant 163 difference in the mean change of R int from baseline between those who reached the PD 20 164 endpoint at that dose step versus those who did not, except at dose step 6 (p=0.014) (figure 165 1B). A combined analysis of all dose steps comparing responders and non-responders, using 166 This study showed that, although there was a significant increase of mean R int values with 187 higher doses of methacholine, individual changes in R int are highly variable during 188 methacholine challenge. The sensitivity and specificity of R int to detect methacholine induced 189 bronchoconstriction is low, which might limit the use of R int in individual patients. 190

Several studies evaluated lung function measures requiring less patient effort than 191 FEV 1 , which could possibly be used as alternative measures of changes in 192 bronchoconstriction. Klug et al showed that R int was significantly less sensitive than specific 193 airway resistance in detecting changes in airway function [START_REF] Klug | Measurement of lung function in awake 2-4-year-old asthmatic 304 children during methacholine challenge and acute asthma: a comparison of the 305 impulse oscillation technique, the interrupter technique, and transcutaneous 306 measurement of oxygen versus whole-body plethysmography[END_REF]. Phagoo et al found that R int was 194 also less sensitive and showed more variability than transcutaneous partial pressure of oxygen 195 measurements (9;10). In a study of Beydon et al. the majority of children with a 20% fall in 196 transcutaneous partial pressure of oxygen (PD 20 P tc O 2 ) showed a significant change of R int 197 during methacholine challenge. However, all children who did not reach the PD 20 P tc O 2 had at 198 least once a significant change from baseline [START_REF] Beydon | Measurements of resistance by the 309 interrupter technique and of transcutaneous partial pressure of oxygen in young 310 children during methacholine challenge[END_REF], resulting in many false positive as well as 199 false negative outcomes. 200 Despite these data R int is still advocated as an alternative measure for airway 201 obstruction in those who are not able to perform forced breathing manoeuvres. Several studies 202 showed significant correlation between gold standard FEV 1 and R int in groups of patients 203 (4;6), and reference values for R int measurement in healthy controls have been published 204 (15;17-20). Although there is convincing evidence on the value of R int measurement as a 205 measure of airway obstruction in groups of patients, the sensitivity of R int to detect changes in 206 airway obstruction in individual patients has been little researched. Only one previous study 207 compared R int with FEV 1 during methacholine challenge testing [START_REF] Bisgaard | Lung function measurement in awake young children[END_REF] (22%) children reached the R int endpoint (15% increase from baseline) and only 6 of them 212 also reached the FEV 1 endpoint (10% decrease from baseline). The children who showed a 213 15% rise in R int but who did not have a 10% decrease in FEV 1 could be misclassified by R int 214 as having BHR. Davies et al. investigated the correlation between R int and FEV 1 in 215 bronchodilator response, and despite a correlation between FEV 1 and R int in a group of 216 patients, they found no significant correlation in individual children (22). 217

To evaluate the sensitivity and specificity of the R int technique to detect changes in 218 airway obstruction in individual children we performed simultaneous measurements of R int 219 and gold standard FEV 1 in a large group of children with asthma. We show that in individual 220 cases the variability of R int measurements is high (mean intra-patient CV 10.6%), precluding 221 proper prediction of changes in FEV 1 . In the present study the ROC curve, describing the 222 sensitivity and specificity of R int in detecting BHR as determined by the primary outcome 223 FEV 1 , had a small AUC of 0.65. This predictive capacity is lower compared to impulse 224 oscillometry (AUC 0.68 to 0.75 (23)). 225 Large individual fluctuations in R int might be due to large intrapatient short-term 226 variation in airway resistance during tidal breathing (11;17;20;24). This variability, resulting 227 in low sensitivity and specificity, could limit the ability to detect thresholds in individual 228 airway obstruction as required in clinical practice. Furthermore the sensitivity might be lower 229 compared to FEV 1 because during (severe) airflow obstruction equilibration of alveolar 230 pressure might take longer than is realised during the shutter closure time during R int 231 measurement (25). This may explain the lower R int values in case of (severe) peripheral 232 airway obstruction and hence lead to misclassifications during methacholine challenge 233 testing. A third explanation of high variability of R int might be the variability of upper airway 234 The findings of our study imply that on group level BHR can be documented, since we 237 showed on average a significant rise in R int with increasing methacholine doses. This may be 238 useful in epidemiologic research. However, R int seems to have insufficient capacity to 239 diagnose BHR in individual patients and therefore it is of limited value in BHR measurements 240 in clinical practice. 241

A major strength of our study is the fact that the methacholine challenge test was 242 repeated after 26 weeks and resulted in a similarly low sensitivity and specificity of R int , 243 which validated the results. Beside this, the present study may have some limitations. Firstly, 244 the study was performed in children with a mean age of 9.3 years, because preschool children 245 are seldom able to perform FEV 1 measurements. It could be questioned if the found results 246 are applicable to younger children. Since R int is a measure of total airway resistance, an 247 induced bronchoconstriction will lead to a relative larger increase in R int in young children 248 compared to older children, because in young children lower airways resistance contributes 249 more to total airway resistance (26). Nevertheless, the fluctuations in R int are expected to be 250 similar as the short term repeatability of R int that has been found to be independent of age 251 (20;27). Additionally we used a 20% fall as 'gold standard' in detecting methacholine induced 252 bronchoconstriction. When we used a 32% increase in R int (the coefficient of repeatability of 253 R int measurements as described by Beydon et al.) as a cut-off value to determine BHR, there 254 were 12 children with a significant change in R int but without a 20% fall in FEV 1 . The 255 pathophysiology of a fall in FEV 1 during a forced manoeuvre might be different from an 256 increase of R int during tidal breathing. Therefore, there is a possibility that FEV 1 might not be 257 the gold standard for bronchial challenges especially in young children. 258 Therefore, there could have been children with BHR, who did not have a 20% fall in 259 FEV 1 but who did have a significant increase in R int . This might have resulted in too low 260 sensitivity because of too many false positives. However, FEV 1 is the primary outcome in 261 methacholine challenge test, and no better 'gold standard' is available. 262

The current study showed on average an increase of R int with subsequent methacholine 263 doses during methacholine challenge tests and therefore it might be useful as a measure of 264 airflow obstruction in groups of patients, e.g. in large epidemiological studies. However, we 265 conclude that R int seems to have insufficient capacity to diagnose BHR in individual patients. 266 According to GINA guidelines bronchial hyperresponsiveness (BHR) is a key feature of 60 asthma (1). To objectify BHR, forced flow volume manoeuvres are required to measure the 61 forced expiratory volume in one second (FEV 1 ), as primary outcome measure in bronchial 62 challenge testing (2). Because of the difficulty of obtaining forced lung function 63 measurements in early childhood the diagnosis of asthma in this age group is challenging and 64 complicated. Several studies have advocated measurement of the interrupter resistance (R int ) 65 as an alternative to quantify airway obstruction in preschool children and in children with 66 developmental delay (3;4). The interrupter technique is non-invasive and can be obtained 67 during tidal breathing (5). R int can be measured in children from two years of age as it 68 requires minimal cooperation (6). The R int technique has been compared to body 69 plethysmography [START_REF] Klug | Measurement of lung function in awake 2-4-year-old asthmatic 304 children during methacholine challenge and acute asthma: a comparison of the 305 impulse oscillation technique, the interrupter technique, and transcutaneous 306 measurement of oxygen versus whole-body plethysmography[END_REF] and transcutaneous partial pressure of oxygen measurements (7-10) 70 during methacholine challenge tests, but most studies made no comparison with the outcome 71 measure FEV 1 . The one study that compared R int with FEV 1 during methacholine challenge 72 testing reported a mean increase in R int with increasing doses of methacholine [START_REF] Bisgaard | Lung function measurement in awake young children[END_REF]. However, 73 this study did not present the changes in R int for individual patients nor how these related to 74 individual changes in FEV 1 . The ability of R int to detect methacholine induced 75 bronchoconstriction in individual patients, as measured by a 20% decrease in FEV 1 , has never 76 been studied. 77

The latest ATS/ERS statement on pulmonary function testing in preschool children 78 recommended the need to determine the role of R int in bronchial challenge tests (5). The 79 current study evaluated the sensitivity and specificity of R int to detect airflow obstruction 80 during methacholine challenge tests in asthmatic children by comparing measurements with 81 FEV 1 as the gold standard. 82

MATERIALS AND METHODS 83

This study was a substudy of the multicentre COMBO-study in children between 6 and 16 84 years with moderate asthma (see online supplement) [START_REF] Vaessen-Verberne | Combination therapy 321 salmeterol/fluticasone versus doubling dose of fluticasone in 322 children with asthma[END_REF]. Nine out of 19 hospitals performed 85 additional R int measurements during methacholine challenge tests. The children from these 86 centres were included in this substudy. It was approved by the local medical ethical 87 committees and written informed consent was obtained from the parents (and the child if ≥12 88 years) before start of the study. For additional information see online supplement. 89 90

Study protocol 91

Methacholine provocation tests were performed twice (at start of study and after 26 weeks) 92 measuring R int and FEV 1 after each methacholine dose. All children abstained from long-93 acting β2 agonists and inhaled steroids for 12 hours before the first methacholine provocation 94 test (and 36 hours before the second methacholine provocation test), and from short acting β2 95 agonists for 6 hours before provocation testing. To prevent possible, unwanted physiologic 96 effects of deep inhalation on R int , R int was measured prior to FEV 1 . Pulmonary function tests 97 were performed to conform with latest ATS/ERS guidelines (2;5;13) and briefly described 98 below. The complete methodology is described in the online supplement. 99 100

Methacholine challenge testing 101

Methacholine challenge tests were performed using the dosimeter method ( 2 (15). Wilcoxon Signed Ranks test was used to investigate 121 whether there was a significant increase in mean change from baseline of R int at each dose 122 step. To quantify the variability of FEV 1 and R int within individuals between the various 123 dosesteps, coefficients of variation (CV) were calculated (see online supplement for 124 calculation). The resulting individual paired CV's of FEV 1 and R int were compared using 125 Wilcoxon's test. Comparison of mean CV's between centers was done using the Kruskal-126 Wallis test. The association between changes in FEV 1 and changes in R int at PD 20 was 127 analysed using Spearman's correlation (r S ). At each separate dose step Mann Whitney tests 128 were computed to compare the difference in changes from baseline between subjects who did 129 and who did not reach the PD 20 endpoint at the dose step. Cox regression, with change from 130 baseline of R int as time-dependent variable, was used to analyse whether these changes were 131 predictive for having reached the PD 20 endpoint at the subsequent dose steps. The sensitivity 132 At the first COMBO-study visit 73 children with moderate asthma (60 % boys) with a median 143 age of 9.2 years (range: 6.3 -13.4 years) performed both R int and FEV 1 measurements during 144 methacholine challenge. Baseline characteristics are shown in Table 1. Fifty-two children 145 reached the PD 20 endpoint and were defined as responders (Table 2). 146

There was a significant increase from baseline of R int starting at the sixth methacholine 147 dose step and all dose steps thereafter (all p<0.01). From the first dose step mean FEV 1 was 148 significantly different from baseline value (all p< 0.01), except for dose step 2. Visual 149 inspection of individual R int profiles during the challenge test showed large fluctuations with 150 rises and falls at subsequent dose steps. In contrast, much smoother profiles with gradual falls 151 were found for FEV 1 (see E-figure 1 in the online supplement). To express the variability in 152 individual curves, the CV of measured values was calculated per patient. For R int the mean 153 CV of all patients was 10.6% (10 th -90 th percentile: 5.5% -16.3%). The mean CV for FEV 1 154 was significantly smaller: 2.9% (1.7% -4.3%, p<0.001). Further analysis of CV R int showed 155 that there was no correlation with age (r S = -0.13, p = 0.26). Also centres did not significantly 156 differ from each other (Kruskal-Wallis test: p=0.17). 157

Analysing the 52 children who reached the PD 20 endpoint, there was no significant 158 correlation between changes from baseline of FEV 1 and changes from baseline of R int at the 159 last dose step at which the PD 20 endpoint was reached (r S =0.11, p = 0.44). 160 Figure 1 demonstrates the mean changes from baseline of FEV 1 and R int at each dose 161 step, while at each dose step patients are grouped according to whether or not the PD 20 162 endpoint was reached at the dose step. At none of the dose steps there was a significant 163 difference in the mean change of R int from baseline between those who reached the PD 20 164 endpoint at that dose step versus those who did not, except at dose step 6 (p=0.014) (figure 165 1B). A combined analysis of all dose steps comparing responders and non-responders, using 166 Cox regression analysis, showed that a greater change of R int was associated with a higher 167 probability of reaching the PD 20 endpoint (p= 0.038). However, when inspecting the 168 individual changes from baseline, there is large overlap of children who did and who did not 169 reach the PD 20 endpoint at the various dose steps (Figure 2). A ROC curve of changes from 170 baseline of R int as a measure to detect a 20% decrease in FEV 1 is shown in Figure 3, resulting 171 in a small AUC of 0.65. 172

When using a 32.1% increase in R int as cut-off value to calculate the sensitivity and 173 specificity of R int to predict a 20% fall in FEV 1 , we found a sensitivity of 50% and a 174 specificity of 43%. This means that R int was able to detect half of the responders as 175 determined by PD20 and that in 12 out of 21 children R int increased with more than 32% 176 This study showed that, although there was a significant increase of mean R int values with 187 higher doses of methacholine, individual changes in R int are highly variable during 188 methacholine challenge. The sensitivity and specificity of R int to detect methacholine induced 189 bronchoconstriction is low, which might limit the use of R int in individual patients. 190

Several studies evaluated lung function measures requiring less patient effort than 191 FEV 1 , which could possibly be used as alternative measures of changes in 192 bronchoconstriction. Klug et al showed that R int was significantly less sensitive than specific 193 airway resistance in detecting changes in airway function [START_REF] Klug | Measurement of lung function in awake 2-4-year-old asthmatic 304 children during methacholine challenge and acute asthma: a comparison of the 305 impulse oscillation technique, the interrupter technique, and transcutaneous 306 measurement of oxygen versus whole-body plethysmography[END_REF]. Phagoo et al found that R int was 194 also less sensitive and showed more variability than transcutaneous partial pressure of oxygen 195 measurements (9;10). In a study of Beydon et al. the majority of children with a 20% fall in 196 transcutaneous partial pressure of oxygen (PD 20 P tc O 2 ) showed a significant change of R int 197 during methacholine challenge. However, all children who did not reach the PD 20 P tc O 2 had at 198 least once a significant change from baseline [START_REF] Beydon | Measurements of resistance by the 309 interrupter technique and of transcutaneous partial pressure of oxygen in young 310 children during methacholine challenge[END_REF], resulting in many false positive as well as 199 false negative outcomes. 200 Despite these data R int is still advocated as an alternative measure for airway 201 obstruction in those who are not able to perform forced breathing manoeuvres. Several studies 202 showed significant correlation between gold standard FEV 1 and R int in groups of patients 203 (4;6), and reference values for R int measurement in healthy controls have been published 204 (15;17-20). Although there is convincing evidence on the value of R int measurement as a 205 measure of airway obstruction in groups of patients, the sensitivity of R int to detect changes in 206 airway obstruction in individual patients has been little researched. Only one previous study 207 compared R int with FEV 1 during methacholine challenge testing [START_REF] Bisgaard | Lung function measurement in awake young children[END_REF] The findings of our study imply that on group level BHR can be documented, since we 237 showed on average a significant rise in R int with increasing methacholine doses. This may be 238 useful in epidemiologic research. However, R int seems to have insufficient capacity to 239 diagnose BHR in individual patients and therefore it is of limited value in BHR measurements 240 in clinical practice. 241

A major strength of our study is the fact that the methacholine challenge test was 242 repeated after 26 weeks and resulted in a similarly low sensitivity and specificity of R int , 243 which validated the results. Beside this, the present study may have some limitations. Firstly, 244 the study was performed in children with a mean age of 9.3 years, because preschool children 245 are seldom able to perform FEV 1 measurements. It could be questioned if the found results 246 are applicable to younger children. Since R int is a measure of total airway resistance, an 247 induced bronchoconstriction will lead to a relative larger increase in R int in young children 248 compared to older children, because in young children lower airways resistance contributes 249 more to total airway resistance (26). Nevertheless, the fluctuations in R int are expected to be 250 similar as the short term repeatability of R int that has been found to be independent of age 251 (20;27). Additionally we used a 20% fall as 'gold standard' in detecting methacholine induced 252 bronchoconstriction. When we used a 32% increase in R int (the coefficient of repeatability of 253 R int measurements as described by Beydon et al.) as a cut-off value to determine BHR, there 254 were 12 children with a significant change in R int but without a 20% fall in FEV 1 . The 255 pathophysiology of a fall in FEV 1 during a forced manoeuvre might be different from an 256 increase of R int during tidal breathing. Therefore, there is a possibility that FEV 1 might not be 257 the gold standard for bronchial challenges especially in young children. 258 Pediatric Pulmonology

  were carried out using the MicroRint (Micro Medical 110 Limited, Kent, UK). A bacterial filter was used (Spirosafe bacterial filter, Micro Medical, 111 Kent, UK). Reference values of Merkus et al. (15) were used. 112 113 Spirometry 114

  positioning of the head and neck and adequate support of the cheeks is 235 highly warranted and might lead to intra-individual variability. 236

Figure 1 . 378 379Figure 2 .Figure 3 .FEV 1 (

 1378231 FIGURE LEGENDS 373

Table 2 :Figure 1 .Figure 1 .Figure 2 .Figure 3 .

 21123 Figure 1. Mean changes from baseline of FEV1 (1A) and Rint (1B). At each dose step patients are grouped according to whether (closed symbols) or not (open symbols) the PD20 endpoint (20% fall in FEV1) was reached at that dose step. Error bars represent SEM. 179x190mm (96 x 96 DPI)

  ;14) with a 102 nebulizer (model 646, DeVilbiss Healthcare, Somerset, USA) attached to a dosimeter 103 (Rosenthal French dosimeter, PDS Instrumentation, Louisville CO, USA). Methacholine 104 chloride was administered in doubling doses and provocation was continued after at least five 105 minutes and until the dose at which at least 20% fall in FEV 1 (PD 20 ) or the maximum dose 106 was reached. 107 were carried out using the MicroRint (Micro Medical 110 Limited, Kent, UK). A bacterial filter was used (Spirosafe bacterial filter, Micro Medical, 111 Kent, UK). Reference values of Merkus et al. (15) were used. 112 113 Spirometry 114 Maximal expiratory flow-volume measurements were performed with standardized 115 equipment, using reference values of Stanojevic (16). 116 117 Analysis 118 Changes in FEV 1 and R int were analyzed as percentages change from baseline. Change in R int 119 was also assessed as change in percentage predicted and change in z-score, computed from 120 the latest R int reference values

  of R int in relation to having reached FEV 1 -based PD 20 was described by 133 receiver operating characteristic (ROC) curves. The area under the curve (AUC) shows 134 whether the change in R int can correctly classify those who do and do not reach the PD 20 135 endpoint. We used the coefficient of repeatability calculated by Beydon et al. (a 32.1% 136 increase in R int ) as a cut-off value to calculate the sensitivity and specificity of R int to predict a

  without a concomitant fall of FEV 1 with 20% . 177 Repeating all analyses with change in R int expressed as change in percentage predicted 178 and change in z-score led to similar results (data not shown). AUC's of ROC curves for 179 change in percentage predicted and for change in z-score were both 0.66. 180In 64 children the methacholine challenge test was repeated after 26 weeks. The 181 analyses to compare R int with FEV 1 (as described above) showed similar results (data not 182 shown). The AUC of the ROC curve was 0.61

Figure 1 . 374 375Figure 2 .Figure 3 .FEV 1 (

 1374231 FIGURE LEGENDS 369

Figure 1 .Figure 1 .Figure 2 .Figure 3 . 1 .E-figure 1 .

 112311 Figure 1. Mean changes from baseline of FEV1 (1A) and Rint (1B). At each dose step patients are grouped according to whether (closed symbols) or not (open symbols) the PD20 endpoint (20% fall in FEV1) was reached at that dose step. Error bars represent SEM. 179x190mm (96 x 96 DPI)

  and found that on 208 average R int increased during methacholine challenge comparable to changes in FEV 1 209 measurements, but individual changes in R int were not shown. Kannisto et al. compared R int 210

	211	with FEV 1 during exercise challenge to diagnose BHR (21). However, in this study only 11
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		w
		11

Table 1 : Baseline characteristics of children with moderate asthma (n=73) who performed a methacholine provocation test with both R int and FEV 1 measurement at first visit. Median (interquartile range)

 1

	Age (years)

  and found that on 208 average R int increased during methacholine challenge comparable to changes in FEV 1 209 measurements, but individual changes in R int were not shown. Kannisto et al. compared R int 210 Proper positioning of the head and neck and adequate support of the cheeks is 235 highly warranted and might lead to intra-individual variability. 236

	211	with FEV 1 during exercise challenge to diagnose BHR (21). However, in this study only 11 resistance.
		F o r (22%) children reached the R int endpoint (15% increase from baseline) and only 6 of them F o r
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
		w w
		11 12

Table 1 : Baseline characteristics of children with moderate asthma (n=73) who performed a methacholine provocation test with both R int and FEV 1 measurement at first visit. Median (interquartile range)

 1

	Age (years)

members of the COMBO-R int research group for recruiting the patients. The COMBO-R int

Berg (Flevo Hospital Almere), P.L.P. Brand (Princess Amalia Children's Hospital Zwolle),

Oswald-Mammosser M, Llerena C, Speich JP, Donata L, Lonsdorfer. Measurements

children. Pediatr Pulmonol 1997 August;24(2):78-85.

Kannisto S, Vanninen E, Remes K, Korppi M. Interrupter technique for evaluation of

exercise-induced bronchospasm in children. Pediatr Pulmonol 1999 March;27(3):203-

John Wiley & Sons, Inc.Pediatric Pulmonology

John Wiley & Sons, Inc.

This study was a substudy performed in some centres participating in the multicentre

spirometer (nSpire Health GmbH, Oberthulba, Germany). Measurements were performed

preschool children. Am J Respir Crit Care Med 2007 June 15;175(12):1304-45.

volume in 1 second (FEV 1 )) is difficult to measure in young patients. This study evaluated the

sensitivity and specificity of the interrupter resistance (R int ) technique, which does not require

also reached the FEV 1 endpoint (10% decrease from baseline). The children who showed a

April;97(4):366-74.

ACKNOWLEDGEMENTS 269

The authors would like to thank the lung function technicians for the measurements and the

ACKNOWLEDGEMENTS 265

The authors would like to thank the lung function technicians for the measurements and the

This study was supported by an unconditional grant from 28 GlaxoSmithKline Pharma Europe. This study was supported by an unconditional grant from 21 GlaxoSmithKline Pharma Europe. This study was supported by an unconditional grant from 28 GlaxoSmithKline Pharma Europe. This study was supported by an unconditional grant from 21 GlaxoSmithKline Pharma Europe.

Authors: Marije Koopman, MD 1 ; Hein J.L. Brackel, MD, PhD 2 ; Anja A.P.H. Verberne, MD, PhD 3 ; Wim C. Hop, PhD 4 ; Cornelis K. van der Ent, MD, PhD 1 ; on behalf of 7 the COMBO-Rint research group. 8

Word count abstract: 190 53

Authors: Marije Koopman, MD 1 ; Hein J.L. Brackel, MD, PhD 2 ; Anja A.P.H. Vaessen-6

Verberne, MD, PhD 3 ; Wim C. Hop, PhD 4 ; Cornelis K. van der Ent, MD, PhD 1 ; on behalf of 7 the COMBO-Rint research group. 8 minutes after each inhalation, R int and FEV 1 were measured. Provocation was continued after 49 at least five minutes and until the PD 20 or the maximum dose was reached. 50 51

Interrupter resistance 52

Airway resistance measurements were carried out using the MicroRint (Micro Medical 53 Limited, Kent, UK) that contains a pneumotachometer, a flow interruption valve and a 54 pressure transducer to measure mouth pressure post-occlusion. A bacterial filter was used 55 (Spirosafe bacterial filter, Micro Medical, Kent, UK). Measurements were performed conform 56 the latest ATS/ERS guideline (5). The child was seated and diverted to ensure quiet tidal 57 breathing. Children were breathing through a mouth piece while the technicians supported the 58 cheeks to reduce the change in upper airway compliance. Ten interruptions of 100 ms were 59 made at peak expiratory flow with a random frequency, with the valve closing within 10 ms. 60

The MicroRint software automatically rejected measurements that showed an artefact on the 61 pressure curve. Additionally measurements that showed tachypnoea or irregular breathing, 62 vocalisation, or hyperextension or flexion of the neck were manually discarded as well as 63 were tracings with a horizontal or declining pressure signal suggesting air leakage around the 64 mouthpiece. R int values were calculated using the two-point linear fit back extrapolation 65 technique to t=15ms (6). A minimum of five successful interruptions was required to calculate 66 the median R int value. Reference values of Merkus et al.