International Journal of Sport Nutrition and Exercise Metabolism, 2014, 24, 188 -195 http://dx.doi.org/10.1123/ijsnem.2013-0136 © 2014 Human Kinetics, Inc.
www.IJSNEM-Journal.com ORIGINAL RESEARCH
Dose Response of Whey Protein Isolate in Addition to a Typical Mixed Meal on Blood Amino Acids and Hormonal Concentrations Scott C. Forbes, Linda McCargar, Paul Jelen, and Gordon J. Bell The purpose was to investigate the effects of a controlled typical 1-day diet supplemented with two different doses of whey protein isolate on blood amino acid profiles and hormonal concentrations following the final meal. Nine males (age: 29.6 ± 6.3 yrs) completed four conditions in random order: a control (C) condition of a typical mixed diet containing ~10% protein (0.8 g·kg–1), 65% carbohydrate, and 25% fat; a placebo (P) condition calorically matched with carbohydrate to the whey protein conditions; a low-dose condition of 0.8 grams of whey protein isolate per kilogram body mass per day (g·kg–1·d–1; W1) in addition to the typical mixed diet; or a high-dose condition of 1.6 g·kg–1·d–1 (W2) of supplemental whey protein in addition to the typical mixed diet. Following the final meal, significant (p < .05) increases in total amino acids, essential amino acids (EAA), branch-chained amino acids (BCAA), and leucine were observed in plasma with whey protein supplementation while no changes were observed in the control and placebo conditions. There was no significant group difference for glucose, insulin, testosterone, cortisol, or growth hormone. In conclusion, supplementing a typical daily food intake consisting of 0.8 g of protein·kg–1·d–1 with a whey protein isolate (an additional 0.8 or 1.6 g·kg–1·d–1) significantly elevated total amino acids, EAA, BCAA, and leucine but had no effect on glucose, insulin, testosterone, cortisol, or growth hormone following the final meal. Future acute and chronic supplementation research examining the physiological and health outcomes associated with elevated amino acid profiles is warranted. Keywords: nutrition, branch-chained amino acids, diet, leucine, essential amino acids, testosterone Protein is an important component of dietary food intake and is needed for growth and a variety of cellular processes (Wu, 2009). This is also underscored by the fact that malnutrition will occur if there is an inadequate dietary intake of protein even with adequate caloric intake (Grover & Ee, 2009; Phillips & Van Loon, 2011). The recommended daily allowance (RDA) for protein intake for sedentary adults is 0.8 g·kg–1.d–1 (Institute of Medicine, 2005). Physically active or athletic individuals are often recommended to ingest a larger amount of dietary protein than the RDA (1.2–1.7 g·kg–1.d–1; Lemon, 1998; Phillips, 2012; Rennie & Tipton, 2000; American Dietetic Association, 2009; Tarnopolsky, 2004). The greater dietary protein recommendations in active or athletic individuals may be associated with elevated exercise induced cellular processes, such as mitochondrial biogenesis, protein synthesis, and endocrine and immune Forbes and Bell are with the Faculty of Physical Education and Recreation, University of Alberta, Edmonton, Canada. McCargar and Jelen are with the Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada. Address author correspondence to Scott C. Forbes at [email protected].
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responses (Lemon, 1991). Furthermore, habitual daily protein intakes of resistance-trained individuals are often greater than 2.0 g·kg–1.d–1 and have been reported to be as high as 3.5 g·kg–1.d–1 (Phillips, 2004). To consume more dietary protein, individuals often use nutritional supplementation containing various amino acids and/ or proteins of which there are a myriad of commercially available products. Whey protein powder is one of these products, which has received considerable attention recently due to its unique properties (Burke et al., 2012). Whey protein is commercially added to certain foods and also available in powder form as a nutritional aid or supplement. Whey protein is a heterogeneous mixture of proteins containing all essential amino acids (EAA), and branch-chain amino acids (BCAA; e.g., leucine, isoleucine, and valine; Stark et al., 2012). Elevated extracellular aminoacidemia following whey protein ingestion is known to enhance skeletal muscle myofibrillar protein synthesis (Bohe et al., 2003; West et al., 2011) which is stimulated primarily by EAAs (Paddon-Jones et al., 2004; Tipton et al., 1999; Volpi et al., 2003) and by the BCAA leucine (Drummond and Rasmussen, 2008). Intravenous administration of BCAAs elevates the phosphorylation and activation of p70S6 kinase and 4E-BP1 (Greiwe et al., 2001); p70S6 kinase and 4E-BP1 which are down-
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stream substrates of the mammalian target of rapamycin (mTOR) pathway (Greiwe et al., 2001; Liu et al., 2006; Manders et al., 2012). Leucine supplementation alone has been shown to directly phosphorylate and activate mTOR (Fujita et al., 2007); however, alterations in the rates of myofibrillar protein synthesis are relatively transient unless EAA are provided in sufficient quantities (Manders et al., 2012). Furthermore, Calbet and MacLean (2002) found that whey protein produced a significant increase in circulating levels of insulin and this insulin response was greater than achieved with a carbohydrate solution. Thus, the physiological response to whey protein ingestion can be quite diverse. Because of this diversity, whey protein is currently added to enhance the quality of particular foods (e.g., yogurts, breads, and as a dietary supplement for active or athletic individuals). However, there is little research examining specific dosages of whey protein powder added to a typical mixed daily intake of food on blood amino acids (e.g., total amino acids, EAA, BCAA, and leucine), hormonal (e.g., insulin, testosterone, growth hormone, and cortisol) and glucose responses following the final meal of the day. Therefore, the primary purpose of this study was to determine the effects of two different doses of a whey protein isolate added to a controlled typical mixed one day diet, containing 10% of energy from protein, on the amino acid profiles following the final meal. Secondary purpose was to examine the hormonal and glucose concentrations in the blood after the final meal of the day with whey protein supplementation compared with a typical controlled diet at the RDA for protein. These doses were selected to represent the current sport nutrition guidelines (Lemon, 1998; Phillips, 2012; Rennie & Tipton, 2000; American Dietetic Association, 2009; Tarnopolsky 2004) a typical composition of athletes, and the current RDA (Institute of Medicine, 2005). It was hypothesized that supplementing a daily diet with a whey protein isolate would cause a greater increase in certain amino acids (e.g., essential amino acids, branched chain amino acids, and leucine) and may stimulate a greater release of particular hormones (e.g., insulin) in a dose dependent manner following the final meal of the day.
Methods Subjects Ten recreationally active male subjects were recruited from a university population. One subject withdrew from the study after the first testing session due to personal time commitment issues and the remaining 9 subjects completed all testing conditions.
Experimental Design Each subject attended an orientation session for an explanation of the study, scheduling and to sign an informed consent that was approved by a university research ethics
committee. At this time each subject was provided with a 3-day dietary record and shown how to complete it accurately. A second session was attended for measurements of height, body mass, resting metabolic rate and body composition. Please note that the experimental design of this study and a small portion of the methods have been previously published (Middleton et al., 2004) but there is no duplication in any of the dependent variables reported in this paper. The 3-day dietary records were entered into a computer software program (The Food Processor II for Windows, Nutritional Analysis Nutritional Software Version 6.11, Salem, OR) to determine the amount of carbohydrate, fat, protein and the number of calories consumed each meal and for each day. Following computer analysis, the nutritional intake of each subject was adjusted for the amount and/or proportion of food to balance the diet to obtain 0.8 g·kg–1.d–1 of mixed food protein (Institute of Medicine, 2005) at the same time as maintaining the total calories equal to the subject’s original diet record for each of the 3-days of the recording. This corresponded to a proportion of 10% protein, 65% carbohydrate and 25% fat. The modified records with the exact type and size of food items were photocopied and returned to each subject to ensure that they consumed this exact diet for the two days before and on the day of all 4 experimental conditions. There were 4 experimental conditions that were completed and all calculations were made based on each individual’s body mass: Control (C)—3 days of controlled dietary intake (2 days before and on the day of the experimental condition). The diet consisted of a typical mixed food intake modified to contain a proportion of 10% protein, 65% carbohydrate and 25% fat and total protein intake of 0.8 g·kg–1.d–1 from a nonwhey protein mixed food diet. Placebo (P)—identical dietary intake as in C condition except that a glucose polymer (Polycose) was added to a noncalorie 1 L flavored drink (Crystal-Lite) that was calorically matched to the extra calories added as a result of the highest whey protein supplementation condition (W2) described below. This was done so that the total caloric intake was controlled and matched to the extra calories from the highest protein supplementation condition to ensure that any observed effect was not due to an increase in total calories consumed. Total protein intake for this condition was 0.8 g·kg–1.d–1. Whey Protein Supplementation Dose 1 (W1)—same diet as the control condition plus 0.8 g·kg–1.d–1 of whey protein powder mixed in the same 1 L volume of noncalorie flavored drink as the placebo condition plus a smaller amount of glucose polymer to match the additional calories consumed with the highest whey protein supplementation condition (W2) described below. Again total caloric intake was matched between the placebo and the highest whey protein dose condition. Total protein intake for this condition was equal to 1.6 g·kg–1.d–1. Whey Protein Supplementation Dose 2 (W2)—same mixed diet as the control condition plus 1.6 g·kg–1.d–1
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of whey protein powder mixed in the same drink as the other conditions for a total protein intake equal to 2.4 g·kg–1.d–1. Each subject acted as his own control, thus all subjects participated in all the experimental conditions (C, P, W1 and W2) in a random, double-blind design. The daily dietary mixed protein intake (0.8 g·kg–1.d–1) and total calories consumed was based on foods that each subject preferred but was modified accordingly so that each subject had the same dietary composition leading up to and including the day of each experimental condition. The whey protein powder was dissolved in a noncaloric flavored drink in an attempt to mask the flavor and was contained in a 1 L colored opaque bottle to mask the appearance. The supplement drinks were consumed in equal portions with each meal during the day. Total volume of fluid (experimental drinks and water intake) was also the same for each condition. No formal exercise training (e.g., running or resistance training) was allowed on the days before and on the days of the experimental conditions but the subjects were allowed to resume their regular workday schedules.
Whey Protein Isolate The whey protein isolate chosen for this study was PowerPro Plus (Land O’ Lakes, St.Paul, MN, USA) and is produced through a proprietary process that includes ultra/microfiltration concentration of the native whey proteins in an attempt to keep each protein in its functional form. The constituents of this product are available from the manufacturer and are reported in our previous study (Middleton et al., 2004). Briefly, the amino acid profile for PowerPro Plus in grams of amino acids per 100 g of product: alanine = 4.6 g, arginine = 2.2 g, aspartic acid = 9.9 g, cysteine = 2.3 g, glutamic acid = 11.4 g, glutamine = 4.4 g, glycine = 1.7 g, histidine = 1.8 g, isoleucine = 5.8 g, leucine = 10.1 g, lysine = 8.5 g, methionine = 1.9 g, phenylalanine = 3.1 g, proline = 5.2 g, serine = 4.4 g, threonine = 6.0 g, tryptophan = 1.6 g, tyrosine = 2.8 g, and valine = 5.4 g.
Measurement of Resting Metabolic Rate (RMR) and Body Composition Assessment Each subject completed a RMR measurement between 7:30 and 8:30 a.m. after an overnight fast (12 hr) on a different day in a rested state. Once in the laboratory, each subject rested on a bed for 20 min followed immediately by a 30-min period of metabolic measurement of expired gases in a canopy designed for such measurements (MedGraphics CPXD, Medical Graphics Corp., Minneapolis, MN, USA). The room was quiet and in dim light to help each subject relax. Each subject was asked not to fall asleep. The metabolic cart was calibrated for volume of air, and known oxygen and carbon dioxide concentrations before and after each RMR measurement. Body composition was assessed using the hydrostatic
weighing procedure in a fasted state immediately after the RMR measurement as previously reported for our laboratory (Biondo et al., 2008). Custom designed software performed all standard calculations and body fat was predicted mathematically according to the formula of Brozek (1963).
Blood Samples Blood (approximately 10 ml per sample) was obtained using a venipuncture from an anticubutal vein or via a cathlon inserted into a forearm vein. Each subject came to the laboratory in the morning (7:30 a.m. to 8:30 a.m.) after an overnight fast and rested while the first blood sample was taken using venipuncture and collected into a plain vacutainer. The blood sample was put on ice until it clotted (~30 min), centrifuged at 1500 xg for 10-min, and 3 equal volumes of the serum were pipetted into separate 1.5-mL microcentrifuge tubes and frozen at –80 °C until analyses. Following the resting sample, the subjects ate their prescribed breakfast, morning snack, lunch, afternoon snack and final meal at their usual times with (P, W1, W2) or without (C) the experimental drink depending on the randomized condition. Each subject consumed the experimental drinks in 3 equal portions at the 3 major prescribed meal times (breakfast, lunch and final meal). The subjects returned to the laboratory before the final meal of the day to have a cathlon placed in a forearm vein by a registered nurse. The cathlon was attached to a three-way stopcock and kept patent with 0.9% sterile saline solution. Blood was drawn in the fasted state (i.e., morning), immediately before the final meal of the day and 30, 60, 120, 180, and 240 min after the final meal. All blood samples were prepared and frozen in the same manner. The subjects were required to remain in the laboratory for the final meal and postmeal blood sampling periods.
Amino Acid Analysis Each amino acid concentration was determined individually in all blood samples in duplicate using a protocol previously published for our laboratory (Sedgwick et al., 1991). The protocol established 19 amino acids that were categorized based on their level of dispensability: dispensable (alanine, aspartate, asparagine, glutamate, serine); conditionally indispensable (arginine, cysteine, glutamine, glycine, tyrosine); and indispensable (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine; Pencharz and Ball, 2003). In addition, taurine, and ornithine were analyzed for a total of 21 amino acids. All the amino acids analyzed (except taurine and ornithine) were present in the whey protein isolate used in this study. The detailed procedures of the amino acids using high pressure liquid chromatography have been previously described by Sedgwick et al. (1991). Briefly, the
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serum blood sample was deproteinized by mixing 25 μL of serum with 50 μL of EA/BABA (Ethanolamine and β-amino-butyric acid; 25 nmol/mL) followed by the addition of 100 μL of 5% wt·vol-1 TCA (trichloroacetic acid) and centrifuged at 1500 xg for 15-min. Subsequently, 100 μL of the deproteinized supernatant was mixed with 150 μL of saturated K2B4O7 to adjust pH to ~9.0 and then 100 μL of HPLC grade H2O was added. One hundred μL of the final supernatant was pipetted into a 1.5 μL vial add added to the autosampler rack of the high performance liquid chromatography (HPLC) machine. A 100 uM amino acid standard solution for each amino acid was prepared and the amino acid samples were then transformed using the o-phthaldialdehyde (OPA) method reported by Jones and Gilligan (1983). Individual amino acid was determined using a Varian 5000 high performance liquid chromatograph and fluorichrom detector. Chromatographic peaks were recorded integrated using a chromatography data system (Hewlett Packard 3353, Mississauga, ON, Canada). The amino acid concentrations were determined by comparison with the proteinfree standard solutions prepared in an identical manner.
Blood Glucose and Hormonal Analysis Glucose and hormonal concentrations were measured in duplicate at every time point. Glucose was analyzed in serum using the glucose oxidase method (Sigma Aldrich, 2004). Insulin, cortisol, growth hormone, and testosterone concentrations were determined with radioimmunoassay, using commercial assay kits (Diagnostic Products Corporation, Los Angeles, CA, USA).
Statistical Analysis A two-way ANOVA with repeated measures was used to compare the four experimental trials (C, P, W1 and W2) and seven sampling time points for all dependent variables (total amino acids, EAA, BCAA, leucine, insulin, glucose, testosterone, growth hormone, and cortisol). Area under the curve (AUC) was calculated using Prism software (Graphpad 4, San Diego, CA, USA) and one-way repeated-measures ANOVA was used to assess the AUC between conditions. Significant F ratios were further analyzed with a Tukey’s pairwise comparison. Statistical analyses were carried out using Statistica, version 10 (StatsSoft Inc., Tulsa, OK, USA). All results are expressed as means ± SD. Statistical significance was set at p ≤ .05.
Results The mean age, body weight, height, percent body fat and resting metabolic rate (RMR) were 29.6 ± 6.3 yrs, 81.2 ± 12.6 kg, 180.1 ± 7.7 cm, 19.6 ± 6.5%, and 1677 ± 197.8 kcals·d–1, respectively. There were no observed or reported side effects with any condition. Resting fasting values for all the dependent variables (total amino acids, EAA, BCAA, leucine, insulin, glucose, testosterone,
growth hormone, and cortisol) were not significantly (p > .05) different between conditions.
Blood Amino Acid Profiles Total blood amino acid concentrations demonstrated an interaction (treatment by time) effect (p < .0001) with W1 and W2 being elevated above the placebo and control conditions at 60 and 120 min following the final meal. In addition, W2 was elevated above placebo and control conditions at the 30 and 180 min time points; however, there were no differences between the two whey protein conditions. Blood EAAs showed a similar interaction (treatment by time) effect (p = .00001; Figure 1). Compared with the placebo and control, W2 was significantly elevated at 30, 60, 120, 180, and 240 min after the final meal, while the lower dose of whey protein (W1) was only elevated at the 60 and 120 min time points. Similarly, there were no differences between the two whey protein supplement conditions. Blood BCAA were significantly (p < .00001) higher 30, 60, 120, 180 min time points during the W2 condition and higher at 60 and 120 min time points during the W1 condition compared with the placebo and control. As shown in Figure 2, leucine was significantly (p < .0001) elevated at 30, 60, 120, and 180 min time points during the W2 condition and elevated at 60 and 120 min time points during the W1 condition compared with the placebo and control trials. There were no differences in leucine between W2 and W1 at any time points. The AUC for total amino acids (, EAA, and BCAA revealed a significant (p < .0001) difference between W1 and W2 compared with the placebo and control conditions. The AUC for leucine revealed greater values for the whey protein doses (W1 and W2); however, W1 was similar to the placebo condition and different than the control condition.
Blood Glucose and Hormones There was a main effect of sampling time for serum glucose (p = .002) concentration revealing a significant increase in serum glucose 30 min after the final meal of the day that was higher than all other time points. There was no significant difference in serum glucose response between conditions. Similarly, serum insulin increased (p < .0001) at the 30, 60, 120, and 180 min after the final meal compared with the fasting sample; however no differences were observed between conditions (Figure 3A). There was a main effect of sampling time for serum testosterone (p < .0001) and cortisol (p < .0001) concentrations revealing significantly higher serum concentrations during the fasted state compared with all other time points, while there were no differences between conditions (Figures 3B and 3C). Growth hormone showed an interaction (treatment by time; p = .03) effect; however, post hoc analysis revealed no significant differences at any time point between all four
Figure 1 — Blood essential amino acid concentrations in each experimental condition and over the different sampling times. Values are means ± SEM a = W2 significantly higher than placebo and control conditions. b = W2 and W1 significantly higher than placebo and control conditions. W2 = whey protein supplementation dose of 1.6 g·kg–1.d–1, W1 = whey protein supplementation dose of 0.8 g·kg–1.d–1.
Figure 2 — Blood leucine concentrations in each experimental condition and over the different sampling times. Values are means ± SEM a = W2 significantly higher than placebo and control conditions. b = W2 and W1 significantly higher than placebo and control conditions. c = W2 significantly different than placebo and control and W1 is different than control but similar to placebo. W2 = whey protein supplementation dose of 1.6 g·kg–1.d–1, W1 = whey protein supplementation dose of 0.8 g·kg–1.d–1.
Figure 3 — Mean ± SEM. Serum (A) insulin (B) testosterone (C) cortisol (D) growth hormone in each experimental condition and over the different sampling times. * = significantly different than all other time points. # = significantly different than fasting.
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conditions (Figure 3D). The AUC was similar between all four conditions for glucose, insulin, testosterone, cortisol, and growth hormone (p > .05).
Discussion The main finding of the current study was that a one day meal plan that contained a mixed dietary intake of 0.8 g·kg–1.d–1 of protein supplemented with whey protein isolate in a low (0.8 g·kg–1.d–1; total of 1.6 g·kg–1.d–1 of protein) or a high dose (1.6 g·kg–1.d–1; total 2.4 g·kg– 1.d–1 of protein) increased total amino acids, EAAs, BCAAs, and leucine concentration in the blood after the final meal of the day and this response was significantly higher than the control and placebo conditions. Despite the elevation in amino acid concentrations there were no differences between conditions for insulin, glucose, testosterone, growth hormone, or cortisol at any time of the day in any experimental condition. The measurement of amino acids in the blood after standardized meals with various doses of whey protein supplementation is one method of determining the absorption and extracellular availability. The extracellular availability of amino acids, facilitated by blood flow, has been shown to increase protein synthesis and decrease protein degradation (Biolo et al., 1995; Bohe et al., 2003) leading to an enhanced net protein balance. Volek et al. (2013) provides evidence that humans fed protein (e.g., whey protein) containing a higher proportion of leucine will result in an elevation of blood leucine concentration and stimulate muscle protein synthesis and this may lead to greater gains in muscle mass over time; however, others do not support this linear association between elevated leucine and enhanced muscle protein synthesis (Churchward-Venne et al., 2012). In addition, changes in muscle protein synthesis are relatively small unless sufficient EAA are provided (Manders et al., 2012). The present study found a significant increase in both EAA and leucine following whey protein supplementation compared with a typical meal. However, the AUC for leucine following the final meal was only significantly different than placebo following the high whey protein dose (1.6 g·kg-1.d-1). This would suggest that the higher whey protein dose condition may be ideal for maximizing leucine uptake but future research is necessary to determine if this elevation in leucine concentration could lead to elevations in muscle protein synthesis and body composition changes over time. Furthermore, diet was only controlled for 3 days (2 days before and including the experimentation day) for each condition, this time period may not have been sufficient to allow for a new metabolic equilibrium, which may require between 5–7 days (Jeacocke and Burke, 2010). Therefore, the current study may represent a transitory period; future research examining longer dietary controls may be required to examine protein kinetics with physiological adaptations (e.g., protein oxidation, muscle protein synthesis, and muscle protein breakdown). Recently, Moore et al. (2012)
examined the effects of daytime patterns of dietary protein intake on whole-body protein turnover in resistance trained males and found that ~20 g at regular intervals (~3 hr) throughout the day achieved the highest net protein balance. This may suggest that not only the total daily amount of protein is important but also the timing pattern of protein ingestion throughout a day. Interestingly, the current study found only minor differences between the low and high dose of whey protein supplementation. This may have been associated with a reduced absorption rate with higher protein ingestion due to competitive inhibition of the transport system that moves amino acids into peripheral blood (Adibi & Gray, 1967). In addition, it has been suggested that the digestion of a large quantity of protein may extend beyond a 4 hr following a meal (Adibi & Mercer, 1973), which was a limitation of the current study. It is also important to note that the current study only examined the amino acids profiles at rest, immediately before and following the final meal of the day. Regardless, the current study was able to exhibit a significant increase in circulating amino acids with whey protein supplementation. Despite this increase in amino acids, there were no differences between conditions on any of the measured hormones. It has been shown that several amino acids (e.g., arginine) can stimulate the secretion of insulin and growth hormone (van Loon et al., 2000; Collier et al., 2005) and it has been suggested that the BCAA may influence the time course of hormonal release (Calbet & MacLean, 2002). Whey protein ingestion (30 g·d–1) has been shown to increase plasma insulin and this response was greater than that achieved with an amino acid or casein meal (Dangin et al., 2001). Calbet and MacLean (2002) also found a greater increase in plasma insulin with ingestion of a whey protein solution when compared with milk, a pea peptide solution or a glucose solution. The physiological consequence of an increased circulating concentration of insulin is to promote cellular glucose and amino acid uptake and to assist in the regulation of protein synthesis within the cell (Graham et al., 1995). As well, diets high in protein (30% protein including milk protein) have been shown to lower postprandial blood glucose levels and modestly increase serum insulin concentration after the final meal of the day in people with Type 2 diabetes (Gannon et al., 2003). The present study in healthy adults does not support these latter studies as we observed no significant difference in insulin or glucose following whey protein supplementation (0.8 g·kg-1.d-1 or 1.6 g·kg-1.d-1) compared with control or placebo conditions.
Conclusion Supplementation of a daily meal plan with whey protein produced an overall increase in the total amino acid, EAA, BCAA, and leucine concentrations following the final meal; however, there were no differences between
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the high and low whey protein dose conditions. Thus, supplementation with whey protein throughout the day with a controlled diet was effective at elevating amino acid concentrations in the blood following the final meal. In addition, the current study was unable to demonstrate any differences in insulin, glucose, testosterone, cortisol, or growth hormone after the final meal of the day with the whey protein powder. Further research should focus on the biological responses (e.g., muscle protein synthesis) of whey protein supplementation and longer-term supplementation with controlled dietary experimentation. Acknowledgments The authors would like to thank Ian MacLean, Neil Middleton, Gary Sedgewick, Stephen Cheetham, and Robert Kell for their assistance with this study.
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Phillips, S.M., & Van Loon, L.J. (2011). Dietary protein for athletes: from requirements to optimum adaptation. Journal of Sports Sciences, 29, S29–S38. PubMed doi:10.10 80/02640414.2011.619204 Rennie, M.J., & Tipton, K.D. (2000). Protein and amino acid metabolism during and after exercise and the effects of nutrition. Annual Review of Nutrition, 20, 457–483. PubMed doi:10.1146/annurev.nutr.20.1.457 Sedgwick, G.W., Fenton, T.W., & Thompson, J.R. (1991). Effect of protein precipitating agents on the recovery of plasma free amino acids. Canadian Journal of Animal Science, 71, 953–957. doi:10.4141/cjas91-116 Stark, M., Lukaszuk, J., Prawitz, A., & Salacinski, A. (2012). Protein timing and its effects on muscular hypertrophy and strength in individuals engaged in weight-training. Journal of the International Society of Sports Nutrition, 9, 54. PubMed doi:10.1186/1550-2783-9-54 Tarnopolsky, M. (2004). Protein requirements for endurance athletes. Nutrition (Burbank, Los Angeles County, Calif.), 20, 662–668. PubMed doi:10.1016/j.nut.2004.04.008 Tipton, K.D., Gurkin, B.E., Matin, S., & Wolfe, R.R. (1999). Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers. The Journal of Nutritional Biochemistry, 10, 89–95. PubMed doi:10.1016/S0955-2863(98)00087-4 van Loon, L.J., Kruijshoop, M., Verhagen, H., Saris, W.H., & Wagenmakers, A.J. (2000). Ingestion of protein hydrolysate and amino acid-carbohydrate mixture increases post exercise plasma insulin responses in men. The Journal of Nutrition, 130, 2508–2513. PubMed Volek, J.S., Volk, B.M., Gómez, A.L., Kunces, L.J., Kupchak, B.R., Freidenreich, D.J.,… Kramer, W.J. (2013) Whey protein supplementation during resistance training augments lean body mass. Journal of the American College of Nutrition, 32(2):122–135. Volpi, E., Kobayashi, H., Sheffield-Moore, M., Mittendorfer, B., & Wolfe, R.R. (2003). Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. The American Journal of Clinical Nutrition, 78, 250–258. PubMed West, D.W., Burd, N.A., Coffey, V.G., Baker, S.K., Burke, L.M., Hawley, J.A., . . . Phillips, S.M. (2011). Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise. The American Journal of Clinical Nutrition, 94, 795–803. PubMed doi:10.3945/ajcn.111.013722 Wu, G. (2009). Amino acids: metabolism, functions, and nutrition. Amino Acids, 37, 1–17. PubMed doi:10.1007/ s00726-009-0269-0
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www.IJSNEM-Journal.com ORIGINAL RESEARCH
Dose Response of Whey Protein Isolate in Addition to a Typical Mixed Meal on Blood Amino Acids and Hormonal Concentrations Scott C. Forbes, Linda McCargar, Paul Jelen, and Gordon J. Bell The purpose was to investigate the effects of a controlled typical 1-day diet supplemented with two different doses of whey protein isolate on blood amino acid profiles and hormonal concentrations following the final meal. Nine males (age: 29.6 ± 6.3 yrs) completed four conditions in random order: a control (C) condition of a typical mixed diet containing ~10% protein (0.8 g·kg–1), 65% carbohydrate, and 25% fat; a placebo (P) condition calorically matched with carbohydrate to the whey protein conditions; a low-dose condition of 0.8 grams of whey protein isolate per kilogram body mass per day (g·kg–1·d–1; W1) in addition to the typical mixed diet; or a high-dose condition of 1.6 g·kg–1·d–1 (W2) of supplemental whey protein in addition to the typical mixed diet. Following the final meal, significant (p < .05) increases in total amino acids, essential amino acids (EAA), branch-chained amino acids (BCAA), and leucine were observed in plasma with whey protein supplementation while no changes were observed in the control and placebo conditions. There was no significant group difference for glucose, insulin, testosterone, cortisol, or growth hormone. In conclusion, supplementing a typical daily food intake consisting of 0.8 g of protein·kg–1·d–1 with a whey protein isolate (an additional 0.8 or 1.6 g·kg–1·d–1) significantly elevated total amino acids, EAA, BCAA, and leucine but had no effect on glucose, insulin, testosterone, cortisol, or growth hormone following the final meal. Future acute and chronic supplementation research examining the physiological and health outcomes associated with elevated amino acid profiles is warranted. Keywords: nutrition, branch-chained amino acids, diet, leucine, essential amino acids, testosterone Protein is an important component of dietary food intake and is needed for growth and a variety of cellular processes (Wu, 2009). This is also underscored by the fact that malnutrition will occur if there is an inadequate dietary intake of protein even with adequate caloric intake (Grover & Ee, 2009; Phillips & Van Loon, 2011). The recommended daily allowance (RDA) for protein intake for sedentary adults is 0.8 g·kg–1.d–1 (Institute of Medicine, 2005). Physically active or athletic individuals are often recommended to ingest a larger amount of dietary protein than the RDA (1.2–1.7 g·kg–1.d–1; Lemon, 1998; Phillips, 2012; Rennie & Tipton, 2000; American Dietetic Association, 2009; Tarnopolsky, 2004). The greater dietary protein recommendations in active or athletic individuals may be associated with elevated exercise induced cellular processes, such as mitochondrial biogenesis, protein synthesis, and endocrine and immune Forbes and Bell are with the Faculty of Physical Education and Recreation, University of Alberta, Edmonton, Canada. McCargar and Jelen are with the Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada. Address author correspondence to Scott C. Forbes at [email protected].
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responses (Lemon, 1991). Furthermore, habitual daily protein intakes of resistance-trained individuals are often greater than 2.0 g·kg–1.d–1 and have been reported to be as high as 3.5 g·kg–1.d–1 (Phillips, 2004). To consume more dietary protein, individuals often use nutritional supplementation containing various amino acids and/ or proteins of which there are a myriad of commercially available products. Whey protein powder is one of these products, which has received considerable attention recently due to its unique properties (Burke et al., 2012). Whey protein is commercially added to certain foods and also available in powder form as a nutritional aid or supplement. Whey protein is a heterogeneous mixture of proteins containing all essential amino acids (EAA), and branch-chain amino acids (BCAA; e.g., leucine, isoleucine, and valine; Stark et al., 2012). Elevated extracellular aminoacidemia following whey protein ingestion is known to enhance skeletal muscle myofibrillar protein synthesis (Bohe et al., 2003; West et al., 2011) which is stimulated primarily by EAAs (Paddon-Jones et al., 2004; Tipton et al., 1999; Volpi et al., 2003) and by the BCAA leucine (Drummond and Rasmussen, 2008). Intravenous administration of BCAAs elevates the phosphorylation and activation of p70S6 kinase and 4E-BP1 (Greiwe et al., 2001); p70S6 kinase and 4E-BP1 which are down-
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stream substrates of the mammalian target of rapamycin (mTOR) pathway (Greiwe et al., 2001; Liu et al., 2006; Manders et al., 2012). Leucine supplementation alone has been shown to directly phosphorylate and activate mTOR (Fujita et al., 2007); however, alterations in the rates of myofibrillar protein synthesis are relatively transient unless EAA are provided in sufficient quantities (Manders et al., 2012). Furthermore, Calbet and MacLean (2002) found that whey protein produced a significant increase in circulating levels of insulin and this insulin response was greater than achieved with a carbohydrate solution. Thus, the physiological response to whey protein ingestion can be quite diverse. Because of this diversity, whey protein is currently added to enhance the quality of particular foods (e.g., yogurts, breads, and as a dietary supplement for active or athletic individuals). However, there is little research examining specific dosages of whey protein powder added to a typical mixed daily intake of food on blood amino acids (e.g., total amino acids, EAA, BCAA, and leucine), hormonal (e.g., insulin, testosterone, growth hormone, and cortisol) and glucose responses following the final meal of the day. Therefore, the primary purpose of this study was to determine the effects of two different doses of a whey protein isolate added to a controlled typical mixed one day diet, containing 10% of energy from protein, on the amino acid profiles following the final meal. Secondary purpose was to examine the hormonal and glucose concentrations in the blood after the final meal of the day with whey protein supplementation compared with a typical controlled diet at the RDA for protein. These doses were selected to represent the current sport nutrition guidelines (Lemon, 1998; Phillips, 2012; Rennie & Tipton, 2000; American Dietetic Association, 2009; Tarnopolsky 2004) a typical composition of athletes, and the current RDA (Institute of Medicine, 2005). It was hypothesized that supplementing a daily diet with a whey protein isolate would cause a greater increase in certain amino acids (e.g., essential amino acids, branched chain amino acids, and leucine) and may stimulate a greater release of particular hormones (e.g., insulin) in a dose dependent manner following the final meal of the day.
Methods Subjects Ten recreationally active male subjects were recruited from a university population. One subject withdrew from the study after the first testing session due to personal time commitment issues and the remaining 9 subjects completed all testing conditions.
Experimental Design Each subject attended an orientation session for an explanation of the study, scheduling and to sign an informed consent that was approved by a university research ethics
committee. At this time each subject was provided with a 3-day dietary record and shown how to complete it accurately. A second session was attended for measurements of height, body mass, resting metabolic rate and body composition. Please note that the experimental design of this study and a small portion of the methods have been previously published (Middleton et al., 2004) but there is no duplication in any of the dependent variables reported in this paper. The 3-day dietary records were entered into a computer software program (The Food Processor II for Windows, Nutritional Analysis Nutritional Software Version 6.11, Salem, OR) to determine the amount of carbohydrate, fat, protein and the number of calories consumed each meal and for each day. Following computer analysis, the nutritional intake of each subject was adjusted for the amount and/or proportion of food to balance the diet to obtain 0.8 g·kg–1.d–1 of mixed food protein (Institute of Medicine, 2005) at the same time as maintaining the total calories equal to the subject’s original diet record for each of the 3-days of the recording. This corresponded to a proportion of 10% protein, 65% carbohydrate and 25% fat. The modified records with the exact type and size of food items were photocopied and returned to each subject to ensure that they consumed this exact diet for the two days before and on the day of all 4 experimental conditions. There were 4 experimental conditions that were completed and all calculations were made based on each individual’s body mass: Control (C)—3 days of controlled dietary intake (2 days before and on the day of the experimental condition). The diet consisted of a typical mixed food intake modified to contain a proportion of 10% protein, 65% carbohydrate and 25% fat and total protein intake of 0.8 g·kg–1.d–1 from a nonwhey protein mixed food diet. Placebo (P)—identical dietary intake as in C condition except that a glucose polymer (Polycose) was added to a noncalorie 1 L flavored drink (Crystal-Lite) that was calorically matched to the extra calories added as a result of the highest whey protein supplementation condition (W2) described below. This was done so that the total caloric intake was controlled and matched to the extra calories from the highest protein supplementation condition to ensure that any observed effect was not due to an increase in total calories consumed. Total protein intake for this condition was 0.8 g·kg–1.d–1. Whey Protein Supplementation Dose 1 (W1)—same diet as the control condition plus 0.8 g·kg–1.d–1 of whey protein powder mixed in the same 1 L volume of noncalorie flavored drink as the placebo condition plus a smaller amount of glucose polymer to match the additional calories consumed with the highest whey protein supplementation condition (W2) described below. Again total caloric intake was matched between the placebo and the highest whey protein dose condition. Total protein intake for this condition was equal to 1.6 g·kg–1.d–1. Whey Protein Supplementation Dose 2 (W2)—same mixed diet as the control condition plus 1.6 g·kg–1.d–1
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of whey protein powder mixed in the same drink as the other conditions for a total protein intake equal to 2.4 g·kg–1.d–1. Each subject acted as his own control, thus all subjects participated in all the experimental conditions (C, P, W1 and W2) in a random, double-blind design. The daily dietary mixed protein intake (0.8 g·kg–1.d–1) and total calories consumed was based on foods that each subject preferred but was modified accordingly so that each subject had the same dietary composition leading up to and including the day of each experimental condition. The whey protein powder was dissolved in a noncaloric flavored drink in an attempt to mask the flavor and was contained in a 1 L colored opaque bottle to mask the appearance. The supplement drinks were consumed in equal portions with each meal during the day. Total volume of fluid (experimental drinks and water intake) was also the same for each condition. No formal exercise training (e.g., running or resistance training) was allowed on the days before and on the days of the experimental conditions but the subjects were allowed to resume their regular workday schedules.
Whey Protein Isolate The whey protein isolate chosen for this study was PowerPro Plus (Land O’ Lakes, St.Paul, MN, USA) and is produced through a proprietary process that includes ultra/microfiltration concentration of the native whey proteins in an attempt to keep each protein in its functional form. The constituents of this product are available from the manufacturer and are reported in our previous study (Middleton et al., 2004). Briefly, the amino acid profile for PowerPro Plus in grams of amino acids per 100 g of product: alanine = 4.6 g, arginine = 2.2 g, aspartic acid = 9.9 g, cysteine = 2.3 g, glutamic acid = 11.4 g, glutamine = 4.4 g, glycine = 1.7 g, histidine = 1.8 g, isoleucine = 5.8 g, leucine = 10.1 g, lysine = 8.5 g, methionine = 1.9 g, phenylalanine = 3.1 g, proline = 5.2 g, serine = 4.4 g, threonine = 6.0 g, tryptophan = 1.6 g, tyrosine = 2.8 g, and valine = 5.4 g.
Measurement of Resting Metabolic Rate (RMR) and Body Composition Assessment Each subject completed a RMR measurement between 7:30 and 8:30 a.m. after an overnight fast (12 hr) on a different day in a rested state. Once in the laboratory, each subject rested on a bed for 20 min followed immediately by a 30-min period of metabolic measurement of expired gases in a canopy designed for such measurements (MedGraphics CPXD, Medical Graphics Corp., Minneapolis, MN, USA). The room was quiet and in dim light to help each subject relax. Each subject was asked not to fall asleep. The metabolic cart was calibrated for volume of air, and known oxygen and carbon dioxide concentrations before and after each RMR measurement. Body composition was assessed using the hydrostatic
weighing procedure in a fasted state immediately after the RMR measurement as previously reported for our laboratory (Biondo et al., 2008). Custom designed software performed all standard calculations and body fat was predicted mathematically according to the formula of Brozek (1963).
Blood Samples Blood (approximately 10 ml per sample) was obtained using a venipuncture from an anticubutal vein or via a cathlon inserted into a forearm vein. Each subject came to the laboratory in the morning (7:30 a.m. to 8:30 a.m.) after an overnight fast and rested while the first blood sample was taken using venipuncture and collected into a plain vacutainer. The blood sample was put on ice until it clotted (~30 min), centrifuged at 1500 xg for 10-min, and 3 equal volumes of the serum were pipetted into separate 1.5-mL microcentrifuge tubes and frozen at –80 °C until analyses. Following the resting sample, the subjects ate their prescribed breakfast, morning snack, lunch, afternoon snack and final meal at their usual times with (P, W1, W2) or without (C) the experimental drink depending on the randomized condition. Each subject consumed the experimental drinks in 3 equal portions at the 3 major prescribed meal times (breakfast, lunch and final meal). The subjects returned to the laboratory before the final meal of the day to have a cathlon placed in a forearm vein by a registered nurse. The cathlon was attached to a three-way stopcock and kept patent with 0.9% sterile saline solution. Blood was drawn in the fasted state (i.e., morning), immediately before the final meal of the day and 30, 60, 120, 180, and 240 min after the final meal. All blood samples were prepared and frozen in the same manner. The subjects were required to remain in the laboratory for the final meal and postmeal blood sampling periods.
Amino Acid Analysis Each amino acid concentration was determined individually in all blood samples in duplicate using a protocol previously published for our laboratory (Sedgwick et al., 1991). The protocol established 19 amino acids that were categorized based on their level of dispensability: dispensable (alanine, aspartate, asparagine, glutamate, serine); conditionally indispensable (arginine, cysteine, glutamine, glycine, tyrosine); and indispensable (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine; Pencharz and Ball, 2003). In addition, taurine, and ornithine were analyzed for a total of 21 amino acids. All the amino acids analyzed (except taurine and ornithine) were present in the whey protein isolate used in this study. The detailed procedures of the amino acids using high pressure liquid chromatography have been previously described by Sedgwick et al. (1991). Briefly, the
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serum blood sample was deproteinized by mixing 25 μL of serum with 50 μL of EA/BABA (Ethanolamine and β-amino-butyric acid; 25 nmol/mL) followed by the addition of 100 μL of 5% wt·vol-1 TCA (trichloroacetic acid) and centrifuged at 1500 xg for 15-min. Subsequently, 100 μL of the deproteinized supernatant was mixed with 150 μL of saturated K2B4O7 to adjust pH to ~9.0 and then 100 μL of HPLC grade H2O was added. One hundred μL of the final supernatant was pipetted into a 1.5 μL vial add added to the autosampler rack of the high performance liquid chromatography (HPLC) machine. A 100 uM amino acid standard solution for each amino acid was prepared and the amino acid samples were then transformed using the o-phthaldialdehyde (OPA) method reported by Jones and Gilligan (1983). Individual amino acid was determined using a Varian 5000 high performance liquid chromatograph and fluorichrom detector. Chromatographic peaks were recorded integrated using a chromatography data system (Hewlett Packard 3353, Mississauga, ON, Canada). The amino acid concentrations were determined by comparison with the proteinfree standard solutions prepared in an identical manner.
Blood Glucose and Hormonal Analysis Glucose and hormonal concentrations were measured in duplicate at every time point. Glucose was analyzed in serum using the glucose oxidase method (Sigma Aldrich, 2004). Insulin, cortisol, growth hormone, and testosterone concentrations were determined with radioimmunoassay, using commercial assay kits (Diagnostic Products Corporation, Los Angeles, CA, USA).
Statistical Analysis A two-way ANOVA with repeated measures was used to compare the four experimental trials (C, P, W1 and W2) and seven sampling time points for all dependent variables (total amino acids, EAA, BCAA, leucine, insulin, glucose, testosterone, growth hormone, and cortisol). Area under the curve (AUC) was calculated using Prism software (Graphpad 4, San Diego, CA, USA) and one-way repeated-measures ANOVA was used to assess the AUC between conditions. Significant F ratios were further analyzed with a Tukey’s pairwise comparison. Statistical analyses were carried out using Statistica, version 10 (StatsSoft Inc., Tulsa, OK, USA). All results are expressed as means ± SD. Statistical significance was set at p ≤ .05.
Results The mean age, body weight, height, percent body fat and resting metabolic rate (RMR) were 29.6 ± 6.3 yrs, 81.2 ± 12.6 kg, 180.1 ± 7.7 cm, 19.6 ± 6.5%, and 1677 ± 197.8 kcals·d–1, respectively. There were no observed or reported side effects with any condition. Resting fasting values for all the dependent variables (total amino acids, EAA, BCAA, leucine, insulin, glucose, testosterone,
growth hormone, and cortisol) were not significantly (p > .05) different between conditions.
Blood Amino Acid Profiles Total blood amino acid concentrations demonstrated an interaction (treatment by time) effect (p < .0001) with W1 and W2 being elevated above the placebo and control conditions at 60 and 120 min following the final meal. In addition, W2 was elevated above placebo and control conditions at the 30 and 180 min time points; however, there were no differences between the two whey protein conditions. Blood EAAs showed a similar interaction (treatment by time) effect (p = .00001; Figure 1). Compared with the placebo and control, W2 was significantly elevated at 30, 60, 120, 180, and 240 min after the final meal, while the lower dose of whey protein (W1) was only elevated at the 60 and 120 min time points. Similarly, there were no differences between the two whey protein supplement conditions. Blood BCAA were significantly (p < .00001) higher 30, 60, 120, 180 min time points during the W2 condition and higher at 60 and 120 min time points during the W1 condition compared with the placebo and control. As shown in Figure 2, leucine was significantly (p < .0001) elevated at 30, 60, 120, and 180 min time points during the W2 condition and elevated at 60 and 120 min time points during the W1 condition compared with the placebo and control trials. There were no differences in leucine between W2 and W1 at any time points. The AUC for total amino acids (, EAA, and BCAA revealed a significant (p < .0001) difference between W1 and W2 compared with the placebo and control conditions. The AUC for leucine revealed greater values for the whey protein doses (W1 and W2); however, W1 was similar to the placebo condition and different than the control condition.
Blood Glucose and Hormones There was a main effect of sampling time for serum glucose (p = .002) concentration revealing a significant increase in serum glucose 30 min after the final meal of the day that was higher than all other time points. There was no significant difference in serum glucose response between conditions. Similarly, serum insulin increased (p < .0001) at the 30, 60, 120, and 180 min after the final meal compared with the fasting sample; however no differences were observed between conditions (Figure 3A). There was a main effect of sampling time for serum testosterone (p < .0001) and cortisol (p < .0001) concentrations revealing significantly higher serum concentrations during the fasted state compared with all other time points, while there were no differences between conditions (Figures 3B and 3C). Growth hormone showed an interaction (treatment by time; p = .03) effect; however, post hoc analysis revealed no significant differences at any time point between all four
Figure 1 — Blood essential amino acid concentrations in each experimental condition and over the different sampling times. Values are means ± SEM a = W2 significantly higher than placebo and control conditions. b = W2 and W1 significantly higher than placebo and control conditions. W2 = whey protein supplementation dose of 1.6 g·kg–1.d–1, W1 = whey protein supplementation dose of 0.8 g·kg–1.d–1.
Figure 2 — Blood leucine concentrations in each experimental condition and over the different sampling times. Values are means ± SEM a = W2 significantly higher than placebo and control conditions. b = W2 and W1 significantly higher than placebo and control conditions. c = W2 significantly different than placebo and control and W1 is different than control but similar to placebo. W2 = whey protein supplementation dose of 1.6 g·kg–1.d–1, W1 = whey protein supplementation dose of 0.8 g·kg–1.d–1.
Figure 3 — Mean ± SEM. Serum (A) insulin (B) testosterone (C) cortisol (D) growth hormone in each experimental condition and over the different sampling times. * = significantly different than all other time points. # = significantly different than fasting.
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conditions (Figure 3D). The AUC was similar between all four conditions for glucose, insulin, testosterone, cortisol, and growth hormone (p > .05).
Discussion The main finding of the current study was that a one day meal plan that contained a mixed dietary intake of 0.8 g·kg–1.d–1 of protein supplemented with whey protein isolate in a low (0.8 g·kg–1.d–1; total of 1.6 g·kg–1.d–1 of protein) or a high dose (1.6 g·kg–1.d–1; total 2.4 g·kg– 1.d–1 of protein) increased total amino acids, EAAs, BCAAs, and leucine concentration in the blood after the final meal of the day and this response was significantly higher than the control and placebo conditions. Despite the elevation in amino acid concentrations there were no differences between conditions for insulin, glucose, testosterone, growth hormone, or cortisol at any time of the day in any experimental condition. The measurement of amino acids in the blood after standardized meals with various doses of whey protein supplementation is one method of determining the absorption and extracellular availability. The extracellular availability of amino acids, facilitated by blood flow, has been shown to increase protein synthesis and decrease protein degradation (Biolo et al., 1995; Bohe et al., 2003) leading to an enhanced net protein balance. Volek et al. (2013) provides evidence that humans fed protein (e.g., whey protein) containing a higher proportion of leucine will result in an elevation of blood leucine concentration and stimulate muscle protein synthesis and this may lead to greater gains in muscle mass over time; however, others do not support this linear association between elevated leucine and enhanced muscle protein synthesis (Churchward-Venne et al., 2012). In addition, changes in muscle protein synthesis are relatively small unless sufficient EAA are provided (Manders et al., 2012). The present study found a significant increase in both EAA and leucine following whey protein supplementation compared with a typical meal. However, the AUC for leucine following the final meal was only significantly different than placebo following the high whey protein dose (1.6 g·kg-1.d-1). This would suggest that the higher whey protein dose condition may be ideal for maximizing leucine uptake but future research is necessary to determine if this elevation in leucine concentration could lead to elevations in muscle protein synthesis and body composition changes over time. Furthermore, diet was only controlled for 3 days (2 days before and including the experimentation day) for each condition, this time period may not have been sufficient to allow for a new metabolic equilibrium, which may require between 5–7 days (Jeacocke and Burke, 2010). Therefore, the current study may represent a transitory period; future research examining longer dietary controls may be required to examine protein kinetics with physiological adaptations (e.g., protein oxidation, muscle protein synthesis, and muscle protein breakdown). Recently, Moore et al. (2012)
examined the effects of daytime patterns of dietary protein intake on whole-body protein turnover in resistance trained males and found that ~20 g at regular intervals (~3 hr) throughout the day achieved the highest net protein balance. This may suggest that not only the total daily amount of protein is important but also the timing pattern of protein ingestion throughout a day. Interestingly, the current study found only minor differences between the low and high dose of whey protein supplementation. This may have been associated with a reduced absorption rate with higher protein ingestion due to competitive inhibition of the transport system that moves amino acids into peripheral blood (Adibi & Gray, 1967). In addition, it has been suggested that the digestion of a large quantity of protein may extend beyond a 4 hr following a meal (Adibi & Mercer, 1973), which was a limitation of the current study. It is also important to note that the current study only examined the amino acids profiles at rest, immediately before and following the final meal of the day. Regardless, the current study was able to exhibit a significant increase in circulating amino acids with whey protein supplementation. Despite this increase in amino acids, there were no differences between conditions on any of the measured hormones. It has been shown that several amino acids (e.g., arginine) can stimulate the secretion of insulin and growth hormone (van Loon et al., 2000; Collier et al., 2005) and it has been suggested that the BCAA may influence the time course of hormonal release (Calbet & MacLean, 2002). Whey protein ingestion (30 g·d–1) has been shown to increase plasma insulin and this response was greater than that achieved with an amino acid or casein meal (Dangin et al., 2001). Calbet and MacLean (2002) also found a greater increase in plasma insulin with ingestion of a whey protein solution when compared with milk, a pea peptide solution or a glucose solution. The physiological consequence of an increased circulating concentration of insulin is to promote cellular glucose and amino acid uptake and to assist in the regulation of protein synthesis within the cell (Graham et al., 1995). As well, diets high in protein (30% protein including milk protein) have been shown to lower postprandial blood glucose levels and modestly increase serum insulin concentration after the final meal of the day in people with Type 2 diabetes (Gannon et al., 2003). The present study in healthy adults does not support these latter studies as we observed no significant difference in insulin or glucose following whey protein supplementation (0.8 g·kg-1.d-1 or 1.6 g·kg-1.d-1) compared with control or placebo conditions.
Conclusion Supplementation of a daily meal plan with whey protein produced an overall increase in the total amino acid, EAA, BCAA, and leucine concentrations following the final meal; however, there were no differences between
194 Forbes et al.
the high and low whey protein dose conditions. Thus, supplementation with whey protein throughout the day with a controlled diet was effective at elevating amino acid concentrations in the blood following the final meal. In addition, the current study was unable to demonstrate any differences in insulin, glucose, testosterone, cortisol, or growth hormone after the final meal of the day with the whey protein powder. Further research should focus on the biological responses (e.g., muscle protein synthesis) of whey protein supplementation and longer-term supplementation with controlled dietary experimentation. Acknowledgments The authors would like to thank Ian MacLean, Neil Middleton, Gary Sedgewick, Stephen Cheetham, and Robert Kell for their assistance with this study.
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