Effects of Acetazolamide on Ventilatory ... - ATS Journals

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Aug 23, 2006 - Luc J. Teppema*, George M. Balanos*, Craig D. Steinback, Allison D. Brown, Glen E. Foster, Henry J. Duff,. Richard Leigh, and Marc J. Poulin.
Effects of Acetazolamide on Ventilatory, Cerebrovascular, and Pulmonary Vascular Responses to Hypoxia Luc J. Teppema*, George M. Balanos*, Craig D. Steinback, Allison D. Brown, Glen E. Foster, Henry J. Duff, Richard Leigh, and Marc J. Poulin Department of Anesthesiology, Leiden University Medical Center, Leiden, The Netherlands; School of Sport and Exercise Sciences, University of Birmingham, Birmingham, United Kingdom; and Departments of Physiology and Biophysics, Medicine, and Clinical Neurosciences, Faculty of Medicine, and Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada

Rationale: Acute mountain sickness (AMS) may affect individuals who (rapidly) ascend to altitudes higher than 2,000–3,000 m. A more serious consequence of rapid ascent may be high-altitude pulmonary edema, a hydrostatic edema associated with increased pulmonary capillary pressures. Acetazolamide is effective against AMS, possibly by increasing ventilation and cerebral blood flow (CBF). In animals, it inhibits hypoxic pulmonary vasoconstriction. Objectives: We examined the influence of acetazolamide on the response to hypoxia of ventilation, CBF, and pulmonary vascular resistance (PVR). Methods: In this double-blind, placebo-controlled, randomized study, nine subjects ingested 250 mg acetazolamide every 8 h for 3 d. On the fourth test day, we measured the responses of ventilation, PVR, and CBF to acute isocapnic hypoxia (20 min) and sustained poikilocapnic hypoxia (4 h). Ventilation was measured with pneumotachography. Hypoxia was achieved with dynamic end-tidal forcing. The maximum pressure difference across the tricuspid valve (⌬Pmax, a good index of PVR) was measured with Doppler echocardiography. CBF was measured by transcranial Doppler ultrasound. Results: In normoxia, acetazolamide increased ventilation and reduced ⌬Pmax, but did not influence CBF. The ventilatory and CBF responses to acute isocapnic hypoxia were unaltered, but the rise in ⌬Pmax was reduced by 57%. The increase in ⌬Pmax by sustained poikilocapnic hypoxia observed after placebo was reduced by 34% after acetazolamide, the ventilatory response was increased, but the CBF response remained unaltered. Conclusions: Acetazolamide has complex effects on ventilation, PVR, and CBF that converge to optimize brain oxygenation and may be a valuable means to prevent/treat high-altitude pulmonary edema. Keywords: pulmonary resistance; cerebral blood flow; altitude sickness; hypoxic responses; mountaineering

Acute mountain sickness (AMS) is one of the three major highaltitude–related diseases seen in people who rapidly ascend to altitudes higher than 2,000–3,000 m. The other two diseases are high-altitude pulmonary edema (HAPE) and high-altitude cerebral edema. Worldwide, an increasing number of sea-level residents (altogether several millions per year) visit areas higher

(Received in original form August 23, 2006; accepted in final form November 5, 2006 ) Supported by the Alberta Heritage Foundation for Medical Research, the Heart and Stroke Foundation of Alberta, Northwest Territories, and Nunavut, and the Canadian Institutes of Health Research. *These authors contributed equally to this article. Correspondence and requests for reprints should be addressed to Marc J. Poulin, Ph.D., D.Phil., Departments of Physiology and Biophysics, and Clinical Neurosciences, Faculty of Medicine and Faculty of Kinesiology, University of Calgary, Calgary, AB, T2N 4N1 Canada. E-mail: [email protected] Am J Respir Crit Care Med Vol 175. pp 277–281, 2007 Originally Published in Press as DOI: 10.1164/rccm.200608-1199OC on November 9, 2006 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Acetazolamide is effective against acute mountain sickness, but the mechanisms for this effect are not completely characterized. What This Study Adds to the Field

Acetazolamide has benefit in alleviating acute and subacute hypoxia-induced pulmonary vasoconstriction and increased pulmonary artery pressures.

than 2,500 m and many of them develop symptoms of AMS that may consist of insomnia, headache, lightheadedness, fatigue, breathlessness, anorexia, and nausea (1). A more serious altitude illness is HAPE, which sometimes is preceded by AMS (1, 2). If oxygen therapy is not available or rapid descent is not possible, the first choice of pharmacologic treatment for AMS is the carbonic anhydrase inhibitor acetazolamide (2–4). The mechanisms by which acetazolamide relieves the cerebral symptoms of this disease are believed to be related to improvement of brain oxygenation mediated via a metabolic acidosis-induced rise in ventilation, and possibly also by a rise in cerebral blood flow (CBF), although the latter has not been clearly established. Another potential beneficial action of acetazolamide may be a reduction of hypoxic pulmonary vasoconstriction (HPV). Recent studies have suggested that the ventilation/perfusion mismatch increases due to the uneven distribution of HPV in the lungs, which results in increased pressure on some capillaries, consequently causing capillary leak and pulmonary edema (5–9). In isolated, perfused rabbit lungs, in rats, and in dogs, acetazolamide has been shown to reduce HPV (10–12). To our knowledge, the effect of acetazolamide on pulmonary vascular resistance has not been studied in humans. A reduction of HPV by acetazolamide in clinical doses could be of considerable clinical importance, because it could not only prevent and/or reduce the potential severe consequences of high pulmonary arterial pressure at altitude but also those of other disease states associated with, or resulting from, noncardiogenic pulmonary hypertension. The aim of the present study was to examine the influence of oral acetazolamide on the response to hypoxia of all three variables that may mediate its beneficial action in AMS—namely, ventilation, pulmonary vascular resistance, and CBF in healthy volunteers. We believe this is the first study in which the hypoxia-induced responses of these three vital physiologic variables were measured simultaneously. Some of the results of this study have been previously reported in abstract form (13).

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METHODS Subjects Nine subjects participated in this study (three women, six men; age, 28.4 ⫾ 7.1 yr, mean ⫾ SD). Subjects were included in the study if they had echo evidence of tricuspid regurgitation during systole, which is not clinically relevant but in fact can be demonstrated in most normal individuals. All subjects were nonsmokers and had no history of any preceding cardiorespiratory disease. Female subjects were receiving oral contraceptives or within the first 14 d of their menstrual cycle. All subjects provided informed, written consent. This study was approved by the Conjoint Health Research Ethics Board at the University of Calgary.

Study Design The study was performed according to a prospective, double-blind, placebo-controlled crossover design, in which subjects were randomized to treatment with either acetazolamide or placebo for 3 d prior to outcome measurements on the fourth day. Each treatment period was separated by a 10-d washout period to overcome any potential crossover effect of acetazolamide. Subjects were instructed to abstain from caffeine, alcohol, and strenuous exercise throughout the testing schedule. Subjects ingested either acetazolamide (250 mg/dose) or placebo every 8 h for 3 d, ending the morning of outcome measurements. Outcome days started with measurements of resting ventilation, end-tidal Po2 (PetO2) and Pco2 (PetCO2). Blood acid-base status was determined from finger capillary samples (ABL700; Radiometer Canada, London, ON, Canada). This was followed by two hypoxic challenges performed in succession, separated by 30 min.

Acute Hypoxia Accurate control of end-tidal gases was achieved with the method of dynamic end-tidal forcing, described elsewhere (14, 15). The protocol consisted of 10 min isocapnic normoxia, followed by 20 min of isocapnic hypoxia (PetO2 ⫽ 50 mm Hg, SpO2 ⬇ 87%), followed by a further 10 min of isocapnic normoxia. PetCO2 was controlled at 1 mm Hg above each subject’s resting levels. The hypoxic ventilatory response was de˙ e) over the associfined as the change in expired minute ventilation (V ated change in arterial saturation (L · min⫺1 · %⫺1). The hypoxic ventila˙ e1) over the ˙ e (V ˙ e2 ⫺ V tory response was calculated as the change in V ˙ e1 represents given change in arterial oxygen saturation. Accordingly, V ˙ e2 represents the 1-min average immediately preceding hypoxia and V the 1-min average during the final minute of hypoxia.

Prolonged Hypoxia Subjects were exposed to 4 h of poikilocapnic hypoxia (PiCO2 ⫽ 0 mm Hg, PetO2 ⫽ 50 mm Hg) while seated in a small custom, sealed, normobaric chamber. PetO2 was controlled via a computerized feedback system as previously described (14, 16).

Tricuspid Valve Maximum Pressure Gradient The maximum pressure gradient across the tricuspid valve (⌬Pmax) was measured using Doppler echocardiography (Agilent Sonos 5500; Philips Medical Systems, Markham, ON, Canada) and a standard Doppler ultrasound technique (17, 18). ⌬Pmax has been shown to be a good index of pulmonary vascular resistance (18). Doppler ultrasound was also used to monitor cardiac output from the aortic outflow.

Middle Cerebral Artery Peak Blood Flow Velocity, Blood Pressure, and Heart Rate ¯ p) in the middle cerebral artery was meaPeak blood flow velocity (V sured using transcranial Doppler ultrasound as described previously (14, 15) and used as an index of CBF (15). Blood pressure (photoplethysmography), heart rate, ECG, and blood oxygen saturation (SpO2, pulse oximetry) were measured on a beat-by-beat basis. Statistical Analysis Resting data were compared between the two treatment periods using paired t test. Data from the acute and prolonged hypoxic exposures were analyzed using a multivariate repeated-measures design with two parallel conditions, compared using preplanned contrasts and adjusted

for multiple comparisons. Statistical analyses were performed using SPSS (version 13.0, SPSS, Inc., Chicago, IL). Significance was set at p ⬍ 0.05. Data are expressed as mean ⫾ SD.

RESULTS All subjects completed both treatment periods. Table 1 lists the average resting values for blood electrolytes and respiratory, cardiovascular, and pulmonary variables on the placebo and acetazolamide study outcome days. The data show that acetazolamide caused a hyperchloremic acidosis, increased the mean PetO2 by 8.5 mm Hg, decreased the mean PetCO2 by 6.3 mm Hg, and increased mean ventilation by 2.3 L · min⫺1. It also caused a small but significant decrease in normoxic ⌬Pmax. The other ¯ p, showed no sigmeasured cardiovascular variables, including V nificant change after acetazolamide. Acute Test

˙ e, ⌬Pmax, and V ¯p Figure 1 illustrates the 1-min averages for V during the acute test. Acute isocapnic hypoxia decreased SpO2 to 87.5 ⫾ 2.0% after placebo and 86.6 ⫾ 1.5% after acetazolamide. The increase in ⌬Pmax was the only response that was clearly altered by acetazolamide: after 20 min of hypoxia, it rose from 20.3 ⫾ 3.5 to 31.5 ⫾ 1.4 mm Hg after placebo and from 17.9 ⫾ 2.1 to 22.3 ⫾ 2.6 mm Hg after acetazolamide (p ⬍ 0.001). The responses of the other cardiovascular variables were not influenced by acetazolamide. After 20 min of hypoxia, the HVRs after placebo and acetazolamide were 0.96 ⫾ 0.95 and 0.57 ⫾ ¯ p increased by 0.45 ⫾ 0.71 L · min⫺1 · %⫺1 (not significant [NS]).V 0.32 and 0.78 ⫾ 0.70 cm · s⫺1 · %⫺1, respectively (NS). Chamber Test

¯ p during Figure 2 shows the changes in PetCO2, ⌬Pmax, and V the chamber exposure. The responses to sustained poikilocapnic hypoxia of the main variables after placebo and acetazolamide, respectively, were as follows: SpO2 decreased to 86.3 ⫾ 0.5% and 87.0 ⫾ 0.8% (NS), whereas PetCO2 fell by 3.2 ⫾ 1.6 and 3.3 ⫾ 1.5 mm Hg (NS). The difference in PetCO2 between conditions was significant across all time points (see Figure 2; note that after acetazolamide, normoxic resting PetCO2 was lower and that the chamber exposure was poikilocapnic, i.e., zero inspired Pco2). After 4 h of hypoxia, ⌬Pmax rose from 20.0 ⫾ 1.5 to 42.0 ⫾ 5.0 mm Hg after placebo and from 18.1 ⫾ 2.1 to 32.5 ⫾ 4.0 mm Hg

TABLE 1. AVERAGE RESTING VALUES* Resting Values

PETO2, mm Hg PETCO2, mm Hg V˙E, L · min⫺1† SpO2, %† pH† [HCO3⫺], mM† [Cl⫺], mM† ⌬Pmax, mm Hg MAP, mm Hg Cardiac output, L · min⫺1 Heart rate, beats/min ¯ P, cm · s⫺1 V

Placebo

Acetazolamide

p Value

85.6 ⫾ 4.0 38.2 ⫾ 3.4 8.2 ⫾ 3.0 94.6 ⫾ 1.5 7.430 ⫾ 0.021 23.8 ⫾ 1.2 108.8 ⫾ 2.0 19.5 ⫾ 2.1 79.5 ⫾ 13.4 4.43 ⫾ 0.6 63.1 ⫾ 7.4 56.3 ⫾ 11.7

94.1 ⫾ 3.9 31.9 ⫾ 2.8 10.5 ⫾ 2.7 95.6 ⫾ 1.4 7.339 ⫾ 0.022 17.7 ⫾ 1.2 114.3 ⫾ 2.4 17.1 ⫾ 2.2 74.6 ⫾ 10.0 4.29 ⫾ 0.73 65.9 ⫾ 10.4 52.6 ⫾ 13.5

⬍ 0.001 ⬍ 0.001 0.017 NS ⬍ 0.001 ⬍ 0.001 0.003 0.016 NS NS NS NS

Definition of abbreviations: MAP ⫽ mean arterial pressure; NS ⫽ not significant; ¯ P ⫽ peak blood flow velocity. V Values are means ⫾ SD. * n ⫽ 9. † Values represent n ⫽ 8 because one individual was hyperventilating during sampling. Blood parameters were measured from arterialized finger samples.

Teppema, Balanos, Steinback, et al.: Acetazolamide and Hypoxia

Figure 1. One-minute averages for (A ) ventilation (V˙E), (B ) pulmonary ¯ P) during vascular resistance (⌬Pmax), and (C ) cerebral blood flow (V acute hypoxia. Solid triangles, placebo; open circles, acetazolamide. Values are means ⫾ SD. Note that in placebo, PETCO2 was maintained ⫾ 1 mm Hg above the normoxic resting value, whereas after acetazolamide it was kept at resting level.

after acetazolamide, demonstrating a significant reduction (34%, p ⬍ 0.001) after acetazolamide. Note that, compared with the normoxic preexposure period, after both treatments the rise in ⌬Pmax was significant across all time points (see Figure 2). ¯ p remained unchanged During 4 h of poikilocapnic hypoxia, V from normoxic baseline after both placebo (57.5 ⫾ 4.0 cm · s⫺1) and acetazolamide (60.7 ⫾ 4.7 cm · s⫺1) ingestion. There were ¯ p between conditions across all no significant differences in V time points.

DISCUSSION We measured the effects of a clinically relevant dose of acetazolamide on the responses to hypoxia of ventilation, pulmonary vascular resistance, and CBF. Acetazolamide increased resting normoxic ventilation but did not influence the ventilatory response to isocapnic hypoxia. Acetazolamide also caused a small decrease in pulmonary vascular resistance during normoxia but a large reduction in its response to both isocapnic and poikilocapnic hypoxia (57 and 34%, respectively). Finally, despite a considerable fall in PetCO2 after acetazolamide, CBF did not fall during normoxia, whereas its response to isocapnic and poikilocapnic hypoxia was not reduced, as one would expect in hypocapnic conditions. Ventilatory Response to Hypoxia

When administered in normoxia, a clinically relevant dose of acetazolamide increases ventilation, which is believed to be

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Figure 2. Changes in (A ) end-tidal O2 (PETO2), (B ) pulmonary vascular ¯ P) during 4 h of resistance (⌬Pmax), (C ) and cerebral blood flow (V poikilocapnic hypoxia. Solid triangles, placebo; open circles, acetazolamide. Time ⫽ N refers to the normoxic period immediately preceding hypoxia, and Time ⫽ 0 refers to the time at which 50 mm Hg PETO2 was first reached. Values are expressed as mean ⫾ SD. *Significant difference between conditions (p ⬍ 0.001, unless noted). †Significantly different from Time ⫽ N (p ⬍ 0.001, unless noted). There are no symbols ¯ P because data were not collected at that at Time ⫽ 0 for ⌬Pmax and V time point.

caused by the ensuing metabolic acidosis due to inhibition of renal carbonic anhydrase (19, 20). Indeed, after ingestion of acetazolamide, our subjects developed an appreciable metabolic acidosis (Table 1). Normally, when the arterial pH decreases, the carotid bodies become more sensitive to hypoxia, resulting in an increased ventilatory response (21). However, as illustrated in Figure 1, our subjects’ acidosis did not result in this expected larger hypoxic response, indicating a powerful inhibitory action of acetazolamide on this O2–H⫹ interaction (see also Reference 22). Metabolic acidosis leads to a lower PetCO2 (higher ventilation; Table 1) and, due to the hyperbolic relationship between alveolar Pco2 and ventilation, this results in a situation in which a given change in ventilation causes a smaller change in Pco2. Our finding that during the chamber exposure the decrease in mean PetCO2 after placebo and acetazolamide was similar (3.2 and 3.3 mm Hg, respectively) indicates that, despite an inhibiting action on the carotid bodies, the rise in ventilation after acetazolamide was greater than that observed after placebo. One possible explanation for this finding may be that, whereas in the acute test we showed that the isocapnic hypoxic ventilatory response was not significantly different after both treatments, the fact that acetazolamide appeared to reduce the normal O2–H⫹ interaction may have resulted in a higher poikilocapnic response after acetazolamide. The fall in PetCO2 (rise in pH) during the chamber exposure would dampen the hypoxic response after placebo but less

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so after acetazolamide. In summary, when ingested at altitude, acetazolamide will cause a moderate increase in ventilation, which will depend on the altitude level (degree of hypoxia), and this agrees with findings in the literature (23). Pulmonary Vascular Response to Hypoxia

In this study, we showed for the first time in humans that a clinically relevant dose of acetazolamide causes a small reduction in the resting ⌬Pmax but a substantial reduction in the ⌬Pmax response to both isocapnic and poikilocapnic hypoxia. In isocapnic hypoxia (acute test), acetazolamide attenuated the increase in ⌬Pmax (as measured after 20 min of hypoxia) by an average of 57%, whereas after 4 h of poikilocapnic hypoxia (chamber test), it was blunted by 34% when compared with placebo. The small decrease in resting ⌬Pmax after acetazolamide could be due to the fall in PetCO2 (24) and/or a small decrease in hydrostatic pressure in lung vessels caused by its diuretic action. Although we speculate that the net effect of the acetazolamide treatment on the ⌬Pmax response to hypoxia is largely due to a direct action on pulmonary vascular smooth muscle cells (see below), our data do not allow us to differentiate between potential contributing factors. In both the acute and chamber tests, the subjects had a lower PetCO2 after acetazolamide, and this could have possibly reduced the hypoxia-induced increase in smooth muscle activity. Theoretically, the decrease in mean PetCO2 by 6.3 mm Hg after acetazolamide (Table 1) could give rise to a decrease in pulmonary arterial tension of about 3.7 mm Hg (25). However, the subjects were also acidotic and this may have intensified HPV with the tendency to counteract this dilating influence of the lower end-tidal Pco2 (26, 27). In addition, since after acetazolamide the end-tidal Po2 was higher by 8.5 mm Hg on average, during both hypoxic tests the inspired Po2 was kept at a lower level, and this could have resulted in a somewhat lower alveolar Po2 (estimated from the alveolar gas equation) despite equal (controlled) end-tidal pressures in placebo and acetazolamide. Theoretically, this could have resulted in a slight underestimation of the dilating action of acetazolamide. Presumably, however, an influence of different alveolar Po2 levels was minimal, since in the Po2 range covered, minimal changes on HPV are observed (11). Altogether, we believe that our finding of a reduced hypoxia-induced pulmonary vasoconstriction is largely due to a direct action of acetazolamide. Our findings are relevant to HAPE because it is a form of hydrostatic edema associated with high pulmonary capillary pressure (1, 6, 9), which could well be prevented and treated with an agent that, by inhibiting HPV, will reduce the hydrostatic pressure in pulmonary capillaries. In humans and animals, HPV shows a heterogeneous spatial distribution pattern tending to cause ventilation/perfusion mismatching in the lung (7, 8). In addition, HAPE-susceptible subjects appeared to show a greater perfusion heterogeneity in the lung during hypoxia compared with HAPE-resistant subjects (8). We suggest that, in HAPEsusceptible individuals, acetazolamide (by acting on pulmonary vasculature) will decrease the alveolar to arterial Po2 difference (AaDo2) in at least two ways. First, by decreasing lung edema, it will facilitate the diffusion of oxygen, and second, it will improve the ventilation/perfusion ratio in the lung. Furthermore, note that acetazolamide has shown, by a yet unknown mechanism, to stimulate alveolar fluid clearance in artificially ventilated rats (28). In humans, acetazolamide decreases the AaDo2 during hypoxic exercise (29). Concerning the mechanism by which acetazolamide may reduce the ⌬Pmax response, we refer to studies in isolated pulmonary arterial smooth muscle cells from rats in which the agent inhibits the hypoxia-induced rise in intracellular Ca2⫹ (30). In rabbit lung and in the dog, acetazolamide dose-dependently in-

hibits HPV (10, 11). Therefore, we believe that the decrease in the ⌬Pmax response in our subjects is mainly due to inhibition of HPV by acetazolamide. Because acetazolamide did not alter the time course of the endothelium-dependent rise in ⌬Pmax during the chamber exposure, we speculate that it may act directly on smooth muscle rather than endothelial cells. Finally, it is interesting that, although at least systemic smooth muscle cells contain carbonic anhydrases (12), the inhibiting effect of acetazolamide on isolated smooth muscle cells and on HPV in conscious dogs occurs independently of inhibition of one of these isoenzymes (31, 32). The molecular mechanism by which acetazolamide acts is currently under study and may include a direct interaction with potassium channels (30, 33). CBF Response to Hypoxia

Despite a much lower PetCO2 after acetazolamide, normoxic resting CBF was not different from that measured after placebo (Table 1; see also Reference 34). In sustained hypocapnia, CBF returns to approximate eucapnic levels (35) and this may have occurred in our subjects when ingesting acetazolamide. However, the ensuing metabolic acidosis could also have contributed to the overall influence on resting CBF. Finally, by directly acting on smooth muscle cells, acetazolamide appears to cause vasodilation, even in small doses (36). Consequently, our data do not allow us to ascribe the absence of a change in resting CBF after acetazolamide treatment to a single mechanism. Normally, the vasodilatory response of brain vessels to hypoxia becomes less at lower PetCO2 levels (14). In our subjects, we could not demonstrate an influence of acetazolamide on the CBF responses to either isocapnic or poikilocapnic hypoxia (see also Reference 34). Consequently, our data do not lend support to the idea that the improvement of symptoms in AMS by acetazolamide may be due to an increased CBF response to hypoxia. However, the absence of a lower CBF response to hypoxia after acetazolamide in the face of a much lower PetCO2 could be due to a direct vascular action of acetazolamide and/or to an influence of the metabolic acidosis that alters CBF control in such a way as to maintain an adequate supply of oxygen to the brain in hypoxic conditions. Conclusions

The findings in this study are of considerable clinical interest regarding the prevention and treatment of AMS and HAPE. AMS and HAPE appear to be progressive manifestations of the same underlying physiologic process that may develop into pulmonary edema (1). Because, depending on its severity, in AMS abnormal lung sounds and gas exchange abnormalities (increase in AaDo2) are not uncommon (both phenomena could be manifestations of mild pulmonary edema), the two diseases may indeed be closely related (4, 37, 38). When exposed to a simulated altitude of approximately 4,880 m, subjects with the lowest AMS scores also had the lowest fluid retention (27). The frequent use of acetazolamide by mountaineers may already lower the incidence of HAPE. Acetazolamide has been shown to prevent HAPE in rats (12), and the agent may also prove to be an efficient therapeutic means in the treatment of this disease as alternatives to calcium channel blockers (39), phosphodiesterase inhibitors (40), or inhaled nitric oxide (41) in humans. Altogether, acetazolamide seems unique in its combined effects on pulmonary ventilation, gas exchange, and, possibly, CBF, which act together to optimize brain oxygenation in a hypoxic environment. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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