INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BRONCHIAL HYPERRESPONSIVENESS is usually evaluated by provocation testing with histamine or methacholine, in connection with a measurement of forced expiratory volume in 1 s (FEV₁). Although guidelines for provocation testing suggest that methacholine and histamine can be used interchangeably for routine clinical practice (21), different mechanisms are thought to underlie the airway narrowing process induced by these agents. Methacholine is thought to act directly on muscarinic receptors on bronchial smooth muscle. Histamine may act through a direct effect on bronchial smooth muscle (6), probably via stimulation of H₁ receptors (5), or indirectly as an irritant stimulating vagal parasympathetic reflex bronchoconstriction (20), although the former effect is thought to dominate over the latter (14). In any case, different mechanisms appear to be reflected in the differential behavior of histamine and methacholine, at least in normal human subjects (22, 26), in whom histamine elicits the greatest response in terms of FEV₁. In asthmatic subjects, the differential behavior of histamine and methacholine is less clear: when considering all dosages in moles (27), some studies do show a difference (7, 11) whereas others do not (2, 10, 18, 26). These conflicting reports in asthmatic subjects may be due to the degree to which some of the protective mechanisms against provocation are deficient in any particular group of asthma patients, e.g., the protective effect of the inhibitory muscarinic receptors present in normal subjects may dysfunction to a greater or lesser degree in asthmatic subjects (1). Such findings underline the need to fully understand the differential action of histamine and methacholine in the normal lung.

It has been argued that the inability to reveal differences between histamine and methacholine in some studies may have been related to the intrinsic noise of the measurement (2, 7) and the gross averaging of lung function indexes such as FEV₁ (3). In contrast to spirometry, which is generally considered to reflect a modification of airway caliber and elastic properties of the lungs as a whole, ventilation distribution tests rely on parallel differences in airway caliber and elastic properties of subtended lung units. We have previously shown how indexes of ventilation heterogeneity originating in the conductive airway zone (S_cond) and acinar airway zone (S_acin), as derived from a multiple-breath washout (MBW) test, are affected by histamine provocation (25). On the basis of the very marked S_cond increases and the absence of S_acin increases observed in hyperresponsive and in nonhyperresponsive subjects (classified according to the 20% FEV₁ criterion), we concluded that histamine induced 1) differences in specific ventilation of lung units larger than acini and 2) flow asynchrony between these units during exhalation. In the histamine provocation study (25), S_cond more or less paralleled the FEV₁ behavior across both groups, indicating that there was a general decrease of airway lumen as well as an inequality in narrowing of parallel airways of the same generation, probably caused by inequalities in bronchial tone of the conductive airways. In the present study, we compared methacholine with histamine in terms of respective spirometry and ventilation heterogeneity in normal subjects.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Two groups of 15 subjects underwent methacholine provocation testing after having been classified as nonhyperresponsive on the basis of a decrease in FEV₁ <20% baseline after a cumulative dose of 2 mg histamine (i.e., 6.52 µmol). All subjects were recruited on a voluntary basis in the framework of a prevalence study on hyperresponsiveness in normal subjects in the Brussels area (using a maximum cumulative dose of 2 mg histamine by default). The methacholine procedure always followed the histamine procedure, separated by at least 1 wk and by no longer than 1 mo. The first group (equal-dose group) received the same cumulative dose of 6.67 µmol methacholine (i.e., 1.6 mg) whereas the second group (double-dose group) included one more doubling dose only in the methacholine provocation protocol, to yield a cumulative dose of 13.33 µmol (i.e., 3.2 mg). By using the dosimeter technique (MEFAR dosimeter MB3; vital capacity breath), histamine was administered in four steps (0.52, 1.56, 3.39, and 6.52 µmol) and methacholine in four or five steps (0.83, 1.67, 3.34, 6.67, and 13.3 µmol). On both occasions, subjects were free of respiratory symptoms.

Spirometric parameters were obtained by means of standardized lung function laboratory equipment (Sensormedics, Bilthoven, Netherlands) according to recommended procedures. MBW tests were carried out with a dedicated breathing assembly identical to the one described in detail in our previous work (25). In brief, this equipment consists of a pneumatic valve system that allows a sequence of 20-25 1-liter inspirations from a bag filled with pure O₂ and exhalations through a separate pathway. Throughout the test, end-inspiratory volume was functional residual capacity (FRC), and the exact number of breaths was dependent on subject performance and on progressive N₂ dilution. Volume and N₂ concentration were acquired at 25 Hz. One MBW run typically takes 2-3 min and is separated by 3-5 min before a subsequent MBW maneuver is started, to allow the subject to wash out completely the inhaled O₂.

Before starting the provocation testing, subjects performed spirometry, involving three forced expiratory maneuvers, from which the best maneuver was selected to provide baseline FEV₁, peak expiratory flow (PEF), and forced expiratory flow after exhaling 75% of the forced vital capacity (FEF₇₅), and three baseline MBW tests. At each successive step of the provocation procedure with histamine (4 steps) or methacholine (4 or 5 steps), spirometry was repeated. For operational reasons, MBW tests could not be carried out at each intermediate step of the provocation procedures. In the equal-dose group, subjects performed two MBW tests immediately after spirometry after the final dose of either histamine (6.65 µmol) or methacholine (6.67 µmol). In the double-dose group, subjects performed two MBW tests immediately after spirometry after the final dose of either histamine (6.65 µmol) or methacholine (13.3 µmol). To account for possible time-dependent effects occurring over the course of the two final-dose MBW tests, these were followed by another spirometry. In this way, spirometric data obtained for the final dose of histamine or methacholine were the average values of FEV₁, PEF, and FEF₇₅ obtained immediately before and after the two final-dose MBW tests.

Data analysis. Because of the importance in understanding the computation of some MBW-derived indexes and how they relate to the mechanisms of ventilation heterogeneity, we reiterate some basic features of the MBW phase III slope analysis. In our previous work (25), a more extensive description and theoretical background of all MBW-derived indexes can be found. Briefly, N₂ phase III slope is computed by linear regression between 0.65 liter and end of expiration (nominally 1 liter) and divided by the mean expired N₂ concentration of that expiration in each subsequent breath of the MBW test. In this way, the normalized N₂ phase III slope represents relative N₂ concentration differences in different parts of the lungs with respect to the prevailing N₂ concentration in the lungs at any instance of the MBW test. Normalized phase III slopes for each breath are then plotted against lung turnover (TO), i.e., cumulative expired volume corresponding to each subsequent breath divided by the subject's FRC. Lung TO is used instead of breath number to account for possible differences in lung dilution due to differences in the subjects' FRC (8).

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View larger version (18K): [in this window] [in a new window]   Fig. 1.   The normalized N2 phase III slope plots obtained in 1 subject from the equal-dose group (A) and in 1 subject from the double-dose group (B). All N2 slopes are expressed as a function of lung turnover [TO, i.e., cumulative expired volume divided by functional residual capacity (FRC)] and are means  SD of 3 (baseline; B) or means + SD of 2 (provocation; P) tests. Triangles, baseline multiple-breath washout (MBW) tests; circles, MBW tests after the final dose of a provoking agent [closed symbols, histamine (His) protocol; open symbols, methacholine (MCh) protocol].

Theoretical background. S_cond and S_acin independently reflect ventilation heterogeneities occurring at branch points located in the conductive and acinar lung zones, respectively (24, 25). In the present context of bronchoprovocation in normal subjects in whom conductive airways are expected to be the main component of ventilation heterogeneity (25), we focus on the principles underlying S_cond. The gas transport mechanism leading to nonzero S_cond values even in normal lungs is convective gas flow. In fact, S_cond is the most straightforward MBW index in that it directly relates to N₂ concentration differences accumulating between any two lung units of which one lung unit is preferentially ventilated at every subsequent inhalation. The only condition for this concentration difference to translate into a sloping phase III during each subsequent exhalation is that there is a sequential emptying pattern between these lung units with the most ventilated one emptying preferentially at the onset of exhalation. In every subsequent breath, these concentration differences will accumulate with respect to the prevailing average concentration in the lung, and if the phase III slope is normalized by the mean expired concentration in each expiration, such a mechanism will cause the normalized slope to rise as in Fig. 1. If this were the only mechanism responsible for the normalized slopes, their extrapolation of the normalized slope curves to TO = 0 would be zero. This is obviously not the case (Fig. 1), and the initial slopes are in fact mainly due to the much more complex mechanism involving simultaneous convective and diffusive gas transport in the lung periphery (which underlies S_acin). However, after only a few breaths (from TO = 1.5 onward) an equilibrium state of convection and diffusion is reached in which relative concentration differences remain constant (and produce a nearly constant contribution to the normalized phase III slope) throughout most of the MBW (16). That is why we compute S_cond only in that part of the normalized alveolar slope curve in which only the conductive airway heterogeneity contributes to the rate of slope increase, i.e., beyond TO = 1.5.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1 shows the baseline values of all pertinent spirometry- and MBW-derived indexes in the equal-dose and double-dose groups, showing no significant difference between histamine and methacholine baseline (Wilcoxon signed-rank test, P > 0.1). Table 2 summarizes the differences induced by 6.52 µmol histamine or 6.67 µmol methacholine in the equal-dose group and by 6.52 µmol histamine or 13.3 µmol methacholine in the double-dose group. Note that impaired spirometry is reflected in FEV₁, PEF, or FEF₇₅ decreases (negative numbers in Table 2), whereas impaired ventilation heterogeneity is indicated by S_cond and S_acin increases and Curv decreases (see Theoretical background). Also indicated in Table 2 are the P values of significantly different changes after either histamine or methacholine. For instance, FEV₁ was invariably less affected by methacholine than by histamine in both groups (P = 0.002 equal-dose group; P = 0.009 double-dose group). By contrast, VD_anat decreased significantly by 10-15 ml after each final dose of either provocation agent, but no significant VD_anat were obtained between histamine and methacholine in the same subjects.

Any parameter that showed a significantly different modification after methacholine with respect to histamine in Table 2 is also graphically represented in Figs. 2-6, where panels A and B represent data obtained from the equal-dose and double-dose groups, respectively. The spirometric indexes are also depicted for the intermediate steps throughout the provocation procedures. Figure 2 indeed shows consistently more marked FEV₁ decreases with histamine than with methacholine for an equal cumulative dosage (Fig. 2) or when doubling the final methacholine dose with respect to the final histamine dose (Fig. 2B). The same pattern was observed for PEF (Fig. 3). However, FEF₇₅ was still affected more by histamine than by methacholine for equal dosage (Fig. 4) but was indistinguishable between both agents for one more doubling methacholine dose (Fig. 4B). Such contrasting behavior with respect to FEV₁ is even more dramatic in the case of S_cond (Fig. 5). Although equal doses of histamine and methacholine only induced slightly smaller S_cond increases for methacholine than for histamine (P = 0.04), a double dose of methacholine (Fig. 5B) actually led to a larger S_cond increase (P = 0.006) in the same subjects that produced a smaller FEV₁ decrease (Fig. 2B). The S_cond pattern was paralleled by that obtained for Curv (Fig. 6).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Mean ± SE changes of forced expiratory volume in 1 s (FEV1) as a function of cumulative doses of histamine () or methacholine (); histamine and methacholine doses are expressed on a log scale (in µmol), and the 100% reference is placed arbitrarily at 0.15 µmol. A: equal-dose group (n = 15). B: double-dose group (n = 15).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Mean ± SE changes of peak expiratory flow (PEF) as a function of cumulative doses of histamine () or methacholine (); histamine and methacholine doses are expressed on a log scale (in µmol), and the 100% reference is placed arbitrarily at 0.15 µmol. A: equal-dose group (n = 15). B: double-dose group (n = 15).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Mean ± SE changes of forced expiratory flow at 75% forced vital capacity exhaled (FEF75) as a function of cumulative doses of histamine () or methacholine (); histamine and methacholine doses are expressed on a log-scale (in µmol), and the 100% reference is placed arbitrarily at 0.15 µmol. A: equal-dose group (n = 15). B: double-dose group (n = 15).

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Mean ± SE values of conductive airway ventilation heterogeneity (Scond; see text for details) obtained baseline and after the final dose of histamine () or methacholine (); histamine and methacholine doses are expressed on a log scale (in µmol), and the baseline Scond values are placed arbitrarily at 0.15 µmol. A: equal-dose group (n = 15). B: double-dose group (n = 15).

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Mean ± SE values of specific ventilation differences represented in the curvilinearity of the N2 washout concentration curve (Curv; see text for details) obtained at baseline and after the final dose of histamine () or methacholine (); histamine and methacholine doses are expressed on a log scale (in µmol), and the baseline Curv values are placed arbitrarily at 0.15 µmol. A: equal-dose group (n = 15). B: double-dose group (n = 15).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study displays an apparent paradox by revealing a greater ventilation maldistribution for the bronchoconstriction agent (methacholine) that provokes the least deterioration of spirometry, at least in terms of FEV₁. As in previous studies (22, 26), the FEV₁ behavior observed here (Fig. 2) would suggest that, in normal subjects, histamine is a more potent bronchoconstrictor than methacholine, even when the molar dose of methacholine is doubled with respect to histamine (Fig. 2B). Noteworthy for comparison with previous studies is that the maximal cumulative weight dose for histamine (2 mg) was intermediate between methacholine weight doses of the last step of the equal-dose group (1.6 mg) and that of the double-dose group (3.2 mg). Although histamine may be more potent than methacholine in the large airways, as indicated by the respective FEV₁ and PEF decreases (Figs. 2-3), this may not be necessarily true for the small airways. Indeed, FEF₇₅ decrease for methacholine tended to mimic FEF₇₅ decrease seen for histamine (Fig. 4), with only a marginally significant difference for an equal molar dose of histamine and no difference at all for one more doubling dose of methacholine (Table 2). Such an observation could suggest that, in the small airways, the basic mechanisms of overall airway narrowing may be the same (direct effect at the level of the bronchial smooth muscle) but merely dose dependent (depending on the number distribution of either H₁ or muscarinic receptors).

The ventilation distribution data provide an interesting new perspective on the subject, because its indexes are linked to heterogeneity of airway narrowing rather than an overall lumen decrease at a given lung depth. The two- to threefold S_cond increases and the absence of S_acin change after histamine or methacholine provocation (Table 2) indicate considerable parallel heterogeneity of airway narrowing in the conductive airway tree. An equal molar dose of histamine or methacholine (equal-dose group in Table 2) shows only marginal significance of the differences in S_cond increase (P = 0.041) in the presence of highly significant differences in FEV₁ decline (P = 0.002). This could have suggested that S_cond is rather variable and not very specific in response to provoking agents that clearly constrict the large airways to a different extent (as evidenced by FEV₁ and PEF behavior). However, a doubling dose of methacholine with respect to histamine (double-dose group; Table 2) reveals a clear-cut divergence between a larger S_cond increase (P = 0.006) and a smaller FEV₁ decline (P = 0.009) for methacholine. Clearly, the latter ventilation distribution and spirometric parameters reflect different phenomena occurring simultaneously.

The parallelism between respective S_cond and Curv patterns seen across Figs. 5 and 6 suggests that the respective S_cond changes for histamine and methacholine can be accounted for to a large extent by differences in inspired gas concentration (or specific ventilation) in addition to flow asynchrony patterns developed by the heterogeneity in airway narrowing. In this respect, the present data could explain earlier observations of dissociation between spirometric decline and ventilation-perfusion mismatch seen in mildly asthmatic subjects after methacholine (17). If, in the case of bronchoprovocation, the increased ventilation-perfusion mismatch can be accounted for mainly by ventilation heterogeneities, the present data predict a more marked dissociation between spirometry and ventilation-perfusion mismatch for methacholine than for histamine, at least in normal subjects.

On the basis of S_cond or Curv, it is a priori not possible to locate the heterogeneity of airway narrowing to proximal or peripheral airways within the conductive airway tree (i.e., distinguish between large and small conductive airways). Alternatively, spirometry may not be able to adequately represent narrowing of the most peripheral airways in the conductive airway tree, i.e., the entrance to the silent zone of the lungs. It is nevertheless tempting to speculate on a possible link between the behavior of ventilation maldistribution and spirometry. Let us consider the double-dose group results, in which FEV₁ decreases less (Fig. 2), FEF₇₅ decreases to the same extent (Fig. 4), and S_cond increases more (Fig. 5) after methacholine than after histamine. If the methacholine-induced S_cond increases originated in the large airways, this agent would need to have provoked a large heterogeneity in airway narrowing among a relatively small number of parallel airways while maintaining a milder overall airway narrowing than with histamine (as indicated by FEV₁ and PEF). The alternative possibility seems more likely: methacholine-induced S_cond originates in the small airways, where it provokes a larger heterogeneity in airway narrowing among a relatively large number of parallel airways, for an overall airway narrowing similar to that obtained with histamine (as indicated by FEF₇₅). We should also not exclude the possibility that S_cond may have originated in conductive airways that are even smaller than those that can be reflected in FEF₇₅. Under this assumption, FEF₇₅ merely showed a transitional behavior between the large airways (reflected in FEV₁ and PEF) and these smaller airways (reflected in S_cond and Curv). Finally, we consider the possibility that heterogeneity may also affect the spirometric indexes, despite experimental data by McNamara et al. (13) showing that the forced expired flow-volume relationship fails to reflect interregional heterogeneity of alveolar pressure, as induced by histamine inhalation in dogs. If heterogeneity were a major contributor to FEV₁, our observations would actually imply that the worst ventilation heterogeneity (as induced by methacholine) causes the least deterioration in FEV₁.

The present data may give new impetus to the search for the mechanisms of histamine and methacholine action along the bronchial tree. The S_cond results strongly suggest that when characterizing any mechanism of airway narrowing (e.g., localization of receptors) or when visualizing airway narrowing itself [e.g., high-resolution computed tomography (HRCT)-derived airway lumen], one should not only look for number of receptors or for overall airway lumen decreases at different lung depths but should quantify the parallel heterogeneity in distribution of these receptors, or heterogeneity in constriction of airways, at any given lung depth as well. Because the differentiation between histamine and methacholine clearly occurs within the conductive lung zone, present HRCT resolution (12) is probably sufficient to quantify heterogeneity of airway narrowing. Such information could then be fed into models such as those proposed by Gillis and Lutchen (9) to estimate resulting distributions of specific ventilation, from which S_cond and Curv values can be computed for quantitative comparison with experimental data. Also, the similarity between S_cond and Curv (i.e., the significant role of specific ventilation differences) suggests that differences between histamine and methacholine could be revealed by three-dimensional imaging of ventilation distribution. If the hypothesis holds that S_cond originates at least in part from the smaller, but still conductive, airways, ventilation maldistribution will be operational between lung units as small as groups of acini, and resolution may be a limiting factor.

Sekisawa et al. (19) previously suggested that methacholine was operational mainly in the large airways, whereas histamine affected both large and small airways, on the basis of the respective changes of VD_anat and respiratory resistance (and considering equal histamine and methacholine concentrations in terms of mg/ml). This conclusion has been challenged by other studies using HRCT in dogs (3) and humans (15), demonstrating at least the ability of methacholine to contract the small airways. In the dog study (3), methacholine was in fact seen to provoke a slightly greater airway narrowing than histamine throughout the airway tree. In contrast to Sekisawa et al. (19), we did not find any significant difference between VD_anat decreases after either histamine or methacholine. In the present study with nonhyperresponsive normal subjects, VD_anat changes were small (~10 ml) and subtle differences may have disappeared in the measurement noise, although FRC and breathing frequency were well controlled and showed no significant intrasubject differences across all baseline and provocation stages.

In summary, the present study illustrates a clear dissociation between outcome of spirometry and ventilation heterogeneity in response to methacholine and histamine challenges in normal subjects. However, such observations are not incompatible if both overall airway narrowing and parallel heterogeneity are considered. The present data therefore suggest that future efforts toward the understanding of histamine and methacholine along the airway tree should be oriented toward quantification of both serial and parallel distribution of airway constriction or the underlying sources of constriction. Our data strongly suggest that these should be found in the conductive airway tree, and we speculate that the larger parallel heterogeneity in constriction seen for methacholine than for histamine may originate in the small conductive airways.