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Original Research Sleep Disorders

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Sex and Acetazolamide Effects on Chemoreflex and Periodic Breathing During Sleep at Altitude Sergio Caravita, MD; Andrea Faini, PhD; Carolina Lombardi, MD, PhD; Mariaconsuelo Valentini, MD; Francesca Gregorini, MSc; Jessica Rossi, MD; Paolo Meriggi, PhD; Marco Di Rienzo, MSc; Grzegorz Bilo, MD, PhD; Piergiuseppe Agostoni, MD, PhD; and Gianfranco Parati, MD, PhD

OBJECTIVE: Nocturnal periodic breathing occurs more frequently in men than in women with various clinical and pathophysiologic conditions. The mechanisms accounting for this sexrelated difference are not completely understood. Acetazolamide effectively counteracts nocturnal periodic breathing, but it has been investigated almost exclusively in men. Our aim was to explore possible determinants of nocturnal periodic breathing in a high-altitude setting both in men and in women. We hypothesized that increased hypoxic chemosensitivity in men could be associated with the development of nocturnal periodic breathing at altitude more frequently than in women, and that acetazolamide, by leftward shifting the CO2 ventilatory response, could improve nocturnal periodic breathing at altitude in a sex-independent manner.

Forty-four healthy lowlanders (21 women), randomized to acetazolamide or placebo, underwent cardiorespiratory sleep studies at sea level off treatment and under treatment on the first night after arrival at a 4,559-m altitude. Hypoxic and hypercapnic chemosensitivities were assessed at sea level.

METHODS:

Men, more frequently than women, exhibited increased hypoxic chemosensitivity and displayed nocturnal periodic breathing at altitude. Acetazolamide leftward shifted the CO2 set point and, at altitude, improved oxygenation and reduced periodic breathing in both sexes, but to a larger extent in men. Hypoxic chemosensitivity directly correlated with the number of apneas/hypopneas at altitude in the placebo group but not in the acetazolamide group.

RESULTS:

The greater severity of periodic breathing during sleep displayed by men at altitude could be attributed to their increased hypoxic chemosensitivity. Acetazolamide counteracted the occurrence of periodic breathing at altitude in both sexes, modifying the apneic threshold and improving oxygenation.

CONCLUSIONS:

TRIAL REGISTRY: EU Clinical Trials Register, EudraCT; No.: 2010-019986-27; URL: https:// www.clinicaltrialsregister.eu CHEST 2015; 147(1):120-131

Manuscript received February 7, 2014; revision accepted August 10, 2014; originally published Online First September 4, 2014. ABBREVIATIONS: AMS 5 acute mountain sickness; HA 5 high altitude; HCVR 5 hypercapnic ventilatory response; HVR 5 hypoxic ventilatory response; PBS 5 periodic breathing during sleep; Petco2 5 partial pressure of end-tidal CO2; SL 5 sea level; Spo2 5 blood oxygen saturation AFFILIATIONS: From Istituto Auxologico Italiano (Drs Caravita, Faini, Lombardi, Valentini, Rossi, Bilo, and Parati and Ms Gregorini), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Department of Cardiovascular, Neural and Metabolic Sciences, S. Luca Hospital; the Department of Health Sciences (Drs Caravita and Parati), University of Milano-Bicocca; Polo Tecnologico (Dr Meriggi and Mr Di Rienzo), Biomedical Technology Department, Fondazione Don Carlo Gnocchi

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Onlus; Centro Cardiologico Monzino (Dr Agostoni), IRCCS; and the Department of Clinical Sciences and Community Health (Dr Agostoni), University of Milan, Milan, Italy. Drs Caravita, Faini, and Lombardi contributed equally to this manuscript. Part of this article has been presented in abstract form (Lombardi C, Faini A, Meriggi P, et al. Acetazolamide effect on high altitude periodic breathing during sleep. The HIGHCARE Alps project. Eur Heart J. 2013;34(S1):312) and presented at the European Society of Cardiology Congress, August 31–September 4, 2013, Amsterdam, The Netherlands. FUNDING/SUPPORT: This study was supported primarily by a research grant from the Italian Ministry of Health. Supplementary financial support was provided by the IRCCS Istituto Auxologico Italiano, Milan, Italy. Tao Med provided the metabolic chart for high altitude recordings.

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Acute high-altitude (HA) exposure induces periodic breathing during sleep (PBS) in healthy subjects1-3 and could be viewed as an experimental condition able to model some of the pathophysiologic mechanisms of PBS observed in cardiac patients. In patients affected by heart failure, for instance, PBS is highly prevalent,4,5 and its occurrence has been linked to worse outcome.6,7 A number of mechanisms may contribute to explaining the occurrence of PBS in patients with heart failure,5 and among them, increased chemoreflex sensitivity has been suggested as playing a role.8,9 An increased chemoreflex sensitivity may also be related to the genesis of PBS in healthy subjects acutely exposed to HA hypoxia.10 In our research on a previous HA expedition on Mount Everest, we showed that healthy men exposed to HA are characterized by a higher frequency of PBS than are women.3 Interestingly, this finding is in-line with the sex-related difference in the epidemiology of sleep-related breathing disorders in patients with chronic heart failure, in which men seem to be at a higher risk of PBS than are women.4-6 However, the mechanisms accounting for the sex-related differences in PBS frequency in patients with heart failure, as well as in hypoxic conditions at HA, are not yet fully understood.

Acetazolamide has been shown to reduce the burden of PBS in different clinical conditions, including heart failure9,11,12 and idiopathic central sleep apnea13,14 and in HA exposure,1,2,15 but although its efficacy has been proven extensively in men, its use in managing central sleep apnea in female subjects has been reported very rarely.1,2,9,11-15 In fact, it is currently unknown whether acetazolamide influences the respiratory control differently in relation to sex, and may, thus, affect PBS differently in men and in women.

Materials and Methods

Resting Ventilation Ventilatory parameters were recorded at rest on three occasions: (1) at SL, off treatment; (2) at SL on the third day of treatment; and (3) on the third day of stay at HA, on randomized treatment, as described in detail in e-Appendix 1. As a composite measure of the acetazolamide effect on ventilatory control, we calculated at SL the ratio between the increase in minute ventilation and the reduction in partial pressure of end-tidal CO2 (Petco2) as an effect of treatment, both corrected for the changes occurring in the placebo group. Because the effects of acetazolamide on minute ventilation and on Petco2 may be dose dependent,21 we further normalized such computation for the acetazolamide daily dose, to allow for comparison with the results from previous studies.

As described previously,17-19 44 healthy lowlander volunteers (21 women) were included in this randomized, double-blind, parallel-group, placebocontrolled study. After recruitment, the subjects were randomly assigned to receive either oral placebo or acetazolamide, 250 mg bid for 3 days at sea level (SL) in Milan (122 m above SL) and then during the entire duration of their stay at HA (Capanna Regina Margherita, 4,559 m above SL, barometric pressure 437-439 Torr), with pill intake being started the day before ascent to HA. Data were collected in rooms at a stable temperature (20°C-22°C). Women were requested to record menstrual cycle in a dedicated diary and to avoid birth-control pills. At HA, acute mountain sickness (AMS) symptoms were evaluated every morning through the Lake Louise Score Questionnaire.20 The Lake Louise Score was used as a dichotomic variable to determine the presence (score . 4) or absence of AMS. The study was approved by the institutional review board of Istituto Auxologico Italiano (project approval number 2010_04_13_01). The study was conducted in accordance with the Declaration of Helsinki. All subjects gave written informed consent to the study procedures. The sample size of this study was calculated on the basis of its primary aims, as discussed in previous publications.17 CORRESPONDENCE TO: Gianfranco Parati, MD, PhD, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, Department of Cardiovascular, Neural and Metabolic Sciences, S. Luca Hospital, Piazzale Brescia 20, 20149 Milan, Italy; e-mail: gianfranco.parati@ unimib.it © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-0317

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Because low Po2 is thought to be the primary trigger for PBS at HA, we hypothesized that in men, an increased chemosensitivity to hypoxia could account for their greater burden of PBS at HA as compared with women. Additionally, we hypothesized that acetazolamide, by leftward shifting the ventilatory response to CO2 (and thus increasing the difference between the prevailing blood CO2 level and the apneic threshold),16 could improve PBS at HA in a sex-independent manner. The aim of this work was to explore some as-yet poorly explored aspects of the pathophysiology of PBS under exposure to hypobaric hypoxia at HA by focusing on sexrelated differences in chemoreflex sensitivity and on the effects of acetazolamide on respiratory control at altitude.

Chemoreflex Tests Chemoreflex testing (ie, the measurement of the ventilatory responses to isocapnic hypoxia [intended as a stimulus for peripheral chemoreceptors] and to hyperoxic hypercapnia [intended as a stimulus for central chemoreceptors]) was performed at SL, both off and on treatment. Details on the methodology for chemoreflex testing22,23 are available in e-Appendix 1. The hypoxic ventilatory response (HVR) was defined as the ratio between the maximal ventilation and the minimal blood oxygen saturation (Spo2) attained during the hypoxic stimulus.24 The hypercapnic ventilatory response (HCVR) during the hypercapnic stimulus was obtained as the slope of the best-fit line that related ventilation to Petco2, calculated by least-squares linear regression analysis.8 Cardiorespiratory Sleep Studies Cardiorespiratory sleep studies were performed (1) at SL, off treatment, and (2) during the first night at HA, under randomized treatment. Methodologic details3 are available in e-Appendix 1. Scoring of sleepdisordered breathing was calculated in accordance with current guidelines, as described in e-Appendix 1.

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Statistical Analysis Continuous variables are reported as mean ⫾ SD. To assess the effects of altitude level, treatment group, and sex, we used linear mixed-effects models with contrasts a posteriori accounting for repeated measurements, and a compound symmetry covariance structure,25 fitting the models by maximizing the restricted log likelihood. For multiple post hoc comparisons, we used the algorithm proposed by Holm26 that is intended to control the familywise error rate. We performed a linear regression with residuals analysis to evaluate the interaction between the apnea-hypopnea index and the HVR. We performed a Poisson regression analysis to assess the effects of sex and

Results Of the 44 enrolled subjects, 22 were randomized to placebo and 22 to acetazolamide. As shown in Figure 1, data collection was completed in 20 subjects receiving acetazolamide (10 women) and in 21 receiving placebo (10 women). At baseline, there were no significant differences in demographics (Table 1) or ventilatory characteristics between the acetazolamide and placebo groups. Resting Ventilation

Resting ventilatory parameters at SL and at HA are shown in Table 2. Individual data of minute ventilation for the three study conditions (SL off treatment, SL on treatment, HA) are presented in Figure 2. Men had slightly higher ventilation than did women at SL off treatment, but not after correction for body height. Acetazolamide, compared with placebo, did not

treatment on AMS, as evaluated with the Lake Louise Score questionnaire filled in the morning after the cardiorespiratory sleep recording. We studied the relationship between the apnea-hypopnea index and AMS in the placebo group with the two-sample t test using Welch approximation to the degrees of freedom. Distribution of the variables in terms of proximity to normal and the homogeneity of variances were detected by the Shapiro-Wilk test and the Bartlett test, respectively. We performed a logarithmic transformation to achieve normal distribution of minute ventilation and HCVR. An a level of 0.05 was used for all hypothesis tests. All data analyses were performed using R Core Team.

significantly increase minute ventilation corrected for body height at SL on treatment. The net effect of acetazolamide on minute ventilation, after correction for the changes that occurred in the placebo group, was an increase of 0.9 L/min, as shown in e-Table 1. Mean Spo2 at SL (both off and on treatment) was not different between the sexes or the study groups. At SL off treatment, Petco2 was not different between men and women. At SL on treatment, acetazolamide reduced Petco2, compared with placebo. The net effect of acetazolamide on Petco2, after correction for the changes occurring in the placebo group was a reduction of 4.2 mm Hg, as shown in e-Table 1. The effects of acetazolamide on minute ventilation and on Petco2 normalized for the daily dosage intake, as well as the comparison with results from previous studies in which a higher acetazolamide dose was used,16,27 are shown in e-Table 2.

Figure 1 – Consort diagram. CRS 5 chemoreflex sensitivity assessment; F 5 female; HA 5 high altitude; M 5 male; PSG 5 polysomnographic assessment; w/o CO2 CRS 5 without hyperoxic hypercapnic test; w/o SLpre 5 without sea level, off treatment.

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TABLE 1

] Subjects’ Demographic and Anthropometric Data, Strati¿ed for Sex and Randomized Treatment

Variable

Men ACZ

No.

Men PL

Women ACZ

P Valuea

Women PL

10

11

10

10

Age, y

34.3 ⫾ 6.1

39.3 ⫾ 10.7

35.2 ⫾ 8.2

35.2 ⫾ 9.5

.538

…

Weight, kg

72.4 ⫾ 9.3

76.6 ⫾ 9.8

54.9 ⫾ 5.9

56.5 ⫾ 5.2

, .001

Height, cm

179.2 ⫾ 7.5

179.2 ⫾ 6.7

166.2 ⫾ 3.8

164.5 ⫾ 5.3

, .001

BMI, kg/m2

22.6 ⫾ 3.0

23.9 ⫾ 2.6

19.9 ⫾ 1.6

20.9 ⫾ 1.7

, .001

Data are presented as mean ⫾ SD. ACZ 5 acetazolamide; PL 5 placebo. There were no differences between treatment groups at baseline. aP value refers to differences between men and women.

At HA, resting ventilation was higher in men than in women, with no differences between acetazolamide and placebo. After correction for body height, minute ventilation at HA had similar results between the sexes and the treatments. The increase in ventilation from SL on treatment to HA (P , .001) was, on average, 4.6 ⫾ 2.1 L/min, irrespective of sex and treatment. Petco2 at HA was lower in those receiving acetazolamide than in those receiving placebo, with no sexrelated differences. The Petco2 reduction from SL on treatment to HA (P , .001) was more marked in those receiving acetazolamide than in those receiving placebo (9.2 ⫾ 3.2 mm Hg vs 7.7 ⫾ 3.3 mm Hg, P 5 .041). The reduction of Spo2 from SL on treatment to HA (P , .001) was more marked in those receiving placebo than in those receiving acetazolamide (20.5% ⫾ 6.8% vs 12.6% ⫾ 3.7%, P , .001), without sex-related differences. TABLE 2

Chemoreflex Testing

Isocapnic Hypoxic Test: Ventilatory parameters related to the isocapnic hypoxic test are shown in Table 3. Individual HVR data are presented in Figure 3. During the test, the average minimal Spo2 at SL (both off and on treatment) was not different between the sexes or the study groups. Maximal minute ventilation during isocapnic hypoxia was higher in men than in women, but not after correction for body height. The HVR was higher in men than in women (P , .001 and P 5 .001 at SL off and on treatment, respectively), as depicted in Figure 4. This difference persisted after correction of minute ventilation for body height (P 5 .023 and P 5 .026 at SL off and on treatment, respectively). Acetazolamide administration at SL on treatment did not affect the HVR or the HVR corrected for body height.

] Resting Ventilatory Parameters in the Three Study Conditions

Study Condition

Men

Women

P Value

ACZ

PL

P Value

SL, off treatment E, L/min

7.7 ⫾ 2.2

6.5 ⫾ 2.0

.042

…

…

…

E/h, L/min/m

4.3 ⫾ 1.2

3.9 ⫾ 1.3

1.000

…

…

…

PETCO2, mm Hg

37.3 ⫾ 5.5

36.0 ⫾ 2.9

.609

…

…

…

SpO2, %

97.4 ⫾ 1.4

98.1 ⫾ 1.0

.081

…

…

…

E, L/min

…

…

…

6.9 ⫾ 2.0

6.7 ⫾ 1.4

.855

E/h, L/min/m

…

…

…

4.0 ⫾ 1.1

3.9 ⫾ 0.7

1.000

SL, on treatment

PETCO2, mm Hg

…

…

…

34.1 ⫾ 3.3

37.6 ⫾ 3.7

.021

SpO2, %

…

…

…

97.8 ⫾ 0.7

97.7 ⫾ 0.8

1.000

12.2 ⫾ 2.8

10.5 ⫾ 1.7

.033

11.7 ⫾ 2.2

11.0 ⫾ 2.7

.258

HA E, L/min

6.8 ⫾ 1.5

6.4 ⫾ 1.9

.344

6.8 ⫾ 1.2

6.4 ⫾ 1.4

.297

PETCO2, mm Hg

27.8 ⫾ 3.8

27.1 ⫾ 3.9

.441

24.9 ⫾ 2.7

29.9 ⫾ 3.0

, .001

SpO2, %

81.0 ⫾ 6.4

81.2 ⫾ 7.3

.932

85.2 ⫾ 3.8

77.1 ⫾ 6.6

, .001

E/h, L/min/m

Men and women are pooled together for the comparison between the ACZ and PL groups at SL on treatment and at HA. HA 5 high altitude; PETCO2 5 partial pressure of end-tidal CO2; SL 5 sea level; SpO2 5 hemoglobin peripheral oxygen saturation; E 5 minute ventilation; E/h 5 minute ventilation normalized for body height. See Table 1 legend for expansion of other abbreviations.

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Figure 2 – Individual subjects’ ventilation at rest for the three study conditions. Data are shown separately for the two treatment groups. A, Acetazolamide, uncorrected. B, Placebo, uncorrected. C, Acetazolamide, corrected for body height. D, Placebo, corrected for body height. SL 5 sea level; VE 5 minute ventilation; VE/h 5 minute ventilation corrected for body height. See Figure 1 legend for expansion of other abbreviation.

Hyperoxic Hypercapnic Test: Ventilatory parameters related to the hyperoxic hypercapnic test are shown in Table 4. At the peak of the stimulus, Petco2 at SL off treatment and at SL on treatment was not different between the study groups. At SL off treatment, maximal ventilation during the stimulus was higher in men than in women, but this difference disappeared after ventilation was corrected for TABLE 3

body height. At SL on treatment, maximal ventilation was higher in those receiving acetazolamide than in those receiving placebo, even after correction for body height. The HCVR did not differ between men and women (P 5 .226 and P 5 .810 at SL off and on treatment, respectively). Moreover, the HCVR was not affected by study treatment; however, this relationship was leftward shifted by acetazolamide, as illustrated in Figure 5. Consequently,

] Ventilatory Responses to the Isocapnic Hypoxic Test SL, Off Treatment

Ventilatory Response Emax, L/min Emax/h, L/min/m SpO2min, %

Men 14.4 ⫾ 3.6

Women 10.5 ⫾ 2.6

SL, On Treatment

P Value , .001

ACZ

PL

13.8 ⫾ 3.5

13.6 ⫾ 5.4

P Value .955

8.0 ⫾ 2.1

6.3 ⫾ 1.5

.414

8.0 ⫾ 2.0

7.9 ⫾ 3.0

1.000

78.9 ⫾ 5.9

78.8 ⫾ 6.4

1.000

78.7 ⫾ 3.6

78.0 ⫾ 4.6

1.000

HVR, L/min/%SpO2

0.18 ⫾ 0.04

0.13 ⫾ 0.03

, .001

0.17 ⫾ 0.04

0.17 ⫾ 0.06

1.000

HVR/h, L/min/ %SpO2/m

0.10 ⫾ 0.02

0.08 ⫾ 0.02

.023

0.10 ⫾ 0.02

0.10 ⫾ 0.04

1.000

Men and women are pooled together for the comparison between ACZ and PL group at SL on treatment. Emax 5 maximal minute ventilation; Emax/h 5 maximal minute ventilation normalized for body height; SpO2min 5 minimal hemoglobin peripheral oxygen saturation; HVR 5 hypoxic ventilatory response; HVR/h 5 hypoxic ventilatory response corrected for body height. See Table 1 and 2 legends for expansion of other abbreviations.

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Figure 3 – Individual subjects’ HVR. Data are shown separately for the two treatment groups. A, Acetazolamide, uncorrected. B, Placebo, uncorrected. C, Acetazolamide, corrected for body height. D, Placebo, corrected for body height. HVR 5 hypoxic ventilatory response; HVR/h 5 hypoxic ventilatory response corrected for body height; SpO2 5 blood oxygen saturation. See Figure 2 legend for expansion of other abbreviations.

minute ventilation for every value of Petco2 in the acetazolamide group was higher than in the placebo group. Cardiorespiratory Sleep Studies

At SL, no subject showed abnormal breathing patterns during sleep. HA exposure significantly affected breathing patterns during sleep (because of the appearance of central sleep apneas/PBS, whereas no obstructive events were observed), with relevant sex- and treatment-related differences, as illustrated in Figure 6. In particular, at HA, men receiving placebo displayed an apnea-hypopnea index that was higher than that of women receiving placebo (40.9 ⫾ 27.8 events/h vs 7.3 ⫾ 4.0 events/h, P 5 .008). Acetazolamide, compared with placebo, was associated with a lower apnea-hypopnea index in treated subjects, both men (3.8 ⫾ 3.8 events/h, P 5 .002) and women (2.9 ⫾ 3.5 events/h, P 5 .069), so that no sex difference for the apnea-hypopnea index under active treatment was evident (P 5 .847). Apneahypopnea index values in men receiving acetazolamide were not different from apnea-hypopnea index values in women receiving placebo (P 5 .241). journal.publications.chestnet.org

The mean nighttime Spo2 was higher in those receiving acetazolamide than in those receiving placebo (78.2% ⫾ 2.6% vs 71.4% ⫾ 4.3%, P , .001), without sex-related differences (P 5 .797). Similarly, the minimal Spo2 during nighttime was higher in the acetazolamide than in the placebo group (69.4% ⫾ 3.9% vs 61.1% ⫾ 5.1%, P , .001), without sex-related differences (P 5 .679). In the placebo group, the effect of the HVR on the incidence of PBS was studied using a linear regression analysis. A significant correlation was found between the apnea-hypopnea index and the HVR (R2 5 0.82, P , .001; b1 5 340.3, P , .001; intercept b0 5 233.9, P 5 .001), as shown in Figure 7, as well as between the apnea-hypopnea index and the HVR normalized for body height (R2 5 0.74, P , .001). The correlation between the HVR and the apnea-hypopnea index remained significant (R2 5 0.56, P 5 .001) even after removing a subject with an extremely high HVR and a high apnea-hypopnea index from the analysis. Conversely, no correlation was found between the apneahypopnea index and the HVR in the acetazolamide group (R2 5 0.02, P 5 .591). 125

those free of AMS (32.1 ⫾ 35.2 events/h vs 20.3 ⫾ 14.7 events/h of sleep, P 5 .403).

Discussion Our study provides two novel findings. First, the chemoreflex sensitivity to hypoxia is higher in male subjects, and this could explain our current and previous3 observations on the sex-related differences in the burden of PBS in hypoxic conditions at HA. Second, the capacity of acetazolamide to influence both sides of the chemoreflex loop (directly, by modifying the apneic threshold, and indirectly, by improving oxygenation at HA) may account for the ability of this drug to reduce PBS at HA, especially in men. Sex, Chemosensitivity, and Periodic Breathing

Figure 4 – HVR values for men and women at SL off treatment. See Figure 2 and 3 legends for expansion of abbreviations.

AMS Assessment

Four subjects (20%) receiving acetazolamide presented with AMS, compared with 10 (56%) of those receiving placebo. In a multivariate analysis, the Lake Louise Score after the first night at HA was significantly higher in the placebo group than in the acetazolamide group (4.7 ⫾ 2.4 vs 3.0 ⫾ 2.4), independent of sex (intercept b0 5 1.00, P , .001; male sex b1 5 0.22, P 5 .193; treatment placebo b2 5 0.41, P 5 .014). In subjects receiving placebo, there were no significant differences in the apnea-hypopnea index between those suffering and

TABLE 4

Our study demonstrates a direct linear correlation between isocapnic hypoxic chemosensitivity, as measured at SL in laboratory conditions, and the incidence of PBS in an HA setting. At first glance, this finding may sound somehow paradoxical, because one may expect that subjects who more intensely respond to a given hypoxic stimulus should also present at HA with higher minute ventilation, which, in turn, would render the occurrence of apneas/hypopneas and/or oxygen desaturations less likely. This reasoning would hold true if ventilation were under the exclusive control of the peripheral (hypoxic) chemoreflex. However, ventilation is known to respond not only to arterial oxygen levels but also to changes in arterial CO2 detected by peripheral and central chemoreceptors, as well as to peripheralcentral chemoreflex reciprocal interactions.28 The chemoreflex control of breathing has been modeled as a complex negative feedback regulatory loop whereby the central and peripheral chemoreceptors exert a feedback control of ventilation; at the same time, ventilation modulates pulmonary gas exchange, thus ultimately

] Ventilatory Responses to the Hyperoxic Hypercapnic Test SL, Off Treatment

Ventilatory Response Emax, L/min

SL, On Treatment

Men

Women

P Value

ACZ

PL

P Value

27.3 ⫾ 11.9

19.7 ⫾ 6.5

.020

34.3 ⫾ 11.5

25.0 ⫾ 10.7

.008

Emax/h, L/min/m

15.3 ⫾ 6.9

11.9 ⫾ 3.8

1.000

19.9 ⫾ 6.8

14.5 ⫾ 6.1

.029

PETCO2max, mm Hg

49.6 ⫾ 3.0

48.9 ⫾ 2.5

1.000

49.2 ⫾ 2.4

49.7 ⫾ 1.9

1.000

HCVR, L/min/mm Hg

5.2 ⫾ 4.3

3.1 ⫾ 1.8

.569

5.2 ⫾ 3.7

4.2 ⫾ 2.6

.612

HCVR/h, L/min/mm Hg/m

2.9 ⫾ 2.4

1.9 ⫾ 1.1

.900

3.1 ⫾ 2.3

2.4 ⫾ 1.5

.900

Men and women are pooled together for the comparison between the ACZ and PL groups at SL on treatment. HCVR 5 hypercapnic ventilatory response; HCVR/h 5 hypercapnic ventilatory response corrected for body height; PETCO2max 5 maximal partial pressure of end-tidal CO2. See Table 1-3 legends for expansion of other abbreviations.

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Figure 5 – Hypercapnic ventilatory response (HCVR). VE/h is plotted as a function of PetCO2 during the hyperoxic hypercapnic test. The mean ⫾ SE of the data recorded during the last 20 s of the first minute of hypercapnic stimulus as well as during the last 20 s (maximal stimulus) of the hypercapnic test are shown for both the PL and the ACZ group. The slope of the dotted line that links the first minute of stimulus with the maximal stimulus approximates HCVR (ie, the relationship between PetCO2 and VE during the test). Compared with placebo, acetazolamide resulted in a leftward shift of HCVR (as indicated by the arrow) without significantly changing the slope of the VE/PetCO2 relationship. ACZ 5 acetazolamide; PetCO2 5 partial pressure of end-tidal CO2; PL 5 placebo. See Figure 2 legend for expansion of other abbreviation.

altering chemoreceptor stimulation.29 This means that hypoxia-driven hyperventilation at HA, beyond increasing oxygen levels, will also lower arterial CO2 tension. CO2 tension is physiologically maintained within a narrow range. In hypocapnic conditions, peripheral chemoreceptors cannot fully respond to a hypoxic stimulus because of concomitant feedback inhibition of ventilation, mainly from central chemoreceptors. Additionally, when CO2 tension goes below a certain threshold (the apneic threshold), the stimulus to breathe is greatly inhibited to the point that ventilation could stop for a while. The ensuing of apnea is especially facilitated during sleep (ie, when volitional drive to breathe is absent and the CO2 threshold is rightward shifted). The occurrence of apnea would restore CO2 blood levels but, causing at the same time a fall in oxygen tension, it would again activate peripheral chemoreceptors to a degree that should be directly proportional to their sensitivity, so that a vicious circle of alternating hyperpnea and hypoventilation may occur.29 Low Po2 at HA represents the primary stimulus to hyperventilation. We may thus hypothesize that subjects journal.publications.chestnet.org

Figure 6 – AHI at high altitude in men and women receiving either acetazolamide or placebo. AHI 5 apnea-hypopnea index. See Figure 5 legend for expansion of other abbreviations.

with increased hypoxic chemosensitivity will also frequently develop hypocapnia, and they will be thus definitely more prone to recurrence of apneic/hypopneic episodes during sleep. Our data, showing a direct linear correlation between the HVR and the apnea-hypopnea index, agree with this hypothesis, which is further supported by an engineering model of loop gain,29,30 as well as by previous experimental data recorded at HA.10 The loop gain theory is an engineering concept that has been applied to the study of sleep respiratory disturbances.29,30 According to this model, the overall stability of the feedback system controlling ventilation depends on the sensitivity of the controller gain to a given stimulus: the higher the magnitude of the corrective action implemented by the feedback system in response to a given stimulus, the higher the probability for periodic breathing to occur. In other words, in a feedback control system (such as the chemoreflex ventilatory control system), a high sensitivity to a given stimulus will render the system unstable and more prone to oscillate. 127

agreement with those of other previous works36-39 and are consistent with the evidence suggesting that testosterone increases peripheral chemoreflex sensitivity to hypoxia.32,40 Similarly, our results point to the absence of sex differences in the HCVR in healthy subjects, as reported previously by other authors.35,41,42 As in our previous report,3 we could not find any correlation between sex and the severity of AMS as evaluated through the Lake Louise Questionnaire. Although it has been suggested that the occurrence of AMS may influence ventilatory drive,43 we could not find any significant difference in the incidence of PBS between subjects free of and those suffering from AMS (although most of the subjects presented with only mild degrees of HA-related illnesses).

Figure 7 – Scatter plot with regression lines between AHI and HVR in men and women receiving placebo. See Figure 3 and 6 legends for expansion of abbreviations.

Also in accordance with our results, Lahiri et al,10 by studying a small group of healthy lowlanders and Sherpa highlanders (only male subjects), previously showed an association between the HVR and PBS at HA. In that study, Sherpa highlanders had a blunted HVR values as well as significantly less PBS than did lowlanders. Our work confirms and extends such preliminary evidence by also analyzing sex-related differences in ventilatory control and respiratory behavior during sleep in healthy lowlanders acutely exposed to HA; HVR was strongly correlated with the development of PBS at HA (at least in the placebo group), and male subjects, who were characterized by higher average HVR values than were women, also presented with an increased incidence of PBS at HA. Whether and how sex hormones influence ventilatory control is a debated issue,31,32 likely because of differences in the experimental designs of the studies conducted so far on this topic and because of the relatively small number of subjects examined. Comparisons among data from previous studies are made even more difficult by sex-related differences in anthropometric and lung function indexes. Although some previous studies, characterized by relatively small sample sizes, could not find significant differences in hypoxic sensitivity between the sexes,33-35 our data, showing that the HVR is greater in men than in women, even after correction for body height, are in 128 Original Research

Acetazolamide, Chemosensitivity, and Periodic Breathing

It is well known that acetazolamide is effective in preventing and treating HA illnesses,21 and that one of the mechanisms accounting for its efficacy could be its influence on respiratory control. As in another study in which similar oral doses of acetazolamide were used,16 we found a leftward shift of the CO2 set point in treated subjects, with an overall neutral effect of the drug on peripheral chemoreceptors (HVR) as well as on central chemoreceptors (HCVR). The change in CO2 set point induced by acetazolamide as a consequence of metabolic acidosis is expected at HA to offset the hyperventilation-induced hypocapnia and to result in greater ventilation and improved oxygenation.21 Indeed, at HA, we found higher values of Spo2 in subjects treated with acetazolamide. Quite unexpectedly, ventilation was nearly the same between the two treatment groups at SL, whereas at HA, ventilation was only 0.7 L higher in those receiving acetazolamide than in those receiving placebo, and this difference did not reach statistical significance. In this regard, we have to acknowledge that variability in individual data may have contributed to producing such an apparent lack of effect of acetazolamide on minute ventilation (which at first glance could appear even more anomalous if we consider that at the same time Petco2 was effectively reduced by the drug). This apparent inconsistency may also have been facilitated by the use of a mouthpiece combined with a nose clip, which may have made ventilation less “natural” in spite of the training period scheduled by our protocol before the start of the experimental session. Indeed, use of a mouthpiece in our study may have somehow interfered

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with ventilation, possibly stimulating it,44 mainly at the time of the first test, when subjects used the mouthpiece for the first time. We have, thus, tried to correct the change in minute ventilation caused by acetazolamide administration with the “adaptation effect” quantified in the placebo group as the difference between on- and off-treatment minute ventilation values at SL. However, even after such correction, the effect of acetazolamide on ventilation, as well as on Petco2, was lower than expected if compared with previous studies.16,27 We have to underline that the dosage of acetazolamide we used, albeit considered efficacious at HA,21 was significantly lower than that used in most of the studies assessing the effects of acetazolamide on ventilatory control. As an additional composite measure of the acetazolamide effect on ventilatory control at SL, we then calculated the ratio between the increase in minute ventilation and the reduction in Petco2 caused by the drug, which was further normalized by the daily acetazolamide dosage. Following such computation, the effects of acetazolamide in the subjects appear to be in line with those reported in previous studies in which a higher daily dose of the drug was administered, and which adopted a time schedule for ventilatory assessment similar to ours.16,27 Lastly, we may also ascribe the lack of between-group differences in minute ventilation at HA to the fact that, because of the complexity of our study design, which was planned to allow collection of a great deal of data, minute ventilation was recorded only on the third day of stay at HA, when subjects receiving placebo may also have displayed some degree of acclimatization. In fact, it has been postulated that acetazolamide at HA may simply accelerate the normal acclimatization process that otherwise, after some days of exposure, would result anyway in an increased urinary bicarbonate excretion.21,45 Nevertheless, it is possible that other pleiotropic effects of acetazolamide (eg, diuretic action with reduction of pulmonary congestion21; inhibition of hypoxic pulmonary vasoconstriction27 which seems to occur through a mechanism independent of carbonic anhydrase inhibition46; or improvement of alveolar-capillary diffusion19) may have resulted in more efficient gas exchange at HA in treated subjects, despite similar basal ventilation. In line with previous data, the cardiorespiratory sleep recordings performed during the first night of stay at HA demonstrated that acetazolamide reduces PBS.1,2,14 What had not been described up to now is how acetazolamide interacts with the sex-related differences in respiratory control. In this journal.publications.chestnet.org

regard, combining our results coming from chemoreflex testing with those from HA data collection may provide an explanation as to why acetazolamide is effective in counteracting the occurrence of PBS at HA in both sexes, but especially in men. First, acetazolamide causes a leftward shift of the CO2 chemoreflex response curve irrespective of sex, implying an increase in the difference between the prevailing CO2 and the apneic threshold for CO2. Second, at HA, the drug reduces the extent of the hypoxic stimulus to peripheral chemoreceptors, because subjects receiving acetazolamide presented with higher Spo2 values than did those receiving placebo. Taken together, these two factors contribute to stabilization of the ventilatory control at HA by acting on both arms of the chemoreflex loop, and could thus explain the significant protection from PBS offered by acetazolamide administration to male subjects. It is noteworthy that acetazolamide also improved PBS in women, who were nevertheless characterized by relatively low apnea-hypopnea index values, even in those receiving placebo. Study Limitations

A few limitations of our study must be acknowledged, although they should be contextualized in the challenging HA conditions where part of the recordings took place and which precluded a crossover design to be adopted. For logistic reasons, there is about a 1-day time lag between cardiorespiratory sleep studies and the recording of ventilatory parameters at HA, which may make comparisons between data more difficult. We wish to emphasize, however, that such a time discrepancy at HA between nocturnal polysomnography and ventilatory data at rest does not affect the main results of our study, which was principally aimed at exploring the possible causal role of chemoreflex sensitivity (as evaluated at SL) in determining the different sex prevalence of PBS at HA, and the effects of acetazolamide on such an interaction. We did not directly measure chemosensitivity at HA. However, owing to the acute HA exposure that characterized our expedition (sleep studies performed during the first night), our results are unlikely to have been significantly influenced by chemoreflex adaptation mechanisms.43,47 We also did not measure the chemoreflex responses directly during sleep, assuming that awake chemoreflex sensitivity could be a useful surrogate.48,49 Nevertheless, we cannot exclude the fact that women may present with a greater reduction of the HCVR during sleep,49 as 129

well as with a relatively more preserved ventilatory motor output during hypocapnia at night,50 than do men. Our study was not designed to evaluate the correlation between chemosensitivity and female menstrual cycle. However, the menstrual cycle phase did not seem to bear any correlation with the occurrence of central apneas at HA3 or to significantly interfere with chemoreflex activity.50-52 Ventilatory control is an integrated rather than a separate compartment system, with possible additive or even hyperadditive interactions between the peripheral and the central chemoreflexes.28 Peripheral chemoreceptors may determine the sensitivity of central chemoreceptors to CO2, and such a peripheral-central chemoreflex interaction may have a role in causing hypocapnia-induced apnea. Human studies, unlike animal models, do not permit a clear-cut separation of stimuli to the central and peripheral chemoreceptors, so we cannot quantify the possible contribution to our results by peripheralcentral interactions at HA. Lastly, we cannot completely exclude the possibility that the lack of between-sex or between-treatment differ-

Acknowledgments Author contributions: G. P. is the guarantor of the manuscript and takes responsibility for the integrity of the data and the accuracy of the data analysis. A. F., C. L., M. V., G. B., P. A., and G. P. contributed to the study concept and design; S. C., A. F., C. L., M. V., F. G., J. R., P. M., M. D. R., P. A., and G. P. contributed to data acquisition and analysis; S. C., A. F., C. L., P. A., and G. P. contributed to data interpretation; S. C., A. F., C. L., P. A., and G. P. contributed to drafting and review of the manuscript for important intellectual content; M. V., F. G., J. R., P. M., M. D. R., G. B., and G. P. contributed to revision of the manuscript; and S. C., A. F., C. L., M. V., F. G., J. R., P. M., M. D. R., G. B., P. A., and G. P. contributed to the final approval of the version to be published. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: We express our gratitude to Club Alpino Italiano, Division of Varallo Sesia, the staff of the HA laboratory Capanna Regina Margherita, and the Alpine Guides of Varallo Sesia for their valuable organizational

130 Original Research

ences in certain variables, as discussed earlier for minute ventilation, may be partly caused by variability in individual data and by the relatively small sample size of our study.

Conclusions The increased peripheral chemoreflex sensitivity to hypoxia in male subjects could account for the more frequent occurrence of PBS in hypoxic conditions, such as at HA. Female subjects seem to be relatively protected from PBS at HA, possibly because of lower ventilatory responses to hypoxia, generating less instability in ventilatory control. Acetazolamide abolishes the correlation between hypoxic chemosensitivity and PBS at HA, through both a leftward shifting of the CO2 set point (which implies a reduced apneic threshold) and a higher blood oxygenation at HA (which implies a reduced stimulus to peripheral chemoreceptors). Therefore, this drug not only was extremely effective in counteracting the occurrence of PBS in men, but also proved useful in women. Our results, obtained under conditions of hypobaric hypoxia, may also be relevant to the pathogenesis and management of sleep-related breathing disorders occurring in patients with a variety of cardiorespiratory disease.

support; to Luca Grappiolo, BBA, for the careful administrative management of the project; and to Licia Pietrobon, MA, for the effective secretarial support. We also thank Gian Piero Babbi (Tao Med) for the support given to this study. Additional information: The e-Appendix and e-Tables can be found in the Supplemental Materials section of the online article.

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