**5. Creatine**

A large number of surveys indicate that creatine (n [aminoiminomethyl]-N-methylglycine) is one of the most widely used nutritional supplements [117-122]. Prevalence studies indicate that the use of creatine is particularly common in athletes and soldiers. Among the athletes population, powerlifters, boxers, weightlifters, and track/field athletes report the higher creatine consumption with prevalence ranging between 45 and 75% [122]. The major deter‐ minant of such a widespread consumption by resistance athletes mainly resides in the known ergogenic aid of creatine when supplementation is associated with repeated bouts of high intensity exercise. This combination leads to increased lean body mass (with no effect on fat mass), muscle strength and performance and accelerated post-exercise recovery [123]. Interestingly more pronounced effects of creatine supplementation have been found in strength trained older adults compared to the young adults [124] and in untrained compared to trained individuals whereas similar changes in muscle creatine content and exercise performance have been found between men and women [125, 126]. Besides no ergogenic effect of creatine has been found in a variety of endurance exercise paradigms [127-129].

quicker muscle recovery from strenuous exercise following supplementation. As a matter of fact early studies by Brass et al. (1993) [110] demonstrated that L-carnitine delays hypoxiainduced fatigue in electrically stimulated rat skeletal muscle in vitro through its key stimula‐ tory role in muscle bioenergetics and antioxidant activity. Further evidence demonstrated that L-carnitine exhibits effective superoxide anion radical and hydrogen peroxide scavenging, total reducing power and metal chelating activities in vitro [111]. In vivo, in presence of high glycolytic rates as during strenuous resistance exercise, the stimulated formation of ATP and AMP from molecules of ADP results in the oxidation of AMP to hypoxanthine which is considered a marker of metabolic stress [112]. This oxidation reaction is mediated by xanthine oxidase. Accumulation of xanthine oxidase in spite of xanthine dehydrogenase is the conse‐ quence of the activation of calcium-dependent proteases, which cleave a portion of xanthine dehydrogenase and convert it into xanthine oxidase. This process is a direct consequence of raised intracellular calcium by inhibition of calcium ATPase pumps induced by insufficient supply of ATP. This response appears to be attenuated by L-carnitine supplementation which reduces intracellular hypoxanthine and xanthine oxidase following resistance exercise bouts [103, 106]. Indeed inhibition of xanthine oxidase with allopurinol during exercise has been shown to result in significantly less generation of ROS, reduced tissue damage after exhaustive exercise [112], and less accumulation of cytosolic enzymes CK and LDH [113, 114]. Further‐ more a direct consequence of high-intensity training is hypoxia. Exercise under hypoxic conditions stimulates muscle glucose transport, increases the concentration of ammonia in blood, and lowers the concentration of free carnitine [115, 116]. It has been found that carnitine supplementation during exercise under hypoxic conditions may also prevent ammonia

In summary, L-carnitine supplementation can beneficially affect postexercise markers of metabolic stress, muscle disruption, and muscle soreness in young and old healthy men and women. The attenuation of the side effects of high-intensity training mainly relate to its antioxidant potential and its capability to reduce the magnitude of exercise-induced hypoxia. Further research is needed to conclusively elucidate the mechanisms underlying its protective effects and whether these responses may also arise in exercised individuals affected by

A large number of surveys indicate that creatine (n [aminoiminomethyl]-N-methylglycine) is one of the most widely used nutritional supplements [117-122]. Prevalence studies indicate that the use of creatine is particularly common in athletes and soldiers. Among the athletes population, powerlifters, boxers, weightlifters, and track/field athletes report the higher creatine consumption with prevalence ranging between 45 and 75% [122]. The major deter‐ minant of such a widespread consumption by resistance athletes mainly resides in the known ergogenic aid of creatine when supplementation is associated with repeated bouts of high intensity exercise. This combination leads to increased lean body mass (with no effect on fat mass), muscle strength and performance and accelerated post-exercise recovery [123].

toxicity mainly through reduction of ROS production.

disorders of different origin as neuromuscular diseases.

**5. Creatine**

70 Muscle Injuries in Sport Medicine

Several mechanisms could explain the effects of creatine supplementation on muscle mass, strength and performance when supplementation is combined with strength training.

The hypertrophic response has been attributed to increased myosin heavy chain protein expression [130], changes in the expression of myogenic regulatory factors (MRF4 and myogenin) [131, 132], increased mitotic activity of satellite cells and swelling-induced protein synthesis [133-137] followed by net protein deposition. The most popular mechanism to explain the efficacy of creatine on muscle performance refers to a better match between ATP supply and fibers demands during physical exercise due to the enhancement of the resting high energy phosphate levels (total creatine, phosphocreatine, creatine and ATP) observed following supplementation. This change allows users to maintain a greater work intensity for longer durations of time (increased total training volume). In particular, the intracellular concentration of phosphocreatine is known to play a major role during the bioenergetic system mostly active during exercise at high intensities and short durations. Overall the dosing regimen that has been found to significantly increase the intracellular phosphocreatine is a loading phase of approximately 20 g/day for 5-7 days followed by a maintenance phase of 5 g/day for a period of several weeks [138, 139].

The known effects of creatine upon muscle cell function, structure and protein metabolism may represent the rationale for its potential use to prevent or treat muscle cell injuries. Nevertheless, although solid studies have examined the ergogenic potential of creatine, the current literature is very preliminary in relation to examining the effects of creatine supple‐ mentation in reducing the severity of exercise-induced muscle damage and/or promote recovery following strength training and endurance paradigms [140].

Considering that high-force eccentric exercise alters myofibre membrane structure and function [9, 141] leading to reductions in force, increased soreness, and impaired muscle function and that membrane stabilization due to decreased membrane fluidity is followed to increased intracellular concentration of phosphocreatine [142], the effects of creatine supple‐ mentation on markers of eccentric exercise damage have been assessed following resistance exercise sessions [143-146]. Initial studies conducted in rodents and humans agreed to demonstrate that creatine supplementation does not decrease muscle damage or enhance recovery after high intensity eccentric contractions. In particular Warren and colleagues [143] demonstrated that recovery of mouse anterior crural muscle strength after damage induced by 150 eccentric contractions was unaffected by creatine supplementation at 0.5 and 1% for two weeks. Following 3 minutes recovery, there was no effect of creatine supplementation on the isometric torque loss or on the torque loss at any eccentric or concentric angular velocity tested [143]. In 2001 Rowson and colleagues [146] evaluated the effects of short time creatine and dextrose supplementation (20 gr d-1 creatine and 28 gr d-1 for 5 days, a protocol previously shown to be effective in elevating muscle creatine and phosphocreatine levels [126]) before

performing 50 maximal eccentric contractions of the elbow flexors on blood markers of muscle damage (CK and LDH), maximal isometric force, range of motion, arm circumference (an index of swelling), and muscle soreness. Despite the initial hypothesis, results showed nearly identical loss of maximal isometric force and range of motion, development of soreness, increase of the biceps circumference and change in blood CK and LDH in supplemented and placebo groups of subjects thus suggesting that creatine supplementation lacked to display significant improvement of membrane stabilization at the conditions analyzed. In a second study by the same authors male participants were supplemented with creatine for 5 days prior to, and 5 days following a hypoxic resistance exercise test (5 sets of 15-20 repetitions at 50% of 1 repetition maximum). Similarly to the first study creatine failed to have positive effects on the same criterion measures of muscle damage following the resistance exercise challenge [145]. More recently differing results have been obtained following creatine and carbohydrate supplementation to untrained male subjects by the scheme 5 days prior to, and 14 days following a resistance exercise training session consisting of 4 sets of 10 eccentric repetitions at 120% of maximum concentric 1-RM on the leg press, leg extension and leg flexion machine. Creatine supplementation produced significantly greater isokinetic and isometric knee extension strength during recovery from exercise-induced muscle damage. Furthermore, plasma CK activity was lower after 48, 72, 96 hrs, and 7 days recovery in the supplemented group [144]. As discussed by Cook such diverse observations could be in part attributed to the duration of supplementation period and/or post-exercise supplementation. In particular in the first study by Rawson the subjects enrolled were supplemented only for 5 days prior to the exercise protocol; with no continuation of supplementation following the exercise bout [146]. As it has been suggested that the effect of creatine on protein synthesis and muscle regeneration may be enhanced during the recovery period post-injury [130, 147], the time schedule of creatine supplementation respect to the exercise bout may be considered a potential limiter of the muscular protection against exercise-induced damage. This hypothesis seems to be confirmed by the observed increase of satellite cell number and myonuclei concentration following creatine supplementation in human skeletal muscle [147]. Indeed it can be hypothe‐ sized that this effect may sum to the known training-induced increase in muscle regeneration.

following prolonged running exercise (30Km run) [149]. Marathon runners were supplement‐ ed for 5 days (20 g/day) prior to a 30km race. Blood samples were collected pre-race, and 24 hours following the end and CK, LDH, prostaglandin E2 (PGE2) and TNFalpha (TNF-α) were

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As expected, prolonged running provoked an increase in concentrations of all plasma markers tested, indicating the appearance of cell injury associated with an inflammatory response [6, 150]. Creatine supplementation was effective in significantly attenuate the observed increase in all muscle soreness markers analyzed, unlike CK, thus pointing this nutritional intervention as an effective strategy in maintaining muscle integrity during and after intense and prolonged endurance exercise. In fact the lack of effect upon plasma CK concentration might not reflect the overall positive effect of creatine on muscle damage as a strong variability of this parameter among athletes, its dependence from the training status and the weak correlation with changes in other markers of muscle damage [151-153] lessen its significance in comparison with other markers of cellular death and lysis as LDH [154]. Similar effects on plasma pro inflammatory markers (Interleukin (IL) 1 beta and IL-6, TNF-α, and Interferon alpha (INF alpha) and PGE2) [155] and on plasma markers of cellular integrity (CK, LDH aldolase (ALD), glutamic oxalo‐ acetic, acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) [156] have been obtained in double blind trials following creatine supplemen‐ tation (20gr day-1) 5 days before a half-ironman and after ironman triathlon competition

These results confirm the hypothesis that creatine may have a protective effect against membrane cell disruption following prolonged and intense muscle contractions [156]. Indeed as skeletal muscle damage during an ironman competition mostly result from eccentric contractions, mainly related to the marathon segment of the race [157], it can be argued that creatine may be effective in preventing eccentric induced muscle injury. In fact exhaustive exercises involving eccentric contractions, as in triathlon competition, lead to more pro‐ nounced muscle damage than strenuous exercises involving concentric contractions [158]. Nevertheless other mechanisms but eccentric damage may contribute to muscle damage during triathlon competition including excessive metabolic workload, muscle fatigue, depletion of intramuscular glycogen, and oxidative stress which are generally implicated in prolonged exercise-induced muscle fiber disruption [157, 159, 160]. The observed reduction in plasma activities of GOT and GPT (markers of liver injury) observed in triathletes after an ironman competition may suggest that creatine supplementation can enhance the metabolic efficiency of skeletal muscle preventing the metabolic workload on the liver which has a critical role on the contractile activity-induced skeletal muscle injury. Indeed when eccentric contrac‐ tion is avoided as in electrically stimulated gastrocnemius muscle of the rat, creatine supple‐ mentation has been found to delay the fatigue appearance, preserve the force development, and prevent the rise of LDH and CK plasma activities and muscle vascular permeability evaluated with Evans blu staining [156]. Furthermore, although it cannot be excluded that in endurance settings the benefits of supplementation in preventing muscle damage may relate to the antioxidant potential that has been attributed to creatine in various oxidative stressassociated diseases, few studies have been published on the relationship between supplemen‐

measured.

respectively.

Notwithstanding supplementation was continued for 5 days after the exercise bout, in the second study by Rawson and colleagues no beneficial effects of creatine on criterion measures of muscle damage were observed [145]. Although it cannot be excluded that the resistance exercise paradigm used by Rawson, designed to be hypoxic in nature, may not have elicited enough muscle damage to unmask the anabolic effects of creatine supplementation, to date available conflicting data from a limited number of experimental works on the topic do not allow to safely draw conclusions on the beneficial effects of oral creatine supplementation on skeletal muscle damage and recovery following eccentric exercise challenge and new, more standardized, experimental works would help unravel this question in the next future.

Based on the fact that cell injury in running depends on cell volume integrity and that creatine potentially stabilizes the cell volume through an increase in cell water content, glycogen stores and/or myofibrillar content [135-137, 148], the effect of oral creatine supplementation has also been examined on markers of muscle damage, i.e. inflammatory and muscle soreness markers, following prolonged running exercise (30Km run) [149]. Marathon runners were supplement‐ ed for 5 days (20 g/day) prior to a 30km race. Blood samples were collected pre-race, and 24 hours following the end and CK, LDH, prostaglandin E2 (PGE2) and TNFalpha (TNF-α) were measured.

performing 50 maximal eccentric contractions of the elbow flexors on blood markers of muscle damage (CK and LDH), maximal isometric force, range of motion, arm circumference (an index of swelling), and muscle soreness. Despite the initial hypothesis, results showed nearly identical loss of maximal isometric force and range of motion, development of soreness, increase of the biceps circumference and change in blood CK and LDH in supplemented and placebo groups of subjects thus suggesting that creatine supplementation lacked to display significant improvement of membrane stabilization at the conditions analyzed. In a second study by the same authors male participants were supplemented with creatine for 5 days prior to, and 5 days following a hypoxic resistance exercise test (5 sets of 15-20 repetitions at 50% of 1 repetition maximum). Similarly to the first study creatine failed to have positive effects on the same criterion measures of muscle damage following the resistance exercise challenge [145]. More recently differing results have been obtained following creatine and carbohydrate supplementation to untrained male subjects by the scheme 5 days prior to, and 14 days following a resistance exercise training session consisting of 4 sets of 10 eccentric repetitions at 120% of maximum concentric 1-RM on the leg press, leg extension and leg flexion machine. Creatine supplementation produced significantly greater isokinetic and isometric knee extension strength during recovery from exercise-induced muscle damage. Furthermore, plasma CK activity was lower after 48, 72, 96 hrs, and 7 days recovery in the supplemented group [144]. As discussed by Cook such diverse observations could be in part attributed to the duration of supplementation period and/or post-exercise supplementation. In particular in the first study by Rawson the subjects enrolled were supplemented only for 5 days prior to the exercise protocol; with no continuation of supplementation following the exercise bout [146]. As it has been suggested that the effect of creatine on protein synthesis and muscle regeneration may be enhanced during the recovery period post-injury [130, 147], the time schedule of creatine supplementation respect to the exercise bout may be considered a potential limiter of the muscular protection against exercise-induced damage. This hypothesis seems to be confirmed by the observed increase of satellite cell number and myonuclei concentration following creatine supplementation in human skeletal muscle [147]. Indeed it can be hypothe‐ sized that this effect may sum to the known training-induced increase in muscle regeneration.

72 Muscle Injuries in Sport Medicine

Notwithstanding supplementation was continued for 5 days after the exercise bout, in the second study by Rawson and colleagues no beneficial effects of creatine on criterion measures of muscle damage were observed [145]. Although it cannot be excluded that the resistance exercise paradigm used by Rawson, designed to be hypoxic in nature, may not have elicited enough muscle damage to unmask the anabolic effects of creatine supplementation, to date available conflicting data from a limited number of experimental works on the topic do not allow to safely draw conclusions on the beneficial effects of oral creatine supplementation on skeletal muscle damage and recovery following eccentric exercise challenge and new, more standardized, experimental works would help unravel this question in the next future.

Based on the fact that cell injury in running depends on cell volume integrity and that creatine potentially stabilizes the cell volume through an increase in cell water content, glycogen stores and/or myofibrillar content [135-137, 148], the effect of oral creatine supplementation has also been examined on markers of muscle damage, i.e. inflammatory and muscle soreness markers, As expected, prolonged running provoked an increase in concentrations of all plasma markers tested, indicating the appearance of cell injury associated with an inflammatory response [6, 150]. Creatine supplementation was effective in significantly attenuate the observed increase in all muscle soreness markers analyzed, unlike CK, thus pointing this nutritional intervention as an effective strategy in maintaining muscle integrity during and after intense and prolonged endurance exercise. In fact the lack of effect upon plasma CK concentration might not reflect the overall positive effect of creatine on muscle damage as a strong variability of this parameter among athletes, its dependence from the training status and the weak correlation with changes in other markers of muscle damage [151-153] lessen its significance in comparison with other markers of cellular death and lysis as LDH [154]. Similar effects on plasma pro inflammatory markers (Interleukin (IL) 1 beta and IL-6, TNF-α, and Interferon alpha (INF alpha) and PGE2) [155] and on plasma markers of cellular integrity (CK, LDH aldolase (ALD), glutamic oxalo‐ acetic, acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) [156] have been obtained in double blind trials following creatine supplemen‐ tation (20gr day-1) 5 days before a half-ironman and after ironman triathlon competition respectively.

These results confirm the hypothesis that creatine may have a protective effect against membrane cell disruption following prolonged and intense muscle contractions [156]. Indeed as skeletal muscle damage during an ironman competition mostly result from eccentric contractions, mainly related to the marathon segment of the race [157], it can be argued that creatine may be effective in preventing eccentric induced muscle injury. In fact exhaustive exercises involving eccentric contractions, as in triathlon competition, lead to more pro‐ nounced muscle damage than strenuous exercises involving concentric contractions [158]. Nevertheless other mechanisms but eccentric damage may contribute to muscle damage during triathlon competition including excessive metabolic workload, muscle fatigue, depletion of intramuscular glycogen, and oxidative stress which are generally implicated in prolonged exercise-induced muscle fiber disruption [157, 159, 160]. The observed reduction in plasma activities of GOT and GPT (markers of liver injury) observed in triathletes after an ironman competition may suggest that creatine supplementation can enhance the metabolic efficiency of skeletal muscle preventing the metabolic workload on the liver which has a critical role on the contractile activity-induced skeletal muscle injury. Indeed when eccentric contrac‐ tion is avoided as in electrically stimulated gastrocnemius muscle of the rat, creatine supple‐ mentation has been found to delay the fatigue appearance, preserve the force development, and prevent the rise of LDH and CK plasma activities and muscle vascular permeability evaluated with Evans blu staining [156]. Furthermore, although it cannot be excluded that in endurance settings the benefits of supplementation in preventing muscle damage may relate to the antioxidant potential that has been attributed to creatine in various oxidative stressassociated diseases, few studies have been published on the relationship between supplemen‐ tation and oxidative stress and controversial and not conclusive results are currently available [161-163]. In particular creatine supplementation associated with resistance training or exhaustive exercise training has been associated either with reduced oxidative stress [162, 164], increased free radical generation and related consumption of antioxidant reserves [161] or no change of lipid peroxidation, resistance of low density lipoprotein to oxidative stress or plasma concentrations of non-enzymatic antioxidants [163]. Taken together these observations show that creatine supplementation before strenuous endurance exercise reduces the increase of markers of cell death/lysis and muscle soreness suggesting a positive effect of the supple‐ mentation strategy in maintaining muscle integrity after intense prolonged exercise. The mechanisms underlying such a protective effect are only partially known.

Studies in animals models and humans showed that essential amino acids (EAA) [170, 171] unlike non EAA [170], are fundamental regulators of MPS and mitochondrial biogenesis [172]. It has been shown that hyperaminoacidemia stimulates amino acid transport and net MPS, unlike carbohydrate administration both in the young [173] and in the elderly [174]. The effects on protein synthesis arise independently of changes in anabolic hormone concentration [175, 176], although insulin is required for the effects of EAA on translation [177]. Among EAA, Branched chain amino acids (BCAA: leucine, isoleucine and valine) play a very important role as nutrient signals that regulates MPS through the stimulation of insulin-independent and rapamycin-sensitive pathways [178, 179]. In particular available data suggest that at least part of the postprandial translational activation is to be attributed to BCAAs through activation of mTOR and downstream signals (elF4G, S6K1 and 4E-BP1). Although mTOR is the key integrator of the anabolic response to BCAA, mTOR itself may not be the direct target of EAA. It has been shown that inhibition by the upstream TSC1/2 complex represents the mechanism

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through which leucine and insulin upregulate mTOR and downstream targets.

exercise [173, 183, 184] and prevented BCAA exercise-induced oxidation [182].

repair of muscle damage induced by resistance exercise.

BCAA group [187].

The scenario arising from available studies indicates that the physiological anabolic response to BCAA may help counteracting the metabolic unbalance induced by exercise and in partic‐ ular resistance training which has been linked to concurrent increase of MPS and MPC [180, 181] and negative changes in circulating free amino acids [182]. In these conditions the exercisetriggered hypercatabolism may be counteracted by amino acids supplementation which in turn has been related with net protein synthesis when combined with bouts of resistance

Recent studies suggest that BCAA supplementation, by promoting MPS, may improve the

In particular Nosaka et al. [185] showed that an amino acid supplement containing around 60% BCAA was effective in reducing muscle damage and soreness when consumed immedi‐ ately before (30 min) and during the four days of recovery following a damaging bout of lengthening contractions of the elbow flexors. Later Jackman and coworkers reported the effects of BCAA supplementation during recovery from intense eccentric exercise consisting in 12 x 10 repetitions of unilateral eccentric knee extension in male untrained subjects. A decrease in flexed muscle soreness was observed in supplemented compared with placebo group at 48 h and 72 h post exercise whereas the degree of force loss and the fluctuation of blood markers of muscle damage appeared unchanged between groups [186]. Similar results were obtained in female untrained young subjects by Shimomura et al. [187] examining the effects of BCAA supplementation on squat-exercise-induced DOMS. In this report the participants ingested either BCAA (isoleucine:leucine:valine = 1:2.3:1.2) or dextrin at 100 mg/kg body weight just before the squat exercise consisting of 7 sets of 20 squats/set with 3 min intervals between sets. The peak of DOMS was reached two or three days post exercise but the level of soreness was significantly lower in the BCAA trial than in the placebo. Interestingly three day post exercise the force decrease observed in the placebo appeared to be prevented by BCAA supplementation. Accordingly plasma myoglobin and elastase (index of neutrophil activation) appeared to be increased by exercise in the placebo but not in the
