J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
Journal of Asthma, 29(5), 331-341 (1992)
Development of Lung Function in Relation to Increased Degree of Bronchial Responsiveness Vibeke Backer and Charlotte Suppli Ulrik Laboratory of Respiratory Physiology Department of Medicine B University Hospital Rigshospitalet DK-2100 Copenhagen, Denmark ABSTRACT
To study the relationship between development of lung function and bronchial responsiveness, we examined 106 subjects recruited from a random sample of 527 subjects, aged 8-18 years, from Copenhagen. Lung function and bronchial responsiveness to inhaled histamine were measured at two occasions, in 1986 and 1988. The participating subjects (n = 106) were divided into three groups: (a) 20 asthmatics with bronchial hyperresponsiveness (BHR), (b) 42 nonasthmatic subjects with BHR in 1986, and (c) 44 controls without BHR. In 1986, FEV, expressed as percentage of predicted value, was found to be similar in the three groups (91%, 94%, and 99%, respectively).The increase in height during the observation period was found to be 5, 6, and 6 cm, respectively, in the three groups. However, at the second examination, in 1988, FEV, was found to be significantly reduced in both asthmatics (87%) and nonasthmatic subjects (85O/,) compared with the controls (103%). In 1988, 16 asthmatics (80%) and 24 (57%) nonasthmatic subjects were found to have BHR, whereas none of the controls were Address reprint requests to : Vibeke Backer, JAGTVEJ 200, DK-2100Copenhagen, Denmark.
331 Copyright 0 1992 by Marcel Dekker, Inc.
Backer and Ulrik
332
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
found to have BHR. A multiple regression analysis was used to determine the correlation between change in FEV, and potential factors of importance. The change in FEV, was highly correlated with the presence of BHR in 1986, however, no correlation was found between change in FEV, and change in bronchial responsiveness. In conclusion, nonasthmatic subjects with former BHR showed signs of airflow obstruction and less increase in lung function during growth irrespective of the change in level of bronchial responsiveness, which may suggest a risk for subsequent development of obstructive lung disease.
INTRODUCTION
BHR, asthma, and longitudinal changes in lung function is limited.
Measurement of nonspecific bronchial responsiveness is a widely used approach for the assessment of airway function in children (1-3). Bronchial hyperresponsiveness (BHR) is found in most subjects with asthma; however, BHR is also found in nonasthmatic subjects (4-6). Longitudinal changes in degree of bronchial responsiveness are well recognized in both asthmatic (7)and nonasthmatic subjects (8,9).Yet, asthmatic subjects rarely reach a normal degree of bronchial responsiveness, whereas nonasthmatic children found to have BHR often subsequently will obtain normal degree of bronchial responsiveness (8,9). Children with persistent BHR have been shown to have reduced expiratory flow rates and slower increase in lung function through childhood and adolescence than children with bronchial responsiveness within the normal range (10).These findings suggest an association between asymptomatic BHR, airflow obstruction and subsequent development of obstructive lung disease. However, this association may not be straightforward, because as found by Josephs et al. (ll), increased bronchial responsiveness seems to be the underlying mechanism in asthma, whereas airflow obstruction seems to be a n abnormality in the airways. At present it is unknown whether nonasthmatic children with BHR, who subsequently obtain a level of bronchial responsiveness within the normal range, experience normal development of ventilatory function. Furthermore, our knowledge about the relationship between
Thus, the purpose of the present study was to investigate longitudinal changes in lung function and bronchial responsiveness in children and adolescents with asthma and asymptomatic bronchial hyperresponsiveness. SUBJECTS
A random sample of 983 children and adolescents living in the area surrounding the University Hospital (Rigshospitalet) in Copenhagen was drawn from the civil registration list and invited to participate in a study concerning asthma in 1986. All invited subjects were born in the first week of each month, all were aged between 7 and 16 years, and all were Caucasian. Five-hundred and twenty-seven (54%)accepted the invitation and were examined (Fig. 1). The description of the nonresponders (n = 456) has been presented in a previous paper (12). Seventynine of the children and adolescents (16%) were found to have BHR (i.e., PC-20 FEVl histamine less than 8 mg/ml), of whom 28 (5%) subjects had asthma (12). All subjects with BHR (n = 79) and 62 consecutively selected control subjects were invited to participate in a re-examination study 18 months later, in 1988. One-hundred and six children and adolescents (75%),55 boys and 51 girls, were examined (Fig. 1). The protocol was evaluated and approved by the local ethical committee and informed consent was obtained from all participating children and their parents.
Lung Function in Bronchial Responsiveness
333
Entrv studv in 1986
-
histamine challenge test
I 495 had histamine
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
challenge test
1 79
with BH
Entrv studv in 1988
I 416 without 6H
1 I l-7 79
invited
62
re-examined
62
invited
k-J 44
re-examined
1I
rn 20
L
asthmatics
42
nonasthmatics
44
normal controls
I
Figure 1. A flow chart of recruitment and allocation of subjects at the two examinations in 1986 and 1988.
All participants were interviewed by one of the authors (VB), and asthma was defined according to Hopp et al. (13).The questions, used at both examinations, were as follows: (1)Have you ever had asthma diagnosed?, (2) Have you ever had wheezy or dry cough?, (3) Do you have attacks of shortness of breath with wheezing?, (4) Have you ever been hospitalized and/or been treated for asthma by a doctor?, (5) Have you ever received medication for your asthma?, (6)Did the medication help?, and (7) Do exertion, stress, cold air, damp weather, or allergen exposure (i.e., provocating factozs), cause you pulmonary symptoms? Definite asthma was defined as a positive response to questions 1to 6; probable
asthma as a positive response to questions 1 to 4, and either 5 or 6; and questionable asthma as a positive response t o any two of questions 1 to 3 and any one of questions 4 t o 7. The participating subjects were grouped as follows. Group 1. BHR Asthmatic Subjects (n = 20)
The mean age was 12.2 years (SD 2.51, serum IgE 255 kU/L (SD SOO), and 10 subjects (50%)had atopic disease, defined as a positive skin prick test (SPT).Sixteen subjects had definite asthma, none had probable asthma, and four subjects had questionable asthma. Six of the asthmatics had a family history of
Backer and Ulrik
334 asthma, six had a history of other allergic symptoms than asthma, whereas eight asthmatics had no family history of allergic diseases in first-degree relatives.
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
Group 2. (n = 42)
BHR+ Nonasthmatic Subjects
The mean age was 11.4 years (SD 2.4), serum IgE 85 kU/L (SD 138),and four subjects (10%)had atopic disease.All subjects had bronchial hyperresponsiveness in 1986,and no earlier or present symptoms defined as asthma. At the examination in 1988,eleven subjects (26%)reported respiratory symptoms, of whom four had occasional dry cough, six had occasional exercise-induced shortness of breath without wheezing, and one had experienced wheezing on one occasion, the remaining 31 subjects claimed no respiratory symptoms. Nine of the nonasthmatics had a family history of asthma, 10 had a history of other allergic symptoms than asthma, whereas 23 nonasthmatics had no family history of allergic diseases in first-degree relatives. Groups 3. Nonasthmatic Subjects (Controls) (n = 44)
The mean age was 13.7 years (SD 2.5), serum IgE 58 k U L (SD 136),and none had atopic disease. The 44 participating control subjects were consecutively invited from the group of subjects (n = 416),who had normal degree of bronchial responsiveness in 1986 and no earlier or present respiratory symptoms suggesting a diagnosis of asthma. Two subjects (5%)claimed occasional dry cough at the examination in 1988.No significant differences in degree of bronchial responsiveness were found between the participating control subjects, the invited subjects (n = 62)and the remaining subjects (n = 354).Fourteen of the controls had a family history af asthma, eight had a history of other allergic or allergy-like symptoms than asthma, whereas 22 controls had no family history of allergic diseases in first-degree relatives.
METHODS Study Design
All subjects were asked not to smoke for 2 hours before the tests and were asked to avoid antiasthmatic medication prior to the examination (14).In case of recent respiratory infection, challenge tests were postponed for at least 6 weeks. Spirometry and a histamine challenge test were performed at both examinations, in 1986 and 1988.In 1986,a skin prick test and measurement of total serum IgE were performed. Furthermore, in 1988, static lung volumes, diffusion capacity and airway resistance were measured. lung Function Tests
SPIROMETRY A maximal forced expiratory flow volume curve (MEF) was obtained with a pneumotachograph (Vitalograph@),and FEVl was obtained with a dry bell spirometer (Vitalograph). The highest of three technically acceptable readings of FEVl was recorded; and MEFZ5was read from the accepted curve of FEVI.
LUNGVOLUMES Total lung capacity (TLC),residual volume
(RV),functional residual capacity (FRC), and vital capacity (VC) were measured with the closed circuit helium dilution rebreathing technique (Godard Bell-spirometero ).
DIFFUSIONCAPACITY The diffusion capacity (TLco) was measured with the single breath technique (Morgan spirometer* ) using a nitrogen mixture containing 0.3% CO, 10% helium, and 20% oxygen. Predicted values of the measured parameters based on the height of the subjects were calculated according to Zapletal et al. (15).
Lung Function in Bronchial Responsiveness
335
Bronchial Histamine Challenge Test
DATA ANALYSIS
The bronchial histamine challenge tests were performed according to the protocol by Cockcroft and ceworkers (16).The bronchial responsiveness was expressed a s doseresponse slope (DRS) (i.e., the percentage reduction in FEVl per pmol of inhaled histamine) (17). Prior to expression in log scale, 1.2% decline in FEVl per pmol was added to each value to eliminate negative and zero values (17).
The differences in degree of BHR and results of lung function tests between groups were analyzed using one-way analysis of variance and, when appropriate, a twosample t-test was used. Furthermore, changes in bronchial responsiveness (A-DRS), lung function (A-FEV11 and height (A-Height) between the two examinations were analyzed using a paired t-test. Multiple regression analysis was used for analyzing a potential relationship between changes in FEVl and DRS and selected background variables. The nonsignificant variables were deleted by backward elimination t o determine those associated with the change in FEVl and DRS.
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
Skin Prick Test and Total Serum IgE
A skin prick test with nine common aeroallergens was performed (by VB); the allergens used were: birch, grass, mugwort, horse, dog, cat, house-dust mite (Dermatophagoides pteronyssinus), and two molds (Alternaria iridis and Cladosporium herbarurn) (18).Blood samples were drawn and the total serum IgE was determined by paper radioimmunosorbent test (PRIST, Pharmacia, Copenhagen, Denmark).
RESULTS The FEV1, L, and %pred, was found to be similar in the three groups at the first examination in 1986 (Table 1). In 1988,
Table 1. Results (Mean +_ SD) in 106 Subjects Tested in 1986 and 1988 BHR+ ASTHMATIC GROUP 1 N = 20 In 1986 FEV, (L) FEUl (pred%) DKS-histamine Height (cm)
2.1 91.5 0.8 148.4
In 1988 FEV, (L) FEV, (%pred) DRS-histamine Height (cm)
2.2 87.2 0.7 153.3
A-f:EVl (L) 88-86 0.2 A-FEVlo,o 88-86 -4.3 A-DRS 88-86 -0.1 A-Height 88-86 5.0
+ + + + + +
+
+
+
+ + +
BHR+ NONASTHMATIC GROUP 2 N = 42
0.5 10.9 0.6 15.0
2.0 94.2 0.5 145.7
0.6 10.6 0.4 14.6
2.1 85.3 0.3 151.7
0.3 9.2a 0.7 3.4b
0.1 -8.9 -0.2 6.0
+
+
+
+ +
+
+ + +
+ + +
BHRCONTROLS GROUP 3 N = 44
0.6 12.6 0.4 13.8
2.6 98.5 0.2 155.5
0.7 10.9 0.2 13.1
3.0 102.9 0.1 161.9
0.2 11.3b 0.4b 3.6b
0.5 4.5 -0.1 6.4
+ +
+ +
+ +
+ + + +
+
+
1 vs. 2
2 vs. 3
1 vs. 3
ANOVA
0.8 11.9 0.1 14.3
NS NS p < 0.05 NS
NS NS p < 0.05 p < 0.01
NS NS p < 0.01 NS
8.5 2.7 27.2 5.2
0.9 11.2 0.1 13.8
NS NS
NS p < 0.001 p < 0.001 p < 0.01
NS
17.1 29.4 49.7 6.1
0.4 9.1 0.1 a 4.6b
p p
p
< 0,001 < 0.05 NS 0.05 NS NS
Journal of Asthma, 29(5), 331-341 (1992)
Development of Lung Function in Relation to Increased Degree of Bronchial Responsiveness Vibeke Backer and Charlotte Suppli Ulrik Laboratory of Respiratory Physiology Department of Medicine B University Hospital Rigshospitalet DK-2100 Copenhagen, Denmark ABSTRACT
To study the relationship between development of lung function and bronchial responsiveness, we examined 106 subjects recruited from a random sample of 527 subjects, aged 8-18 years, from Copenhagen. Lung function and bronchial responsiveness to inhaled histamine were measured at two occasions, in 1986 and 1988. The participating subjects (n = 106) were divided into three groups: (a) 20 asthmatics with bronchial hyperresponsiveness (BHR), (b) 42 nonasthmatic subjects with BHR in 1986, and (c) 44 controls without BHR. In 1986, FEV, expressed as percentage of predicted value, was found to be similar in the three groups (91%, 94%, and 99%, respectively).The increase in height during the observation period was found to be 5, 6, and 6 cm, respectively, in the three groups. However, at the second examination, in 1988, FEV, was found to be significantly reduced in both asthmatics (87%) and nonasthmatic subjects (85O/,) compared with the controls (103%). In 1988, 16 asthmatics (80%) and 24 (57%) nonasthmatic subjects were found to have BHR, whereas none of the controls were Address reprint requests to : Vibeke Backer, JAGTVEJ 200, DK-2100Copenhagen, Denmark.
331 Copyright 0 1992 by Marcel Dekker, Inc.
Backer and Ulrik
332
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
found to have BHR. A multiple regression analysis was used to determine the correlation between change in FEV, and potential factors of importance. The change in FEV, was highly correlated with the presence of BHR in 1986, however, no correlation was found between change in FEV, and change in bronchial responsiveness. In conclusion, nonasthmatic subjects with former BHR showed signs of airflow obstruction and less increase in lung function during growth irrespective of the change in level of bronchial responsiveness, which may suggest a risk for subsequent development of obstructive lung disease.
INTRODUCTION
BHR, asthma, and longitudinal changes in lung function is limited.
Measurement of nonspecific bronchial responsiveness is a widely used approach for the assessment of airway function in children (1-3). Bronchial hyperresponsiveness (BHR) is found in most subjects with asthma; however, BHR is also found in nonasthmatic subjects (4-6). Longitudinal changes in degree of bronchial responsiveness are well recognized in both asthmatic (7)and nonasthmatic subjects (8,9).Yet, asthmatic subjects rarely reach a normal degree of bronchial responsiveness, whereas nonasthmatic children found to have BHR often subsequently will obtain normal degree of bronchial responsiveness (8,9). Children with persistent BHR have been shown to have reduced expiratory flow rates and slower increase in lung function through childhood and adolescence than children with bronchial responsiveness within the normal range (10).These findings suggest an association between asymptomatic BHR, airflow obstruction and subsequent development of obstructive lung disease. However, this association may not be straightforward, because as found by Josephs et al. (ll), increased bronchial responsiveness seems to be the underlying mechanism in asthma, whereas airflow obstruction seems to be a n abnormality in the airways. At present it is unknown whether nonasthmatic children with BHR, who subsequently obtain a level of bronchial responsiveness within the normal range, experience normal development of ventilatory function. Furthermore, our knowledge about the relationship between
Thus, the purpose of the present study was to investigate longitudinal changes in lung function and bronchial responsiveness in children and adolescents with asthma and asymptomatic bronchial hyperresponsiveness. SUBJECTS
A random sample of 983 children and adolescents living in the area surrounding the University Hospital (Rigshospitalet) in Copenhagen was drawn from the civil registration list and invited to participate in a study concerning asthma in 1986. All invited subjects were born in the first week of each month, all were aged between 7 and 16 years, and all were Caucasian. Five-hundred and twenty-seven (54%)accepted the invitation and were examined (Fig. 1). The description of the nonresponders (n = 456) has been presented in a previous paper (12). Seventynine of the children and adolescents (16%) were found to have BHR (i.e., PC-20 FEVl histamine less than 8 mg/ml), of whom 28 (5%) subjects had asthma (12). All subjects with BHR (n = 79) and 62 consecutively selected control subjects were invited to participate in a re-examination study 18 months later, in 1988. One-hundred and six children and adolescents (75%),55 boys and 51 girls, were examined (Fig. 1). The protocol was evaluated and approved by the local ethical committee and informed consent was obtained from all participating children and their parents.
Lung Function in Bronchial Responsiveness
333
Entrv studv in 1986
-
histamine challenge test
I 495 had histamine
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
challenge test
1 79
with BH
Entrv studv in 1988
I 416 without 6H
1 I l-7 79
invited
62
re-examined
62
invited
k-J 44
re-examined
1I
rn 20
L
asthmatics
42
nonasthmatics
44
normal controls
I
Figure 1. A flow chart of recruitment and allocation of subjects at the two examinations in 1986 and 1988.
All participants were interviewed by one of the authors (VB), and asthma was defined according to Hopp et al. (13).The questions, used at both examinations, were as follows: (1)Have you ever had asthma diagnosed?, (2) Have you ever had wheezy or dry cough?, (3) Do you have attacks of shortness of breath with wheezing?, (4) Have you ever been hospitalized and/or been treated for asthma by a doctor?, (5) Have you ever received medication for your asthma?, (6)Did the medication help?, and (7) Do exertion, stress, cold air, damp weather, or allergen exposure (i.e., provocating factozs), cause you pulmonary symptoms? Definite asthma was defined as a positive response to questions 1to 6; probable
asthma as a positive response to questions 1 to 4, and either 5 or 6; and questionable asthma as a positive response t o any two of questions 1 to 3 and any one of questions 4 t o 7. The participating subjects were grouped as follows. Group 1. BHR Asthmatic Subjects (n = 20)
The mean age was 12.2 years (SD 2.51, serum IgE 255 kU/L (SD SOO), and 10 subjects (50%)had atopic disease, defined as a positive skin prick test (SPT).Sixteen subjects had definite asthma, none had probable asthma, and four subjects had questionable asthma. Six of the asthmatics had a family history of
Backer and Ulrik
334 asthma, six had a history of other allergic symptoms than asthma, whereas eight asthmatics had no family history of allergic diseases in first-degree relatives.
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
Group 2. (n = 42)
BHR+ Nonasthmatic Subjects
The mean age was 11.4 years (SD 2.4), serum IgE 85 kU/L (SD 138),and four subjects (10%)had atopic disease.All subjects had bronchial hyperresponsiveness in 1986,and no earlier or present symptoms defined as asthma. At the examination in 1988,eleven subjects (26%)reported respiratory symptoms, of whom four had occasional dry cough, six had occasional exercise-induced shortness of breath without wheezing, and one had experienced wheezing on one occasion, the remaining 31 subjects claimed no respiratory symptoms. Nine of the nonasthmatics had a family history of asthma, 10 had a history of other allergic symptoms than asthma, whereas 23 nonasthmatics had no family history of allergic diseases in first-degree relatives. Groups 3. Nonasthmatic Subjects (Controls) (n = 44)
The mean age was 13.7 years (SD 2.5), serum IgE 58 k U L (SD 136),and none had atopic disease. The 44 participating control subjects were consecutively invited from the group of subjects (n = 416),who had normal degree of bronchial responsiveness in 1986 and no earlier or present respiratory symptoms suggesting a diagnosis of asthma. Two subjects (5%)claimed occasional dry cough at the examination in 1988.No significant differences in degree of bronchial responsiveness were found between the participating control subjects, the invited subjects (n = 62)and the remaining subjects (n = 354).Fourteen of the controls had a family history af asthma, eight had a history of other allergic or allergy-like symptoms than asthma, whereas 22 controls had no family history of allergic diseases in first-degree relatives.
METHODS Study Design
All subjects were asked not to smoke for 2 hours before the tests and were asked to avoid antiasthmatic medication prior to the examination (14).In case of recent respiratory infection, challenge tests were postponed for at least 6 weeks. Spirometry and a histamine challenge test were performed at both examinations, in 1986 and 1988.In 1986,a skin prick test and measurement of total serum IgE were performed. Furthermore, in 1988, static lung volumes, diffusion capacity and airway resistance were measured. lung Function Tests
SPIROMETRY A maximal forced expiratory flow volume curve (MEF) was obtained with a pneumotachograph (Vitalograph@),and FEVl was obtained with a dry bell spirometer (Vitalograph). The highest of three technically acceptable readings of FEVl was recorded; and MEFZ5was read from the accepted curve of FEVI.
LUNGVOLUMES Total lung capacity (TLC),residual volume
(RV),functional residual capacity (FRC), and vital capacity (VC) were measured with the closed circuit helium dilution rebreathing technique (Godard Bell-spirometero ).
DIFFUSIONCAPACITY The diffusion capacity (TLco) was measured with the single breath technique (Morgan spirometer* ) using a nitrogen mixture containing 0.3% CO, 10% helium, and 20% oxygen. Predicted values of the measured parameters based on the height of the subjects were calculated according to Zapletal et al. (15).
Lung Function in Bronchial Responsiveness
335
Bronchial Histamine Challenge Test
DATA ANALYSIS
The bronchial histamine challenge tests were performed according to the protocol by Cockcroft and ceworkers (16).The bronchial responsiveness was expressed a s doseresponse slope (DRS) (i.e., the percentage reduction in FEVl per pmol of inhaled histamine) (17). Prior to expression in log scale, 1.2% decline in FEVl per pmol was added to each value to eliminate negative and zero values (17).
The differences in degree of BHR and results of lung function tests between groups were analyzed using one-way analysis of variance and, when appropriate, a twosample t-test was used. Furthermore, changes in bronchial responsiveness (A-DRS), lung function (A-FEV11 and height (A-Height) between the two examinations were analyzed using a paired t-test. Multiple regression analysis was used for analyzing a potential relationship between changes in FEVl and DRS and selected background variables. The nonsignificant variables were deleted by backward elimination t o determine those associated with the change in FEVl and DRS.
J Asthma Downloaded from informahealthcare.com by Mcgill University on 12/10/14 For personal use only.
Skin Prick Test and Total Serum IgE
A skin prick test with nine common aeroallergens was performed (by VB); the allergens used were: birch, grass, mugwort, horse, dog, cat, house-dust mite (Dermatophagoides pteronyssinus), and two molds (Alternaria iridis and Cladosporium herbarurn) (18).Blood samples were drawn and the total serum IgE was determined by paper radioimmunosorbent test (PRIST, Pharmacia, Copenhagen, Denmark).
RESULTS The FEV1, L, and %pred, was found to be similar in the three groups at the first examination in 1986 (Table 1). In 1988,
Table 1. Results (Mean +_ SD) in 106 Subjects Tested in 1986 and 1988 BHR+ ASTHMATIC GROUP 1 N = 20 In 1986 FEV, (L) FEUl (pred%) DKS-histamine Height (cm)
2.1 91.5 0.8 148.4
In 1988 FEV, (L) FEV, (%pred) DRS-histamine Height (cm)
2.2 87.2 0.7 153.3
A-f:EVl (L) 88-86 0.2 A-FEVlo,o 88-86 -4.3 A-DRS 88-86 -0.1 A-Height 88-86 5.0
+ + + + + +
+
+
+
+ + +
BHR+ NONASTHMATIC GROUP 2 N = 42
0.5 10.9 0.6 15.0
2.0 94.2 0.5 145.7
0.6 10.6 0.4 14.6
2.1 85.3 0.3 151.7
0.3 9.2a 0.7 3.4b
0.1 -8.9 -0.2 6.0
+
+
+
+ +
+
+ + +
+ + +
BHRCONTROLS GROUP 3 N = 44
0.6 12.6 0.4 13.8
2.6 98.5 0.2 155.5
0.7 10.9 0.2 13.1
3.0 102.9 0.1 161.9
0.2 11.3b 0.4b 3.6b
0.5 4.5 -0.1 6.4
+ +
+ +
+ +
+ + + +
+
+
1 vs. 2
2 vs. 3
1 vs. 3
ANOVA
0.8 11.9 0.1 14.3
NS NS p < 0.05 NS
NS NS p < 0.05 p < 0.01
NS NS p < 0.01 NS
8.5 2.7 27.2 5.2
0.9 11.2 0.1 13.8
NS NS
NS p < 0.001 p < 0.001 p < 0.01
NS
17.1 29.4 49.7 6.1
0.4 9.1 0.1 a 4.6b
p p
p
< 0,001 < 0.05 NS 0.05 NS NS
