Differences in the control of breathing between Andean highlanders and lowlanders after 10 days acclimatization at 3850m

Transcription

1 J Physiol () pp Differences in the control of breathing between Andean highlanders and lowlanders after days acclimatization at 385m Marat Slessarev, Alexandra Mardimae,DavidPreiss,AlexVesely 3, Dahlia Y. Balaban,RichardGreene 4, James Duffin and Joseph A. Fisher Departments of Anesthesia, Medicine and Physiology, University Health Network, University of Toronto, Toronto, Canada, M5G C4 Boston University School of Medicine, Boston, MA 8, USA 3 Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada V6T Z3 4 New Mexico Highlands University, Las Vegas, NM 877, USA We used Duffin s isoxic hyperoxic (P O = 5 mmhg) and hypoxic (P O = 5 mmhg) rebreathing tests to compare the control of breathing in eight (7 male) Andean highlanders and six (4 male) acclimatizing Caucasian lowlanders after days at 385 m. Compared to lowlanders, highlanders had an increased non-chemoreflex drive to breathe, characterized by higher basal ventilation at both hyperoxia (.5 ±.7 vs. 4.9 ±.5 l min, P =.) and hypoxia (3.8 ±.4 vs. 5.7 ±.9 l min, P <.). Highlanders had a single ventilatory sensitivity to CO that was lower than that of the lowlanders (P <.), whose response was characterized by two ventilatory sensitivities (VeS and VeS) separated by a patterning threshold. There was no difference in ventilatory recruitment thresholds (VRTs) between populations (P =.9). Hypoxia decreased VRT within both populations (highlanders: 36.4 ±.3 to 3.7 ±.7 mmhg, P <.; lowlanders: 35.3 ±.3 to 8.8 ±.9 mmhg, P <.), but it had no effect on basal ventilation (P =.) or on ventilatory sensitivities in either population (P =.684). Within lowlanders, VeS was substantially greater than VeS at both isoxic tensions (hyperoxic: 9.9 ±.7 vs..8 ±., P =.5; hypoxic: 3. ±.9 vs..8 ±.5, P <.), although hypoxia had no effect on either of the sensitivities (P =.9). We conclude that the control of breathing in Andean highlanders is different from that in acclimatizing lowlanders, although there are some similarities. Specifically, acclimatizing lowlanders have relatively lower non-chemoreflex drives to breathe, increased ventilatory sensitivities to CO, and an altered pattern of ventilatory response to CO with two ventilatory sensitivities separated by a patterning threshold. Similar to highlanders and unlike lowlanders at sea-level, acclimatizing lowlanders respond to hypobaric hypoxia by decreasing their VRT instead of changing their ventilatory sensitivity to CO. (Received 4 December 9; accepted after revision March ; first published online 5 March ) Corresponding author J. A. Fisher: Toronto General Hospital, Department of Anesthesiology 3EN, Elizabeth St, Toronto, Canada, M5G C4. joe.fisher@utoronto.ca Abbreviations HVR, hypoxic ventilatory response; P ET,CO, end-tidal P CO ; P ET,O, end-tidal P O ; T, second ventilatory threshold; VeB, sub-ventilatory recruitment threshold basal ventilation; VeS, ventilatory sensitivity to CO in highlanders; VeS, first ventilatory sensitivity to CO in lowlanders; VeS, second ventilatory sensitivity to CO in lowlanders; VRT, ventilatory recruitment threshold. Introduction Ventilatory acclimatization to hypoxia in lowlanders has a complicated time course that results in a progressive increase in resting ventilation occurring over hours to days (Powell et al. 998). This acclimatization is accompanied by an increased hypoxic ventilatory response (HVR) (Sato et al. 99; Howard & Robbins, 995). In contrast, Andean highlanders are known to have lower alveolar ventilation compared to acclimatized lowlanders (Chiodi, 957; Severinghaus et al. 966; Lahiri, 968; Cudkowicz et al. 97; Beall et al. 997; Moore, ), and blunted HVR (Severinghaus et al. 966; Sorensen & Severinghaus, 968; Velasquez et al. 968; Lahiri et al. 969; Cudkowicz C The Authors. Journal compilation C The Physiological Society DOI:.3/jphysiol

2 68 M. Slessarev and others J Physiol et al. 97; Beall et al. 997; Gamboa et al. 3; Brutsaert et al. 5) despite their chronic exposure to hypoxia. The mechanisms responsible for these differences are unclear, but they probably involve alterations in the control of breathing (Moore, ; Brutsaert, 7). The control of breathing can be divided into chemoreflex and non-chemoreflex drives to breathe (Fig. ) (Lloyd & Cunningham, 963). Non-chemoreflex drives (e.g. wakefulness drive, voluntary cortical drive, hormonal factors) have never been measured in highlanders, while a single study of acclimatization in lowlanders showed that these drives remain unchanged from sea-level values after 5 days of high altitude exposure (Somogyi et al. 5). Chemoreflex drives to breathe can be further divided into central and peripheral drives. Both central and peripheral chemoreceptors respond to changes in hydrogen ion concentration ([H + ]) in their immediate environments (Torrance, 996; Nattie & Li, 9), while peripheral chemoreceptors are also responsive to changes in P O via hypoxia-mediated changes in their [H + ]ion sensitivity (Cunningham, 987; Torrance, 996; Kumar & Bin-Jaliah, 7). As a result, most studies in acclimatizing lowlanders and Andean highlanders have induced acute changes in P O and P CO and measured the ensuing changes in ventilation to assess the chemoreflex control of breathing (Duffin, 7). These studies suggest that acclimatization to high altitude in lowlanders affects both central and peripheral chemosensitivity. However, the findings are contradictory, such that central chemosensitivity is either increased (Schoene et al. 99; Fatemian & Robbins, 998; Ainslie et al. 3) or remains unchanged (Sato et al. 99; Somogyi et al. 5; Ainslie & Burgess, 8) and peripheral chemosensitivity is either increased (Ainslie & Burgess, 8) or remains unchanged (Somogyi et al. 5). On the other hand, Andean highlanders have been shown to have higher total and peripheral chemosensitivity to CO during euoxia ( mmhg) compared to sea-level lowlanders, with this difference abolished during hypoxia (5 mmhg) (Fatemian et al. Non-chemoreflex drive Chemoreflex drive Central drive Peripheral drive Respiratory control centre Ventilatory drive Central chemoreceptors + Brain tissue P CO / H + Peripheral chemoreceptors + + +/- Pulmonary ventilation CBF Arterial P O Arterial P CO / H + Figure. The control of breathing model (Lloyd & Cunningham, 963) The total ventilatory drive is the sum of chemoreflex and non-chemoreflex drives to breathe, which are integrated in the respiratory centre. The ventilatory drive exerts its action on the respiratory muscles that affect pulmonary ventilation and result in changes in arterial P CO and P O. Arterial [H + ] is sampled by the peripheral chemoreceptors located in the carotid bodies, where it determines the peripheral chemoreflex drive. Hypoxia exerts its effect on ventilation via peripheral chemoreceptors, where it acts indirectly via increasing the ventilatory sensitivity to [H + ] in most individuals but may also act directly by increasing the overall activity of the receptor. Central chemoreceptors respond to changes in the local [H + ] environment, which is affected by brain tissue P CO.Brain tissue P CO is a function of both arterial P CO and cerebral blood flow, which acts to decrease the brain tissue P CO at a higher flow (Berkenbosch et al. 989; Mohan et al. 999). The central and peripheral chemoreflex drives add together to form a total chemoreflex drive to breathe. C The Authors. Journal compilation C The Physiological Society

3 J Physiol Respiratory control at altitude 69 Table. Resting anthropomorphic and arterial blood gas data from all subjects Age Height Weight Hb Hct ph P a,co P a,o BE HCO 3 TCO S a,o Lac Subject (yrs) (cm) (kg) (g dl ) (%) (mmhg) (mmhg) (mequiv l ) (mmol l ) (mmol l ) (%) (mmol l ) Highlanders (H) H H H H H H n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a H H n Mean (S.D.) (8.6) (8.5) (4.9) () (3) () (3.7) (4.6) (.) (.) (.4) (.5) (.3) Lowlanders (L) L n/a n/a L n/a n/a L n/a n/a L L L n/a n/a n/a n Mean (S.D.) (3.8) (3.6) (7) (.) (3.7) () (.9) (4) (.9) (.7) (.) (4.) (.) Note that in one subject (H7) no arterial blood gas data were obtained due to inability to obtain a blood sample on multiple attempts. Also note that arterial oxygen saturation and lactate values are missing (n/a) in some lowlander subjects due to malfunction of blood analyser cartridges. Hb, haemoglobin concentration; Hct, haematocrit; P a,co, arterial P CO ; P a,o,arterialp O ; BE, base excess; HCO 3, bicarbonate ion concentration; TCO, total CO ; S a,o, arterial oxygen saturation. 3). The comparison of these findings in acclimatizing lowlanders and Andean highlanders is complicated by the different methodologies used (Moore, ; Duffin, 7). To date, there have been no studies that specifically compared both chemoreflex and non-chemoreflex drives to breathe between highlanders and acclimatizing lowlanders. To resolve these conflicting findings we used Duffin s rebreathing technique (Duffin et al. ) to compare chemoreflex and non-chemoreflex drives to breathe between acclimatizing Caucasian lowlanders following days at 385 m and Andean highlanders. Methods Subjects The study was approved by the Ethics Review Board of the University Health Network, Toronto, Canada and Comisión de Ética de la Investigación (CEI) del Comité Nacional de Bioética, La Paz, Bolivia. All studies were performed in accordance with the Declaration of Helsinki of the World Medical Association (4). For acclimatized lowlander studies, six healthy Caucasian subjects (4 male) were recruited from the members of the 8 Bolivia expedition and were all members of a respiratory research laboratory at the University of Toronto in Canada. All six lowlander subjects were also authors on the present manuscript. Their mean ± S.D. age, height and body mass were 3.7 ± 3.8 years, 74. ± 3.6 cm and 7. ± 7. kg, respectively (Table ). For highlander studies, 8 healthy (7 male) long term residents of La Paz, Bolivia (altitude 385 m) were recruited. The highlander subjects identified themselves as belonging to the native Aymara population. However, since no studies were done to determined the extent of their Aymara heritage, we are not able to comment on the degree of European admixture among our highlander subjects. The duration of residence at high altitude of our highlander subjects is unknown, but in general it is thought that the Andean highlanders have resided at altitude for at least, years (Aldenderfer, 3). Their mean ± S.D. age, height and body mass were 38. ± 8.6 years, 58.8 ± 8.5 cm and 63.8 ± 4.9 kg, respectively (Table ). Resting arterial blood gas data were obtained from all subjects (except highlander 7, in whom we were unable to obtain an arterial blood sample). Anthropomorphic and resting arterial blood gas data are summarized in Table. Each subject gave an informed written consent before commencing the study. General protocol Prior to experiments, all lowlander and highlander subjects were familiarized with the rebreathing apparatus C The Authors. Journal compilation C The Physiological Society

4 6 M. Slessarev and others J Physiol and were allowed to complete familiarization tests. For highlanders, the familiarization protocol entailed first watching one of the research scientists perform both hypoxic and hyperoxic rebreathing tests on themselves, and then breathing on the rebreathing apparatus and performing several trial rebreathing runs. At the same time, the nature of the experiments and specific instructions were provided in the highlanders native language through an interpreter, and all their questions were answered. All of the lowlander subjects were members of the respiratory research laboratory and were familiar with the rebreathing experiments. All tests were conducted in the laboratory at the High Altitude Pathology Institute, Clinica IPPA, La Paz, Bolivia (altitude 385 m, ambient barometric pressure 484 mmhg, ambient temperature 8 5 C; temperature and pressure were measured with a standard thermometer and barometer, respectively). Each subject performed a hypoxic and a hyperoxic test in random order, with at least min of rest time between the tests. Lowlanders were tested days after arrival in La Paz (Altitude 385 m) from sea-level (Toronto, Canada, altitude 5 m). Chemoreflex assessment Testing method. Duffin s rebreathing tests (Mohan & Duffin, 997) were used to assess subjects chemoreflexes. Theirinterpretationisgivenin(Duffinet al. ), and they have been extensively discussed (Duffin et al. ; Mahamed & Duffin, ; Mateika et al. 4). In this modification of Read s original method (Read, 967), the rebreathing stage is preceded by 5 min of hyperventilation and isoxia is maintained throughout the test. The hyperventilation ensures that the rebreathing stage starts at a CO level below the ventilatory recruitment threshold (VRT), which enables both the VRT and the sub-threshold (basal) ventilation to be measured (Mohan et al. 999). The latter measures the contribution of the non-chemoreflex drives to breathe (Shea, 996), such as the wakefulness drive (Fink, 96). Rebreathing is repeated at two different isoxic P O tensions: once during hyperoxia (P O = 5 mmhg), to minimize the contribution of the peripheral chemoreceptors and preferential measurement of the central chemoreceptor response (Lloyd & Cunningham, 963), and once during hypoxia (P O = 5 mmhg), to allow measurement of the combined central and peripheral chemoreceptor responses. The difference between the hypoxic and hyperoxic responses represents the contribution of the peripheral chemoreflex and can be used to calculate HVR at any given isocapnic end-tidal P CO (P ET,CO )(Duffin, 7). Testing protocol. All subjects were seated comfortably in an upright position during the rebreathing tests and wore a finger pulse-oximetry probe. The testing environment was quiet, with minimal distractions. Subjects wore nose clips while breathing via a bacterial viral filter (Allegiance, Healthcare Corp., McGaw Park, IL, USA) connected to one side of a Series 87 three-way sliding valve (Hans Rudolph Inc., Kansas City, MO, USA) that allowed the operator to alternate the subject s breathing between ambient air and the rebreathing bag. The rebreathing bag had a volume of 5 litres and an oxygen inlet. It was primed with a CO O mixture to ensure appropriate equilibration at the beginning of the rebreathing test (Mohan & Duffin, 997). Inspired and expired partial pressures of CO and O were sampled at the mouth and monitored throughout the test using infra-red CO and polarographic O sensors (RespirAct, Thornhill Research Inc, Toronto, Canada). The linearity of both gas analysers in the measurement range used in the current study is ±% full scale for the O analyser and ±.% full scale for the CO analyser. Ventilation was measured using a mass flow sensor (AWM7P Airflow, Honeywell, Freeport, IL, USA). Analog data were digitized (DAQCard-64E, National Instruments, Austin, TX, USA) and entered continuously into a computer. A custom-written program (LabVIEW, National Instruments; source code available on request) analysed the data to provide a file of breath-by-breath end-tidal P CO (P ET,CO ), end-tidal P O (P ET,O ), and ventilation. In addition, the program operated a solenoid valve controlling the flow of oxygen to the rebreathing bag to maintain desired isoxic tensions during rebreathing: a P O of 5 mmhg (hyperoxic) or 5 mmhg (hypoxic). The P CO and P O analysers were calibrated using gas from cylinders of analysed medical grade compressed gases, and ventilation was calibrated using a 3 litre calibration syringe (Model R553B, Vacumed, Ventura, CA, USA). A portable pulse oximeter (Autocorr Plus Vital Signs Monitor, BCI international, USA) was used to measure arterial oxygen saturation (S a,o )andheart rate. Each rebreathing test began with a 5 min hyperventilation of ambient air, with subjects coached to maintain their P ET,CO between and 5 mmhg. Subjects then exhaled completely and were switched to the rebreathing bag, from which they took three deep breaths to facilitate rapid equilibration of P CO in the bag, lungs and arterial blood with that of mixed venous blood. This equilibration was verified by observing a plateau in the end-tidal P CO, and was a prerequisite for continuation of the test. The rebreathing test ended when either ventilation exceeded l min, P ET,CO exceeded 6 mmhg, ventilatory discomfort was no longer tolerable, or sufficient data were obtained to show the ventilatory response. The majority of the rebreathing tests were terminated when sufficient data were obtained or when discomfort was indicated by the subject. C The Authors. Journal compilation C The Physiological Society

5 J Physiol Respiratory control at altitude 6 Rebreathing test analysis. Rebreathing test data were analysed using a custom-written program (LabVIEW, National Instruments; source code available on request). After eliminating the initial three equilibration breaths, as well as sighs, swallows and breaths incorrectly detected by the acquisition software, breath-by-breath P ET,CO values were plotted against time and fitted with a least squares regression line. This line provided a predicted value of P ET,CO as a function of time, thereby minimizing inter-breath variability. Tidal volume (ml BTPS), respiratory rate (breaths min ) and ventilation (l min BTPS) were then plotted against the predicted P ET,CO (mmhg). In highlanders (Fig. A), these plots were fitted by dividing the data into two segments separated by a breakpoint corresponding to the VRT, defined as the P ET,CO where ventilation started to increase in response to rising P ET,CO (Duffin et al. ). In lowlanders (Fig. B), the plots were fitted by dividing the data into three segments separated by two thresholds: the VRT as in highlanders and a second threshold (T) defined as the P ET,CO at which the slope of the ventilatory response to P CO changed. In both populations, the first segment was fitted with either an exponential decline to a final value, or a mean, and measured sub-vrt ventilation (VeB) that represented non-chemoreflex ventilation drive. In the present study, all first segments were fitted using means. Note that all responses were fitted with a physiological model in mind rather than an empirical curve-fitting technique. In highlanders, the second segment was fitted with a straight line, whose slope represented the ventilatory sensitivity (VeS). In lowlanders, the second and third segments were also fitted with straight lines, with slopes representing the first (VeS) and second (VeS) ventilatory sensitivities to CO, respectively. Model fitting was based on minimizing the sum of least squares for non-linear regression using the LabVIEW software (Levenberg Marquardt algorithm). The furthest outlying points were automatically discarded until an r value of >.95 was achieved. Statistical analyses All results are reported as mean ± S.E.M. (exceptwhere noted), with significance set at P <.5. A two-way repeated measures ANOVA, with factors population (highlanders vs. lowlanders) and isoxia (hypoxic vs. hyperoxic), and post hoc analysis using Tukey s test were used to detect differences in VeB, VRT and VeS. The highlander VeS was tested against both the VeS and VeS of lowlanders. In lowlanders, a two-way repeated measures ANOVA, with factors sensitivity (VeS vs. VeS) and isoxia (hypoxic vs. hyperoxic),and post hoc analysis using Tukey s test were used to detect differences between VeS and VeS. A Andean highlander B Acclimatizing lowlander VEB VRT VES = VE PCO VRT VES = VE VEB PCO T VES = VE PCO Figure. Breath-by-breath ventilation vs. end-tidal P CO during a representative Duffin s rebreathing test for one highlander (A) and one lowlander (B) illustrating fitting of the plots In highlanders (A), the plots were fitted by dividing them into segments, separated by a breakpoint corresponding to the ventilatory recruitment threshold (VRT). In lowlander subjects (B), the plots were fitted by dividing them into 3 segments, separated by two breakpoints, VRT and T. T was defined as the P ET,CO at which there was a change in the slope of the ventilatory response to CO above VRT. In both populations, the first segment was fitted with either an exponential decline to a final value, or a mean, and measured sub-vrt ventilation (VeB) that represents non-chemoreflex ventilation drive. In highlanders, the second segment was fitted with a straight line where the slope measured the sensitivity (VeS). In lowlanders, the second and third segments were also fitted with straight lines where the slopes measured the first (VeS) and second (VeS) ventilatory sensitivities to CO. C The Authors. Journal compilation C The Physiological Society

6 6 M. Slessarev and others J Physiol Table. Individual subject values for non-chemoreflex drives to breathe (VeB), ventilatory recruitment thresholds (VRT) and sensitivities to CO (VeS in highlanders; VeS and VeS in lowlanders) for all rebreathing tests Lowlanders after days at 385 m Isoxic hyperoxic tests (P O = 5 mmhg) Isoxic hypoxic tests (P O = 5 mmhg) VeB VRT VeS T VeS VeB VRT VeS T VeS Subject (l min ) (mmhg) (l min mmhg ) (mmhg) (l min mmhg ) (l min ) (mmhg) (l min mmhg ) (mmhg) (l min mmhg ) L L L L L L Mean 4.9 (.5) 35.3 (.3).8 (.) 4. (.7) 9.9 (.7) 5.7 (.9) 8.8 (.9).8 (.5) 33.6 (.8) 3. (.9) (S.E.M.) Andean highlanders Isoxic hyperoxic tests (P O = 5 mmhg) Isoxic hypoxic tests (P O = 5 mmhg) VeB VRT VeS VeB VRT VeS Subject (l min ) (mmhg) (l min mmhg ) (l min ) (mmhg) (l min mmhg ) H H H H H H H H Mean (S.E.M.).5 (.7) 36.4 (.3).8 (.) 3.8 (.4) 3.7 (.7) (.) Mean ± S.E.M. values for each condition and subject populations are compared with ( ) values indicating differences between hypoxic and hyperoxic tests within populations and ( ) values indicating differences between populations determined by repeated measures ANOVA (P <.5). VeS in highlanders was compared against both VeS and VeS of lowlanders. Results All subjects completed both rebreathing protocols. Table shows values of VeB, VRT and VeS for all highlander and VeB, VRT, VeS, T and VeS for all lowlander subjects. Figure 3 summarizes these findings for both populations. Highlanders had greater basal ventilation (hyperoxic:.5 ±.7 vs. 4.9 ±.5 l min, P =.; hypoxic: 3.8 ±.4 vs. 5.7 ±.9 l min, P <.), similar ventilatory recruitment thresholds (P =.9), and lower ventilatory sensitivities to CO than lowlanders, even when compared to first (VeS, P =.38) and second (VeS, P <.) lowlander sensitivities. Hypoxia decreased VRT within both populations (highlanders: 36.4 ±.3 to 3.7 ±.7 mmhg, P <.; lowlanders: 35.3 ±.3 to 8.8 ±.9 mmhg, P <.), but it had no statistically detectable effect on basal ventilation (P =.) or ventilatory sensitivities in either population (P =.684). Within lowlanders, VeS was substantially greater than VeS at both isoxic tensions (hyperoxic: 9.9 ±.7 vs..8 ±. l min mmhg, P =.5; hypoxic: 3. ±.9 vs..8 ±.5lmin mmhg, P <.), although hypoxia had no statistically detectable effect on either of the sensitivities (P =.9). Discussion This study is the first direct comparison of all three components of the Oxford control of breathing model (Fig. ) (Lloyd & Cunningham, 963), specifically looking at the central and peripheral chemoreflex drives to breathe and the non-chemoreflex drive to breathe in acclimatized lowlanders and Andean highlanders. As Fig. 3 illustrates, the control of breathing in acclimatized lowlanders differed from that of Andean highlanders in several aspects that are discussed below. Non-chemoreflex drives to breathe Basal ventilation in acclimatized lowlanders was lower than that in highlanders at both isoxic tensions. Since basal sub-vrt ventilation represents non-chemoreflex drives to breathe (Mohan et al. 999; Duffin et al. ), our findings suggest that the non-chemoreflex drive to breathe is either increased in Andean highlanders compared to lowlanders, or that the acclimatization process results in an early suppression of the non-chemoreflex drives to breathe in lowlanders. The latter hypothesis is unlikely, as five days of hypoxic exposure have been shown to have no effect on basal ventilation in acclimatizing C The Authors. Journal compilation C The Physiological Society

7 J Physiol Respiratory control at altitude 63 lowlanders (Somogyi et al. 5); and basal ventilations measured by Somogyi et al. (hyperoxic:.9 ±.7lmin, hypoxic: 4.5 ±. l min ) are comparable to those measured in the present study (hyperoxic: 4.9 ±.5lmin, hypoxic: 5.7±.9lmin ). Furthermore, studies using 3 h of isobaric, isocapnic hypoxia (Mahamedet al. 3) and min of intermittent isobaric, isocapnic hypoxia for weeks (Mahamed & Duffin, ) also found no changes in basal ventilation in lowlanders residing at sea-level. Unfortunately, the lack of sea-level control data in our lowlanders does not allow for direct comparison between their resting sea-level basal ventilations and those following days of altitude exposure. The purpose of this study was to compare control of breathing in Andean highlanders to that of acclimatized lowlanders, so sea-level baseline data for acclimatized lowlanders was not collected during the present study. An investigation of acclimatization and the control of breathing parameters in lowlanders has been previously reported (Somogyi et al. 5) and found no change in basal ventilation. We therefore cannot conclusively rule out the possibility of acclimatization-induced depression of the non-chemoreflex drive to breathe in lowlanders as a cause of their lower basal ventilations relative to highlanders. Alternatively, the relatively high basal ventilation in highlanders can be explained by their higher non-chemoreflex drives to breathe compared to acclimatizing lowlanders. One source of this drive to breathe could be the carotid body, which may increase its basal level of activity in response to chronic hypoxia. Evidence for this notion is the carotid body hypertrophy observed in highlanders (Arias-Stella & Valcarcel, 976; Kay & Laidler, 977; Heath et al. 985; Khan et al. 988; Lahiri et al. ). The change in carotid body function can be the result of chronic exposure to hypobaric hypoxia and, if found to be true in a larger case series and in other high altitude populations, could represent a form of adaptation to chronic hypoxia. Another source of the increased non-chemoreflex drive to breathe in Andean highlanders could be related to their experimental anxiety due to unfamiliarity with respiratory experiments, especially since all lowlander subjects were members of the respiratory research laboratory who were familiar with respiratory experiments. Interestingly, the basal ventilations observed in Andean highlanders in the present study are comparable to those measured in Himalayan highlanders and in sea-level lowlanders (university students) who are naive to respiratory experiments (Jensen et al. 5, ; Slessarev et al. ). Furthermore, all Andean highlanders were familiarized with the rebreathing experiments on their first visit to the laboratory (see Methods), which should have reduced their experimental anxiety. Since acclimatized lowlanders in the present study were experienced respiratory physiologists, it is possible that repeated exposure to rebreathing experiments has resulted in the attenuation of their experimental anxiety and basal ventilations to levels below those achieved during the brief familiarization protocol used with highlanders in the present study. However, recent repeatability studies failed to show such attenuation in experimentally naive sea-level lowlanders (Jensen et al. ). Lastly, other methods of studying the control of breathing, including classic HVR tests, are affected by experimental anxiety to the same extent as the current experiments. As a result, the reported basal ventilations are likely to be the best representation of the non-chemoreflex drives to breathe in the studied populations during the described testing conditions. Further studies with larger sample sizes may offer an alternative explanation for the observed phenomena. Pattern of ventilatory response to CO There was a distinct difference in the pattern of ventilatory response to CO between the two populations; in lowlanders it was characterized by two distinct sensitivities (VeS and VeS) separated by a threshold (T), as compared to a single sensitivity (VeS) in highlanders (Fig. ). Interestingly, VeS was greater than VeS in lowlanders, and both of these were greater than the VeS of highlanders (Table ). The occasional presence of two ventilatory sensitivities separated by a threshold has been previously reported in lowlanders residing at sea-level PO level (mmhg) Lowlanders Andeans PCO (mmhg) Figure 3. Mean breath-by-breath ventilation vs. P CO during isoxic hyperoxic and hypoxic Duffin s rebreathing tests for all Andean highlander (continuous lines) and lowlander (dashed lines) subjects Note the presence of two ventilatory sensitivity slopes, VeS and VeS, in acclimatized lowlanders, while highlander responses have only one slope. C The Authors. Journal compilation C The Physiological Society

8 64 M. Slessarev and others J Physiol (Duffin et al. ; Duffin & Mahamed, 3), where it has been attributed to a change in breathing pattern, with tidal volume reaching a plateau and further increase in ventilation with increasing CO driven by an increase in respiratory rate. In such a case the second threshold, T, has therefore been termed a patterning threshold (Duffin et al. ). The presence of T and two sensitivities in acclimatizing lowlanders has only been observed once previously (Burgess et al. 8), with another study identifying only the VRT and one ventilatory sensitivity (Somogyi et al. 5). Further analysis of our data shows that T corresponds to a change in pattern of breathing in all of our subjects. However, only one subject (L) showed the classic pattern of tidal volume plateau and an augmented increase in respiratory rate (Fig. 4 pattern A). The rest of the subjects displayed an augmentation in the rate of rise in both tidal volume and respiratory rate at T (Fig. 5), such that sensitivity post T (VeS) is greater than prior to T (VeS) (Fig. 4 pattern B). The reason for this newly observed pattern of ventilatory change at T is unclear and may be simply a characteristic of our particular small group of subjects. With T marking a simultaneous increase in the rate of rise of both tidal volume and respiratory rate in response to increasing CO, it appears that there is an overall increase in the ventilatory drive that occurs at T. One possible explanation for this may be that the acclimatizing lowlanders in the group that we tested have different central and peripheral chemoreflex thresholds (Mohan & Duffin, 997), with the central threshold Pattern A (limitation of V T and increase in RR at T) Pattern B (increase in V T and RR at T) VRT T 4 VRT T 3 Tidal Volume (Litres).5.5 Tidal Volume (Litres) Breaths/min Breaths/min Figure 4. Two types of patterning threshold (T) were identified in acclimatizing lowlanders Pattern A has been previously seen in lowlanders at sea-level (Duffin & Mahamed, 3) and is characterized by a decrease in the rate of rise of tidal volume (V T ) and an increase in the rate of rise in respiratory rate (RR), leading to overall increase in ventilatory sensitivity to CO above T. Pattern B is characterized by an increased rate of rise of both V T and RR above T, leading to an overall increase in ventilatory sensitivity to CO above T. Most of the acclimatizing lowlander subjects (5 out of 6) displayed pattern B. C The Authors. Journal compilation C The Physiological Society

9 J Physiol Respiratory control at altitude 65 corresponding to VRT and the peripheral threshold corresponding to T. The lack of T in our highlander population may suggest either that their central and peripheral thresholds are similar, as is the case in most lowlanders studied at sea-level, or that the peripheral threshold is absent altogether due to lack of peripheral chemosensitivity to CO. The latter hypothesis is supported by the absence of a hypoxia-induced increase in ventilatory CO sensitivity in highlanders (Fig. 3), which is a characteristic pattern observed in lowlanders at sea-level (Mohan & Duffin, 997). This absence may be attributed to a change in function of the carotid body and related to the observed hypertrophy and histological changes of this organ in highlanders (Arias-Stella & Valcarcel, 976; Kay & Laidler, 977; Heath et al. 985; Khan et al. 988; Lahiri et al. ). Ventilatory sensitivity to CO A third difference in the control of breathing between acclimatized lowlanders and Andean highlanders was the relatively greater ventilatory sensitivity to CO in acclimatized lowlanders both below (VeS) and above (VeS) T, although the difference between VeS in lowlanders and VeS in highlanders was significant only during hyperoxic (P =.9), but not hypoxic (P =.) tests (Table ). However, this non-significance may have been the result of a type- error due to the small number of subjects in the study (see Methodological considerations below). Since hyperoxia effectively silences the peripheral chemoreceptors (Lloyd & Cunningham, 963; Mohan & Duffin, 997), the ventilatory sensitivities during hyperoxic rebreathing tests primarily represent central chemoreceptor sensitivity (Duffin, 7). Our results suggest that this sensitivity is greater in acclimatized lowlanders than in highlanders, although the mechanism responsible for this difference is unclear. Previous studies of acclimatizing lowlanders showed either no change (Somogyi et al. 5) or an increase (Ainslie & Burgess, 8) in the sensitivity of the ventilatory response to CO following acclimatization to high altitude. However, these two studies differed in the duration and altitude of hypoxic exposure (5 days at 348 m in Somogyi et al.,and9daysat>5 m in Ainslie & Burgess), which could explain the observed difference in the results. Our study was done at an altitude (385 m) similartothatinsomogyietal. but the duration of hypoxic exposure in our study ( days) was closer to that in Ainslie & Burgess, and the ventilatory CO sensitivities measured in our study (hyperoxic:.8 ±.lmin mmhg ; hypoxic:. ±.lmin mmhg ) are closer to those measured by Somogyi et al. (hyperoxic:.3 ±.5 l min mmhg ; hypoxic: 3.7±.8lmin mmhg ) than those measured by Ainslie & Burgess (hyperoxic: 4.8 ± 3.5lmin mmhg ; hypoxic: 5.4 ±.lmin mmhg ). This comparison suggests that the change in ventilatory CO sensitivity in acclimatizing lowlanders is affected more by the absolute altitude of exposure (with higher altitude increasing ventilatory sensitivity to CO ) than by its duration. Ventilatory recruitment thresholds There was no difference in the VRT between the acclimatizing lowlanders and the Andean highlanders. A previous study (Somogyi et al. 5) showed that acclimatization to high altitude in lowlanders is accompanied by a decrease in the VRT from sea-level values. This finding is also supported by the previously observed leftward shift in the x-axis intercept of the ventilation P CO relationship with acclimatization (Ainslie & Burgess, 8). The leftward shift of the VRT can be attributed to an altered [H + ] P CO relationship that results from respiratory alkalosis caused by hypoxia-induced hyperventilation. The change in the [H + ] P CO relationship is such that the same [H + ] corresponds to different P CO values at sea-level and at altitude, with the altitude value being lower (Duffin, 5). The VRTs measured in our study were similar between acclimatized lowlanders and Andean highlanders at both isoxic tensions (hyperoxic: 35.3 ±.3 vs ±.3 mmhg; hypoxic: 8.8 ±.9 vs. 3.7 ±.7 mmhg, P =.9), while both were lower than those measured by Somogyi et al. (5) (hyperoxic: 4. ±.8 mmhg; hypoxic: 33. ±.6 mmhg). Since lowlanders in our study were exposed to a similar altitude, but for a longer duration of hypoxia ( vs. 5days), compared to the lowlanders in the study by Somogyi et al. the observed results suggest that the increased duration of hypoxic exposure results in progressive lowering of VRTs in acclimatizing lowlanders so that they are similar to those of adapted highlanders after days. It is therefore likely that an ongoing correction of respiratory-induced changes in acid base status is responsible for the observed changesinvrt;however,thishypothesisrequiresfurther investigation. Ventilatory response to hypoxia The ventilatory response to hypoxia in both populations was marked by a decrease in VRT (highlanders: 36.4 ±.3 to 3.7 ±.7 mmhg, P <.; lowlanders: 35.3 ±.3 to 8.8 ±.9 mmhg, P <.), with no change in basal ventilation (P =.) or ventilatory sensitivities in either population (P =.684). This is distinctly different from the response seen in lowlanders at sea-level, who were previously shown to respond to hypoxia by increasing their ventilatory sensitivity to CO C The Authors. Journal compilation C The Physiological Society

10 66 M. Slessarev and others J Physiol Lowlander PO level 5 5 PO =5 PO = PO level 5 5 PO =5 PO =5 Lowlander Lowlander PO level 5 5 PO =5 PO =5 Tidal Volume (L).5.5 PO =5 PO =5 Lowlander Tidal Volume (L).5.5 PO =5 PO =5 Lowlander Tidal Volume (L).5.5 PO =5 PO =5 Lowlander PO =5 Frequency (b/min) PO =5 Frequency (b/min) PO =5 PO =5 Frequency (b/min) 3 PO =5 PO =5 Tidal Volume (L) PO level Lowlander Predicted PCO.5 (mmhg) PO =5 PO = Lowlander Lowlander Lowlander PO =5 PO =5 Lowlander 4 Tidal Volume (L) PO level 5 5 Lowlander PO =5 PO =5 PO =5 PO =5 Tidal Volume (L) Lowlander Lowlander 5 Lowlander PO level 5 5 PO =5 PO =5 PO =5 PO =5 Frequency (b/min) 3 PO =5 PO =5 Frequency (b/min) PO =5 PO =5 Frequency (b/min) PO =5 PO =5 Lowlander Lowlander Lowlander Figure 5. Patterns of ventilatory response to hypoxic and hyperoxic rebreathing from all lowlander subjects illustrating the patterning thresholds Note that only Lowlander displayed the classic patterning threshold with tidal volume reaching a maximum so that a further increase in ventilation was driven by an increase in respiratory rate alone. The rest of lowlanders increased both their tidal volume and respiratory rate at T. C The Authors. Journal compilation C The Physiological Society

11 J Physiol Respiratory control at altitude 67 (Mohan & Duffin, 997). Interestingly, Andean highlanders, like Himalayan highlanders from our companion study (Slessarev et al. ), displayed marked heterogeneity in their ventilatory response to hypoxia (Fig. 6). While most highlanders seemed to decrease their VRT in response to hypoxia (the only change that was statistically significant), some also increased their basal ventilation without change in sensitivity or VRT, while others increased their sensitivity in addition to a change in VRT. Our limited sample size prevents us from commenting on whether these observations represent certain subsets within the highlander population, but it demonstrates that the ventilatory response to hypoxia in highlanders may be heterogeneous, which could further confound the interpretation of ventilatory control studies. More studies with larger sample sizes are required to further elucidate the nature of this heterogeneity. A decrease in VRT with hypoxia without an increase in sensitivity has been reported previously (Somogyi et al. 5), suggesting that this pattern of hypoxic response is ubiquitous in acclimatizing lowlanders. In our rebreathing tests the difference in ventilatory CO sensitivities between hyperoxic and hypoxic tests represents the contribution of the peripheral chemoreflex. The absence of a hypoxia-induced change in ventilatory CO sensitivity in acclimatized lowlanders and Andean highlanders therefore suggests that the hypoxic enhancement of peripheral CO chemosensitivity is blunted. Summary Our findings suggest that the control of breathing in acclimatizing lowlanders is distinctly different from that in Andean highlanders. Specifically, acclimatizing lowlanders have lower basal ventilations and relatively lower non-chemoreflex drives to breathe, higher ventilatory CO sensitivities, and show a patterning threshold. The nature of higher basal ventilations in highlanders is not clear and further dedicated studies are required to identify its source. However, there are also some similarities between Andean highlanders and acclimatizing lowlanders. For example, both populations lack the hypoxia-induced increase in ventilatory CO sensitivity seen in sea-level lowlanders, which suggests that peripheral CO O interaction is blunted in these populations. Instead, hypoxia induces adecreaseintheirvrt. Comparison of our data with previous studies suggests that a longer duration of hypoxic exposure in lowlanders results in a progressive decrease in VRTs, such that those of acclimatizing lowlanders are similar to those of Andean highlanders following days at high altitude, while exposure of only 5 days results in VRTs that are higher than those of Andean highlanders, but lower than those of sea-level lowlanders (Somogyi et al. 5). It is not clear which of the observed differences or similarities between the two populations are adaptive to life at high altitude. However, further studies and comparison of this data with other high altitude populations may help to identify specific changes in the control of breathing that may represent adaptive strategies. Methodological considerations The absence of statistically significant changes in basal ventilations and ventilatory sensitivities with hypoxia should be interpreted with caution, since the power of the performed within-population tests was relatively low due to small sample size in the present study. For instance, in highlanders a change in basal ventilation of 3% and in ventilatory sensitivity of 45% with hypoxia would have been required to produce tests with a power greater than 8%. Similarly, in lowlanders a change in basal ventilation of 5%, VeS of 36% and VeS of 84% with hypoxia would have been required to produce tests with a power greater than 8%. We therefore suggest that the general patterns of change should be taken into account when interpreting results. We used Duffin s rebreathing technique (Mohan et al. 999; Duffin et al. ) to measure the chemoreflex and non-chemoreflex drives to breathe. In contrast, previous studies of ventilatory control in acclimatizing lowlanders and highlanders have employed either steady-state methods or Read s rebreathing; the latter only measures the hyperoxic ventilatory response to CO. Although these methods provide useful information about ventilatory sensitivities to O and CO, the separation of the contributions of individual components of the control of breathing model is difficult (Fig. ), thereby complicating comparison between studies and between populations (Duffin, 7). Furthermore, increasing cerebral blood flow secondary to hypoxia or hypercapnia results in washout of CO and lower [H + ] at the central chemoreceptors with steady-state methods, thereby attenuating the measured ventilatory response (Berkenbosch et al. 989; Mohan et al. 999). Since control of cerebral blood flow in highlanders may be altered (Norcliffe et al. 5; Appenzeller et al. 6; Claydon et al. 8), the effects of this attenuation are hard to predict, which may further complicate the comparison of results between populations (Ainslie & Duffin, 9). We only studied Andean highlanders residing at one altitude (385 m). Previous studies have shown that the level of altitude residence may affect control of breathing in highlanders (Curran et al. 995), and this factor requires further evaluation, possibly with the rebreathing technique employed in the current study. Moreover, the C The Authors. Journal compilation C The Physiological Society

12 68 M. Slessarev and others J Physiol PO level 5 5 Highlander Highlander Highlander Highlander Highlander Highlander Highlander 7 3 Highlander Figure 6. Hypoxic and hyperoxic rebreathing responses from all highlander subjects Note the different patterns of responses between hypoxic and hyperoxic rebreathing (see text for more detailed discussion). C The Authors. Journal compilation C The Physiological Society

13 J Physiol Respiratory control at altitude 69 ancestry of highlanders, which may have an effect on the control of breathing (Curran et al. 997; Brutsaert et al. 5; Beall, 7), was not established in the present study. Future studies using Duffin s rebreathing method in larger samples of highlanders with a known pedigree of highlander ancestry may help identify specific aspects of the control of breathing model that are adaptive to life at high altitude. Lastly, recent studies suggest that there are several patterns of adaptation to life at high altitude (Moore, ; Beall, 7), with probable differences in the control of breathing between different high altitude populations. It would be interesting to compare the results of the present study with rebreathing studies from other high altitude populations in an attempt to identify specific differences in the control of breathing between populations. Conclusion The control of breathing in acclimatizing lowlanders is distinctly different from that in Andean highlanders, although some similarities exist. Compared to Andean highlanders, acclimatized lowlanders have relatively lower non-chemoreflex drives to breathe and a greater ventilatory response to CO. Both highlanders and acclimatized lowlanders display blunted peripheral CO chemosensitivity and respond to hypoxia with a decrease in ventilatory recruitment threshold. References Ainslie PN & Burgess KR (8). Cardiorespiratory and cerebrovascular responses to hyperoxic and hypoxic rebreathing: effects of acclimatization to high altitude. Respir Physiol Neurobiol 6, 9. Ainslie PN & Duffin J (9). Integration of cerebrovascular CO reactivity and chemoreflex control of breathing: mechanisms of regulation, measurement, and interpretation. Am J Physiol Regul Integr Comp Physiol 96, R473 R495. Ainslie PN, Kolb JC, Ide K & Poulin MJ (3). Effects of five nights of normobaric hypoxia on the ventilatory responses to acute hypoxia and hypercapnia. Respir Physiol Neurobiol 38, Aldenderfer MS (3). Moving up in the world. Am Sci 9, Appenzeller O, Claydon VE, Gulli G, Qualls C, Slessarev M, Zenebe G, Gebremedhin A & Hainsworth R (6). Cerebral vasodilatation to exogenous NO is a measure of fitness for life at altitude. Stroke 37, Arias-Stella J & Valcarcel J (976). Chief cell hyperplasia in the human carotid body at high altitudes; physiologic and pathologic significance. Hum Pathol 7, Beall CM (7). Two routes to functional adaptation: Tibetan and Andean high-altitude natives. Proc Natl Acad Sci U S A 4 (Suppl ), Beall CM, Strohl KP, Blangero J, Williams-Blangero S, Almasy LA, Decker MJ, Worthman CM, Goldstein MC, Vargas E, Villena M, Soria R, Alarcon AM & Gonzales C (997). Ventilation and hypoxic ventilatory response of Tibetan and Aymara high altitude natives. Am J Phys Anthropol 4, Berkenbosch A, Bovill JG, Dahan A, DeGoede J & Olievier IC (989). The ventilatory CO sensitivities from Read s rebreathing method and the steady-state method are not equal in man. JPhysiol4, Brutsaert TD (7). Population genetic aspects and phenotypic plasticity of ventilatory responses in high altitude natives. Respir Physiol Neurobiol 58, 5 6. Brutsaert TD, Parra EJ, Shriver MD, Gamboa A, Rivera-Ch M & Leon-Velarde F (5). Ancestry explains the blunted ventilatory response to sustained hypoxia and lower exercise ventilation of Quechua altitude natives. Am J Physiol Regul Integr Comp Physiol 89, R5 R34. Burgess K, Burgess K, Subedi P, Ainslie P, Topor Z & Whitelaw W (8). Prediction of periodic breathing at altitude. In Integration in Respiratory Control: From Genes to Systems,ed. Poulin MJ & Wilson RJA. Springer. Chiodi H (957). Respiratory adaptations to chronic high altitude hypoxia. JApplPhysiol, ClaydonVE,GulliG,SlessarevM,AppenzellerO,ZenebeG, Gebremedhin A & Hainsworth R (8). Cerebrovascular responses to hypoxia and hypocapnia in Ethiopian high altitude dwellers. Stroke 39, Cudkowicz L, Spielvogel H & Zubieta G (97). Respiratory studies in women at high altitude (3,6 m or, ft and 5, m or 7, ft). Respiration 9, Cunningham DJ (987). Studies on arterial chemoreceptors in man. JPhysiol384, 6. Curran LS, Zhuang J, Droma T, Land L & Moore LG (995). Hypoxic ventilatory responses in Tibetan residents of 44 m compared with 3658 m. Respir Physiol, 3 3. Curran LS, Zhuang J, Sun SF & Moore LG (997). Ventilation and hypoxic ventilatory responsiveness in Chinese-Tibetan residents at 3658 m. JApplPhysiol83, Duffin J (5). Role of acid-base balance in the chemoreflex control of breathing. JApplPhysiol99, Duffin J (7). Measuring the ventilatory response to hypoxia. JPhysiol584, Duffin J & Mahamed S (3). Adaptation in the respiratory control system. Can J Physiol Pharmacol 8, Duffin J, Mohan RM, Vasiliou P, Stephenson R & Mahamed S (). A model of the chemoreflex control of breathing in humans: model parameters measurement. Respir Physiol, 3 6. Fatemian M, Gamboa A, Leon-Velarde F, Rivera-Ch M, Palacios JA & Robbins PA (3). Selected contribution: Ventilatory response to CO in high-altitude natives and patients with chronic mountain sickness. JApplPhysiol94, Fatemian M & Robbins PA (998). Human ventilatory response to CO after 8 h of isocapnic or poikilocapnic hypoxia. JAppl Physiol 85, C The Authors. Journal compilation C The Physiological Society