Branched Chain Amino Acids: Functions in Exercise   El Khoury Dalia Department of Nutritional Sciences, Faculty of Medicine University of Toronto, Toronto, Canada

   

 

1

Introduction

A well balanced diet is the foundation upon which optimal performance and training can be established (Bishop, 2010). Adequate nutrition is considered an essential component of exercise due to its recognized influence on health, body weight, body composition, substrate availability and ultimately overall performance. It is an important prerequisite for effective replenishment of water, improvement of endurance, enhancement of muscle strength, prevention of injury and fatigue and maintenance of immunity during exercise periods. However, athletes have always attempted to improve muscle adaptation and performance by ingesting a variety of nutritional substances. Various dietary supplements have been globally promoted among athletes and exercising individuals as an affordable and effective means to improve nutritional intake, enhance strength and physical performance, promote muscle building and prevent future illnesses. Global supplement use is estimated to range from 40% to 88% among adult athletes as well as athletes at high school and collegiate levels (Molinero & Márquez, 2009; Sobal & Marquart, 1994; Sobal & Marquart, 1994). Since amino acids and proteins are essential for the synthesis of structural proteins and are involved in various metabolic pathways of significant relevance to exercise, protein and amino acid supplements have been widely promoted among athletes for muscular growth and repair. A special role of essential amino acids, which cannot be produced in sufficient amounts in the body and must be supplied by the diet, was described in exercise with special emphasis on branched chain amino acids as they are actively metabolized in skeletal muscle as energy sources during exercise. The current chapter elucidates the role of branched chain amino acids in exercise, and discusses the potential benefits of branched chain amino acid supplements to athletes. In the first section, various components of exercise are explored, and then followed by a description of the distinct functions of branched chain amino acids in optimizing those components.

2

Definition of Exercise and Related Components

The term “physical activity” is often interchanged with the term “exercise”, although each has its separate components (Romeo, et al., 2010). Physical activity is defined as any body movement produced by an action of the skeletal muscle that results in increased energy expenditure, while exercise is a planned, structured and repetitive physical activity. Skeletal muscle, which constitutes 50-75% of body proteins and approximately 40% of total body weight, is the generator of force during physical activity and exercise. It is formed of long multinucleate, cylindrical cells called muscle fibers. Each muscle fiber contains many myofibrils, which are bundles of actin and myosin filaments organized into a chain of repeating units called sarcomeres (Cooper, 2000). Skeletal muscle is a mixture of slow- and fast-twitch muscle fibers, which are all recruited during exercise activity. Muscle fiber types differ in the metabolic pathways used for the production of adenosine triphosphate (ATP) as a major source of energy (Hochachka, 1994). Fast-twitch fibers possess capability for high rates of ATP synthesis through the anaerobic pathways high-energy phosphate transfer and glycolysis. On the other hand, slow-twitch fibers are specialized in aerobic ATP production; they are characterized with low potentials for high-energy phosphate transfer and glycolysis and a high potential for oxidative phosphorylation. Muscle movement is characterized with three types of contraction, including shortening when the muscle is activated and shortened, lengthening when the muscle is activated and lengthened and isometric when the muscle is activated and maintained at the same length (Close, et al., 2005). Steps of muscle

contraction are well established and depicted as follow: When myosin binds to actin, the actomyosin is still in a weekly bound low-force state; with the release of inorganic phosphate (Pi) upon ATP breakdown and utilization as a source of mechanical energy, the cross-bridge transforms into strongly bound high-force state and goes through the power stroke; adenosine diphosphate (ADP) is then released, returning the cross bridge to the rigor complex (Geeves, et al., 2005). During maximal contraction, the strongly bound high-force states are the dominant forms. However, during isotonic shortening, skeletal muscle myosin is only 5% of the cycle time in strongly-bound states. Peak rate of force development depends on the rate of transition from the weakly bound low-force state to the strongly-bound high-force state. Calcium (Ca2+) within the myoplasm is an essential determinant of muscle contraction; it allows the cycling of cross bridges, and consequently results in force development (Ashley, et al., 1991). Physical activity and exercise are typical situations during which muscle contraction and force generation take place. Exercise is classified according to the aerobic capacity or VO2max required for energy and force production (Pollock & Wilmore, 1990). VO2max is defined as the maximum oxygen volume that a person can attain for ATP generation, and is one of the factors that determines an athlete’s capacity to perform sustained exercise. The classification of exercise intensity based on 20 to 60 minutes of endurance training was reported as follows: very light (30% VO2max), light (30-49% VO2max), moderate (50-74% VO2max), intense (75-84% VO2max) and very intense (85% VO2max). Exercise places a high demand, which varies in amplitude by exercise type, duration and intensity, on muscle’s finite endogenous reserves (Betts & Williams, 2010). If reserves are not adequately replenished and exercise is continued for prolonged duration, various sports-related aspects are deteriorated comprising exercise performance, onset of fatigue, short-term recovery and immunity. In the following, some of these components are discussed including muscle kinetics, fatigue and immunology more precisely in relation to exercise of high intensities. 2.1

Exercise and Muscle Kinetics

Muscle kinetics, including muscle protein turnover, muscle strength and muscle damage, are highly influenced by exercise and depend to a great extent on the type, duration and intensity of exercising bouts. 2.1.1

Exercise and Muscle Protein Turnover

Muscle protein turnover is a continuous cellular process, and is dictated by the balance between muscle protein synthesis and muscle protein breakdown (Glynn, et al., 2010). In general, exercise training results in increased protein synthesis concomitant with a corresponding elevation in protein degradation. Postprandial muscle protein synthesis rates were found to be higher after exercise than after rest, independent of age (Pennings, et al., 2011). Exercise improved the use of dietary-derived amino acids for de novo muscle protein synthesis both in young and elderly men. Furthermore, both muscle protein breakdown (Phillips, et al., 1997) and proteolytic gene expression (Louis, et al., 2007) were shown to be elevated following an acute bout of high intensity resistance exercise. Resistance exercise was found to induce skeletal muscle protein synthesis to a greater extent than skeletal muscle protein breakdown, leading to muscle hypertrophy. In a study by (Biolo, et al., 1995), muscle protein synthesis increased by 108% whereas muscle protein breakdown by 51% 3 hours following resistance training. The acute increases in muscle protein synthesis after resistance exercise has been linked to enhanced mRNA translation, more precisely to the activation of the mammalian target of rapamycin complex (mTORC) 1 pathway as a regulator of mRNA translation and of muscle protein synthesis (Dreyer, et al., 2010). Although minimal, the effect of aerobic exercise of moderate to high intensi-

ty on muscle protein metabolism has received attention. Various studies reported a stimulation of muscle protein synthesis rate in the fasted and fed states following acute aerobic exercise bouts (Harber, et al., 2010), whereas chronic aerobic exercise elicited an increase in resting rates of muscle protein synthesis (Pikosky, et al., 2006). Acute aerobic exercise stimulates mitochondrial protein synthesis with minimal influence on myofibrillar protein synthesis, through upregulation of various signalling molecules in the mTORC1 pathway (Camera, et al., 2010). 2.1.2

Exercise and Muscle Strength

Muscle strength, being proportional to muscle cross-sectional area, has been strongly linked to muscle bulk. Since muscle tissue is mainly composed of proteins (actin and myosin) and water, increasing muscle bulk occurs by increasing protein content through stimulation of protein synthesis and/or attenuation of protein breakdown. Resistance exercise training was established as a strong determinant of muscle bulk and muscle strength. Changes in anabolic hormones can partly account for the increases in muscle mass and muscle strength observed following resistance training (Kraemer, et al., 1990). Sex hormones do not play a role, as various studies did not report significant effects of physical training on sex-related hormone levels (Crewther, et al., 2006). On the other hand, human growth hormones appear as a more probable mediator of exercise-induced increases in muscle mass and muscular function. Production of human growth hormone, a physiological regulator of body composition and physical performance, was found to increase in response to regular strength training and physical fitness in middle-aged and elderly people (Frystyk, 2010). 2.1.3

Exercise and Muscle Damage

Muscle damage can take place in response to high intensity heavy resistance training of prolonged duration. Lengthening contractions have been found to be the most damaging to skeletal muscles. Ultrastructural muscle damage becomes progressively worse with the days following resistance exercise, with more damage observed 24-48 hours or 3 days later. Exercise-induced muscle damage has a number of consequences including delayed onset of muscle soreness, increased release of intramuscular enzymes such as creatine kinase and of myoglobin into the plasma, and decrements in muscle performance (Cockburn, et al., 2008). In fact, both creatine kinase and myoglobin have been defined as indicators of muscle damage. Acute inflammation might take place in response to muscle tissue trauma, as a way to limit tissue damage while activating the repair process (Buckley, et al., 2001). Chronic inflammation might though develop if the initial damage is not resolved possibly due to repetitive applications of the injury stimulus such as following intense athletic training or long-distance competition. Chronic form of tissue trauma may lead to persistent local inflammatory reactions and might drive immunity cells such as neutrophils, macrophages and natural killer cells to inappropriate and prolonged migration stimulus. Muscle damage could be subdivided into primary and secondary damage. Although the exact mechanisms that underlie initial phases of muscle damage are not completely understood, there are most likely contributions from both metabolic and mechanical mechanisms. A major component of metabolic muscle damage is increased production of reactive oxygen species. During contractile activity, a number of reactive oxygen species are released into muscular extracellular space (McArdle, et al., 2001) inducing structural abnormalities, membrane damage and inflammatory responses. However, it is still not evident whether repeated exposure to oxidants may lead to permanent damage to the muscle tissue. In fact, two cytoprotective responses were identified in skeletal muscle, including increased muscle activity of antioxidant enzymes and increased muscle production of heat shock proteins (Close, et al., 2005). On the

other hand, mechanical damage occurs as a direct consequence of the mechanical loading on the myofibers. Lengthening of sarcomeres is non-uniform, and result in some myofilaments being stretched and no longer overlapping within the sarcomere (Proske & Allen, 2005). Following the primary phase of damage, sequential processes are initiated by disruption of the intracellular Ca2+ homeostasis that consequently leads to secondary myofibrillar damage in skeletal muscle (Yasuda, et al., 1997). 2.2

Exercise and Fatigue

Fatigue, unlike muscle injury, is a reversible decline in muscle performance during activity, and recovery usually occurs within the first hour (Allen, et al., 2008). It is commonly described after high-intensity exercise of short-duration (2-7 minutes), predominantly in fast-twitch muscle fibers that are highly susceptible to fatigue contrary to slow-twitch fibers. Fatigue is characterized with reduced peak force, which can be explained by either a decline in the force generated per cross bridge or a decrease in the number of cross bridges in the high-force states or both (Geeves, et al., 2005). Knowing that muscles are activated by both central pathways starting in the cortex and spinal cord and peripheral signals initiated at the peripheral nerves, neuromuscular junctions and skeletal muscle itself, muscle fatigue can arise at any of these points and is divided into central and peripheral fatigue. Central fatigue is the inability to maintain power output due to alterations in the concentrations of certain neurotransmitters in the central nervous system (Newsholme & Blomstrand, 2006). Among these neurotransmitters, serotonin was found to play a major role in triggering fatigue and debilitating performance. Increased concentrations of brain serotonin occur in response to increased brain levels of the amino acid tryptophan, which is an amino acid precursor of serotonin. There is enough evidence that brain serotonin activity increases during prolonged exercise and that this response is associated with fatigue both in rats and humans. Unlike central fatigue, peripheral fatigue is confined to the contracting skeletal muscle itself and has various contributing factors characterized by either disturbance in the availability of metabolic substrates or decreased content of energy substrates such as glycogen or both during exercise (Wildman, 2004). Inorganic phosphate is one of the contributing factors to fatigue (Westerblad, et al., 2002). During periods of high energy demand, creatine phosphate and not ATP is utilized and breaks down to creatine and Pi. Accumulation of inorganic phosphate, unlike creatine with little effect on contractile functions, was reported to inhibit force production through inhibition of transition to high-force cross-bridge state (Allen, et al., 2008); however, the contribution of Pi-induced decrease in cross-bridge force production is small and occurs at early stages of fatigue in fast-twitch fibers (~10% of maximum force). At later stages of fatigue, increases in Pi have a larger impact on force production by reducing myofibrillar Ca2+ sensitivity. Depletion of intramuscular ATP and creatine phosphate was suggested as another determinant of fatigue; however, exercise was found to continue beyond the point at which no further changes in these metabolites occur. In fact, creatine phosphate breakdown can contribute to ATP generation for more than 20s, instead of 10s as originally measured, since ATP is supplied from other energy sources and because energy expenditure decreases within few seconds of muscular contraction during exercise (Sahlin, et al., 1998). Acidosis within active muscle fibers, resulting from increased rates of anaerobic glycolysis which take place during intense exercise, was also suggested to be strongly associated with fatigue process. Lactic acid, the main byproduct of intramuscular glycogen upon glycolysis, has been always considered a fatigue-inducing agent either through a direct effect on the contractile process (Spriet, et al., 1987) or through inhibition of phosphofructokinase which leads to a reduction in the rate of energy production by anaerobic glycolysis (Sahlin, 1983). However, it should be noted that the direct inhibitory effect of acido-

sis on the contractile machinery is negligible as it was shown that near complete recovery in force took place despite muscle pH remaining depressed (Sahlin & Ren, 1989). In addition, there is a growing literature establishing the production of reactive oxygen species in exercising muscles as an underlying mechanism of fatigue (Reid, 2001). Increased muscle activity and increased muscle temperature, which usually occur during exercise, accelerate reactive oxygen species’ generation. Evidence on the contribution of reactive oxygen species to fatigue is derived from experiments in which exogenously added reactive oxygen species’ scavengers reduced the rate of fatigue in isolated muscles or intact muscles in animals (Allen, et al., 2008). In humans, on the other hand, improvements in performance in response to dietary antioxidants have been rarely observed. Various observations emphasized the importance of muscle glycogen for optimal endurance exercise performance. Elevated pre-exercise muscle glycogen availability has been associated with increases in exercise time during protocols requiring participants to cycle at a fixed work rate until exhaustion (Balsom, et al., 1999). As glycogen stores are gradually depleted during exercise, fatigue progressively develops. Marked depletion of glycogen stores was reported to affect individual’s capacity to sustain maximal sprint effort. Both lactate and amino acids were reported as the major endogenous carbon sources that can be mobilized for the resynthesis of muscle glycogen during recovery from exercise; however, their relative contribution depends on the type of exercise and duration of recovery (Fournier, et al., 2002). Among amino acids, there is some evidence that glutamine supplementation may assist in glycogen resynthesis in the first hours of recovery following exercise. The ingestion of 8 g of glutamine in addition to 61 g of glucose polymer after a glycogen-depleted bout of exhaustive exercise resulted in a 25% increase in whole body glucose disposal in the 2 hour-recovery period in comparison to glucose polymer alone (Bowtell, et al., 1999). Moreover, lactate was also reported as a major carbon source for muscle glycogen resynthesis after high-intensity exercise, not only in lower vertebrates such as fish, amphibians and reptiles but also in mammals including humans (Bangsbo, et al., 1997; Palmer & Fournier, 1997). The conversion of lactate to glycogen can occur either through direct intramuscular lactate glyconeogenesis in type II (fast-twitch) but not type I (slow-twitch) muscle fiber or via an indirect lactate conversion to glucose by hepatic/renal gluconeogenesis followed by glucose storage as glycogen in both type I and type II muscle fibers (Palmer & Fournier, 1997). However, if exercising under fasting conditions during which nutrients are not available for glycogen replenishment, the limited amount of circulating lactate becomes the major source for glycogen resynthesis. Indeed, lactate oxidation in muscles was found to be accelerated following exercise of moderate intensity performed immediately after a bout of intense physical activity to exhaustion (Bangsbo, et al., 1994). Such increased lactate oxidation rate may lower the amount of lactate available for the resynthesis of muscle glycogen and thus impair glycogen repletion in fasting individuals. 2.3

Exercise and Immunology

With all its branches, including innate and acquired, immunity system was found to be variably affected by exercise. Immunity is divided into cellular innate immunity and acquired immunity, although these branches are inextricably linked with each other (Walsh, et al., 2011). Innate immunity is the first line of defense against pathogens, and is involved in tissue repair and remodeling. Innate immunity does not strengthen upon repeated exposures, and is less specific in terms of pathogen recognition. The innate branch of the immune system includes both soluble factors and cells, comprising neutrophils, dendritic cells, macrophages and natural killer cells as well as proteins mediating phagocytosis, controlling inflammation and interacting with antibodies and anti-microbial peptides. Acquired immunity, also known

as adaptive or specific immunity, is designed to combat infections by preventing pathogen colonisation and destroying invading microorganisms. Helper T (Th) cells, type 1 and 2, are major components of this immunity. While Th1 cells are essential for defense against intracellular pathogens such as viruses and stimulate the release of cytokines interferon-γ and interleukin (IL)-2 that stimulate T cell activation, Th2 cells release various cytokines including IL-4, IL-5, IL-6 and IL13 which are mainly involved in protection against extracellular parasites and can activate the proliferation and differentiation of B lymphocytes into memory cells and plasma cells. An “Inverted J Hypothesis” was suggested in exercise immunology, where infection susceptibility is increased both in sedentary as well as in over-trained individuals in comparison to regular moderate training (Woods, et al., 1999). It should be noted that immune adaptation to physical activity and exercise depend on variable factors, including intensity, duration and type of exercise, environment temperature, nutrition and hydration status, body composition and baseline concentration of immunity markers. While moderate physical activity was found to be inversely associated with upper respiratory tract infection, there is enough evidence stating that athletes and sportsmen are at increased risk of infections during periods of heavy training (Moreira, et al., 2007). It has been well established that moderate physical activity can enhance immune responses. Animal studies showed beneficial effects of moderate exercise training on natural cytotoxicity and T lymphocyte proliferation as well as on enhanced counts of T cells, B cells and immunoglobulins (Elphick, et al., 2003). In elderly people, moderate-intensity exercise training upregulated Th-cell-mediated immune functions and reduced autoimmune diseases and infection risk in addition to increasing lymphocyte proliferation, T cell subsets and IL-2 production (Shimizu, et al., 2008). On the other hand, intense and long-duration competition and training sessions are associated with impairments in the immune system. Most pronounced depression of post-exercise immune function was reported when the exercise is continuous and prolonged (>1.5h), of moderate to high intensity (55-75% of aerobic capacity) and performed without food intake (Gleeson, 2006). Increases in training load among well-trained athletes was found to be associated with decreases in circulating numbers of type 1 T cells, inhibition of type 1 T cell cytokine production, reduced T cell proliferative responses and falls in B cell immunoglobulin and salivary secretory immunoglobulin A (SIgA) synthesis. Salivary secretory immunoglobulin A (SIgA) is, to date, the only immune variable associated with increased incidence of infections. An association between increased risk of upper respiratory tract infection and lower IgA concentrations and secretion rates has been consistently reported in high-performance endurance athletes undertaking intensive training (Fahlman & Engels, 2005). Prolonged intense rowing training over a period of 5 months lowered as well the concentrations of salivary lactoferrin and lysozyme, humoral factors of the innate immune system, compared to sedentary individuals and negatively impacted competitive and training performance of athletes (West, et al., 2010). This temporary decrease in cell function can turn into a chronic depression of acquired immunity if recovery between exercise sessions is insufficient such as during prolonged periods of intensified training. Exercise-induced immune dysfunction has been mainly attributed to the immunosuppressive actions of stress hormones such as adrenaline, cortisol and catecholamines. Indeed, elevated levels of catecholamines following a bout of acute exercise affect both circulating leukocyte numbers and functions (McCarthy & Dale, 1988). Oxidative stress was also suggested as an underlying mechanism. Substantial increases in oxygen consumption and production of reactive oxygen species, strongly associated with detrimental immune functions, were described following sessions of heavy exercise (Marzatico, et al., 1997). Strenuous exercise may also induce depletion and oxidation of glutathione, an important factor in the maintenance of tissue antioxidant defenses.

Although nutritional information, knowledge, beliefs and practices have been extremely variable in the sports field, various macronutrients and micronutrients were found to modulate responses of muscle kinetics, fatigue and immunity to exercise. Dietary proteins, more precisely branched chain amino acids, have drawn the most attention with respect to performance and recovery phases of exercise and led to controversial findings.

3

Branched Chain Amino Acid Metabolism

Branched chain amino acids are a group of hydrophobic essential amino acids with aliphatic non-linear side chains, and include leucine, isoleucine and valine. They all present high protein digestibility corrected amino acid scores, which evaluate protein quality based on age-related amino acid requirements of humans plus estimates of the digestibility of protein (Darragh & Hodgkinson, 2000). Both ileal and fecal nitrogen measures appeared to appropriately assess amino acid digestibility in humans. An important role of branched chain amino acids has been described in protein metabolism and protein structure (Creighton, 1993). In addition, branched chain amino acids showed a significant impact on the survival of animals even more effectively than caloric restriction. In fact, a mixture enriched with branched chain amino acids increased the average life span of middle-aged mice (D'Antona, et al., 2010). The anti-aging role of branched chain amino acids was found to be mediated by mitochondrial biogenesis both in cardiac and skeletal muscles but not in adipose tissues and liver, and by increased expression of genes involved in antioxidant defense and reductions of reactive oxygen species’ production. Branched chain amino acids account for 35% of the essential amino acids in muscle proteins. Unlike other essential amino acids which are primarily oxidized in the liver, they are most actively oxidized in skeletal muscle cells (Shimomura, et al., 2006). After passing through the hepatic bed into systemic circulation, more than 60% of branched chain amino acids were reported to undergo metabolism by the skeletal muscle. The first two enzymes in the catabolism of branched chain amino acids, including the aminotransferase and the flux-generating dehydrogenase, are common to the three amino acids. This explains the strong correlations among the plasma levels of the three branched chain amino acids in a variety of situations (Brosnan & Brosnan, 2006). The first step in metabolism is characterized by a transamination step for the formation of branched chain keto acids (BCKAs) (reversible reaction), followed by oxidative decarboxylation by branched chain keto acid dehydrogenase (BCKDH) complex (irreversible reaction) into succinyl CoA, acetyl CoA and acetoacetate which are intermediates of the Kreb’s cycle (Shimomura, et al., 2004). BCKDH enzyme exists both in active (dephosphorylated) and inactive (phosphorylated) forms (Harris, et al., 1997). The kinase responsible for the phosphorylation and inactivation of this enzyme is the BCDH kinase, which is located exclusively in the mitochondria. Exercise was found to activate BCKDH complex in human and rat skeletal muscle by inducing conformational changes (Shimomura, et al., 1995). Exercise training increases not only BCKDH total enzyme activity but also the amount of enzyme protein and mRNA expression for this enzyme. The metabolism of branched chain amino acids is a critical component of exercise metabolism and endurance. Disrupted branched chain amino acid metabolism in mice impaired various components of exercise metabolism and affected exercise capacity (She, et al., 2010). Exercise was found to promote amino acid catabolism generally and branched chain amino acid catabolism in specific. Indeed, plasma concentrations of branched chain amino acids were reported to be decreased following prolonged exercise but returned to pre-exercise levels within 16 hours (Castell,

et al., 1997). However, it should be noted that, although branched chain amino acids are important energy sources for skeletal muscle, their contribution follows that of carbohydrates and fat during exercise. Using 13C-labelling technique, branched chain amino acid oxidation increased only by 2- to 3-fold during exercise compared to a 10- to 20-fold increase in the oxidation of carbohydrates and fat (Wolfe, et al., 1982). When availability of other fuels is limited such as in states of glycogen depletion, contribution of amino acid, more precisely branched chain amino acid, oxidation to energy provision becomes more important. For instance, the rate of leucine oxidation was found to increase up to fivefold during sessions of strenuous training (Wolfe, et al., 1982). Although branched chain amino acids share similar metabolic pathways, enriching diets with varied amounts of each of the three amino acids does not necessarily produce similar results. For instance, a special role of leucine, unlike isoleucine and valine, has been described in protein synthesis in several tissues including skeletal muscle, liver and adipose tissue (Anthony, et al., 2000; Anthony, et al., 2001; Lynch, et al., 2002). On the other hand, functions of isoleucine and valine were reported in glucose and lipid metabolism respectively (Chida, et al., 2003; Doi, et al., 2007). The fact that individual branched chain amino acids act as different nutritional signals in the body could be attributed to differences in the shape, size and hydrophobicity of their side chains. Leucine is more common in α-helices, whereas valine and isoleucine occur more in β-sheets (Chou & Fasman, 1978). Thus, although the three branched chain amino acids have been evaluated in exercise as one entity in most studies, they are not the same and this should be carefully addressed when discussing their functions in exercise.

4

Branched Chain Amino Acids and Exercise

Based on the hypothesized major contribution of branched chain amino acids to energy metabolism during exercise, claims have been made that increased intakes of branched chain amino acids during prolonged exercise may attenuate muscle protein breakdown, muscle soreness and muscle damage, delay the onset of central fatigue, accelerate recovery phases and enhance performance. The theoretical utility of branched chain amino acid supplementation during exercise has been supported by a series of studies, but negated by others. These contradictory findings are illustrated in the following sections. 4.1

Branched Chain Amino Acids and Muscle Kinetics

Maintaining muscular pool and circulating amino acids, which are substrates for the synthesis of muscle proteins, at adequate levels was found to maximize the anabolic effects of resistance training. The ingestion of dietary protein and/or amino acid supplements alongside resistance training has been proven effective in increasing protein synthesis rates and consequently skeletal muscle hypertrophy over time. Although intake of essential and non-essential amino acids increases plasma amino acid concentrations up to 3 hours, the availability of essential amino acids is the primary promoter of muscle protein synthesis (Volpi, et al., 2003). The effect on net protein synthesis was similar when subjects were given 40 g of a balanced amino acid mixture (21.4 g of essential amino acids and 18.6 g of non-essential amino acids) or 40 g of only essential amino acids (Tipton, et al., 1999). Whether supplied by amino acid mixtures or by whole proteins, the intake of essential amino acids following resistance training was found to be an important factor in promoting muscle protein synthesis. Studies have demonstrated greater increases in muscle protein synthesis rates when essential amino acids were ingested after resistance exercise rather than when essential amino acids ingested at rest or when resistance exercise performed in the fasting

state, with minor increases in muscle protein breakdown (Dreyer, et al., 2008). Such anabolic stimulus has been mainly attributed to branched chain amino acids and more precisely to leucine. The efficacy of individual branched chain amino acids in modulating protein synthesis in cardiac and skeletal muscles of neonatal pigs was compared (Escobar, et al., 2006). Physiological increases in circulating leucine, and not isoleucine and valine, stimulated protein synthesis both in skeletal muscles containing either fasttwitch glycolytic or slow-twitch oxidative muscle fibers as well as in the left ventricular wall. The benefit of leucine in stimulating muscle protein synthesis has been established in wellcontrolled cell culture and animal studies (Pasiakos & McClung, 2011). The anabolic properties of leucine are primarily mediated by its positive effects on specific markers of the protein synthetic pathway (mTOR pathway) (Greiwe, et al., 2001). Leucine activates mTOR signaling pathway by increasing intracellular Ca2+, which in turn activates a class III PI3 kinase. However, inconsistencies in findings were reported in humans. Under resting conditions, the consumption of an essential amino acid mixture (10 g) containing a higher concentration of leucine (3.5 g) did not further improve net protein anabolism beyond that of the mixture containing the lower amount of leucine (1.85 g) in young men and women (Glynn, et al., 2010). Even after resistance exercise, the consumption of 20 g of whole egg protein (~8.6 g essential amino acids and ~1.7 g leucine) increased muscle protein synthesis above that observed with both 5 g and 10 g of protein but was not further stimulated with doses greater than 40 g of protein (Moore, et al., 2009). In addition, the ingestion of a whey protein drink providing a balanced mixture of essential amino acids (69 g total protein) and containing either an adequate amount (4.7 g) or an increased amount (17.6 g) of leucine, after a 30-minute bout of moderate exercise, enhanced muscle protein synthesis to a similar extent when consumed over a 6-hour period in older adults (Koopman, et al., 2008). Thus, when sufficient doses of essential amino acids and/or leucine are provided, no visible additional beneficial effects of leucine on muscle protein kinetics are reported (Glynn, et al., 2010; Koopman, et al., 2008). On the other hand, there is growing evidence supporting the anti-catabolic effects and the repair-accelerating effects of muscle damage by branched chain amino acids during and after exercise although not strong. 4.2

Branched Chain Amino Acids and Muscle Damage

When high forces are generated such as during resistance exercise, lengthening contractions in the skeletal muscle may precipitate into temporary exercise-induced muscle damage that can last for several days after the initial exercise bout. Muscle damage usually manifests as reductions in neuromuscular function and range of motion, increased muscle soreness, limb swelling and elevations of intramuscular proteins in circulation. Among nutritional interventions, branched chain amino acids have showed significant efficacy in reducing the negative impact of exercise-induced muscle damage. Anti-catabolic properties of branched chain amino acids have been revealed in various studies, when consumed immediately before, during and/or after exercise sessions. A reduced release of 32% of aromatic amino acids, tyrosine and phenylalanine, from the legs was described during the 2-hour recovery period upon the ingestion of 100 mg/kg body weight of branched chain amino acids 15 min before exercise, immediately before exercise, at 15, 30, 45 and 60 min during exercise and at 15, 30, 60 and 90 min of recovery (Blomstrand & Saltin, 2001). Following damaging resistance exercise, amino acid supplementation, containing 60% branched chain amino acids, reduced effectively muscle damage and soreness when consumed immediately before and during the four recovery days after a damaging bout of lengthening contractions (Nosaka, et al., 2006). By reducing the leak of creatine kinase from skeletal muscle into the blood, indicative of damage in the sarcolemma, branched chain amino acids were suggested to maintain membrane integrity and thus protect against muscle damage (Howatson, et al., 2012).

It was also suggested that reduction in muscle soreness and muscle damage following branched chain amino acid supplementation may be related to reduced inflammation of the connection tissue elements, which is associated with decreased sensations of pain by the sensitive nocireceptors of the muscle. However, no differences in the acute inflammatory responses, including IL-6, to exercise were reported between the branched chain amino acid and the placebo groups despite reductions in muscle soreness with branched chain amino acid supplementation (Jackman, et al., 2010). 4.3

Branched Chain Amino Acids and Fatigue

In addition to attenuating exercise-induced muscle damage, benefits of branched chain amino acids have been reported on ratings of perceived exhaustion and fatigue sensations during prolonged exercise. The administration of branched chain amino acids prior to graded exercise performed until volitional exhaustion improved psychomotor performance, which was reflected by shortening of the reaction time (Mikulski, et al., 2002). Even during treadmill exercise of changing intensity simulating a soccer game in male soccer players, the ingestion of branched chain amino acids (7 g) an hour before exercise shortened reaction time by an average of 10% before and during exercise compared to the placebo treatment (Wisnik, et al., 2011). Reaction time, an indicator of psychomotor performance, reflects the time a subject takes to react to single or multiple stimuli. By entering Kreb’s cycle as acetoacetate, acetyl CoA and/or succinyl CoA without passing through the glycolytic pathway, lactate is not produced during branched chain amino acid catabolism. Branched chain amino acid supplementation was found to suppress increases in lactate concentrations and lactate release from muscle during exercise both in humans and rats by decreasing lactate production (MacLean, et al., 1996) and was reported to increase lactate threshold during incremental exercise tests in humans (Matsumoto, et al., 2009). Lactate threshold, inversely related to blood lactate concentrations, has been always used as an index of endurance exercise capacity. In exercise intolerant mice with disrupted branched chain amino acid metabolism, increased rates of lactate release from skeletal muscle during exercise were described (She, et al., 2010). In addition, the ingestion of a branched chain amino acid drink (0.4% branched chain amino acids, 4% carbohydrates; 1500 mL/day) over 6 days did improve lactate threshold during an incremental exercise test on the 7th day in comparison to an isocaloric placebo drink composed of dextrin in trained male subjects (Matsumoto, et al., 2009). Branched chain amino acids were also suggested to be involved in opposing central fatigue developed during prolonged training sessions. Both tryptophan, a precursor of serotonin, and large neutral amino acids such as branched chain amino acids compete for transport across the blood-brain barrier into the brain (Blomstrand, et al., 1988). Serotonin is a determinant of mood and aggression and plays an essential role in the onset of sleep. Due to increased utilization of branched chain amino acids as energy sources during prolonged exercise, the ratio of tryptophan to branched chain amino acids is altered in favour of tryptophan entry into the brain and onset of fatigue. Upon ingestion of branched chain amino acids, increased concentrations of branched chain amino acids in circulation were suggested to lead to less tryptophan entry into the brain and consequently to a delay in the onset of fatigue. However, the hypothesis of central fatigue was not supported as various studies failed to demonstrate ergogenic properties of several forms of branched chain amino acid administration (infusion, oral, and with and without carbohyrates) on exercise capacity or ratings of perceived exertion (Blomstrand, et al., 1997; Madsen, et al., 1996; van Hall, et al., 1995; Varnier, et al., 1994).

4.4

Branched Chain Amino Acids and Immunity

All forms of immunity are affected by protein malnutrition in humans, with particular depression in the number of mature fully-differentiated T lymphocytes and impairment of in vivo proliferative response to mitogens (Daly, et al., 1990). Immune defenses are dependent on proteins for rapid cell replication and for the production of immunoglobulins, acute phase proteins and cytokines. Deficiencies in protein are common among vegetarian athletes and in sports in which leanness is required to confer a performance advantage or to meet certain body weight categories. Avoiding protein deficiencies was suggested essential for maintaining an effective immune system following prolonged and intense exercise bouts. Certain amino acids, including branched chain amino acids, were reported to enhance immunity responses if consumed before or during exercise. There is a lack of studies regarding branched chain amino acid supply and immunological status, pointing out the need for further investigations. Although animal studies showed that insufficient availability of branched chain amino acids impairs certain aspects of immunity, there is little evidence about upregulation of immune function by supplementation with branched chain amino acids (Calder, 2006). Furthermore, studies in humans are also inconclusive. Supplementation of branched chain amino acids (6 g/day for 15 days) prior to a triathlon or a 30-km run in male triathletes and marathoners respectively prevented any decline in mitogen-stimulated lymphocyte proliferation and increased production of lymphocyte, IL-2 and interferon-γ as compared to the placebo group (Bassit, et al., 2002). By increasing skeletal muscle glutamine output into circulation, branched chain amino acid ingestion was suggested to prevent post-exercise decreases in plasma glutamine concentrations, maintain a constant supply of energy for lymphocytes and macrophages and attenuate deteriorations of immunological functions following intense and prolonged exercise sessions. Glutamine is an important energy source for immune cells including leukocytes, lymphocytes and other rapidly dividing cells such as gut mucosa and bone marrow stem cells (Gleeson, 2006). Branched chain amino acids were reported to serve as a major source of nitrogen for glutamine formation in muscle cells via the alanine-glucose cycle during fasting and exercise (Darmaun & Déchelotte, 1991). However, there is no evidence that such reported changes in a number of biological parameters reflect per se beneficial physiological effects of branched chain amino acids on immunity. In summary, despite the vast amounts of papers addressing branched chain amino acid supplementation and exercise, there is still no consensus on the net benefits of these amino acids on various components of physical activity and exercise.

5

Branched Chain Amino Acid Supplements in Exercise

Despite the lack of consensus regarding the necessity to increase intakes of dietary proteins in general and branched chain amino acids in specific for ergogenic purposes during exercise, there is a strong belief among athletes that increased ingestion of branched chain amino acids stimulates muscle mass, prevents muscle damage, prolongs time to fatigue and enhances overall performance. Although human daily requirements for branched chain amino acids were estimated to be 68 to 144 mg/kg body weight (Baker, 2005), consumption of branched chain amino acids in large amounts was described in various groups of athletes. Intakes of branched chain amino acid supplements were even reported among exercising individuals at gyms. In Brazil, 6% of 405 supplement users of people exercising in gyms reported taking branched chain amino acid supplements (Goston & Correia, 2010). In addition, 2.2% of 186 individuals

exercising in gyms in Beirut city and using dietary supplements on regular basis reported the intake of branched chain amino acids as supplements (El Khoury & Antoine-Jonville, 2012). Branched chain amino acids are typically consumed in doses of 10-20 g with a standard 2:1:1 Leucine/Isoleucine/Valine ratio. This stoichiometric ratio was reported to be the most beneficial to exercising individuals and athletes. A mixture of branched chain amino acids provided in this ratio was found to improve various aspects of exercise performance such as heart frequency, VO2max and power production peak compared to creatine and placebo groups, when consumed by a group of athletes (Dioguardi, 2003). The availability of branched chain amino acids in this ratio promoted amino acid and free fatty acid oxidation both in muscle cells and in cardiomyocites. These metabolic changes suggested a reduced dependence on glycolysis of at least 30% and reflected that lactate production from pyruvate would be rapidly and more largely converted to pyruvate in animals treated with this ratio, leading consequently to maximized power generation and delayed onset of fatigue. However, we should keep in mind that, when given in a mixture, the clearance of individual amino acids could occur at rates of uptake that do not correspond directly to their composition in the ingested mixture (Borsheim, et al., 2002). 5.1

Sources of Branched Chain Amino Acids

Branched chain amino acids are usually obtained from whole food proteins, solutions of protein hydrolysates and free amino acids (Gleeson, 2005). Effects of these different sources on muscle protein synthesis, skeletal muscle hypertrophy and performance have been never directly compared. There is a general consensus that, during times of recovery, pre-digested proteins or free amino acids are more advantageous in promoting muscle protein synthesis and reducing muscle damage and soreness compared to whole intact proteins due to the accelerated absorption and utilization of their amino acids (Buckley, et al., 2010; Nosaka, et al., 2006). The faster rate of amino acid appearance and greater peak of amino acid concentrations following casein hydrolysate compared to intact casein magnified muscle protein synthesis response to exercise (Koopman, et al., 2009). However, data on intact whey and whey protein hydrolysate is less clear. Since whey is already an effectively rapidly-absorbed short-acting protein source, its hydrolysis may not further augment its gastric emptying and substantially accelerate appearance of amino acids in circulation (Power, et al., 2009). Such hydrolysis may though ameliorate kinetic responses to some of the larger whey peptides (Ha & Zemel, 2003). Indeed, in one acute training study, peak isometric torque was fully recovered by 6 hours post-fatiguing eccentric exercise in subjects consuming 25g of whey protein hydrolysate, whereas it was still depressed by 24 hours post-exercise both in the whey protein isolate and placebo groups (Buckley, et al., 2010). Although hydrolyzed proteins and free amino acids are the most promoted among athletes, beneficial impact of whole protein sources on muscle protein synthesis, skeletal muscle hypertrophy as well as muscle adaptations during resistance exercise have been also reported. Amongst high-quality proteins, a superiority of dairy proteins in increasing muscle protein synthesis is revealed in the literature. Supplementation with milk (Rankin, et al., 2004) or whey protein (Burke, et al., 2001) immediately following exercise resulted in greater gains in strength measures compared to an isocaloric carbohydrate drink during a 10-12 week resistance training program. Furthermore, the consumption of fat-free milk after resistance exercise, over 12 weeks, increased lean mass and type II myofibrillar crosssectional area to a greater extent than soy or carbohydrate in untrained young men (Hartman, et al., 2007). The functional properties of dairy may be attributed to the large concentrations of branched chain amino acids embedded in their proteins.

Based on these findings, the role of branched chain amino acids, which are identified in dairy products and in whey protein hydrolysates, in promoting muscle protein growth during exercise seems to be visible irrespective of the source. According to the European Food Safety Authority, evidence is still lacking for the scientific substantiation of health claims regarding the superior effects of branched chain amino acids, when consumed in addition to adequate protein intakes, on growth and maintenance of muscle mass, attenuation of muscle power decline during exercise, recovery from muscle fatigue after exercise, improvement of cognitive function after exercise, reduction in perceived exertion during exercise and enhancement of immunity health above the established role of high quality proteins on the claimed effects (EFSA Panel on Dietetic Products, 2010). 5.2

Safety Aspects of Branched Chain Amino Acids

Most studies associating high protein intake with metabolic strain on kidneys and with increased risk of osteoporosis were based either on isolated tissues exposed to physiologically-irrelevant amounts of proteins or on individuals with predisposition to renal and bone-related impairments (Maughan, et al., 2007). Branched chain amino acids were generally considered to be safe. When consumed as part of a diet, ingestion of the three branched chain amino acids up to 450 mg/kg/day, which is around three times the estimated average requirement, caused no adverse effects in healthy adults (Gleeson, 2005). Even an acute dose of 450 mg/kg/day, or around 30 g/day, of branched chain amino acid supplements was well tolerated by adults. However, it should be noted that oral exposure to branched chain amino acids as free amino acids may be harmful although not yet well explored. A mild concern was raised regarding the possible interference of branched chain amino acids with the absorption of other amino acids and their potential association with gastrointestinal distress (Gleeson, 2005). Moreover, the three branched chain amino acids may compete among each others for transport across membranes. In animals, high dietary intakes of leucine decreased plasma isoleucine and valine concentrations (Harper, et al., 1984). In humans, on the other hand, no significant interactions were reported within the physiological range of branched chain amino acid intake (Pelletier, et al., 1991; Pelletier, et al., 1991). However, upon administration of a given branched chain amino acid or a mixture of branched chain amino acids at large non-physiological doses similar to doses typically consumed by athletes, the intake of one amino acid may influence the oxidation rates and the requirements of the other two amino acids (Hutson, et al., 2005). Furthermore, large doses of branched chain amino acids were hypothesized to promote tumor growth due to their modulation of anabolic hormonal signaling (Nair & Short, 2005). Upregulated signaling through PI3 kinase and mTOR pathways, which are the pathways of action of branched chain amino acids, was described in some cancers (Vogt, 2001). In addition, based on findings of the Vascular Interaction with Age in Myocardial Infarction (VINTAGE MI) clinical trial, the addition of L-arginine at 3 g 3 times per day over 6 months to standard postinfarct medications did not reduce non-invasive measures of vascular stiffness or improve ejection fraction and other clinical outcomes (Schulman, et al., 2006). On the contrary, an increased risk of death in older patients was noted with L-arginine supplementation compared to those taking a placebo. Although this study investigated arginine and not branched chain amino acids, findings reflect how exogenous introduction of amino acids may have dramatic negative impact on health. Due to the potential risks associated with the intake of branched chain amino acids as free amino acid supplements, foods rich in branched chain amino acids may offer affordable and risk-free alternatives of branched chain amino acid supplements for athletes. In comparison to a typical branched chain amino acid supplement sold in tablet form and providing 200 mg valine, 200 mg isoleucine and 400 mg leucine, egg white (200 kCal)

provides 3371 mg valine, 2754 mg isoleucine and 4233 mg leucine which is equivalent to 11 tablets of branched chain amino acid supplements.

6

Conclusion

To date, literature does not support any superior benefits of the addition of branched chain amino acids to a well-balanced diet meeting requirements for essential amino acids on improved muscle hypertrophy, delayed onset of fatigue, enhanced performance and ameliorated immunological responses in athletes and exercising individuals with the exception of a minimal attenuation in muscle tissue damage which is induced by an intense training of prolonged duration. Increased intakes of branched chain amino acids, within safe limits, through foods, supplements or both may be though needed in vulnerable groups including low energy consumers for reasons related to competition requirements, vegetarian athletes with limited choice and student performers with limited budget. Future studies should address the impact of chronic branched chain amino acid supplementation, alone or in combination with other amino acids, on muscle functionality, repair and regeneration, immunological functions as well as performance during exercise of varied types, intensities and duration and explore the underlying mechanisms.

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