Bronchial Clearance of DTPA Is Increased in Acute Asthma but Not in Chronic Asthma 1 - 3
PATRICIA LEMARCHAND, THIERRY CHINET, MARIE-ANNE COLLIGNON, GUILLERMO URZUA, LIONEL BARRITAULT, and GERARD J. HUCHON
Introduction
Epithelial damage of the airway mucosa is a feature of asthma, both in the interval state as wellas during acute attacks (1, 2). It has been suggested that bronchial epithelial damage and the related increased permeability, by allowing higher concentrations of inhaled material to reach irritant receptorsand airwaysmooth muscle, might cause bronchial hyperresponsiveness (3). Such a mechanism could explain enhanced bronchial hyperresponsiveness in normal subjects after mucosal injury induced by viral infections (4) or inhaled pollutants (5), or in asthmatics after antigen challenge (6). The most frequently used method of assessing in vivo epithelial permeability in humans has been the measurement of the clearance of aerosolized radiolabeled diethylenetriaminepentaacetate (DTPA), using inhaled particles with diameters less than 2 urn, so that most of the deposition occurs in terminal respiratory units. In stable asthmatics the epithelial clearance of DTPA (presumably from the alveoli) has been found to be normal and did not correlate with bronchial hyperresponsiveness (7). In an attempt to assess bronchial mucosal clearance rather than clearance from terminal respiratory units, Elwood and coworkers (8) used a radiolabeled aerosol with particles 6 urn in diameter to enhance deposition in bronchi and not in terminal respiratory units; they observed that the bronchial clearance of DTPA in subjects with stable asthma was similar to that of control subjects. Bennett and Ilowite (9) attempted to calculate the rate of DTPA transport across the bronchial epithelium by measuring the clearance of aerosolized 99ffiTc-DTPA and correcting the overall value for the amount removed by mucociliary clearance, which they evaluated by the clearance of 99ffiTc-Iabeled albumin; using this method, they found that after correcting for slower mucociliary clearance, 99ffiTc_DTPA clearance in stable asthmatics was increased (10). However,
SUMMARY To investigate bronchial permeability In asthma, we measured the bronchial clearance of 113mln-DTPA In seven asthmatics during and after an acute attack of asthma, seven asthmatics with chronic airflow limitation, and seven asthmatics without airflow limitation but with bronchial hyperresponslveneas to methacholine. We compared these resulta with those from seven normal subjects, seven patlenta with chronic bronchitis and bronchial Infection, and seven patlenta with emphysema. An aerosol of 113mln_DTPA was produced with a spinning disc to ensure a predominantly bronchial deposition of Inhaled particles (6.3 11m MMAD). Radioactivity over the chest was recorded with a gamma-camera for 10 min after the subject Inhaled the aerosol. central regions of Interest were selected, and the logarithm of the radioactivity was plotted against time; bronchial clearance of 113mln·DTPA was calculated as the negative slope of the regreaslon line. Clearance was substantially higher In asthmatics during their acute attacks than In all other groupa (p < 0.0001), and It decreased toward normal levels after recovery from the acute episode. The bronchial clear· ance of 113mln·DTPA In all other groups did not differ from normal. We conclude that the bronchial clearance of 113mln_DTPA Is Increased In asthmatics during attacks of asthma but In the stable state Is not related either to bronchlsl hyperresponslveneas or to airflow limitation. Our findings are best explained by an Increase In permeability of the bronchial mucosa of asthmatics during acute attacks. AM REV RESPIR DIS 1992; 145:147-152
because the mass median diameter of the 99ffiTc_DTPA aerosol was 2.0 urn and the studies were done in stable asthmatics, it is still not known whether there is a change in the bronchial clearance of DTPA during attacks of asthma. The purpose of our study was to evaluate the bronchial clearance of DTPA in asthmatics during and after attacks. For this reason, we used an aerosol of larger particles designed to assess bronchial rather than alveolar clearance. To clarify the relationship of the bronchial clearance of DTPA to bronchial hyperresponsiveness, to the effects of abnormal lung function, and to airflow limitation, we also studied groups of subjects with various chronic obstructive pulmonary diseases: stable asthma, chronic bronchitis, and emphysema. Methods The bronchial clearance of 113mln_DTPA and pulmonary function tests (PFT) were determined in each subject on the same day. Measurements of bronchial clearance were repeated 1 to 4 wk later in the asthmatics who had been studied during an acute attack. The study was approved by the Hospital Ethics Committee, and written consent was obtained from all subjects after the aims, procedures, and
side effects of the study had been explained to them.
Subjects Six groups of seven subjects each were studied. Patients included three groups of asthmatic subjects (defined below), all of whom fulfilled the American Thoracic Society criteria for asthma; in addition, we studied one group of sevensubjects with criteria for chronic bronchitis, and one group of seven subjects with criteria for emphysema (11). Seven healthy nonsmokers were studied as control subjects. Anthropometric data and results of PFT in each group are shown in table 1. Asthmatics All subjects with asthma were nonsmokers. Acute asthma: seven subjects had a history (Received in original form April 9, 1990 and in revised form June 28, 1991) I From the Universite de Paris Rene Descartes: Hopital Ambroise Pare, Boulogne, and Hopital Laennec, Paris, France. 2 Supported by grants from the Association pour l'Etude de la Respiration et de l'Environnement and from the Fonds Special des Comites Departementaux contre les Maladies Respiratoires et la
Tuberculose, 3 Correspondence and requests for reprints should be addressed to Gerard J. Huchon, M.D., Hopital Ambroise Pare, 9 AvenueCharles de Gaulle, F-92104 Boulogne cedex, France.
147
148
LEMARCHAND, CHINET, COLLIGNON, URZUA, BARRITAULT, AND HUCHON
TABLE 1 ANTHROPOMETRIC DATA AND PULMONARY FUNCTION TESTS IN STUDY GROUPS·
Control Asthma: Acute airflow limitation Chronic airflow limitation Without airflow limitation Chronic bronchitis Emphysema
Age
Sex
Weight
Height
(yr)
(MIF)
(kg)
(em)
FEV, (% pred)
FEV,/FVC (% pred)
29 ± 7
5/2
71 ± 12
175 ± 4
110 ± 13
109 ± 6
3/4
66 66 57 70 66
53 43 31 68 52
± 18 ± 14 ± 11 ± 12 ± 12
4/3 4/3 6/1 7/0
± 10 ± 9 ± 13
± 9 ± 8
169 166 169 170 172
± 10 ± 8
56 51 110 46 28
± 11 ± 4 ± 8
± 22 ± 16 ± 11 ± 13 ± 14
79 67 107 72 45
16 13 11 10 ± 16
± ± ± ±
FRC (% pred) 98 ± 25 123 108 113 115 186
± 31 ± 24 ± 19
± 27 ± 49
RV (% pred) 92 ± 41 130 129 115 129 217
± 62 ± 38 ± 32 ± 48 ±
rr
• Values are mean ± SO.
of an attack for less than 3 days, and had clinical symptoms of acute asthma at the time of the study. Their treatment and arterial blood gas values at the time of the first and the second studies are shown in table 2. Pulmonary function tests and measurement of DTPA bronchial clearance were performed during attack 10 min after inhalation of 200 !lg to 5 mg salbutamol. Chronic asthma: 14 clinically stable subjects had no recent history of respiratory infection and had not received inhaled or oral corticosteroids for at least 6 months. Among the subjects with stable asthma, seven had no airflow limitation at the time of the study but had bronchial hyperresponsiveness to inhaled methacholine; the seven other stable asthmatics had chronic airflow limitation partially reversible after inhalation of betas-agonist (mean increase in FEV" 32 ± 31070, mean ± SD). No subject with chronic asthma received inhaled beta.agonist within 4 h of the measurement of DTPA bronchial clearance.
Subjects with Chronic Bronchitis and Intercurrent Bronchial Infection Seven subjects with chronic bronchitis had clinical symptoms of acute bronchial infection at the time of the study, including cough and production of purulent sputum. They did not receive any treatment before the study. All had airflow limitation (table 1),which was not reversed after inhalation of betas-agonist (mean increase in FEV" 9 ± 3%). All were
past smokers (41 ± 33 pack-years), but all had stopped smoking for at least 3 months before the study.
Patients with Emphysema Seven subjects had emphysema: four subjects had PiZZ alpha.-antitrypsin deficiency, one had had a pulmonary biopsy that showed lesions of panlobular emphysema; moreover, all had evidence of bullae on chest roentgenograms and computed tomography, and of severe airflow limitation by spirometry. All but one were past smokers (42 ± 23 pack-years) but had stopped smoking for at least 3 months and were clinically stable without cough or purulent sputum. Measurement of DTPA Bronchial Clearance The bronchial clearance of solutes was assessed using 113mln-DTPA. To avoid the dissociation of the DTPA from its radiolabel, we used 113mln-DTPA, which has a low dissociation constant (12). 113mln_DTPA (MW, 504 daltons) was obtained from 13 mg of DTPA labeled with 20 mCi of 113mln from a 113Sn generator (Oris-Cis, Saclay, France) using 5 ml of 0.04 N hydrochloric acid. An aerosol of 113mln_DTPA was generated by a spinning disc usiag compressed air (13). To ensure a predominantly bronchial deposition of the aerosol, we used particles with a mass median diameter (MMAD) of 6.3 urn, and a geometric standard deviation (GSD) of 2.1,
TABLE 2 INDIVIDUAL TREATMENT AND ARTERIAL BLOOD GAS VALUES AT THE TIME OF THE FIRST STUDY AND OF THE SECOND STUDY IN PATIENTS WITH ACUTE ASTHMA Subject No.
First study Inhaled steroids Inhaled bata,-adrenergic drugs pso., mm Hg Paco" mm Hg Second study Oral sterOids/day (prednisolone) Inhaled steroids Inhaled bata,-adrenergic drugs Pao., mm Hg Paco., mm Hg • Not measured.
2
3
4
5
6
7
Yes Yes 59 33
Yes Yes 72 38
No Yes
No Yes
No Yes 53 50
Yes Yes 64 41
No Yes 58 46
50 mg No Yes 82
30 mg No Yes
20 mg No Yes
No Yes Yes
40 mg No Yes 70
No Yes Yes 75 39
30 mg No Yes
33
40
83 40
as measured by a Mercer Impactor (14). Subjects breathed the aerosol at tidal volume through a nonrebreathing T-piece system for about 8 min. After inhalation, the subjects rinsed their mouths and gargled with water to remove any radioaerosol deposited in the oropharynx and drank water to clear the esophagus. None of the subjects coughed during the 30-min observation period. Counts of radioactivity from the entire thorax in the anterior projection were acquired in 30-s frames by a gamma scintillation camera (pHO/Gamma HP3; Nuclear Chicago, Chicago, IL), during aerosolization and for 22 min thereafter. Counts were dynamically recorded on a matrix 64 x 64 through a computer (Scintigraphic Data Analyzer; HewlettPackard, Cupertino, CA). Both the entire lung fields and central airway regions of interest were selected as shown in figure 1; the maximal value of the counts in the central region of interest was about 2,000 counts/min. After correction for radionuclide decay, the logarithm of the counts in the central region was plotted against time, from the peak of radioactivity to the end of the experiment. The regression line of the counts in the central region of interest (log-activity curve) was calculated by the least-squares method for the 10min after the peak ofradioactivity; DTPA bronchial clearance was the negative slope of this line, expressed as a percentage decline of activity per minute (15).Central airway deposition was defined by the ratio of the radioactivity in the central region to the radioactivity in the entire lungs, calculated at the peak of radioactivity.
Pulmonary Function 'Tests and Bronchial Responsiveness FEV, and FVC were measured using a dryseal spirometer (VG2000;Ninjaart, Holland), the residual volume (RV),FRC, and TLC were measured using the helium dilution method. Results were expressed as a percentage of predicted values from the European Coal and Steel Community Survey (16). Bronchial responsiveness was assessed by inhaled methacholine (17) in control subjects and in stable asthmatics without airflow limitation, just after the measurement oflJfPA bronchial clearance. Methacholine (1%) was prepared by diluting stock solution in distilled water. A metered nebulizer dosimeter (Mefar) deliv-
BRONCHIAL CLEARANCE OF OTPA IN ASTHMA
149
Results
Fig. 1. Deposition pattern of 11.mln· DTPA aerosol in entire lungs and central airway regions of interest.
ered the methacholine from a Mefar ampule by means of an air compressor (driving pressure, 1.5 kg/cm). Inhalation time was set at 1.1 s, every inhalation delivering 150 I1g of methacholine. Phosphate-buffered saline was inhaled first, followed at 5-min intervals by cumulative doses of methacholine (150, 300, 600, and 1,200I1g), delivered with increasing number of inhalations. The response was measured as the change in FEV 1 from the lowest postsaline value, 0.5 and 1.5min after each inhalation. Inhalations were discontinued when FEV 1 had fallen by 20070 or more in asthmatics, or when a cumulative dose of 2,250 I1g of methacholine had been aerosolized in control subjects.Resultswereexpressed as challenge dose of methacholine necessary to cause a 20% fall in FEV1 (PD 20 ) , obtained from the log dose-response curve by linear interpolation of the last two points. The PD 20 valuesfor three control subjectswereobtained by extrapolation of the last two points of the dose response curve, and four control subjects were nonresponders to methacholine. Effect of Acute Increase in Lung Volume on DTPA Bronchial Clearance Five healthy nonsmokers participated in an additional study of the effect of acute increase in lung volume on DTPAbronchial clearance. Increase in lung volume was achieved using continuous positive airway pressure (CPAP). CPAP was provided by immersion of the expiratory tubing in a vertical cylinder containing water. Expiratory pressure was set at 12 em H20. To determine the degree of CPAPinduced hyperinflation, lung volumes were measured in the sitting position during relaxed and CPAP breathing. For the measurement of DTPAbronchial clearanceat high lung volume, CPAP wasapplied after the end of aerosolization and continued during the whole period of counting.
Statistical Analysis Resultsare expressedas mean ± SD.Betweengroups comparisons weredone by analysis of variance and comparison between the mean values by Student-Newman-Keul tests when the F statistic was significant; repeated values of DTPA bronchial clearance werecompared usingStudent's paired ttest; least squareswere used to calculate linear correlation coefficients. We considered a p value of < 0.05 as significant (18).
Results of central airway deposition and of the DTPA bronchial clearance in each group are shown in table 3; figure 2 shows individual values of the DTPA bronchial clearance. There was no difference in the deposition rate of the labeled aerosol among the groups, but DTPA bronchial clearance was higher in all seven subjects during their attacks of asthma than in subjects with symptom-free asthma, with or without airflow limitation, in subjects with chronic bronchitis, or in subjects with emphysema (p < 0.0001). There was no correlation between DTPA bronchial clearance and FEV 1 (figure 3), RV, and FRC in asthmatics and in nonasthmatics. Values for PDzo were 2.9 ± 1.1 mg in the three control subjects who were reactive to methacholine, and 0.7 ± 0.6 mg in asthmatics without airflow limitation. There was no correlation between DTPA bronchial clearance and PD2 0 in asthmatics (p = 0.84). DTPA bronchial clearance and FEV 1 during the initial evaluation in subjects with acute asthma, and 1 to 4 wk later, are shown in figure 4. All subjects but one received steroids orally between the two measurements, and five subjects were still receiving steroids orally at the time of the second study (table 2). DTPA bronchial clearance decreased significantly
TABLE 3 EFFECT OF CPAP ON FRC AND BRONCHIAL CLEARANCE OF 11.mln·DTPA (DTPA BRONCHIAL CLEARANCE)'
FRC, ml DTPA bronchial clearance,
%/min
NO CPAP
CPAP
2,588 ± 427
3,782 ± 1,025
0.81 ± 0.39
0.56 ± 0.18
p Value
p
< 0.03 NS
• Results are expressed as mean ± SO. Comparisons were made using Studenfs paired t test.
• •
• •• Fig. 2. Individual values of the bronchiaI clearance of 11.mln·DTPA (DTPA bronchial clearance) in the stUdy groups.
•
• • I •I • ..... airflcwlimitation I
""""01,
• • ••• I """"~
•
•
,I,
I
I
•••
•
• I
-.out
airflowlimitation airflow limitation I I chroniC as1hmatics bronchitis
emphysema
150
LEMARCHAND, CHINET, COLLIGNON, URZUA, BARRITAULT, AND HUCHON
140
•
120
~ >
.O~
100
8
80
•
•••
0 0
0
W
u,
0
• • • o •••
60
0
• 0
40
Ie
20
•
0
0
0 0
•
Fig. 3. Individual values of the clearance of 113mln-DTPA (DTPA bronchial clearance) and of FEV, (open circles = asthmatics; closed circles = nonasthmatics).
0
0
•
o 0
•
0
0 0
,
,
,
3
4
6
SG-In-DTPA ('Iojmin)
~ -
6-
120
5
100
4
80
C
0~
;?
W
40
2
OJ
20
o
evaluation # 1
evaluation #
2
o
evaluation #
1
evaluation #
2
Fig. 4. Individual values of the bronchial clearance of 113mln_DTPA (DTPA bronchial clearance) and of FEV, in SUbjectsduring acute attacks of asthma during the initial evaluation (Evaluation 1) and 1 to 4 wk later (Evaluation 2). Each subject is designated by a symbol.
(p < 0.01), but it remained increasedcompared with that in control subjects (p < 0.05). FEV 1 increased significantly between the two measurements (p < 0.05). There wasno correlation between the decrease in DTPA bronchial clearance and the increase in FEV 1 • The effect of CPAP on FRC and DTPA bronchial clearance in five normal subjects is shown in table 3.
Discussion
Compared with control subjects and with stable asthmatics, DTPA bronchial clearance was increased in asthmatics during acute attacks; furthermore, clearance values decreased within 4 wk after treatment of the attacks in six of seven asthmatics, but remained increased compared with values in the control groups. The increase in DTPA bronchial clearance during attacks did not correlate with the severity ofairflow limitation at the time, as measured by FEV 1 • Bronchial hyper-
responsiveness, present in asthmatic subjects without airflow limitation, was not associated with an increase in DTPA bronchial clearance. Several potential technical problems might have caused increases in the measured rates of clearance using this technique, and thus might lead to a misinterpretation of the results.
Dissociation of the Compound The results of several studies of healthy smokers have shown that the alveolar clearance of 99mTc-DTPA was increased (19); however, part of this increase may be explained by dissociation of the 99mTc-DTPA complex, leading to the formation of compounds with fast epithelial transfer (20). To avoid this potential problem, we used 113mIn, which is more tightly bound to DTPA than is 99mTc (21). Therefore, dissociation of 113mIn from DTPA cannot explain the increase in DTPA bronchial clearance observed in our study.
Deposition Site The site of deposition of the labeled DTPAin the lung wascrucial because this determined whether weassessedbronchial or respiratory clearanceor both. Visual evaluation of the scintiphoto demonstrated a pattern of predominantly central deposition (figure 1), similar to that obtained by Yeates and coworkers (22), who used an aerosol with similarMMAD. Some deposition occurred in the periphery of the lungs, as shown by the ratio of counts from the central regions to counts from the entire lungs (table 4), but this deposition probably mainly occurred in airways and to a lesser extent in terminal respiratory units because the MMAD of the aerosol was 6.3 urn, Furthermore, to ensure that we measured the DTPA clearance in the airways and not in the terminal respiratory units, we calculated DTPA bronchial clearance from the decreasing radioactivity over the central regions of interest and not from the increasing radioactivity in blood, which comes from both airways and terminal respiratory units. In addition, DTPA bronchial clearance was not affected by CPAP-induced lung hyperinflation; because lung hyperinflation markedly increases DTPA respiratory clearance (21), the method we used to measure DTPA clearance reflects predominantly bronchial clearance, with a minimal participation of respiratory clearance. Finally, the increased DTPA bronchial clearance found in subjects with acute asthma was probably not due to differences in deposition of the aerosol among the groups because the ratio of the radioactivity in central regions to the radioactivity in the entire lungs was similar in all groups (table 3). Binding to Mucus Glycoproteins Cheema and coworkers (23) suggested that the absence of an increase in airway mucosal clearance in stable asthmatics, as compared with that in normal subjects, might be due to binding of 99mTc_ DTPA to glycoproteins in the mucus. However, we have shown that, compared with control subjects, DTPA bronchial clearance was four times faster in subjects with acute attacks of asthma, and that clearance in subjects with chronic bronchitis and intercurrent bronchial infection was similar to that in control subjects. Because subjects with bronchial infections had more mucus in their bronchi than did control subjects, even if DTPA binds to mucus, this does not appear to affect the values of DTPA bronchial clearance.
151
BRONCHIAL CLEARANCE OF OTPA IN ASTHMA
TABLE 4 BRONCHIAL DEPOSITION AND BRONCHIAL CLEARANCE OF 113mln_DTPA (DTPA BRONCHIAL CLEARANCE) IN STUDY GROUPS' Central Airways Deposition (0/0 total deposition) Control Asthma Acute airflow limitation Chronic airflow limitation Without airflow limitation Chronic bronchitis Emphysema
DTPA Bronchial Clearance (%/min)
42.5 ± 7.1
0.9 ± 0.6
43.9 39.2 47.1 50.4 43.5
3.9 0.9 0.9 0.8 0.7
± ± ± ± ±
7.4 10.1 14.7 16.9 10.2
± 1.4 ± 0.5 ± 0.7 ± 0.5 ± 0.5
• Values are mean ± SD.
Mucociliary Clearance Two potential routes of elimination may account for DTPA bronchial clearance: transepithelial transfer and mucociliary clearance. As mentioned previously, Bennett and Ilowite (9) showed by dynamic studies of the deposition scan that mucociliary clearance of DTPA may be a significant component of total DTPA clearance from the airways; in addition, they calculated the rate of 99mTc_DTPA transport across bronchial epithelium by correcting for the removal of DTPA by the mucociliary clearance, which was evaluated with 99mTc-Iabeled albumin. These studies rely on the assumption that 99mTc_DTPA, a small compound with a molecular weight of 492 daltons, has the same mucociliary clearance as human serum albumin, which has a much higher molecular weight of 66,000 daltons and different physicochemical properties. In companion studies, Ilowite and coworkers (10)used a polydispersed aerosol with a MMAD of 2.0 J.1m, which therefore was likely to deposit mainly in the peripheral regions of the lungs; to enhance central deposition, they modified the pattern of ventilation of their subjects, but they included the whole right lung field in their analysis, a method that presumably measured a combination of both respiratory and bronchial clearances of DTPA. Because Ilowite and coworkers (10) found a normal total DTPA clearance in stable asthma, which included a component of mucociliary removal, and because our results are similar to theirs, some mucociliary transport of DTPA may have occurred during our measurements of DTPA bronchial clearance. If so, the increase in DTPA bronchial clearance during attacks of asthma could conceivably have been due to a marked increase in DTPA mucociliary clearance. However, the results of many studies have shown decreased mucociliary clearance in stable asthma, even during remission (9, 10,
24, 25). To our knowledge, mucociliary clearance has not yet been studied during acute asthmatic attacks in humans, but after bronchial challenge with specific antigen (ragweed), tracheal mucus transport rate decreased to 720/0 of baseline (26). It is also possible that mucociliaryclearance could have been increased in our asthmatics by the betas-agonist drugs they were receiving. However, the effect of beta.-agonists on mucociliary clearance has usually been measured in stable asthmatics and did not exceed a 100% increase from a baseline of 10 to 20% of normal control values (24), in contrast with the 4000/0 increase in DTPA bronchial clearance documented in our subjects. Furthermore, a recent in vivo study in humans failed to find an increase in mucociliary clearance with inhaled betaj-agonists (27). Therefore, we believe it is unlikely that the entire increase in DTPA bronchial clearance observed in our asthmatics during their acute attacks can be explained by an increase in mucociliary clearance, although this may have contributed to some extent to the observed values.
Age The subjects in all the groups except the asthmatics without airflow limitation were older than the control subjects (table 1). However, only the asthmatics with acute airflow limitation had increased DTPA bronchial clearance. Therefore, it is unlikely that differences in the ages of our subjects could explain the increased bronchial clearance in asthmatics with acute airflow limitation.
Airflow Limitation and Hyperinflation To determine whether an increase in DTPA bronchial clearance could be attributable to differences in airflow limitation or hyperinflation, we measured clearance in asthmatics with chronic air-
flow limitation, in subjects with chronic bronchitis, and in subjects with emphysema who had equal or lower values of FEV I and FEVl/FVC and more severe hyperinflation (increased residual volume) than did patients with acute attacks of asthma (table 1). Because DTPA bronchial clearance was increased only in asthmatics during acute attacks, the increase was unlikely to be due either to airflow limitation or to chronic hyperinflation. In addition, we ruled out an effect of acute hyperinflation, which occurs during asthma attacks, on DTPA bronchial clearance: no increase in DTPA bronchial clearance was observed during CPAP breathing in five control subjects, which indicates that acute increase in lung volume does not increase DTPA bronchial clearance. After considering the potential technical problems, discussed previously, we believe that the main results of our investigations - that bronchial clearance is increased in asthmatics during acute attacks and not in the other groups which were studied - are valid and, moreover, that the observations can be explained by present concepts about the pathophysiology of asthma, especially the role of mucosal permeability. An increase of bronchial clearance of aerosolized 2-J.1mdiameter histamine has been shown in allergic monkeys after inhalation of a specific antigen (28), and to horseradish peroxidase in allergic guinea pigs after specific challenge (29). It has been postulated that this increase is mediated by increased paracellular as well as transcellular movement of large solutes across the epithelial barrier (29); however, the exact mechanisms are unknown. Our data suggest that in humans the increase in DTPA bronchial clearance is related to events that occur in the airways during attacks of asthma. The decrease of DTPA bronchial clearance after treatment, including the administration of corticosteroids, shows that transbronchial movement of solutes decreases toward normal as asthma remits, and suggests that the increase in DTPA bronchial clearance during acute episodes could be linked to inflammation of the airway mucosa. Inflammatory stimuli induce an increase in permeability of the tracheobronchial microvessels and epithelium in asthma (30), and DTPA bronchial clearance may increase in concert with the increase of vascular permeability during attacks. All subjects with acute asthma inhaled betas-agonists before the study, which might modify vascular permeability and thus affect DTPA bronchial clear-
152
LEMARCHAND, CHINET, COLLIGNON, URZUA. BARRITAULT, AND HUCHON
ance. However, studies in vitro in humans and in vivo in guinea pigs have shown that these drugs reduce plasma leakage from the tracheobronchial microcirculation (30) and thus act in the opposite direction to the increase noted in our asthmatics. Although our method of measuring DTPA bronchial clearance cannot determine if a mild degree of residual airway inflammation and epithelial damage persists in the interval state, the present data suggest that abnormalities in bronchial clearance of DTPA exist in some asthmatics, particularly during acute attacks. References 1. Laitinen LA, Heino M, Laitenen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 2. Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma: an ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989; 140:1745-53. 3. Hogg JC. Bronchial mucosal permeability and its relationship to airways hyperreactivity. J Allergy Clin Immunol 1981; 67:421-5. 4. Busse WW. Respiratory infections and bronchial hyperreactivity. J Allergy Clin Immunol1988; 81:770-5. 5. Sheppard D. Mechanisms of acute increases in airway responsiveness caused by environmental chemicals. J Allergy Clin Immuno11988; 81:128-32. 6. O'Byrne PM. Allergen-induced airway responsiveness. J Allergy Clin Immunol1988; 81:119-27. 7. O'Byrne PM, Dolovich M, Dirks R, Roberts
RS, Newhouse MT. Lung epithelial permeability: relation to nonspecific airway responsiveness. J Appl Physiol 1984; 57:77-84. 8. Elwood RK, Kennedy S, Belzberg A, Hogg JC, Pare PD. Respiratory mucosal permeability in asthma. Am Rev Respir Dis 1983; 128:523-7. 9. Bennett WD, Ilowite JS. Dual pathway clearance of 99mTc_DTPA from the bronchial mucosa. Am Rev Respir Dis 1989; 139:1132-8. 10. Ilowite JS, Bennett WD, Sheetz MS, Oroth ML, Nierman DM. Permeability of the bronchial mucosa to 99mTc_DTPA in asthma. Am Rev Respir Dis 1989; 139:1139-43. 11. American Thoracic Society. Chronic bronchitis, asthma, and pulmonary emphysema. Am Rev Respir Dis 1962; 85:762-9. 12. Moerlein SM, Welch MJ. The chemistry of gallium and indium as related to radiopharmaceutical production. Int J Nucl Med Bioi 1981; 8: 277-87. 13. May KR. An improved spinning top homogeneous spray apparatus. J Appl Phys 1949;20: 932-8. 14. Mercer TT, Tillery MI, Newton OJ. A multistage low flow rate cascade impactor. Aerosol Sci 1970; 1:9-15. 15. Dusser OJ, Minty BD, Collignon MA, Hinge 0, Barritault LG, H uchon OJ. Regional respiratory clearance of aerosolized ••mTc-DTPA: posture and smoking effects. J Appl Physiol 1986; 60: 2000-6. 16. Standardized lung function testing. Interim report of the working party "standardization of lung function tests" of the European Community for Coal and Steel. Luxembourg: European Community for Coal and Steel, 1981. 17. Eiser NM, Kerrebijn KF, Quanjer PH. Guidelines for standardization of bronchial challenges with (nonspecific) bronchoconstricting agents. Bull Eur Physiopathol Respir 1983; 19:495-514. 18. Zar JM. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall, 1974. 19. Jones JO, Minty BD, Lawler P, Hulands 0,
Crawley JCW, Veall N. Increased alveolar epithelial permeability in cigarette smokers. Lancet 1980; 1:66-8. 20. Eckelman WC, Volkert WA. In vivo chemistry of 99mTc-chelates. Int J Appl Radiat Isot 1982; 33:945-51. 21. Huchon OJ. Respiratory clearance of solutes. Bull Eur Physiopathol Respir 1986; 22:495-510. 22. Yeates DB, Aspin N, Levison H, Jones MT, Bryan AC. Mucociliary tracheal transport rates in man. J Appl Physiol 1975; 39:487-95. 23. Cheema MS, Oroth S, Marriott C. Binding and diffusion characteristics of I4C-EDTA and 99mTc_DTPA in respiratory tract mucus glycoprotein from patients with chronic bronchitis. Thorax 1988; 43:669-73. 24. Wanner A. Clinical aspects of mucociliary transport. Am Rev Respir Dis 1977; 116:73-126. 25. Pavia D, Bateman JRM, Sheahan NF, Agnew JE, Clarke SW. Tracheobronchial mucociliary clearance in asthma: impairment during remission. Thorax 1985; 40:171-5. 26. Mezey RJ, Cohn MA, Fernandez RJ, Januszkiewicz AJ, Wanner A. Mucociliary transport in allergic patients with antigen -induced bronchospasm. Am Rev Respir Dis 1978; 118:677-84. 27. Isawa T, Teshima T, Hirano T, et al. Does a betas-stimulator really facilitate mucociliary transport? A study with Procaterol. Am Rev Respir Dis 1990; 141:715-20. 28. Boucher RC, Pare PO, Hogg JC. Relationship between airway hyperreactivity and hyperpermeability in ascaris-sensitive monkeys. J Allergy Clin Immunol 1979; 64:197-201. 29. Ranga V, Powers MA, Padilla M, Strope OL, Fowler L, Kleinerman J. Effect of allergic bronchoconstriction on airways epithelial permeability to large polar solutes in the guinea pig. Am Rev Respir Dis 1983; 128:1065-70. 30. Persson CO. Plasma exudation and asthma. Lung 1988; 166:1-23.
PATRICIA LEMARCHAND, THIERRY CHINET, MARIE-ANNE COLLIGNON, GUILLERMO URZUA, LIONEL BARRITAULT, and GERARD J. HUCHON
Introduction
Epithelial damage of the airway mucosa is a feature of asthma, both in the interval state as wellas during acute attacks (1, 2). It has been suggested that bronchial epithelial damage and the related increased permeability, by allowing higher concentrations of inhaled material to reach irritant receptorsand airwaysmooth muscle, might cause bronchial hyperresponsiveness (3). Such a mechanism could explain enhanced bronchial hyperresponsiveness in normal subjects after mucosal injury induced by viral infections (4) or inhaled pollutants (5), or in asthmatics after antigen challenge (6). The most frequently used method of assessing in vivo epithelial permeability in humans has been the measurement of the clearance of aerosolized radiolabeled diethylenetriaminepentaacetate (DTPA), using inhaled particles with diameters less than 2 urn, so that most of the deposition occurs in terminal respiratory units. In stable asthmatics the epithelial clearance of DTPA (presumably from the alveoli) has been found to be normal and did not correlate with bronchial hyperresponsiveness (7). In an attempt to assess bronchial mucosal clearance rather than clearance from terminal respiratory units, Elwood and coworkers (8) used a radiolabeled aerosol with particles 6 urn in diameter to enhance deposition in bronchi and not in terminal respiratory units; they observed that the bronchial clearance of DTPA in subjects with stable asthma was similar to that of control subjects. Bennett and Ilowite (9) attempted to calculate the rate of DTPA transport across the bronchial epithelium by measuring the clearance of aerosolized 99ffiTc-DTPA and correcting the overall value for the amount removed by mucociliary clearance, which they evaluated by the clearance of 99ffiTc-Iabeled albumin; using this method, they found that after correcting for slower mucociliary clearance, 99ffiTc_DTPA clearance in stable asthmatics was increased (10). However,
SUMMARY To investigate bronchial permeability In asthma, we measured the bronchial clearance of 113mln-DTPA In seven asthmatics during and after an acute attack of asthma, seven asthmatics with chronic airflow limitation, and seven asthmatics without airflow limitation but with bronchial hyperresponslveneas to methacholine. We compared these resulta with those from seven normal subjects, seven patlenta with chronic bronchitis and bronchial Infection, and seven patlenta with emphysema. An aerosol of 113mln_DTPA was produced with a spinning disc to ensure a predominantly bronchial deposition of Inhaled particles (6.3 11m MMAD). Radioactivity over the chest was recorded with a gamma-camera for 10 min after the subject Inhaled the aerosol. central regions of Interest were selected, and the logarithm of the radioactivity was plotted against time; bronchial clearance of 113mln·DTPA was calculated as the negative slope of the regreaslon line. Clearance was substantially higher In asthmatics during their acute attacks than In all other groupa (p < 0.0001), and It decreased toward normal levels after recovery from the acute episode. The bronchial clear· ance of 113mln·DTPA In all other groups did not differ from normal. We conclude that the bronchial clearance of 113mln_DTPA Is Increased In asthmatics during attacks of asthma but In the stable state Is not related either to bronchlsl hyperresponslveneas or to airflow limitation. Our findings are best explained by an Increase In permeability of the bronchial mucosa of asthmatics during acute attacks. AM REV RESPIR DIS 1992; 145:147-152
because the mass median diameter of the 99ffiTc_DTPA aerosol was 2.0 urn and the studies were done in stable asthmatics, it is still not known whether there is a change in the bronchial clearance of DTPA during attacks of asthma. The purpose of our study was to evaluate the bronchial clearance of DTPA in asthmatics during and after attacks. For this reason, we used an aerosol of larger particles designed to assess bronchial rather than alveolar clearance. To clarify the relationship of the bronchial clearance of DTPA to bronchial hyperresponsiveness, to the effects of abnormal lung function, and to airflow limitation, we also studied groups of subjects with various chronic obstructive pulmonary diseases: stable asthma, chronic bronchitis, and emphysema. Methods The bronchial clearance of 113mln_DTPA and pulmonary function tests (PFT) were determined in each subject on the same day. Measurements of bronchial clearance were repeated 1 to 4 wk later in the asthmatics who had been studied during an acute attack. The study was approved by the Hospital Ethics Committee, and written consent was obtained from all subjects after the aims, procedures, and
side effects of the study had been explained to them.
Subjects Six groups of seven subjects each were studied. Patients included three groups of asthmatic subjects (defined below), all of whom fulfilled the American Thoracic Society criteria for asthma; in addition, we studied one group of sevensubjects with criteria for chronic bronchitis, and one group of seven subjects with criteria for emphysema (11). Seven healthy nonsmokers were studied as control subjects. Anthropometric data and results of PFT in each group are shown in table 1. Asthmatics All subjects with asthma were nonsmokers. Acute asthma: seven subjects had a history (Received in original form April 9, 1990 and in revised form June 28, 1991) I From the Universite de Paris Rene Descartes: Hopital Ambroise Pare, Boulogne, and Hopital Laennec, Paris, France. 2 Supported by grants from the Association pour l'Etude de la Respiration et de l'Environnement and from the Fonds Special des Comites Departementaux contre les Maladies Respiratoires et la
Tuberculose, 3 Correspondence and requests for reprints should be addressed to Gerard J. Huchon, M.D., Hopital Ambroise Pare, 9 AvenueCharles de Gaulle, F-92104 Boulogne cedex, France.
147
148
LEMARCHAND, CHINET, COLLIGNON, URZUA, BARRITAULT, AND HUCHON
TABLE 1 ANTHROPOMETRIC DATA AND PULMONARY FUNCTION TESTS IN STUDY GROUPS·
Control Asthma: Acute airflow limitation Chronic airflow limitation Without airflow limitation Chronic bronchitis Emphysema
Age
Sex
Weight
Height
(yr)
(MIF)
(kg)
(em)
FEV, (% pred)
FEV,/FVC (% pred)
29 ± 7
5/2
71 ± 12
175 ± 4
110 ± 13
109 ± 6
3/4
66 66 57 70 66
53 43 31 68 52
± 18 ± 14 ± 11 ± 12 ± 12
4/3 4/3 6/1 7/0
± 10 ± 9 ± 13
± 9 ± 8
169 166 169 170 172
± 10 ± 8
56 51 110 46 28
± 11 ± 4 ± 8
± 22 ± 16 ± 11 ± 13 ± 14
79 67 107 72 45
16 13 11 10 ± 16
± ± ± ±
FRC (% pred) 98 ± 25 123 108 113 115 186
± 31 ± 24 ± 19
± 27 ± 49
RV (% pred) 92 ± 41 130 129 115 129 217
± 62 ± 38 ± 32 ± 48 ±
rr
• Values are mean ± SO.
of an attack for less than 3 days, and had clinical symptoms of acute asthma at the time of the study. Their treatment and arterial blood gas values at the time of the first and the second studies are shown in table 2. Pulmonary function tests and measurement of DTPA bronchial clearance were performed during attack 10 min after inhalation of 200 !lg to 5 mg salbutamol. Chronic asthma: 14 clinically stable subjects had no recent history of respiratory infection and had not received inhaled or oral corticosteroids for at least 6 months. Among the subjects with stable asthma, seven had no airflow limitation at the time of the study but had bronchial hyperresponsiveness to inhaled methacholine; the seven other stable asthmatics had chronic airflow limitation partially reversible after inhalation of betas-agonist (mean increase in FEV" 32 ± 31070, mean ± SD). No subject with chronic asthma received inhaled beta.agonist within 4 h of the measurement of DTPA bronchial clearance.
Subjects with Chronic Bronchitis and Intercurrent Bronchial Infection Seven subjects with chronic bronchitis had clinical symptoms of acute bronchial infection at the time of the study, including cough and production of purulent sputum. They did not receive any treatment before the study. All had airflow limitation (table 1),which was not reversed after inhalation of betas-agonist (mean increase in FEV" 9 ± 3%). All were
past smokers (41 ± 33 pack-years), but all had stopped smoking for at least 3 months before the study.
Patients with Emphysema Seven subjects had emphysema: four subjects had PiZZ alpha.-antitrypsin deficiency, one had had a pulmonary biopsy that showed lesions of panlobular emphysema; moreover, all had evidence of bullae on chest roentgenograms and computed tomography, and of severe airflow limitation by spirometry. All but one were past smokers (42 ± 23 pack-years) but had stopped smoking for at least 3 months and were clinically stable without cough or purulent sputum. Measurement of DTPA Bronchial Clearance The bronchial clearance of solutes was assessed using 113mln-DTPA. To avoid the dissociation of the DTPA from its radiolabel, we used 113mln-DTPA, which has a low dissociation constant (12). 113mln_DTPA (MW, 504 daltons) was obtained from 13 mg of DTPA labeled with 20 mCi of 113mln from a 113Sn generator (Oris-Cis, Saclay, France) using 5 ml of 0.04 N hydrochloric acid. An aerosol of 113mln_DTPA was generated by a spinning disc usiag compressed air (13). To ensure a predominantly bronchial deposition of the aerosol, we used particles with a mass median diameter (MMAD) of 6.3 urn, and a geometric standard deviation (GSD) of 2.1,
TABLE 2 INDIVIDUAL TREATMENT AND ARTERIAL BLOOD GAS VALUES AT THE TIME OF THE FIRST STUDY AND OF THE SECOND STUDY IN PATIENTS WITH ACUTE ASTHMA Subject No.
First study Inhaled steroids Inhaled bata,-adrenergic drugs pso., mm Hg Paco" mm Hg Second study Oral sterOids/day (prednisolone) Inhaled steroids Inhaled bata,-adrenergic drugs Pao., mm Hg Paco., mm Hg • Not measured.
2
3
4
5
6
7
Yes Yes 59 33
Yes Yes 72 38
No Yes
No Yes
No Yes 53 50
Yes Yes 64 41
No Yes 58 46
50 mg No Yes 82
30 mg No Yes
20 mg No Yes
No Yes Yes
40 mg No Yes 70
No Yes Yes 75 39
30 mg No Yes
33
40
83 40
as measured by a Mercer Impactor (14). Subjects breathed the aerosol at tidal volume through a nonrebreathing T-piece system for about 8 min. After inhalation, the subjects rinsed their mouths and gargled with water to remove any radioaerosol deposited in the oropharynx and drank water to clear the esophagus. None of the subjects coughed during the 30-min observation period. Counts of radioactivity from the entire thorax in the anterior projection were acquired in 30-s frames by a gamma scintillation camera (pHO/Gamma HP3; Nuclear Chicago, Chicago, IL), during aerosolization and for 22 min thereafter. Counts were dynamically recorded on a matrix 64 x 64 through a computer (Scintigraphic Data Analyzer; HewlettPackard, Cupertino, CA). Both the entire lung fields and central airway regions of interest were selected as shown in figure 1; the maximal value of the counts in the central region of interest was about 2,000 counts/min. After correction for radionuclide decay, the logarithm of the counts in the central region was plotted against time, from the peak of radioactivity to the end of the experiment. The regression line of the counts in the central region of interest (log-activity curve) was calculated by the least-squares method for the 10min after the peak ofradioactivity; DTPA bronchial clearance was the negative slope of this line, expressed as a percentage decline of activity per minute (15).Central airway deposition was defined by the ratio of the radioactivity in the central region to the radioactivity in the entire lungs, calculated at the peak of radioactivity.
Pulmonary Function 'Tests and Bronchial Responsiveness FEV, and FVC were measured using a dryseal spirometer (VG2000;Ninjaart, Holland), the residual volume (RV),FRC, and TLC were measured using the helium dilution method. Results were expressed as a percentage of predicted values from the European Coal and Steel Community Survey (16). Bronchial responsiveness was assessed by inhaled methacholine (17) in control subjects and in stable asthmatics without airflow limitation, just after the measurement oflJfPA bronchial clearance. Methacholine (1%) was prepared by diluting stock solution in distilled water. A metered nebulizer dosimeter (Mefar) deliv-
BRONCHIAL CLEARANCE OF OTPA IN ASTHMA
149
Results
Fig. 1. Deposition pattern of 11.mln· DTPA aerosol in entire lungs and central airway regions of interest.
ered the methacholine from a Mefar ampule by means of an air compressor (driving pressure, 1.5 kg/cm). Inhalation time was set at 1.1 s, every inhalation delivering 150 I1g of methacholine. Phosphate-buffered saline was inhaled first, followed at 5-min intervals by cumulative doses of methacholine (150, 300, 600, and 1,200I1g), delivered with increasing number of inhalations. The response was measured as the change in FEV 1 from the lowest postsaline value, 0.5 and 1.5min after each inhalation. Inhalations were discontinued when FEV 1 had fallen by 20070 or more in asthmatics, or when a cumulative dose of 2,250 I1g of methacholine had been aerosolized in control subjects.Resultswereexpressed as challenge dose of methacholine necessary to cause a 20% fall in FEV1 (PD 20 ) , obtained from the log dose-response curve by linear interpolation of the last two points. The PD 20 valuesfor three control subjectswereobtained by extrapolation of the last two points of the dose response curve, and four control subjects were nonresponders to methacholine. Effect of Acute Increase in Lung Volume on DTPA Bronchial Clearance Five healthy nonsmokers participated in an additional study of the effect of acute increase in lung volume on DTPAbronchial clearance. Increase in lung volume was achieved using continuous positive airway pressure (CPAP). CPAP was provided by immersion of the expiratory tubing in a vertical cylinder containing water. Expiratory pressure was set at 12 em H20. To determine the degree of CPAPinduced hyperinflation, lung volumes were measured in the sitting position during relaxed and CPAP breathing. For the measurement of DTPAbronchial clearanceat high lung volume, CPAP wasapplied after the end of aerosolization and continued during the whole period of counting.
Statistical Analysis Resultsare expressedas mean ± SD.Betweengroups comparisons weredone by analysis of variance and comparison between the mean values by Student-Newman-Keul tests when the F statistic was significant; repeated values of DTPA bronchial clearance werecompared usingStudent's paired ttest; least squareswere used to calculate linear correlation coefficients. We considered a p value of < 0.05 as significant (18).
Results of central airway deposition and of the DTPA bronchial clearance in each group are shown in table 3; figure 2 shows individual values of the DTPA bronchial clearance. There was no difference in the deposition rate of the labeled aerosol among the groups, but DTPA bronchial clearance was higher in all seven subjects during their attacks of asthma than in subjects with symptom-free asthma, with or without airflow limitation, in subjects with chronic bronchitis, or in subjects with emphysema (p < 0.0001). There was no correlation between DTPA bronchial clearance and FEV 1 (figure 3), RV, and FRC in asthmatics and in nonasthmatics. Values for PDzo were 2.9 ± 1.1 mg in the three control subjects who were reactive to methacholine, and 0.7 ± 0.6 mg in asthmatics without airflow limitation. There was no correlation between DTPA bronchial clearance and PD2 0 in asthmatics (p = 0.84). DTPA bronchial clearance and FEV 1 during the initial evaluation in subjects with acute asthma, and 1 to 4 wk later, are shown in figure 4. All subjects but one received steroids orally between the two measurements, and five subjects were still receiving steroids orally at the time of the second study (table 2). DTPA bronchial clearance decreased significantly
TABLE 3 EFFECT OF CPAP ON FRC AND BRONCHIAL CLEARANCE OF 11.mln·DTPA (DTPA BRONCHIAL CLEARANCE)'
FRC, ml DTPA bronchial clearance,
%/min
NO CPAP
CPAP
2,588 ± 427
3,782 ± 1,025
0.81 ± 0.39
0.56 ± 0.18
p Value
p
< 0.03 NS
• Results are expressed as mean ± SO. Comparisons were made using Studenfs paired t test.
• •
• •• Fig. 2. Individual values of the bronchiaI clearance of 11.mln·DTPA (DTPA bronchial clearance) in the stUdy groups.
•
• • I •I • ..... airflcwlimitation I
""""01,
• • ••• I """"~
•
•
,I,
I
I
•••
•
• I
-.out
airflowlimitation airflow limitation I I chroniC as1hmatics bronchitis
emphysema
150
LEMARCHAND, CHINET, COLLIGNON, URZUA, BARRITAULT, AND HUCHON
140
•
120
~ >
.O~
100
8
80
•
•••
0 0
0
W
u,
0
• • • o •••
60
0
• 0
40
Ie
20
•
0
0
0 0
•
Fig. 3. Individual values of the clearance of 113mln-DTPA (DTPA bronchial clearance) and of FEV, (open circles = asthmatics; closed circles = nonasthmatics).
0
0
•
o 0
•
0
0 0
,
,
,
3
4
6
SG-In-DTPA ('Iojmin)
~ -
6-
120
5
100
4
80
C
0~
;?
W
40
2
OJ
20
o
evaluation # 1
evaluation #
2
o
evaluation #
1
evaluation #
2
Fig. 4. Individual values of the bronchial clearance of 113mln_DTPA (DTPA bronchial clearance) and of FEV, in SUbjectsduring acute attacks of asthma during the initial evaluation (Evaluation 1) and 1 to 4 wk later (Evaluation 2). Each subject is designated by a symbol.
(p < 0.01), but it remained increasedcompared with that in control subjects (p < 0.05). FEV 1 increased significantly between the two measurements (p < 0.05). There wasno correlation between the decrease in DTPA bronchial clearance and the increase in FEV 1 • The effect of CPAP on FRC and DTPA bronchial clearance in five normal subjects is shown in table 3.
Discussion
Compared with control subjects and with stable asthmatics, DTPA bronchial clearance was increased in asthmatics during acute attacks; furthermore, clearance values decreased within 4 wk after treatment of the attacks in six of seven asthmatics, but remained increased compared with values in the control groups. The increase in DTPA bronchial clearance during attacks did not correlate with the severity ofairflow limitation at the time, as measured by FEV 1 • Bronchial hyper-
responsiveness, present in asthmatic subjects without airflow limitation, was not associated with an increase in DTPA bronchial clearance. Several potential technical problems might have caused increases in the measured rates of clearance using this technique, and thus might lead to a misinterpretation of the results.
Dissociation of the Compound The results of several studies of healthy smokers have shown that the alveolar clearance of 99mTc-DTPA was increased (19); however, part of this increase may be explained by dissociation of the 99mTc-DTPA complex, leading to the formation of compounds with fast epithelial transfer (20). To avoid this potential problem, we used 113mIn, which is more tightly bound to DTPA than is 99mTc (21). Therefore, dissociation of 113mIn from DTPA cannot explain the increase in DTPA bronchial clearance observed in our study.
Deposition Site The site of deposition of the labeled DTPAin the lung wascrucial because this determined whether weassessedbronchial or respiratory clearanceor both. Visual evaluation of the scintiphoto demonstrated a pattern of predominantly central deposition (figure 1), similar to that obtained by Yeates and coworkers (22), who used an aerosol with similarMMAD. Some deposition occurred in the periphery of the lungs, as shown by the ratio of counts from the central regions to counts from the entire lungs (table 4), but this deposition probably mainly occurred in airways and to a lesser extent in terminal respiratory units because the MMAD of the aerosol was 6.3 urn, Furthermore, to ensure that we measured the DTPA clearance in the airways and not in the terminal respiratory units, we calculated DTPA bronchial clearance from the decreasing radioactivity over the central regions of interest and not from the increasing radioactivity in blood, which comes from both airways and terminal respiratory units. In addition, DTPA bronchial clearance was not affected by CPAP-induced lung hyperinflation; because lung hyperinflation markedly increases DTPA respiratory clearance (21), the method we used to measure DTPA clearance reflects predominantly bronchial clearance, with a minimal participation of respiratory clearance. Finally, the increased DTPA bronchial clearance found in subjects with acute asthma was probably not due to differences in deposition of the aerosol among the groups because the ratio of the radioactivity in central regions to the radioactivity in the entire lungs was similar in all groups (table 3). Binding to Mucus Glycoproteins Cheema and coworkers (23) suggested that the absence of an increase in airway mucosal clearance in stable asthmatics, as compared with that in normal subjects, might be due to binding of 99mTc_ DTPA to glycoproteins in the mucus. However, we have shown that, compared with control subjects, DTPA bronchial clearance was four times faster in subjects with acute attacks of asthma, and that clearance in subjects with chronic bronchitis and intercurrent bronchial infection was similar to that in control subjects. Because subjects with bronchial infections had more mucus in their bronchi than did control subjects, even if DTPA binds to mucus, this does not appear to affect the values of DTPA bronchial clearance.
151
BRONCHIAL CLEARANCE OF OTPA IN ASTHMA
TABLE 4 BRONCHIAL DEPOSITION AND BRONCHIAL CLEARANCE OF 113mln_DTPA (DTPA BRONCHIAL CLEARANCE) IN STUDY GROUPS' Central Airways Deposition (0/0 total deposition) Control Asthma Acute airflow limitation Chronic airflow limitation Without airflow limitation Chronic bronchitis Emphysema
DTPA Bronchial Clearance (%/min)
42.5 ± 7.1
0.9 ± 0.6
43.9 39.2 47.1 50.4 43.5
3.9 0.9 0.9 0.8 0.7
± ± ± ± ±
7.4 10.1 14.7 16.9 10.2
± 1.4 ± 0.5 ± 0.7 ± 0.5 ± 0.5
• Values are mean ± SD.
Mucociliary Clearance Two potential routes of elimination may account for DTPA bronchial clearance: transepithelial transfer and mucociliary clearance. As mentioned previously, Bennett and Ilowite (9) showed by dynamic studies of the deposition scan that mucociliary clearance of DTPA may be a significant component of total DTPA clearance from the airways; in addition, they calculated the rate of 99mTc_DTPA transport across bronchial epithelium by correcting for the removal of DTPA by the mucociliary clearance, which was evaluated with 99mTc-Iabeled albumin. These studies rely on the assumption that 99mTc_DTPA, a small compound with a molecular weight of 492 daltons, has the same mucociliary clearance as human serum albumin, which has a much higher molecular weight of 66,000 daltons and different physicochemical properties. In companion studies, Ilowite and coworkers (10)used a polydispersed aerosol with a MMAD of 2.0 J.1m, which therefore was likely to deposit mainly in the peripheral regions of the lungs; to enhance central deposition, they modified the pattern of ventilation of their subjects, but they included the whole right lung field in their analysis, a method that presumably measured a combination of both respiratory and bronchial clearances of DTPA. Because Ilowite and coworkers (10) found a normal total DTPA clearance in stable asthma, which included a component of mucociliary removal, and because our results are similar to theirs, some mucociliary transport of DTPA may have occurred during our measurements of DTPA bronchial clearance. If so, the increase in DTPA bronchial clearance during attacks of asthma could conceivably have been due to a marked increase in DTPA mucociliary clearance. However, the results of many studies have shown decreased mucociliary clearance in stable asthma, even during remission (9, 10,
24, 25). To our knowledge, mucociliary clearance has not yet been studied during acute asthmatic attacks in humans, but after bronchial challenge with specific antigen (ragweed), tracheal mucus transport rate decreased to 720/0 of baseline (26). It is also possible that mucociliaryclearance could have been increased in our asthmatics by the betas-agonist drugs they were receiving. However, the effect of beta.-agonists on mucociliary clearance has usually been measured in stable asthmatics and did not exceed a 100% increase from a baseline of 10 to 20% of normal control values (24), in contrast with the 4000/0 increase in DTPA bronchial clearance documented in our subjects. Furthermore, a recent in vivo study in humans failed to find an increase in mucociliary clearance with inhaled betaj-agonists (27). Therefore, we believe it is unlikely that the entire increase in DTPA bronchial clearance observed in our asthmatics during their acute attacks can be explained by an increase in mucociliary clearance, although this may have contributed to some extent to the observed values.
Age The subjects in all the groups except the asthmatics without airflow limitation were older than the control subjects (table 1). However, only the asthmatics with acute airflow limitation had increased DTPA bronchial clearance. Therefore, it is unlikely that differences in the ages of our subjects could explain the increased bronchial clearance in asthmatics with acute airflow limitation.
Airflow Limitation and Hyperinflation To determine whether an increase in DTPA bronchial clearance could be attributable to differences in airflow limitation or hyperinflation, we measured clearance in asthmatics with chronic air-
flow limitation, in subjects with chronic bronchitis, and in subjects with emphysema who had equal or lower values of FEV I and FEVl/FVC and more severe hyperinflation (increased residual volume) than did patients with acute attacks of asthma (table 1). Because DTPA bronchial clearance was increased only in asthmatics during acute attacks, the increase was unlikely to be due either to airflow limitation or to chronic hyperinflation. In addition, we ruled out an effect of acute hyperinflation, which occurs during asthma attacks, on DTPA bronchial clearance: no increase in DTPA bronchial clearance was observed during CPAP breathing in five control subjects, which indicates that acute increase in lung volume does not increase DTPA bronchial clearance. After considering the potential technical problems, discussed previously, we believe that the main results of our investigations - that bronchial clearance is increased in asthmatics during acute attacks and not in the other groups which were studied - are valid and, moreover, that the observations can be explained by present concepts about the pathophysiology of asthma, especially the role of mucosal permeability. An increase of bronchial clearance of aerosolized 2-J.1mdiameter histamine has been shown in allergic monkeys after inhalation of a specific antigen (28), and to horseradish peroxidase in allergic guinea pigs after specific challenge (29). It has been postulated that this increase is mediated by increased paracellular as well as transcellular movement of large solutes across the epithelial barrier (29); however, the exact mechanisms are unknown. Our data suggest that in humans the increase in DTPA bronchial clearance is related to events that occur in the airways during attacks of asthma. The decrease of DTPA bronchial clearance after treatment, including the administration of corticosteroids, shows that transbronchial movement of solutes decreases toward normal as asthma remits, and suggests that the increase in DTPA bronchial clearance during acute episodes could be linked to inflammation of the airway mucosa. Inflammatory stimuli induce an increase in permeability of the tracheobronchial microvessels and epithelium in asthma (30), and DTPA bronchial clearance may increase in concert with the increase of vascular permeability during attacks. All subjects with acute asthma inhaled betas-agonists before the study, which might modify vascular permeability and thus affect DTPA bronchial clear-
152
LEMARCHAND, CHINET, COLLIGNON, URZUA. BARRITAULT, AND HUCHON
ance. However, studies in vitro in humans and in vivo in guinea pigs have shown that these drugs reduce plasma leakage from the tracheobronchial microcirculation (30) and thus act in the opposite direction to the increase noted in our asthmatics. Although our method of measuring DTPA bronchial clearance cannot determine if a mild degree of residual airway inflammation and epithelial damage persists in the interval state, the present data suggest that abnormalities in bronchial clearance of DTPA exist in some asthmatics, particularly during acute attacks. References 1. Laitinen LA, Heino M, Laitenen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 2. Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma: an ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989; 140:1745-53. 3. Hogg JC. Bronchial mucosal permeability and its relationship to airways hyperreactivity. J Allergy Clin Immunol 1981; 67:421-5. 4. Busse WW. Respiratory infections and bronchial hyperreactivity. J Allergy Clin Immunol1988; 81:770-5. 5. Sheppard D. Mechanisms of acute increases in airway responsiveness caused by environmental chemicals. J Allergy Clin Immuno11988; 81:128-32. 6. O'Byrne PM. Allergen-induced airway responsiveness. J Allergy Clin Immunol1988; 81:119-27. 7. O'Byrne PM, Dolovich M, Dirks R, Roberts
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