CREATINE AND β-alanine SUPPLEMENTATION IN STRENGTH/POWER ATHLETES

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1 CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 8, No. 1, pp , 2010 ISSN print, Copyright 2010 by New Century Health Publishers, LLC All rights of reproduction in any form reserved CREATINE AND β-alanine SUPPLEMENTATION IN STRENGTH/POWER ATHLETES Jay R. Hoffman The College of New Jersey, Ewing, NJ [Received December 31, 20089; Accepted February 13, 2010] ABSTRACT: Creatine and ß-alanine are two of the most popular sport supplements used by strength/power athletes today. The popularity of creatine has resulted in more than 600 studies examining the physiology, efficacy and safety of its use among various athletic populations. Recently, ß-alanine has become as popular a supplement for the anaerobic athlete due to its unique ability to enhance muscle buffering capacity. This review will examine the studies that have been conducted on the efficacy of these supplements. In addition, the physiology that underlies the mechanisms of action behind these supplements will be described and provide an understanding to the potential ergogenic benefits that they hold for strength and power athletes. Finally, discussion will also examine the potential adverse effects associated with each supplement. KEY WORDS: Body Mass Changes, Creatine Side Effects, Physiology, Skeletal Muscle Adaptation Corresponding Author: Jay R. Hoffman, Ph.D., FACSM, FNSCA, Department of Health and Exercise Science, The College of New Jersey, PO Box 7718, Ewing, New Jersey 08628; Tel: ; Fax: ; hoffmanj@tcnj.edu INTRODUCTION For the past 20 years the most popular supplement among strength/power athletes has been creatine. This is in part related to the efficacy that has been demonstrated in the hundreds of studies that have been published to date. In addition, creatine use has been shown to be quite safe in a number of studies examining both short and long term effects in young, healthy athletic populations. β-alanine is a relatively new supplement that is starting to gain in popularity due to its ability to enhance the quality of training sessions and its impact on sport performance by delaying fatigue. This paper will examine the physiology of both creatine and β-alanine, their mechanism of action, dosing patterns, effects on performance, and health issues associated with their use. In addition, the benefit of combining these two supplements is also explored. PHYSIOLOGY OF CREATINE Creatine is a nitrogenous organic compound that is synthesized naturally in the body primarily in the liver. It can also be synthesized in smaller amounts in both the kidneys and pancreas. The amino acids arginine, glycine and methionine are the precursors for the synthesis of creatine in those organs. Creatine can also be consumed in the diet with high concentrations found in both meat and fish. Approximately 98% of creatine is stored within skeletal muscle in either its free form (40%) or in its phosphorylated form (60%) (Heymsfield et al., 1983). It is the phosphorylated form of creatine, referred to as phosphocreatine (PCr), that the muscle cell uses to provide energy to fuel high intensity exercise. Small amounts of creatine are also stored in the heart, brain and testes. Creatine is transported from its site of synthesis to the skeletal muscle via the circulation. Creatine combined with a phosphate group has a critical role in energy metabolism. It acts as a substrate in the formation of adenosine triphosphate (ATP) by rephosphorylating adenosine diphosphate (ADP). This becomes quite important during short duration, high intensity exercise. The ability to rapidly rephosphorylate ADP to ATP is dependent upon the enzyme creatine kinase and the availability of PCr within the muscle. As PCr concentrations become reduced, the ability to maintain or produce high intensity exercise will decline. During short duration, high intensity activity (i.e. 50m - 100m sprint) the energy needed to perform that activity is derived primarily through the hydrolysis of PCr (Gaitanos et al., 1993; Hirvonen et al., 1987). As duration of high intensity exercise increases, the extent that PCr serves as the primary source of energy is reduced. For example, during a 6- second bout of maximal exercise (e.g., a set of 6 repetitions in the squat exercise, or a 50 m sprint) PCr concentrations within the muscle are reduced between 35 57% from resting levels (Gaitanos et al., 1993, Boobis et al., 1987). As exercise duration increases to 30-seconds (e.g. a 200 m sprint), PCr levels in the muscle are reduced to 64 80% of resting levels (Bogdanis et al., 1996; Boobis et al., 1987; Cheetham et al., 1986), and during repetitive high intensity exercise PCr levels in the muscle become almost completely depleted (McCartney et al., 1986). As muscle PCr concentrations decrease the ability to perform maximal exercise is

2 20 Creatine and β-alanine supplementation reduced (Hirvonen et al., 1992). However, if muscle PCr concentrations are elevated the ability to sustain high intensity exercise will be maintained. This is the basis behind creatine supplementation. Creatine supplementation has been demonstrated to elevate muscle creatine content by approximately 20% (Febbraio et al., 1995; Hultman et al., 1996). Interestingly, there does appear to be a ceiling effect in which once muscle creatine concentrations are saturated, any further intake will be unable to increase muscle creatine concentrations. Once creatine concentrations in skeletal muscle reach mmol.kg -1 dry weight additional supplementation does not appear to be able to further increase muscle creatine concentrations (Balsom et al., 1994; Greenhaff, 1995). This helps coaches, nutritionists and sport scientists assist athletes by helping develop proper and realistic dosing schemes, and prevents the if a little is good, then more must be better philosophy that is often employed by athletes in the use of nutritional supplements. CREATINE DOSING SCHEMES To maximize the muscle creatine concentrations athletes will typically use a dosing regimen of grams daily for 5 days, or 0.3 g. kg -1 body mass if an individual wishes to dose relative to their body weight (Hultman et al., 1996). This is generally termed the loading phase. To maintain muscle creatine content a maintenance dose of 2-5 g.day -1 or g.kg -1 day -1 is often used following the loading phase. However, if athletes supplement with creatine without an initial loading dose, muscle creatine content can still reach similar levels that are seen in athletes that initially used a loading dose. The only difference is that it will take longer to reach that same muscle creatine concentration ( 30 days versus 5 days). Muscle creatine levels will remain elevated as long as the maintenance dose is taken. Once creatine supplementation is stopped muscle creatine concentrations will return to original baseline levels within approximately 4-weeks (Febbraio et al., 1995; Hultman et al., 1996). EFFICACY OF CREATINE SUPPLEMENTATION Creatine supplementation is one of the most widely studied ergogenic aids to date. The results of these investigations have been consistent in showing significant ergogenic benefits (Bemben et al., 2001; Brenner et al., 2000; Eckerson et al., 2004; Haff et al., 2000; Hoffman et al., 2006; Kirksey et al., 1999; Lehmkuhl et al., 2003; Pearson et al., 1999; Volek et al., 1999). Although the efficacy of creatine is primarily seen during prolonged supplementation, varying durations of dosing regimens have been studied with varying results. Short-term creatine supplementation protocols (3 6 days) have generally not resulted in significant performance improvements during a single, acute bout of explosive exercise (Cooke et al., 1995; Dawson et al., 1995; Mujica et al., 1996; Odland et al., 1997; Snow et al., 1998). However, one study did report that a 3-day loading dose (20 g of creatine monohydrate per day) in NCAA Division I strength/power athletes was able to significantly improve repeat sprint cycle performance (Ziegenfuss et al., 2002). Considering that the greatest increases in muscle creatine concentrations occur within the first 2 3 days of a loading phase, it is likely that the differences between this latter study and the others may be related to the exercise protocol used. The study that showed a positive effect used a multiple set high intensity sprint protocol (6 x 10 sec loaded sprints) with a 60-s rest interval between each sprint. The majority of the other studies used an isolated bout of exercise or a longer rest interval that would allow greater muscle phosphagen restoration. Other studies demonstrating the efficacy of short-term (5-6 days) creatine supplementation have used a loading dose (20 g d -1 ) as their supplement regime (Cottrell et al., 2002; Cox et al., 2002; Mujica et al, 2000; Preen et al., 2001; Stout et al., 2000) and have used a more appropriate testing regimen (e.g. repeated or prolonged high intensity exercise). Only two investigations are known that have shown ergogenic benefits from a low dose creatine supplementation (Burke et al., 2000; Hoffman et al., 2005). One study used a dosing scheme of 7.7 g d -1 (0.1 g kg -1 ) during 28 days of supplementation (Burke et al., 2000). Although this was a much longer supplementation period than typically seen during short-term studies, it was the first study to show efficacy with a lowdose, relatively short duration supplement regimen. Hoffman and colleagues (2005) demonstrated that even using a low-dose (6-g day -1 ), short duration (6 days) supplementation regimen in active, collegeaged men changes in fatigue rate may be seen, although no improvements were demonstrated in maximal power performance. It is important to note that the typical creatine dosing schedule for strength/power athletes is generally longer than 3 7 days. Studies examining typical dosing regimens (28 84 days) have consistently demonstrated significant improvements in strength and power performance (Bemben et al., 2001, Haff et al., 2000; Hoffman et al., 2006; Kirksey et al., 1999; Pearson et al., 1999; Volek et al., 1999; Kreider et al, 1998). The magnitude of strength improvements has been reported to be nearly 2-3 fold greater in athletes supplementing with creatine compared to a placebo (see Figure 1). Clearly, the benefit of creatine supplementation is expressed by enhancing the quality of workouts. By improving the quality of each training session the training stimulus to the muscle will ultimately yield greater physiological adaptation resulting in improved performance. FIGURE 1. Typical Strength Improvements Resulting from Creatine Supplementation in Strength/Power Athletes. Adapted from Hoffman and colleagues (2006). Kg Bench Press Placebo Creatine Squat

3 Creatine and β-alanine supplementation 21 THE EFFECT OF CREATINE SUPPLEMENTATION ON BODY MASS CHANGES Creatine supplementation has also been generally associated with increases in body weight. During prolonged supplementation increases in body mass are primarily seen as fat free mass. Increases in weight gain appear to occur relatively quickly as individuals supplement with creatine. This is likely related to an increase in total body water. As creatine content within skeletal muscle increases the intracellular osmotic gradient that results causes water to fill the cell (Volek and Kraemer, 1996). Increases in lean body mass are likely related to the enhanced stimulus to the muscle from a higher quality workout resulting in an increased synthesis of muscle contractile proteins (Balsom et al., 1993; Bessman and Savabi, 2000). CREATINE SUPPLEMENTATION AND SKELETAL MUSCLE ADAPTATION As discussed earlier, creatine supplementation is associated with increases in lean body mass, strength and power. These improvements are in part related to the physiological adaptations that occur within skeletal muscle as a result of the training stimulus, but are also aided by the creatine intervention. A 12-week examination of creatine supplementation during a periodized resistance training program revealed a significantly greater increase in the crosssectional area of Type I, Type IIa and Type IIab fibers (see Figure 2) in subjects ingesting creatine compared to a placebo (Volek et al., 1999). It was suggested that the greater gains in muscle cross-sectional area could be attributable to the higher quality workouts associated with creatine supplementation. These results were confirmed by another study examining a 12-week training and dosing protocol (Willoughby and Rosene, 2001). Figure 3 depicts the effect that creatine supplementation in conjunction with a heavy resistance training program can have on amplifying the response of myosin heavy chain protein synthesis (these are the microfilaments comprising muscle that are involved in muscle contraction). Again, this is likely due to the greater training stimulus that is associated with the higher creatine content within skeletal muscle. It appears that creatine is effective in eliciting muscle morphological changes, most probably as in indirect manner by providing the athlete the ability to sustain a higher quality workout that stimulates the greater physiological response. In contrast, some investigators have indicated that creatine may have a direct % improvment mg-ml -1 FIGURE 2. Effect of 12-Weeks of Creatine Supplementation on Increases in Muscle Cross-Sectional Area. * = p < Adapted from Volek and colleagues (1999) Type l effect on changing skeletal muscle morphology. Vierck and colleagues (2003), showed that when creatine is added to a cell culture of satellite cells it had the potential to increase satellite cell proliferation and differentiation. Satellite cells are myogenic stem cells that lie dormant between the basal lamina of the myofiber and the plasma membrane. When activated these cells proliferate, differentiate and fuse with existing myofibers within muscle to permit DNA accretion and cause muscle hypertrophy. The fusion of the satellite cells appears to be a necessary occurrence for muscle differentiation (Dayton and Hathaway, 1989). Thus, it does appear that creatine may both a direct and indirect role in stimulating muscle morphological changes during supplementation. * * Creatine Type lla Placebo * * Type llab FIGURE 3. Creatine Supplementation and Change in Myofibrillar Protein Content. Con = untrained control; P = placebo; Cr = creatine; * = significantly different (p < 0.05) than Con; # = significantly different than P. Adapted from Willoughby and Rosene (2001). *,# CON P CR

4 22 Creatine and β-alanine supplementation CREATINE SUPPLEMENTATION: RESPONDERS VS. NON-RESPONDERS For creatine to provide an ergogenic benefit it is believed that muscle creatine concentrations need to be increased by close to 20 mmol kg -1 of dry weight (Greenhaff, 1997). If not, then the benefits of creatine may not be realized. Greenhalf and colleagues (1994) have suggested that between 20 30% of subjects that ingest creatine may not respond. Non-responders are defined as subjects that achieve a less than 10 mmol kg -1 of dry weight increase in muscle creatine content following 5 days of creatine supplementation at 20 g d -1. This has been confirmed by other investigators as well (Syrotuik and Bell, 2004). Approximately 30% of individuals that supplement with creatine appear to be non-responders (Greenhaff et al., 1994; Syrotuik and Bell, 2004). Interestingly, subjects that were deemed non-responders also had lower strength performance gains compared to responders during the 5-days supplementation period (Syrotuik and Bell, 2004). Interestingly, the majority of subjects that supplemented were labeled as quasi-responders; individuals whose muscle creatine concentrations increased greater than 10 mmol kg -1 of dry weight, but less than 20 mmol kg -1 of dry weight. These individuals exhibited greater strength scores than the non-responders, but slightly less than that seen in the responders. Whether a strength/power athlete will respond to creatine supplementation or not appears to be dependent on initial muscle creatine content (Syrotuik and Bell, 2004; Ekblom, 1996; Harris et al, 1992). Athletes with low initial muscle creatine or phosphocreatine concentrations benefit the most from creatine supplementation, while those athletes with high initial levels benefit the least. While initial muscle creatine content appears to be the most important factor determining the effectiveness of creatine supplementation, muscle fiber composition may also contribute to the potential ergogenic benefit of creatine supplementation. Syrotuik and Bell (2004) reported that responders have the greatest percentage of type II fibers, followed by the quasi-responders and the non-responders had the lowest percentage of type II fibers. It appears that individuals who have predominantly fast twitch fibers with low initial levels of muscle creatine may reap the greatest benefit from creatine supplementation. SIDE EFFECTS ASSOCIATED WITH CREATINE SUPPLEMENTATION An increase in body mass associated with creatine supplementation has been suggested to be an unwanted side effect by some individuals (Schilling et al., 2001). For most athletes supplementing with creatine, weight gain is often a desired outcome. Side effects generally refer to potentially debilitating effects. There have been a host of anecdotal reports regarding creatine supplementation and gastrointestinal, cardiovascular and muscular problems. Muscle cramps has been the most frequently mentioned side-effect associated with creatine ingestion. However, controlled prospective studies have been unable to document any significant side effects from creatine supplementation. Even during prolonged supplementation (10 12 weeks), in competitive athletes or recreationally trained individuals, the frequency of side effects reported by individuals supplementing with creatine were no different than those seen in individuals ingesting a placebo (Hoffman et al., 2006; Volek et al., 1999; Kreider et al., 1998). Even studies of longer duration supplement protocols appear to support the notion that creatine is relatively safe. Schilling and colleagues (2001) retrospectively examined current and former competitive athletes that had used creatine for up to 4-years in duration. They reported only occasional gastrointestinal upset during the loading phase, primarily described as gas to mild diarrhea. Other major concerns during creatine supplementation include kidney strain due to the high nitrogen content of creatine and reported increases in creatinine excretion during short-term ingestion (Harris et al., 1992). However, no renal dysfunction has been reported in either short- (5 days) or long-term (up to 5 years) creatine use (Poortsman and Francaux, 1999; Poortsman et al., 1997). One of the greatest myths associated with creatine supplementation is that it can increase the athlete s risk for heat illness, especially when it is consumed in hot environments. It has been suggested that creatine supplementation should be avoided by athletes during activities being performed in the heat (Terjung et al., 2000). The basis behind this was that creatine is an osmotically active substance that will increase the solute content of skeletal muscle during supplementation (Ziegenfuss et al., 1998). As the concentration of creatine within skeletal muscle increases it will increase the osmotic gradient drawing water into the cell through the process of osmosis. As a result the water content of the muscle cell increases. It is thought that the osmotic pull of water into skeletal muscle, due to an increase in creatine content, may exacerbate the plasma volume loss generally seen during exercise in the heat (Lopez et al., 2009). However, a number of scientific studies have actually examined this specific question and have clearly indicated that creatine supplementation does not increase the risk for dehydration or reduce the ability of athletes to thermoregulate (Lopez et al., 2009; Mendel et al., 2005; Vogel et al., 2000; Watson et al., 2006; Wright et al., 2007). In one study examining the effect of creatine supplementation and fluid regulation, a 5 -day creatine loading protocol in subjects dehydrated to 2.5% and 4.0% of their body weight elicited a ~ - 7% and a ~-9% loss in plasma volume, respectively (Vogel et al., 2000). Each subject group exercised 75 min on a cycle ergometer. Results of the study demonstrated that creatine ingestion in hypohydrated subjects did not negatively impact hydration status. In addition, no increase in muscle cramping was reported as well. Additional studies examining the effect of creatine supplementation on thermoregulation during exercise in the heat, in both euhydrated and dehydrated subjects, have been consistent in finding no reduction in the ability of subjects to tolerate both heat and exercise stresses (Watson et al., 2006; Wright et al., 2007). Recently a meta-analysis (e.g., a statistical examination of the scientific literature in a particular subject area) on creatine supplementation and exercise in the heat concluded that creatine supplementation neither hinders nor negatively affects the athlete s ability to exercise in the heat or body fluid balance (Lopez et al., 2009).

5 Creatine and β-alanine supplementation 23 WHAT TYPE OF CREATINE IS MOST EFFECTIVE? The vast majority of research that has demonstrated the efficacy of creatine supplementation has used the creatine monohydrate form. Several laboratories have shown that the intestinal absorption of creatine when it is supplied in the monohydrate form is nearly 100% (Chanutin, 1926; Deldicque et al., 2008). However, there are a number of sport nutrition companies that have used various formulations of creatine with exaggerated claims of greater efficacy. It has caused some confusion among athletes and coaches alike as to the best creatine to use. This has generated several investigations by sport nutrition scientists to compare the effects of these various creatine formulations. Jäger and colleagues (2007) examined the change in plasma creatine concentrations and pharmokinetics of creatine absorption following ingestion of equal concentrations of creatine monohydrate, tri-creatine citrate and creatine pyruvate. Their results indicated that these different forms of creatine can result in slightly altered kinetics of plasma creatine absorption with the highest plasma concentrations seen from the ingestion of creatine pyruvate. The researchers though did not believe that there were any differences in the bioavailability of these different forms of creatine since the absorption of creatine monohydrate is practically 100% to begin with. In addition the use of effervescent powders containing di-creatine citrate has been shown to have similar bioavailability as creatine monohydrate (Dash and Sawhney, 2002; Ganguly et al., 2003), and is often found in a number of creatine supplements Another form of creatine that is being marketed is creatine ethyl ester. Sport nutrition companies that promote this product often claim that creatine ethyl ester can enhance creatine absorption, delay its degradation and simply increase its bioavailability. How this can be accomplished when creatine monohydrate absorption is already close to 100% has left many sport biochemists shaking their heads. The suggested hypothesis is that the esterification of creatine will decrease its hydrophilicity, and bypass the creatine transporter due to an enhanced sarcolemmal permeability towards creatine (Spillane et al., 2009). Although creatine ethyl ester has been shown to be rapidly degraded to creatinine in the gut due to the low ph (Child and Tallon, 2007; Mold et al., 1955), companies continue to make their claims. Spillane and colleagues (2009) compared 5-days of creatine ethyl ester supplementation to creatine monohydrate and placebo and found it to be inferior to both creatine monohydrate and placebo in changes in body mass and lean body mass (Spillane et al., 2009). The use of creatine ethyl ester showed no significant increase in serum and muscle creatine content suggesting that a large portion of that supplement was degraded within the gut following ingestion. PHYSIOLOGY OF β-alanine β-alanine is a non-essential, non-proteogenic amino acid that is synthesized in the liver (Matthews and Traut, 1987). It is also common in many foods that we eat, such as beef, chicken and turkey. In the diet β-alanine is consumed as a histidine containing dipeptide such anserine, balenine and carnosine, however, it is carnosine that is the principle histidine containing dipeptide found in human skeletal tissue. The hydrolysis of these dipeptides yields β-alanine, which is taken up into skeletal muscle for the re-synthesis of carnosine. By itself, the ergogenic properties of β-alanine are very limited, and it appears to be only effective as an ergogenic aid by its involvement in the synthesis of carnosine (Dunnett and Harris, 1999). Carnosine itself has important ergogenic potential for strength and power athletes by its ability to act as an intracellular buffer to high intensity exercise. Carnosine is comprised of the amino acids â-alanine and histidine. It is unable to be absorbed from the circulation by muscle (Bauer and Schulz, 1994), so it needs to be synthesized within skeletal muscle. Skeletal muscle has a relatively low concentration of β-alanine (Skaper et al., 1973), but a high concentration of histidine and carnosine synthetase, the enzyme responsible for carnosine synthesis. As a result, it is β-alanine that is the rate limiting step in carnosine synthesis. Supplementing with β-alanine (4 to 6 grams per day) for 4 weeks has resulted in a mean increase of 64% in carnosine within skeletal muscle (Harris et al., 2006). In humans, carnosine is found primarily in fast-twitch skeletal muscle, and is estimated to contribute up to 40% of skeletal muscle buffering capacity of H + produced during high intensity exercise (Harris et al., 2006; Hill et al., 2007). Similarly, histidine containing dipeptides (anserine, balenine and carnosine) is found in great concentrations in a number of vertebrate species that perform highly anaerobic activites. Whales, that can spend prolonged time under water are reported to have concentrations of 400 mmol kg - 1 of dry muscle, while thoroughbred race horses and greyhound race dogs have histidine containing dipeptide concentrations of 110 mmol kg -1 of dry muscle and 90 mmol kg -1 of dry muscle, respectively (Abe, 2000; Artioli et al., 2009; Harris et al., 1990) Interestingly, human sprinters have been reported to have muscle carnosine concentrations ranging between mmol kg -1 of dry muscle (Harris et al., 1990). As expected, these concentrations are higher than that typically found in endurance athletes, untrained individuals, and the elderly (Harris et al., 2006; Suzuki et al., 2002). Training does appear to have a profound effect on muscle carnosine concentrations. One of the primary physiological adaptations for anaerobic athletes during their training is the development of an enhanced buffering capacity (Hoffman, 2002). Parkhouse and colleagues (1985) demonstrated that highly trained anaerobic athletes have a greater buffering capacity and a significantly greater skeletal muscle concentration of carnosine than endurance athletes and untrained subjects. Tallon and colleagues (2005) compared trained bodybuilders to untrained control subjects and reported that muscle carnosine concentrations in the vastus lateralis were significantly greater in the bodybuilders, which could not be explained by differences in the muscle size between the groups. The nature of the training program of bodybuilders does suggest that high intensity resistance training can stimulate endogenous changes in muscle carnosine concentrations. Other investigators have examined the relationship between skeletal muscle carnosine content and high intensity exercise performance in trained cyclists (Suzuki et al., 2002). A positive relationship between carnosine concentration and the mean power from a 30

6 24 Creatine and β-alanine supplementation second maximal sprint on a cycle ergometer was found. Thus, adding support to the theory that skeletal muscle carnosine levels have a positive correlation to anaerobic exercise performance due to the relationship of carnosine and muscle buffering capacity. These cross-sectional studies though do not provide a clear understanding of the effects of training on muscle carnosine concentrations. Although Suzuki and colleagues (2004) were able to see a 100% elevation in muscle carnosine concentrations following 8-weeks of high intensity training, the majority of longitudinal training studies (lasting 4 16 weeks) investigating high intensity exercise and muscle carnosine changes have been unable to provide support (Kendrick et al., 2008; 2009; Kim et al., 2006; Mannion et al., 1994). Furthermore, the results of Tallon et al., (2005) may be suspect as the bodybuilders examined in their study did selfadmit to using anabolic steroids. Carnosine synthesis has been shown to be up-regulated by circulating testosterone concentrations (Penafiel et al., 2004), however whether exogenous androgens can elevate muscle carnosine content is not clear. Considering that most of the available evidence to date indicates that neither short nor long term high intensity training regimens can elevate muscle carnosine concentrations, the method that appears to best increase muscle carnosine content is supplementing with β-alanine. β-alanine DOSING SCHEME Harris and colleagues (2006) examined the effect of three different dosing regimens (10 mg kg -1, 20 mg kg -1 and 40 mg kg -1 body weight). The two highest doses yielded the greatest increase in plasma β- alanine concentrations (see Figure 4), but were also associated with uncomfortable side effects (e.g. paresthesia; a tingling-like sensation felt in the skin) that prohibits those dosages from being used. The 10 mg kg -1 (equivalent to an approximate 800 mg dose) resulted in an elevation in plasma β-alanine concentrations, albeit significantly lower than the higher doses but without the associated side effect. The kinetics of the β-alanine response to the low dosing scheme was a time to peak in plasma β-alanine concentration of minutes following ingestion, a concentration half-life (time at which 50% reduction of peak concentration) of 25 minutes, and a return to baseline concentration by 3 hours post-ingestion. According to this kinetic profile the appropriate dosing regimen should be 800 mg of β-alanine taken every three to four hours. This would provide a daily dosing regimen of 4.8 g 6.4 g per day. However, there is some evidence that indicates that dosing should be relative to an FIGURE 4. Plasma β-alanine Concentrations. Adapted from Harris et al., 2006 Plasma ß- alanineµmol.l individual s body mass (Hoffman et al., 2008a). Maximal performance benefits appear to occur when daily dosing is between mg kg - 1 body mass per day (this would involve multiple feedings). Hill and colleagues (2007) using a comparable dosing protocol (4.0 g a day for the first week of supplementation and then 6.4 g per day for an additional 9 weeks (this was equivalent to the mg kg -1 body mass per day) measured muscle carnosine concentrations at weeks 0, 4, and 10. In addition, subjects performed a cycling to exhaustion test at 110% of max power. Muscle carnosine concentrations increased by 58% at week 4 and an additional 15% at week 10. Additionally, there was a 13% and 16% increase in total work done at weeks 4 and 10, respectively. Although the majority of studies have found positive results using this dosing scheme, there is some evidence that suggests that muscle carnosine concentrations may also be elevated (37%) in untrained subjects following only two weeks of supplementation (Harris et al., 2007). In addition, recent studies have examined the use of time-release capsule technology and have demonstrated that dosages of 1600 mg per ingestion four times per day can also be consumed without any side effects and result in a 40% increase in muscle carnosine concentrations (Derave et al., 2007; Harris et al., 2009). This latter method appears to be a more practical dosing pattern that has the same ergogenic potential without any accompanying side effects. Based upon the available evidence, it is not clear whether a ceiling effect exists regarding muscle carnosine content and β- alanine supplementation. It does appear though that the limits on supplementation dose may be more related to the side effects associated with higher dosing regimens. Future investigations will need to examine whether the use of time-release capsules can result in greater muscle carnosine content than powder or regular capsule ingestion. Regarding the time course of muscle carnosine concentrations returning to baseline concentrations following cessation of β-alanine supplementation, the information is not clear. 10 mg.kg -1 bwt 20 mg.kg -1 bwt 40 mg.kg -1 bwt Time (min) A recent study investigated subjects consuming 4.8 g of β-alanine per day for 6 weeks (Baguet et al., 2009). Following three weeks of supplement cessation, muscle carnosine concentrations decreased by only 30%. By nine weeks of cessation muscle carnosine concentrations returned to baseline levels. Additional evaluation of the data suggested that the time course to a return to baseline levels may be dependent upon the effectiveness of the supplementation program. Those subjects that were deemed high

7 Creatine and β-alanine supplementation 25 responders (greater accumulation of muscle carnosine content) required a greater washout time to return to baseline levels (~15 weeks), while low responders saw a return to baseline levels within 6 weeks. EFFICACY OF β-alanine SUPPLEMENTATION The physiological mechanism described concerning β-alanine supplementation suggests that it would be most effective in performances involving high intensity activity. The investigations that have focused on this aspect of athletic performance have consistently reported positive results in the ergogenic benefit of β- alanine supplementation (Derave et al., 2007; Hill et al., 2007; Hoffman et a., 2006; Hoffman et al., 2008a, b; Stout et al., 2006; 2007; Van Thienen et al. 2009). Stout et al. (2006) examined the effects of β-alanine supplementation on physical working capacity at fatigue threshold (PWC FT ) in untrained young men. Subjects consumed either 1.6 g of β-alanine or a placebo four times per day for six days, then 3.2 grams per day for 22 additional days. Prior to, and following supplementation, the subjects performed an incremental cycle ergometry test to determine PWC FT, which was determined from bipolar surface electromyography recorded from the vastus lateralis muscle. The PWC FT provides a measure for the highest exercise intensity a person can maintain without signs of fatigue and has been highly correlated with anaerobic threshold measurements (lactate and ventilatory thresholds). The results of that study demonstrated a significantly greater increase in PWC FT (9%) in the β alanine group compared to no change in the placebo. In a follow-up study, Stout s research group examined the effect of 28 days of β-alanine supplementation in untrained college-age women. They confirmed their previous results by demonstrating a significantly greater PWC FT (12.6%), ventilatory threshold (13.9%) and time to exhaustion (2.5%) during a graded exercise cycle ergometry test than the placebo supplemented group (Stout et al., 2007). These findings indicated that 28 days of β-alanine supplementation in untrained subjects can delay fatigue during intense exercise. The efficacy of β-alanine supplementation has also been demonstrated in trained strength/power athletes. Hoffman and colleagues (2008a) examined the effect of two weeks of supplementation (4.5 g day -1 ) prior to the onset of training camp in college football players. Supplementation continued for an additional two weeks during pre-season practices. Performance testing which occurred following two weeks of supplementation revealed no ergogenic effect in sprint times or fatigue rates during performance of repeated line drills (an approximate second shuttle run performed three times with 2-min rest between each sprint). In addition, no significant differences were observed in peak power, mean power and total work in a 60-sec Wingate anaerobic power test. However, a trend (p = 0.07) towards a reduced rate of fatigue was found in the football players consuming the supplement versus the placebo. Examination of the resistance training logs (performed during training camp) revealed no significant difference in the average training intensity in either the bench press or squat exercises, but did indicate a trend (p = 0.09) towards a higher (9.2 %) volume of training (both bench press and squat combined) seen for the athletes supplementing with β- alanine compared to the placebo. In addition, subjective feelings of fatigue during camp were significantly lower in athletes using the supplement compared to the placebo. The inability to see any effect on repeated sprints of approximately seconds but, a trend towards an improved fatigue rate in a 60-sec maximal intensity bout of exercise provides support to the effect that β-alanine supplementation has on improved buffering capacity during prolonged high intensity exercise. Interestingly, Derave and colleagues (2007) reported that 4-weeks of β-alanine supplementation in 400-m sprinters could delay fatigue in repeated isokinetic bouts (5 sets) of exercise, but not improve 400-m race time. It appears that prolonged high intensity exercise bouts (~60 sec) benefit the most from improved buffering capacities brought about by increases in muscle carnosine concentrations. However, high intensity exercise performed immediately following a prolonged bout of endurance exercise may also benefit from β-alanine supplementation. A recent study demonstrated that trained cyclists supplementing for 8-weeks could improve 30-second sprint performance following a 110-min time trial (Van Thienen et al., 2009). A 4-week training study, employing a double-blind cross-over design, examined the effect of 4.8 g per day of β-alanine on the acute endocrine response to a resistance training session in eight experienced, resistance trained athletes (Hoffman et al., 2008b). When consuming β-alanine the Δ total number of repetitions performed in the squat exercise per workout (expressed as the difference between workouts performed at week 0 and week 4) was significantly greater than when using a placebo (9.0 ± 4.1 and 0.3 ± 7.8, respectively). In addition, a significant difference between the groups was also seen in Δ mean power. The greater volume of training though did not correspond to any significant change to the acute testosterone or growth hormone response to the exercise protocol. In addition, no difference was seen in improvement of squat strength following 4-weeks of supplementation between subjects supplementing with β-alanine (5.9 ± 4.3 kg) versus the placebo (3.9 ± 4.1 kg). The lack of significant strength improvement is consistent with other studies that have failed to show significant improvements in strength following β-alanine supplement durations lasting between 4 10 weeks (Kendrick et al., 2008; 2009). These results are not surprising considering the physiological mechanism that is affected by elevations in muscle carnosine concentrations. An improved intra-muscular buffering system would have a greater effect on fatiguing type exercises by extending duration of exercise. As a result it does not have a direct effect on strength development. Thus, it is not surprising that in relatively short duration training protocols no significant improvements in strength are seen in trained individuals. In contrast, the window of strength improvements for untrained individuals is so large during the onset of a resistance training program (Hoffman, 2002), that supplements are generally not recommended for novice resistance trained populations. The ability of β-alanine supplementation to enhance strength performance is likely more effective during prolonged durations of training. Improvements in muscle

8 26 Creatine and β-alanine supplementation buffering capacity appears to improve the quality of a resistance training workout by increasing the number of repetitions that can be performed at a given intensity of training (Hoffman et al., 2006; 2008a,b). The greater training volume would potentially provide a greater stimulus for muscle adaptation, but likely following a longer duration of training. THE EFFECT OF β-alanine SUPPLEMENTATION AND BODY MASS CHANGES Several of the training studies examining β-alanine supplementation have been unable to see any change in body mass in studies ranging from 4 15 weeks (Hoffman et al., 2006; 2008b). This has been attributed to the low daily caloric intake of subjects in those studies. However, the greater volume of training seen in the resistance training program of trained strength/power athletes supplementing with β- alanine appeared to stimulate significant decreases in 8000 fat mass and increases in lean body mass compared to subjects consuming creatine only or a placebo (Hoffman et al., 2006). The higher training volume 6000 associated with β-alanine supplementation is typical of a resistance training program designed to enhance muscle hypertrophy adaptations, similar to that 4000 performed by bodybuilders. Improvements in lean muscle mass and decreases in fat mass are desirable 2000 outcomes for individuals whose primary training goals are improvements in body composition. SIDE EFFECTS ASSOCIATED WITH β- ALANINE SUPPLEMENTATION The only known side effect associated with β-alanine supplementation is paresthesia. Paresthesia is a sensation of numbing or tingling in the skin. It appears when high doses of β- alanine are ingested. It generally disappears within one hour following ingestion (Harris et al., 2006). Interestingly, when β- alanine is mixed with a carbohydrate and electrolyte drink the appearance of this side effect appears negligible (Hoffman et al., 2006). Studies examining potential side effects from prolonged (greater than 15 weeks) supplementation durations have not been performed. However, considering that β-alanine is an amino acid with an important physiological role in the body, in dosages studied that are similar to that consumed regularly in the diet, it is likely a very safe supplement to use (Artioli et al., 2009). CREATINE AND β-alanine COMBINATION Hoffman and colleagues (2006) were the first research team to examine the combination of both creatine and β-alanine supplements. The hypothesis was that this combination of supplements would provide a significant benefit for strength/ power athletes. Results of their study demonstrated that this combination significantly improved the quality of the workout more so than creatine alone. Specifically, improvements in training volume was found (see Figure 5) that was associated with significantly greater gains in lean body mass and decreases in fat Kg mass. Interestingly, creatine and creatine + β-alanine resulted in significantly greater strength gains in 1-RM bench press and 1- RM squat than the placebo. However, the addition of β-alanine did not provide any further benefit to strength improvement. Similarly, Stout et al., (2006) also compared the combination of creatine and β-alanine to creatine and β-alanine alone. Significant improvements were seen, but no additive benefits were noted in physical work capacity in previously untrained men. FIGURE 5. Comparison of Creatine to Creatine + β-alanine on Training Volume in the Squat Exercise. * = significantly different from P and C. Adapted from Hoffman et al., P CA C SUMMARY Creatine is the most widely studied ergogenic aid to date. Evidence is convincing that it is both safe and effective in enhancing performance in strength/power activities. Significant improvements in size, strength and power are associated with creatine supplementation. Similarly, the evidence examining the efficacy of β-alanine supplementation for athletes participating in high intensity exercise is also quite convincing. However, the additive benefits of combining β-alanine to creatine in enhancing strength performance is not clear, but may become more relevant for enhancing the quality of the workout. For the experienced resistance trained athlete this may stimulate greater strength and power adaptations in training durations greater than that presently studied. In addition, the benefits may also be more prevalent during actual athletic performance when athletes will perform repeated, prolonged duration high intensity exercise. REFERENCES Abe, H. (2000) Role of histidine-related compounds as intracellular proton buffering constituents in vg.ertebrate muscle. Biochemistry (Moscow). 65, Artioli, G.G. Gualano, B. Smith, A. Stout, J.R. and Junior A.H.L. (2009) The Role of β-alanine Supplementation on Muscle *

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