CERMAK, NAOMI M.1; SOLHEIM, AMY S.1; GARDNER, MELANIE S.1; TARNOPOLSKY, MARK A.2; GIBALA, MARTIN J.1
It is well established that CHO ingestion during prolonged endurance exercise delays fatigue and improves the power output that can be maintained (8,16). The beneficial effect of CHO supplementation in most instances is attributable to the maintenance of euglycemia and a high rate of CHO oxidation, but other mechanisms may also be involved (8,16). Based on the wealth of evidence available, sports nutrition experts generally recommend that athletes ingest 30 to 60 g of CHO per hour during exercise to optimize endurance performance and subsequent training adaptations (1). An efficacious way for athletes to meet this recommendation and also to satisfy their hydration needs is to drink 600 to 1400 mL·h−1 of a 4% to 8% CHO solution, preferably in small, frequent doses from the onset of activity (1).
In contrast to CHO ingestion, exogenous provision of protein (PRO) during exercise is not generally regarded as important for endurance athletes (1). However, several studies have reported that coingestion of PRO with CHO during prolonged exercise improved time to exhaustion compared with CHO alone (14,25,26). This is an equivocal area of research, with other studies showing no difference between 6% CHO + 2% PRO (CHO + PRO) and CHO alone on endurance performance (21,23,28,29), and direct comparisons between studies are hampered by differences in research designs. Nonetheless, researchers who have reported an ergogenic effect of CHO + PRO compared with CHO alone have identified several potential mechanisms (14,25,26), including a reduced rate of muscle glycogen use and less reliance on nonoxidative metabolism, secondary to better maintenance of the muscle pool of tricarboxylic acid cycle intermediates (TCAI). However, no study has directly examined this issue, and thus mechanisms proposed to explain the improved endurance capacity after CHO + PRO ingestion (14,25,26) remain speculative.
The primary purpose of the present study was to determine whether coingestion of PRO with CHO would alter selected markers of skeletal muscle metabolic control during moderate-intensity exercise as compared with CHO alone. Using a double-blind, repeated-measures crossover design, we recruited trained cyclists and had them perform 90 min of constant-load cycling at 69% ± 1% V˙O2peak while ingesting either a 6% CHO or a 6% CHO + 2% PRO solution at a rate of 1 L·h−1. This drinking strategy was adopted to ensure a CHO delivery rate of 60 g·h−1 in both trials, which is the upper limit generally recommended to improve endurance performance (1). We tested the hypotheses that compared with CHO alone, CHO + PRO ingestion during exercise would favorably affect muscle energy metabolism as evidenced by 1) a reduced rate of glycogen catabolism, 2) an increased muscle pool of TCAI, and 3) a reduced phosphocreatine use. We (10) and others (6,20) have previously shown that nutritional perturbations can induce measurable changes in glycogen, TCAI, and phosphocreatine (PCr) metabolism during exercise at approximately 70% V˙O2peak in human skeletal muscle. A secondary purpose was to examine the influence of CHO versus CHO + PRO ingestion during exercise on markers of skeletal muscle recovery. Specifically, we measured the activity of creatine kinase (CK) in blood, an indirect marker of skeletal muscle membrane disruption (31), and 20-km time trial performance approximately 24 h after the first exercise bout.
Eight healthy men (mean ± SE: age = 29 ± 3 yr; weight = 79 ± 3 kg; height = 181 ± 2 cm) with a background in road cycling, triathlon, or duathlon were recruited. All subjects had been engaged in regular cycle exercise training for at least 2 yr before the study (mean = 4.8 yr, SE = 0.8 yr) and were cycling an average of 12 h·wk−1 or approximately 300 to 400 km·wk−1 at the time of the study. Their peak oxygen uptake (V˙O2peak) determined using on-line gas collection system (Moxus Modular V˙O2 System; AEI Technologies, Inc., Pittsburgh, PA) during a ramp test to exhaustion on an electronically braked cycle ergometer (Excalibur Sport V2.0; Lode BV, Groningen, The Netherlands) was 55 ± 2 mL·kg−1·min−1. After being advised of the purpose and the potential risks of the study, all subjects provided written, informed consent. The experimental protocol was approved by the Hamilton Health Sciences/Faculty of Health Sciences Research Ethics Board.
The main phase of the study was a randomized, double-blind comparison of the effect of ingesting 6% CHO or 6% CHO + 2% PRO during a 90-min bout of constant-load cycling that elicited 69% ± 1% V˙O2peak on selected markers of skeletal muscle metabolic control. Subjects arrived at the laboratory in the morning after ingesting a breakfast of their own choosing to simulate their habitual prerace practice. A catheter was inserted into an antecubital vein for blood sampling, and the area over one thigh was prepared for the extraction of a needle muscle biopsy sample. Briefly, the lateral portion of one thigh was anaesthetized (1% xylocaine) and a small incision was made through the skin and the underlying fascia to obtain a tissue sample from the vastus lateralis muscle. After removal from the leg, the muscle sample was immediately frozen in liquid nitrogen. After resting blood and biopsy samples were obtained, subjects initiated the cycling bout and ingested either the CHO or the CHO + PRO drink at a rate of 250 mL every 15 min to ensure a CHO delivery rate of 60 g·h−1, with or without 20 g of PRO per hour. Cardiorespiratory data and RPE were collected and averaged over the 5-min intervals beginning at 20, 40, 60, and 80 min into the exercise bout. Rates of whole-body CHO and fat oxidation were calculated based on the equations published by Peronnet and Massicotte (22). A muscle biopsy and a blood sample were also obtained immediately after exercise. Approximately 24 h after the constant-load test, subjects returned to the laboratory and performed a simulated 20-km cycling time trial on a Computrainer (RacerMate, Inc., Seattle, WA) using their own bicycle as previously described (29). Subjects received no temporal, verbal, or physiological feedback during the time trial. Subjects collected their urine into a 4-L container during the period between the first and the second rides, and upon return to the laboratory, a blood sample was obtained by venipuncture before the time trial. The two experimental trials were performed in random order separated by at least 7 d. Subjects also visited the laboratory on several occasions before the main experiment to establish the appropriate workload for the constant-load test and to perform a familiarization time trial.
The two experimental beverages were formulated by Gatorade (Barrington, IL) and contained the same amount of electrolytes and were similarly flavored (Table 1). The only difference between the beverages was that one contained 6% CHO in the form of sucrose (CHO) and one contained 6% CHO plus 2% whey PRO (CHO + PRO). The source of the whey PRO isolate was Lacprodan (Arla Foods, Basking Ridge, NJ). The two beverages were delivered as dry powders in sealed packages, identified by code numbers to ensure blinding, and were subsequently stored in sealed and locked containers at room temperature in the laboratory. Aliquots of test beverages were carefully weighed and dissolved in water according to the manufacturer's instructions on the day of each experimental trial. The drinks were stored in translucent containers, each containing 250 mL of fluid, and served slightly chilled.
Physical activity and dietary controls.
Subjects were asked to keep their weekly training schedule as consistent as possible over the course of the experiment, but given the within-subject design (i.e., each subject served as their own control), training was not standardized across subjects. Subjects were specifically instructed to standardize their final workout performed 48 h before each experimental trial and to perform no physical activity, aside from activities of daily living, for 24 h before the start of each trial. Subjects were also advised to maintain their habitual diet over the course of the study and were required to maintain food records during the 24 h before the constant-load test and the 24-h period between the constant-load test and the time trial. After the first experimental trial, subjects were instructed to replicate their individual nutritional pattern over the course of the second trial and again record food intake, noting any deviations from the first trial. Food records were subsequently analyzed (Nutritionist Five dietary analysis software; First Data Bank, San Bruno, CA), and results confirmed no difference in total energy intake or macronutrient composition between trials (Table 2).
After initial freezing in liquid nitrogen, muscle samples were subsequently freeze dried, powdered, dissected free of nonmuscle elements, and stored at −80°C before metabolite analyses using standard methods in our laboratory. For glycogen determination, 2- to 3-mg aliquot of freeze-dried muscle was incubated in 2.0 N HCl and heated for 2 h at 100°C to hydrolyze the glycogen to glucosyl units. The solution was neutralized with an equal volume of 2.0 N NaOH and analyzed for glucose using an enzymatic assay adapted for fluorometry (22). A second 5- to 10-mg aliquot of freeze-dried muscle was extracted on ice using 0.5 M perchloric acid (PCA) containing 1 mM ethylenediaminetetraacetic acid, neutralized with 2.2 M KHCO3, and the resulting supernatant was analyzed for adenosine triphosphate (ATP), phosphocreatine, creatine, lactate, and TCAI citrate and malate using enzymatic assays adopted for fluorometry (22).
Venous blood samples were collected into commercial tubes that contained either no additive or heparin. Nonheparinized blood was allowed to clot, then centrifuged, and the serum stored for subsequent analysis of CK (CK-NAC 2-part liquid reagent set; Pointe Scientific Inc., Canton, MI). Heparinized blood was used for the immediate determination of glucose (Ascensia Contour Blood Glucose Monitor; Bayer Health Care, Toronto, ON) and lactate (Accutrend Lactate; Roche Diagnostics, Mannheim, Germany). The remaining heparinized blood was centrifuged, and the resulting supernatant was removed and stored at −20°C for the subsequent analysis of plasma insulin using an immunoassay kit (Insulin EIA; Alpco Diagnostics, Salem, NH) and plasma amino acids using a high-performance liquid chromatography method described by Wilkinson et al. (32). Briefly, extracts were derivatized before injection using Waters™ AccQ·Fluor™ reagent kit (Milford, MA) by heating for 10 min at 55°C to form the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivative of all physiologic amino acids. Samples and standards (Sigma, St. Louis, MO) were run on a Waters™ 2695 high-performance liquid chromatography separations module through a 4-mm AccQ·Tag column (Water, Nova-Pak C18, bonded silica) to separate the amino acids. The amino acids were detected using Waters 2475 scanning fluorescence detector excitation and emission wavelengths of 250 and 395 nm, respectively. Amino acid peak areas were integrated compared with known standards and analyzed using a Waters Millenium32 software package.
After total urine volume was measured, an aliquot was stored at −86°C for subsequent analysis of urea and creatine using commercial kit assays (Pointe Scientific Inc.).
All muscle and blood data were analyzed using a two-factor (condition × time) repeated-measures ANOVA (Sigma Stat 3.1, Point Richmond, CA). All diet, time trial performance, and urine data were analyzed using a one-way repeated-measures ANOVA. The level of significance for all analyses was set at P < 0.05, and significant interactions and main effects were subsequently analyzed using a Tukey post hoc test. All data are presented as mean ± SE (n = 8). One subject did not adhere to the physical activity, and nutritional controls after the constant-load test in one trial and hence CK activity, diet, and time trial data are based on n = 7.
Mean V˙O2, averaged over the 90-min bout of constant-load cycling, corresponded to an intensity of 69% ± 1% V˙O2peak in both experimental trials (Table 3). There was no difference between treatments in V˙O2, HR, expired ventilation, respiratory exchange ratio, whole-body substrate oxidation, or RPE during exercise (Table 3).
The plasma concentration of branched chain amino acids (Fig. 1) and total essential amino acids (Table 4) was higher after exercise in CHO + PRO versus CHO (P < 0.05). Blood insulin decreased during exercise compared with rest (main effect, P < 0.05), but there were no differences between treatments. Changes in blood lactate and glucose were also similar between trials (Table 4). Serum CK measured 24 h after the constant-load exercise bout was higher compared with baseline (main effect, P < 0.05), but there was no difference between treatments (Fig. 2).
Muscle glycogen decreased during exercise (main effects, P < 0.05), but there was no difference between the CHO and the CHO + PRO trials (Fig. 3). The TCAI citrate and malate increased during exercise, but there was no difference between trials in their either individual or sum concentration (Fig. 4). Similarly, there were no treatment effects for muscle ATP, phosphocreatine, creatine, or lactate (Table 4).
There was no difference between treatments in creatinine (CHO + PRO = 1.2 ± 0.6 vs CHO = 1.3 ± 0.6 g·24 h−1) or urea (CHO + PRO = 10.5 ± 1.3 vs CHO = 12.5 ± 1.6 g·24 h−1).
Time trial performance.
Time to complete the 20-km time trial was not different between conditions (Fig. 5). Average HR (CHO + PRO = 157 ± 3 vs CHO = 158 ± 3 beats·min−1) and mean power output (CHO + PRO = 286 ± 24 vs CHO = 284 ± 22 W) were also similar between treatments.
FIGURE 5-Individual ...Image Tools
The major novel finding from the present study was that coingestion of PRO with CHO during 90 min of cycling at 69% ± 1% V˙O2peak did not alter selected markers of skeletal muscle metabolic control compared with CHO alone. We also found no effect of the nutritional intervention on the activity of CK measured in venous blood or next-day time trial performance. The relative concentration of CHO and PRO in the drinks used in the present study was similar to previous studies that have reported CHO + PRO ingestion improves endurance capacity compared with CHO alone (14,25,26). However, the overall rate of energy intake in the present study was higher because of this greater volume of fluid ingested because our goal was to ensure that CHO intake was on the upper end of the range commonly recommended by sports nutrition experts (1). We also demonstrated that the blood concentration of specific amino acids was increased after CHO + PRO ingestion compared with CHO, which is presumably important for the intervention to induce a metabolic effect in skeletal muscle or other tissues (14).
Many studies have reported that CHO + PRO ingestion during exercise improves cycle endurance capacity compared with CHO alone (14,25,26). For example, Ivy et al. (14) reported that time to exhaustion during cycling at 85% V˙O2peak was increased by 36% when trained cyclists ingested approximately 2% PRO with approximately 8% CHO (at a rate of ∼600 mL·h−1) during a standardized 3-h variable intensity cycling bout performed immediately before the timed ride to fatigue. Similar performance improvements were reported by Saunders et al. (25,26), who showed that trained cyclists rode longer to exhaustion at an intensity of 75% V˙O2peak when they ingested approximately 2% PRO with approximately 7% CHO compared with CHO alone (also at a rate of ∼600 mL·h−1). Various theories have been advanced to explain these observations (14,25,26), and the present study specifically addressed two mechanisms originally proposed by Ivy et al. (14). Specifically, these authors (14) postulated that the improved performance after CHO + PRO ingestion "could be related to the sparing or more efficient use of muscle glycogen (or) anaplerotic reactions and the retention of Krebs cycle intermediates."
The precise manner by which PRO ingestion during exercise could attenuate the rate of muscle glycogen use has not been clearly elucidated. However, the general theory (14) is based on the findings from some studies that showed PRO ingestion with CHO after prolonged moderate-intensity exercise augmented insulin secretion (3,4,15,33) and rate of muscle glycogen resynthesis (2,13,33). In fact, the potential for PRO to influence postexercise glycogen resynthesis is an equivocal topic and appears to depend on the rate and the amount of CHO ingested (7). As summarized by Burke et al. (7), "Most evidence suggests that feeding a high amount of CHO at frequent intervals negates the benefits of added PRO … (but) coingestion of PRO with CHO will increase the efficiency of muscle glycogen storage when the amount of CHO ingested is below the threshold for maximal glycogen synthesis." Irrespective of the data on recovery from exercise, there is little evidence to suggest that ingesting PRO with CHO during prolonged exercise alters blood insulin concentration compared with CHO alone, and the present results are consistent with other recent studies in this regard (14,29). Indeed, as recognized by Ivy et al. (14) in their original study, an elevation in blood insulin represents a questionable hypothesis to explain the purported glycogen sparing effect of CHO + PRO ingestion. The present study is the first to address this topic directly, and we found no difference in the rate of muscle glycogen use during exercise at an intensity of 69% ± 1% V˙O2peak when subjects ingested CHO or CHO + PRO.
A second mechanism proposed to explain the finding of longer time to exhaustion after CHO + PRO ingestion compared with CHO alone relates to changes in the skeletal muscle pool of TCAI (14). Several investigators have proposed that the increase in muscle TCAI is crucial to achieve high rates of mitochondrial respiration during exercise (24,30), and a common interpretation is that a given concentration of TCAI is required to sustain a given rate of oxidative phosphorylation during exercise (14). However, as recently reviewed by Bowtell et al. (5), a substantial body of evidence from both human and animal studies conducted in several laboratories has questioned this theory from a basic science perspective. For example, we (9) combined measurements of muscle TCAI, amino acids, and key energy metabolites with direct measurements of limb oxygen uptake during a 90-min bout of moderate-intensity exercise. After a threefold expansion during the initial minutes of exercise, the muscle TCAI pool rapidly declined such that the value after 60 and 90 min was not different from the resting concentration. Despite the decline in muscle TCAI, the capacity for aerobic energy provision was not compromised, as evidenced by stable limb oxygen uptake during exercise and no change in muscle phosphocreatine content, which is a sensitive indicator of mitochondrial respiration.
In the present study, we found no difference between the CHO and the CHO + PRO treatments on the total concentration of malate and citrate, which account for approximately 70% of the muscle TCAI pool in humans (9), or the rate of muscle phosphocreatine degradation. Paradoxically, according to the theory originally proposed by Wagenmakers et al. (30), one would predict that an increased rate of PRO oxidation during exercise (i.e., secondary to PRO ingestion) could potentially impair aerobic energy provision by reducing the muscle concentration of TCAI. Regardless, a reasonable interpretation of the available literature is that changes in muscle TCAI are not causally related to the capacity for aerobic energy provision in human skeletal muscle (5).
In addition to our primary focus on skeletal muscle metabolism, we also measured markers of skeletal muscle recovery approximately 24 h after the first bout. Several studies have reported that PRO ingestion during prolonged exercise attenuates the postexercise rise in CK (17,25,26,28), but our data are consistent with other reports that have failed to show a difference in this regard (11,19). Despite the widespread use of these markers (11,12,17,19,23,25,26,28), blood levels of myofiber enzymes correlate poorly with changes in muscle function, and many investigators have recommended deemphasis on the use of these methods to quantify the magnitude and the time course of muscle injury (31). Future research that incorporates other techniques including functional measurements, noninvasive imaging, and direct muscle sampling to evaluate histological changes may clarify our understanding of the potential for PRO ingestion to attenuate muscle disruption after exercise. We also found no treatment effect on our other marker of skeletal muscle recovery, the 20-km time trial performance. These data are in contrast to a study that showed improved time to exhaustion at 85% V˙O2peak 12 to 15 h after a bout of exercise when subjects ingested CHO + PRO compared with CHO alone but consistent with recent data from Betts et al. (4), who found no difference between the CHO and the CHO + PRO drinks ingested during recovery on subsequent run performance.
In summary, the present study found that, as compared with CHO alone, coingestion of PRO with CHO during cycling exercise at 69% ± 1% V˙O2peak did not alter the magnitude of muscle glycogen or phosphocreatine use, the net expansion of the tricarboxylic acid cycle pool, the blood CK activity, or the next-day time trial performance. These data therefore suggest that when trained men ingest CHO at a rate on the upper end of the range generally recommended to improve endurance performance, coingestion of PRO does not alter specific markers proposed to reflect an enhanced capacity for skeletal muscle energy delivery. PRO ingestion during exercise has been reported to induce other acute changes that could facilitate exercise capacity, including reduced muscle proteolysis (18) and improved fluid retention (27). It is also possible that the potential metabolic and performance effects of PRO ingestion are influenced by relative exercise intensity, and this could explain some of the equivocal findings in the literature. Regardless, additional work is warranted to establish a viable mechanism to explain the finding by some authors that adding PRO to a CHO-based sport drink improved acute endurance performance.
The authors thank the participants for their time and effort and Todd Prior and Tracy Rerecich for their technical assistance. This project was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) to MG. NC held an NSERC doctoral scholarship (PGS-D), and AS was the recipient of an NSERC Undergraduate Student Research Award. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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