INTRODUCTION

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A recent supplement of this journal, dedicated to the small airways in asthma (1), summarizes a number of hypotheses concerning peripheral airway involvement in asthma, and also highlights the continuing need for in vivo measurements to substantiate the occurrence of any such peripheral effects. In this work we provide a physiological measurement of acinar airway function in asthma by means of a multiple-breath N₂ washout (MBW) technique, which takes the diffusion front as the cutoff between events occurring proximally and peripherally to it. By considering that under tidal breathing conditions the O₂-N₂ diffusion front is situated at the level of the acinar entrance, we refer to the MBW indices as S_cond and S_acin to account for, respectively, conductive and acinar airway contribution to ventilation inhomogeneity (2). Both S_cond and S_acin indices are derived from curves of normalized N₂ phase III slope throughout an N₂ MBW test, a tool that was used experimentally for the first time by Crawford and coworkers (3). It was not until more recently that similar analyses of the MBW normalized slope curves were used for clinical applications (4, 5).

Although S_cond and S_acin are derived from the same MBW normalized slope curve, they reflect events at different levels of the airway tree, and concomitantly different gas transport mechanisms (2). In the conductive airways convective gas transport predominates and results in an increased S_cond if there is an increased concentration difference between lung units subtended from these conductive airways and/or if there is a increased degree of unequal emptying of these units. These changes can arise from uneven conductive airway narrowing and/or changes in P-V characteristics of lung units subtended by branch points proximal to the diffusion front. Anatomically, such lung units comprise at least several acini. In the acinar airways, the gas-mixing mechanism is far more complex as to how concentration differences develop and are maintained during expiration (6, 7). Briefly, we can state that a marked change in acinar lung structurebringing about a change in asymmetry of cross-sectional area of two daughter airways or changing the respective volumes of lung units subtended by any given branch pointis likely to result in increased S_acin. In summary, S_cond and S_acin are expected to reflect alterations of lung structure proximal and peripheral to approximately the fifteenth lung generation, respectively.

After having used this technique for the study of conductive and acinar ventilation inhomogeneity in asymptomatic subjects undergoing histamine provocation (during which only S_cond increased) and in patients with chronic obstructive pulmonary disease (COPD) in the baseline condition (where both S_cond and S_acin were elevated), we now apply this technique to study conductive and acinar ventilation distribution in patients with asthma, as well as their respective responses to inhaled salbutamol. The effect of salbutamol in patients with asthma, is compared with that in patients with COPD. The acinar airway response to salbutamol is particularly relevant because reports on deposition patterns of directly radiolabeled salbutamol (8) and measurements of salbutamol plasma levels (9) have shown that, even without a spacer, metered dose inhaler (MDI)-delivered salbutamol does reach the lung periphery.

	METHODS

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Patients with well-established but stable asthma were selected according to the following criteria: no smoking history, presence of atopy (positive skin allergy tests), baseline forced expired volume in 1 s (FEV₁) to forced vital capacity (FVC)  70%, and an increase in FEV₁ after salbutamol inhalation equaling 10% or more of the predicted value. Two other groups (designated Normal and COPD) were also included in part of the measurement procedures, and were selected to fulfill the following criteria: (1) Normal: about 40 yr of age, nonsmoking, no respiratory history or symptoms, and normal lung function, (2) COPD:  45 yr of age, smoking history ( 10 pack-years), absence of atopy (negative skin allergy tests), FEV₁/FVC  70%, and an increase in FEV₁ after salbutamol inhalation of less than 9% of the predicted value. All patients with asthma and COPD were stable on individualized medical treatment according to the international guidelines for treatment and diagnosis of asthma (10) and COPD (11). None of the participating subjects took any medication in the 12-h period before the time of study, which was usually carried out between 9:00 and 10:00 A.M.

The Normal group was included to provide baseline MBW results representing a group of asymptomatic normal subjects that were age-matched [38 ± 4 (SD) yr, n = 10; 4 females and 6 males] with the Asthma group [39 ± 13 (SD) yr, n = 20; 6 females and 14 males]. The COPD group was included to compare the effect of inhaled salbutamol with respect to that in the Asthma group. The patients with COPD are those from a previous study (5), where baseline MBW and lung function results are reported, whereas the salbutamol data have not been published elsewhere. With the age limit ( 45 yr) on the part of the COPD selection the subject age range was between 45 and 75 yr, and the generally younger subjects in the Asthma group ranged between 19 and 69 yr.

A number of tests were performed before and 10 min after salbutamol inhalation (400 µg of Ventolin via an Inhalation Aid [Boehringer Ingelheim, Ingelheim am Rhein, Germany] small-volume spacer). Baseline tests included body plethysmographic volume measurements (for functional residual capacity, FRC_pl), a single-breath carbon monoxide diffusing capacity test (for diffusing capacity, DL_CO), three forced expiration maneuvers (for FEV₁; FVC; peak expiratory flow, PEF; forced expiratory flow after exhalation of 75% of FVC, FEF₇₅) and three MBW tests (for functional residual capacity, FRC_mbw; conductive ventilation inhomogeneity index, S_cond; acinar ventilation inhomogeneity index, S_acin; Fowler dead space of the first breath, VD_F). Postsalbutamol measurements involved three forced expiration maneuvers and two MBW tests.

Lung function parameters were obtained by means of standardized lung function laboratory equipment (SensorMedics, Bilthoven, The Netherlands) and according to recommended procedures (12). For the MBW tests, the subjects were aided by a computer-controlled breathing assembly, which has been described in detail elsewhere (2). The procedure involved some air breathing, allowing the patient to practice 1-L inhalations above FRC, after which a valve is switched to substitute pure oxygen for air and the MBW test actually starts. Throughout the MBW test, involving twenty to twenty-five 1-L breaths of pure oxygen, volume and N₂ signals are continuously recorded.

The MBW analysis procedure consists of plotting N₂ concentration as a function of cumulative inspired and expired volume, determining the N₂ phase III slope of each subsequent expiration, and normalizing each N₂ slope by the mean expired N₂ concentration. If the normalized slope is then plotted as a function of lung turnover (TO), i.e., cumulative expired volume divided by functional residual capacity, this results in plots such as those shown in Figure 1, i.e., steadily increasing normalized slope as a function of lung turnover. The closed symbols in Figure 1 correspond to the average of three baseline MBW tests in a typical subject from the COPD group (triangles) or the Asthma group (squares), and the open symbols are the average of two postsalbutamol MBW tests obtained in the same subjects. The dotted line is the normalized alveolar slope curve reconstructed from previously reported normal subjects (2).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Representative results of MBW tests performed by one patient from the Asthma group (squares) and one from the COPD group (triangles). Normalized alveolar slopes (Sn) are expressed as a function of lung turnover (TO). The symbols represent the average Sn values (± SD) of three MBW tests in the baseline condition (closed symbols) and of two MBW tests after salbutamol administration (open symbols). Also represented is the curve (dotted line) reconstructed from equivalent MBW data obtained in a group of asymptomatic, nonsmoking, nonhyperresponsive subjects (Normal; retrieved from reference 2), with illustration of indices Scond and Sacin (see text for details).

From the normalized slope curves such as those in Figure 1, indices S_cond and S_acin are derived, representing, respectively, the conductive airway and acinar airway contribution to the ventilation inhomogeneity. S_cond is the normalized slope difference per unit TO, which is determined in the part of the MBW where only conductive airways are known to contribute to the rate of rise of normalized slope, i.e., between TO = 1.5 and TO = 6 (2). S_acin is determined by subtracting from the slope of the first breath, the part that is attributed to the conductive airways, i.e., S_cond multiplied by the TO value of the first breath (which is ~ 0.3 in the case of Figure 1). In the examples in Figure 1, the baseline MBW test for the subject with COPD corresponds to S_cond = 0.10 L¹ and S_acin = 0.67 L¹ [= 0.70 L¹  (0.3)(0.10 L¹)] and for the subject with Asthma, S_cond = 0.06 L¹ and S_acin = 0.24 L¹ [= 0.26 L¹  (0.3)(0.06 L¹)]. Only in the subject with Asthma did postsalbutamol values differ from baseline, yielding S_cond = 0.047 L¹ and S_acin = 0.12 L¹. The dotted line corresponds to S_cond = 0.033 L¹ and S_acin = 0.075 L¹ (2).

For the detailed explanation of S_cond and S_acin computation, the reader is referred to previous work (2). We only summarize here that the normalized slope of the first breath is predominantly generated by diffusion-convection-dependent ventilation inhomogeneity within the peripheral acinar lung units. The rate of rise of the normalized slope originates from convection-dependent ventilation inhomogeneity, which is generated by concentration differences and flow sequencing between lung units larger than acini. As such, S_cond and S_acin should be considered as two independent indices of ventilation inhomogeneity in the lungs.

Statistical Analysis

Using Statistica 5.1 (StatSoft, Tulsa, OK), nonparametric tests were performed to detect differences in baseline values and salbutamol changes between Asthma and COPD groups (Mann-Whitney), and to test for the significance of changes after salbutamol inhalation within each group (Wilcoxon). The significance level was set at p = 0.05.

	RESULTS

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Table 1 lists the average baseline and postsalbutamol values of lung function and MBW parameters obtained in the Asthma and COPD groups, each group consisting of 20 patients. Figure 2 is a graphical representation of the average S_cond and S_acin values in Table 1, also showing the individual baseline and postsalbutamol values. The horizontal marks in Figure 2 depict the baseline S_cond and S_acin values previously reported (2) in a group of 10 nonhyperresponsive, nonsmoking normal subjects [S_cond = 0.033 ± 0.003 (SEM) L¹, S_acin = 0.075 ± 0.007 (SEM) L¹], and newly obtained values from the 10 subjects in the Normal group [S_cond = 0.031 ± 0.005 (SEM) L¹, S_acin = 0.067 ± 0.008 (SEM) L¹]. Concerning the latter group, which was age matched with the Asthma group, lung function and MBW values can be found in Table 2.

View larger version (25K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   Figure 2.   (A) Average (± SEM) and individual Scond values before and after salbutamol administration in Asthma (solid lines) and COPD groups (dotted lines). The horizontal marks (± SEM) at baseline refer to normal Scond values obtained previously (2) and in a new group of age-matched unaffected subjects (Normal group), the latter group corresponding to the lowest horizontal mark. (B) Average (± SEM) and individual Sacin values before and after salbutamol administration in Asthma (solid lines) and COPD groups (dotted lines). The horizontal marks (± SEM) at baseline refer to normal Scond values obtained previously (2) and in a new group of age-matched unaffected subjects (Normal group), the latter group corresponding to the lowest horizontal mark.

In the Asthma group, baseline forced expiration parameters, but not DL_CO, were abnormal with respect to predicted values (Table 1). Also, both S_cond and S_acin were significantly higher than the previously reported and newly obtained normal values (Tables 1 and 2, Figure 2). In the COPD group, baseline forced expiration parameters as well as DL_CO were abnormal and all of these values, except PEF, were significantly lower than in the Asthma group (Table 1). Also, S_acin in the COPD group was significantly higher than in the Asthma group, whereas S_cond was impaired to the same extent. FRC_mbw was normal in both groups, whereas FRC_pl values were on average 690 and 1,058 ml larger than respective FRC_mbw values in the Asthma and COPD groups, such that FRC_pl was not significantly different between both groups. VD_F was 30 ml lower in the Asthma than in the COPD group.

After salbutamol administration, all lung function and MBW indices measured in the Asthma group improved significantly, except for FRC_mbw, which remained unchanged (Table 1). In the COPD group, FEV₁ and FEV₁/FVC also increased significantly after salbutamol inhalation, but these changes were much smaller than in the Asthma group. In fact, the last column of Table 1 shows whether relative change, i.e., salbutamol minus baseline value expressed as a percentage of baseline, was significantly different between both groups. The result is that basically all indices except FRC_mbw and VD_F changed significantly more in the Asthma than in the COPD group. The most striking feature of the salbutamol measurements is the absence of significant S_cond and S_acin decreases in the COPD group despite larger baseline S_cond and S_acin values with respect to the Asthma group (where both S_cond and S_acin decrease).

The average age in Asthma and COPD groups was significantly different (Table 1) and could have contributed to the observed S_cond and S_acin differences. We first checked whether, within either group, S_cond or S_acin was age related. This was not the case (p > 0.1 for correlations of all parameters with age). We also examined Asthma and COPD subgroups corresponding to the overlapping age range between both groups (i.e., between 45 and 69 yr of age). These Asthma and COPD subgroups (Table 2) had forced expiratory parameters that were no longer significantly different from each other, whereas DL_CO and S_acin remained significantly different between both groups.

	DISCUSSION

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In this study we obtained evidence of impaired acinar ventilation distribution in asthma through physiological measurement. We also demonstrated that in this group of patients, this acinar airway alteration is partly reversible after salbutamol inhalation. Both acinar and conductive airway components of ventilation inhomogeneity in asthma are now discussed in comparison with those observed in patients with COPD, and also as related to results previously obtained in asymptomatic hyperresponsive and nonhyperresponsive subjects using the same MBW technique (2).

Acinar Airways Contribution to Ventilation Inhomogeneity (S_acin)

Under baseline conditions, the severity of acinar ventilation inhomogeneity in the Asthma group (S_acin = 0.195 L¹) was intermediate between that of Normal (S_acin = 0.067 L¹) and COPD (S_acin = 0.433 L¹) groups. One could argue that part of the baseline S_acin differences between the Asthma and COPD groups was attributable to the age difference. However, we found no correlations of S_acin with age within the Asthma group or within the COPD group, and when considering Asthma and COPD subgroups with overlapping age ranges, the respective S_acin values shifted to 0.264 ± 0.152 (SD) L¹ (Asthma) and to 0.440 ± 0.172 (SD) L¹ (COPD), i.e., S_acin values were still significantly different.

The relevance of the abnormal baseline S_acin in the Asthma group is that impaired acinar ventilation distribution was detected in patients with normal diffusion capacity. This could suggest either that S_acin is more sensitive to alveolar structure alteration than DL_CO, or that the acinar spaces involved are the more proximal acinar airways, while the alveolated structure remains largely unaltered. In the COPD group the combination of a more elevated baseline S_acin and decreased diffusion capacity confirms the presence of emphysematous lesions. The presence of such lesions could also explain the fact that after salbutamol inhalation, acinar ventilation inhomogeneity is not reversible in the COPD group (S_acin in Table 1). Despite the improvement of acinar ventilation inhomogeneity in the Asthma group after inhalation of 400 µg of salbutamol, S_acin values in the Asthma group had not returned to Normal values (Tables 1 and 2). Whether S_acin could be further reduced by higher doses of salbutamol remains an open question.

In a previous study with hyperresponsive but otherwise asymptomatic subjects, we found a slightly, but significantly, larger baseline S_acin [0.107 ± 0.025 (SD) L¹] than in nonhyperresponsive age-matched normal subjects [0.075 ± 0.022 (SD) L¹]. However, in neither of these groups was S_acin affected by histamine provocation, or by subsequent salbutamol inhalation (2). Concerning salbutamol inhalation, one of the reasons could have been that even in the hyperresponsive group S_acin was not sufficiently elevated to be reversible at all. Although it is true that the baseline S_acin value of the Asthma group was indeed more elevated than in the above-mentioned hyperresponsive subjects, the even larger baseline S_acin value and absence of significant S_acin change in the COPD group shows that the magnitude of baseline S_acin is not the only prerequisite for S_acin reversibility. In the COPD group, where the baseline acinar ventilation distribution is most impaired, the underlying structural alteration is clearly nonreversible.

Conductive Airway Contribution to Ventilation Inhomogeneity (S_cond)

Baseline conductive airway ventilation distribution is abnormal in the Asthma group, i.e., S_cond is about three times the value obtained in the Normal group (Table 2). More interesting is that the average S_cond value in the Asthma group is not significantly different from that found for the COPD group, whether entire groups (Table 1) or age range-overlapped subgroups (Table 2) are considered. In fact, the average baseline S_cond values in the Asthma (0.076 ± 0.026 L¹) and COPD groups (0.085 ± 0.041 L¹) are similar to the S_cond value we previously obtained in subjects with bronchial hyperresponsiveness in the state of histamine provocation (0.091 ± 0.035 L¹) when their average FEV₁ had decreased to 74% of their baseline value.

Taken together, these S_cond data indicate a limitation in the extent of impaired conductive ventilation inhomogeneity, whether in stable patients with Asthma or COPD or in histamine-provoked hyperresponsive subjects. Because S_cond depends on (1) end-inspiratory concentration differences between relatively large lung units subtended by conductive airways, and (2) unequal emptying between them, this means that either or both of these effects are somehow limited as to how much ventilation inhomogeneity can be generated. It is possible that the similar baseline S_cond in Asthma and COPD group patients is in part due to the similar degree of trapped gas in both groups, as inferred from the difference between FRC_pl and FRC_mbw (Table 1). It is conceivable that the trapped gas volume is a determinant factor of the relative P-V characteristics of large lung units, thereby influencing the degree of unequal emptying between them. However, the poor accuracy of FRC_pl and FRC_mbw measurement in obstructive patients (13) hinders a quantitative evaluation of the possible link with trapped gas volume.

At least some of the baseline conductive ventilation impairment must be due to different mechanisms in Asthma and COPD group patients, because part of it can be reversed by salbutamol only in the Asthma group (S_cond in Table 1). Among patients in the COPD group, the absence of change in S_cond could be explained if the emphysematous lesions also affected the P-V characteristics of lung units larger than acini. In the Asthma group, the observed decrease is S_cond suggests a reduction in unequal narrowing of conductive airways. However, as in the case for acinar ventilation inhomogeneity, a residual conductive component of ventilation impairment persisted after inhalation of 400 µg of salbutamol, i.e., S_cond remained significantly higher than in the Normal group (Tables 1 and 2). Taken together, the residual S_cond and S_acin abnormality in the Asthma group patients after 400 µg of inhaled salbutamol points to residual structural alteration both at the conductive and acinar level of the airway tree. This could be due to a combination of factorsthat cannot be reversed simply by means of a ₂-adrenergic agonistsuch as edema, inflammation, cellular infiltration, and epithelial shedding, or it could correspond in part to the airway remodeling reported in chronic asthma (14).

Relation to Previous Studies of Peripheral Airway Involvement in Asthma

The involvement of the peripheral airways in patients with asthma under baseline conditions and after inhaling ₂-mimetic drugs has been a subject of considerable interest in past publications, where the terminology "peripheral airways" may cover a large range of airway generations depending on the measurement method used. Wagner and coworkers (15) measured peripheral resistance, thought to reflect the combined resistance of collateral channels and airways as small as 0.5 mm in diameter, and found it to be increased in patients with asthma with respect to unaffected subjects, but did not detect any change after isoproterenol treatment. Yanai and coworkers (16) measured relative changes in Fowler dead space and specific conductance after fenoterol inhalation by patients with asthma, concluding that an airway response could be elicited both in the central and peripheral airways. Using density dependence of flow, Fairshter and colleagues (17) in fact showed that the predominant sites of bronchodilatation in patients with asthma are related to the baselineperipheral or centralsite of air flow limitation. The abnormal baseline S_cond and S_acin, and their respective responses to salbutamol in our Asthma group, are consistent with such a pattern in terms of ventilation distribution as well, namely, that both conductive and acinar airway baseline impairment are partly reversible at both these levels of the airway tree.

Ventilation distribution has been previously investigated in patients with asthma in terms of N₂ phase III slope of the single-breath washout (17) or end-tidal concentration curves of the multiple-breath washout (22), with a general observation of decreased overall lung ventilation inhomogeneity after inhalation of ₂-mimetic drugs. Olofsson and coworkers (18) actually found salbutamol dose-response curves of decreasing N₂ phase III slope paralleling the FEV₁ increase, but in a subsequent study by the same group (19) it was concluded that these N₂ decreases were essentially due to concomitant decrease in preinspiratory lung volume, i.e., residual volume in their case. As is the case for washout tests in general and the MBW procedure in particular, it is indeed crucial that the preinspiratory ventilated lung volume be unchanged when comparing phase III slopes or derived indices such as S_cond and S_acin between, e.g., baseline and dilatation measurements. Using the MBW computation of FRC as was done here, Lutchen and colleagues (22) did not observe significant FRC change after albuterol inhalation. Tables 1 and 2 show that FRC_mbw was indeed not significantly different between baseline and postsalbutamol measurements, nor between Asthma, COPD, and Normal groups. We are therefore confident that in this study the observed S_cond and S_acin differences between groups and after salbutamol administration do reflect lung structural differences.

A modified single-breath washout maneuver used by Cooper and coworkers (20) in children with asthma started the O₂ inhalation from FRC rather than from residual volume, a maneuver that was indeed expected to enhance detection of lung structural alterations (23). Cooper and colleagues (20) found that (1) the patients with asthma with the steepest baseline N₂ phase III slopes also showed the largest decreases after isoproterenol inhalation, and (2) postisoproterenol N₂ phase III slopes were still elevated with respect to normal values. The present study shows that after salbutamol inhalation, both of these observations hold at conductive airway and acinar airway levels separately, as exemplified by the S_cond and S_acin behavior shown in Figure 2. Finally, Peces-Barba Romero and colleagues (21) used the respective behavior of helium and SF₆ phase III slopes of a vital capacity single-breath washout to demonstrate considerable intraacinar contribution to baseline and postdilatation ventilation distribution in patients with asthma. On the basis of N₂ phase III slope analysis of the MBW test, our elevated baseline S_acin values and the significant S_acin decreases after salbutamol inhalation in our Asthma group lend further support to these findings.