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(Chest. 1999;116:1653-1658.)
© 1999 American College of Chest Physicians

Effect of Mild Hypoxia on Airway Responsiveness to Methacholine in Subjects With Airway Hyperresponsiveness*

Hiroshi Saito, MD, FCCP; Masaharu Nishimura, MD; Hideki Shinano, MD; Fumihiko Sato, MD; Kenji Miyamoto, MD and Yoshikazu Kawakami, MD, FCCP

* From the First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan.

Correspondence to: Corresponding Author; Masaharu Nishimura, MD, First Department of Medicine, Hokkaido University School of Medicine, Kita 15-Jou Nishi 7-choume, Kita-ku, Sapporo, 060-0015, Japan


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Study objectives: The inhalation of hypoxic gas has been reported to enhance the airway responsiveness to methacholine in some animal models. However, the data on humans have so far been conflicting. We attempted to examine the effect of hypoxic gas inhalation on the airway responsiveness to methacholine.

Design: We evaluated the airway responsiveness to methacholine by continuously measuring respiratory conductance with the forced oscillation method under normoxia or hypoxia in a single-blind, randomized, crossover fashion (2 days for each).

Participants: Twelve asymptomatic male volunteers (mean ± SD age, 27 ± 4 years) with airway hyperresponsiveness to methacholine. Two of the 12 volunteers had a history of bronchial asthma.

Setting: The participants inhaled either normoxic or hypoxic gas with continuous inhalation of aerosolized methacholine in incremental doses with a sustained respiratory rate of 15 breaths/min. The arterial oxygen saturation was kept to 90% on the hypoxic days.

Results: There were no significant differences in any indexes of airway responsiveness to methacholine (the cumulative dose of methacholine at the threshold and the point of 35% decrease of the respiratory conductance, and the slope factor of the dose-response curve) between the hypoxic days and the normoxic days.

Conclusion: The inhalation of mildly hypoxic gas does not enhance the airway responsiveness to methacholine in humans with airway hyperresponsiveness.

Key Words: airway responsiveness • human • hypoxia • methacholine


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The inhalation of moderately hypoxic gas (fraction of inspired oxygen [FIO2], 0.10 to 0.13) is reported to enhance the airway responsiveness to methacholine in canine,1 sheep,2 and rabbits.3 Possible local mediators that explain this hypoxia-induced airway hyperresponsiveness include mast cell-derived chemical mediators such as leukotriene-C42 4 5 and adenosine,6 a degraded nucleoside from adenosine triphosphate under hypoxic conditions. Another possible mechanism is hypoxic stimulation of the peripheral chemoreceptors, which in turn influences the nervous control of airway caliber.7

Despite these animal studies, the data on humans have so far been scanty and conflicting. Although there has been, to our knowledge, only one study that demonstrated the enhancing effect of mild hypoxia (FIO2, 0.155) on airway responsiveness to methacholine,8 it was not confirmed in two other studies.9 10 The issue is of clinical importance because if hypoxia enhances airway responsiveness, it may play some role in the airway hyperresponsiveness observed in those patients with hypoxemia for any reason.

The purpose of this study is to examine in clinically stable subjects with airway hyperresponsiveness whether the inhalation of mildly hypoxic gas really enhances the airway responsiveness to methacholine. To achieve this goal, we used a more sophisticated apparatus and a more carefully designed protocol than was used in any previous studies.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Subjects
Twelve male volunteers (mean ± SD age, 27 ± 4 years) served as subjects for this study with informed consent. They were medical students or doctors recruited from Hokkaido University School of Medicine. The protocol of this study was approved by the Ethics Committee of the Hokkaido University School of Medicine. The subjects included 2 patients with a history of bronchial asthma and 10 asymptomatic volunteers with airway hyperresponsiveness evidenced by the bronchial provocation test (described below), but who had been clinically stable and free from any medication for bronchial asthma for, at least, the 3 previous months.

Apparatus
Respiratory resistance (Rrs) was continuously measured using the forced oscillation method with a commercially available system (Astograph TCK-6000M; Chest; Tokyo, Japan).11 This system can display a dose-response curve of Rrs for continuous inhalation of the aerosolized drug while the subject is under tidal breathing. Briefly described, the system consists of an aerosol delivery part, a loudspeaker box system generating a constant-amplitude sine wave pressure at 3 Hz, and another part for measuring Rrs automatically from mouth flow and mouth pressure. Aerosols containing 4 mL solution are driven with a constant airflow of 6 L/min by an air compressor to elicit an output of approximately 0.15 mL/min. The particle size ranges from 0.5 to 4.0 mm.11 Figure 1 shows a block diagram of the experimental setup that consists of the above-mentioned system and a gas controller system. Inhalation gas was made by a gas controller system using a gas blender (Gas Blender No. 10; Taiyo Sanso; Tokyo, Japan). After being properly humidified and warmed, the gas was sent to both the compressor of the nebulizers and the loudspeaker box via a reservoir bag. The advantages of this system are continuous measurement and noninvasiveness.



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  		Figure 1. A block diagram of the experimental setup. Rrs was continuously measured using the forced oscillation method. The experimental setup consists of an aerosol delivery part, a loudspeaker box system generating a constant-amplitude sine wave pressure at 3 Hz, and another part for measuring Rrs automatically from mouth flow and mouth pressure. Inspiratory and expiratory gas fractions of carbon dioxide and oxygen were monitored at the mouthpiece using a mass spectrometer. The thoracic and abdominal movements were recorded using a RIP. SaO2 and heart rate (HR) were continuously monitored using pulse oximetry applied to the fingertip. RR = respiratory rate.

 
PCO2 and PO2 of inspiratory and expiratory gas were monitored at the mouthpiece with a mass spectrometer (Medical Gas Analyzer 1100A; Marquette; Milwaukee, WI). In order to compare the respiration rate and the tidal volume (VT) between normoxia and hypoxia, the thoracic and abdominal movements were recorded with a respiratory inductance plethysmograph ([RIP]; Respitrace System, Model 10.5100-5; Ambulatory Monitoring; Ardsley, NY). Change of ventilation was calculated by a simultaneous equation method.12 Arterial oxygen saturation (SaO2) and heart rate were continuously monitored using pulse oximetry applied to the fingertip (Biox 3740; Ohmeda; Louisville, CO).

Protocol
The subjects underwent four series of trials on 4 separate days, with an interval of at least 3 days between trials. The 4 days consisted of 2 days for the normoxic condition and 2 days for the hypoxic condition in a single-blind, randomized, crossover fashion. Accordingly, six subjects were studied in the order hypoxia-normoxia-normoxia-hypoxia, and the other six subjects were studied in the order normoxia-hypoxia-hypoxia-normoxia. The mean values of the 2 days were adopted as representative of each condition.

On each experimental day, we first measured a flow-volume curve and a baseline Rrs to ensure that there were no significant differences in baseline pulmonary function among the experimental days for a given subject and that the subjects were all clinically stable.

During an experimental run, the subject was requested to breathe through a mouthpiece at a rate of 15 breaths/min with use of a metronome. Inhaled gas was continuously adjusted either for normoxia (FIO2, 0.21) or for hypoxia (SaO2, 90%). The gas was carbon dioxide free and appropriately warmed and humidified. After the ventilation and SaO2 leveled off for at least 5 minutes, the subject underwent a methacholine provocation test that consisted of continuous measurement of Rrs for repeated administration of methacholine in incremental doses.

Methacholine was prepared in 0.9% saline solution in twofold increasing concentrations ranging from 0.78 to 400 mg/mL. After it was confirmed that a 2-min inhalation of saline solution did not change the baseline Rrs, each concentration of methacholine solution was inhaled for 1 min until Rrs reached about twice the baseline value or until the maximum concentration was administered. A solution of 10 mg of salbutamol diluted in 0.9% saline solution was inhaled for 2 min after provocation was stopped.

Analysis
In this study, we used the following three parameters to assess the airway responsiveness to methacholine11 13 : (1) the cumulative dose of methacholine at the point where respiratory conductance (Grs; the reciprocal of Rrs) starts to decrease linearly (Dmin; unit); (2) bronchial reactivity (per minute), the slope of the dose-response curve of Grs against inhalation time (L/s/cm H2O/min) per Grs control (L/s/cm H2O); and (3) the cumulative dose of methacholine when Grs decreased by 35% from the initial value (PD35; unit).14 The cumulative dose of methacholine was calculated in terms of a unit defined as 1-min inhalation of 1 mg/mL of methacholine during tidal breathing. Dmin and PD35 were obtained using log-transformed data before analysis; thus, statistics were expressed as the geometric mean and per-cent standard error. All values represent the average of two separate trials for each experimental condition. Airway hyperresponsiveness in this study was defined as PD35 < 200 U (logarithm of PD35 < 2.30) and/or Dmin < 50 U (logarithm Dmin < 1.70).11 13 Wilcoxon’s signed-rank test was used to assess the statistical significance between hypoxic day and control day, or before methacholine challenge and the point of PD35. All data except for age and spirographic measures are presented as means ± SEM.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Subjects’ profiles and mean pulmonary function data for the 4 experimental days are summarized in Table 1 . All parameters taken from the flow-volume curve were within normal limits. There were no significant differences in the baseline pulmonary function tests among the 4 experimental days, and the coefficient variances were not > 5% in every subject.


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  		Table 1. Subjects’ Profile (n = 12) and Mean Spirographic Data*

 
On the day when the subjects inhaled hypoxic gas, SaO2 monitored using pulse oximetry was 89.7 ± 0.6% before methacholine inhalation and 89.5 ± 0.4% at the point of PD35; on the control day, these measures were 97.4 ± 0.2% and 96.9 ± 0.2%, respectively (Table 2 ). To keep SaO2 at about 90% throughout the experiment on the hypoxic days, FIO2 (mean ± SD) varied from 0.144 ± 0.006 to 0.152 ± 0.008 for all subjects. The time from the start of hypoxia to PD35 was from 5 to 10 min (mean ± SD, 8.1 ± 1.6).


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  		Table 2. Mean Physiologic Parameters Under Each Experimental Condition*

 
PCO2 in the end-tidal expiratory gas (PETCO2) at the point of PD35 was significantly lower than that before methacholine challenge (36.1 ± 1.2 mm Hg vs 34.0 ± 1.3 mm Hg for the control and 35.7 ± 0.9 mm Hg vs 34.3 ± 1.2 mm Hg for hypoxia (p < 0.01 on both experimental days; Table 2 ). Thus, although we requested that the subjects sustain normal tidal breathing at a fixed rate, they must have had significantly larger VT breathing at the point of PD35 compared to the baseline condition. There was, however, no significant difference in the mean value of PETCO2 between the control day and the hypoxic day (36.1 ± 1.2 mm Hg vs 35.7 ± 0.9 mm Hg before methacholine challenge, and 34.0 ± 1.3 mm Hg vs 34.3 ± 1.2 mm Hg at the point of PD35; Table 2 ). Similarly, there was no significant difference in VT measured using RIP between the 2 experimental days.

Concerning the parameters of airway hyperresponsiveness, there were no significant differences in logarithm of Dmin (0.99 ± 0.15 vs 0.97 ± 0.13), logarithm of PD35 (1.50 ± 0.16 vs 1.44 ± 0.14), and bronchial reactivity (0.21 ± 0.01/min vs 0.22 ± 0.02/min) between the control day and the hypoxic day, respectively (Fig 2 ).



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  		Figure 2. A comparison of airway responsiveness to methacholine between normoxia and hypoxia. There were no significant differences in any of Dmin, PD35, or bronchial reactivity between the normoxic days and the hypoxic days. Each value represents the average of two separate trials for each experimental condition. See text for the definition of PD35, Dmin, and bronchial reactivity. The closed circles and bars are mean ± SEM. N.S. = not significant.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this study, we demonstrated that inhalation of mildly hypoxic gas did not enhance airway responsiveness to methacholine in asymptomatic male subjects who had a history of bronchial asthma or who had airway hyperresponsiveness evidenced by previous provocation tests.

Although some animal studies previously demonstrated a positive enhancing effect of hypoxia on the airway responsiveness,1 2 3 and the issue of their studies seemed to be the mechanism by which inhalation of hypoxic gas caused such enhancement in airway hyperresponsiveness in animals, it seems to be an open question as to whether the hypoxic enhancement of airway hyperresponsiveness holds true in humans. Although there has been, to our knowledge, only one study that demonstrated hypoxic enhancement of airway responsiveness to methacholine in human subjects,8 it was not confirmed in two other studies.9 10 In the study by Tam et al,9 although they challenged relatively severe hypoxia (FIO2, 0.07 to 0.10), they could not detect any effect of hypoxia on airway hyperresponsiveness. However, it could be argued that severe hypoxia might increase sympathetic output and thereby cancel the enhancement of airway responsiveness. In addition, the inhalation gas they used was not humidified, so it might have affected the bronchomotor tone in various ways. In the second study by Alberts et al,10 although these problems were overcome, their method of evaluation for airway hyperresponsiveness needed a forced expiratory maneuver, which in turn might influence the baseline level of bronchomotor tone. Moreover, as the subject’s maximal effort was required for all the repeated measurements throughout the study, the intrasubject variation might be wide, thus masking any small difference.

In the study by Denjean et al,8 they demonstrated a significant enhancement of hypoxic gas inhalation on the airway hyperresponsiveness to methacholine in 10 subjects with bronchial asthma. They confirmed that the level of mild hypoxia (FIO2, 0.15) did not affect plasma catecholamines. The differences between their study and ours may be several fold. First, while we measured Rrs as indexes of airflow obstruction, they used lung resistance instead. For the measurement of lung resistance, they had to insert an esophageal balloon into each subject. The discomfort caused by the esophageal balloon may destabilize the autonomic nervous control of airways. Second, the breathing pattern during the tests should be different between the two studies. We instructed the subjects to breathe at a fixed rate (15 breaths/min) as much as possible, because we wanted to avoid "the rapid shallow breathing" that might affect the airway responsiveness independent of the hypoxic stimuli itself. The difference in the degree of hyperventilation may produce the alteration of the airway responsiveness when the temperature of the inhalation gas was not equal to body temperature. Although Denjean et al8 allowed the subjects to use any breathing pattern at the time of study, they unfortunately did not monitor the ventilation and the respiratory pattern. Third, the SaO2 level may be different between the two studies. We continuously attempted to keep the SaO2 at 90% throughout the experiment (FIO2, 0.144 to 0.152), while they used the fixed level of oxygen fraction (FIO2, 0.155) so that SaO2 during the experiment was unpredictable due to variable level of ventilation in response to hypoxia and bronchoconstriction.

The method we applied was noninvasive in nature, and the data obtained were not modified by the change in VT and/or intrathoracic pressure. There were no differences between normoxia and hypoxia at PD35 for heart rate, respiratory rate (15 breaths/min), PETCO2 (ie, minute ventilation) and VT except for SaO2 (96.9 ± 0.2% vs 89.5 ± 0.4%, respectively). It meant that ventilation certainly increased at the level of PD35 on both experimental days, but there was no hypoxia-induced ventilatory augmentation at the SaO2 level of 90%. Accordingly, our data could be interpreted to examine the pure effect of hypoxemia on the airway hyperresponsiveness that was independent of respiratory pattern changes.

The findings of this study have a couple of clinical implications. First, one does not have to be concerned about the possible detrimental effect of mild hypoxia caused either by exercise or by nocturnal arterial oxygen desaturation on airway responsiveness in those who have bronchial asthma. Second, if the worsening of bronchial asthma occurs under hypoxic conditions for any reason, it should not be due to hypoxia itself, but due to the consequence of the pathophysiologic changes causing hypoxia or due to hyperventilation caused by hypoxia.

Mention should be made of the possibility that severe hypoxia may enhance the airway responsiveness to methacholine. The level of hypoxia used in this study may not be severe enough to cause any inherent effect of hypoxia on the airway responsiveness. As is suggested in several animal studies, severe hypoxia may induce mast cell-derived chemical mediators such as leukotriene-C42 4 5 and/or adenosine in the airways, causing hyperresponsiveness also in humans. Alternatively, more vigorous hypoxia via the stimulation of the peripheral chemoreceptors may influence the nervous control of airway hyperresponsiveness in humans.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
We demonstrated in human subjects with airway hyperresponsiveness that mild hypoxia has no enhancing effect on the airway responsiveness to methacholine under conditions where ventilation is not increased.


   Footnotes
 
Abbreviations: Dmin = cumulative dose of methacholine at the point where reciprocal of respiratory resistance starts to decrease linearly; FIO2 = fraction of inspired oxygen; Grs = reciprocal of respiratory resistance; PD35 = cumulative dose of methacholine when Grs decreased by 35% from the initial value; PETCO2 = end-tidal carbon dioxide pressure; RIP = respiratory inductance plethysmograph; Rrs = respiratory resistance; SaO2 = arterial oxygen saturation; VT = tidal volume

This study was funded by The Ministry of Education, Science, Sports and Culture of Japan.

Received for publication January 8, 1999. Accepted for publication July 8, 1999.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Vidruk, EH, Sorkness, RL (1985) Histamine-induced reflex tracheal constriction is attenuated by hyperoxia and exaggerated by hypoxia. Am Rev Respir Dis 132,287-291[ISI][Medline]
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  6. Cushley, MJ, Tattersfield, AE, Holgate, ST (1984) Adenosine-induced bronchoconstriction in asthma. Antagonism by inhaled theophylline. Am Rev Respir Dis 129,380-384[ISI][Medline]
  7. Denjean, A, Canet, E, Praud, JP, et al (1991) Hypoxia-induced bronchial responsiveness in awake sheep: role of carotid chemoreceptors. Respir Physiol 83,201-210[CrossRef][ISI][Medline]
  8. Denjean, A, Roux, C, Herve, P, et al (1988) Mild isocapnic hypoxia enhances the bronchial response to methacholine in asthmatic subjects. Am Rev Respir Dis 138,789-793[ISI][Medline]
  9. Tam, EK, Geffroy, A, Myers, DJ, et al (1985) Effect of eucapnic hypoxia on bronchomotor tone and on the bronchomotor response to dry air in asthmatic subjects. Am Rev Respir Dis 132,690-693[ISI][Medline]
  10. Alberts, WM, Colice, GC, Hammond, MD, et al (1990) Effect of mild hypoxemia on bronchial responsiveness. Ann Allergy 65,189-193[ISI][Medline]
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  12. Cohn, MA, Watson, H, Weisshaut, R, et al (1978) A transducer for non-invasive monitoring of respiration. Stott, FD Raftery, EB Sleight, P Goulding, L eds. Proceedings of the Second International Symposium on Ambulatory Monitoring ,119-128 Academic Press London, UK.
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