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Am J Physiol Endocrinol Metab 278: E316-E329, 2000;
0193-1849/00 $5.00
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Vol. 278, Issue 2, E316-E329, February 2000

Effect of induced metabolic alkalosis on human skeletal muscle metabolism during exercise

M. G. Hollidge-Horvat, M. L. Parolin, D. Wong, N. L. Jones, and G. J. F. Heigenhauser

Department of Medicine, McMaster University Medical Centre, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the study was to examine the roles of active pyruvate dehydrogenase (PDHa), glycogen phosphorylase (Phos), and their regulators in lactate (Lac-) metabolism during incremental exercise after ingestion of 0.3 g/kg of either NaHCO3 [metabolic alkalosis (ALK)] or CaCO3 [control (CON)]. Subjects (n = 8) were studied at rest, rest postingestion, and during constant rate cycling at three stages (15 min each): 30, 60, 75% of maximal O2 uptake (VO2 max). Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ALK resulted in significantly (P < 0.05) higher intramuscular Lac- concentration ([Lac-]; ALK 72.8 vs. CON 65.2 mmol/kg dry wt), arterial whole blood [Lac-] (ALK 8.7 vs. CON 7.0 mmol/l), and leg Lac- efflux (ALK 10.0 vs. CON 4.2 mmol/min) at 75% VO2 max. The increased intramuscular [Lac-] resulted from increased pyruvate production due to stimulation of glycogenolysis at the level of Phos a and phosphofructokinase due to allosteric regulation mediated by increased free ADP (ADPf), free AMP (AMPf), and free Pi concentrations. PDHa increased with ALK at 60% VO2 max but was similar to CON at 75% VO2 max. The increased PDHa may have resulted from alterations in the acetyl-CoA, ADPf, pyruvate, NADH, and H+ concentrations leading to a lower relative activity of PDH kinase, whereas the similar values at 75% VO2 max may have reflected maximal activation. The results demonstrate that imposed metabolic alkalosis in skeletal muscle results in acceleration of glycogenolysis at the level of Phos relative to maximal PDH activation, resulting in a mismatch between the rates of pyruvate production and oxidation resulting in an increase in Lac- production.

glycogen phosphorylase; pyruvate dehydrogenase; lactate metabolism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INDUCED METABOLIC ALKALOSIS by sodium bicarbonate (NaHCO3) ingestion in humans has previously been shown to increase blood Lac- concentration ([Lac-]) during exercise (10, 33, 37, 46, 63). Unfortunately, the majority of studies exploring the effects of metabolic alkalosis on Lac- metabolism have focussed on the possible performance-enhancing capabilities of "bicarbonate loading" (31). Ingestion of NaHCO3 is thought to enhance performance by buffering the lactic acid produced with exercise, thereby limiting the effects of the decreased intramuscular pH (pHi; see Ref. 31). Metabolic alkalosis through ingestion or infusion of NaHCO3 has been shown to enhance performance for short-duration, high-intensity exercise, but the mechanisms have not been elucidated. Different mechanisms have been postulated to explain this, including an increase in muscle Lac- production (10, 63) and/or enhanced Lac- efflux from the muscle (44). Lactate accumulation results from the conversion of nonoxidized pyruvate to Lac- by lactate dehydrogenase (LDH) and as such will be influenced by both pyruvate production from glycogen via glycogen phosphorylase (Phos) and pyruvate oxidation by pyruvate dehydrogenase (PDH; see Refs. 18, 38).

In an effort to discern the possible mechanisms responsible for the increased blood [Lac-] with metabolic alkalosis, we chose an oral dose of NaHCO3, previously shown to induce a significant metabolic alkalosis, to influence plasma [Lac-], and to enhance performance (37). Continuous, dynamic constant-rate exercise at low, moderate, and high intensity was chosen to follow the metabolic effects, compare fuel utilization with previously described carbohydrate (CHO) and free fatty acid (FFA) contributions at these power outputs (50), and to maintain the ATP turnover rate constant between conditions. This is the first human in vivo study to examine the key regulatory enzymes, their controllers, and fuel utilization during continuous dynamic constant rate exercise under alkalotic conditions.

The aim of the present study was not to examine the performance effect of an induced metabolic alkalosis during exercise. Rather, the first aim was to determine the effect of metabolic alkalosis on the key regulatory enzymes Phos and PDH and their allosteric regulators. The second aim was to measure the effect of metabolic alkalosis on glycolytic intermediates, muscle pyruvate production, and pyruvate oxidation. The third aim was to measure muscle lactate accumulation, production, and efflux. The last aim was to determine if alkalosis has any effects on glucose uptake and FFA utilization during exercise.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Eight healthy male volunteers participated in the study [age 23 ± 1.8 (SE) yr; height 173 ± 3.8 cm; weight 75.3 ± 4.4 kg]. Written consent was obtained from each subject after explanation of the purposes and associated risks of the study protocol. The study was approved by the Ethics Committees of both McMaster University and McMaster University Medical Center.

Preexperimental Protocol

All subjects completed an initial incremental maximal exercise test on a cycle ergometer to determine VO2 max and maximal work capacity using a metabolic measurement system (Quinton Q-Plex 2; Quinton Instruments, Seattle, WA). Mean VO2 max for the group was 3.2 ± 0.2 l/min. None of the subjects was well trained, but all participated in some form of regular activity. Each subject was instructed to refrain from caffeine, alcohol, and exercise for 24 h before each trial, and studies were carried out at the same time of day.

Experimental Protocol

Each subject participated in two experimental trials separated by 2-3 wk and was randomized to receive capsules containing either 0.3 g/kg of NaHCO3 [metabolic alkalosis (ALK)] or 0.3 g/kg of CaCO3 [control (CON)]. On the morning of each trial, the subjects reported to the laboratory after consumption of a standard light meal consisting primarily of CHO. The exercise portion of the protocol consisted of three levels of continuous, constant-rate exercise on a cycle ergometer at 30, 60, and 75% of VO2 max, each maintained for 15 min, which began after insertion of arterial and femoral venous catheters and ingestion of the required capsules (Fig. 1).


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  		Fig. 1.   Experimental study protocol. Total of two trials were completed per subject; 0.3 g/kg of either CaCO3 [control (CON)] or NaHCO3 [alkalosis (ALK)] was ingested over the time indicated, each constituting one trial. Leg blood flow and arterial and femoral venous blood samples were taken at the times indicated. Exercise bouts refer to three continuous power outputs of 15 min each. 30, 60, and 75% indicate cycling intensity of 30%, 60% and 75% maximal O2 uptake (VO2 max), respectively. Muscle biopsies were taken at the times indicated. Respiratory measurements were taken immediately after blood sample/blood flow.

A radial artery was catheterized with a Teflon catheter (20 gauge, 3.2 cm; Baxter, Irvine, CA) percutaneously after anesthetizing the area with 0.5 ml of 2% lidocaine without epinephrine (6). A femoral vein was catheterized percutaneously for insertion of the thermodilution catheter (model 93-135-6F; Baxter) using the Seldinger technique (6) after administration of 3-4 ml of lidocaine without epinephrine. Both the arterial and femoral venous catheters were maintained patent with sterile, nonheparinized, isotonic saline solution. Arterial and femoral venous blood samples were simultaneously taken at rest, rest postingestion, and during each of the three exercise bouts at 6 and 11 min. Single leg blood flow measurements were made after blood sampling at the same time points. Single leg blood flow was determined using the thermodilution technique, as described by Andersen and Saltin (1). Nonheparinized isotonic saline (10 ml) was injected, and leg blood flow was calculated by a portable CO monitor (Spacelab, Redmond, VA). At least three measurements were recorded at each time point and then averaged.

A total of five percutaneous needle biopsies of the vastus lateralis were taken (1 at rest, 1 at rest postingestion, and 3 during exercise at the end of each power output). The resting biopsies were obtained with the subject lying on a bed. The resting and exercise biopsies were obtained on opposite legs and then were reversed for the second trial. Biopsy sites were prepared by making an incision through the deep fascia under local anesthetic (2% lidocaine without epinephrine), as described by Bergström et al. (5). Respiratory measurements of ventilation (VE), O2 uptake (VO2), CO2 production (VCO2), and respiratory exchange ratio (RER) were measured at 5 and 11 min of each exercise stage.

Muscle Analysis

Muscle samples were immediately frozen in liquid N2. A small piece (10-35 mg) was chipped from each biopsy (under liquid N2) for determination of the fraction of PDH in the active form (PDHa), as previously described (20, 51). The remainder of the sample was freeze-dried, dissected free of blood and connective tissue, and powdered. One aliquot was analyzed for Phos activity according to the methods of Young et al. (72). Briefly, a 3- to 4-mg sample of muscle was homogenized at -20°C for 0.2 ml in 100 mM of Tris/HCl (pH 7.5) containing glycerol, potassium fluoride, and EDTA. Homogenates were then diluted with 0.8 ml of the same buffer without glycerol and homogenized further at 0°C. Total (a + b) Phos activity (measured in the presence of 3 mM AMP) and Phos in the active a form (Phos a; measured in the absence of added AMP) were measured at 30°C with a spectrophotometer. Maximum velocity (Vmax) was derived from the equation described by Lineweaver and Burke (41)
1/<IT>V</IT> = (<IT>K</IT><SUB>m</SUB>/<IT>V</IT><SUB>max</SUB>)(1/S) + (1/<IT>V</IT><SUB>max</SUB>)
where V is the initial reaction rate expressed as millimoles dry wt per kilogram per min, S is the Pi concentration ([Pi]) in millimoles per liter, and Km is the Michaelis constant (26.2 mmol/l). The mole fraction of Phos a is presented as a percentage and is calculated as Vmax a/Vmax(a + b) × 100. Phos a measurements were made only on exercise samples for two reasons. First, resting samples must be kept at room temperature for ~30 s before freezing for accuracy, which would have required two additional biopsies (53). Ethically, this was not acceptable for the provision of one measurement. Second, the changes at rest and rest postingestion were not a main focus of this study. A second aliquot was used to determine muscle glycogen, fluorometrically, using the enzymatic end-point method described by Bergmeyer (4). A third aliquot of dry muscle was extracted in 0.5 M perchloric acid (PCA) and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3, and analyzed for acetyl-CoA, free CoASH, total CoA, acetylcarnitine, free carnitine, and total carnitine according to the methods of Cederblad et al. (11). A fourth aliquot was used to determine ATP, pyruvate, Lac-, phosphocreatine (PCr), creatine, glucose, glucose 6-phosphate (G-6-P), glucose 1-phosphate (G-1-P), fructose 6-phosphate (F-6-P), and glycerol 3-phosphate (Gly-3-P) concentrations using the methods described by Bergmeyer (4) and were adapted for fluorometry. All muscle metabolites were normalized to the highest total creatine content for a given individual (mean total creatine = 121.7 ± 6.2 mmol/kg dry wt) to correct for nonmuscle contamination. Free contents of ADP (ADPf) and AMP (AMPf) were calculated as described by Dudley et al. (23), using the reactants and equilibrium constants of the near-equilibrium reactions catalyzed by creatine kinase (CK) and adenylate kinase. ADPf was estimated using the measured ATP, PCr, and creatine contents and an estimated H+ concentration {[H+], calculated indirectly from muscle [Lac-] and pyruvate concentration ([pyruvate]) using the regression equation of Sahlin et al. (55)}. From this information, the concentration of AMPf was determined assuming a Keq of 1.05 for the adenylate kinase reaction. Free Pi content was calculated from the sum of the estimated resting free [Pi] of 10.8 mmol/kg dry wt (23) and the Delta PCr - Delta G-6-P - Delta F-6-P - Delta Gly-3-P between rest and each time point during exercise. For the purposes of ADPf, AMPf, and free Pi calculations, no differences were observed between the rest and rest postingestion values; therefore, the mean of the two values was taken as the resting value.

Blood Sampling and Analysis

Arterial and femoral venous blood samples (~10 ml) were collected in heparinized plastic syringes and placed on ice. One portion (1-2 ml) of each blood sample was analyzed for blood gas determination (AVL 995 Automatic Blood Gas Analyzer), O2 and CO2 content (Cameron Instrument, Port Arkansas, TX), and hemoglobin (OSM3 Hemoximeter; Radiometer, Copenhagen, Denmark). A second portion of each sample was deproteinized with 6% PCA and stored at -20°C until analysis for glucose, Lac-, and glycerol according to the methods of Bergmeyer (4) adapted for fluorometry. The third portion of blood was immediately centrifuged at 15,900 g for 2 min, and the plasma supernatant was frozen and later analyzed for FFA (Wako, NEFA C test kit; Wako Chemical, Montreal, Canada). Hematocrit was determined on blood samples using a heparinized microcapillary tube centrifuged for 5 min at 15,000 g.

Leg Uptake and Release of Metabolites, VO2, and VCO2

Uptake and release of metabolites (glucose, glycerol, Lac-) were calculated from their whole blood measurements in arterial and femoral venous blood and leg blood flow according to the Fick equation. Because there were differences in the hematocrit over time within a condition and between matched arterial and femoral venous samples, venous samples were corrected for fluid shifts. Fluid shifts for the whole blood measurements were corrected using the differences in hemoglobin (Hb) to calculate a percent change in blood volume (%Delta BV), as calculated by the equation (assuming no change in intravascular hemoglobin; see Ref. 30)
%&Dgr;BV = [(Hb<SUB>arterial</SUB>/Hb<SUB>venous</SUB>) − 1] × 100
This value was then multiplied by the measured venous value to yield a corrected value that was used in determining uptake/release for that metabolite. Uptake and release of plasma FFA were determined as above, but venous values were corrected using changes in plasma protein concentration to correct for changes in plasma water (30). The leg VO2 and VCO2 were calculated from their respective arterial and femoral venous content differences and blood flow.

Subjects exercised at a constant rate, and because no significant differences occurred in blood flows or metabolite concentrations between the 6- and the 11-min sampling points at each power output, the two values were averaged to obtain one value for each power output. Reported values are for the single leg only.

Calculations

Flux through Phos and therefore glycogenolysis was calculated from the differences in glycogen utilization divided by time. PDHa flux was estimated from the PDHa as measured in wet tissue and converted to dry tissue using the wet-to-dry ratio. Pyruvate production was calculated from the sum of the rates of glycogen breakdown and glucose uptake minus the sum of the rates of accumulation of muscle glucose, G-6-P, and F-6-P. Lac- production was calculated from the sum of the rates of muscle Lac- accumulation and Lac- release. Pyruvate oxidation was calculated as pyruvate production minus Lac- production. All values are reported in millimoles per kilogram per minute dry weight and are for a single leg only. All values were calculated in three carbon units and assume a wet muscle mass of 5 kg.

Intramuscular pH. Intramuscular pH (pHi) was calculated from the [Lac-] and [pyruvate] according to the methods of Sahlin et al. (55).

Lac- gradient and [H+] gradient. The Lac- gradient between the plasma and muscle for both trials was calculated for arterial and femoral venous blood at 75% VO2 max only. The Lac- gradient between the muscle and arterial plasma was calculated as the difference between the wet weight intramuscular [Lac-] and arterial plasma [Lac-]. The Lac- gradient from muscle-to-femoral venous plasma was calculated as above using the noncorrected venous values. The arterial-to-muscle and femoral venous-to-muscle [H+] gradients were calculated as the difference between the respective blood compartment [H+] and the calculated intramuscular [H+].

Statistical Analysis

Data were analyzed using two-way ANOVA with repeated measures (treatment × time), except where otherwise stated. When a significant F ratio was found, the Newman-Keuls post hoc test was used to compare means. The following data were analyzed using a two-tailed paired dependent-sample Student's t-test: Phos a and glycogen utilization at each power output. Data are presented as means ± SE. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle Metabolism

Phos. Phos a activity did not change with power output during CON. However, with ALK, Phos a progressively decreased with power output and was significantly lower at 75% VO2 max with ALK compared with CON (ALK 37.9 ± 5.1 vs. 48.8 ± 5.0 mmol dry wt · kg-1 · min-1; Fig. 2).


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  		Fig. 2.   Phosphorylase (Phos) a, mole fraction % during cycling at the various power outputs. + Significantly different from CON at matched time points. There was a significant main effect of time with each condition. Values are means ± SE.

Glycogen. Resting and rest postingestion muscle glycogen concentrations were not different between conditions (Table 1). Muscle glycogen content decreased with increasing power output, but to a greater degree with ALK (Table 1). During the complete exercise study, total muscle glycogen utilization was 25% greater with ALK compared with CON (305 ± 9 vs. 229 ± 10 mmol/kg dry wt). No differences in muscle glycogen utilization were observed at 30% VO2 max, but muscle glycogen utilization was significantly higher at both 60 and 75% VO2 max with ALK compared with CON (60% -132 ± 15 vs. 75 ± 6 mmol/kg dry wt; 75% -133 ± 15 vs. 113 ± 12 mmol/kg dry wt; Fig. 3).

                              
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  		Table 1.   Muscle glycogen and glycolytic intermediate contents in vastus lateralis at rest, rest postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK



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  		Fig. 3.   Glycogen utilization for each power output. Data are means ± SE. + Significantly different from CON at matched time points.

Glucose, G-6-P, F-6-P, G-1-P, and Gly-3-P. Intramuscular accumulation of glucose increased with exercise similarly between conditions (Table 1). Intramuscular G-6-P concentration ([G-6-P]) and F-6-P concentration ([F-6-P]) increased with increasing power output for the first and second power outputs, but each was significantly lower with ALK during 75% VO2 max only (Table 1). Muscle G-1-P and Gly-3-P were similar between conditions, increasing with each power output (Table 1).

Lactate and pyruvate. Intramuscular [Lac-] increased with each power output but was significantly higher with ALK at both 60% (ALK 40.9 ± 8.3 vs. CON 26.2 ± 5.0 mmol/kg dry wt) and 75% (ALK 72.8 ± 11.8 vs. CON 65.2 ± 10.1 mmol/kg dry wt) VO2 max (Fig. 4). Muscle [pyruvate] increased with each power output and was similar between conditions at 30 and 75% VO2 max and significantly higher with ALK during the second power output (Fig. 4).


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  		Fig. 4.   Muscle lactate (Lac-) and pyruvate concentrations with ALK and CON at rest and during each power output. + Significantly different from CON at matched time points. Values are means ± SE. dw, Dry weight. There was a significant main effect of time for both Lac- and pyruvate, specifically at 60 and 75% VO2 max.

PDHa. Resting (ALK 0.55 ± 0.01 vs. CON 0.55 ± 0.08 mmol wet wt · kg-1 · min-1) and rest postingestion (ALK 0.55 ± 0.11 vs. CON 0.53 ± 0.05 mmol wet wt · kg-1 · min-1) PDHa were not different between conditions. Under both conditions, PDHa increased progressively with each power output but was significantly higher at 60% VO2 max (4.17 ± 0.23 vs. 3.77 ± 0.27) with ALK compared with CON, respectively (Fig. 5).


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  		Fig. 5.   Muscle active pyruvate dehydrogenase (PDHa) activity with ALK and CON at rest and during each power output. + Significantly different from CON at matched time points. Data are means ± SE. ww, Wet weight. There was a significant main effect of time for each condition.

CoA, carnitine, and acetylated forms. Total muscle CoA was not different between conditions at rest or during exercise (Table 2). The acetyl-CoA concentration ([acetyl-CoA]) increased with each power output similarly between conditions at 30%, but during both 60 and 75% VO2 max the [acetyl-CoA] was significantly lower with ALK (Table 2). Free CoASH declined equally between conditions with exercise (Table 2). The acetyl-CoA-to-CoASH ratio was also significantly lower at 75% VO2 max with ALK (0.26 ± 0.02 vs. 0.37 ± 0.06; Table 2). Acetylcarnitine followed a similar pattern to acetyl-CoA (increasing with each power output) but was not different between conditions (Table 2). Muscle total carnitine content increased significantly from rest to 75% VO2 max to the same degree in each condition, whereas free carnitine decreased in a reciprocal manner with increasing power output. There were no differences in total carnitine between conditions, but free carnitine was significantly lower at 60% VO2 max during ALK compared with CON (Table 2).

                              
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  		Table 2.   Muscle acetyl group content in vastus lateralis at rest, rest postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK

ATP, ADPf, AMPf, free Pi, and PCr. Muscle ATP concentration ([ATP]) was unaltered by exercise or as a result of ALK. Muscle [ADPf] and [AMPf] increased with each power output, but both were significantly higher with ALK at 60 and 75% VO2 max (Table 3). Free Pi increased with each power output, but to a significantly greater degree with ALK (Fig. 6). The [PCr] decreased with increasing power output but was significantly more depleted at each power output during ALK compared with CON (Fig. 6).

                              
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  		Table 3.   Muscle high energy phosphate content in vastus lateralis at rest, rest postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK



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  		Fig. 6.   Phosphocreatine (PCr) and free Pi with ALK and CON at rest and at each power output. There was a significant main effect of time for each condition. + Significantly different from CON at matched time points. Values are means ± SE.

Pyruvate production and oxidation and lactate production and oxidation. Pyruvate production increased with exercise but was significantly higher during both the second and third power outputs with ALK. Pyruvate oxidation increased with exercise similarly between conditions at both 30 and 75% VO2 max. At 60% VO2 max, pyruvate oxidation was significantly higher during ALK (Table 4). Relative pyruvate oxidation expressed as the percentage of pyruvate produced that was oxidized was similar between conditions for both 30 and 75% VO2 max. However, relative pyruvate oxidation was significantly higher during 60% VO2 max with ALK (Table 4). Lactate production was similar between conditions during the first two power outputs but was significantly higher at 75% VO2 max during ALK (Table 4).

                              
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  		Table 4.   Muscle pyruvate production, oxidation, and lactate production at 30, 60, and 75% VO2 max after either CON or ALK

Blood Metabolites, Blood Flow, and Exchange Across the Leg

Blood pH, PCO2, and HCO-3. Arterial pH and HCO-3 (Fig. 7) and venous pH and HCO-3 (Table 5) were all significantly higher during ALK compared with CON at rest postingestion and each of the three power outputs.


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  		Fig. 7.   Arterial blood pH, PCO2, and HCO-3 during ALK and CON at rest and during each power output. + Significantly different from CON at matched time points. Values are means ± SE.


                              
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  		Table 5.   Arterial concentration of bloodborne substrates during rest, rest postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK

Leg blood flow and leg respiratory quotient. Leg blood flow increased progressively with exercise similarly between conditions (Table 6). Leg VO2, VCO2, and leg respiratory quotient were not different between conditions, increasing with each power output (Table 6).

                              
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  		Table 6.   Leg blood flow, RQ, CO2 production, and O2 uptake at rest, rest postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK

Blood lactate and flux. Arterial [Lac-] increased progressively with each power output but was significantly higher at both 60 and 75% VO2 max with ALK (Table 5). Net Lac- release across the leg increased with each power output but was significantly higher during 75% VO2 max with ALK (Fig. 8).


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  		Fig. 8.   Glucose uptake and lactate release across the leg during ALK and CON at rest and for each power output. + Significantly different from CON at matched time points. There was a significant main effect of time for glucose uptake and Lac- release. Values are means ± SE.

Blood FFA and glycerol. Arterial plasma [FFA] declined progressively with exercise similarly between conditions (Table 5). FFA release across the leg occurred at rest postingestion and at each of the power outputs during CON. However, with ALK, a significantly lower net release occurred at rest postingestion, whereas during exercise at each of the three power outputs, a net uptake occurred (Fig. 9). Arterial [glycerol] increased with each power output similarly between conditions (Table 5). Glycerol release across the leg occurred at rest postingestion and 30% VO2 max similarly between conditions. During 60% VO2 max, a net release occurred with CON, whereas a net uptake occurred with ALK. At 75% VO2 max, a net uptake across the leg occurred for both conditions but was significantly lower with ALK (Fig. 9).


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  		Fig. 9.   Glycerol flux and free fatty acid (FFA) flux across the leg during ALK and under CON conditions at rest and during each power output. + Significantly different from CON at matched time point. There was a significant main effect of time with each condition. Values are means ± SE.

Blood glucose and flux. Arterial [glucose] (Table 5) and leg glucose uptake (Fig. 8) were similar at all power outputs between conditions.

Lactate Gradient, H+ Gradient, and pHi

The lactate gradient between both the arterial and femoral venous plasma and muscle was not different between conditions at 75% VO2 max (Table 7). The [H+] gradient between the arterial blood and muscle and the femoral venous blood and muscle were both significantly elevated with ALK compared with CON during 75% VO2 max (Table 7). Intramuscular [H+] was significantly elevated during both the second and third power outputs with ALK (Table 7).

                              
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  		Table 7.   Lactate and hydrogen ion gradients from arterial to muscle and femoral venous to muscle during cycle ergometry at 75% VO2 max only after either CON or ALK

Respiratory Gas Exchange Variables

Whole body VO2 increased similarly between conditions with each power output (Table 8). Whole body VCO2 was significantly higher during 60% (2.39 ± 0.09 vs. 2.28 ± 0.13) and 75% (3.18 ± 0.11 vs. 3.01 ± 0.22) VO2 max in ALK compared with CON, respectively. RER was also significantly higher during ALK at both 60 and 75% VO2 max (Table 8). VE increased similarly between conditions (Table 8).

                              
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  		Table 8.   Respiratory variables during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ALK


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined the effects of induced alkalosis on the metabolic responses of skeletal muscle during continuous, dynamic, constant-rate exercise at three power outputs (30, 60, and 75% VO2 max). The main effects of alkalosis during this type of exercise occurred during the two higher power outputs and included an enhanced glycogen utilization with a concomitant increase in pyruvate production, increased intramuscular Lac- accumulation, enhanced Lac- efflux from the exercising leg, and a greater relative activation of Phos than PDHa.

Lactate production within the muscle is dependent on the balance between the rates of pyruvate production and oxidation. Intramuscular Lac- is formed from pyruvate by the action of the near-equilibrium enzyme LDH (47). Although greater pyruvate production was observed at the two highest power outputs, Lac- production was elevated over CON values only during 75% VO2 max (Table 4). Lactate production at the highest power output resulted from a significant degree of mismatch between the rates of glycogenolysis/glycolysis and maximal PDHa activation. At 60% VO2 max, the absence of an increase in lactate production despite an increased pyruvate production resulted from an enhanced pyruvate oxidation relative to production due to greater PDHa with ALK. These changes resulted from the effect of alkalosis on the rate-limiting enzymes Phos, phosphofructokinase (PFK), and PDH. The main control points for glycogenolysis/glycolysis involve Phos and PFK, respectively, whereas entry into the oxidative pathway is controlled by PDHa (47).

Phos

Phos is the flux-generating enzyme responsible for glycogenolysis within skeletal muscle and is subject to both covalent and allosteric regulation. Phos a, considered the active form, is active in the absence of AMPf, whereas Phos b, the less active form, requires AMPf (12). Covalent b-to-a transformation is mediated by Phos kinase a, which is activated by either an increase in epinephrine or cytosolic Ca2+ concentration ([Ca2+]) via cAMP-dependent and -independent mechanisms, respectively (54). Posttransformational allosteric activation of Phos b is mediated by AMP and IMP, whereas inhibition is mediated by ATP and G-6-P. Substrate regulation of both forms of Phos by the free Pi and glycogen concentrations is equally important (12, 18).

Phos a was significantly reduced at the highest power output with ALK. This reduction presumably reflects the inhibition of Phos kinase a by the increased [H+] (Table 3). Previous studies have demonstrated this relationship between [H+] and Phos a in intensely exercising muscle (13). However, the mole fraction of Phos in the a form is not the sole determinant of glycogenolytic flux, as previous studies examining the relationship between Phos a transformation and glycogenolysis have demonstrated (14, 18, 34, 54). The present results of a higher glycogen utilization at 75% VO2 max (133 ± 6 vs. 113 ± 12; Fig. 3) despite a lower Phos a (Fig. 2) and the observed greater glycogen utilization (132 ± 15 vs. 75 ± 6; Fig. 3) despite similar Phos a (Fig. 2) at 60% VO2 max with ALK are in agreement with these previous studies. Additionally, there may have been a hormonal effect throughout exercise due to a decrease in the circulating epinephrine concentration ([epinephrine]) with ALK, which may have contributed to the lower observed Phos a. Bouissou and colleagues (10) observed a 34% reduction in the plasma [epinephrine] and higher intramuscular [Lac-] with alkalosis in humans who cycled to exhaustion. The enhanced glycogenolysis despite similar or lower Phos a transformation with ALK likely resulted from posttransformational modulation by increases in the AMPf, and IMP concentrations and an increase in its substrate free Pi.

AMP and IMP have been shown to stimulate Phos a (2, 54) and Phos b (2, 15). AMP acts on Phos a by reducing the Km for Pi from 26.8 to 11.8 mM in the presence of as little as 0.01 mM AMP (2, 54). AMP acts in a similar manner on Phos b but with a higher Km. Both the [AMPf] and free [Pi] were significantly greater with ALK during the second and third power outputs and were well above the required concentrations for activation (Table 3 and Fig. 6). In support of the close relationship between Phos activity, glycogenolytic flux, and the [AMPf], other studies using caffeine ingestion (17), increased FFA availability (24, 48), and short-term training (16) have demonstrated glycogen sparing during exercise associated with blunted AMPf accumulation. The increased [AMPf] may have augmented activation of both Phos a and Phos b. IMP activates Phos b with a Km of 1.2 mM (2). IMP concentration ([IMP]) was not measured in the present study but has been shown previously to increase with exercise and when the [ADPf] increases (64). The [ADPf] was significantly elevated with ALK, which may have led to an increase in the [IMP], which in turn may have activated Phos b.

ATP and G-6-P are both inhibitors of Phos b. ATP has an inhibitory constant (Ki) of ~2 mM, whereas G-6-P has a Ki of ~0.3 mM (25). In the present study, [ATP] remained constant throughout exercise and between conditions at levels above the Ki. [G-6-P] was significantly lower with ALK at 75% VO2 max but remained above the Ki. These combined results should favor a reduction in Phos activity and glycogenolysis. However, it has been previously demonstrated that the inhibition of Phos b by both ATP and G-6-P can be overcome when the [AMPf] increases sufficiently (18). In addition, previous studies have demonstrated that glycogenolytic flux is closely tied to the availability of its substrate, free Pi, and the allosteric regulator, AMPf, both of which were elevated with ALK (16, 17).

In summary, Phos activity and therefore glycogenolytic flux results from the combination of covalent, allosteric, and substrate regulation. During ALK, despite a lower transformation of Phos a, glycogenolytic flux was enhanced, and glycogen utilization increased during the second and third power outputs due to the maintenance of flux through posttransformational allosteric activation of Phos a + b by an increased [AMPf], and possibly [IMP], and an increase in the concentration of its substrate, free Pi.

PFK

PFK plays a key role in the regulation of glycolysis and therefore pyruvate production. PFK catalyzes the conversion of F-6-P to fructose 1,6-bisphosphate with the use of ATP (47), with the relative enzyme activity reflected by changes in the [F-6-P] and [G-6-P], with which it is in equilibrium. PFK is subject to regulation by a large number of metabolites that function to either inhibit or activate the enzyme complex. ATP and H+ inhibit, whereas ADP, AMP, Pi, and F-6-P activate, the enzyme complex, with the net enzyme activity resulting from the combination of these inputs (66). These are the most potent regulators, which reflect energy state and fuel utilization within the cell and thereby provide feedback regulation to adjust glycolytic flux.

[ATP] remained constant between trials and across power outputs, which provided a small degree of inhibition. Also, during the highest power output, intramuscular [H+] was significantly elevated for both trials (Table 3), which would provide inhibition, as previous in vitro studies using constant [ATP] with declining pH have demonstrated (22, 65). Human exhaustive exercise protocols have found similar changes reflecting reductions in PFK activity with reduced intramuscular [H+] (36, 59). The magnitude of the pH inhibition can be modulated by increases in [F-6-P] and the activators ADP, AMP, and free Pi. Alkalosis led to increases in [F-6-P] and [G-6-P] during the third power output but to a significantly lower magnitude than CON. At this power output, pHi was significantly lower compared with CON but may have failed to inhibit PFK activity due to positive modulation by the significantly elevated [AMPf], [ADPf], and [Pi]. AMP acts by augmenting PFK's affinity for its substrate, F-6-P (9). The increased [F-6-P], although lower than CON, was elevated above the Km of 0.1-0.2 mM, which could have opposed the pH inhibition by decreasing the affinity of the ATP binding site (39). The accompanying rise in the [G-6-P], although above the Ki for Phos, may have been overridden by the positive modulation of Phos by the increased [AMPf] and [Pi]. Previous in vitro studies have also demonstrated substantial acceleration of glycolysis with the lowering of the ATP-to-ADP ratio, a situation present with ALK at both 60% (CON 132 vs. ALK 87) and 75% VO2 max (CON 80 vs. ALK 55) due to the increase in [ADPf] (71). The stimulatory effect of an increase in [ADPf] on PFK activity has been shown to substantially increase with as little as a 2 mM increase in Pi, which also occurred in the present study with ALK (71).

The combined results demonstrate enhanced glycogenolytic/glycolytic flux with ALK due to allosteric upregulation of both Phos and PFK activity, leading to the increased glycogen utilization and pyruvate production at the two highest power outputs.

PDH

PDHc is a mitochondrial enzyme complex that catalyzes the decarboxylation of glycolytically derived pyruvate and therefore reflects the rate of CHO entry into the tricarboxylic acid (TCA) cycle. PDHc transformation between the active (PDHa) and inactive (PDHb) forms is regulated by the balance between PDH kinase (PDHK; deactivating) and PDH phosphatase (PDHP; activating; see Refs. 52, 68). The relative phosphatase/kinase activity is controlled by the mitochondrial acetyl-CoA-to-CoASH, ATP-to-ATP, and NADH-to-NAD+ ratios and the allosteric regulators Ca2+, pyruvate, and H+. Increases in the ratios decrease PDHa transformation, whereas decreases in the ratios have the opposite effect. Increases in [pyruvate] inhibit the kinase only, increases in [Ca2+] inhibit the kinase and activate the phosphatase, and increases in [H+] activate the phosphatase only (51, 52, 68). Due to the complex interaction of regulators and the observed differences in PDHa between conditions, the changes occurring at 60 and 75% VO2 max will be discussed separately.

Changes in PDHa at 60% VO2 max. In the present study, PDHa increased with each power output as a result of contraction-induced increases in the [Ca2+] (19, 21, 34). However, increases in the [Ca2+] cannot be the sole mechanism responsible for the increased PDHa with ALK, since the power outputs were identical between trials. The elevated PDHa with ALK resulted from changes in the allosteric regulators acetyl-CoA, ADPf, H+, pyruvate, and the NADH-to-NAD+ ratio.

The [acetyl-CoA] was significantly lower during this power output with ALK (Table 2) and probably reflects reduced FFA utilization in the face of increased glycogen utilization and pyruvate production (Table 4). Because acetyl-CoA inhibits PDHP, the reduced concentration would serve to activate the phosphatase and therefore contribute to the greater PDHa observed with ALK (68).

The ATP-to-ADP ratio was significantly reduced with ALK (87 vs. 132) at this power output due to a significant elevation in the [ADPf] without changes in [ATP] (Table 3). This ratio effects PDHK only, as ATP is the substrate for the reaction and therefore competes with its product ADP, which inhibits catalytic activity (68). The lower ATP-to-ADP ratio observed with ALK could have resulted in lower PDHK activity and therefore contributed to the greater PDHa observed.

Hydrogen ion has been shown to activate PDHa in acidotic perfused rat hearts (49) and has been attributed to the differences in pH optimum of PDHK and PDHP. PDHK has a pH optimum of 7.0 ~ 7.2, with increased inhibition as pH falls, whereas PDHP has a pH optimum of 6.7 ~ 7.1 (35). In the present study, the calculated intramuscular [H+] was significantly elevated at this power output with ALK compared with CON (Table 3), which may have resulted in greater activation of the phosphatase and contributed to the increased PDHa observed.

The intramuscular [pyruvate] was also significantly elevated with ALK at this power output (Fig. 4). Pyruvate is a potent stimulator of PDHa, since it is both substrate and an inhibitor of PDHK with a Ki of 0.5-2.0 mM (42). In the present study, the intramuscular [pyruvate] measured at this power output with ALK is below the Ki. However, the [pyruvate] was determined from a biopsy taken at the end of the exercise bout, and given that both glycogen utilization and pyruvate production were significantly elevated, it is possible that the [pyruvate] rose above the Ki during the initial stages of exercise (20, 51). In addition, the lactate production rate remained similar to CON despite the elevation in pyruvate production, suggesting that most of the pyruvate made available to PDHa was oxidized and therefore may not be reflected by the intramuscular [pyruvate] in the sample taken at the end of exercise. This is further supported by the observation that the amount of pyruvate oxidized relative to that produced was significantly elevated with ALK at this power output (Table 4).

Neither the NADH concentration ([NADH]) nor the NADH-to-NAD+ ratio was measured in the present study. However, previous studies using indirect techniques have shown that the [NADH] decreases and therefore the NADH-to-NAD+ ratio declines with high-intensity exercise (29, 60), which would favor an increase in PDHa. In addition, the markedly increased glycogen utilization with ALK may have led to reduced FFA utilization functioning as the "glucose-fatty acid cycle reversed," as previously demonstrated by Sidossis et al. (56, 57). These researchers have demonstrated in exercising humans that the intracellular availability of CHO (rather than FFA) determines the nature of substrate oxidation when both CHO and FFA are made available during exercise. The mechanism whereby enhanced CHO availability reduces FFA oxidation is not precisely known, but findings from in vitro studies using human tissue (61) and human exercise studies (56) point to the inhibition of long-chain fatty acid entry into the mitochondria by inhibition of carnitine palmitoyltransferase I (CPT-1). The mechanism responsible for CPT-1 inhibition is not clear but may be mediated by a pH effect, as it has been shown in isolated rat muscle preparations that CPT-1 is inhibited by a low pH (62). In humans, the pH inhibition has recently been shown to be more sensitive than that of rats, with inhibition at a pHi of ~6.8 (61). The reduced FFA utilization would lead to reduced intramitochondrial [NADH]. Cytosolic [NADH] would also decrease, as NAD+ would be required for the maintenance of glycolytic flux and would be provided from the conversion of pyruvate to Lac-. In support of this is the observation that intramuscular [Lac-] increased with ALK in the absence of an increase in Lac- production (Fig. 4 and Table 4). Because the mitochondrial and cytosolic compartments are thought to be in equilibrium, the overall result would be a reduction in PDHK activity due to decreased [NADH] and an increase in PDHP due to increased [NAD+] and therefore contribute to the elevated PDHa seen with ALK at this power output. The absence of a difference in both the leg RQ and mouth RER reflecting a change in the relative CHO and FFA utilization is not surprising. Previous authors have demonstrated the lack of sensitivity of both measures in detecting small changes in fuel utilization during high-intensity exercise (50).

In summary, the significant increase in PDHa observed with ALK at 60% VO2 max can be attributed to the combined inhibitory effects of a decrease in the [acetyl-CoA], an increase in the [ADPf], [pyruvate], and [Ca2+] on PDHK, and the stimulatory effects of the elevated [H+] on PDHP.

Changes in PDHa at 75% VO2 max. At this power output, PDHa transformation was similar between conditions and reflects the attainment of maximal PDHa (Fig. 5). Previous studies have shown maximal activation at this power output (34, 50). The increased pyruvate production with ALK resulted from a slightly higher glycogen utilization. The absence of a difference in the relative pyruvate oxidation rates between conditions was due to the similar rates of PDHa. The higher lactate production rate observed with ALK resulted from the higher glycogenolytic/glycolytic rate. However, at this power output with ALK, the major fate of the lactate produced was efflux from the muscle and not intramuscular accumulation, which will be discussed later.

Cellular energetics. The rate of mitochondrial ATP production is regulated by O2 availability and the [NADH]-to-[NAD+] and the [ATP]-to-[ADP] × [Pi] ratios (70). O2 availability was not limiting in the present study in either trial, as neither the mouth VO2 nor O2 uptake across the leg was different (Tables 6 and 8). However, differences in glycogen and FFA utilization were apparent. During the two higher power outputs with ALK, there was a decrease in FFA utilization, as evidenced by decreased [acetyl-CoA] and the markedly higher glycogen utilization. During CON, the significantly lower glycogen utilization necessitated an increase in FFA utilization to match energy production to ATP demand. The reduced FFA utilization with ALK may have decreased the mitochondrial [NADH], which would necessitate a higher [ADPf] and [Pi] to drive oxidative phosphorylation according to the equation (70)
NADH<SUB>i</SUB> + 2c<SUP>3+</SUP> + 2 ADP<SUB>e</SUB> + 2 P<SUB>ie</SUB> ↔ NAD<SUP>+</SUP><SUB>i</SUB> 

+ 2<SUP>2+</SUP><SUB>c</SUB> + 2 ATP<SUB>e</SUB> + H<SUP>+</SUP> (1)
where c3+ and c2+ are the oxidized and reduced forms of mitochondrial cytochrome c, respectively, and the subscripts i and e refer to the intramitochondrial and extramitochondrial pools of reactants, respectively. This phenomenon of an obligatory increase in the [ADPf] and [Pi] was observed with ALK during the higher power outputs (Table 3 and Fig. 6). The CK reaction and PCr play key roles in the regulation of oxidative phosphorylation and other metabolic processes as an "energy buffer" and an "energy transport" system between the sites of ATP production and ATP utilization (67). The CK/PCr system is very sensitive to changes in intracellular [ADPf] and serves to keep this concentration low to prevent the inactivation of cellular ATPases and the net loss of cellular adenine nucleotides (67). In addition to functioning as a "barometer" for the intracellular [ADPf] and therefore mitochondrial respiration, the CK/PCr system acts as a proton buffer, since the production of ATP consumes both ADP and H+, which are both products of ATP hydrolysis
MgADP<SUP>−</SUP> + PCr<SUP>2−</SUP> + H<SUP>+</SUP> ⇆ MGATP<SUP>2−</SUP> + Cr (2)
The coupling of CK with the ATPases at the site of utilization prevents the local acidification at the initiation of exercise before activation of glycogenolysis. The hydrolysis of PCr also liberates free Pi at the onset of exercise, which is essential for the activation of glycogenolysis and glycolysis (3, 67). The increased degradation of PCr usually reflects a lack of mitochondrial-derived ATP from oxidative phosphorylation (70). In the present study, the increased PCr breakdown observed during ALK (Fig. 6) may have resulted from either a reduced [NADH] that accompanied a reduction in FFA utilization (Eq. 1) or may have resulted from a change in the [H+] with ALK (Eq. 2). Unfortunately, it is not clear from the present results which mechanism occurred first or what the exact mechanism is. Regardless of the mechanism, it is clear that the increased degradation of PCr led to elevation in the free [Pi], which contributed to the increased glycogenolysis observed with ALK, and is in agreement with previous studies examining the effect of increased FFA availability in rats (26) and humans (24), which have found that the concentrations of ADP, Pi, PCr, and NADH have direct effects on TCA cycle activity, mitochondrial respiration, and glycogen metabolism.

Lactate Metabolism and Transport

Intramuscular lactate accumulation reflects the balance between the rates of lactate production and efflux from the muscle (7, 38). Previous studies in humans employing metabolic alkalosis have focused on the effects of alkalosis on blood [Lac-] and have demonstrated similar results to the present study, i.e., an increase in the blood [Lac-] (10, 27, 33, 37, 46, 69). Only one study has investigated the effects of alkalosis on intramuscular lactate accumulation and, as in the present study, an increase in the muscle [Lac-] was found (63). Only one study has examined lactate efflux during alkalosis, and similar results to the present study, i.e., an increase in lactate efflux, were found (33). However, none of the studies was able to elucidate the mechanisms responsible for these observations during induced metabolic alkalosis.

Lactate production during ALK at 75% VO2 max was increased compared with CON and resulted from the mismatch between the rates of glycogenolysis and PDHa flux, as evidenced by enhanced pyruvate production and significantly higher glycogen utilization in the absence of a difference in PDHa between trials (Fig. 3 and Table 4). The similar PDHa between trials reflects maximal activation and is supported by the similar rates of pyruvate oxidation at this power output (Table 4). Therefore, the only difference between trials at this power output was the augmented CHO utilization and thus glycogenolytic flux, which greatly exceeded the maximal PDHa flux. Previous studies have demonstrated that a mismatch does exist between the maximal rates of Phos and PDHa at higher power outputs, resulting in a significant increase in lactate production (34, 51).

Intramuscular lactate accumulation is also a function of the rate of efflux from the muscle (38). ALK significantly increased arterial whole blood [Lac-] and plasma [Lac-] (Table 5) during both the second and third power outputs and enhanced efflux from the exercising leg during the highest power output only (Fig. 8). Blood [Lac-] represents the balance of lactate entry from muscle and uptake by inactive tissue (7). The present results can be explained by both enhanced lactate transport out of the exercising leg and possibly reduced uptake by nonexercising tissue.

Lactate transport across the sarcolemma occurs via a monocarboxylate lactate-proton cotransport protein and as such is the rate-limiting step in lactate efflux (38, 45). Kinetic studies of the transporter using isolated sarcolemmal vesicle preparations have shown it to have a high affinity for L-lactate and to be sensitive to changes in both the Lac- and H+ concentration gradients (38).

In the present study with ALK, there was an increase in the intramuscular [Lac-], but the [Lac-] gradient between the muscle and both the arterial plasma and femoral venous plasma compartments was similar between conditions (Table 7). Therefore, the enhanced lactate efflux observed with ALK was not a function of the increase in the intramuscular [Lac-]. This means that some other factor likely had an effect on the transporter (8). The most plausible effector is H+, as the [H+] gradient between muscle and both the arterial and femoral venous blood was significantly elevated with ALK (Table 7; see Refs. 38, 40). ALK also induced a significant elevation in extracellular HCO-3 concentration ([HCO-3]) that may have contributed to the enhanced lactate efflux from the muscle. The importance of external [HCO-3] on lactate efflux has been demonstrated in isolated muscle preparations with low external [HCO-3] yielding reduced lactate efflux (32, 43, 58). In addition, studies employing metabolic alkalosis in exercising humans (10, 27, 33, 37, 46, 63, 69) have consistently demonstrated increases in the appearance of lactate in the plasma when accompanied by increases in [HCO-3]. During ALK in the present study, extracellular [HCO-3] was significantly elevated in both arterial and femoral venous blood at rest postingestion and remained elevated throughout each of the three power outputs, which is an agreement with the above evidence (Table 5). Additionally, decreased lactate uptake by inactive tissue may have contributed to the higher arterial [Lac-] with ALK. Uptake by inactive tissue has been shown to occur during exercise (28). Normally, with exercise, the increased blood [H+] and [Lac-] create an inwardly directed [H+] and [Lac-] gradient, which facilitates uptake into inactive tissue. However, alkalosis decreases the [H+] gradient and therefore may reduce uptake compared with control conditions.

Summary and Conclusions

Induction of a metabolic alkalosis results in a complex series of metabolic effects during exercise reflecting changes in the activity of key regulatory enzymes and fuel utilization. The main findings of the study demonstrate that alkalosis during 60% VO2 max leads to increased glycogen utilization and pyruvate production as a result of posttransformational allosteric activation of Phos mediated by increases in ADPf, AMPf, and free Pi concentrations. Greater PDHa transformation also occurs with alkalosis at this moderate intensity as a result of the combined inhibitory effects of a decrease in [acetyl-CoA], an increase in the ADPf, pyruvate, and Ca2+ concentrations on PDHK, and the stimulatory effects of an increase in the [H+] on PDHP. The net result is an enhanced pyruvate oxidation and therefore a lack of an increase in lactate production. The increase in intramuscular [Lac-] observed with ALK at this power output results from the necessary regeneration of cytosolic NAD+ to maintain glycolytic flux in the face of markedly increased CHO utilization.

High-intensity exercise (75% VO2 max) with metabolic alkalosis leads to significantly increased lactate production, intramuscular accumulation, and efflux. The increased lactate production and increased intramuscular accumulation results from the absence of downregulation of glycogenolysis and glycolysis that typically occurs as pHi declines. Instead, the increased ADPf, AMPf, and free Pi concentrations competed with and/or negated the pH effect, resulting in the maintenance of glycogenolysis and therefore pyruvate production. However, the glycogenolytic rate exceeded the maximal PDHa rate, resulting in increased lactate production.

The elevated blood [Lac-] accompanying alkalosis likely resulted from the effects of an altered [H+] gradient on the transporters and is not due to changes in the [Lac-] gradient. Reduced uptake of lactate by inactive tissue may also have contributed to the increased arterial [Lac-] with ALK, but this contribution was not assessed in the present study.

The present data demonstrate that the increased blood [Lac-] commonly observed with metabolic alkalosis results from a complex series of events that modulate the activities of the key regulatory enzymes Phos, PFK, and PDHa.


    ACKNOWLEDGEMENTS

We acknowledge G. Obminski, Dr. M. Ganagaragah, T. M. Bragg, and J. Otis for technical assistance.


    FOOTNOTES

This work was supported by grants from the Medical Research Council of Canada (MRC). M. G. Hollidge-Horvat was supported by an MRC Studentship, M. L. Parolin was supported by a Natural Science and Engineering Council studentship, and G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. J. F. Heigenhauser, Dept. of Medicine, McMaster University Medical Centre, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: heigeng{at}fhs.csu.McMaster.ca).

Received 5 May 1999; accepted in final form 15 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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