Applied Physiology, Nutrition, and Metabolism

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1 Aminoacidemia following ingestion of native whey protein, micellar casein, and a whey-casein blend in young men Journal: Manuscript ID Manuscript Type: Date Submitted by the Author: Complete List of Authors: Is the invited manuscript for consideration in a Special Issue? : apnm r1 Brief communication 16-Jul-2018 Traylor, Daniel; McMaster University Department of Kinesiology Gorissen, Stefan ; McMaster University, Kinesiology Hopper, Hannah; McMaster University Department of Kinesiology Prior, Todd; McMaster University Department of Kinesiology McGlory, Chris ; McMaster University, Kinesiology Phillips, Stuart; McMaster University, Not applicable (regular submission) Keyword: Amino acid, Leucine, muscle, Supplementation, protein synthesis

2 Page 1 of 15 1 Aminoacidemia following ingestion of native whey protein, micellar casein, and a wheycasein blend in young men Daniel A. Traylor, Stefan H.M. Gorissen, Hannah Hopper, Todd Prior, Chris McGlory, Stuart M. Phillips Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada Corresponding author: Professor Stuart Phillips Ivor Wynne Centre, Room E Main Street West Hamilton, Ontario, L8S 4K1, Canada Phone: ext phillis@mcmaster.ca Authors last names for PubMed indexing: Traylor, Gorissen, Hopper, Prior, McGlory, Phillips Word count: 2920 Number of Figures: 1 Number of Tables: 1 Financial support: Funding for this work was from Leprino Foods Company. SMP acknowledges the National Science and Engineering Research Council (NSERC) of Canada, the Canadian Institutes for Health Research, and the Canada Research Chairs program for support. List of abbreviations: EAA, essential amino acids; MPS, muscle protein synthesis

3 Page 2 of 15 2 ABSTRACT We examined the aminoacidemic, glycemic, and insulinemic responses following ingestion of 25 g of native whey protein, micellar casein, and a 1:1 blend of whey and casein in randomized order in young adult men. Blood samples were drawn at baseline and at regular intervals for six hours following ingestion. Area under curve and peak plasma essential amino acid concentrations after the ingestion of the protein blend were similar to whey and greater compared with casein. Key words: Amino acid, Leucine, Muscle, Supplementation, Protein synthesis

4 Page 3 of 15 3 INTRODUCTION Protein ingestion, and the subsequent aminoacidemia, stimulates muscle protein synthesis (MPS) in humans (Bohe et al. 2003; Volpi et al. 2003). The amplitude and duration of the postprandial MPS response is largely dependent on protein digestion and amino acid absorption kinetics as well as the amino acid composition of the protein consumed (Morton et al. 2015). For example, whey protein has a high leucine content, is acid soluble, and therefore rapidly digested, resulting in a rapid but transient rise in circulating essential amino acids (EAA), and leucine in particular (Boirie et al. 1997). Leucine plays a central role in stimulating skeletal muscle anabolism (Katsanos et al. 2006) and a certain dose of leucine is required to activate the MPS machinery (Atherton et al. 2010). In contrast to whey protein, micellar casein precipitates in the stomach and is more slowly digested, resulting in a moderate but protracted aminoacidemia (Boirie et al. 1997; Pennings et al. 2011; Burd et al. 2012). Whey protein is often considered superior in stimulating MPS, although the postprandial MPS response to whey ingestion may be shorter when compared to casein (Reidy et al. 2013; Gorissen et al. 2016). Casein, due to its slow digestion kinetics (Boirie et al. 1997), may not result in a rapid hyperleucinemia and thus be less effective in activating the MPS machinery (Pennings, 2011; Burd et al. 2012); however, casein could provide EAA in the later postprandial period to maintain elevated rates of MPS (Gorissen et al. 2016). No study has addressed whether the ingestion of a 1:1 blend of whey protein and micellar casein results in a synergistic effect on both amplitude and duration of postprandial aminoacidemia. We aimed to assess postprandial aminoacidemia following the ingestion of equivalent amounts of protein (25 g) as native whey protein, micellar casein, or a 1:1 blend of whey protein and micellar casein. We hypothesized that the ingestion of casein, protein blend, and

5 Page 4 of 15 4 whey would result in a stepwise increase in peak plasma EAA levels with no difference in area under the curve (AUC) over a 6 h postprandial period. MATERIALS AND METHODS Participants. Ten healthy, non-smoking, non-diabetic men ([mean±sem] age 24±1 y; body mass 83.4±5.4 kg; BMI 25.7±1.5 kg/m 2 ; lean body mass 62.5±2.2 kg; body fat 21±2%) provided written consent after being informed of the purpose, protocol, and risks of the study. Exclusion criteria were: use of analgesic or anti-inflammatory drugs, history of neuromuscular problems or muscle and/or bone wasting diseases, any acute or chronic illness, metabolic disorders, use of corticosteroids. Experimental procedures were approved by the Hamilton Integrated Research Ethics Board. This study was registered at clinicaltrials.gov as NCT Experimental design. This randomized double-blind crossover design study consisted of three testing days separated by a minimum of 48 hours. Participants underwent a DXA scan at baseline and were randomized (using an online randomization plan generator; to ingest a single 25 g bolus of native whey protein isolate (Whey), micellar casein (MCas), or a 1:1 ratio of Whey and MCas (Blend) (all proteins were provided by Leprino Foods, Denver, CO) dissolved in 300 ml of water. Whey provided 3.0 g leucine and 12.1 g EAA, MCas provided 2.2 g leucine and 11.0 g EAA, and Blend provided 2.6 g leucine and 11.5 g EAA (see Supplementary Table S1 for complete amino acid profile). The amount of protein selected has been shown to result in a robust stimulation of MPS (Churchward-Venne et al. 2012; Churchward-Venne et al. 2014). Participants were instructed to refrain from alcohol consumption and any sort of strenuous physical activity and to keep their diet as consistent as possible for 2 d before each trial. The evening before each trial, participants consumed a standardized meal (19.6±1.3 kj/kg body mass) providing 19% of

6 Page 5 of 15 5 energy as fat, 24% of energy as protein, and 57% of energy as carbohydrate (Heart-to-Home Meals, Cambridge, ON, Canada). On each testing day, participants reported to the laboratory after an overnight fast. A catheter was placed in the antecubital vein that was kept patent with a saline drip. Arterialized venous blood samples were obtained before (t=0 min) and at 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, and 360 min after protein ingestion. Protein beverages were consumed within 5 min. Sample collection and analyses. Blood samples were collected in EDTA-containing tubes, centrifuged at 1000 g for 10 min at 4 C, and plasma was aliquoted and stored at -80 C until analysis. Plasma AA concentrations were measured using EZ:faast TM amino acid analysis kit for gas chromatography-mass spectrometry (Torrance, CA, USA). Samples were analyzed using an Agilent 5975C GC/MS (Source 240 C; Quad 180 C; MS Transfer Line 310 C). The instrument was configured to use electron impact ionization and was set to run in Scan mode using the column supplied by Phenomenex (Phenom cgo-7169zb-aaa; 325 C; 10 m x 250 µm x 0.25 µm). Aliquots of 2 µl of the derivatized sample/standard were delivered by autosampler into the MM inlet set to run a 15:1 split mode injection at 250 C. The GC was operated in constant pressure mode at approximately 2.9 psi producing and starting flow rate of 1.4 ml/min with an initial oven temperature of 110 C. The temperature ramp used was as follows: 110 C with no hold, followed by a ramp of 30 C/min to 320 C, followed by 1 min hold and 1 min post run at 320 C for a total run time of 8 min. Chromatographs were quantified using the enhanced data analysis software provided by Agilent in conjunction with the method instructions and EI ion database provided with the Phenomenex kit. Plasma glucose concentrations were analyzed using Hexokinase/G-6-PDH methodology and plasma insulin concentrations were analyzed by Chemiluminescent Microparticle Immunoassay. Statistical analyses. For plasma concentration curves, a two-way repeated-measures ANOVA was used to identify differences between treatments over time. When a significant

7 Page 6 of 15 6 interaction effect was observed, Fischer s least significant difference post hoc analysis was performed to locate these differences. For peak concentration (C max ), time to peak concentration (T max ), and area under the curve (AUC), one-way ANOVAs were used to identify differences between treatments. AUC was calculated using the trapezoid method. Significance was set at P<0.05. All calculations were performed by using IBM SPSS Statistics (version 23). RESULTS Plasma amino acid concentrations increased after protein ingestion (Time P<0.001; Figure 1). Plasma leucine, BCAA, and EAA increased to a greater extent after the ingestion of Whey and Blend compared with MCas (time-by-treatment P<0.001; Figure 1A-C). Blend ingestion resulted in a more prolonged increase in plasma BCAA and EAA when compared with Whey. Total amino acid concentrations did not differ between treatments (time-by-treatment P=0.21, Treatment P=0.17; Figure 1D). Of the individual EAA, leucine (P<0.001), isoleucine (P<0.001), valine (P=0.004), and tryptophan (P=0.001) showed a significant interaction effect (data not shown) with isoleucine and valine showing similar trends as leucine. Tryptophan increased to a lower, intermediate, and greater extent after the ingestion of MCas, Blend, and Whey, respectively. Peak postprandial plasma leucine, BCAA, EAA, and total AA concentrations were higher after the ingestion of Whey and Blend when compared with MCas (P<0.05; Table 1). The AUC over the entire 6 h postprandial period for plasma leucine and BCAA concentrations were higher after the ingestion of Whey and Blend when compared with MCas (P<0.05). The AUC of plasma EAA concentrations was greater after the ingestion of Blend when compared with MCas (P=0.007), while both treatments did not differ from Whey (P>0.05). Plasma insulin concentrations increased after protein intake (Time P<0.001)

8 Page 7 of 15 7 reaching peak concentrations of 13.0±0.5 mu/l with no differences between groups. Plasma glucose concentrations remained between 4.5 and 5.1 mmol/l throughout the postprandial period. DISCUSSION Our goal was to examine the aminoacidemic responses following ingestion of a blend of casein and whey protein that each have unique digestion and absorption kinetics (Boirie et al. 1997). We observed that the consumption of a 1:1 blend of native whey protein and micellar casein elicited a rapid increase in plasma EAA concentrations. In contrast to our hypothesis, peak EAA concentrations did not differ between the Blend and Whey treatments and both resulted in greater net aminoacidemia (AUC), as compared with the MCas treatment. In addition, EAA concentrations in the late (2-6 h) postprandial period were highest following the ingestion of Blend. In the present study, peak leucinemia was not different between ingestion of Blend (~475 µm) and Whey (~550 µm) but both were greater when compared with MCas (~350 µm). The observed leucinemia with ingestion of Blend and Whey has previously been shown sufficient to stimulate MPS (West et al. 2011; Churchward-Venne, 2012). In addition, the increases in plasma leucine concentrations reported in the present study were similar to those reported to increase MPS following the ingestion of a single dose of leucine-enriched EAA or suboptimal amounts of protein in young and older adults (Katsanos et al. 2006; Churchward- Venne et al. 2012). Our lab has recently demonstrated that plasma leucine concentrations peaking at ~450 µm following co-ingestion of 5 g crystalline leucine with breakfast, lunch, and dinner, resulted in enhanced integrated MPS rates over 3 days (Murphy et al. 2016). Thus, the current findings suggest that the amplitude of leucinemia following consumption of Blend and Whey was sufficient to stimulate MPS.

9 Page 8 of 15 8 Upon activation of the MPS machinery, which is largely driven by the rapid postprandial increase in plasma leucine concentrations (Atherton et al. 2010), a sustained supply of all EAA could maintain elevated rates of MPS (Reidy et al. 2013). Our data indicate that ingestion of Blend resulted in a greater net EAA exposure than MCas as indicated by the AUC. In addition, plasma EAA concentrations were higher following the ingestion of Blend when compared with Whey in the late (2-6 h) postprandial period (Figure 1). Optimizing protein intake to allow for a rapid leucinemia and a prolonged elevation in plasma EAA concentrations may enhance the amplitude and duration of the postprandial MPS response. This point becomes more significant when considering that mixed meals often contain a blend of both fast and slow digestible protein sources. Here, we show that a 1:1 ratio of whey protein and micellar casein resulted in a rapid and equivalent peak aminoacidemia and leucinemia as Whey, and was able to sustain aminoacidemia. Our data provide proof-of-concept that protein formulations that take advantage of differing digestion and absorption kinetics (slow versus fast), as well as providing high levels of EAA and leucine and the highest protein quality score, may better elicit an initial rapid followed by a sustained hyperaminoacidemia. Specifically, our data are novel in that they show that a 1:1 ratio of whey protein and casein induces the highest plasma concentrations of EAA, BCAA, and leucine during the later postprandial phase in healthy young men (Figure 1). In conclusion, the ingestion of 25 g of a 1:1 ratio of native whey protein and micellar casein resulted in a similar peak leucinemia to Whey and greater peak leucinemia and EAA AUC than MCas. Blend also resulted in greater EAA concentrations at later time points postingestion versus Whey. Future studies are needed to examine the minimum amount of the present blend formulation needed to stimulate MPS in the early (0-2 h) and late (2-6 h) postprandial periods and thereby promote muscle mass retention.

10 Page 9 of 15 9 ACKNOWLEDGEMENTS We thank Dr. Amy Hector for her analytical support. DAT and SMP designed the research; DAT, SHMG, HH, and TP conducted the research; DAT and SHMG performed the statistical analysis; and DAT, SHMG, CM, and SMP wrote the manuscript. All authors read and approved the final manuscript. Conflicts of interest: SMP reports having received competitive research funding, travel expenses, and honoraria from the US National Dairy Council (NDC). Some studies discussed in this paper may have been funded by the US NDC. No other authors report conflicts of interest.

11 Page 10 of REFERENCES Atherton, P.J., Smith, K., Etheridge, T., Rankin, D., and Rennie, M.J Distinct anabolic signalling responses to amino acids in C2C12 skeletal muscle cells. Amino Acids, 38: PMID: Bohe, J., Low, A., Wolfe, R.R., and Rennie, M.J Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study. J. Physiol. 552: PMID: Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrere, B Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc. Natl. Acad. Sci. U S A, 94: PMID: Burd, N.A., Yang, Y., Moore, D.R., Tang, J.E., Tarnopolsky, M.A., and Phillips, S.M Greater stimulation of myofibrillar protein synthesis with ingestion of whey protein isolate v. micellar casein at rest and after resistance exercise in elderly men. Br. J. Nutr. 108: PMID: Churchward-Venne, T.A., Breen, L., Di Donato, D.M., Hector, A.J., Mitchell, C.J., Moore, D.R., et al Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial. Am. J. Clin. Nutr. 99: PMID: Churchward-Venne, T.A., Burd, N.A., Mitchell, C.J., West, D.W., Philp, A., Marcotte, G.R., et al Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men. J. Physiol. 590: PMID: Gorissen, S.H., Horstman, A.M., Franssen, R., Crombag, J.J., Langer, H., Bierau, J., et al Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial. J. Nutr. 146: PMID:

12 Page 11 of Katsanos, C.S., Kobayashi, H., Sheffield-Moore, M., Aarsland, A., and Wolfe, R.R A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly. Am. J. Physiol. Endocrinol. Metab. 291: E PMID: Morton, R.W., McGlory, C., and Phillips, S.M Nutritional interventions to augment resistance training-induced skeletal muscle hypertrophy. Front. Physiol. 6: 245. PMID: Murphy, C.H., Saddler, N.I., Devries, M.C., McGlory, C., Baker, S.K., and Phillips, S.M Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study. Am. J. Clin. Nutr. 104: PMID: Pennings, B., Boirie, Y., Senden, J.M., Gijsen, A.P., Kuipers, H., and van Loon, L.J Whey protein stimulates postprandial muscle protein accretion more effectively than do casein and casein hydrolysate in older men. Am. J. Clin. Nutr. 93: PMID: Reidy, P.T., Walker, D.K., Dickinson, J.M., Gundermann, D.M., Drummond, M.J., Timmerman, K.L., et al Protein blend ingestion following resistance exercise promotes human muscle protein synthesis. J. Nutr. 143: PMID: Volpi, E., Kobayashi, H., Sheffield-Moore, M., Mittendorfer, B., and Wolfe, R.R Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am. J. Clin. Nutr. 78: PMID: West, D.W., Burd, N.A., Coffey, V.G., Baker, S.K., Burke, L.M., Hawley, J.A., et al Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise. Am. J. Clin. Nutr. 94: PMID:

13 Page 12 of TABLES Table 1. Plasma amino acid and insulin concentrations. MCas Whey Blend Leucine C max (µm) 354 ± 24 a 545 ± 44 b 475 ± 42 b T max (min) 36 ± 2 a 62 ± 7 b 57 ± 9 b AUC (µm 6 h) 1286 ± 48 a 1594 ± 66 b 1570 ± 60 b BCAA C max (µm) 1303 ± 88 a 1731 ± 128 b 1682 ± 113 b T max (min) 38 ± 3 a 62 ± 7 b 54 ± 8 a,b AUC (µm 6 h) 5308 ± 224 a 5960 ± 355 b 6435 ± 298 b EAA C max (µm) 2198 ± 112 a 2883 ± 200 b 2795 ± 193 b T max (min) 36 ± 2 89 ± ± 8 AUC (µm 6 h) 9083 ± 300 a ± 613 a,b ± 501 b Total AA C max (µm) 4447 ± 140 a 5200 ± 307 b 5237 ± 324 b T max (min) 38 ± 3 80 ± ± 10 AUC (µm 6 h) ± ± ± 1047 Insulin C max (mu/l) 13.1 ± ± ± 1.2 T max (min) 50 ± 6 48 ± 3 42 ± 3 AUC (mu/l 6 h) 43.2 ± ± ± 0.8 Glucose C max (mm) 5.5 ± ± ± 0.1 T max (min) 93 ± ± ± 33 AUC (mm 6 h) 29.1 ± ± ± 0.2 Values are mean ± SEM, n=10 per treatment. Treatments without a common superscript letter differ, P<0.05. MCas, 25 g micellar casein; Whey, 25 g whey protein; Blend, 25 g of a 1:1 micellar casein/whey protein blend; BCAA, branched-chain amino acids; EAA, essential

14 Page 13 of amino acids; AA, amino acids; C max, maximal concentration; T max, time to reach maximal concentration; AUC, area under curve.

15 Page 14 of FIGURE CAPTIONS Figure 1. Plasma leucine (A), branched-chain amino acid (BCAA; B), essential amino acid (EAA; C), and total amino acid (AA; D) concentrations (µm) in the fasting state and after the ingestion of 25 g: micellar casein (MCas), native whey protein (Whey), or a 1:1 micellar casein:native whey protein blend (Blend) in healthy young men. Values are mean±sem, n=10 per treatment. * Whey significantly different from MCas. Blend significantly different from MCas. # Blend significantly different from Whey.

16 Page 15 of 15 MCas Blend Whey A Plasma leucine (µm) 600 * # B Plasma SBCAA (µm) * # C 3000 * D 6000 Plasma SEAA (µm) # Plasma SAA (µm) Time (min) Time (min)