Respiratory Physiology & Neurobiology

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1 Respiratory Physiology & Neurobiology 162 (2008) Contents lists available at ScienceDirect Respiratory Physiology & Neurobiology journal homepage: Effect of acetazolamide on ventilatory response in subjects with chronic mountain sickness Maria Rivera-Ch a,, Luis Huicho b, Patrick Bouchet c, Jean Paul Richalet d, Fabiola León-Velarde a a Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Instituto de Investigaciones de Altura, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 31, Peru b Departamento Académico de Pediatría, Universidad Nacional Mayor de San Marcos, Universidad Peruana Cayetano Heredia, and Instituto de Salud del Niño, Lima 05, Peru c INSERM U280, Lyon, France d Université Paris 13, Laboratoire Réponses cellulaires et fonctionnelles à l hypoxie, EA2363, ARPE, 74 rue Marcel Cachin, Bobigny Cedex, France article info abstract Article history: Accepted 9 June 2008 Keywords: Acetazolamide Hypoxia Hematocrit Chronic mountain sickness Although the effects of acetazolamide (ACZ) on ventilation during acute mountain sickness are well known, there are no studies assessing its effect on ventilatory response in chronic hypoxia. We studied this effect in patients with chronic mountain sickness (CMS). Subjects with CMS, living permanently at 4300 m, were assigned in a randomized, double-blind study to 250 mg/day (n = 9) or to 500 mg/day (n =9)ofACZ. Resting end-tidal PET O2 and end-tidal PET CO2 were measured before and after 3 weeks of acetazolamide. Ventilatory responses were evaluated by the determination of sensitivity to hypoxia and to CO 2.After treatment ventilatory response to hypoxia increased, resting PET CO2 decreased, and ACZ caused a leftward shift in the position, but not a change in the slope of the ventilation ( VE) versus PET CO2 relationship. There were no differences between the two doses used. ACZ administration provides a beneficial effect on respiratory function of high altitude natives with CMS and thus it can be an effective therapy for the disease Elsevier B.V. All rights reserved. 1. Introduction High altitude (HA) natives living at altitude settings have higher minute ventilation ( VE) and lower end-tidal PCO 2 (PET CO2 ) resting values than sea-level natives living at sea level. Furthermore, HA natives show acute hypoxic ventilatory responses (AHVR) considerably lower than those seen in sea-level natives (Milledge and Lahiri, 1967; Velasquez et al., 1968; Lahiri and Edelman, 1969; Weil et al., 1971). There is no direct proof that alterations in carotid bodies physiology cause the low values for AHVR in HA natives, but interestingly, carotid bodies of HA natives show great hypertrophy (Arias-Stella, 1969). With regard to sensitivity to CO 2, under conditions of euoxia (PET O2 = 100 Torr), total ventilatory sensitivity to CO 2 and peripheral (fast) chemoreflex sensitivity to CO 2 in HA natives is higher than in SL subjects. Hypoxia causes a lower increase in total CO 2 sensitivity in HA subjects. Mean values for the time constant of response to CO 2 for HA individuals are lower than for SL residents (Fatemian et al., 2003). In addition, HA natives show increased hemoglobin and hematocrit values as part of a normal response to chronic hypoxia. Corresponding author. Tel.: ; fax: address: mrivera@upch.edu.pe (M. Rivera-Ch). However, HA natives may lose their adaptation to high altitude and display a variety of clinical symptoms, as well as excessive erythrocytosis. The combination of these features has been defined as chronic mountain sickness (CMS) (Hurtado, 1942; Monge and Whittembury, 1976). This syndrome has primarily been observed in Andean natives living at altitudes above 3,000 m, but it also occurs in almost all the high altitude regions of the world (Winslow and Monge, 1987; Ward et al., 1999). Compared with healthy high altitude natives, patients with CMS living at the same altitude are relatively hypoxic and hypercapnic (Severinghaus et al., 1966; Kryger et al., 1978). In the physiopathological sequence of CMS, these respiratory characteristics of the patients provoke hypoxemia, leading to excessive erythrocytosis (Winslow and Monge, 1987; Monge et al., 1992). We reasoned that these impaired respiratory characteristics may be pharmacologically modified with acetazolamide (ACZ), an inhibitor of carbonic anhydrase. Carbonic anhydrases belong to a family of ubiquitous metalloenzymes that catalyse predominantly the reversible hydration dehydration reaction of carbon dioxide to carbonic acid. They play an important role in a broad range of physiological functions including but not limited to respiration, CO 2 transport, urinary acidification and ion transfer (Maren, 1967). Inhibition of renal carbonic anhydrase with ACZ leads to an increased urinary excretion of HCO 3 -, sodium and potassium, and to a decreased urinary /$ see front matter 2008 Elsevier B.V. All rights reserved. doi: /j.resp

2 M. Rivera-Ch et al. / Respiratory Physiology & Neurobiology 162 (2008) excretion of titratable acids and ammonium. The loss of base precedes the development of metabolic acidosis, leading to increase of ventilation, a well-established effect (Skatrud and Dempsey, 1983; Teppema and Dahan, 1999). Thus, ACZ is useful in the prophylaxis of chronic obstructive pulmonary diseases, sleep-disordered breathing syndromes, and acute mountain sickness (Hackett et al., 1987; Burki et al., 1992; Swenson and Hughes, 1993; Bales and Timpe, 2004). By enhancing resting ventilation and ventilatory drive, which is secondary to metabolic acidosis, ACZ improves the blood oxygenation. Although the effects of ACZ on ventilation in subjects exposed to acute hypoxia and in patients with acute mountain sickness are well documented (Burki et al., 1992; Swenson and Hughes, 1993), there are no studies on its effects on the ventilatory response in subjects with or without CMS who are exposed to chronic hypoxia. A previous double-blind placebo-controlled study by our group showed that ACZ decreased hematocrit and serum erythropoietin, increased nocturnal arterial O 2 saturation, and decreased mean nocturnal heart rate and the number of apnea/hypopnea episodes during sleep in CMS treated subjects (Richalet et al., 2005) when compared to those who received placebo. The overall decrease in erythropoietin was thought to be partly an indirect effect of ACZ through the increase in ventilation and thus in arterial O 2 saturation (Richalet et al., 2005). For patients with CMS, it is important to improve the chemical drive to breath. Thus we aimed to document the effect of clinical doses of ACZ on resting end-tidal PO 2 (PET O2 ), end-tidal PCO 2 (PET CO2 ) and minute ventilation ( VE), and on the sensitivity of chemoreceptors to decreasing concentrations of inspired O 2 fraction and to increasing concentrations of inspired CO 2 fraction in subjects with CMS. We specifically aimed to extend our previous findings (Richalet et al., 2005), by comparing now the efficacy of two acetazolamide doses. 2. Methods The study was performed in Cerro de Pasco, Peru, at an altitude of 4300 m. It was approved by the ethics committee of Cayetano Heredia University in Lima, Peru. A written informed consent was obtained from all participants before performing any study procedure. This was a randomized doubled-blind study that compared the effect of two doses of acetazolamide in subjects with CMS, who had undergone a parallel study (Richalet et al., 2005). Subjects were assigned to 250 mg/day or to 500 mg/day of acetazolamide for 3 weeks. Eighteen male subjects born and living in Cerro de Pasco were investigated. Their last stay at lower altitudes had to be at least 6 months before enrolment in the study. CMS presence was determined through the CMS score (León-Velarde et al., 2005). This scoring system includes clinical symptoms found consistently in CMS (dizziness, physical and mental fatigue, anorexia, paresthesia of hands and feet, cyanosis of lips, face, or hands, vasodilatation of capillaries of conjunctives, hands and feet, sleep disturbance, breathlessness or palpitations, tinnitus, and headache). It also considers as pathological Hb value above 21 g/dl (León-Velarde et al., 2005). A medical history and examination were conducted to exclude major diseases. The subjects also underwent spirometry (Hill Med-Specialist, Miami, FL), hematocrit measurement by microcentrifugation, and arterial oxygen saturation (SaO 2 ) through a pulse-oximeter (Nellcor Incorporated, Pleasanton, CA). The end-tidal PO 2 (PET O2 ) and end-tidal PCO 2 (PET CO2 ) of each subject were determined during daytime, using a fast gas analyzer (Normocap Oxy; Datex-Ohmeda Division, Instrumentarium, Hatfield, UK). A fine nasal catheter was used so as to disturb the subject as little as possible. An instantaneous value for the respiratory quotient was calculated as a further verification procedure to ensure that the subject was not hyperventilating. In general, experiments were performed in equal numbers on each experimental day. Subjects were seated comfortably and breathed through a mouthpiece with the nose occluded. A pulse-oximeter was attached to the ear lobe to monitor the SaO 2. The mouthpiece assembly contained low-resistance inspiratory and expiratory valves. In this implementation, the gas was mixed in a small mixing chamber close to the subject, and inspiratory and expiratory volumes were recorded via a turbine volume-measuring device (VMM 400, Interface Associates, Laguna Niguel, CA) for determinations of VE. Gas at the mouth was sampled continuously and analyzed for PCO 2 and PO 2 (Normocap Oxy, Datex). PET O2 and PET CO2 values associated with each of the inspiratory gas mixtures were recorded in the last 30 s of exposure to each gas mixture, once the breathing pattern of subjects reached a steady state, as previously reported (Gamboa et al., 2003) Experimental assessment of sensitivity to hypoxia The protocol for assessing sensitivity to hypoxia involved administering 10 different gas mixtures. A settling period of 10 min was employed, during which PET O2 was held at 100 Torr, and at a free inspired CO 2 in breathing air. The subsequent nine mixtures contained a progressively decreasing FI O2, corresponding to a decrease in PET O2 from 75.2 to 38.1 Torr. PET O2 values for creating the nine steps had been calculated so as to provide approximately even reductions in saturation and consequently, approximately even increase in ventilation between steps. Each exposure level lasted 1 min Experimental assessment of sensitivity to CO 2 The ventilatory response to CO 2 was tested in conditions of relative hyperoxia (PET O2 = 100 Torr), with a settling period of 10 min that was similar to the protocol for assessing sensitivity to hypoxia. We employed a background of high O 2 to minimize the activity of the peripheral chemoreceptors. After the settling period, seven different gas mixtures with increasing FIC O2 were administered progressively, each step lasting 1 min. The FIC O2 values (in percentage) were increased by 0.2 above the resting PET CO2 values for each individual and for each step (each individual at rest was breathing no CO 2 ). We kept the PET CO2 change small ( 6 Torr) to minimize the extent of the Bohr effect on the constancy of PaO 2 and of Hb oxygen saturation. The gases were mixed during each experiment by using a column rotameter. The total gas flow of 50 l/min was in excess of the mean inspiratory flow rate of the subjects at their highest level of stimulated breathing. A change in inspired gas composition normally took approximately 10 s to complete (Vargas et al., 1998) Data analysis In both protocols, the average VE was calculated for the last 30 s of exposure to each gas mixture. Hyperoxic CO 2 ventilatory response (HCVR) was calculated as VE/ PET CO2 for each individual and as VE at a common PET CO2 to all the ventilatory response measurements. This HCVR was calculated through interpolation of individual VE values versus PET CO2, and it represents a set point for the ventilatory response (Swenson and Hughes, 1993). Twoway ANOVA with Bonferroni post hoc tests was used for comparing between group differences, one factor being before and after treatment values, and the other factor low and high ACZ dosage. Mean

3 186 M. Rivera-Ch et al. / Respiratory Physiology & Neurobiology 162 (2008) Table 1 Baseline physiological parameters of patients with chronic mountain sickness ACZ, 250 mg/day (n =9) ACZ, 500 mg/day (n =9) P-Value a Age (years) ± ± Weight (kg) ± ± Height (m) 1.60 ± ± Hematocrit (%) ± ± RBC (millions/mm 3 ) 6.79 ± ± VC (l/(min m 2 )) ± ± FEV ± ± Values are mean ± S.D. RBS, number of red blood cells; VC, vital capacity; FEV1, forced expiratory volume at 1 s. a When compared the two dose groups (independent-samples t-test). data obtained were normalized through natural logarithmic transformation to account for wide ranges of standard deviation in outcome variables shown in Table 2, and thus improve the fitness of the data and the reliability of the analyses (Bland and Altman, 1996). In addition, linear regression was employed within each subject groups to determine the slope of the relationship of VE versus PET CO2. Differences were considered significant if p < All the data were analyzed with the statistical package SPSS for Windows, version Results Subjects in both study groups were closely matched for baseline anthropometric and hematologic parameters (Table 1). From the original 20 subjects enrolled, two patients withdrew prematurely, one in each group, because of reasons not related to the study medication. Thus 18 subjects, 9 in each group, were finally studied. The mean data for resting ventilation, VE, PET O2, PET CO2, and SaO 2 for all subjects before and after ACZ treatment are shown in Table 2. The drug increased resting ventilation ( VE), and resulted in a lower resting PET CO2 and in a higher PET O2 and SaO 2 (Table 3). There were not significant differences among the two dose groups (Table 3) Sensitivity to hypoxia The average ventilatory response values ( VE as a function of SaO 2 ) before ACZ treatment did not differ from those seen after treatment (Fig. 1), and neither the slope (l/min, %) nor the intercept values for AHVR were significantly different. In regard to the comparison among dose groups, some subjects from 500 mg dose showed a trend to higher values after treatment, whereas variation between subjects was lower in those from 250 mg group. However, there was no significant difference between average values between each dose group. Fig. 1. Ventilatory responses to acute hypoxia in patients with chronic mountain sickness (CMS): (A) subjects treated with 250 mg/day of acetazolamide (G250); (B) subjects treated with 500 mg/day of acetazolamide (G500). (Filled circles: before acetazolamide; open circles: after acetazolamide) Mean ± S.E. values Sensitivity to CO 2 The effects of ACZ on ventilatory responsiveness to CO 2 (HCVR) are expressed as the slope of the VE versus PET CO2 relationship ( VE/ PET CO2 ) and as the interpolation of the individual plots of VE versus PET CO2 (Fig. 2). The HCVR slope was unchanged with ACZ administration in the group receiving 250 mg/day (Pre, 1.83 ± 1.00; Post, 2.06 ± 1.07 l/(min Torr) as well as in the subjects receiving 500 mg/day of ACZ (Pre, 1.68 ± 1.05; Post, 2.32 ± 1.32 l/(min Torr). However, ACZ increased the set point ( VE at PET CO2 equal to 31 Torr) from 10.2 to 19.3 l/min (p < ) in the subjects receiving the lower dose, and from 10.1 to 21.9 l/min (p < ) in the group given the higher dose, without significant difference within the two ACZ doses. 4. Discussion Table 2 Effects of acetazolamide (ACZ) on resting respiratory status, after log transformation (for significance of comparisons, see Table 3) ACZ (250 mg/day) ACZ (500 mg/day) Before (n = 9) After (n = 9) Before (n = 9) After (n = 9) Log VE (l/min) 2.30 ± ± ± ± 0.04 Log PET O2 (Torr) 3.90 ± ± ± ± Log PET CO2 (Torr) 3.33 ± ± ± ± 0.07 Log SaO 2 (%) 4.44 ± ± ± ± 0.03 Log RR (resp/min) 2.90 ± ± ± ± 0.12 Log RQ 0.15 ± ± ± ± 0.01 Values are mean ± S.D. VE, minute ventilation; PET O2, end-tidal PO 2 ; PET CO2, endtidal PCO 2 ; SaO 2, oxygen saturation; RR, respiratory rate; RQ, respiratory quotient. To our knowledge, this is the first study to assess the chronic effect of ACZ on ventilation and ventilatory control in subjects with CMS. We sought to determine if the chronic inhibition of carbonic anhydrase might show a similar therapeutic efficacy in chronic hypoxia, as it does in subjects exposed to acute hypoxia, due to its effects in improving the blood oxygenation. Our most important findings are that ACZ administered regularly during 3 weeks: (1) increased significantly the ventilatory response to hypoxia and resulted in a lower resting PET CO2, (2) caused a leftward shift in the position, but not a change in the slope of VE versus PET CO2 relationship, and (3) there was no difference between 250 and 500 mg/day treatments.

4 M. Rivera-Ch et al. / Respiratory Physiology & Neurobiology 162 (2008) Table 3 P-Values when comparing before after and higher lower dose groups (two-way ANOVA) ACZ 250 before, with ACZ 500 before, with ACZ 500 after, with ACZ 500 after ACZ 250 after ACZ 250 before ACZ 500 after ACZ 250 after ACZ 250 after Log VE Log PET O Log PET CO Log SaO Log RR Log RQ A significant increase in the ventilatory response to hypoxia and a significant decrease in PET CO2 were observed in both dose groups, although we must acknowledge that we did not use a placebo control group in our study. ACZ has multiple effects on the ventilatory reflexes. Thus the metabolic acidosis induced by ACZ has a stimulatory effect on central chemoreceptors, while ACZ may blunt the carotid body response to hypoxia (Teppema et al., 1988; Leaf and Goldfarb, 2007), although the mechanism of this inhibitory effect has not been elucidated yet. In cats, ACZ reduces carotid body output and baseline CO 2 sensitivity in vivo (Wagenaar et al., 1996) and abolishes the hypoxic ventilatory response (Teppema et al., 1992). Previous studies in chronic hypoxia in humans showed no increase in the AHVR with carbonic anhydrase inhibitors (Tojima et al., 1986; Hackett et al., 1987; Teppema et al., 2007). In regard to the effect of ACZ on the CO 2 related responses, Teppema and Dahan (1999), found in human subjects an unchanged peripheral CO 2 sensitivity and time constant after 250 mg of oral ACZ every 8 h during 3 days. Swenson and Hughes (1993) observed that ACZ increases resting ventilation and the ventilatory response to CO 2, with a decrease of the CO 2 response slope during hypoxia after acute administration. Indeed, length of exposure, species dif- Fig. 2. Ventilatory responses to CO 2 in patients with chronic mountain sickness (CMS): (A) subjects treated with 250 mg/day of acetazolamide (G250); (B) subjects treated with 500 mg/day of acetazolamide (G500). (Filled circles: before acetazolamide; open circles: after acetazolamide) Mean ± S.E. values. ferences and variations in methodology and dose to determine the slope of the CO 2 response curve, may all account for these varying study results. However, in our study, we did not show an additional beneficial effect of 500 mg/day of ACZ, neither in the resting PET CO2 nor in the position or the slope of VE versus PET CO2 relationship, when compared with 250 mg/day. This finding would suggest that a highest level of metabolic acidosis might have been attained with the lower dose, and that 250 mg/day of ACZ is enough to cause an effective inhibition of systemic carbonic anhydrase. One weakness of our study is that the acid base status of the subjects was not measured. However, Richalet et al. (2005) calculated those values from the resting values of PET O2 and PET CO2 and the plasma bicarbonate concentration for those subjects. In that study, as expected, ACZ-induced metabolic acidosis, which is supposed to participate not only in the stimulation of ventilation, but also in producing a rightward shift of the oxyhemoglobin dissociation curve that might allow a better oxygenation of renal cells producing erythropoietin. ACZ is an efficient pharmacological tool to lower plasma bicarbonate concentration and arterial PCO 2. For patients with CMS it is essential that they are able to lower their PCO 2 (and increase their PO 2 ), because these subjects are not only hypoxic, but also hypercapnic. As ACZ decreases the resting PET CO2, it could also decrease PaCO 2 with a minor increase in ventilation. This possibility is in line with the effects of ACZ in patients with chronic pulmonary obstructive disease. In treated patients it caused an improvement of blood gases without a significant rise in ventilation (Vos et al., 1994). In fact, CMS subjects treated with 250 mg/day of ACZ showed a decrease in mean PET CO2 of approximately 4.1 Torr upon a rise in PET O2 of 4.2 Torr and an increase in mean SaO 2 of approximately 6.3%. Sensitivity to CO 2 and the time constant of response to CO 2 are lower in HA individuals than in SL residents (Fatemian et al., 2003). The time constant depends on the cerebral blood flow per unit volume of brain tissue together with the ratio between blood and brain for the buffering capacity of CO 2 (Robbins, 1984). Of these factors, it is well recognized that cerebral blood flow is decreased in HA natives (Milledge and Sorensen, 1972; Sorensen et al., 1974; Brugniaux et al., 2007). In these circumstances, the increase in hematocrit at high altitude will increase the buffering capacity of the blood, but, on the other hand, will reduce the plasma flow, and hence the space for ion exchange in the blood (Fatemian et al., 2003). Administration of ACZ is known to increase cerebral blood flow in different species (Laux and Raichle, 1978; Hauge et al., 1983). However, this seems to be strongly dependent on the dose used (Huang et al., 1988). In spite of the latter, in the face of a lower PCO 2, it cannot be ruled out that a direct vascular action of ACZ and/or an influence of the metabolic acidosis that alters the cerebral blood flow control may occur in order to maintain an adequate supply of oxygen to the brain in hypoxic conditions (Teppema et al., 2007), although this possibility remains speculative. In addition, a lowered cerebrovascular CO 2 sensitivity may play a role in the genesis of periodic breathing at high altitude (Ainslie

5 188 M. Rivera-Ch et al. / Respiratory Physiology & Neurobiology 162 (2008) et al., 2007). In fact, CMS patients have lower saturations at night (Bernardi et al., 2003; Richalet et al., 2005). We know from Dempsey (2005), and Swenson and Teppema (2007), that breathing during sleep is more stable with ACZ, because it increases the CO 2 reserve, reduces the ventilatory increase required for a given reduction in PaCO 2, and reduces the ventilatory responsiveness to CO 2 above eupnea. Thus it is likely that CMS patients will have additional benefits with the regular use of ACZ. It must also be taken into account that with hypoventilation, the ph of the cerebrospinal fluid (CSF) increases by accumulating HCO 3,asCO 2 dissociates into HCO 3 and H+. Therefore, a reduction of intracellular ph or a diminution of HCO 3 concentration might be needed in order to reset the chemoreceptors to operate around the HA resting PCO 2. Because of the logarithmic relationship between ph and C O2, the CSF ph change produced by a PCO 2 change is greater at low PCO 2 than at high PCO 2. In this sense, Severinghaus et al. (1966) have shown that normal HA natives and CMS patients may display a 30% and 18% rise in CSF ph, respectively, as related to sea-level natives, in order to maintain ventilation at 15 l/(min m 2 ) with a Pa O2 equal to 40 Torr. Thus CMS subjects might accumulate HCO 3. However, a potential role for HCO 3 as a chemosensitive signal has not been extensively investigated. HCO 3 could affect neuronal activity in several ways. The exchange of HCO 3 and Cl across the bloodbrain barrier is involved in the response of the brain extracellular space to respiratory acidosis in the intact organism (Ahmad and Loeschcke, 1982). HCO 3 -dependent transporters are involved in the regulation of intracellular ph of neurons in chemosensitive and nonchemosensitive areas of the brain (Putnam, 2001). HCO 3 has also the capacity to reduce free radical production, which was shown to activate chemosensitive neurons within the nucleus tractus solitarius (Mulkey et al., 2003). In addition, the GABA A channel has been shown to be permeable to HCO 3 (Kaila and Voipio, 1987), which can result in an efflux of HCO 3 that leads to an intracellular acidification. Taken as a whole, this information suggests a role for HCO 3 in chemosensitive signaling (Putnam et al., 2004). However, with the current evidence, it is not possible to establish its role in resetting the resting PCO 2 of CMS patients to normal HA natives values. In conclusion, the beneficial effects of ACZ in CMS patients by reducing erythropoiesis (Richalet et al., 2005) are due, in part, to increased blood oxygenation, which is revealed by elevated PET O2 and SaO 2 after 3 weeks of treatment. 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