INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The effect of deep inspiration (DI) on airway caliber has long been a subject of debate. In 1961, Nadel and Tierney (1) showed that although DI had no effect on airway resistance in healthy subjects in the control state, it reduced resistance when induced bronchoconstriction was present. In 1968, Froeb and Mead (2), studying healthy subjects, found an increase in anatomical dead space, suggesting dilatation of the airways after DI. Fish and coworkers (3) compared the effect of DI on methacholine-induced bronchoconstriction in asthmatic subjects with that in nonasthmatic subjects. They showed that the reduction of airway resistance after DI seen in normal subjects was diminished or absent in asthmatic subjects. Later studies have shown variously that the bronchodilating effect of DI is diminished, absent, or even reversed in asthmatic subjects (4), with some studies showing an inverse relation between the bronchodilating effect of DI and the severity of asthma, as measured by baseline airway caliber or reactivity (4, 6). There is general agreement that the bronchodilating effect of DI is enhanced in the context of methacholine induced bronchoconstriction in both asthmatic (5, 7) and nonasthmatic subjects (1, 10).

The relative attenuation of the bronchodilating effect of DI seen in asthmatics led Fish and coworkers (3) to hypothesize that hyperresponsiveness in asthma is caused by impaired ability of inspiration to stretch airway smooth muscle. Skloot and coworkers (11) reasoned that if this hypothesis were true, the sensitivity to inhaled methacholine of both normal and asthmatic subjects should be the same if the challenge was carried out under conditions where DI was prohibited. They performed methacholine challenge under such conditions in asthmatic and control subjects and found no apparent differences in the responses of the two groups. As we had theoretical reservations that their index of bronchoconstriction might obscure differences between the responses of the two groups, we retested the hypothesis using an index not prone to such an error.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We studied 10 mildly asthmatic and 10 normal subjects (Table 1). None of the asthmatic subjects was receiving more than 400 µg of inhaled steroid (beclomethasone) per day. Normal subjects were all hospital employees. They reported no symptoms of asthma, had never received a diagnosis of asthma from a physician, and had normal responses to a standard methacholine challenge. All subjects were non-smokers and had had no recent upper respiratory tract infection. Approval was obtained from the local Ethics Committee, and written informed consent was obtained.

Subjects performed a modified version of a standard methacholine challenge (12). Methacholine aerosol was administered in doubling cumulative doses (3,125 to 6,400 µg) from a Mefar dosimeter (MB3; Mefar, Borezzo, Italy) at 5 min intervals until a 40% decrement in FEV₁ was recorded or the dose sequence completed. The aerosol was released electronically (using a thermistor) in 10-µl (± 10%) aliquots over 1.5 s as the subject began to inhale from end tidal expiration. He or she continued to inhale (or breathhold after full inspiration) for a further 4 s. Five aliquots inhaled in rapid succession constituted a single challenge dose, further doses being administered at 5-min intervals. Each dose was administered immediately on completion of the measurements following the previous dose. After each dose of methacholine subjects were asked to avoid taking a deep breath. A period of approximately 4 min elapsed between the last DI associated with methacholine administration and measurement of airway function. During the final minute prior to measurement, tidal breathing was monitored with the subject breathing through a flow sensor to confirm the absence of DI. Subjects then performed a forced expiration from the end of a normal tidal inspiration to residual volume (RV). A full inspiration to TLC completed the maneuver. Assuming that TLC did not change (13, 14), this allowed measurement of flow at the same absolute lung volume at each stage of challenge. As the volume at which subjects commenced their partial expiration inevitably varied, the lung volume (expressed as percentage baseline VC) at which partial flow (p) was measured was selected such that in all subjects at all stages of challenge, the partial expiration was commenced above that point. The greatest volume that satisfied this condition was 35% baseline VC (65% expired). The expiratory flow at this volume during a partial expiration (p₃₅), was extracted using software linked to the flow sensor (max; SensorMedics, Anaheim, CA). In addition we calculated the index  used by Skloot and coworkers (11) (defined as: FET_25-75/ ln3, where FET_25-75 is the time to expire the middle 50% of the partial VC and ln3 is natural log of 3) from the same forced partial expiratory maneuvers. The volume at the end of tidal inspiration (EILV) just prior to the forced partial expiration was determined as a percentage of baseline VC using the subsequent TLC as a reference point.

All control subjects completed the challenge sequence to the final total cumulative dose of 6,400 µg methacholine. For a number of the asthmatic subjects the challenge was terminated by a 40% fall in FEV₁ before the dose sequence was complete. The highest delivered dose common to all subjects (asthmatic and control) was 50 µg. Comparison of response between the two groups was therefore made at this dose. All comparisons between two groups were made using Student's two-tailed t test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

As expected, there was a clear difference in the standard response to challenge as measured by FEV₁ between the two groups (Table 2). In addition, however, the highly significant difference in response as measured by p₃₅ shows that even in the absence of DI the asthmatic subjects were more responsive. The difference in response as measured by the  index, which had been derived from the same forced expiratory maneuvers as p₃₅, failed to reach statistical significance. We found a greater rise in EILV and RV between baseline and 50 µg methacholine in asthmatic subjects than in control subjects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The hypothesis under investigation is that "asthmatic hyperresponsiveness is due to a problem of smooth muscle relaxation with deep inspiration" (11). This idea was first mooted by Fish and coworkers in 1981. At the heart of the hypothesis is that the asthmatic and healthy responses to DI differ. Fish and coworkers compared the effect of DI on methacholine-induced bronchoconstriction in asthmatic and nonasthmatic subjects. The reduction of airway resistance after DI in control subjects was less or absent in asthmatic subjects. The results were attributed to a failure, in asthmatic subjects, of DI to stretch (and relax) airway smooth muscle. Subsequently, a number of researchers have investigated the response to DI in asthmatic subjects. Although attenuation of the bronchodilator effect in asthma has been a common finding (4), several investigators have found a bronchoconstrictor effect of DI in some asthmatics, particularly in those with more severe disease (4, 9, 15, 16). Such findings do not contradict the proposed mechanism of Fish and coworkers, but they cannot be entirely explained by it as an additional mechanism to account for the bronchoconstriction is required. The mechanism(s) involved have been the subject of much debate. No final consensus has been achieved, though some data support a mechanism involving an altered balance between airway and parenchymal hysteresis (17). Whatever the mechanism, the diminution (or reversal) of the bronchodilating effect of DI on induced bronchoconstriction in asthmatics implies that if DI is prohibited the difference in responsiveness between asthmatic and normal subjects is inevitably reduced. It is therefore important to determine whether, in the absence of DI, there is complete loss of asthmatic hyperresponsiveness or only the relative diminution that would be predicted by previous studies. Only complete loss would support the revised (and more limited) hypothesis that hyperresponsiveness in asthma can be accounted for entirely by the altered response to DI.

Studies (18) using specific airway conductance (SGaw) as the index of airway function have consistently shown hyperresponsiveness in asthmatic subjects. In principle such measurements are independent of DI but it is not clear from these reports to what extent, if any, DI was prohibited prior to the measurement of SGaw. The influence of any preceding DI therefore could not be excluded.

In the present study we have measured the responses of asthmatic and control subjects to methacholine in the absence of DI. To do this requires an index of bronchoconstriction that is independent of both the effects of DI and any change in EILV that occurs as bronchoconstriction progresses. Our results show that the latter is relevant as a greater increase in EILV during challenge was seen in the asthmatic subjects. Independence from DI was achieved by prohibiting DI for approximately 4 min prior to measurement of the partial flow. Volume independence was ensured by measuring p at the same absolute lung volume at each stage of challenge, in this case 35% baseline VC. The assumption that p₃₅ is measured at isovolume relies on the assumption that TLC remains unchanged during induced bronchoconstriction. The data of Kirby and coworkers (13) and Lougheed and colleagues (14) support the validity of this assumption as both studies showed no change in TLC with methacholine-induced airway narrowing.

Using partial flow we found a highly significant difference between the asthmatic and control responses to methacholine challenge, implying that even in the absence of DI, asthmatics are more responsive to methacholine than are control subjects. This leads us to the conclusion that hyperresponsiveness in asthma cannot be attributed entirely to an abnormal response to DI. This conclusion would appear to contradict the results of Skloot and coworkers (11) who, using a different protocol, found that in the absence of DI asthmatic and control responses were similar. There are, however, two essential methodologic differences between our study and that of Skloot and coworkers: (1) We used isovolume flow as the main index of bronchoconstriction, whereas they derived an index in the time domain; (2) Skloot and coworkers prohibited deep breaths for the entire duration of the challenge, whereas our protocol allowed DI during the challenge, although partial maneuvers were preceded by a 4-min DI-free period. Let us consider each of these in turn.

The  Index

Skloot and coworkers (11) derived an index of bronchoconstriction from partial forced expiratory maneuvers. Because the lung volume (EILV) at which forced expiration is initiated varies, they defined an index in the time domain, , which equals the forced expiratory time between 25 and 75% of the partial expiration divided by the natural log of 3. They showed that the response to methacholine as measured by  was similar in asthmatic and control subjects and concluded that asthmatic hyperresponsiveness was attributable to a lack of smooth muscle relaxation with DI. The volume independence of the index relies on the assumption that the volume-time relation during forced expiration approximates to a monoexponential function. Under this assumption Skloot and coworkers argued that the  index is "equal to the reciprocal of the mean slope of the flow volume curve between 25% and 75% of the forced expiration" (11). In fact the assumption implies that the descending limb of the flow volume curve is rectilinear and that  is equal to the reciprocal of the gradient of the entire slope of the flow volume curve. Whereas this is a reasonable assumption in healthy young subjects, with airway narrowing the flow volume curve is characteristically concave and therefore the volume time curve is not monoexponential. The more severe the airway narrowing, the farther the deviation from this assumed curve. This implies that  is not independent of volume, rather, it will decrease if tidal breathing occurs over a higher volume range. For a given degree of bronchoconstriction therefore, an increase in EILV (or RV) will result in a lower value of , suggesting less bronchoconstriction. The rise in EILV as bronchoconstriction progresses therefore tends to mask the change in . As the rise in EILV was greater in our asthmatic subjects than in our healthy subjects (mean, 15 versus 2.4% of baseline FVC, respectively), the masking effect would be greater in that group. The net effect would therefore be to underestimate the difference in responsiveness between the two groups. When assessed in a typical asthmatic subject, a 15% rise in EILV reduced the observed increase in  over the course of challenge by 54%. A change in EILV in healthy subjects has less effect on  because, as described above, the volume-time relation of forced expiratory flow more closely approximates to a monoexponential; FET_25-75 and  are therefore less volume dependent. When assessed in a typical healthy subject a 2.4% increase in EILV at the end point of challenge was found to have no measurable effect on .

We found a clear difference in the response as measured by p₃₅ between the two groups. When  was applied to the same partial expiratory maneuvers, we found, as did Skloot and coworkers (11), a difference in responsiveness, which was not statistically significant. This suggests that the absence of a significant difference in  in both studies may be due to an inherent flaw in the index, rather than to a true absence of difference.

In the current study the responses of the two groups were compared after a cumulative methacholine dose of 50 µg, at which point the mean fractional changes in  in asthmatic and healthy subjects were 1.77 and 0.84, respectively (a difference that did not achieve statistical significance, p = 0.075). At the point of comparison in the study by Skloot and coworkers, the mean fractional changes in  in both asthmatic and healthy subjects were considerably less at 0.263 and 0.245, respectively. We have therefore also compared the responses of the two subject groups in the current study after a dose of methacholine, which in our healthy subjects produced a similar mean change in  to that seen in the study of Skloot and coworkers. After 3,125 µg of methacholine the mean fractional changes in  in asthmatic and healthy subjects were 0.34 and 0.25, respectively (p = 0.62), i.e., as in the study of Skloot and coworkers, at this stage of challenge the asthmatic and healthy responses are indistinct.

We would argue, therefore, that the apparent difference between the results of the two studies (the current demonstrating a distinction between asthmatic and healthy responses, whereas the Skloot study did not) is due to a difference in the index of airway function used rather than to a difference in either subject selection or challenge protocol.

Duration of the DI-free Period

It has been our experience that prohibition of DI for the entire duration of the challenge is impracticable. Even if DI can be consciously resisted for prolonged periods the cough frequently provoked by methacholine effectively ends the DI-free period. In addition to the practical difficulties, we had reservations as to whether the prohibition of DI for such an extended period was actually desirable. The available evidence on the duration of the effect of DI (7, 21, 22) suggests that this is brief. Using SGaw, Lim and colleagues (7) attempted to quantify the duration of the effect of DI separately in two groups, one that showed bronchodilatation after DI, the other bronchoconstriction after DI. The group showing bronchodilatation had a quicker recovery from the effect of DI than did the group demonstrating bronchoconstriction. Even with the slower recovery of the second group there was effectively complete restoration of baseline caliber well within a 4-min period. Pellegrino and coworkers (21), also using SGaw, found almost complete restoration of baseline caliber 10 s and 60 s after DI in control and asthmatic subjects, respectively. Green and Mead (22), measuring partial flow at various time intervals after deep inspiration in healthy subjects, found most of the bronchodilating effect of DI had worn off after only 5 s.

This short-term bronchodilating effect is probably related to (though perhaps not entirely accounted for by) stretching of smooth muscle, as an enhanced bronchodilating effect to DI is seen in the presence of smooth muscle constrictors (1, 5, 7, 10) and a diminished bronchodilating effect of DI in the presence of beta-2-agonists (5, 6, 23). On the other hand, prolonged inhibition of DI, particularly in the context of induced bronchoconstriction, may have other effects, in particular widespread micro-atelectasis. This in itself could affect expiratory flow. If we wish to study the effect of DI on smooth muscle tone, such potentially confounding effects are better avoided.

On the basis of the evidence available we would suggest that the 4-min DI free period used in our study is sufficient to identify the short-term effects of DI while not being so long as to induce unwanted effects, unrelated to smooth muscle stretching, on expiratory flow.

In summary, less difference in the responsiveness of asthmatic and control subjects to methacholine when DI is prohibited is implicit in the results of earlier studies. At issue is whether, in the absence of DI, the asthmatic and control responses to methacholine are actually the same. Our results suggest that the responses remain different even if DI is avoided, thus refuting the hypothesis that asthmatic hyperresponsiveness can be accounted for entirely by an altered response to DI. These results differ from those of Skloot and coworkers (11), and we suggest that the main reason for this difference is that the  index does not allow valid comparison between asthmatic and control responses when the volume time relation during forced expiration does not conform to a monoexponential and the relative volume ranges of tidal breathing differ.

We conclude, therefore, that airway hyperresponsiveness in asthma cannot be accounted for solely by an abnormal response to deep inspiration.