Ventilatory responses to isocapnic and poikilocapnic hypoxia in humans

Respiratory Physiology & Neurobiology 155 (2007) 104–113

Ventilatory responses to isocapnic and poikilocapnic hypoxia in humans Craig D. Steinback a,b , Marc J. Poulin a,b,c,∗ a

Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada b Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada c Faculty of Kinesiology, University of Calgary, Calgary, Alberta T2N 4N1, Canada Accepted 24 May 2006

Abstract We examined the hypoxic ventilatory response (HVR) including breathing frequency (fR ) and tidal volume (VT ) responses during 20 min of step isocapnic (IH) and poikilocapnic (PH) hypoxia (45 Torr). We hypothesized an index related to PETCO2 (pHPR) may be more robust during PH. Peak HVR was suppressed during PH (P < 0.001), and mediated by VT during PH and both VT and fR during IH. The relative magnitude of HVD remained similar between conditions indicating a suppressive role of hypocapnia in development of the HVR unrelated to the degree of subsequent HVD, implying a primarily O2 dependant mechanism. Post-hypoxic frequency decline was observed following both IH (3.4 ± 3.7 bpm, P < 0.05) and PH (3.6 ± 3.1 bpm, P < 0.01), despite no fR response during exposure to PH. Use of pHPR improved the signal to noise ratio during PH, though failed to detect the peak ventilatory response, and therefore may not be appropriate when describing peak responses. © 2006 Elsevier B.V. All rights reserved. Keywords: Hypoxia; Isocapnia; Poikilocapnia; Ventilation; Breathing frequency; Tidal volume

1. Introduction The ventilatory response to the hypoxia of altitude involves the complex interaction of multiple mechanisms. The peripheral chemoreflex sensitivity to a decrease in the partial pressure of oxygen in the arterial blood (PaO2 ) drives an initial increase in ventilation (Weil and Zwillich, 1976). However, this increase in ventilation causes a concomitant decrease in arterial CO2, which counters the hypoxic drive to breathe (Lahiri and Gelfand, 1981; Bisgard and Neubauer, 1995). Further, during short periods of sustained hypoxia, ventilation is characterized by an initial increase followed by a subsequent decline, reaching a new steady state by approximately 20 min. This secondary component, termed hypoxic ventilatory decline (HVD), is not fully understood but is thought to represent peripheral chemoreceptor desensitization and/or other centrally mediated mechanisms as well as the initial stages of acclimatization (Bascom et al., 1990). As such, the initial ventilatory response to hypoxia is biphasic over time (Easton et al., 1986). ∗

Corresponding author at: Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Heritage Medical Research Building, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4C2, Canada. Tel.: +1 403 220 8372; fax: +1 403 270 8928. E-mail address: [email protected] (M.J. Poulin). 1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.05.006

The complexities associated with the measurement of the hypoxic ventilatory response (HVR), have led the majority of past investigations of the HVR to focus on the initial peripheral chemoreceptor response to hypoxia per se by utilizing protocols of brief isocapnic exposures to hypoxia, including single breath exposures to hypoxia, ramp-like decreases, multi-step decreases and single step decreases in blood oxygen saturation, PaO2 or FIO2 . While some investigations of the HVR have sought to utilize the HVR for applied (e.g. susceptibility to high altitude illness, Moore et al., 1986; Bartsch et al., 2001) and clinical (e.g. relationship to chronic obstructive pulmonary disease, Kobayashi et al., 1996) purposes, these studies have yielded mixed results partly due to the limitations of abbreviated HVR measurements. One way to address this issue involves the measurement of the acute HVR, including the initial HVR response and the ensuing HVD, under conditions of both isocapnic and poikilocapnic hypoxia. This would help separate the effects of hypoxia per se from the respiratory alkalosis that arises secondary to the increase in ventilation with hypoxia. Further, broadening the analysis of the HVR to include ventilatory timing and individual ventilatory components (i.e. VT and fR ) may provide additional information which may shed light on the specific adaptations to either isocapnic or poikilocapnic hypoxia and how these adaptations may differ between individuals. This may be particularly important during poikilocapnic hypoxia, where the

C.D. Steinback, M.J. Poulin / Respiratory Physiology & Neurobiology 155 (2007) 104–113

ventilatory suppression that occurs decreases the signal to noise ratio, making the determination of the HVR more difficult. Therefore, the main objectives of the present study were as follows. First, this study aimed to quantify the dynamic characteristics of the HVR during both isocapnic and poikilocapnic hypoxia in the same individuals. Second, as tidal volume (VT ) and breathing frequency (fR ) are under separate neural control, we sought to identify their independent contributions in an effort to better understand the mechanisms underlying HVR. Finally, this study determined the usefulness of a novel index that measures the change in PETCO2 as a surrogate for ventilatory changes and HVR during poikilocapnic hypoxia (Steinback et al., in press). This index is derived from the change in PETCO2 for a given desaturation, similar to the HVR. It is hypothesized that this index is a more sensitive marker of the hypoxic response than the HVR by improving the signal to noise ratio because small changes in ventilation should cause relatively larger changes in PETCO2 as dictated by the metabolic hyperbola.

105

MO, USA). Accurate control of end-tidal gases was achieved using the technique of dynamic end-tidal forcing (Robbins et al., 1982a,b; Ainslie and Poulin, 2004) and dedicated software (BreatheM v2.35, University Laboratory of Physiology, Oxford, UK). After 10 min of euoxia (PETO2 = 88 Torr), PETO2 was decreased within ∼2–3 breaths to 45 Torr. During an isocapnic hypoxia (IH) protocol, PETCO2 was held constant at +1 Torr above resting (Khamnei and Robbins, 1990). During a poikilocapnic hypoxia (PH) protocol, PETCO2 was allowed to vary naturally. Hypoxia was maintained for 20 min, after which period PETO2 was returned to 88 Torr for 10 min. Blood pressure (photoplethysmography, Portapress, TPD Biomedical Instrumentation, Delft, The Netherlands) and heart rate (standard three-lead ECG, Micromon 7142B, Kontron Medical, Milton Keynes, UK) were monitored continuously throughout testing. Blood oxygen saturation (SpO2 ) was measured using a pulse-oximeter situated on the left index finger (Model 3900, Datex-Ohmeda, Louisville, CO, USA).

2. Methods 2.1. Subjects Ten healthy male subjects (25.7 ± 4.2 years, mean ± S.D.) participated in this study. All subjects provided informed written consent after receiving verbal and written instructions outlining the experimental procedures. Participants were not taking any medications, all were non-smokers, and none had any history of cardiovascular or respiratory disease. This study was approved by the Conjoint Health Research Ethics Board at the University of Calgary (Grant ID: 15671) and conforms to the standards set by the Declaration of Helsinki. 2.2. Protocol Experiments were conducted at an elevation of 1103 m and a barometric pressure of 665 ± 5 Torr. Subjects abstained from caffeine, alcohol and strenuous exercise for 12 h prior to testing. During experimentation, subjects took part in two randomized protocols separated by a 40 min recovery period. Prior to each protocol, the subject’s resting PETO2 and PETCO2 were measured for ∼10 min with the subject in a comfortable semi-supine position. Respired gas was sampled continuously (20 ml min−1 ) via a fine catheter and analyzed for PO2 and PCO2 by mass spectrometer (AMIS 2000, Innovision, Odense, Denmark). Values for PO2 and PCO2 were sampled by computer every 10 ms, and PETO2 and PETCO2 were identified and recorded for each breath using a computer and dedicated software (Chamber v2.10, University Laboratory of Physiology, Oxford, UK). Each protocol began with a 10 min baseline period during which the subject breathed normally through a facemask which allowed for natural mouth and/or nasal breathing (Model 16709, ResMed Corp., Poway, CA, USA). Respiratory volumes were measured using a turbine device (VMM-400, Interface Associates, Laguna Niguel, CA, USA), while flow direction and timing were determined using a pneumotachograph and differential pressure transducer (RSS100-HR, Hans Rudolph, Kansas City,

2.3. Data analysis Minute ventilation (V˙ E ), tidal volume (VT ), breathing frequency (fR ), and the phase durations of inspiration (TI ) and expiration (TE ) were determined from the ventilatory records by the BreatheM collection software. Ventilatory data are expressed in BTPS. One minute averages were calculated for all variables immediately prior to the onset of hypoxia (time = −1 min), every 5 min during hypoxia (time = +5, +10, +15 and +20 min) and 1 and 5 min post-hypoxia (+21 and +25 min). Peak V˙ E and its time of occurrence were calculated as 1 min averages centered around the visually identified time of maximal ventilation. To express HVR as a linear function of the hypoxic stimulus, PETO2 was converted to a calculation of O2 saturation (ScO2 ) using the transform described by Severinghaus (1979): −1

ScO2 = (((PO2 3 + 150PO2 )

−1

× 23400) + 1)

× 100

(1)

The use of ScO2 in lieu of SpO2 also avoided the inherent temporal delay of pulse oximetry (Trivedi et al., 1997) and ensured the appropriate O2 saturation was used in the calculation of the HVR. The isocapnic HVR was calculated as the change in V˙ E divided by the change in ScO2 (iHVR). The poikilocapnic HVR was calculated in two different ways, first using the change in V˙ E (pHVR), and second, suing the change in PETCO2 divided by the change in ScO2 (pHPR). Hypoxic ventilatory decline was calculated as the % decrease of V˙ E from peak HVR at time t for both iHVR and pHVR, and as the % return to control PETCO2 in pHPR. iHVR and pHVR (L min−1 %−1 ) = pHPR (mmHg %−1 ) =

PETCO2 ScO2

V˙ E ScO2

(2)

(3)

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 HVD (%) = 100 1 −



HVRt HVRpeak

 (4)

Both pHVR and pHPR were utilized to analyze poikilocapnic data. The change in PETCO2 may be more robust and less susceptible to variability compared to the ventilatory response under these conditions (Steinback et al., in press). All delta values were calculated in relation to the time point occurring immediately prior to the onset of hypoxia (time = −1 min). 2.4. Mathematical modeling A first order, dynamic mathematical model was used to fit the ventilatory response to isocapnic hypoxia (Liang et al., 1997). From this model, a second estimation of the acute HVR was obtained as well as the magnitude and time course of HVD. This model, described in detail by Painter et al. (1993), can be written in the form: V˙ E = Vc + Vp   dVp 1 (gp (1 − S(t − dp ) + kp ) − Vp ) = dt Tp   dgp 1 (g100 − gh (1 − S(t − dp )) − gp ) = dt Th

(5) (6)

(7)

where Vc and Vp represent the central and peripheral chemoreceptor contributions to ventilation, respectively. Tp and gp represent the time constant and sensitivity of the peripheral chemoreceptor response to the hypoxic stimulus, defined by (1 − S), where S is arterial oxygen saturation. This response is also described by a peripheral time delay (dp ) and a non-negative peripheral offset (kp ). In Eq. (7), Th describes the time constant associated with the development of HVD, and g100 describes the steady-state chemoreflex sensitivity when S = 1.0. The term gh denotes the ratio of sensitivity decrease to the decrease in arterial oxygen

saturation during the development of HVD. Both g100 and gh are constrained to be positive values. 2.5. Statistical analysis Respiratory data, iHVR and pHVR were analyzed using a multivariate repeated measures design with two parallel conditions compared using pre-planned contrasts. To account for multiple comparisons (c), the comparison-wise error rate (α, 0.05) was adjusted using the experiment-wise error rate (αe ) (Hinkle et al., 2003): αe (8) α1 = c αe = 1 − (1 − α)c

(9)

The number of multiple comparisons (c) differed depending on the variable analyzed. pHPR was analyzed using a one-way repeated measured ANOVA, and between-condition analysis of HVD was conducted using a student paired T-test. Pearson correlations were used to determine relationships between variables. All statistical analyses were performed using SPSS (v13.0, SPSS Inc.). Significance was set at P < 0.05. Data are expressed as mean ± S.D. 3. Results 3.1. General pattern of respiratory responses End-tidal values and blood oxygen saturation data are shown in Table 1. PH caused a decrease in PETCO2 which was significant across all time points (P < 0.001). During IH, peak ventilation was markedly higher (40.6 ± 11.6 L min−1 ) than during PH (19.6 ± 6.2 L min−1 , P < 0.001). The peak response time during IH (144 ± 88 s) was not significantly different than during PH (80 ± 42 s) (P = 0.092). The peak increase in V˙ E was mediated by increases in both VT (P < 0.001) and fR (P < 0.05) during IH. However, the response during PH

Table 1 End-tidal and oxygen saturation values during isocapnic and poikilocapnic hypoxia −1 min

+5 min

+10 min

+15 min

+20 min

+25 min

PETCO2 (mmHg) Isocapnia Poikilocapnia

88.9 ± 1.7 88.1 ± 0.8

44.7 ± 0.5†,a 44.0 ± 0.5a,e

44.6 ± 0.5a 44.0 ± 1.6a,e

45.2 ± 0.9a 44.5 ± 1.9a

45.4 ± 2.3a 46.1 ± 2.0a

90.1 ± 1.8 87.3 ± 4.2

PETCO2 (mmHg) Isocapnia Poikilocapnia

37.2 ± 1.8 36.4 ± 2.5

37.3 ± 1.9 31.8 ± 2.4‡,a,f

37.3 ± 1.9d 32.1 ± 2.7‡,a,f

37.1 ± 2.0 32.3 ± 2.6‡,a,e

37.3 ± 1.9 32.8 ± 2.6‡,a

36.9 ± 2.3 35.9 ± 2.0

SpO2 (%) Isocapnia Poikilocapnia

96.9 ± 1.5 96.3 ± 1.0

82.6 ± 2.6a 80.6 ± 3.9*,a

81.8 ± 2.7a,c 78.5 ± 4.8†,a,b

81.5 ± 3.2a 78.2 ± 5.7*,a

81.7 ± 3.0a,b 77.3 ± 5.2†,a,b

96.9 ± 1.6 96.6 ± 1.9

ScO2 (%) Isocapnia Poikilocapnia

96.8 ± 0.2 96.7 ± 0.1

80.4 ± 0.5a 79.7 ± 0.5*,a,e

80.3 ± 0.4a 79.6 ± 1.8a,e

80.9 ± 0.9a 80.1 ± 2.1a

80.9 ± 1.9a 81.6 ± 1.8a

97.0 ± 0.2 96.6 ± 0.5

Values are expressed as mean ± S.D. Times are in reference to the onset of hypoxia. Significant difference between conditions: * P < 0.05, † P < 0.01, ‡ P < 0.001. Significantly different from baseline, within condition: a P < 0.001. Significantly different from time = +5 min, within condition: b P < 0.05, c P < 0.01. Significantly different from time = +15 min, within condition: d P < 0.05. Significantly different from time = +20 min, within condition: e P < 0.05, f P < 0.01.

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Fig. 2. The ventilatory response to hypoxia, expressed as the relationship between ventilation and blood oxygen desaturation (L min−1 %−1 ), during isocapnic hypoxia and poikilocapnic hypoxia. Values are expressed as mean ± S.D. Significant difference between conditions, * P < 0.05, † P < 0.01, ‡ P < 0.001. Within-condition comparisons are listed in Table 3.

3.2. Hypoxic ventilatory responses Peak iHVR (1.80 ± 0.71 L min−1 %−1 ) was significantly higher than peak pHVR (0.60 ± 0.27 L min−1 %−1 ) (P < 0.001). iHVR and pHVR were significantly correlated (R = 0.67, P < 0.001, Fig. 2). At time +20 iHVR fell to 0.41 ± 0.32 L min−1 %−1 (P < 0.001) and pHVR to 0.11 ± 0.11 L min−1 %−1 (P < 0.001). This is illustrated in Fig. 2, and all numerical values are listed in Table 2. pHPR was 0.27 ± 0.11 Torr %−1 at the time of peak ventilation and was not different across time (Fig. 3; Table 3). However, pHPR remained weakly but significantly correlated to both iHVR (R = 0.32, P < 0.05) and pHVR (R = 0.36, P < 0.01). Fig. 1. Mean traces of end-tidal gases and associated ventilation responses to isocapnic (solid lines) and poikilocapnic (dotted lines) hypoxia. (Panel A) Endtidal PO2 (PETO2 ). (Panel B) End-tidal PCO2 (PETCO2 ). (Panel C) Arterial blood oxygen saturation (SP O2 ). (Panel D) Ventilation (V˙ E ). (Panel E) Tidal volume (VT ). (Panel F) Breathing frequency (fR ). (Panel G) Inspiration time (TI ). (Panel H) Expiration time (TE ).

was due solely to an increase in VT (P < 0.01). Over time, a large HVD was observed during both conditions. During IH, V˙ E declined 21.8 ± 11.0 L min−1 (76 ± 20%) from peak values by time +20 min and 7.9 ± 4.3 L min−1 (80 ± 22%) from peak values during PH. HVD was mediated by both decreases in VT (P < 0.001) and fR (P < 0.05) during IH but only decreases in VT (P < 0.01) during PH. At the offset of hypoxia, there was a small but significant depression in ventilation below baseline values during both IH (20.0 ± 16.7%, P < 0.01) and PH (17.7 ± 18.7%, P < 0.05). This depression was mediated by a post-hypoxic frequency decline (PHFD) of 3.4 ± 3.7 breaths min−1 during IH (−19.8 ± 23.0%, P < 0.05), and 3.6 ± 3.1 breaths min−1 during PH (−21.7 ± 21.7%, P < 0.01). Fig. 1 shows the temporal profile of V˙ E and its components in response to hypoxia, with all respiratory values shown in Table 2.

3.3. Modeled response to isocapnic hypoxia Mathematical modeling of the response to isocapnic hypoxia provided a secondary objective confirmation of the calculated

Fig. 3. The ventilatory response to poikilocapnic hypoxia, expressed as the relationship between change in end-tidal PCO2 and blood oxygen desaturation (Torr %−1 ). Values are expressed as mean ± S.D. Within condition comparison are listed in Table 3.

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Table 2 Ventilation and its components during isocapnic and poikilocapnic hypoxia −1 min

Peak

V˙ E (L min−1 ) Isocapnia Poikilocapnia

12.6 ± 2.4† 10.0 ± 2.7

40.6 ± 11.6‡,c,d,i,l,o 32.7 ± 12.0‡,c,h,k,n 24.0 ± 7.2‡,c,j,m 12.7 ± 2.6b 19.6 ± 6.2c,e,h,k,o 13.4 ± 2.8c,m

VT (L) Isocapnia Poikilocapnia

0.9 ± 0.3 0.78 ± 0.2

fR (bpm) Isocapnia Poikilocapnia

14.7 ± 4.6 14.2 ± 4.4

TI (s) Isocapnia Poikilocapnia

2.0 ± 1.1 2.0 ± 0.8

TE (s) Isocapnia Poikilocapnia

2.6 ± 0.8 2.8 ± 1.2

2.0 ± 0.4†,c,i,l,o 1.3 ± 0.4b,e,g,k,n 19.2 ± 4.5†,a,m 14.2 ± 5.1

+5 min

1.8 ± 0.6†,c,g,k,m 0.9 ± 0.1b

+10 min

+15 min

+20 min

+21 min

+25 min

19.9 ± 5.1†,b,m 12.5 ± 4.0a

18.8 ± 4.9†,b 11.7 ± 2.7a

10.1 ± 2.6*,b 8.1 ± 2.1a

11.0 ± 3.4 9.4 ± 3.4

1.2 ± 0.2*,a 0.9 ± 0.2

1.2 ± 0.3† 0.9 ± 0.1a

0.9 ± 0.4 0.8 ± 0.3

0.7 ± 0.2 0.7 ± 0.3

1.4 ± 0.3†,c,j 1.0 ± 0.2a

17.7 ± 4.6* 13.5 ± 3.5

16.5 ± 3.8† 12.6 ± 3.7

16.5 ± 2.9† 13.4 ± 2.8

14.9 ± 3.4† 12.0 ± 2.7

11.4 ± 3.9a 10.6 ± 2.9b

15.0 ± 4.0 13.4 ± 4.2

1.6 ± 0.5* 2.0 ± 0.6

1.7 ± 0.8 1.9 ± 0.6

1.7 ± 0.5* 2.2 ± 0.8

1.7 ± 0.4* 1.9 ± 0.6

1.8 ± 0.5* 2.3 ± 0.9

2.5 ± 1.0 2.4 ± 0.9

1.8 ± 0.5 2.0 ± 1.0

1.7 ± 0.3†,a,d,g,j,m 2.7 ± 1.0

1.9 ± 0.5*,a 2.9 ± 0.9

2.2 ± 0.7† 3.0 ± 0.9

2.1 ± 0.5‡ 2.7 ± 0.5

2.5 ± 0.8* 2.9 ± 0.5

3.4 ± 0.9a 3.7 ± 1.2a

2.5 ± 0.6 3.0 ± 1.0

Values are expressed as mean ± S.D. Peak values correspond to the values occurring at the time associated with the maximal V˙ E . Times are in reference to the onset of hypoxia. Significant difference between conditions, at same time point: * P < 0.05, † P < 0.01, ‡ P < 0.001. Significant difference from baseline, within condition: a P < 0.05, b P < 0.01, c P < 0.001. Significantly different from time = +5min, within condition: d P < 0.05, e P < 0.01, f P < 0.001. Significant difference from time = +10 min, within condition: g P < 0.05, h P < 0.01, i P < 0.001. Significantly different from time = +15 min, within condition: j P < 0.05, k P < 0.01, l P < 0.001. Significantly different from time = +20 min, within condition: m P < 0.05, n P < 0.01, o P < 0.001. Table 3 Hypoxic ventilatory responses during isocapnic and poikilocapnic hypoxia Peak

+5 min

+10 min

+15 min

+20 min

HVR (L min−1 %−1 ) Isocapnia Poikilocapnia

1.80 ± 0.71‡,a,d,g,j 0.60 ± 0.27b,c,f,j

1.23 ± 0.72†,c,f,i 0.20 ± 0.11

0.69 ± 0.39†,e,h 0.15 ± 0.09

0.45 ± 0.28* 0.15 ± 0.17

0.41 ± 0.32* 0.11 ± 0.11

HPR (mmHg %−1 ) Poikilocapnia

0.27 ± 0.11

0.27 ± 0.07

0.25 ± 0.09

0.25 ± 0.10

0.24 ± 0.10

Values are expressed as mean ± S.D. Times are in reference to the onset of hypoxia, with “Peak” denoting the time of peak ventilation. Significant difference between conditions, at same time point: * P < 0.05, † P < 0.01, ‡ P < 0.001. Significantly different from time = +5min, within condition: a P < 0.05, b P < 0.01. Significant difference from time = +10 min, within condition: c P < 0.01, d P < 0.001. Significantly different from time = +15 min, within condition: e P < 0.05, f P < 0.01, g P < 0.001. Significantly different from time = +20 min, within condition: h P < 0.05, i P < 0.01, j P < 0.001.

results, as well as a broader description of the acute HVR and subsequent HVD that unfolded during IH. The ventilatory sensitivity to hypoxia derived using mathematical modeling (gp ) was 2.17 ± 0.89 L min−1 %−1 with a time constant (Tp ) of 18.1 ± 8.0 s. Fig. 4 shows the model fit and residuals for one

individual and a complete description of all model parameters is included in Table 4. Though gp was consistently higher than iHVR (P < 0.01), correlation analysis showed a strong significant relationship between the two values of sensitivity (R = 0.92, P < 0.001), supporting our results. The HVD calculated from the modeled data (77 ± 19% at 20 min) correlated strongly with the HVD calculated from the raw data (R = 0.94, P < 0.001) and had a time constant of 589 ± 265 s. 4. Discussion 4.1. Main findings

Fig. 4. Ventilation (circles), modeled data (solid line) and residuals representing goodness of fit for one subject during isocapnic hypoxia.

The present study examined the dynamics of the ventilatory response to steady-state isocapnic and poikilocapnic hypoxia. We documented a biphasic pattern of ventilation during both protocols, with the response mediated by both VT and fR components during IH but only VT during PH. However, despite the suppressive effect of hypocapnia the relative magnitude of the subsequent ventilatory decline remained similar between

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Table 4 Values for model parameters in response to isocapnic hypoxia Subject

Vc (l min−1 )

gp (l min−1 %−1 )

Tp (s)

dp (s)

kp (l min−1 )

g100 (l min−1 %−1 )

gh (l min−1 %−1 )

Th (s)

005 043 051 075 080 089 091 092 093 102

5.5 12.1 5.9 1.2 4.2 5.5 5.6 2.3 6.1 5.6

1.75 1.88 3.43 3.82 2.28 1.04 1.56 1.26 2.37 2.29

19.3 23.7 20.6 17.2 5.5 11.6 24.3 5.8 26.2 26.9

6.7 2.5 4.9 5.0 7.7 5.1 6.8 19.8 5.0 3.8

0.0 0.0 0.0 0.0 0.7 0.0 0.0 7.4 0.5 0.0

1.76 1.90 3.46 3.86 2.30 1.05 1.57 1.27 2.39 2.31

0.05 0.08 0.17 0.18 0.10 0.06 0.06 0.03 0.09 0.08

999.0 786.0 601.5 445.9 446.8 867.3 738.4 539.1 116.4 353.6

5.4 2.9

2.17 0.89

18.1 8.0

6.7 4.9

0.9 2.3

2.19 0.90

0.09 0.05

589.4 264.3

Mean S.D.

Definition of terms: Vc , the central chemoreceptor contribution to ventilation; gp , the peripheral chemoreceptor sensitivity to hypoxia; Tp , the peripheral chemoreflex time constant; dp , the time delay associated with the peripheral response; kp , non-negative peripheral offset; g100 , the steady-state chemoreceptor sensitivity at a blood oxygen saturation of 100%; gh , the ratio of sensitivity change to change in saturation as HVD develops; Th , the time constant of HVD development.

protocols, pointing to a mechanism specific to hypoxia. Also, the termination of hypoxia (both IH and PH) resulted in a pronounced frequency decline. To our knowledge, this is the first documentation and quantification of this response in humans. While our measures of the HVR are higher than other data from previous literature, the influence of modest altitude may play a role. In an effort to better describe the response during PH, we calculated the relationship between PETCO2 decline and ScO2 . While this index (pHPR) had a better signal to noise ratio it is less sensitive when determining the peak response than the traditional measure of V˙ E versus arterial oxygenation or PaO2 . As such this index may not be a suitable alternative to the traditional measure relating to ventilation. 4.2. Respiratory dynamics Exposure to hypoxia, whether IH or PH, elicited a biphasic response, characterized by an initial rapid increase in V˙ E followed by a progressive decline over the following minutes. The acute HVR was blunted during PH, and this is likely due to a reduction in PCO2 and/or H+ stimulation of peripheral and central chemoreceptors (Dempsey and Forster, 1982; Bisgard and Neubauer, 1995; Cunningham et al., 2002; Corne et al., 2003) as well as a decreased response to hypoxia per se (Dempsey and Forster, 1982; Cunningham et al., 2002). The blunted response during PH was mediated by a decrease in VT and abolition of the fR response (see below). Though non-significant, there was a trend for the peak ventilatory response to occur earlier during PH, with the ventilatory rise time, expressed as the change in ventilation per unit time, showing no difference between protocols. Despite peak V˙ E responses of different magnitudes, the subsequent relative decline in V˙ E seen during hypoxia was the same between conditions. Further, the absolute decline in ventilation was highly correlated with the initial increase, regardless of condition. These findings are similar to those of Easton (Easton et al., 1986; Easton and Anthonisen, 1988), and support the theory that HVD arises as an adaptation to hypoxia independent of

PaO2 /pH status, and is influenced by individual variations in the sensitivity to hypoxia. With the termination of hypoxia, V˙ E during both IH and PH exhibited a temporary decrease below baseline, caused by a significant PHFD. While there is some evidence of ventilatory depression following hypoxia in awake humans (Bascom et al., 1992; Gleeson and Sweer, 1993) as well as PHFD following isocapnic hypoxia (Gardner, 1980), this is, to our knowledge, the first documentation of PHFD following poikilocapnic hypoxia. This is particularly interesting as no fR response was observed during exposure to hypoxia under poikilocapnic conditions. While the mechanisms of this response have been proposed to be similar to those underlying HVD, Day and Wilson (2005) showed recently that hypoxia specific to the carotid body is sufficient to elicit PHFD as measured by phrenic nerve output. Together with the findings of Bascom et al. (1990) regarding the mechanism of HVD this supports the theory of a primary role of the arterial chemoreceptors in the ventilatory response to sustained hypoxia. 4.3. Contribution of ventilatory components During IH, both fR and VT contributed to the development of the ventilatory response, while the contribution of VT dominated under poikilocapnic conditions. This is supported by the findings of Reynolds and Milhorn (1973), who documented a similar pattern in response to 10 min isocapnic hypoxia (PETO2 ∼ 47 Torr), with an increased contribution of fR with lower PETO2 stimuli. However, this is inconsistent with the work of Easton et al., who failed to measure any increase in fR in two studies (Easton et al., 1988; Easton and Anthonisen, 1988), and documented only a small (∼1.5 bpm) increase in a third (Easton et al., 1986), in response to step decreases in saturation (∼80%). Likewise, Fregosi (1991) showed no increase in fR in resting humans in response to rapidly lowered FIO2 , and Bender’s reanalysis of several studies (1987) indicated the predominance of the VT contribution to V˙ E during ramp and constant hypoxic stimuli.

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However, studies have shown that increased V˙ E after extended periods at altitude is due to increased frequency, with VT returning to near baseline levels (Burki, 1984; Bender et al., 1987). These findings indicate an important role of fR in the acclimatization process perhaps not seen at lower altitudes. While this may explain, at least in part, the disparity between our work and that of Easton (Easton et al., 1986, 1988; Easton and Anthonisen, 1988), the supportive findings of Reynolds and Milhorn (1973) were documented at low altitude (∼80 m) with other conflicting data coming from similar (Fregosi, 1991) or higher (Bender et al., 1987) altitudes. Thus, the influence of relative altitudes in studies of acute hypoxic exposure remains unclear. The appearance of a fR response may also be influenced by the magnitude of the ventilatory response elicited. Rebuck et al. (1976) documented a strong relationship between the fR and the magnitude of the ventilatory response to graded isocapnic hypoxia. A review of their data indicates that the predicted fR from regression analysis is similar to the measured response from our own data. Further, VT and fR increase in a curvilinear fashion with respect to V˙ E , typified by a break point, below which VT dominates the response, and above which fR dominates. Clark and von Euler (1972) documented this break point at approximately 35 L min−1 during hyperoxic rebreathing. During IH, peak ventilation was above this threshold (∼40 L min−1 ), though not during PH (∼25 L min−1 ). As such, it is possible that the fR response did not develop during PH because V˙ E was below the threshold for recruitment. A review of the aforementioned studies reveal that those not observing a fR response are below the proposed ventilatory threshold for fR recruitment (Easton et al., 1986, 1988; Easton and Anthonisen, 1988; Fregosi, 1991). However, it is worth noting that during the study by Clark and von Euler (1972) there was a progressive rise in CO2 , which may also play a role in the development of the fR response. The HVD observed as a result of prolonged hypoxia was also mediated by decreases in both VT and fR during IH and VT during PH. While the proposed standard terminology put forth by Powell et al. (1998) separates the contributions of VT and fR into distinct and separate responses, it is obvious that they interact to produce an overall reduced hypoxic ventilatory gain. Their interaction does not necessitate a shared mechanism, though the stimulus may be similar. In light of previous findings documenting negligible rises in fR , is it conceivable that in these instances the signal to noise ratio would be elevated thus masking any appreciable decline in the fR response over time, whereas with a larger rise in fR as we have documented, a decrease would be observed. 4.4. Ventilatory sensitivity to hypoxia Previous studies investigating the HVR have predominantly used three specific experimental procedures: (a) progressive decreases (Kronenberg et al., 1972; Hirshman et al., 1975; Moore et al., 1984; van Klaveren and Demedts, 1998; Zhang and Robbins, 2000; Bartsch et al., 2002; Foster et al., 2005), (b) incremental step or sawtooth decreases (Zhang and Robbins, 2000; Ainslie et al., 2003; Ainslie and Poulin, 2004) or (c) single immediate step decreases (Sato et al., 1992, 1994; Poulin

and Robbins, 1998; Zhang and Robbins, 2000; Hupperets et al., 2004) in PaO2 /SpO2 , with only a few poikilocapnic designs (Edelman et al., 1973; Moore et al., 1984; Bartsch et al., 2002; Ainslie and Poulin, 2004). Data from these studies (recalculating where necessary) indicates an average pHVR of ∼0.53 L min−1 , similar to the present study. The mean literature value for iHVR was ∼1.2 (0.4–3.2) L min−1 , similar to the normative values proposed by Rebuck and Slutsky (1981), ∼1.0 L min−1 from step protocols alone. Our reported iHVR is within the normal range of values found, but it is substantially higher than the mean. Though the discrepancy may be due to permutations of the step protocol or other variations between studies, methodological considerations put forth by consensus (Cherniack et al., 1977; Powell et al., 2005) were utilized during the planning of the experimental protocol, for instance, to minimize the influence of outside stimuli (see Section 2). Therefore, this seems an unlikely explanation for higher values of HVR, and points to other physiologic determinants. Variation of the HVR between subjects and between days within the same subject has been reported previously (Hirshman et al., 1975; Weil and Zwillich, 1976; Sahn et al., 1977; Zhang and Robbins, 2000), and may be expected to carry forward between studies as well. Data from a previous study from our laboratory (Kolb et al., 2004) showed a 15.6% coefficient of variation when measuring iHVR. We expect that similar variability may exists in the present data as well, though the study design did not allow an assessment of this. The use of a mathematical model designed to describe the ventilatory response to isocapnic hypoxia (Liang et al., 1997) served as a secondary objective confirmation of our results. The strong agreement between our calculated values of the HVR and the model parameters discounts any observer error or bias. No model exists to assess the response under poikilocapnic conditions, though we can speculate that as per the isocapnic values, that measurement of the pHVR was free from subjective bias and subject to a similar degree of variability in this subject group. Studies of altitude acclimatization have indicated an increase of the HVR from sea level values, which is maintained for days to weeks (Sato et al., 1992, 1994; Ainslie et al., 2003; Hupperets et al., 2004). Conversely, the phenomenon of hypoxic desensitization that appears in high altitude natives and residents, may take years to decades to develop (Weil et al., 1971; Forster et al., 1971; Dempsey and Forster, 1982; Weil, 1986). Also unknown is the effect of repeated exposures to lower altitudes on the development of hypoxic desensitization. Seven of 10 subjects taking part in this study were native to altitudes lower than 500 m, and or returned to near sea level altitudes multiple times yearly. As such, it is possible that the subjects in the present study, though residing at a moderate altitude, still exhibit an elevated HVR similar to high altitude sojourners. A third method of assessing the response to poikilocapnic hypoxia was used to address the low signal to noise ratio associated with the blunted ventilatory response under such conditions. As the increase in ventilation unfolds during poikilocapnic hypoxia, CO2 is blown off. This reduction in PETCO2 was theorized to be a more robust indication of responsiveness during poikilocapnic hypoxia due to the shape of the shape of the metabolic hyperbola. We showed an improved signal to noise

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ratio as evident from the standard deviations, with our values agreeing with previously collected data (Ainslie and Poulin, 2004). However, the present data suggest that the alterations in PETCO2 may not be suitable to detect appreciable changes across time, as evident when one compares the peak pHVR and pHPR responses. The use of pHVR detected substantial peak response and a decline in responsiveness overtime associated with the development of HVD, while pHPR values remained unchanged across the hypoxic exposure. 4.5. Methodological considerations The present study sought to investigate the ventilatory response to hypoxia by including the contributions of fR and VT . We also sought to address the role of hypocapnia, in the development of the HVR and how this response adapted over time. This required a prolonged and finely controlled stimulus. The use of dynamic end-tidal forcing, allowed for precise control of end-tidal (∼arterial) PO2 and PCO2 on a breath-by-breath basis. Recent work using similar methods has shown this particular technique to be precise to within ±0.6 Torr for PETCO2 and ± 2.0 Torr for PETO2 (Vantanajal, 2004). In this way, we were certain that the delivered stimulus was consistent and accurate, avoiding oscillations which could interfere with analysis and interpretation. The curvilinear relationship between V˙ E and PETO2 was linearized by calculating blood oxygen saturation. ScO2 (calculated) was utilized instead of SpO2 (pulse oximetry) due to limitations of the technique (see Section 2) and an unexpected progressive decline in SpO2 during PH. This decline was counter to the expected leftward shift the oxygen dissociation curve as predicted by an alkaline blood pH and was further unexplained by the slow rise in PETCO2 over time during hypoxia as no differences in saturation were observed early in hypoxia despite a reduced PETCO2 . However, due to technical limitations, we were unable to assess blood pH status during experimentation and cannot comment conclusively on the effects of this. Alternatively, a hypocapnic induced decrease in peripheral blood flow, due to increased vascular resistance (Kontos et al., 1972; Richardson et al., 1972), might explain this disparity. This illustrates a limitation of using SpO2 in the calculation of HVR where potentially spurious changes in SpO2 could over- or under-estimate HVR and calls into question the common use of SpO2 as the controlled variable. However, it is recognized that the calculation of ScO2 used here may also be limited by its non-inclusion of a pH correction factor. While many distinct methodologies are often used to assess the HVR (Rebuck and Slutsky, 1981), in the present study we chose a single hypoxic step (PETO2 = 45 Torr) for 20 min allowing for a more complete description of the acute response to hypoxia. Under isocapnic conditions, single breaths or brief pulses (Kronenberg et al., 1972; Edelman et al., 1973) of hypoxia specifically isolate the momentary sensitivity of the carotid chemoreceptors to a specific PO2 stimulus and may fail to develop a complete response and are susceptibility to random ventilatory fluctuations (Sugimori et al., 1996; Zhang and Robbins, 2000). Ramped decreases in PaO2 or blood oxygen

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saturation (Weil and Zwillich, 1976; Rebuck and Slutsky, 1981; Zhang and Robbins, 2000) produce response curves over a range of stimuli, the curvilinear relationship between PaO2 and V˙ E can be fit by a hyperbolic function while the slope of the linear relationship between SaO2 and V˙ E represents the gain of the carotid chemoreceptors. However, this technique is influenced by the rate of PETO2 change and HVD which is not quantified (Sugimori et al., 1996; Powell et al., 2000). Further, these abbreviated techniques reduce the description of the response to hypoxia to a single or limited few values, limiting the interpretations which can be made from them. As such, we chose a constant stimulus applied in a rapid manner and sustained over a prolonged duration, similar to previous investigations (Easton et al., 1986, 1988; Easton and Anthonisen, 1988; Bascom et al., 1990). Though more complicated, this approach allowed for a broader characterization of the complete ventilatory response to hypoxia, including dynamic adaptive components. However, one limitation of this experimental approach is its use of one specific hypoxic stimulus. As such, data from this particular protocol may not be representative of the response at more extreme or less severe levels of hypoxia. Another important consideration is the addition of a poikilocapnic protocol. While such protocols produce results which are more complicated due to the interactive effects of hypoxia and concomitant hypocapnia, they are more representative of the high altitude environments. Therefore, though the determination of the mechanisms contributing to the responses becomes more difficult to tease out, the responses themselves are more readily assessed for their applied significance. 4.6. Summary The results from the present study describe the dynamic ventilatory response to hypoxia, during both controlled isocapnic and natural poikilocapnic conditions. We have shown that the ventilatory response to hypoxia is mediated through increases in both frequency and tidal volume components during isocapnia but only tidal volume during poikilocapnia. Further, the phenomenon of post-hypoxic frequency decline was documented following both isocapnic and poikilocapnic hypoxia despite no fR response during the poikilocapnic exposure. These findings point to mechanisms related specifically to hypoxia, but unrelated to the development of the HVR, possibly mediated by higher brain centre adaptations. Finally, while the use of an index relating to the decease in PETCO2 during poikilocapnic hypoxia improves the signal to noise ratio associated with suppressed ventilation it fails to detect the ventilatory peak response occurring early in hypoxia. Acknowledgments We thank Dr. John W. Severinghaus, who first introduced the Consensus Statement at the 13th Hypoxia Symposia in Banff Canada (2003), and for his assistance throughout this project. We also thank Dr. P.N. Ainslie and Dr. J.C. Kolb for their input and feedback throughout the study. Finally, we extend our gratitude to Professor P.A. Robbins for assistance in setting up the

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