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INTEGRATIVE |
1 Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0623, USA
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Abstract |
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30, 50 and 90%
in normoxia (FIO2
= 0.2093) and acute hypoxia (FIO2
= 0.125). In a double-blind, order-balanced, crossover design, six healthy, trained men (normoxic 
= 59 ml kg–1 min–1) exercised at both FIO2 values after ACZ (3 doses of 250 mg, 8 h apart) and placebo. One week later this protocol was repeated using the other drug (placebo or ACZ). We measured cardiac output 
, leg blood flow (LBF), and muscle and pulmonary gas exchange, the latter using the multiple inert gas elimination technique. ACZ did not significantly affect 
, 
, LBF or muscle gas exchange. As expected, ACZ led to lower arterial and venous blood [HCO3–], pH and lactate levels (P < 0.05), and increased ventilation (P < 0.05). In both normoxia and hypoxia, ACZ resulted in higher arterial PO2 and saturation and a lower alveolar–arterial PO2 difference (AaDO2) due to both less 
mismatch and less diffusion limitation (P < 0.05). In summary, ACZ improved arterial oxygenation during exercise, due to both greater ventilation and more efficient pulmonary gas exchange. However, muscle gas exchange was unaffected.
(Received 11 September 2006;
accepted after revision 9 January 2007;
first published online 11 January 2007)
Corresponding author P. D. Wagner: University of California, San Diego, Department of Medicine 0623A, 9500 Gilman Drive, La Jolla, CA 92093-0623, USA. Email: pdwagner{at}ucsd.edu
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Introduction |
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Exercise is an intrinsic part of mountaineering and recreational activities at altitude and is well known to worsen lung function at altitude as a result of O2 diffusion limitation and increased ventilation–perfusion 
mismatching (Gale et al. 1985; Torre-Bueno et al. 1985; Wagner et al. 1986). The development of pulmonary oedema at altitude (‘high altitude pulmonary edema’
– HAPE) will further impair lung function. Because hypoxic pulmonary vasoconstriction and increased pulmonary artery pressure are important contributors to the development of HAPE and are known to be attenuated by carbonic anhydrase inhibition (Swenson, 2006), acetazolamide could affect gas exchange at altitude through this mechanism.
The effects of acetazolamide on exercise capacity in hypoxia and normoxia have been studied, but the results have been contradictory (Schoene et al. 1983; Hackett et al. 1985; McLellan et al. 1988; Stager et al. 1990; Garske et al. 2003). Moreover, while the influence of acetazolamide during exercise has been studied extensively with respect to acid–base regulation (Hollidge-Horvat et al. 1999; Scheuermann et al. 2000a,b; Gladden, 2004), the effect on gas exchange is less clear. Some studies examining pulmonary gas exchange efficiency at rest in normoxia have found a smaller alveolar–arterial PO2 difference (AaDO2) with acetazolamide (Berthelsen & Dich-Nielsen, 1987; Frans et al. 1993; Swenson et al. 1993). Yet, in these studies, either no effect on 
mismatch (Frans et al. 1993) or even an increase in inequality was found (Berthelsen & Dich-Nielsen, 1987; Swenson et al. 1993). Since diffusion limitation is normally not present at rest, this component was not studied and the decreased AaDO2 was attributed to the Bohr effect. In terms of exercise performance, the Bohr effect is potentially important since a right shift of the oxyhaemoglobin dissociation curve (induced by metabolic acidosis) would be expected to make pulmonary O2 loading less efficient (i.e. impede O2 loading), but at the same time should improve O2 unloading at the muscle (Schoene et al. 1983). Surprisingly, the effect of acetazolamide on pulmonary gas exchange efficiency during hypoxic exercise has yet to be fully examined, and to our knowledge no literature is available on the influence of acetazolamide on skeletal muscle gas exchange.
It is therefore apparent that acetazolamide could alter O2 transport to the mitochondria through a number of interacting effects in both the lungs and muscle. We hypothesized that especially during hypoxic exercise, the metabolic acidosis caused by acetazolamide will, in addition to increasing ventilation, affect lung function by (1) inducing a right-shift in the oxyhaemoglobin dissociation curve which will impede diffusive loading of O2 in the pulmonary capillary; (2) reducing the beneficial effect of CO2 unloading on O2 loading (lessen the Bohr effect); and (3) improving 
relationships and reducing diffusion limitation through lower pulmonary artery pressures and possibly less interstitial oedema, which may occur due to reduced hypoxic pulmonary vasoconstriction. The net effect of these diverse processes is hard to predict.
Similarly, in the skeletal muscle we hypothesized that metabolic acidosis per se would right-shift the oxyhaemoglobin curve, and thus be expected to facilitate O2 unloading and improve muscle gas exchange. However, if ventilation were increased and PCO2 reduced, and if blood lactate levels were lower, the in vivo dissociation curve might not in fact undergo a right shift, negating the effect of the metabolic acidosis itself. In addition, if carbonic anhydrase activity inhibited the CO2 loading/O2 unloading process in the muscle capillaries (i.e. lessened the Bohr effect), then we hypothesized that acetazolamide would act to impede O2 unloading at the muscle.
In order to separately examine the importance of all these mechanisms, we combined the multiple inert gas elimination technique (MIGET) and femoral vein-based measurements (leg O2 extraction, blood flow and O2 consumption) to allow discrete quantification of pulmonary diffusion limitation, 
inequality, effects of hyperventilation, effects of acidosis on the Hb dissociation curve, and any potential differences in muscle O2 extraction and transport. We applied these methods to normal, fit subjects during near-maximal exercise in both normoxia and acute hypoxia.
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Methods |
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This study was approved by the Human Research Protection Program at the University of California, San Diego, and conducted in accordance with the Declaration of Helsinki. Each subject was informed of potential risks and discomforts and signed an informed consent prior to participation. A self-reporting medical history was obtained which was followed up by a physical examination (which included three-lead electrocardiogram, spirometry, and venous blood draw for haemoglobin and haematocrit assessment) to exclude participants with evidence of cardiac, pulmonary and haematological abnormalities.
Subjects performed two incremental exercise tests to exhaustion on an electronically braked cycle ergometer (Excaliber, Lode, Groningen, the Netherlands), equipped with a racing saddle and the subject's own pedals, in order to determine maximal oxygen consumption 
and maximal workload in both normoxia (FIO2
= 0.2093)) and acute hypoxia (FIO2
= 0.125). In both conditions, subjects warmed up at 150 W for 5 min after which the workload was increased (30 W min–1) until they were unable to continue. Exercise intensities corresponding to 30%, 50%, and 
90% of 
(from each respective FIO2) were used during exercise performed during experimental phase (i.e. same workloads for acetazolamide and placebo at the respective FIO2). The two exercise tests were assigned in balanced order of FIO2 and were separated by an hour's rest. During the tests, heart rate and rhythm were continuously monitored by a cardiac monitor (Lifepak 6, Physio-Control, Redmond, WA, USA), and arterial O2 saturation by pulse oximetry (Nellcor N395, Pleasanton, CA, USA)
Subjects breathed through a non-rebreathing valve with a deadspace of 100 ml (model 2700, Hans Rudolph, Kansas City, MO, USA), while expired gas was continuously sampled from a heated mixing chamber, and O2 and CO2 concentrations were measured with a mass spectrometer (model 1100, Perkin-Elmer, Pomona, CA, USA). Expired gas flow was measured using a pneumotach (Hans Rudolph 3813, Hans Rudolph Inc, Kansas City, MO, USA). Minute ventilation, 
and CO2 production (
) were measured and calculated using a commercially available system (TrueOne 2400, Parvo Medics, Salt Lake City, UT, USA). Determination of 
was based on the average of the two highest consecutive 15 s measurements of 
obtain during the highest workload reached by each subject.
Experimental design and data collection
This study was performed using a double blind, order-balanced, crossover approach, such that all subjects performed exercise after having taken either acetazolamide or placebo on each of two separate days, one week apart. Subjects performed identical exercise protocols during the same time of the day on each visit. Before exercising testing, subjects were instructed to ingest three separate capsules (each taken 8 h apart commencing 24 h prior to the study) of either 250 mg acetazolamide or placebo. Drug dosing was scheduled so that the first exercise test was performed 1–2 h after the ingestion of the final (third) capsule. A local hospital pharmacy supplied the identically appearing drug/placebo capsules in generically marked tubes such that both subjects and investigators were blinded to drug/placebo. However, drug dosing design was set such that half the subjects would begin with acetazolamide (and the other half with placebo) in order to control for potential ordering effects.
On each study day, participants exercised at workloads corresponding to approximately 30%, 50% and 90% of the previously determined 
while breathing either room air (20.93% O2) or normobaric hypoxic gas (12.5% O2). Five minutes before the start of the hypoxic test, subjects were connected to a 150 l reservoir bag containing 12.5% O2 in nitrogen (PIO2
–95 mmHg). Arterial O2 saturation was monitored continuously by pulse oximetry (N395, Nellcor) with the use of a forehead sensor (RS-10, Nellcor).
The ordering of the two exercise tests was the same as during the subject's prescreening, and they were separated by 1 h of rest. Each workload had a duration of approximately 5 min, except for 13–14 min at 50%
. This longer time was needed to clear acetylene, used at 1% concentrations to measure cardiac output, in order to prevent chromatographic interference with the inert gases (present at p.p.m. levels) used for the MIGET at subsequent, higher workloads. Heart rate and rhythm was monitored by a cardiac monitor (Lifepak 6, Physio-Control) and minute ventilation, and 
and 
were measured in the same manner as during prescreening (as previously described). Additionally, arterial and femoral venous blood samples, expired gas samples, and leg blood flow (via the thermodilution technique, see below) data were collected during steady state rest and exercise conditions.
Subject preparation
Three catheters were placed using sterile technique under local anaesthesia (1% lidocaine) while the subject rested comfortably in the supine position: (a) in a superficial vein of the dominant arm (18 gauge), (b) in the radial artery of the non-dominant arm (20 gauge), and (c) in the left femoral vein pointing distally (4F multiport catheter, Cook Critical Care, Bloomington, IN, USA). In addition to the catheters, a sterile 19 gauge thermistor (IT-19P, Physitemp Instructments Inc., Clifton, NJ, USA) was inserted into the left femoral vein pointing proximally. The thermistor tip was located 10–12 cm from the tip of the 4F catheter. After placement of the catheters and thermistor, subjects rested briefly prior to commencing the infusion of inert gases for the initial (resting) gas exchange measurements.
Pulmonary gas exchange
The multiple inert gas elimination technique (MIGET) was applied in the usual manner (Gale et al. 1985) to measure arterial and mixed expired inert gas concentrations. Briefly, an inert gas solution (containing sulphur hexafluoride, ethane, cyclopropane, isoflurane, ether and acetone) was prepared in sterile 0.9% saline solution and infused in the superficial arm vein for 20 min before the collection of resting samples. At rest, the infusion rate was 3 ml min–1, and this was increased during exercise to a rate proportional to ventilation (0.3 ml min–1 l–1 min–1). Duplicate samples of mixed expired gas (15 ml) and arterial blood (5 ml) were obtained in gas-tight glass syringes at rest and 90%
, while at 50%
a single set of samples was taken. The concentrations of the six inert gases obtained from mixed expired gas and arterial blood were determined by gas chromatography (Hewlett-Packard 5890, Wilmington, DE, USA) (Wagner et al. 1974). 
distributions were computed using a least squares best fit regression analysis with enforced smoothing (Evans & Wagner, 1977). 
heterogeneity is represented by the second moments (log scale) of the blood flow 
and ventilation distributions 
as described by Evans & Wagner (1977). The residual sum of squares (RSS) was used as an indicator of the adequacy of the data.
Immediately after collecting samples for MIGET analysis, 2 ml arterial and femoral arterial blood samples were collected anaerobically, debubbled and kept on ice until measured using an IL Synthesis 45 analyser and IL682 Co-oximeter (Instrumentation Laboratories, Lexington, MA, USA), usually within 15 min. PO2, PCO2, pH, saturation, Hb and haematocrit were determined at 37°C and corrected to femoral venous temperature measured at the time of collection. Blood lactate levels were measured using a YSI 2300D Stat Plus blood lactate analyser (Yellow Springs Instrument Co. Inc., Yellow Springs, OH, USA).
Efficiency of pulmonary gas exchange was also assessed by the alveolar–arterial PO2 difference, AaDO2. The role of diffusion limitation was assessed by comparing the measured AaDO2 (AaDO2(m)) to the AaDO2 predicted from the inert gas elimination data (AaDO2(p)). In the absence of diffusion limitation, AaDO2(m) and AaDO2(p) should agree, such that their difference, AaDO2(m–p), is statistically zero. But in the presence of diffusion limitation, the measured AaDO2 will exceed the predicted value, so that AaDO2(m–p) > 0. This is because, unlike O2, inert gases are essentially invulnerable to diffusion limitation and thus reflect the effects of 
inequality only (Hammond & Hempleman, 1987). When O2 was diffusion limited by these criteria, whole-lung diffusing capacity for oxygen (DLO2) was calculated by the method of Hammond & Hempleman (1987). In brief, the Hammond–Hempleman algorithm iteratively finds the value of DLO2 that, combined with the MIGET-measured degree of 
inequality, quantitatively explains the actual arterial PO2 by a combination of the measured 
inequality and diffusion limitation.
Calculation of in vivo P50
Taking the measured arterial PO2, PCO2 and pH, all corrected to body temperature, together with Hb, Hct and standard P50 (the PO2 corresponding to 50% saturation of haemoglobin at pH 7.40, PCO2 40 mmHg and temperature 37°C), we use the Kelman computerized O2 (Kelman, 1966) and CO2 (Kelman, 1967) dissociation curves to iteratively find the PO2 value that corresponds to a saturation of 50%. This is the ‘in vivo lung P50’ because arterial blood best represents conditions at the end of the pulmonary capillaries. The same procedure was applied using femoral venous data (i.e. PO2, PCO2 and pH, corrected to body temperature) to determine ‘in vivo muscle P50’ because femoral venous blood best represents conditions at the end of the muscle capillaries.
Cardiac output 
Cardiac output was measured at rest and during steady state exercise at 50% and 90%
, using the non-invasive open-circuit acetylene uptake method as previously described by Barker et al. (1999). Briefly, subjects breathed a gas mixture containing 1% acetylene, 7% helium, and either 20.93% O2 (normoxia) or 12.5% O2 (hypoxia), with the balanced nitrogen. The end-tidal concentrations of acetylene and helium were measured by mass spectrometer (Perkin Elmer MGA 1100, Pomona, CA, USA). Cardiac output was calculated from inspired and helium-corrected end-tidal acetylene concentrations, end-tidal PCO2, mixed expiratory PCO2 and the blood-gas partition coefficient (
) of acetylene based on mass balance principles (Barker et al. 1999). On both study days, was determined for each subject in duplicate by use of gas chromatography from venous blood samples containing C2H2 (Wagner et al. 1974).
Leg blood flow
Leg blood flow measurements were made in duplicate at 30%, 50% and 90%
using the thermodilution method (Jorfeldt & Wahren, 1971). This was done during the fourth and fifth minute of each workload, using short-term steady state infusion (15 s) of iced 0.9% saline into the femoral vein of the left leg. The infusion rate was set with a continuous flow roller pump to give 1.0°C drop in femoral vein temperature. Femoral vein temperature and saline infusion rate were recorded and displayed on a personal computer using the AcqKnowledge data acquistion system (Biopac Systems Inc., Goleta, CA, USA). Leg blood flow was calculated from these data by the thermal balance equation detailed by Andersen & Saltin (1985).
Statistical analysis
Data presented are expressed as the mean ± S.E.M., unless otherwise noted. Three-factor repeated measures ANOVA (StatView5.0, SAS Institute, Inc., Cary, NC, USA) was used for determination of significant differences due to acetazolamide, FIO2 and exercise intensity. When overall significant main effects or a significant interaction was found for acetazolamide, post hoc analysis (Student's t test) was performed to identify the exercise intensities where significant effects occurred. Correction for multiple t test comparisons was not necessary in our analysis since the t test was only performed post hoc when a significant overall main effect was observed. Significance was accepted at P < 0.05 (two tailed).
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Results |
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Table 2 shows the mean values of the metabolic variables and minute ventilation at each exercise intensity, FIO2 and drug condition. The 
/workload relationship was unaffected by acetazolamide. Likewise, the respiratory exchange ratio was not significantly different between acetazolamide and placebo. At rest, acetazolamide had no significant effect on minute ventilation. However, during exercise, minute ventilation was significantly higher with acetazolamide (P < 0.001). Since respiratory rate remained the same, tidal volume was larger. Acetazolamide had no effect on cardiac output at rest or during exercise. Cardiac output increased with exercise as expected, but was not significantly different between normoxic and hypoxic exercise at the same relative intensity.
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The effects of acetazolamide on acid–base status are presented in Table 3. At rest and moderate exercise, arterial and femoral venous [HCO3–] were significantly lower with acetazolamide than placebo (P < 0.001), but no significant differences were found during heavy exercise. However, at all exercise intensities arterial and femoral venous pH were significantly lower with acetazolamide (P < 0.001). Arterial and venous lactate concentrations were lower with acetazolamide than placebo at rest and during exercise (P < 0.001, arterial and P < 0.01, venous), but the calculated net lactate efflux rate was unaffected by acetazolamide. Also, arterial and femoral venous base deficit values were significantly greater with acetazolamide (P < 0.05), demonstrating the well known metabolic acidosis the drug produces.
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Arterial blood gas data and variables derived from the MIGET are shown in Figs 1 and 2. SaO2 (P < 0.05) and PaO2 (P < 0.01) were significantly higher with acetazolamide than placebo, but no significant differences in arterial O2 content (CaO2) were observed. Standard P50 was slightly but significantly lower after acetazolamide (26.9 ± 0.6 mmHg) than placebo (27.8 ± 0.4 mmHg) (P < 0.001). In vivo P50, based on arterial blood pH and PCO2, and therefore relevant to the lungs, was slightly higher after acetazolamide (35.3 ± 0.08 versus placebo values of 34.7 ± 1.0 mmHg in normoxia, P < 0.05; 31.9 ± 0.09 versus 30.7 ± 1.1 mmHg in hypoxia, P = 0.18).
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As expected, the measured AaDO2 increased with increasing exercise intensity, but the increase was significantly less with acetazolamide than placebo (P < 0.001) (Fig. 2). With placebo, the average AaDO2 increased from 3.0 mmHg (at rest) to 17.5 mmHg (at heavy exercise) in normoxia and from 4.4 to 18.4 mmHg in hypoxia, whereas with acetazolamide, AaDO2 increased from 0.5 to 9.9 mmHg in normoxia, and from 2.0 to 14.7 mmHg in hypoxia. No significant overall drug effect on femoral venous PO2 or saturation was found, but there was a significant drug–workload interaction and femoral venous PCO2 was significantly higher with acetazolamide during light and heavy exercise (Table 4, both P < 0.05).
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, data not shown) and perfusion (
, Fig. 2), the latter of which is commonly used as a measure of 
inequality, were significantly less with acetazolamide than placebo (P < 0.05). The improvement in 
, in the absence of separate populations of low or high 
units, reflects a general narrowing of the unimodal 
distribution seen under control conditions. During moderate and heavy exercise in hypoxia, and during heavy exercise in normoxia, the measured AaDO2 (AaDO2(m)) exceeded the AaDO2 predicted (AaDO2(p)) from the MIGET, indicating the presence of diffusion limitation and/or extrapulmonary shunt contributing to the AaDO2. It should be noted that in some cases the AaDO2(p) may be found to be greater than AaDO2(m) and thus result in a negative AaDO2(m–p), as seen in Fig. 2. However, physiologically speaking a negative AaDO2(m–p) is not possible. One reason for a negative AaDO2(m–p) may be error propagation in the calculation of AaDO2. For example, the accumlulation of even a 1% error in each of these variables (
, 
, PaO2 and Pa,CO2) could result in a negative AaDO2. Added to this is the potential of random errors in the estimation of 
inequality obtained from MIGET, as well as the fact the MIGET predicted AaDO2, i.e. AaDO2(p), is constrained to be 0 (it is not allowed to be negative). However, together these errors only results in an uncertainity of the AaDO2(m–p) on the order of 5 mmHg. Thus, when AaDO2(m) and AaDO2(p) values are close (such as during rest or light exercise) it may be difficult to separate out error from physiological effects, but when the AaDO2(m–p) is large there is little question regarding the presence of O2 diffusion limitation. As seen in Fig. 2, the level of diffusion limitation (and/or extrapulmonary shunting) during exercise (as idicated by the size of the portion of the total AaDO2 not due to 
inequality) was significantly lower with acetazolamide than placebo (P < 0.05). However, acetazolamide did not signifcantly affect O2 diffusing capacity (DLO2) in either hypoxia (90.3 ± 3.9 versus 85.0 ± 3.0 ml min–1 mmHg–1, acetazolamide and placebo, respectively) or normoxia (74.9 ± 6.4 versus 74.6 ± 8.3 ml min–1 mmHg–1). The quality of the MIGET measurements was assessed by the adequacy of fit of the experimental data, where the residual sum of squares (RSS) is expected to be 5.3 or less half the time (50th percentile) and 10.6 or smaller 90% of the time (90th percentile) according to the Chi-square distribution with six degrees of freedom (Roca & Wagner, 1994). In our experiment, 58% of the RSS were less than 5.3 and 100% were less than 10.6, indicating good data quality.
Effect of acetazolamide on leg blood flow and muscle gas exchange
Leg blood flow and muscle gas exchange data are shown in Table 4. No differences in leg blood flow or leg O2 delivery (the product of leg blood flow and arterial O2 content) were found with acetazolamide compared to placebo. Muscle gas exchange efficiency as defined by muscle O2 diffusion conductance (DM,O2, computed as the ratio of 
to mean capillary PO2 at maximal work load) was also not changed by acetazolamide. The in vivo P50, based on femoral venous pH and PCO2, which is more relevant to the muscles, was unaffected by acetazolamide (43.5 ± 1.0 versus placebo 43.3 ± 1.4 mmHg in normoxia, P
= 0.61; 37.6 ± 0.8 versus 37.5 ± 1.4 mmHg in hypoxia, P
= 0.74). Leg 
(the product of leg blood flow and the arterial–femoral venous O2 content difference (Ca-fv,O2) and leg fractional O2 extraction were also not affected by acetazolamide (Table 4).
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Discussion |
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matching and less O2 diffusion limitation. Despite changes in acid–base status, no net influence of acetazolamide on muscle gas exchange was found. Acid–base and ventilatory responses with acetazolamide
Acetazolamide was administered in a commonly used dose for the prevention of acute mountain sickness (three 250 mg acetazolamide capsules 8 h apart, or 3.1–3.8 mg kg–1) (Barry & Pollard, 2003). This dose is believed to generate a modest renal effect with minimal red cell carbonic anhydrase inhibition (Swenson, 1998). The metabolic acidosis (as indicated by a significant decrease in pH and [HCO3–]) developed as expected (Table 3), and the reduction in pH consequently increased minute ventilation (Table 2) and contributed to a generally higher PaO2 and SaO2 and a tendency (main effect of drug on Pa,CO2, P = 0.13) for reduced Pa,CO2 (Fig. 1). These findings are comparable to other studies that examined the effects of acetazolamide on acid–base regulation and minute ventilation (McLellan et al. 1988; Stager et al. 1990; Kowalchuk et al. 1994; Swenson, 1998; Wagenaar et al. 1998; Hollidge-Horvat et al. 1999; Scheuermann et al. 2000b; Garske et al. 2003). Interestingly, the recent observation that antioxidants can prevent acetazolamide-induced reductions on the hypoxic ventilatory response suggests that regulation of redox state in the carotid body may also be important in regulating ventilation (Teppema et al. 2006). However, the extent to which this influences our results is unclear, as the resting ventilatory response to hypoxia in our subjects was not different between acetazolamide and placebo (Table 2), which is likely explained by the differing acute versus chronic effect of acetazolamide usage on the hypoxic ventilatory response (Teppema et al. 1992; Swenson & Hughes, 1993).
A lower plasma lactate is commonly found with acetazolamide (McLellan et al. 1988; Stager et al. 1990; Kowalchuk et al. 1992, 1994; Hollidge-Horvat et al. 1999; Kowalchuk et al. 2000; Scheuermann et al. 2000a,b) and can be explained by the influence of extracellular acidosis on lactate efflux (McLellan et al. 1988; Kowalchuk et al. 2000; Wetzel et al. 2001). Extracellular acidosis causes accumulation of H+ on the muscle membrane and diminishes the transmembrane H+ gradient, which thereby impairs lactate and H+ removal from the muscle (Kowalchuk et al. 2000; Wetzel et al. 2001). However, as can be seen in Table 3, our data are not conclusive about the influence of acetazolamide on lactate efflux. To draw a definite conclusion about the cause of the lower plasma lactate concentrations with acetazolamide, muscles biopsies or use of magnetic resonance spectroscopy or imaging would be necessary to follow the pathway of lactate production, turnover and clearance more closely.
The changes in acid–base status by acetazolamide observed in our study might influence muscle function (Gladden, 2004). Low plasma [HCO3–] and base excess values may limit the glycolytic capacity of the muscle as exercise intensity progressively increases (Stager et al. 1990; Scheuermann et al. 2000a) and an elevated muscle [H+] could depress muscle function (Fulco et al. 2006). Interestingly, the notion that lactate is simply a metabolic waste product from glycolysis and impairs muscle function during exercise has been challenged lately (Gladden, 2004). In the present study, the capacity to exercise was not significantly different between placebo and acetazolamide in spite of roughly 3 mM differences in lactate levels. Furthermore, participants did not distinguish any difference in symptoms exercising with placebo versus acetazolamide. However, it should be pointed out that the design of our study would not have uncovered small differences in exercise capacity.
Pulmonary gas exchange efficiency with acetazolamide
Separate from its effects in increasing alveolar ventilation, acetazolamide improved pulmonary gas exchange efficiency under both resting and exercise conditions, as indicated by a smaller AaDO2 (Fig. 2). This agrees with the work of Frans et al. (1993) who used hydrochloric acid infusion to create a metabolic acidosis in anaesthetized dogs. However, the authors found no improvement in 
relationships, in contrast to the current MIGET data showing both decreased 
mismatch and less O2 diffusion limitation with acetazolamide (Fig. 2). Since diffusion limitation is not seen at rest, and since ventilation was controlled, the reduced AaDO2 found by Frans et al. is difficult to explain. On the other hand, Swenson et al. (1993) reported impaired 
matching with acetazolamide. However, this study also involved dogs under anaesthesia, at rest, and under controlled ventilation. These differences in study design make Swenson's results also hard to compare with findings from the present study in exercising humans.
One possible explanation for improved 
homogeneity with acetazolamide in our study is attenuated pulmonary vasoconstriction (Deem et al. 2000; Berg et al. 2004; Hohne et al. 2004; Swenson, 2006). Normally, vasoconstriction occurs rapidly in response to low alveolar PO2 (Jensen et al. 1992; Podolsky et al. 1996; Hohne et al. 2004), which would potentially explain our hypoxic data. However, increased vascular resistance will increase pulmonary arterial pressure (Deem et al. 2000) irrespective of FIO2, and studies have shown an inverse relationship between pulmonary arterial pressure and pulmonary gas exchange efficiency. For example, HAPE-susceptible (but otherwise healthy) individuals show both increased 
inequality and higher pulmonary artery pressures during exercise, even in normoxia (Podolsky et al. 1996). An increase in pulmonary arterial pressure may also cause interstitial fluid accumulation, which could impair 
matching (Wagner et al. 1987; Loeppky et al. 1992; Podolsky et al. 1996), an event that might be mitigated if acetazolamide attenuates increases in pulmonary artery pressure. Consistent with this is the recent observation that sildenafil, a phosphodiesterase V inhibitor which reduces pulmonary artery pressure, improves PaO2 and SaO2 and reduces AaDO2 in subjects at rest and during exercise at high altitude (Richalet et al. 2005). Indeed, acetazolamide-induced hyperventilation leading to a higher alveolar PO2 could further reduce vasoconstriction and contribute to decreased 
mismatch.
Slightly less O2 diffusion limitation was found during heavy exercise with acetazolamide in both normoxia and hypoxia, as shown by a smaller difference between the measured and MIGET-predicted values for the AaDO2 (i.e. AaDO2(m–p); Fig. 2). Perhaps the best way to understand the basis of this is to examine the determinants of diffusion equilibration as presented by Piiper & Scheid (1983). Using an analytical approach, they showed that the alveolar–arterial PO2 difference caused by diffusion limitation (as a fraction of the alveolar to mixed venous PO2 difference) is given by the expression 1 – exp[–DLO2/(
)], where DLO2 is lung diffusing capacity for O2, is the average slope of the Hb dissociation curve between the arterial and venous points and 
is cardiac output. This expression is conceptually helpful because of its analytical form, but application quantitatively is limited by the assumption of a linear O2 dissociation curve. As mentioned in the results, neither computed DLO2 nor cardiac output was significantly different between acetazolamide and placebo, but DLO2 averaged 90.3 ml min–1 mmHg–1 after acetazolamide compared to 85.0 ml min–1 mmHg–1 with placebo. In addition, in vivo P50 (as opposed to standard P50) was slightly higher after acetazolamide (due to lower lactate levels and higher ventilation more than offsetting the metabolic acidosis), implying a slightly smaller value of . Thus, despite a small rightward shift in the O2 dissociation curve which would make it more difficult for O2 loading, DLO2/(
) was numerically higher after acetazolamide, which could explain less diffusion limitation (even though individually none of DLO2, 
and was significantly different). Thus, the explanation for the slightly lower degree of diffusion limitation after acetazolamide may lie both in the O2 dissociation curve and in possibly less interstitial oedema leading to higher values for DLO2. However, the data do not permit statistically definitive conclusions on this component of the acetazolamide effect. Also, as previously stated, the application of this model assumes a linear O2 dissociation curve. Thus it is useful quantitatively only when PaO2 is on the steep part of the O2 dissociation curve (e.g. during hypoxia). In normoxia its limitations must be recognized and conclusions must be limited as a result.
It should be noted that MIGET analysis found no intrapulmonary shunting with or without acetazolamide, but the MIGET technique is not able to differentiate the relative contributions of pulmonary O2 diffusion limitation and extrapulmonary shunting (e.g. from bronchial and thebesian veins) towards the overall AaDO2(m–p), and thus we cannot dismiss the possibility that extrapulmonary shunting may also have contributed to the increase in the AaDO2. In healthy subjects, pulmonary shunting at rest via the bronchial and thebesian veins is believed to comprise < 2% of total 
(Torre-Bueno et al. 1985; Hammond et al. 1986; Wagner et al. 1986). In our study, the change in PaO2 during the normoxic exercise (> 90% of 
) is equivalent to 1% shunt, and therefore could account for all of the AaDO2(m–p) we observed. In hypoxia, however, an extrapulmonary shunt > 10% of 
would have been necessary to account for the AaDO2(m–p) we observed. As it is highly unlikely that such a shunt would develop in normal healthy young subjects, we reasoned that pulmonary O2 diffusion limitation is the primary component contributing to the increasing AaDO2(m–p) during hypoxic exercise, and that extrapulmonary shunting comprises a very small component of the overall AaDO2(m–p).
Muscle gas exchange with acetazolamide
As can be seen in Table 4, acetazolamide did not significantly alter muscle gas exchange or leg blood flow. Classically, the metabolic acidosis with acetazolamide should induce a rightward shift of the oxyhaemoglobin dissociation curve at the muscle level, and thereby facilitate O2 unloading. However, when calculating in vivo P50 at the muscle (based on femoral venous blood) in normoxia (placebo 43.3 ± 1.4 mmHg versus acetazolamide 43.5 ± 1.0 mmHg) or hypoxia (placebo 37.5 ± 1.4 mmHg versus acetazolamide 37.6 ± 0.08 mmHg), it appears that there is no net shift in the dissociation curve seen at the muscle as a result of acetazolamide. Incidentally, it is worth mentioning that our in vivo P50 values are similar to those found in other studies examining muscle gas exchange during exercise (Calbet et al. 2005; Lundby et al. 2006). Moreover, when analysing the data in a manner similar to those for the lungs in the preceding section, (the Piiper et al. (1984) approach can be applied to muscles as well as lungs), we found no differences in the muscle diffusional conductance of oxygen (i.e. DMO2; 44.3 ml min–1 mmHg–1 placebo versus 44.2 ml min–1 mmHg–1 acetazolamide, Table 4). Leg blood flow was also unaffected by acetazolamide. The tendency of metabolic acidosis to shift the dissociation curve rightwards was therefore completely balanced by opposing factors. Consequently, the determinants of diffusive O2 unloading in the muscle were closely matched between placebo and acetazolamide and likely account for the lack of effect of the drug in spite of the metabolic acidosis.
It should be noted that determination of DM,O2 is dependent on subjects exercising maximally when mitochondrial PO2 is presumed to be very close to zero. Thus we have calculated DM,O2 only at the heaviest workload, which in fact is likely to generate a 
greater than at the set target of 90% of 
because of the time spent (4 min) exercising at this heavy workload. If the work performed by our subjects was not maximal, leg blood flow may have been slightly less and therefore have led to a slightly lower DM,O2 value compared to that at true 
. Nevertheless, our intent was to compare DM,O2 under the same conditions at (or near) maximal exercise with both acetazolamide and placebo in each subject. To that end, our interpretation of the effect of acetazolamide on muscle gas exchange during exercise would not be different, even if we may have slightly underestimated the DM,O2 values under both conditions.
Conclusion
The principal finding of this study is that acetazolamide improves arterial oxygenation in part by improving pulmonary gas exchange efficiency (reduced 
inequality and less diffusion limitation) and not only by increased minute ventilation. No changes in muscle gas exchange were found. The improvement in pulmonary gas exchange efficiency may in part reflect shifts in the O2 dissociation curve but may also be due to attenuation of pulmonary vasoconstriction, resulting in improved distribution in blood flow and possibly less interstitial fluid accumulation.
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, respectively) in hypoxia (12.5% O2) and normoxia 
Significant main effect acetazolamide versus placebo, P < 0.05. 
Significant main effect of exercise, P < 0.05. *Post hoc analysis identifying individual group differences between acetazolamide and placebo, P < 0.05.
, respectively) in hypoxia (12.5% O2) and normoxia. Inert gas data were not obtained during light exercise (i.e. 30% of 
), and therefore measured minus predicted AaDO2
[AaDO2(m–p)] and log standard deviation of the distribution of perfusion 
values are not shown at this level of exercise. Values are expressed as means ±
S.E.M.