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The effects of macro- and micro- nutrient timing on post-exercise hepcidin response in elite and professional… Dahlquist, Dylan Timothy 2016

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THE EFFECTS OF MACRO- AND MICRO- NUTRIENT TIMING ON POST-EXERCISE HEPCIDIN RESPONSE IN ELITE AND PROFESSIONAL ATHLETES  by  DYLAN TIMOTHY DAHLQUIST BS, Western Washington University, 2013   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCEINCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   June 2016  © Dylan Timothy Dahlquist, 2016   ii  Abstract BACKGROUND INFORMATION Iron deficiency (ID) has debilitating effects on athletic performance, causing significant reductions (-34%) in VO2max. Inflammation caused by exercise has been shown to impede iron absorption in the digestive tract by up-regulating the expression of the iron regulatory protein, hepcidin. To date, nutritional interventions to blunt hepcidin response have been few and equivocal. We investigated the effects of nutrient timing with essential macro- and micro- nutrients to potentially attenuate the post-exercise rise in hepcidin in highly trained athletes.  PURPOSE To determine if a post-exercise drink consisting of whey protein isolate (25g) and carbohydrates (75g) with the addition of vitamins, D3 (5,000 IU) and K2 (1,000 mcg) (VPRO) or without D3 and K2 (PRO), following a bout of high-intensity interval exercise has an effect on the acute post-exercise hepcidin responses in athletes as compared to a non-caloric placebo drink (PLA). Our hypothesis was that both VPRO and PRO will significantly decrease hepcidin following a bout of high intensity exercise as compared to PLA, with VPRO supplementation having a greater effect on hepcidin versus PRO supplementation alone.  METHODS Ten elite male cyclists (age: 26.9 ± 6.4 yrs; VO2max: 67.4 ± 4.4 ml/kg/min) partook in four cycling sessions. A randomized, placebo-controlled, single-blinded triple crossover design was utilized. Experimental days consisted of an 8-min warm-up at 50% pVO2max, followed by 8 x 3 min intervals at 85% pVO2max with, 1.5 min at 60% pVO2max between each interval. Blood samples were collected pre-exercise, post-exercise and three hours post-exercise. Three varying drinks (PRO, VPRO or PLA) were consumed immediately after the post-exercise blood sample.   RESULTS/CONCLUSIONS The results from the investigation demonstrate that following a fatiguing interval-based cycling exercise in highly-trained athletes, subjects experienced a significant time-dependent increase in all biomarkers measured independent of post-exercise drink composition. In conclusion, the post-exercise drinks had no significant effect on any biomarker. The findings could potentially be related to the dosage of nutrients, the timing of blood samples, or the training status of individuals. The lack of an effect in either of the drinks on hepcidin and other biomarkers are contrary to our hypothesis.     iii  Preface Collaborators of this thesis study are as follows:  Dr. Michael Koehle, MD, PhD, supervised the project, assisted in the ethics application, research design, funding application to Own The Podium (OTP) and provided guidance throughout the entire data collection and writing process.  Dr. Trent Stellingwerff, PhD, helped in formulating the research design, aided with writing the proposal and obtaining funding via Own The Podium (OTP) grant in conjunction with the Canadian Sport Institute – Pacific (CSI-P), provided advice during the data collection and assisted in writing the final thesis.  Dr. Don McKenzie, MD, PhD, assisted in trouble shooting potential issues with data collection, provided guidance for obtaining proper resources for sample analysis and aided in writing the final thesis.  Dr. Brad Dieter, PhD, assisted with data collection, data analysis, troubleshooting, and provided ample amounts of feedback and ideas during the writing process.  Dylan Dahlquist, BS, developed the research design, obtained funding from Own The Podium (OTP), the Canadian Sport Institute – Pacific (CSI-P) and the UBC Faculty of Education, created and completed the application for ethics approval, recruited subjects, performed all the data collected, purchased equipment for testing, assisted in assay analysis, analyzed the data and wrote the final thesis document.   No manuscripts resulting from the work presented in this thesis have been published to date. An abstract and poster will be submitted for a poster presentation at the SPort INnovation (SPIN) Summit organized by OTP in September 2016 held in: Calgary, Canada.  This study involved human subjects and was granted full board approval from the University of British Columbia Clinical Research Ethics Board (H15-00721).     iv  Table of Contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii Acknowledgements ......................................................................................................................... x Introduction ..................................................................................................................................... 1 Overview ..................................................................................................................................... 1 Oxygen Delivery and Iron Metabolism ....................................................................................... 2 Hepcidin, IL-6 & Iron Absorption .............................................................................................. 4 Carbohydrate Feeding ................................................................................................................. 6 Protein and Inflammation ............................................................................................................ 7 Vitamin D3 and K2 on Inflammation ........................................................................................... 9 Summary & Potential Significance ........................................................................................... 12 Effect of a Post-exercise Drink With or Without Additional Vitamins on Hepcidin ................... 13 Introduction ............................................................................................................................... 13 Purpose, Objectives and Hypothesis ......................................................................................... 14 Research Question(s)................................................................................................................. 15 Objective(s) ............................................................................................................................... 15 Primary Research Outcomes ..................................................................................................... 15 Secondary Research Outcomes ................................................................................................. 15 Hypothesis ................................................................................................................................. 15 Experimental Design / Description of Project .............................................................................. 16 Subject Recruitment .................................................................................................................. 16 Sample Size Determination ....................................................................................................... 16 Methods ..................................................................................................................................... 17 Study Protocol ....................................................................................................................... 17 Screening Day: ...................................................................................................................... 17 Visit 1, Familiarization Day ...................................................................................................... 17 Incremental Exercise Test (IET) ............................................................................................ 17 Visits 2 to 4 Experimental Days ................................................................................................ 19   v  Post-exercise Recovery Drink Consumption ............................................................................ 20 Experimental Drink Contents ................................................................................................ 20 Laboratory Procedures .............................................................................................................. 21 Blood Collection .................................................................................................................... 21 Blood Analysis ...................................................................................................................... 21 Statistical Analysis .................................................................................................................... 22 Results ........................................................................................................................................... 23 Subject Characteristics .............................................................................................................. 23 Physiological Responses ........................................................................................................... 23 Serum Interleukin-6................................................................................................................... 23 Hepcidin-25 & Baseline Vitamin D .......................................................................................... 25 Iron Parameters ......................................................................................................................... 26 Hemoglobin & Hematocrit ........................................................................................................ 28 Discussion ..................................................................................................................................... 32 Conclusions ................................................................................................................................... 40 Strengths, Limitations, Future Directions ................................................................................. 40 Conclusions Regarding Thesis Hypotheses .............................................................................. 41 Conclusion & Practical Application .......................................................................................... 42 References ..................................................................................................................................... 43 Appendix A ................................................................................................................................... 55 Appendix B ................................................................................................................................... 56 Appendix C ................................................................................................................................... 57 Appendix D ................................................................................................................................... 61 Appendix E ................................................................................................................................... 62          vi  List of Tables  Table 1: Subject characteristics, baseline measurements and VO2max results…………….……..23 Table 2: Summary for heart rate (HR), power output (Watts), energy expenditure (Kcal), rating of perceived exertion (RPE), pre- and post-exercise mass for VPRO, PRO, and PLA conditions. Expressed in mean ± SD………………………………………………………………….………31 Table 3: Mean ± SD for serum interleukin-6 (IL-6), hepcidin-25, serum iron, serum ferritin, hemoglobin and hematocrit levels at baseline, post-exercise, and three hours post-exercise in VPRO, PRO, and PLA conditions………………………………………………………..………31                       vii  List of Figures  Fig 1: The effects of exercise and post-exercise hepcidin response on iron metabolism...…...…….3 Fig 2: Timeline of data collection (pre-screening and visit 1)…..………………………………...18 Fig 3: Timeline of data collection (experimental visits 2-4)………………….……………...........19 Fig 4: Mean ± SD of serum interleukin-6 levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions…………………………………………………....24 Fig 5: Mean ± SD of hepcidin-25 levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions………………………………………………...…………...26 Fig 6: Mean ± SD of serum ferritin levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions……………………………………………………………..27 Fig 7: Mean ± SD of serum iron levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions………………………………………………………………...28 Fig 8: Mean ± SD of serum hemoglobin levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions……………………………...…………………….29 Fig 9: Mean ± SD of hematocrit percent levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions……………………………...…………………….30                viii  List of Abbreviations ID: iron deficiency FPN1: ferroportin FECH: ferrochelatase RE: reticuloendothelial RES: reticuloendothelial system gp130: glycoprotein 130 VO2max: maximal oxygen uptake pVO2max: power output (W) at maximal oxygen uptake (VO2max) vVO2max: velocity at maximal oxygen uptake (VO2max) VO2kinetics: characteristics of oxygen uptake W: watts CBC: complete blood count ELISA: enzyme-linked immunosorbent assay EDTA: ethylenediaminetetraacetic acid SD: standard deviation RPE: rating of perceived exertion HR: heart rate HAMP: hepcidin antimicrobial peptide gene JAKs: janus kinases STAT: signal transducers and activators of transcription proteins HIF: hypoxic inducible factors ERFE: erythroferrone MPS: muscle protein synthesis  mTORC1: mammalian target of rapamycin complex1 PI3K-mTOR: phosphatidylinositol 3-kinase, mammalian target of rapamycin pathway IL-1B: interleukin-1B  IL-6: interleukin-6  IL-12: interleukin-12 IL-10: interleukin-10 TNF-α: tumor necrosis factor alpha Hbmass: hemoglobin (Hb) mass CKD: chronic kidney disease ALT: alanine AST: aspartate  RANKL: receptor activator nuclear factor-KB ligand CRP: C-reactive protein PAR-Q: physically active readiness questionnaire LPS: lipopolysaccharide K1: phylloquinone   ix  K2, MK-4: menaquinone-4 K3: menadione DMK: 2,3-dimethoxy-1,4-naphthoquinone KCAT: 2-methyl, 3-(2’methyl)-hexanoic acid-1,4-naphtoquinone LCHF: low-carbohydrate, high-fat EPO: erythropoietin RBCs: red blood cells  Hb: hemoglobin ATP: adenosine triphosphate GI: gastrointestinal tract Fe3+: ferric  Fe2+: ferrous Dcytb: duodenal cytochrome B STEAP: human 6-transmembrane epithelial antigen of prostate proteins  DMT1: divalent metal transporter 1 HPC1: heme protein carrier 1 25(OH)D: 25-hydroxyvitamin D  CHO: carbohydrate PRO: protein and carbohydrate drink VPRO: protein and carbohydrate with vitamin D3 and K2 drink PLA: placebo drink min: minute g: gram kg: kilogram hr: hour mL: milliliter L: liter UVB: ultraviolet B EE: energy expenditure Kcal: kilocalorie             x  Acknowledgements I have done a lot of crazy but amazing things in my life, such as coming face-to-face with a mountain lion, being chased by a bear, running a marathon, to even running on a broken leg for a good two months…but this Masters project is certainly on my top 10 list for one of my most proudest moments in life. To say it was challenging is an understatement, but I am thankful for everything (good and bad) I have faced along the way. It has given me a new perspective on research and how hard it is to actually carry out a study, let alone controlling for all the confounding variables when working with elite and professional athletes.  If it weren’t for some key people however, this roller coaster ride would not have been anywhere near as enjoyable as it was. A special thanks goes out to the following people:  …to all three of my committee members, Dr. Michael Koehle, Dr. Trent Stellingwerff, and Dr. Don McKenzie. I do not think I can ever put into words how much I owe them. It has been an absolute honour to have learned from them over these past two years and I will forever be thankful for this experience. The wisdom they passed on to me will be used for greatness.  …to Dr. Brad Dieter who has become a close friend and a mentor to me. Our daily chats have helped me in so many ways; from providing gracious support when I was lost in data collection, to the daily comradery, to putting up with my crazy rambles. He is a wealth of knowledge and has helped me grow as a critical researcher and person. I am forever grateful to be able to call him a lifelong friend. #Brofist.  ...to Alan Aragon, who if it weren't for you being so epically awesome, your 3 part blog post on "Directions Toward a Career in Fitness", and your amazing posts on bodybuilding (dot) com back in 2008, I don't think I would be typing this right now. You have helped inspire me (along with many others) to become great in this industry, and I thank you for that. I owe you more then you will ever know. #UltimateBroFist.    xi  …to James Brotherhood for allowing me to work with him from the start of this journey. He opened the door to so many amazing experiences and became an impeccable friend along the way. Here are too many more summers of watching epic volleyball at Kits Beach.  …to Joe McCullum and Lisa Bonang for giving me a sense of community when I first came to Vancouver, BC. Even before I started my academic career here, I was brought in as a Strength & Conditioning (S&C) intern to aid in making our varsity athletes excel in their sport. Joe and Lisa, along with my fellow interns, became a second family to me, and I thank them deeply for allowing the American to be part of it.  …to my lab mates, Sarah Koch, Eric Carter, Maha Elashi, Sean Sinden, Elliott Boake, Nadine Sinnen and Kimberly Bowman for their friendship, knowledge and putting my mind at ease when something goes haywire in lab.  …to Martin MacInnis, Kristin MacLeod and Margaux Chan who provided excellent examples and helpful advice.  …to my friends James Matson, Jordan Sahlberg, Joana Houplin, Sheryl Gilmore, Lindsay Aarseth and Dr. Erik DeRoche for giving me the courage to pursue this Master’s degree and becoming an exceptional support team. You all are epic.  …to all my undergraduate professors who had a huge impact on my life and academic career. They are exceptional mentors and phenomenal friends. A special thank you to Dr. Dave Suprak, Dr. Lorrie Brilla and Dr. Julie Vieselmeyer.  …to Kym van Duynhoven, Chris Krammer, and Tiffany Johns for helping me out when my brain was on the frits and keeping me plugging away at this massive project.  …to all of the legendary subjects who partook in this study. Without all of you willing to come in and spend over 15 hours of your time to partake in it, this study would not have come into existence.   xii   …to Helen Luk, Kathy Manson, and Maral Khoshkholgh for their amazing assistance.  …to Dr. Ted Steiner and the impeccable work done by May Wong.  …to all of the amazing individuals at the Canadian Sport Institute – Pacific (CSI-P) and Own The Podium (OTP).  …and to most importantly, to my Mom and Dad. For without them, I would not have been able to do any of this. I owe you both the world and I graciously appreciate all the love you bestow upon me, even though I’m a bit crazy from time to time. Love you two.       1  Introduction Overview Iron has central roles in oxygen delivery, heme structure, enzymatic transfers of electrons, cardiovascular health, oxidative and glucose metabolism, exercise-induced inflammation, neurological function, bone health and skeletal muscle tissue. All are critical components to athletic performance and animal and human studies have revealed that even without anemia, ID can decrease endurance performance and the oxygen carrying capacity of the blood y up to ~30% (Brownlie, Utermohlen, Hinton, & Haas, 2004; Davies et al., 1984; Davies, Maguire, Brooks, Dallman, & Packer, 1982; Dellavalle & Haas, 2011). Despite this, iron deficiency (ID) is a global issue and the most prevalent nutrient deficiency in the world (Cogswell et al., 2009; DellaValle & Haas, 2014), affecting roughly 20% of the world’s population (Schumann, Elsenhans, & Maurer, 1998) and athletes are even more susceptible to becoming ID (Koehler et al., 2012); with ~60 percent of all female athletes and ~4-50% suffering from some form of ID in a given year (Brandy, Christine, Robert, & Stephanie, 2003).  Although it seems plausible that ID can be prevented in athletes by meeting the recommended dietary intake of elemental iron (males: 8 mg/day; pre-menopausal females: 18 mg/day (Sim, Dawson, Landers, Trinder, & Peeling, 2014)), athletes need a higher intake of iron due to some of the deleterious outcomes of prolonged physical activity performed at very high intensities (Dopsaj et al., 2013); in addition to an increased iron turnover rate, gastrointestinal bleeding, foot strike hemolysis, iron malabsorption, sweat losses, inadequate hydration and, for women, menstruation (Brandy et al., 2003; Chatard, Mujika, Guy, & Lacour, 1999). Accordingly, recent data have shown that increases in inflammation caused by exercise (Varamenti et al., 2013) down-regulates iron absorption in the human digestive tract (Przybyszewska & Zekanowska, 2014) by up-regulating the gene expression of the iron regulatory protein hepcidin (Roecker, Meier-Buttermilch, Brechtel, Nemeth, & Ganz, 2005). Hepcidin is an iron-regulatory protein (Roecker et al., 2005) that inhibits iron absorption, and has been shown to have both an acute and chronic phase in its increase post-exercise (Dellavalle & Haas, 2012). It has been reported that three hours post-exercise, hepcidin levels peak, and return to pre-exercise baseline by 24 hours (Wachsmuth, Aigner, Volzke, Zapf, & Schmidt, 2015), potentially as long as athletes have not subsequently supplemented with iron (Moretti et al., 2015). This response has been demonstrated both in urine collection (Peeling et al., 2009) and blood analysis (Sim et al., 2012). It has been   2  shown that baseline serum ferritin levels correlate with resting hepcidin concentrations (Peeling et al., 2014) and the rise in hepcidin post-exercise correlates with the increase in inflammation via anti-inflammatory cytokines, specifically interleukin-6 (IL-6) (Wachsmuth et al., 2015). Oxygen Delivery and Iron Metabolism An athlete’s maximal oxygen uptake (VO2max) is one of the best indicators, and most widely accepted measurements of an individual’s cardiorespiratory (aerobic) and fitness levels (Bassett & Howley, 1999). Trained individuals have been seen to have an enhanced ability to transport and utilize oxygen that is being carried throughout the bloodstream by the globular protein, hemoglobin (Hb), located on red blood cells (RBCs) (Hoffbrand, Moss, & Pettit, 2006). In order for Hb to properly function, it must have adequate supplies of macro- and micro- nutrients and minerals, which act as key regulators for a number of biochemical reactions in the body, including metabolic function and adenosine triphosphate (ATP) production. During strenuous exercise, metabolic adenosine triphosphate (ATP) production increases up to 1,000 times (Baker, McCormick, & Robergs, 2010), signifying the importance of and an increased nutrient demand for physically active individuals or athletes training at high intensities. Although multiple physiological, biological and external factors can negatively affect the number of circulating Hb molecules in the bloodstream, and/or the quaternary protein structure of Hb (which disturbs the oxygen-hemoglobin dissociation curve) (Bottomley & Fleming, 2014), one of the most prevalent factors is dietary iron. It is well recognized that the trace mineral iron plays multiple roles in the human body. Dietary iron enables oxygen to properly bind to Hb at the alveoli, to transport it from the lungs through the blood and then unload it at the peripheral tissues (McLellan, 2004).  Iron derived from the diet goes through a complex absorption and exportation matrix that takes place in the human gastrointestinal (GI) tract, specifically, the proximal segment of the small intestine, (i.e. the jejunum and the duodenum) (Brie, Christopher, & Gregory, 2012; Okhee, 2011). There are two main membranes in the transportation process that the iron must cross to get from the GI into the enterocytes and then finally the blood (see Figure 1): the apical membrane and the basolateral membrane (Waldvogel-Abramowskia et al., 2014). Glycoproteins located in the duodenum called mucins (Przybyszewska & Zekanowska, 2014), are synthesized by GI mucosa and bind to iron ions (both ferric (Fe3+) and ferrous (Fe2+) forms), enabling absorption from the duodenum. Non-heme iron (commonly found in the Fe3+ state) is reduced to the more stable form   3  of iron, Fe2+ (Brie et al., 2012) by three different mechanisms: (1) the acidic environment of the gut, (2) reductase activities performed by duodenal cytochrome B (Dcytb) (Jeehyea et al., 2012) and STEAP proteins (Ohgami, 2006) located on the brush border (apical membrane) of the duodenum, and (3) reduction by ferrireductase (Ohgami et al., 2005). The reduced Fe2+ molecule interacts with a multitude of transporter proteins (Brie et al., 2012), mainly divalent metal transporter 1 (DMT1) (Przybyszewska & Zekanowska, 2014), to be transported into the enterocytes where it is stored as ferritin until later use. Fig 1: The effects of exercise and post-exercise hepcidin response on iron metabolism.  Heme iron absorption is less well understood, but enters the enterocyte via heme protein carrier 1 (HPC1) and is then cleaved of its hemoproteins to Fe2+ by heme oxygenase to then be added to the same pool of iron within enterocytes (Krishnamurthy, Xie, & Schuetz, 2007; Solange Le, Michael, & Miguel, 2012). Once in the enterocytes, the fate of the stored ferritin is multifaceted and is tightly regulated by the size of the iron pool and demands of iron within the body (Aisen,   4  2001). Primarily, if iron is required by the body for erythropoiesis, ferritin will be converted into Fe2+ and will exit the enterocyte via the main protein transporter, ferroportin (FPN1) (Ward & Kaplan, 2012), located on the basolateral membrane to reach the circulation.  Once in the bloodstream Fe2+ must reach the mitochondria. To do so it is transported there via two main proteins, transferrin (which contains roughly 90% of iron in the body) and transferrin receptor 1 (TfR1) (Chatard et al., 1999; Hoffbrand et al., 2006). One or two iron molecules bind to transferrin and are transported through the blood to interact with TfR1 located on cell membranes of erythroblasts in bone marrow (Punnonen, Irjala, & Rajamaki, 1997). Here is where iron is incorporated to synthesize a mature Hb protein (Hoffbrand et al., 2006). The protein Hb is largely synthesized in the mitochondria of a cell and is comprised of four polypeptide (globin) chains, α1β1 and α2β2, and a heme group. The polypeptide chains are derived from amino acids and the heme group is synthesized after protoporphyrin (derived from bone marrow) combines with iron in the Fe2+ state by the rate-limiting enzyme, ferrochelatase (FECH) (Bottomley & Fleming, 2014). However, without enough circulating iron, Hb synthesis is attenuated which will subsequently decrease oxidative metabolism, lactate threshold and aerobic performance (Dellavalle & Haas, 2012). Physical activity performed at very high intensities and/or other illnesses producing an inflammatory state impairs iron absorption from the gut (Przybyszewska & Zekanowska, 2014) by up-regulating the gene expression of the iron regulatory protein hepcidin (Roecker et al., 2005).   Hepcidin, IL-6 & Iron Absorption Hepcidin is an iron-regulatory protein (Roecker et al., 2005) that inhibits iron absorption, and has been shown to have both an acute and chronic phase in the post-exercise window (Dellavalle & Haas, 2012). The 25 amino acid cysteine-rich peptide is largely synthesized in hepatocytes (Munoz, Villar, & Garcia-Erce, 2009) and secreted into the blood from the liver (Edina et al., 2013). It is expressed in response to various stimuli (Schmidt & Prommer, 2010); such as liver iron levels, inflammation due to injury/illness (Auersperger et al., 2013), hypoxia (Haase, 2010; Liu, Davidoff, Niss, & Haase, 2012) and exercise (Auersperger et al., 2013). Upon stimulation, the hepcidin antimicrobial peptide (HAMP) gene encodes hepcidin production after the prohormone version of hepcidin, pro-hepcidin, binds to and transcribes the expression of HAMP (Edina et al., 2013). This suggests the hepcidin gene expression acts as an autoregulatory   5  loop, regulating its own production (Edina et al., 2013). Hepcidin then acts as an antimicrobial protein and controls the rate of iron exportation from the enterocytes and macrophages by binding to the main protein transporter located on the cell membranes, FPN1 (Ward & Kaplan, 2012). It has been reported that three hours post-exercise, hepcidin levels peak, and return to baseline 24 hours later (Wachsmuth et al., 2015). This time course has been in both urine and blood analysis (Peeling et al., 2009; Sim, Dawson, Landers, Trinder, et al., 2014). The increase in hepcidin concentration happens secondary to the rise of the anti-inflammatory cytokine, IL-6 (Wachsmuth et al., 2015). IL-6 is stimulated by acute inflammation and binds to IL-6 receptor alpha and gp130 (Heinrich et al., 2003). Once bound to the complex, IL-6 activates Janus kinases (JAKs) and in turn stimulates the proliferation of signal transducers and activators of transcription (STAT) proteins. Out of the STAT family, STAT3 has been shown to bind to the hepcidin promoter gene and in turn upregulates hepcidin expression (Wrighting & Andrews, 2006). Furthermore, hepcidin levels are also increased during periods of iron overload (Xinggang et al., 2012) and reversed in cases of anemia or ID (Nemeth & Ganz, 2006). Both mechanisms result in an attenuation of stored ferritin being released from enterocytes, macrophages and the liver (Munoz et al., 2009); inhibiting the body’s ability to facilitate erythropoiesis (Gammella et al., 2015). In addition to the above, hepcidin concentrations express a diurnal rhythm and are innately affected by periods of prolonged fasting (Troutt et al., 2012) and iron supplementation (Moretti et al., 2015). Multiple studies have shown that hepcidin levels are the highest in the morning, progressively decline throughout the day and then increase again in the late evening (Dale, Burritt, & Zinsmeister, 2002; Sinniah, Doggar, & Neil, 1969; Wiltink, Kruithof, Mol, Bos, & Van Eijk, 1973). Additionally, supplementation with oral iron increases hepcidin concentrations within a 24-hour period and thus further impedes iron absorption the following day (Moretti et al., 2015). Recent work has focused on different recovery manipulations in order to elucidate ways to mitigate the acute post-exercise hepcidin response in the athletic population. The use of carbohydrate nutrient partitioning on the transient shift of hepcidin following a training session will be the first part of the following section. We then elaborate on two areas that warrant further research and are the focus of this proposal, the specific nutrient timing with protein and carbohydrates, and vitamins D3 and K2.     6  Carbohydrate Feeding  Supplementation with carbohydrates (CHO) to prolong exercise performance has been an established practice in events lasting at least one hour in duration (Stellingwerff & Cox, 2014). Recent work has shown that not only does CHO intake have an effect on sparing muscle glycogen (Stellingwerff et al., 2007) leading to an increase in time to exhaustion performance, but large doses of CHO solution (6%, 250 mL every 15 minutes [4 mL kg-1]) during a 2.5-hour run can decrease exercise-induced inflammation, specifically IL-6 by 40% (Nehlsen-Cannarella et al., 1985; Nieman et al., 1998). It has been shown that depleted intramuscular glycogen content drives IL-6 response (Keller et al., 2001) which subsequently influences hepatic glucose metabolism (Steensberg et al., 2000), and the consumption of exogenous CHO in and around a bout of exercise prevents muscle glycogen depletion (Steensberg et al., 2001; Stellingwerff et al., 2007; Tsintzas, Williams, Boobis, & Greenhaff, 1995, 1996).  It has therefore been postulated that CHO intake could blunt the post-exercise hepcidin response by modulating the IL-6 response.  Robson-Ansley and colleagues (2011) utilized a much lower dosage of CHO, to prevent potential GI discomfort (Rehrer, van Kemenade, Meester, Brouns, & Saris, 1992; Smith et al., 2013; Stellingwerff et al., 2007), to see the effects of both pre- and intra- CHO ingestion on circulating hepcidin post-exercise. In a randomized double-blind crossover design, participants consumed a beverage immediately before and every 20 minutes during a 120-minute run at 60% VO2max followed by a five-km time trial. The beverages were either an 8% CHO (2 mL kg-1) solution or a matched placebo (PLA) (Robson-Ansley, Walshe, & Ward, 2011). Results showed CHO supplementation significantly reduced post-exercise IL-6 concentrations, but had no effect on serum hepcidin concentrations when compared to the PLA group,  (Robson-Ansley et al., 2011). This finding could be due to the fact that the authors only measured hepcidin immediately and 24 hours after cessation of the 120-minute run and five-km time trial, and not three hours post-exercise when levels have been shown to peak (Dellavalle & Haas, 2011). Furthermore, hepcidin levels return to baseline measurements 24 hours thereafter. However, recent work by Sim and colleagues (2012) further built on the model that CHO supplementation during exercise could blunt the post-exercise hepcidin response, and strengthened the design by looking at hecpidin responses both immediately, three hours and 24 hours post-exercise. The researchers concluded that ingestion of a 6% CHO solution at 3 mL kg-1 every 20 minutes during the endurance run (90 minutes at 75% peak oxygen uptake velocity (vVO2peak))   7  had no effect on inflammatory biomarkers (e.g., IL-6) or serum hepcidin levels when compared to consuming water alone (Sim et al., 2012). Ihalainen and colleagues (2014) supported the above findings, reporting that post-exercise CHO supplementation at various doses (0.0%, 1.5% and 7.0%) had no effect on IL-6 concentrations following a continuous 18- to 20- km run at 75% of VO2max in seven recreational runners (4 men and 3 women) (Ihalainen, Vuorimaa, Puurtinen, Hamalainen, & Mero, 2014). Badenhorst and colleagues (2015) showed in 11 well-trained endurance athletes that early (immediately post-exercise and two hours post-exercise) or late (two hours post-exercise and four hours post-exercise) post-exercise feeding with 1.2 g kg-1 of CHO supplementation did not significantly alter post-exercise IL-6 and hepcidin responses in either condition after 8 x 3 minute high intensity interval runs at 85% vVO2max. The authors did show a time-dependent increase in hepcidin concentrations both three and five hours post-exercise compared to baseline, but there was no significant difference between the early or late feeding strategies (Badenhorst et al., 2015b). Recently, low-carbohydrate, high-fat (LCHF) diets have gained in popularity in the athletic population. Although controversy exists whether one can actually increase performance by adapting to a LCHF diet (Burke, 2015), recent finding from Badenhorst and colleagues (2015) showed that over the course of 24 hours, a moderate CHO diet (3 g kg/day-1) in well-trained endurance athletes (VO2peak: 68.9 ± 7.2 mL kg-1 min-1) resulted in significantly higher pre- and post- exercise measurements of hepcidin when compared to a high CHO diet (10 g kg-1). The authors report that the reason for the increase hepcidin levels are due to the upregulation of gluconeogenic signaling of the liver caused by the impaired glycogen stores from the LCHF diet (Badenhorst et al., 2015a).   Protein and Inflammation The common practice of elite athletes training multiple times per day, is to consume some form of high-quality protein in order to maximize training adaptations and initiate the remodeling of skeletal muscle tissue (Phillips & Van Loon, 2011). Moreover, an athlete’s ability to recover from strenuous exercise relies on the ability to upregulate net muscle protein synthesis (MPS). Multiple physiological, biological and morphological factors can negatively affect that rate of protein synthesis in skeletal muscle tissue (Schoenfeld, 2010). The mammalian target of rapamycin complex 1 (mTORC1) is commonly accepted as one of the master regulators for cellular growth   8  and regeneration (Knight, Schmidt, Birsoy, Tan, & Friedman, 2014). Inhibition of mTORC1 will lead to a potential decrease in hypertrophic and strength adaptations to resistance training  (Atherton & Smith, 2012). More specifically, the PI3K-mTOR pathway has been shown to modulate the expression of pro- and anti- inflammatory cytokines involved in the acute phase response of exercise (Weichhart & Saemann, 2009). Mutations in PI3K-mTOR leads to an increase inflammatory state and atrophy to muscle tissue (Weichhart & Saemann, 2009), which could be mediated by chronic elevations IL-6 (Haddad, Zaldivar, Cooper, & Adams, 2005). However, the initial release of IL-6 from working skeletal muscle plays a pinnacle role in the growth and regeneration of muscle tissue via the stimulation of myosatellite cells and the incorporation of new myonuclei into pre-existing muscle fibers (Morgan & Partridge, 2003; Serrano, Baeza-Raja, Perdiguero, Jardi, & Munoz-Canoves, 2008). Additionally, the JAK/STAT pathway described earlier, has a dual role in proliferation and differentiation of muscle cells (Sun et al., 2007; Wang, Wang, Xiao, Wang, & Wu, 2008). First, the JAK1/STAT1/STAT3 pathway prevents premature myoblast differentiation and fusion by blocking the expression of MyoD and MEF2 (Fernando, Kelly, Balazsi, Slack, & Megeney, 2002; Hunt, Upadhyay, Jazayeri, Tudor, & White, 2011). Alternatively, the JAK2/STAT2/STAT3 pathways augments the expression of the above proteins, in addition to increasing growth factors related to hypertrophy, such as insulin-like growth factor (IGF) (Glass, 2010; Jacquemin, Furling, Bigot, Butler-Browne, & Mouly, 2004). The PI3K-mTOR pathway is activated in response to a favorable energy status (eucaloric or hypocaloric), growth factors (e.g., IGF), amino acids and cellular stress (Howell & Manning, 2011; Sengupta, Peterson, & Sabatini, 2010). Consequently, mTORC1 located in erythrocytes is down-regulated in response to low iron (both in vivo and in vitro), low energy availability and deoxyribonucleic acid (DNA) damage (Dunlop & Tee, 2009; Soliman, 2005); which indicates that mTORC1 is, in part, mediated by the availability of iron in the blood (Knight et al., 2014). Furthermore, it has been demonstrated that mTOR plays a part in regulating the expression of hepcidin by direct inhibition of HAMP gene expression (Mleczko-Sanecka et al., 2014). Thus, being ID and having chronically elevated levels of IL-6 could be deleterious to the human body. By upregulating mTORC1 by maximizing MPS could potentially lead to the suppression of hepcidin either indirectly (e.g., blunting chronic elevations of IL-6) or directly (e.g., inhibiting HAMP gene expression).    9  Three grams of the essential amino acid, leucine, maximizes muscle protein synthesis (Pennings et al., 2011; Tang, Moore, Kujbida, Tarnopolsky, & Phillips, 2009). Whey protein has been shown to have a greater effect on net protein synthesis in the muscle when compared to casein and soy (Pennings et al., 2011; Tang et al., 2009), due to in part the high leucine content (Churchward-Venne et al., 2014; Rowlands et al., 2015). Evidence shows that when ample amounts of carbohydrates (CHO) are added to a protein-rich meal post-exercise, it further potentiates muscle glycogen re-synthesis and recovery (Berardi, Price, Noreen, & Lemon, 2006; Howarth, Moreau, Phillips, & Gibala, 2009; Ivy et al., 2002). Thus, it could be postulated that combining ample amounts of CHO and protein around a bout of exercise, could blunt hepcidin by putting an individual in an energy positive state while supplying ample amounts of amino acids to stimulate mTOR. Kerasioti et al (2013) tested this hypothesis by taking nine physically-active men (age: 28 ± 2 years, VO2max: 4.1 ± 0.2 L/min) and subjecting them to a two-hour exhaustive cycling bout. Immediately post-exercise, subjects either consumed a special calorically matched CHO-whey cake with high protein (23.92 ± 0.40 g) or low protein (9.20 ± 0.40 g). Compared to the low protein cake, the high protein cake significantly (p < 0.05) reduced levels of IL-6 (-50%) and CRP (-46%) four hours post-exercise (Kerasioti et al., 2013). Furthermore, a recent intra-exercise supplementation study by Schroer et al (2013) showed a significant decrease in IL-6 activity following a 120-minute constant load cycle (55% of peak power) when subjects supplemented with 90 g of whey protein hydrolysate (45 g hour-1) compared to a noncaloric sweetened (Splenda™) placebo drink (Schroer, Saunders, Baur, Womack, & Luden, 2014). The authors concluded that the differences in IL-6 activity following the session were likely due to the protein ingestion and a reduction of metabolic damage. Given the associations with hepcidin and inflammation, one can theorize that an ingestion of a protein-rich drink following a bout of fatiguing exercise may be able to blunt the hepcidin rise three hours following cessation. This hypothesis has yet to be tested in the current literature.  Vitamin D3 and K2 on Inflammation Vitamin D, a fat-soluble vitamin (McCollum, Simmonds, Becker, & Shipley, 1922),  is an essential vitamin acting as a precursor steroid to a host of metabolic and biological processes in the human body. Insufficient levels in the major circulating metabolite of vitamin D, 25-  10  hydroxyvitamin D [25(OH)D] and the active hormonal form, 1,25-dihydroxyvitamin D have been shown to significantly (p < 0.001) decrease hemoglobin concentrations in a linear fashion (Patel et al., 2010). Patel and colleagues (2010) reported that insufficient levels were associated with a higher prevalence of anemia (2-3 fold increase) when compared to subjects with higher levels (Patel et al., 2010). Recent research has focused on the benefits of vitamin D supplementation to decrease inflammatory cytokines (Nonn, Peng, Feldman, & Peehl, 2006), when induced by exercise (Barker, Schneider, Dixon, Henriksen, & Weaver, 2013), in healthy individuals (Bacchetta et al., 2014) and in chronically ill patients with chronic kidney disease (CKD) (Zughaier, Alvarez, Sloan, Konrad, & Tangpricha, 2014). More explicitly, vitamin D supplementation has been shown to blunt hepcidin via the direct reduction of HAMP expression and up-regulating FPN1 (Bacchetta et al., 2014). Two recent studies have shown that dosages of 50,000 IU (7,143 IU/day for three months) and 100,000 IU (single dose) of vitamin D can significantly reduce hepcidin levels (28% and 34%, respectively) in healthy individuals and patients suffering from CKD (which affects over 40% of adults ages 65 and older) (Bacchetta et al., 2014; Zughaier et al., 2014). Zughaier and colleagues (2014) further demonstrated a dose-dependent response in lipopolysaccharide (LPS) stimulated THP-1 cells treated with 1,25-dihydroxyvitamin D from 5 nm to 40 nm lead to a subsequent decrease in pro-hepcidin cytokines (Zughaier et al., 2014).  Furthermore, at a much lower dosage and in physically-active healthy male subjects, Barker and his colleagues (2013) showed an inverse relationship with vitamin D supplementation and exercise-induced inflammation in a randomized, double-blind, placebo-controlled study. The authors demonstrated that 4,000 IU/day of vitamin D over 28 days, significantly (p < 0.05) decreases the inflammatory biomarkers alanine (ALT) and aspartate (AST) after 10 sets of 10 repetitions of peak isometric force eccentric-concentric jumps 24, 48, 72 and 168 hours later. The literature regarding toxicity levels of vitamin D remain inconclusive (Heaney, 2008; Holick, 2005). In a recent review by our lab, a relationship has been seen with vitamin D acting synergistically with vitamin K in order to prevent toxicity and calcification (Dahlquist, Dieter, & Koehle, 2015). Specifically, a vitamin K2 variant, MK-4, has been shown to regulate osteoclastogenesis. Vitamin K2 prevents vascular calcification and hypervitaminosis by carboxylating ostecalcin proteins formed in mature bone cells stimulated by receptor activator nuclear factor-kB ligand (RANKL) (Gundberg, Lian, & Booth, 2012). RANKL is produced via   11  the stimulation of vitamin D. If vitamin K is not supplemented with vitamin D, ostecalcin proteins remain un-carboxylated, which potentially causes vascular calcification and toxicity (Koul et al., 2011). Additionally, un-carboxylated osteocalcin cells has been shown to increase IL-6 and C-reactive protein (CRP) concentrations in the elderly from poor plasma levels of vitamin K (Shea, Dallal, et al., 2008). This suggests that increasing vitamin K stores may reduce inflammatory cytokine activity, in addition to preventing vitamin D toxicity.  Redi and colleagues (1995) demonstrated that various vitamin K variants inhibit the production of IL-6 via lipopolysaccharide (LPS) stimulated human fibroblasts, in vitro (Reddi et al., 1995). Each compound tested, phylloquinone (K1), menaquinone-4 (K2, MK-4), menadione (K3), 2,3-dimethoxy-1,4-naphthoquinone (DMK), and 2-methyl, 3-(2’methyl)-hexanoic acid-1,4-naphtoquinone (KCAT) [synthetically derived] were capable of inhibiting IL-6 production at varying degrees. Out of the commonly found vitamin K variants in supplements and food, K1 and K2, MK-4 decreased IL-6 production to a greater extent than K1 (~195 pg/mL and ~95 pg/mL, respectively) (Reddi et al., 1995). Rat models have demonstrated that vitamin K-rich diets can decrease LPS-induced inflammation when compared to vitamin K-deficient diets (Koul et al., 2011; Ohsaki et al., 2006). Furthermore, higher concentrations of circulating vitamin K are significantly (p < 0.05) associated with lower concentrations of inflammatory markers, specifically CRP (Shea, Booth, et al., 2008). In a study of 510 elderly patients with type II diabetes mellitus evaluated after a one-year follow-up, individuals consuming a vitamin K-enriched (70.5 to 767.5 mcg/day) diet had lower concentrations of IL-6 (-27.9%) and other inflammatory cytokines when compared to those with insufficient (≤ 69.4 mcg/day) vitamin K diets (Juanola-Falgarona et al., 2013). However, it is uncertain whether vitamin K1 supplementation at dosages of 500 mcg/day are sufficient enough to decrease IL-6 activity over a 3-year time period in obese individuals (Shea, Dallal, et al., 2008) and standard American diets have been shown to have far less than the RDA (Booth, Webb, & Peters, 1999), such as 12-24 mcg/day in all age groups (2-70+ years of age)  (Booth, Pennington, & Sadowski, 1996a) with the exception of infants fed baby formula (Booth, Pennington, & Sadowski, 1996b; Shearer, 2009; von Kries & Gobel, 1992). The above findings suggest the need for further studies with higher dosages of vitamin K and different vitamin K variants.  Approximately 4,000 to 5,000 IU/day of vitamin D3 in combination with 50 to 1,000 mcg/day of vitamin K1 and K2 seems to be a safe dose and has the potential to aid athletic   12  performance. Additionally, based on the above literature, dosages in this range can potentially blunt the post-exercise hepcidin response if timed appropriately.  Summary & Potential Significance Athletes, both female and male, are susceptible (~60% and ~4 to 50%, respectively) to the adverse effects of ID. In individuals with ID, VO2max can drop by up to 34% (Booth et al., 1996a; Woodson, Wills, & Lenfant, 1978). When it comes to the elite few, marginal gains of a mere 1% can dictate whether or not an Olympic Athlete will place on the podium (10,000 m Final, London Olympics 2012, 3rd and 4th), this 34% reduction is problematic. There are currently many strategies to correct ID non-anemic and ID anemic athletes (DellaValle & Haas, 2014; Hinton & Sinclair, 2007; Wachsmuth et al., 2015); which have all shown to augment performance measurements when compared to a placebo. In contrast, research shows inconsistent results when trying to prevent ID from occurring by mitigating the rise of hepcidin following training. As previously mentioned, both macro- and micro- nutrients have potent effects on post-exercise inflammatory biomarkers which are associated with hepcidin. Intervention trials manipulating CHO alone have had equivocal findings (Badenhorst et al., 2015b; Ihalainen et al., 2014; Nehlsen-Cannarella et al., 1985; Nieman et al., 1998; Robson-Ansley et al., 2011; Sim et al., 2012), and more so, restricting CHO intake over a 24-hour period can increase one’s susceptibility to ID by elevating baseline hepcidin concentrations (Badenhorst et al., 2015a). Protein ingested during (Schroer et al., 2014) and after with additional CHO (Kerasioti et al., 2013) has been shown to down regulate IL-6 activity both immediately after exercise and four hours after exercise. Furthermore, recent research has focused on the benefits of vitamin D supplementation to decrease inflammatory cytokines (Nonn et al., 2006), when induced by exercise (Barker et al., 2013), in chronically ill patients with CKD (Zughaier et al., 2014) and healthy individuals (Bacchetta et al., 2014). Lastly, vitamin K has been shown to decrease IL-6 concentrations (Ohsaki et al., 2006; Reddi et al., 1995) and prevent vitamin D toxicity (Dahlquist et al., 2015).  Given the associations of hepcidin release with inflammation (e.g., IL-6), an ingestion of a CHO plus protein rich drink with or without the addition of vitamin D3 and K2 following a bout of fatiguing exercise may be able to blunt the post-exercise hepcidin response. This hypothesis has yet to be tested.    13  Effect of a Post-exercise Drink with or without Additional Vitamins on Hepcidin Introduction Iron deficiency (ID) is disturbingly prevalent in elite endurance athletes (~60% of all female athletes (Brandy et al., 2003) and ~4 to 50% of all male athletes (Hinton, 2014) suffer from some form of ID in a given year), with significant potential for deleterious effects on performance. Iron-repletion studies in ID and ID non-anemic athletes have shown significant (p < 0.05) reductions in fat mass and increases in lactate threshold, energetic efficiency and expenditure, ventilatory threshold, and in severe ID cases when an athlete takes an iron supplement, studies have shown a 7.4% increase in maximal oxygen uptake (VO2max) and Hbmass (DellaValle & Haas, 2014; Hinton & Sinclair, 2007; Wachsmuth et al., 2015). Hbmass is an indicator of aerobic capacity in elite field hockey players (Hinrichs et al., 2010), and every one gram increase in Hbmass results in a 2.3 mL/min increase in VO2max (Wachsmuth et al., 2015).  Some initial and preliminary research has focused on different recovery modalities and nutritional interventions in order to try and mitigate the increase in acute post-exercise hepcidin response in athletic populations (Badenhorst et al., 2014; Robson-Ansley et al., 2011; Sim et al., 2012), which would mechanistically increase the bioavailability of iron absorption and recycling (Ganz & Nemeth, 2012). Large doses of carbohydrate (CHO) solution (6%, 250 mL every 15 minutes [4 mL kg-1]) during a 2.5-hour run can decrease exercise-induced inflammation, specifically IL-6, by 40% (Nehlsen-Cannarella et al., 1985; Nieman et al., 1998). Despite this, four recent studies in trained endurance athletes have shown intra- or post- CHO supplementation has no effect on hepcidin following high intensity runs (Badenhorst et al., 2015b; Ihalainen et al., 2014; Robson-Ansley et al., 2011; Sim et al., 2012). Furthermore, low CHO availability over a 24 hour period has been shown to increase baseline and post-exercise hepcidin levels in trained men (Badenhorst et al., 2015a).  A recent intra-exercise protein supplementation study by Schroer et al. (2014) showed a significant (p < 0.05) decrease in IL-6 activity following a 120 minute constant load cycle (55% of peak power) performance when subjects supplemented with 90 g of whey protein hydrolysate over two hours (45 g hour-1) compared to a noncaloric sweetened (Splenda) placebo drink (Schroer et al., 2014). The differences in IL-6 activity in this study design were postulated to be contributed to the protein ingestion (Schroer et al., 2014). Furthermore, consumption of a special CHO-whey protein cake has shown a favorable effect on inflammation following an exhaustive bout of cycling   14  (two hours) (Kerasioti et al., 2013). Compared to the low protein cake (Total Intake: 101.20 ± 3.40 g of CHO and 9.20 ± 0.4 g of protein), the high protein cake (Total Intake: 82.80 ± 3.40 g of CHO and 23.92 ± 0.40 g of protein) significantly (p < 0.05) reduced the inflammatory levels of IL-6 (-50%) and C-reactive protein (CRP) (-46%) four hours post-exercise (Kerasioti et al., 2013). Given the associations of hepcidin release with inflammation (and IL-6), an ingestion of a CHO plus protein rich drink following a bout of fatiguing exercise may be able to blunt the post-exercise hepcidin response. Furthermore, this mimics that of a more common practice of elite athletes post-training, which is to co-ingest CHO and protein in order to maximize training adaptations and initiate the remodeling of skeletal muscle tissue (Phillips & Van Loon, 2011). Lastly, recent research has focused on the benefits of vitamin D supplementation to decrease inflammatory cytokines (Nonn et al., 2006), when induced by exercise (Barker et al., 2013), in chronically ill patients with chronic kidney disease (CKD) (Zughaier et al., 2014) and healthy individuals (Bacchetta et al., 2014). Additionally, vitamin K has been shown to decrease IL-6 (Ohsaki et al., 2006; Reddi et al., 1995) and prevent vitamin D toxicity (Dahlquist et al., 2015).  Therefore, instead of focusing on the iron supplementation side of therapy which in essence, is repairing something that is already broken, the main objective of the study is to reverse the hepcidin mediated interference with iron absorption through specific nutrient timing with essential macro- and micro- nutrients. Purpose, Objectives and Hypothesis Based on the literature presented above, the primary objective of the project was to employ a novel yet practical method to address iron malabsorption and ID by down-regulating the exercise-induced protein hepcidin following a training session with a CHO and protein-rich post-exercise drink with or without the addition of vitamins D3 and K2. Decreasing the activity of hepcidin via this method could help increase iron absorption in athletes and prevent issues of ID. Lastly, we aimed to investigate whether there was a further benefit from adding additional vitamins (D3 and K2) to the post-exercise drink to determine whether hepcidin was not further blunted when compared to the consumption of protein and CHO alone.       15  The Aims and Objectives of This Study are Listed Below  Research Question(s) To determine if a post-exercise drink consisting of whey protein isolate (~25g) and CHO (~75g) with the addition of vitamins, D3 (5,000 IU) and K2 (1,000 mcg) (VPRO) or without vitamin addition (PRO) following a bout of exercise has an effect on the acute post-exercise hepcidin response in athletes as compared to a placebo drink (zero calorie; PLA). Objective(s) 1) To investigate the effects of PLA vs. VPRO vs. PRO drink following a high intensity exercise bout on inflammation and hepcidin, 2) To test a novel recovery modality that can be utilized by athletes to mitigate iron malabsorption. Primary Research Outcomes The specific aim of this project was to address ways athletes can manipulate their recovery period from training in order to optimize iron absorption and distribution by blunting the hepcidin response. Secondary Research Outcomes To determine if a post-exercise drink consisting of high CHO and protein content derived from whey protein immediately following a bout of an exhaustive training session had an effect on the acute post-exercise inflammatory markers. Hypothesis (1) That both VPRO and PRO will significantly decrease hepcidin following a bout of high intensity exercise as compared to PLA; and (2) VPRO supplementation will have a greater effect on hepcidin versus PRO supplementation alone.     16  Experimental Design / Description of Project Subject Recruitment Ten male elite endurance athletes were recruited for the study with the following inclusion criteria: endurance or endurance-power sport athletes who were healthy, physically active, currently or previously competing at a high level (e.g., Elite, Professional or CAT 1/2/3 for Cycling, International or National Level, etc.), were not consuming anti-inflammatories on a regular basis, 18-45 years of age, and training 5-6 times a week with at least ≥ 1 year of training experience. Furthermore, athletes were required to have baseline serum ferritin levels of ≥ 30 mcg/L in order to partake in the study (Auersperger et al., 2013; Peeling et al., 2014), and subjects refrained from taking vitamin D, vitamin C, vitamin K, and multi-vitamins 24 hours prior to experimental days were. Furthermore, subjects refrained from using fish oil, krill oil, turmeric and serrapeptidase supplements for 48 hours prior to experimental days. Female participants were excluded from the study due to the effects of the menstrual cycle and estradiol on hepcidin (Sim, Dawson, Landers, Trinder, et al., 2014; Yang, Jian, Katz, Abramson, & Huang, 2012). Once the criteria were met, subjects completed a Physically Active Readiness Questionnaire (PAR-Q) and provided written consent. The University of British Columbia Clinical Research Ethics Board granted full approval for the study (H15-00721).  Sample Size Determination A conservative sample size of n = 10 was determined using data from (Badenhorst et al., 2014).  In this study, 10 well-trained male endurance athletes completed two 8 x 3 minute intervals running sessions at 85% of their maximal aerobic velocity (vVO2max) on a motorized treadmill before being randomly allocated to a three-hour recovery period in a hypoxic (~2,900 m above sea level) or normoxic environment. There was a significant time (p = 0.01) and interaction effect (p = 0.049) between hypoxic and normoxic conditions on hepcidin-25. Based on the differences of mean and standard deviations between baseline measurements and three hours post-exercise for the hypoxic intervention (2.18 and 1.26, respectively) on the biomarker, hepcidin-25, an effect size of (dz = 1.730159) and a minimum sample size of n = 9 subjects were needed.    17  Methods Study Protocol A randomized, placebo-controlled, single-blinded triple crossover design was used. Ten elite and professional male cyclists (age: 26.9 ± 6.4) with a VO2max of 67.4 ± 4.4 mL/kg/min partook in four separate cycling sessions. A 48-hour washout was used and each session took place at the same time of day. Subjects recorded their standard diet and daily activity the day prior to the experimental visits and fasted for 10 hours overnight before reporting to the laboratory. Subjects were provided a standardized meal, which they consumed 60 minutes prior to arrival. Each meal was provided to the subjects on the previous visit (e.g., meal for visit 2 was given to the subjects after visit 1). The standardized meal consisted of a choice of the following food items: 3 packs of granola bars (Natures Valley, Crunchy – Honey & Oats; Minneapolis, MN, US). Subjects had the option to consume as much or as little as they wanted, and were required to repeat the exact selection on subsequent visits (3 and 4) (e.g. if they ate only 1 granola bar on visit 2, they had to eat only 1 granola bar on visits 3 and 4). Screening Day Prior to the familiarization phase (visit 1), subjects reported to a local laboratory in order to measure baseline serum ferritin and serum vitamin D, in order to rule out ID and ID anemia (Figure 2). Subjects with ferritin levels of ≥ 30 mcg/L were excluded from the study.  Visit 1, Familiarization Day Visit 1 lasted two hours and consisted of equipment familiarization and VO2max testing using an incremental cycle test (IET). Subjects then performed a familiarization to the exercise challenge, consisting of an 8-minute warm-up (cycle) at 50% power output (Watts) at VO2max (pVO2max), followed by 2 x 3 minute intervals at 85% pVO2max. Each interval was separated by a 1.5 minute of active recovery at 60% pVO2max (Figure 2). Incremental Exercise Test (IET) Participants performed a 10-minute self-selected warm-up protocol on their own bike on a cycle trainer (Wahoo KICKR, Wahoo Fitness, Atlanta, GA, US). Upon completion of the warm-up, the IET began immediately. Subjects were required to pedal at a cadence of ≥ 60 revolutions per minute (rpm) and work rate began at 0 Watts (W) (Wilkie, Dominelli, Sporer, Koehle, & Sheel, 2015). Work rate increased by 30 W every minute until volitional exhaustion. Subjects were fitted with a heart rate monitor (Wahoo TICKR, Wahoo Fitness, Atlanta, GA, US) and a face-mask (Oro-  18  Nasal 74550 V2 Mask, Hans Rudolph,  Shawnee, KS) connected to a two-way non-rebreathing valve (2700 T-shape, Hans Rudolph; Shawnee, KS) attached to a metabolic cart (TrueOne, Parvo Medics; Sandy, UT) in order to assess VO2kinetics throughout the IET. The subjects’ VO2max was then utilized to determine cycling power output (W) at percent (%) VO2max (pVO2max) for subsequent cycling tests.  Fig 2: Timeline of data collection (pre-screening and visit 1). After subjects passed pre-screening, they arrived to the laboratory to take part in visit 1. Visit 1 consisted of a self-selected warm-up directly into an incremental exercise test (IET) to exhaustion to determine pVO2max for experimental visits (visits 2-4). Following the IET, subjects commenced a familiarization protocol. After familiarization, subjects began a self-selected cool-down and left the laboratory.    19  Visits 2 to 4 Experimental Days  During the three experimental days, venous blood samples were collected upon arrival, immediately post-exercise, and three hours post-exercise. Body mass (kg) was recorded both pre- and post-exercise. Subjects were allowed to drink water ad libitum during the trial. They were positioned on their own bike (used in visit 1) attached to the ergometer and fitted with a heart rate monitor. After an 8-minute warm-up at 50% pVO2max, subjects began the exercise test of 8 x 3 minute intervals at 85% pVO2max with a 1.5 minute active recovery (60% pVO2max) separating the bouts. Heart rate (HR) was collected throughout and rating of perceived exertion (RPE) was collected after each 3-minute interval. Following the final interval and 8-minute cool-down cycle at 50% pVO2max, a blood sample was taken. Immediately following the blood sample, subjects consumed one of the three experimental recovery drinks in a randomized single-blind triple crossover fashion (see below). Participants then rested in the laboratory for three hours, a venous blood sample was collected, and then subjects departed (see Figure 3). Total time for experimental days was approximately 6 hours.   20  Fig 3: Timeline of data collection (experimental visits 2-4). After baseline blood sampling and anthropometrics were taken, subjects were set up on their own bicycle on the cycle trainer and fitted with a heart rate monitor. They then commenced the warm-up followed by the high intensity cycling task and subsequently the cool-down immediately following the final interval. Once subjects were off the bicycle, body mass (kg) was recorded and a second blood sample was taken. They then consumed one of the three post-exercise drinks (VPRO, PRO, or PLA) within 10 minutes following the end of the cool-down, then rested in the laboratory for three hours (recovery period). Following the recovery period, a final blood sample was taken and then participants departed. Post-exercise Recovery Drink Consumption One of three post-exercise drinks was immediately consumed within 10 minutes after the cessation of the cool-down in a randomized single-blind triple crossover fashion consisting of the following ingredients: 1) PLA: non-nitrogenous, zero calorie control drink (artificial flavour, sweetener and water). 2) VPRO: 75g of CHO (maltodextrin), 25g of protein (whey protein supplement), 10 droplets of a vitamin D complex containing 5,000 IU of vitamin D3 and 1,000 mcg of vitamin K2 (as menatetrenone), artificial flavour, sweetener and water. 3) PRO: 75g of CHO (maltodextrin), 25g of protein (whey protein supplement), artificial flavour, sweetener and water. Experimental Drink Contents Experimental drinks consisted of a whey protein supplement (Whey Protein Isolate – Vanilla - Restore, EXOS Fuel; Sandpoint, ID, US) containing whey protein isolate, sunflower lecithin, evaporated cane juice sugar, carboxymethylcellulose gum, xanthan gum, and stevia extract (leaf). 32g of dry protein powder (~1 scoop), equating to ~25g of protein, was weighed using a digital food scale and mixed with 550 mL of water for both PRO and VPRO conditions. Unflavoured maltodextrin (Cytocarb2, CytoMax - CytoSport; Benicia, California, US) was used as the carbohydrate (CHO) source for each drink (1g of dry powder = 1g of CHO), and was sweetened with a commercially-available artificial flavour and sweetener (Kraft Foods, Crystal Light; Northfield, IL, US). Additionally, 10 droplets of a vitamin D + K complex (Vitamin D + K Complex, EXOS Fuel; Sandpoint, Idaho, US) containing 5,000 IU of vitamin D3 and 1,000 mcg of vitamin K2 was placed in the VPRO drink only. The placebo (PLA) drink consisted of 550 mL of water and the same artificial flavour and sweetener utilized for PRO and VPRO. All drinks   21  looked, smelled, and tasted similar and were mixed in identical opaque bottles by a designated team member to blind the subject. Laboratory Procedures Blood Collection  Venous blood samples were taken at three different time periods, prior to exercise, pre-supplementation (post-exercise), and three hours post-exercise to measure serum ferritin, serum iron, serum interleukin-6 (IL-6), serum hemoglobin (Hb), hematocrit and hepcidin-25. All venous blood samples were taken after the subject rested in a seated position for five minutes in order to minimize the confounding effects of plasma volume changes due to posture. Blood was sampled with a 21-gauge needle into two 5 mL SST Gel separator tubes, a 3 mL SST Gel separator tube, a 4.5 mL PST Gel separator tube, and a 3 mL EDTA collection tube for blood sampling. 20.5 mL of blood was collected with each sample, for a total blood collection of 61.5 mL (~1% of total blood volume). Immediately following blood collection, the fresh samples (3 mL SST, 4.5 mL PST, and 3 mL EDTA) were taken to the University of British Columbia Hospital laboratory to perform a complete blood count (CBC), serum iron and serum ferritin. The two 5.0 mL SST samples were allowed to clot for 60 minutes at room temperature and then centrifuged at 10 oC and 1,500 G for 10 minutes. The centrifuged samples were separated into 1 mL aliquots and stored at -80 oC until later use. Once the blood samples were ready to be analyzed, they were transferred to a paid service lab where they carried out enzyme-linked immunosorbent assay (ELISA) on the blood biomarkers described above. Blood Analysis Blood was analyzed within a 24-hour period for Hb (CBC), serum iron and serum ferritin. The remaining frozen serum samples were used to measure the bioactive form of hepcidin, hepcidin-25 via c-ELISA, and IL-6. Serum IL-6 was analyzed using a commercially-available ELISA (Quantikine HS, R&D Systems; Minneapolis, Minnesota, USA) with a sensitivity of 0.11 pg/mL and range of 0.2-10 pg/mL. Hepcidin-25 was assessed using a commercially-available ELISA (Quantikine HS, R&D Systems; Minneapolis, Minnesota, USA) with a sensitivity of 3.81 pg/mL and a detection range of 15-1,000 ng/mL.     22  Statistical Analysis Results were analyzed utilizing Microsoft Excel (Microsoft, Office – Excel; Redmond, WA, US) and SPSS software (IBM North America, New York, NY). Data and results were reported as mean ± standard deviation (SD). A 3x3 repeated measures analysis of variance (supplement type X time) for each of hepcidin-25, Hb, IL-6, serum ferritin and serum iron was performed to assess the differences between the three conditions. Both IL-6 and hepcidin-25 were tested for order effect. Paired sample t-tests were performed to determine significance between treatments. A multivariate general linear model was performed in order to determine if starting baseline vitamin D measurements had a significant effect on individual post-exercise hepcidin responses to the VPRO drink only. The data that did not pass normality were transformed using the natural logarithm. Data that were statistically analyzed utilizing log transformed data are presented as mean ± standard deviation (SD) of non-transformed data within tables and results. Data presented in figures are either transformed or non-transformed (indicated on figures). F-ratios will be found significant at p ≤ 0.05.       23  Results Subject Characteristics Ten healthy, highly-trained male elite and professional athletes volunteered for the study. The subject pool consisted of Category 1 and 2 road and track cyclists, a national level track cyclist, two professional mountain bikers and one professional triathlete. Subject characteristics, baseline measurements, and VO2max results are shown in Table 1. Table 1: Subject characteristics, baseline measurements and VO2max results. Physiological Responses Mean ± standard deviations (SD) of HR (bpm), RPE [1-10 Borg scale] (G. Borg, 1998; G. A. Borg, 1981), energy expenditure (Kcal) (Haakonssen, Martin, Burke, & Jenkins, 2013; Vogt et al., 2005), pre- and post-exercise body mass (kg) and mean power output (Watts) for the interval cycling trials are presented in Table 2. There were no significant differences between HR, RPE, pre- and post-exercise mass and mean power output between conditions. There was a significant difference between pre- and post-exercise body mass for PRO (p = 0.001), VPRO (p = 0.001) and PLA (p < 0.001), where mass decreased from pre- to post-exercise. Serum Interleukin-6 There was a significant time effect (F[1.901, 15.205] = 53.638, p < 0.001), for PLA, VPRO and PRO (see Figure 4).  However, there was no significant condition (F[1.241, 9.929] = 0.012, p = 0.948) effect on IL-6 (Table 3). There was no significant difference in baseline measurements of IL-6 for VPRO v PRO (t[-1.586], p = 0.147), VPRO v PLA (t[-0.407], p = 0.694), or PRO v PLA Subject (#) Age (years) Weight (kg) Ferritin (ug/L) Vitamin D (nmol/L) VO2max (mL/kg/min) Wmax (WATTS) @ VO2 Watts / kg1 21 75.5 102 101 64.3 480 6.42 24 65.0 82 132 71.9 460 7.13 22 73.2 129 86 62.4 417 5.74 24 66.3 70 54 69.1 429 6.55 21 74.5 53 86 69.9 471 6.36 22 75.3 313 59 61.3 457 6.17 36 69.4 123 67 70.8 435 6.38 38 75.9 81 34 61.9 430 5.79 30 83.5 44 72 70.0 527 6.310 31 61.7 94 54 72.5 410 6.6Mean 26.9 72.0 109 75 67.4 452 6.3SD 6.4 6.4 77 28 4.4 35 0.4Min 21.0 61.7 44 34 61.3 410 5.7Max 38.0 83.5 313 132 72.5 527 7.1  24  (t[0.733], p = 0.482). There was a significant increase from baseline to post-exercise for VPRO (2.869 ± 1.367 pg/mL, p = 0.001), PRO (2.304 ± 1.649 pg/mL, p = 0.001) and PLA (2.060 ± 1.604 pg/mL, p = 0.001), and then a significant decrease from post-exercise to three hours post-exercise for VPRO (-2.153 ± 1.432 pg/mL, p = 0.001), PRO (-1.832 ± 1.309 pg/mL, p = 0.001) and PLA (-1.396 ± 1.467 pg/mL, p = 0.001). IL-6 was significantly higher at three hours post-exercise than at baseline for each VPRO (p = 0.002), PRO (p = 0.042) and PLA (p = 0.004) (0.716 ± 0.623 pg/mL, 0.472 ± 0.707 pg/mL and 0.664 ± 0.540 pg/mL, respectively). VPRO was significantly (p = 0.034) higher than PRO immediately post-exercise. There was no order effect for IL-6 Values based on visit day.  Fig 4: Mean ± SD of serum interleukin-6 levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9.   25  Hepcidin-25 & Baseline Vitamin D There was a significant effect of time (F[1.234, 9.874] = 33.237, p < 0.001), but not type of drink (F[1.987, 15.897] = 0.911, p = 0.421) on hepcidin-25 for all conditions (see Figure 5). 3x3 repeated measures ANOVA revealed a significant increase from baseline to post-exercise (11.07 ± 41.72 ng/mL), baseline to three hours post-exercise (89.16 ± 57.40 ng/mL) and post-exercise to three hours post-exercise (78.82 ± 74.16 ng/mL) on hepcidin-25 levels in each condition (see Figure 2). There were no significant differences observed in the rate of change from baseline to post-exercise or post-exercise to three hours post-exercise or baseline to three hours post-exercise in VPRO vs PRO (p = 0.912, 0.961, and 0.894), VPRO vs PLA (p = 0.753, 0.615, and 0.589), or PRO vs PLA (p = 0.955, 0.902, and 0.542). Paired sample t-tests indicated a significant increase from baseline to post-exercise in VPRO and PRO (p = 0.031, 0.021, respectively) but no significant increase in PLA (p = 0.185). There was a significant increase from post-exercise to three hours post-exercise for VPRO, PRO and PLA (p = 0.110, 0.002, 0.000, respectively). Hepcidin-25 concentrations were significantly higher at three hours post-exercise than at baseline for VPRO, PRO and PLA (p = 0.110, 0.002, < 0.001, respectively). Furthermore, there was no order effect for hepcidin values based on visit day. A multivariate ANOVA revealed starting vitamin D levels (74.50 ± 28.00 nmol/L, MIN = 34 nmol/L, MAX = 132 nmol/L) did not affect hepcidin-25 response in the VPRO condition (p = 0.225).   26   Fig 5: Mean ± SD of hepcidin-25 levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9. Iron Parameters Repeated measures ANOVA revealed a significant time (p = 0.001) effect on serum iron and serum ferritin for all conditions (see Table 3 and Figure 6-7). There was no effect of drink on iron (F[2, 14] = 1.518, p = 0.253). After log transforming the non-normally distributed data of serum ferritin, results indicated no significant difference between VPRO, PRO and PLA for serum ferritin (F[1.187, 9.494] = 4.733, p = 0.051). Follow-up paired sample t-tests revealed a significant difference between VPRO when compared to PRO and PLA for serum ferritin at all time periods (p < 0.05). Paired sample t-tests demonstrated a significant increase from baseline to post-exercise for both serum ferritin and serum iron in VPRO (p = 0.001 [ferritin], 0.017 [iron]) and PRO (p = 0.001 [ferritin], 0.028 [iron]) and PLA (p = 0.001 [ferritin], 0.004 [iron])). There was a significant   27  decrease from post-exercise to three hours post-exercise for serum ferritin and serum iron in VPRO (p = 0.004 [ferritin], 0.001 [iron]) and PRO (p = 0.002 [ferritin], 0.002 [iron]). There was no significant difference from post-exercise to three hours post exercise in serum ferritin for PLA (p = 0.090). However, there was a significant decrease from post-exercise to three hours post-exercise in serum iron for PLA (p = 0.004). Serum ferritin levels were significantly higher for baseline (p = 0.001, 0.037), post-exercise (p = 0.002, 0.035) and three hours post-exercise (p = 0.001, 0.016) in VPRO when compared to PRO and PLA, respectively. Serum ferritin was significantly higher three hours post-exercise compared to baseline for PRO (p = 0.003), VPRO (p = 0.001) and PLA (p = 0.014). Serum iron was significantly lower three hours post-exercise compared to baseline in PRO (p = 0.019) and VPRO (p = 0.032) but not PLA (p = 0.065).   Fig 6: Mean ± SD of serum ferritin levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr   28  Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9.  Fig 7: Mean ± SD of serum iron levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9. Hemoglobin & Hematocrit Significant time (p = 0.001) effects were recorded for Hb and hematocrit. No significant difference was observed for either Hb (F[2, 14] = 0.777, p = 0.479) or hematocrit (F[2, 14] = 0.424, p = 0.663) between VPRO, PRO and PLA (see Table 3 and Figure 8-9). Paired sample t-tests demonstrated a significant increase from baseline to post-exercise for both Hb and hematocrit in VPRO (p = 0.022 [Hb], 0.019 [hematocrit]) and PRO (p = 0.005 [Hb], 0.007 [hematocrit]). There was no significant difference from baseline to post-exercise for Hb (p = 0.066) or hematocrit (p = 0.154) in PLA. There was a significant decrease from post-exercise to three hours post-exercise in all conditions   29  for both Hb (p = 0.004 [VPRO], 0.002 [PRO], 0.001 [PLA]) and hematocrit (p = 0.001 [VPRO], 0.002 [PRO], 0.001 [PLA]). Hb was significantly lower three hours post-exercise compared to baseline in PRO (p = 0.003) only. Hematocrit was significantly lower three hours post-exercise compared to baseline in PRO (p = 0.001) and PLA (p = 0.041).  Fig 8: Mean ± SD of serum hemoglobin levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9.   30   Fig 9: Mean ± SD of hematocrit percent levels at baseline, post-exercise and three hours post-exercise for VPRO, PRO and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9.         31    Table 2: Summary for heart rate (HR), power output (Watts), energy expenditure (Kcal), rating of perceived exertion (RPE), pre- and post-exercise mass for VPRO, PRO, and PLA conditions. Expressed in mean ± SD. § = Significant difference (p < 0.05) from Baseline.    Table 3: Mean ± SD for serum interleukin-6 (IL-6), hepcidin-25, serum iron, serum ferritin, hemoglobin and hematocrit levels at baseline, post-exercise, and three hours post-exercise in VPRO, PRO, and PLA conditions. § = Significant difference (p < 0.05) from Baseline to Post-exercise. * = Significant difference (p < 0.05) from Post-exercise to 3 hr Post-exercise. δ = Significant difference (p < 0.05) from Baseline to 3 hr Post-exercise. n=10 except for PLA post-exercise where n=9.Drink HR (BPM) Average Power (Watts) Watts/kg Mass (kg) Pre Mass (kg) Post Mass (kg) ChangeVPRO 170 11 278 31 3.8 0.3 971 107 430 46 8.6 0.9 72.3 6.6 71.7 6.4 -0.60 0.36PRO 169 9 269 33 3.7 0.3 942 117 427 43 8.7 1.3 72.2 6.2 71.7 6.2 -0.58 0.38PLA 169 10 270 31 3.8 0.3 946 109 427 55 9.0 1.3 72.1 6.4 71.6 6.4 -0.51 0.29Energy Expenditure (Kcal)MAX Power (Watts)Rating of Perceived Exertion (1-10)§§§Serum Iron (mcg mol L-1) 21.30 5.08 23.00 5.39 § 18.80 5.22 * δ 20.70 7.51 23.00 8.82 § 17.90 5.88 * δ 23.80 8.90 25.44 9.32 § 21.10 6.79 * δSerum Ferritin (mcg L-1) 101.10 85.17 108.30 85.38 § 104.20 84.27 * δ 88.20 68.91 87.50 77.86 § 91.90 70.51 * δ 87.00 81.74 84.60 89.19 § 90.70 79.52Serum IL-6 (pg mL-1) 0.92 1.70 3.79 2.94 § 1.64 1.98 * δ 0.81 0.74 3.12 2.32 § 1.29 1.31 * δ 0.73 0.66 3.10 1.99 § 1.40 0.89 * δHemoglobin (mg L-1) 151.50 8.18 154.40 7.68 § 150.44 10.36 * 150.20 10.37 155.56 10.73 § 147.40 10.02 * δ 149.10 7.91 153.22 9.68 146.30 9.88 *Hematocrit 0.43 0.02 0.44 0.02 § 0.43 0.02 * 0.43 0.02 0.45 0.02 § 0.42 0.03 * δ 0.43 0.01 0.44 0.02 0.42 0.02 * δHepcidin-25 (nmol L-1) 14.18 14.90 17.84 19.84 § 25.44 11.91 * δ 9.94 8.93 11.78 10.24 § 22.27 13.41 * δ 10.44 14.62 10.09 7.68 22.57 15.57 * δBlood Biomarker PRO VPRO PLA3 hr Post-exercise Baseline Post-exercise 3 hr Post-exerciseIronBaseline Post-exercise 3 hr Post-exercise Baseline Post-exerciseInflammatoryHemoglobin & HematocritHepcidin  32  Discussion The present study examined the effects of a post-exercise drink consisting of whey protein isolate and carbohydrates (CHO) with vitamins D3 and K2 (VPRO) or without (PRO) as compared to a no-calorie placebo (PLA) following a bout of exercise on the acute post-exercise hepcidin response in highly-trained athletes. The results from the investigation demonstrate that following a fatiguing interval cycling exercise (8 x 3 min intervals at 85% pVO2max), subjects experienced a significant time-dependent increase in hepcidin, IL-6, Hb, hematocrit and iron biomarkers independent of the post-exercise drink composition (see Table 3). Contrary to our hypothesis, VPRO, PRO or PLA post-exercise drink compositions had no significant (p > 0.05) effect on any biomarker measured. The hepcidin levels in the current study mimic that of previous literature where peak levels were seen three hours post-exercise for all conditions (see Figure 5). This response is secondary to that of peak activity of IL-6 (Kemna, Pickkers, Nemeth, van der Hoeven, & Swinkels, 2005). There was no significant difference between either of the conditions from post-exercise to three hours post-exercise in the current study. This peak level in hepcidin three hours post-exercise has been correlated with altered iron metabolism within the athletic population (Wachsmuth et al., 2015), partly due to hepcidin interference with the main exporter of iron from the enterocyte and macrophage, FPN1 (Ward & Kaplan, 2012). Thus, individuals may be more susceptible to iron malabsorption near and around three hours post-exercise when hepcidin levels are at their highest. Furthermore, baseline measurements of hepcidin were not significantly different in any of the conditions, indicating that individuals did not come into the testing day in an already inflamed state. Hepcidin response in the current study is therefore characteristic of normal post-exercise inflammatory responses in combination with the rise of IL-6.  Following a bout of exercise, IL-6 has been reported to drastically increase five- to 100-fold (Ostrowski, Rohde, Asp, Schjerling, & Pedersen, 1999; Peeling et al., 2009), and peak levels are attained immediately following the cessation of exercise (Pedersen, 2000). IL-6 acts as both an anti-inflammatory (during exercise) and pro-inflammatory (post-exercise) cytokine (Wachsmuth et al., 2015), while also regulating glucose uptake into the cell as a glucoregulatory hormone (Steensberg et al., 2001). Our results demonstrated IL-6 significantly increasing from baseline to post-exercise in each condition (see Figure 4). The timing and quantity of CHO ingestion in and around a bout of exercise in relation to IL-6 changes has been well-studied, but the impact is   33  equivocal. The consumption of large quantities of CHO-rich beverages before and during exercise can blunt plasma IL-6 concentrations (Nehlsen-Cannarella et al., 1985; Nieman et al., 1998; Robson-Ansley et al., 2011). However, dosages more practical to prevent GI issues have yet to show a significant effect on IL-6 (Ihalainen et al., 2014; Sim et al., 2012).  There are a limited number of studies showing the effects of both protein and CHO consumption in and around a bout of exercise on inflammatory markers, and the results remain equivocal. Corsio-lima et al (2012) took six well trained (VO2max: 66 ± 2.7 mL/kg/min) cyclists and subjected them to two separate 2.5-hour cycling sessions at 75% VO2max in a hot environment (35 degrees Celsius and 60% relative humidity). Participants were given either a 6% CHO solution (4 mL kg-1 bodyweight) or a 4:1 CHO-protein solution (4 mL kg-1 bodyweight) in a randomized, crossover design every 15 minutes during the cycling sessions (Cosio-Lima, Desai, Stelzer, & Schuler, 2012). Total caloric ingestion over the 2.5-hour cycle was 1,040 calories or 416 calories/hour (CHO: 210 g and Protein: 50 g) for the 4:1 CHO-protein solution and 1,120 calories or 448 calories/hour (CHO: 280 g and Protein: 0 g) for the CHO only solution. There was a significant increase in IL-6 concentrations post-exercise, but no significant difference (p > 0.05) between conditions (Cosio-Lima et al., 2012). Another study took 12 well-trained cyclists and subjected them to a battery of high-intensity cycling tests on two different visits (Rowlands et al., 2008). Following the tests, subjects were given either a high protein (0.7 g kg-1 hour-1), moderate CHO (0.26 g kg-1 hour-1) beverage or a low protein (0.1 g kg-1 hour-1), high CHO (2.1 g kg-1 hour-1) beverage with equal fat content during a four hour recovery period. There was only a trivial effect on TNF-α and IL-6 despite the drink composition (Rowlands et al., 2008). These findings are in contrast of a more recent study in nine physically active men (age: 28 ± 2, VO2max: 4.2 ± 0.2 L/min) who completed a two-hour cycle at 60-65% VO2max followed by a four-hour recovery period. During the recovery period, both immediately after and two hours into the recovery period, subjects ingested either a high protein, moderate CHO cake (Total Intake, Protein: 23.92 g and CHO: 82.8 g, Calories: 426.9) or a low protein, high CHO cake (Total Intake, Protein: 9.2 g and CHO: 101.2 g, Calories: 441.6) (Kerasioti et al., 2013). There was a significant decrease in IL-6 concentrations by ~50% when subjects consumed the high protein, moderate CHO cake compared to the low protein, high CHO cake. Furthermore, it has been demonstrated in individuals cycling at a constant load (55% peak power) for 120 minutes that the consumption of 45 g of protein per hour significantly decreases IL-6 concentrations immediately post-exercise, when compared to a   34  non-caloric sweetened (Splenda™) placebo drink (Schroer et al., 2014). Contrary to Kerasioti et al (2013) and Schroer et al (2014), our results are in line with findings by Corsio-Lima et al (2012) and Rowlands et al (2008), where the protein and CHO dosages of the PRO and VPRO drinks had no significant effect on IL-6 levels when compared to the PLA drink.  Our findings could potentially be related to the timing of the blood sample and/or the timing of nutrient intake. Kerasioti and colleagues (2013) reported that IL-6 was significantly different four hours after the cessation of exercise. Thus, we may have seen a significant difference between conditions if we had measured IL-6 and hepcidin levels four hours and not three hours post-exercise. Additionally, Schroer et al (2014) utilized a peri-workout feeding protocol, where subjects consumed 45 g of protein per hour over a two-hour cycling session. The protein source utilized by Schorer and colleagues (2014) was high in BCAA. Branched chain amino acids supplemented pre- and intra-workout have been shown to attenuate muscle soreness, muscle damage and inflammatory markers related to metabolic stress. Only water was utilized during the cycling intervals for the study at hand. Thus, metabolic and oxidative stress may have been attenuated if we chose to utilize a different feeding strategy (Schroer et al., 2014).  In addition to the above, the artificial (sucralose) and natural (Stevia) sweeteners utilized in the drinks may have had a small influence on post-exercise inflammation. Sucralose, commonly known as SplendaTM, has been shown to have a immunosuppressant effect and decreases IL-6 and IL-10 (Rahiman & Pool, 2014). Rats fed 100, 300, 500, or 1,000 mg/kg of SplendaTM for 12 weeks demonstrated altered gut microflora an increased gut permeability in a dose dependent manor (Abou-Donia, El-Masry, Abdel-Rahman, McLendon, & Schiffman, 2008). Furthermore, Stevia has antioxidant, anti-inflammatory and antimicrobial effects (Muanda, Soulimani, Diop, & Dicko, 2011). Cultured LPS-activated macrophage cells treated with Stevia suppressed LPS-induced nuclear factor-KB and IL-6 in a dose:dependent fashion (6.25, 12.5, 25, and 50 mcg/mL) (Jeong et al., 2010). These finding are further supported by Kim and colleagues (2013), showing Stevia inhibited nuclear factor-KB gene expression in LPS-stimulated cells. The authors concluded that Stevia may have anti-inflammatory effects (Kim et al., 2013). Collectively, it could be assumed that both the sucralose and Stevia used in our study may have affected the inflammatory biomarkers three hours post-exercise. Despite the above findings to suggest this, neither the VPRO or PRO drinks which contained both sucralose and Stevia, nor the PLA drink which contained   35  only sucralose, had any significant effect on IL-6 or hepcidin-25. Additionally, there was no significant difference between any of the conditions three hours post-exercise. This suggests that the artificial and natural sweeteners used in our study may not have had a large enough effect to show discrepancies between treatments; despite there being significant effects in rat models and in vitro (Abou-Donia et al., 2008; Jeong et al., 2010; Kim et al., 2013; Rahiman & Pool, 2014). Significant increases in serum ferritin and serum iron were reported from baseline to post-exercise in the current study (see Figure 6 and 7). This response mimics that of previous literature showing significant increases in serum ferritin levels and serum iron levels following the cessation of exercise (Badenhorst et al., 2015a; Badenhorst et al., 2014; Marc et al., 2013; Peeling, Dawson, Goodman, Landers, & Trinder, 2008; Peeling et al., 2009; Peeling et al., 2014; Sim et al., 2012). Exercise-induced inflammation has a robust effect on the reticuloendothelial system (RES), which is comprised of monocytes, macrophages and precursor cells (Knutson & Wessling-Resnick, 2003) contributing to iron recycling and storage. As erythrocytes become damaged, the cell membrane containing stored ferritin becomes impaired thus causing a leakage into circulating plasma (Pattini, Schena, & Guidi, 1990). This leads to an increase in ferritin reuptake and reticuloendothelial (RE) cells then recycle ferritin via the spleen, liver and bone marrow (Knutson & Wessling-Resnick, 2003). In addition to the above, exercise-induced dehydration has been shown to decrease plasma volume which subsequently increases the concentration of circulating proteins (Jimenez et al., 1999; Reljic, Hassler, Jost, & Friedmann-Bette, 2013). Our subjects lost a total of 0.6 ± 0.4, 0.6 ± 0.4 and 0.5 ± 0.3 kg for the VPRO, PRO and PLA visits, respectively. Every one g of body mass lost equates to roughly 1 mL of water (Cheuvront, Haymes, & Sawka, 2002; Sawka, Cheuvront, & Kenefick, 2012). Based on that assumption, our subjects would have lost approximately ~500-600 mL of water during each cycling session, and could have altered plasma volume and further explain why we saw a significant increase in serum Hb, hematocrit, serum ferritin post-exercise in all conditions. However, because we did not monitor fluid intake pre-exercise, intra-exercise and/or during the three hour recovery period, we cannot fully assess the effects of fluid intake and rehydration on the blood biomarkers measured in the current study; due to the fact the hydration status of the individual can greatly affect hematological responses to exercise (Maughan & Shirreffs, 2010).   36  Following post-exercise, there was a significant decrease in serum ferritin, Hb and hematocrit seen three hours post-exercise (see Figure 6, 8 and 9) could be attributed to this clearance mechanism of both serum ferritin and free Hb from the circulating plasma, and thus, increasing ferritin stores in the liver (Nemeth & Ganz, 2006; Nemeth et al., 2004). Furthermore, elevated levels of hepcidin have been shown to prevent iron recycling by macrophages (Nemeth et al., 2004), which could also further contribute to the marked decrease in serum iron and serum ferritin levels three hours post-exercise in the current study for VPRO, PRO and PLA, due to ferritin being unable to be released from the macrophages. Additionally, different modes of exercise can have a greater effect on hemolysis. High impact forces and exercise-induced hemolysis lead to an increase in serum ferritin and serum iron (Peeling et al., 2008). Running has been shown to have the greater impact on hemolytic responses when compared to cycling (Sim, Dawson, Landers, Swinkels, et al., 2014; Telford et al., 2003). Despite this, Sim et al (2012) reported that there are no significant differences on IL-6 or hepcidin concentrations if intensity and duration are matched between running and cycling. However, it has been shown that muscle glycogen depletion can significantly augment IL-6 activity produced by skeletal muscle tissue (Keller et al., 2001; Steensberg et al., 2001), and both intensity and duration of exercise are important factors to determine the rate of muscle glycogen utilization (Ivy, Katz, Cutler, Sherman, & Coyle, 1988). It may take upwards to 2-3 hours of exercise at 70-80% VO2max in order to fully deplete muscle glycogen stores (Coyle et al., 1983). Keller and colleagues (2001) demonstrated that depleted muscle glycogen stores leads to a ~40- and ~60- fold increase in IL-6 gene activation after 90 and 180 minutes of dynamic knee extensor exercises performed at 50-60% maximal workload when compared to a controlled (adequate supply) muscle glycogen content group. IL-6 was two-fold higher in the glycogen-depleted group then in the control group two hours post-exercise (Keller et al., 2001). With that in mind, the pre-workout meal containing roughly ~1.2 g CHO kg-1 consumed one hour prior to the beginning of the high-intensity exercise trial, which has been shown to be the optimal range of CHO consumption to replete muscle glycogen stores (Burke, 2010; Ivy, 2004), and the nature of elite endurance athletes normal diet being high CHO (8 to 11 g/kg = 576.24 ± 51.52 to 792.33 ± 70.84) (Burke, 2001; Jeukendrup, 2011; Jeukendrup, Jentjens, & Moseley, 2005; Robins, 2007; Vogt et al., 2005) could have fully replenished muscle glycogen stores which subsequently decreases the post-exercise IL-6 response. Additionally, the nature of the exercise task at hand (8 x 3 min intervals at 85% pVO2max = ~900   37  Kcal), and the study population being elite and professional athletes who have been shown to have a greater capacity to store glycogen and replenish it at accelerated rates when compared to untrained individuals (Gollnick, Armstrong, Saubert, Piehl, & Saltin, 1972; Piehl, Adolfsson, & Nazar, 1973), muscle glycogen stores may not have been sufficiently taxed (depleted) in the PLA condition to begin to significantly influence IL-6 activity when compared to the VPRO and PRO conditions. Lastly, the post-exercise inflammatory response has been reported to be different in trained versus untrained individuals (Evans et al., 1986; Schild et al., 2016). Chronic adaptations to both resistance (Calle & Fernandez, 2010) and endurance (Farney et al., 2012) training leads to a marked reduction in reactive oxygen species and a subsequent decrease anti- and pro- inflammatory cytokines following a bout of high intensity exercise. Thus, the findings of the study could be in part because our population was highly-trained athletes (VO2max of 67.4 ± 4.4 mL/kg/min). This combination of factors could possibly explain the current findings, where there was no significant differences in IL-6 or hepcidin activity for any condition immediately post-exercise and three hours post-exercise. Vitamin D supplementation in healthy individuals (Bacchetta et al., 2014) and individuals suffering from CKD (Zughaier et al., 2014) have led to a marked decrease in hepcidin response following acute and chronic dosing. Bacchetta et al (2014) utilized a dosage 20-fold higher (100,000 IU) than that of the single dose used in our study. Furthermore, hepcidin measurements were monitored immediately before and 24 hours after supplementation (Bacchetta et al., 2014), and showed a 34% reduction in hepcidin concentrations. Results from Zughaier et al (2014) were obtained after a three-month follow up, with a dosing protocol of 7,143 IU/day of vitamin D3. The authors reported a 28% decrease in hepcidin concentrations in CKD patients (Zughaier et al., 2014). Furthermore, Barker and his colleagues (2013) showed that 4,000 IU/day of vitamin D supplementation over the course of 28 days significantly (p < 0.05) attenuated the inflammatory biomarkers alanine (ALT) and aspartate (AST) after 10 sets of 10 repetitions of peak isometric force eccentric-concentric jumps in modestly active and healthy adult males 24, 48, 72 and 168 hours following the exercise bouts. The study at hand looked at the acute effects of vitamin D3 and K2 supplementation within a three-hour time frame and at a much lower dose. No differences in IL-6 or hepcidin concentrations for our VPRO drink when compared to PRO or PLA. Thus, the supplements may not have been able to take effect within the small time frame and results could   38  have varied if IL-6 and hepcidin levels were re-assessed 24 hours after the ingestion of the drinks or if we utilized a chronic supplementation intervention. Baseline measurements were taken for serum vitamin D in order to determine if starting levels had an effect on VPRO drink response (see Table 1). This measurement was taken since it has been shown that individuals with insufficient vitamin D stores have a higher risk of anemia (Patel et al., 2010) and low levels of ferritin blunts the post-exercise hepcidin response (Auersperger et al., 2013; Peeling et al., 2014). Thus, the authors made the assumption that baseline values of vitamin D may also effect the post-exercise hepcidin response in a similar fashion. Results from the study showed that baseline starting values of vitamin D had no significant effect on post-exercise hepcidin response three hours after the completion of the cycling protocol in the VPRO condition. Although a few of our subjects were classified as deficient (< 50 nmol/L, n=1) and insufficient (51-74 nmol/L, n=5) stores of 25(OH)D (Dahlquist et al., 2015), and Patel and colleagues (2010) reported that insufficient levels of 25(OH)D are associated with a higher prevalence of anemia (2-3 fold increase), none of our participants with low levels of 25(OH)D were classified with ID (see Table 1). Furthermore, only 10% of our subjects fell below 25(OH)D cut off set by Statistics Canada as compared with the mean value for Canadians in this age group (41%) (Canada, 2015). The higher than normal values seen with 25(OH)D could possibly be related to when the study commenced, which was right after summer cycling season for the athletes. It has been shown that 15 min of adequate sun exposure (290-315 nm of ultraviolet B [UVB] radiation) (Lim et al., 2005; Wolpowitz & Gilchrest, 2006) during the summer months produces 10,000 to 20,000 IU of vitamin D3 (Heaney, 2008). It could be that sustained outdoor training and its concomitant increase in synthesis of vitamin D3 (Zughaier et al., 2014), could have potentially elevated 25(OH)D concentrations in our study population and subsequently affected the blood biomarkers in the current study. Lastly, vitamin K supplementation has been shown to blunt the inflammatory cytokine IL-6 (Ohsaki et al., 2006; Reddi et al., 1995; Shea, Booth, et al., 2008; Shea, Dallal, et al., 2008). Thus, it was postulated that hepcidin could be down-regulated by vitamin K supplementation. Unfortunately, our results did not show any significant difference in IL-6 or hepcidin when vitamin K2 was combined with vitamin D3 supplementation. In vitro studies in rat models, isolated cell cultures and obese humans demonstrate a marked reduction in IL-6 after vitamin K   39  supplementation with various dosages (Ohsaki et al., 2006; Reddi et al., 1995; Shea, Booth, et al., 2008; Shea, Dallal, et al., 2008). Ohaski and colleagues (2006) showed a marked reduction in LPS and a subsequent decrease in IL-6 when rats were fed a vitamin K-rich diet (75 mg/kg/day) compared to a vitamin K-deficient diet. Furthermore, 0.5 mL of vitamin K2 supplementation to cell cultures treated with LPS have shown to decrease IL-6 (-190%) concentrations compared to cell cultures not treated with vitamin K (Reddi et al., 1995). Lastly, two studies conducted in elderly and obese individuals, have reported that those who had higher circulating concentrations of vitamin K and vitamin D had lower levels of inflammatory markers related to disease (Shea, Booth, et al., 2008; Shea, Dallal, et al., 2008). However, one of the studies indicated that further supplementation of 500 mcg/day of vitamin K showed no further impact on inflammatory markers, and the authors reported that the causation of lower levels of inflammation cannot solely be linked to vitamin K or vitamin D (Shea, Dallal, et al., 2008); because it could be assumed that diets rich in vitamin K are associated with healthier eating patterns. Thus, extrapolation to healthy and highly fit males may not be warranted based on the findings of the study (Reagan-Shaw, Nihal, & Ahmad, 2008; Sharma & McNeill, 2009; Tappy et al., 1994).             40  Conclusions Strengths, Limitations, Future Directions This is the first study to compare the effects of carbohydrates and protein with or without the addition of vitamin D3 and K2 against a placebo drink on the exercise-induced hepcidin response in elite and professional athletes in a randomized, placebo-controlled, single-blinded triple crossover design. The protein, CHO, and vitamin D + K supplements are all commercially-available and the acute intervention protocol utilized in the study is a practical method to be utilized for athletes training or competing. For the design of the experiment, safe dosages of vitamin D3, K2, CHO and protein were utilized for the intervention drinks to restore muscle glycogen, maximize MPS and prevent GI upset (Rehrer et al., 1992; Smith et al., 2013; Stellingwerff et al., 2007).  Although we did not report any significant effect on hepcidin, IL-6, Hb, hematocrit or iron biomarkers in any condition, other studies have utilized different nutrient timing protocols with similar CHO and protein dosages with varying results. Thus, future studies should revisit the effectiveness of pre- and peri-workout consumption of both CHO and protein with or without the addition of vitamin D3 and K2 on the exercise-induced hepcidin response. Other supplements with known effects on pro- and anti-inflammatory properties in the acute phase response also warrant further investigation (e.g., fish oil) (Li, Huang, Zheng, Wu, & Li, 2014). Limitations to the study included: (1) the exclusion of females, (2) he lack of measurements of testosterone, (3) that subject diets were uncontrolled 24 hours prior to experimental visits, (4) that daily activity prior to experimental visits was not tightly controlled, (5) the lack of hematological and iron parameters 24 hours after cessation of exercise, and (6) the pre-workout meal. Sixty percent of all female athletes suffer from some form of ID within a given year. With such staggering statistics, research needs to emphasize ways to mitigate this issue. Since the menstrual cycle and estradiol affect the post-exercise hepcidin response (Sim, Dawson, Landers, Trinder, et al., 2014), the limited time frame in which the researchers had to complete the study made it impractical to include female participants. Future research needs to include females and consider the phase of the menstrual cycle during the time of testing in order to control for confounding variables. In response to heavy training loads, male elite endurance athletes have been shown to have significantly lower levels of circulating reproductive hormones (Banfi, Marinelli, Roi, & Agape, 1993; Cumming, Wheeler, & McColl, 1989; Ishigaki et al., 2005; Lin & Chang, 2008;   41  Lucia et al., 2001; MacConnie, Barkan, Lampman, Schork, & Bieitins, 1986; Weimann, 2002). Of the main reproductive hormones, testosterone has an intricate role in regulating hepcidin (Bachman et al., 2010; Bachman et al., 2014; Guo et al., 2013). Low testosterone levels act in a similar dose:response manner to high estrogen and/or progesterone levels to blunt hepcidin concentrations (Sim, Dawson, Landers, Trinder, et al., 2014; Yang et al., 2012). Since our subjects were highly trained elite and professional endurance athletes, testosterone levels could have potentially been low which would have inflated the hepcidin response of our study. Future studies looking at the effects of testosterone on hepcidin concentrations three hours post exercise in this population is warranted. Twenty-four-hour diet control is also suggested for future research. Although our results indicated that baseline IL-6 and hepcidin concentrations were not significantly different between testing days, it has been shown that low glycogen availability can increase these markers at baseline. Since diet was not controlled for within a 24-hour period leading into experimental visits, subjects might have started the study in a slightly depleted state, confounding the results. Additionally, the reverse could have happened, and muscle glycogen stores could have been topped out, especially due to the CHO heavy pre-workout meal consumed 60 minutes prior to the cycling test, subsequently effecting the post-exercise inflammatory response. Furthermore, daily activity was not monitored; elite and professional athletes train multiple times per week (Knechtle, Rust, Rosemann, & Martin, 2014; Stellingwerff, 2012, 2016; Storen, Bratland-Sanda, Haave, & Helgerud, 2012), thus in order to make the study and adherence protocol mimic that of real life training, athletes were allowed to continue their normal training throughout the experimental visits. Lastly, we did not monitor any hematological changes four or 24 hours after the ingestion of the post-workout drink, significant effects on IL-6 activity have been demonstrated four hours after the cessation of exercise following the consumption of a CHO-protein mixture (Rowlands et al., 2008).  Conclusions Regarding Thesis Hypotheses Hypothesis 1: I reject our hypothesis that VPRO and PRO would significantly decrease hepcidin following a bout of high intensity cycle as compared to PLA. There was no significant difference between all three conditions.   42  Hypothesis 2: I reject our hypothesis that VPRO supplementation would have a greater effect on decreasing the post-exercise hepcidin response when compared to PRO supplementation. There was no significant difference between VPRO and PRO and hepcidin concentrations post-exercise and three hours post-exercise.  Conclusion & Practical Application Although previous research has demonstrated that consuming a CHO and protein-rich beverage post-exercise will help to accelerate recovery, increase subsequent performances in the same day and enhance muscle repair mechanisms, the consumption of a CHO and protein-rich drink with or without the addition of vitamin D3 and K2 had no significant effects on hepcidin, IL-6, Hb, hematocrit, serum ferritin or serum iron in the present study. Athletes, nutritionists and coaches should take this into consideration when structuring nutrient partitioning, and may consider avoiding the three-hour post-exercise window when supplementing iron in order to enhance iron absorption. The effects of different recovery modalities, nutrient timing strategies and exercise interventions on the post-exercise hepcidin and IL-6 response in the athletic population has continued to gain interest among researchers and sport medical practitioners, as we try to determine the optimal paradigm to maximize iron absorption in athletes. Future research needs to focus on the timing, the constituents and dose of nutrition and supplement protocols from both an acute and chronic standpoint.          43  References Abou-Donia, M. B., El-Masry, E. M., Abdel-Rahman, A. A., McLendon, R. E., & Schiffman, S. S. (2008). Splenda alters gut microflora and increases intestinal p-glycoprotein and cytochrome p-450 in male rats. J Toxicol Environ Health A, 71(21), 1415-1429. doi:10.1080/15287390802328630 Aisen, P. (2001). Chemistry and biology of eukaryotic iron metabolism. The International Journal of Biochemistry & Cell Biology, 33(10), 940-959. doi:10.1016/S1357-2725(01)00063-2 Atherton, P. 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