The ingestion of carbohydrate–salt solutions during prolonged exercise can improve performance (14). Recently, interest in the role of protein coingestion with carbohydrate has led to the findings of increased endurance in blind end point time-to-exhaustion cycling tests (10,12,27,28) but equivocal outcomes in known end point time trials (6,18,22,29,32,35,36). Although sample size remains limited, Stearns et al. (32) in a recent meta-analysis linked the improvement in time to exhaustion with protein coingestion to a possible central mechanism. Other possible mechanisms have been suggested including elevated circulating insulin, increasing glucose uptake and carbohydrate metabolism (1,2,12), and additional energy (32).
Furthermore, there is some evidence from animal studies (8,38) and one human study (26) that increased dietary amino acid concentrations in the small intestine may increase fluid, electrolyte, and glucose absorption, which may support some of the mechanisms associated with the independent effects of fluid and carbohydrate on performance (7). Although amino acids may stimulate additional fluid and glucose absorption by activating supplementary cotransporters (16), the additional calories (20) and formation of casein clots in the stomach from native proteins (5)—although possibly of only minor consequence at low concentration (e.g., 2%)—could, on the other hand, slow gastric emptying, retarding the delivery of coingested carbohydrate and increasing the risk of gastrointestinal distress that could offset any benefit of the beverage (21).
Therefore, we asked whether protein coingestion could increase exogenous carbohydrate delivery as measured by exogenous glucose oxidation rate and if there was a negative effect on gut comfort or lowering of perceived exertion. Although we recognize that higher ingestion rates are probably optimal for performance, we provided exogenous carbohydrate at 12 g·15 min−1 (0.8 g·min−1), which is below the saturation rate for exogenous glucose oxidation of ∼1.0–1.1 g·min−1 (14), to prevent intestinal–epithelial site saturation from obscuring possible effects of an interaction between increased luminal amino acid concentration and glucose absorption. Therefore, delivery below intestinal saturation provided an unobtrusive design to provide evidence for or against a substantial effect of protein coingestion on exogenous carbohydrate metabolism.
MATERIALS AND METHODS
Eight male cyclists and triathletes aged (mean ± SD) 34 ± 7 yr and weighing 83 ± 6 kg completed the study. Maximal oxygen uptake (V˙O2max) and power (Wmax) were 4.7 ± 0.3 L·min−1 and 354 ± 24 W. Cyclists were screened for contraindications assessed by a general health questionnaire and provided written consent to participate in the study approved by the Massey University Human Ethics Committee.
The design was a double-blind, randomized, crossover one used to compare the effect of adding protein to a glucose solution on exogenous glucose and endogenous carbohydrate and fat oxidation rates, plasma substrate, gut comfort, and perceived exertion. Each cyclist made four visits to the laboratory on a weekly basis: visit 1 consisted of an incremental test to volitional exhaustion and visits 2 to 4 comprised a 150-min steady-state ride ingesting the experimental solutions. To reduce endogenous 13C background, participants were asked to omit foods with components derived from plants with a C4 photosynthetic cycle, which are naturally high in 13C (maize, sugar cane, or sugar beet) for at least 7 d before the first experimental trial to reduce background 13C-enrichment. Participants recorded their diet and training during week 1 and were asked to replicate it on a daily basis for the following weeks to minimize effects on outcome measures. For a given participant, all laboratory tests were conducted at the same time of the day beginning between 6:00 and 8:00 a.m. after an overnight fast. No strenuous activity or alcohol consumption was undertaken for 24 h before a laboratory test.
Incremental test and familiarization.
V˙O2max and Wmax were measured using a progressive exercise protocol on an electronically braked cycle ergometer (VeloTron Racer Mate, Seattle, WA) as described elsewhere (33). V˙O2max was measured online (SensorMedics Vmax; SensorMedics Corp., Yorba Linda, CA) and taken as the highest attained 20-s average oxygen uptake. After the incremental test, participants cycled for another 15–20 min during which time they were familiarized with the psychometric scales used to rate gastrointestinal distress and ratings of relative perceived exertion during the experimental trials.
On reporting to the laboratory, participants were asked to urinate/defecate before a 20-gauge catheter was inserted into an antecubital vein. A two-way stopcock valve was connected to the cannula to allow for serial blood sampling at rest and during exercise. Participants cycled at 50% Wmax for 150 min on the Velotron ergometer. Beginning 3.5 min before each 15-min point of exercise, samples were collected in the following order: psychometric parameters, expired gas (∼90-s volume into a Douglas bag) for the calculation of oxygen consumption and carbon dioxide production rates, breath drawn into evacuated tubes (Exetainer, Labco Ltd., High Wycombe, UK) from a 5-L mixing chamber (connected in series between the participant and Douglas bag) for 13C-enrichment and subsequent calculation of exogenous glucose oxidation rate (see below), and, finally, blood from 15 min and, subsequently, every 30 min after 60 min until the end of exercise. During all rides environmental conditions were maintained at 21°C–22°C by air conditioning, and participants were cooled by airflow from a fan.
The experimental solutions were protein plus glucose (protein–glucose), glucose only (glucose), and noncaloric placebo (water). Immediately before exercise and every 15 min throughout shortly after sample collection, cyclists ingested 150 mL of test solution. In each trial, a total of 1.5 L of solution (600 mL·h−1) were ingested. Each 150-mL bolus of the glucose solution contained 12 g (ingestion rate: 0.8 g·min−1; 266 mmol·L−1) glucose (Bronson and Jacobs Pty. Ltd., NSW, Australia), 0.59 g of sodium citrate (Sigma-Aldrich Inc., St. Louis, MO; [Na+] 60 mmol·L−1), 15 μL of orange flavor (U24096; Danisco Ltd., Wellingborough Northants, UK), and filtered water. The noncaloric placebo control solution contained all the ingredients of the glucose solution except for glucose. The protein–glucose solution contained the same ingredients as the glucose plus 2% (3 g) milk protein concentrate (MPC 70; Fonterra, Auckland, New Zealand). Solution osmolalities (mOsm·kg−1) were 41 (water), 469 (glucose), and 516 (protein–glucose).
The glucose powder was maize derived and further enriched with the addition of 0.040 g of [U-13C] glucose (D-glucose U-13C6, 99%; Cambridge Isotope Laboratories, Inc., Andover, MA) per 1 L of solution. Samples of these solutions were retained and later dehydrated in a slow oven (40°C). The 13C enrichment of the glucose powder was 34.9 ± 1.2 δ‰ versus Pee Dee Bellemnitella (PDB) determined by elemental analysis (Dumas, Europa Scientific ANCA-SL Crewe, UK) stable isotope ratio mass spectrometry (Europa Scientific 20-20 Stable Isotope Analyser, Crewe, UK).
Perceived exertion (effort, muscle tiredness) and gastrointestinal comfort markers (nausea, fullness, abdominal cramps) were measured using Likert scales: 0, nothing; 2, mild; 4, moderate; 6, high; 8, very high; 10, maximum (21,25,33). Participants made a pen mark on the scale according to the strength of their exertion or comfort. The numerical value for each verbal anchor was not displayed on the scale charts so as not to distract the participants from their qualitative rating.
Five milliliters of blood was drawn for analysis, transferred into prechilled lithium heparin vacutainers (Becton Dickinson and Co., Franklin Lakes, NJ), and then centrifuged at 2000g, 4°C for 12 min, with the resulting plasma aspirated into Eppendorf tubes and stored at −80°C until electrochemical analysis of plasma lactate and glucose concentrations using an automated analyzer (Bayer Rapidlab 800; Bayer HealthCare LLC, Tarrytown, NY).
The fractions of oxygen and carbon dioxide of expired gas collected into Douglas bags were measured in calibrated analyzers (SensorMedics Corp.). Expired gas volume was measured using PowerLab 4/20 spirometer (ADInstruments, Sydney, Australia). Breath samples collected into the evacuated tubes were analyzed for the 13C/12C ratio by gas chromatography continuous flow isotope ratio mass spectrometry (Finnigan Delta XP, Bremen, Germany).
Total fat and carbohydrate oxidation rates (g·min−1) were calculated using the equations of Jeukendrup and Wallis (13): carbohydrate oxidation (g·min−1) = 4.210 V˙CO2 – 2.962 V˙O2; fat oxidation (g·min−1) = 1.695 V˙O2 – 1.701 V˙CO2. Oxidation rates (g·min−1) of exogenous glucose were calculated from breath and ingested carbohydrate 13C enrichments and indirect calorimetry. Isotopic enrichment of expired breath was the δ‰ difference between the 13C/12C ratio of the sample and the reference standard (Vienna Pee Dee Belemnite; V-PDB) according to the formula: δ13C = [(13C/12C ratio sample/13C/12C ratio standard) − 1] × 103 ‰, where, 13C/12C standard = 0.011237 (9). The rate of exogenous glucose oxidized (g·min−1) was equal to V˙CO2 [(δExp − δbkg)/(δIng − δbkg)] / k, where δbkg is the 13C enrichment of the CO2 in expired breath in the background (water) trial, δExp is the 13C enrichment of expired CO2 during the 2.5-h rides with 13C-enriched carbohydrate ingestion, δIng is the 13C enrichment of the carbohydrate, and k is the amount of CO2 (L) produced via oxidation of 1 g of glucose (k = 0.7467 L CO2·g−1 glucose).
Calculation of exogenous substrate oxidation rate is affected by the delayed equilibration of 13CO2 with the large endogenous HCO3− pool (24). However, during exercise, CO2 turnover increased 7- to 10-fold from rest expediting the flux through the bicarbonate pool and time course to reach the tissue to expired air CO2 steady-state. Under these conditions, recovery of 13CO2 from oxidation of 13C-enriched carbohydrate will approach 100% after 60 min (23). As a consequence, the main outcome measures for substrate oxidation were calculated from 60 to 150 min of exercise.
The effects of protein on blood parameters, psychometric outcomes, and substrate oxidation during exercise were estimated with mixed modeling (Proc Mixed, SAS Version 9.1; SAS Institute, Cary, NC). With the exception of psychometric outcomes, all metabolic variables were log-transformed before modeling to reduce nonuniformity of error and to express outcomes as percent (11). From examination of means, all data exhibited linear trends over time, so linear modeling was chosen as the method to estimate overall and treatment effects over time. Time as a linear numeric effect was interacted with treatment to model a different rate of change of the dependent variable for each of the three levels of treatment. The baseline score was included as a covariate adjustment for the analysis of the psychometric data. Outcomes were determined from 0 to 150 min for psychometric data, from 15 to 150 min for blood, and from 60 for 150 min for substrate oxidation. The random effect was subject identity. Inferences about the population values of statistics were made via magnitude-based inference (11) using the method described recently (25). Uncertainty was presented as 90% confidence limits (CLs).
Stable isotope enrichment.
The mean ± SD breath 13CO2 enrichment (δ‰ vs PDB) during the 60-to 150-min period of exercise was −15.8 ± 1.2, −16.8 ± 1.4, and −26.6 ± 0.1 with the protein–glucose, glucose, and water solutions, respectively (Fig. 1). Enrichment was moderately increased by 1.0 δ‰ versus PDB (±90% CL ±0.6 δ‰ vs PDB) and 10.4 δ‰ versus PDB (±0.6 δ‰ vs PDB) with the protein–glucose solution, relative to the glucose and water solutions, respectively. Enrichment in the glucose control was 9.4 ± 0.6 δ‰ versus PDB higher than water.
Substrate oxidation rates are shown in Figure 2, with a summary of mean effects presented in Table 1. The addition of protein had trivial effect on the exogenous glucose oxidation rate but led to a small increase in endogenous carbohydrate oxidation rate relative to the glucose control solution and a small increase in total carbohydrate oxidation compared with water. Protein coingestion resulted in a small reduction in fat oxidation relative to water, but the difference relative to glucose was trivial. Consumption of the glucose solution led to a small increase in total carbohydrate oxidation rate, coupled with small decreases in endogenous carbohydrate and fat oxidation rates, when compared with water (Table 1).
Plasma glucose and lactate.
Plasma glucose and lactate concentrations during exercise are shown in Figure 3. Relative to water (15–150 min mean ± SD concentration: 5.2 ± 0.5 mmol·L−1), moderate and large increases in plasma glucose concentration were observed in the protein–glucose (9.2%; 90% CL ±3.4%) and glucose (15.9% ± 3.6%) solutions, respectively. Relative to glucose (6.1 ± 0.8 mmol·L−1), protein–glucose (5.6 ± 0.5 mmol·L−1) caused a small reduction in plasma glucose concentration (−5.8% ± 3%). There were no clear differences in lactate or glucose concentrations for the other comparisons.
Perceptions of physical exertion (perceived exertion, muscle tiredness) and gastrointestinal distress (nausea, abdominal cramp, fullness) in response to solution composition are illustrated in Figure 4. With all solutions, physical exertion and leg muscle tiredness were within the mild to moderate range (Fig. 4), but the addition of protein had trivial effect on overall ratings relative to glucose and water.
Gastrointestinal comfort ratings ranged from nil to mild (Fig. 4). There was a small decrease in nausea detected with the protein–glucose solution relative to water (−0.14; 90% CL ±0.08 U); all other comparisons were unclear. A small reduction in abdominal cramps was observed with glucose compared with water (−0.19 ± 0.10 U); other comparisons were unclear or trivial. Finally, all comparisons for fullness were also trivial or inconclusive.
The purposes of this study were to determine whether the coingestion of protein with carbohydrate during endurance exercise could influence exogenous carbohydrate delivery as measured by end point oxidation and to explore if increased exogenous carbohydrate delivery or alterations in gut comfort and perceived exertion could provide a possible mechanism for improved endurance capacity reported previously with added protein (10,12,27,28), concurred by meta-analysis (32). We report that the addition of 2% protein offered no clear benefit, was not detriment to exogenous carbohydrate oxidation, and had no clear effect on gastrointestinal comfort or physical exertion compared to a solution containing glucose only.
The negligible effect of protein coingestion on the exogenous glucose oxidation rate suggests that it is unlikely to be the mechanism for improved endurance exercise capacity. Nevertheless, protein did have some effect on metabolism via lowering of plasma glucose concentration by 0.5 mmol·L−1 and increased endogenous carbohydrate oxidation rate by a small 0.15 g·min−1. These small changes in metabolism, although interesting, are unlikely to be large enough to affect endurance capacity. In one of the few studies to attempt to quantify the effect of exogenous carbohydrate ingestion dose and oxidation rate on endurance performance, Smith et al. (30) reported a likely substantial (magnitude-based inference) mean 2.3% and 3.1% increase in 20-km time trial performance after a preload with the ingestion of glucose at 60 g·h−1, relative to glucose ingested at 30 and 15 g·h−1. The exogenous glucose oxidation rate increased with ingestion rate (0.17, 0.33, and 0.52 g·min−1 for 15, 30, and 60 g·h−1 glucose), but endogenous carbohydrate oxidation rate was reduced only with 30 and 60 g·h−1 (small, 8%–14%) because of the progressive inhibition of glucose released from the liver without evidence for muscle glycogen sparing. Therefore, a very to extremely large 37%–67% increase in exogenous carbohydrate oxidation rate was required to observe a likely substantial increase in performance; the effects on endogenous substrate metabolism did not seem important. Others have noted no significant change in substrate oxidation when performance effects of protein coingestion have been positive (10,12,28), negative (34), or indifferent (18). Although more highly powered studies (greater measurement reliability and sample size) are required, together, these data suggest that large changes in metabolism are required to affect small changes in performance and that other physiological mechanisms may be responsible for ergogenic action of protein (e.g., central mechanism, placebo via inadequate blinding to solution composition).
The lower blood glucose concentration suggests either slower absorption of glucose from the gut or faster absorption of glucose from the plasma into tissues. Protein has been shown to have blood glucose–moderating and insulin-enhancing effects when coingested with carbohydrate relative to carbohydrate only at rest (31) and during exercise (15). We were unable to measure insulin concentration because of sample degradation, and without plasma glucose, turnover data can only speculate about the balance between splanchnic glucose appearance and peripheral glucose uptake, but the current small increase in endogenous carbohydrate oxidation is consistent with the effect of increased insulin concentration (17). During exercise, a reduced rate of glucose appearance into the circulation has also been suggested to explain the attenuated blood glucose concentration (34), but the similar exogenous glucose oxidation rate points against this explanation because intestinal absorption seems to limit the oxidation rate of ingested glucose during exercise (14).
A methodological point with respect to the metabolic outcomes was that we observed an increased 13C-breath enrichment with the protein–glucose solution suggesting enhanced oxidation rate. However, because of the small size of the difference in enrichment and the normal error associated with measurement of external respiration (the between-trial sample CV for V˙CO2 in the current study was 5.1%), this finding was not carried through to the exogenous carbohydrate oxidation rate. The error of measurement of the mass spectrometer was 0.02 δ‰ versus PDB. If we assume no error in V˙CO2, the mean difference in exogenous glucose oxidation rate between protein–glucose and glucose would have been 0.05 g·min−1, which is likely to be biologically trivial.
Protein coingestion had no substantial effect on gut comfort during exercise. Gut comfort has been formally evaluated in several recent studies as a potential mechanism covariate affecting high-intensity endurance performance in repeated-sprint models via intervention in carbohydrate solution composition (21,25,33). In previous works, we reported that small elevations in nausea associated with small differences in carbohydrate composition of a beverage were associated with small to moderate increases in performance (21,33), but there was no association between the small 0.1-g·min−1 increase in exogenous carbohydrate oxidation rate and performance (21). In the current study, nausea showed a small decrease with protein coingestion compared with the water placebo, but this was not the case compared to glucose alone. Therefore, the negligible effect of protein coingestion on both gut comfort and exogenous glucose oxidation rate provide further evidence to suggest protein coingestion is unlikely to be detrimental to performance, which is verified in the performance literature (6,10,12,18,22,27–29,32,35,36).
In the current study, protein consumption had no effect on measures of perceived exertion but delaying the onset of central fatigue has been proposed as a possible mechanism by which the coingestion of protein can be beneficial during exercise (2). During exercise, plasma branched-chain amino acids (BCAAs) decrease, causing a subsequent unloading of tryptophan from albumin as a result of the rise in plasma free fatty acids. Tryptophan and BCAAs compete for the same transporter across the blood–brain barrier, thus the change in tryptophan/BCAA ratio enhances brain uptake of tryptophan—a precursor to serotonin, which potentially lowers brain activity and causes central fatigue (2). Protein provides a dietary source of BCAAs, favorably altering the plasma ratio of free tryptophan/BCAA. Indeed, when BCAAs have been supplied to exercising humans, they have been shown to enhance endurance performance and reduce feelings of perceived exertion in both marathon runners and endurance cyclists (4,19); however, this finding is not universal, and the inclusion of BCAAs has also been found to not affect performance (2,37). Moreover, Blomstrand et al. (3) suggested that coingestion of carbohydrate during exercise can delay possible effects of BCAAs on fatigue, thus providing a possible explanation as to why the addition of protein did not reduce perceived exertion in the current and other studies (10,22).
To conclude, protein coingestion with glucose during exercise had a neutral effect on exogenous carbohydrate oxidation, gut comfort, and perceived exertion. Our data suggest that protein coingestion will not adversely affect gut comfort or perceived effort or reduce the rate of carbohydrate delivery to the muscle during prolonged endurance exercise, suggesting that previously reported effects of protein coingestion on endurance capacity were unlikely to be due to increased exogenous carbohydrate provision.
This study was funded by income obtained from services provided by the Exercise Physiology and Metabolism Laboratory, Massey University Wellington.
The authors thank Megan Thorburn and Rhys Thorp for laboratory assistance.
The authors declare no conflict of interest.
The results of the study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:©2012The American College of Sports Medicine
STABLE ISOTOPE; GLUCOSE OXIDATION; ENDURANCE EXERCISE; GUT COMFORT