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Creatine Monohydrate Supplementation, but not Creatyl-L-Leucine, Increased Muscle Creatine Content in Healthy Young Adults: A Double-Blind Randomized Controlled Trial (2022)
Andrew T. Askow, Kevin J.M. Paulussen, Colleen F. McKenna, Amadeo F. Salvador, Susannah E. Scaroni, Jade S. Hamann, Alexander V. Ulanov, Zhong Li, Scott A. Paluska, Kayleigh M. Beaudry, Michael De Lisio, and Nicholas A. Burd
Creatine (Cr) supplementation is a well-established strategy to enhance gains in strength, lean body mass, and power from a period of resistance training. However, the effectiveness of creatyl-L-leucine (CLL), a purported Cr amide, is unknown. Therefore, the purpose of this study was to assess the effects of CLL on muscle Cr content. Twenty-nine healthy men (n = 17) and women (n = 12) consumed 5 g/day of either Cr monohydrate (n = 8; 28.5 ± 7.3 years, 172.1 ± 11.0 cm, 76.6 ± 10.7 kg), CLL (n = 11; 29.2 ± 9.3 years, 170.3 ± 10.5 cm, 71.9 ± 14.5 kg), or placebo (n = 10; 30.3 ± 6.9 years, 167.8 ± 9.9 cm, 69.9 ± 11.1 kg) for 14 days in a randomized, double-blind design. Participants completed three bouts of supervised resistance exercise per week. Muscle biopsies were collected before and after the intervention for quantification of muscle Cr. Cr monohydrate supplementation significantly increased muscle Cr content with 14 days of supplementation. No changes in muscle Cr were observed for the placebo or CLL groups. Cr monohydrate supplementation is an effective strategy to augment muscle Cr content while CLL is not.
Creatine (Cr) is one of the most researched dietary supplements and is considered to have good-to-strong evidence for its efficacy to serve as an ergogenic aid by the International Olympic Committee (Maughan et al., 2018). Cr is a naturally occurring nonprotein amino acid derivative that can be synthesized endogenously from glycine, arginine, and methionine in the liver and kidneys. In addition, Cr can be obtained from dietary sources, such as meat and seafood, and the typical omnivorous diet is estimated to yield ∼1–2 g of Cr per day (Ostojic, 2021). While relatively small quantities of the total body Cr pool are in other tissues, ∼95% of total Cr is in skeletal muscle tissue
During short-term, high-intensity muscle actions, the phosphorylated form of Cr, phosphocreatine (PCr) is degraded to Cr and a phosphate group to regenerate adenosine triphosphate via the energy and phosphate group released from PCr degradation. PCr is then replenished when Cr is bonded with another phosphate group via the reversible enzymatic action of Cr kinase. Thus, maintenance of intramuscular Cr and PCr concentration is important for sustaining muscular effort. While de novo synthesis and dietary sources of Cr are typically sufficient to replenish the ∼1%–2% of the body’s Cr pool that is degraded to creatinine per day (Hoberman et al., 1948; Picou et al., 1976), muscular Cr content can be augmented by ∼30% via dietary supplementation with Cr. This increase in Cr allows for greater PCr resynthesis and enhanced performance in high-intensity, repetitive exercise bouts (Harris et al., 1992). Over time, this performance enhancement has been shown to lead to greater gains in muscular strength, lean body mass (LBM), and muscular endurance after a period of resistance training and other high-intensity exercises in both young and older adults (Branch, 2003; Devries & Phillips, 2014).
Indeed, most studies demonstrating the effectiveness of Cr supplementation have used Cr monohydrate (CrM). However, several purported analogs of Cr have been introduced into the consumer market since the late 1990s in an attempt to improve the efficacy of Cr supplementation on skeletal muscle content compared with CrM. Many of these molecules are purported to increase the bioavailability, solubility, or safety of Cr supplementation compared with CrM. Despite previous studies demonstrating that many of these putative forms of Cr fail to improve the ergogenic effects of CrM and augment muscle Cr content (Jagim et al., 2012; Kreider et al., 2022; Spillane et al., 2009), novel supplements continue to be introduced to the market. For example, one such purported analog of Cr, creatyl-L-leucine (CLL), is currently on the market as part of a multi-ingredient blend and is sold under the name “Super Creatine®.” Scientifically, it is not entirely clear what anabolic properties, if any, may manifest from consuming CLL. However, it is important to investigate the potential ergogenic effects of other analogs of Cr within the consumer market given the potential benefits of Cr to enhance muscle mass and strength for all adult ages. While the toxicological effects of CLL have been studied in rodents (Reddeman et al., 2018), to our knowledge, no studies to date have investigated the effects of CLL supplementation on muscle Cr content in humans. Therefore, the purpose of this study was to compare the effects of 2 weeks of dietary supplementation with CrM, CLL, or a placebo (PLA) on muscle Cr content in healthy males and females. We tested the hypothesis that both CrM and CLL would increase muscle Cr content compared with PLA with CrM supplementation yielding enhanced Cr content compared with CLL.
Supplementation Protocol
The supplementation period lasted a total of 14 days and began on the day of participants’ preliminary trial. During this time, participants consumed 5 g of either CrM (Creapure®, Bare Performance Nutrition), CLL, or PLA (maltodextrin, BulkSupplements) in a double-blind manner. CLL was provided by the sponsor (lot no. 201907016). Upon arrival of the supplements to the research facility, the powders were given to a researcher that was not involved in the study for transfer into identical containers labeled A, B, or C. The individual then recorded the contents of each container on a piece of paper, folded the paper, and subsequently sealed the blinding key with a signed and dated piece of tape. The blinding key was then stored in the office of the principal investigator (N.A. Burd) until all analysis was completed. Once blinded, 5 g portions of each condition were weighed to the nearest 0.1 g and transferred to plastic bottles for distribution to participants. Participants were instructed to consume the contents of one of these bottles at the same time daily by filling the bottle with ∼12 oz of warm water, mixing until all the powder was dissolved, and promptly consuming the mixture (within 3 min of preparation). To promote compliance, participants returned empty supplement bottles on exercise days.
Discussion
The identification of more effective Cr supplementation regimens has important implications for performance and clinical nutrition when the goal is to support muscle mass and strength. In this study, we examined the impact of CLL and CrM supplementation (5 g/day for 14 days) on muscle Cr content in healthy males and females against the backdrop of resistance training. The results presented herein demonstrated that 2 weeks of supplementation with CLL did not statistically significantly increase muscle Cr while CrM supplementation significantly increased muscle Cr content. Indeed, a past study evaluated a product (mixed ingredient supplement) containing CLL on strength and LBM outcomes (Schwarz et al., 2019). However, this particular study could not reach any firm conclusions on the potential independent ergogenic effects of CLL. Our study is the first to directly compare CLL versus Cr in isolation on muscle Cr and body composition in humans.
Previous studies on Cr supplementation have reported an increase of 10%–40% in muscle Cr content after a period of supplementation (Greenhaff et al., 1994; Hultman et al., 1996; Jagim et al., 2012). While there is significant heterogeneity in the response to Cr supplementation, this can be explained, in part, by differing supplementation strategies and durations of observation. Most studies use a loading phase (typically 20 g/day for 5–7 days) followed by a lower maintenance dose (∼3–5 g/day). However, CLL, to our knowledge, is currently only available commercially in a multi-ingredient energy drink or pre-workout supplement formulation. Hence, the typical use of CLL as a component of a beverage does not allow for a traditional loading phase. Thus, we elected to forgo the loading phase in our study. While this likely reduced the rate at which muscle Cr accumulated, foundational work in this area demonstrated that this strategy is a valid approach to saturate muscle Cr (Hultman et al., 1996). Along those lines, Hultman et al. (1996) report a ∼12% increase in muscle Cr following 14 days of supplementation with CrM (3 g/day). This is slightly lower than the ∼24% increase we observed in our study for the CrM group, which is to be expected considering participants in the current investigation consumed a higher dose of CrM (5 g/day) compared with Hultman et al. (1996). Moreover, participants in our study completed supervised bouts of resistance exercise 3 days/week throughout the supplementation period. Exercise in combination with Cr supplementation has previously been shown to enhance muscle Cr above supplementation alone (Harris et al., 1992)
The mean increase in muscle Cr was ∼24% for the CrM group with no increase for the CLL group. While no study has characterized the digestive fate of ingested CLL, the absorption of CrM is nearly 100% (Deldicque et al., 2008; Harris et al., 1992), and thus, the potential benefit of consuming a purported creatyl amide, such as CLL, for enhanced solubility and subsequent increase in muscle Cr content is not entirely clear. Future studies could test higher-dose CLL supplementation or a longer supplementation period to enhance muscle Cr. However, based on the results presented from the current study, it seems more straightforward to simply supplement with CrM, especially given we did not observe a statistically significant increase in muscle Cr content in the CLL condition.
We did not observe any changes to LBM as measured by dual-energy X-ray absorptiometry in any groups. While Cr supplementation is known to enhance the training-induced gain in LBM (Branch, 2003; Devries & Phillips, 2014), most demonstrating this effect employ a longer duration of supplementation (i.e., >8 weeks) in combination with a progressive resistance training program. One study reports a significant increase in fat-free mass following 3 days of loading and 7 days of maintenance supplementation at a dose of 20 and 5 g/day, respectively (Safdar et al., 2008). However, this gain in fat-free mass is most likely due to increased fluid retention following Cr supplementation (Bone et al., 2017; Powers et al., 2003) rather than a true increase in myofibrillar protein content. As such, the lack of a significant gain in LBM for our study is in line with what is expected in response to 2 weeks of Cr supplementation
In conclusion, we demonstrated that CrM supplementation significantly increased muscle Cr content by ∼24%, whereas there was no statistically significant change in muscle Cr content in the PLA and/or the CLL conditions. As such, this work does not support the use of CLL as an alternative dietary supplementation strategy to CrM to increase muscle Cr content in healthy males and females.
Andrew T. Askow, Kevin J.M. Paulussen, Colleen F. McKenna, Amadeo F. Salvador, Susannah E. Scaroni, Jade S. Hamann, Alexander V. Ulanov, Zhong Li, Scott A. Paluska, Kayleigh M. Beaudry, Michael De Lisio, and Nicholas A. Burd
Creatine (Cr) supplementation is a well-established strategy to enhance gains in strength, lean body mass, and power from a period of resistance training. However, the effectiveness of creatyl-L-leucine (CLL), a purported Cr amide, is unknown. Therefore, the purpose of this study was to assess the effects of CLL on muscle Cr content. Twenty-nine healthy men (n = 17) and women (n = 12) consumed 5 g/day of either Cr monohydrate (n = 8; 28.5 ± 7.3 years, 172.1 ± 11.0 cm, 76.6 ± 10.7 kg), CLL (n = 11; 29.2 ± 9.3 years, 170.3 ± 10.5 cm, 71.9 ± 14.5 kg), or placebo (n = 10; 30.3 ± 6.9 years, 167.8 ± 9.9 cm, 69.9 ± 11.1 kg) for 14 days in a randomized, double-blind design. Participants completed three bouts of supervised resistance exercise per week. Muscle biopsies were collected before and after the intervention for quantification of muscle Cr. Cr monohydrate supplementation significantly increased muscle Cr content with 14 days of supplementation. No changes in muscle Cr were observed for the placebo or CLL groups. Cr monohydrate supplementation is an effective strategy to augment muscle Cr content while CLL is not.
Creatine (Cr) is one of the most researched dietary supplements and is considered to have good-to-strong evidence for its efficacy to serve as an ergogenic aid by the International Olympic Committee (Maughan et al., 2018). Cr is a naturally occurring nonprotein amino acid derivative that can be synthesized endogenously from glycine, arginine, and methionine in the liver and kidneys. In addition, Cr can be obtained from dietary sources, such as meat and seafood, and the typical omnivorous diet is estimated to yield ∼1–2 g of Cr per day (Ostojic, 2021). While relatively small quantities of the total body Cr pool are in other tissues, ∼95% of total Cr is in skeletal muscle tissue
During short-term, high-intensity muscle actions, the phosphorylated form of Cr, phosphocreatine (PCr) is degraded to Cr and a phosphate group to regenerate adenosine triphosphate via the energy and phosphate group released from PCr degradation. PCr is then replenished when Cr is bonded with another phosphate group via the reversible enzymatic action of Cr kinase. Thus, maintenance of intramuscular Cr and PCr concentration is important for sustaining muscular effort. While de novo synthesis and dietary sources of Cr are typically sufficient to replenish the ∼1%–2% of the body’s Cr pool that is degraded to creatinine per day (Hoberman et al., 1948; Picou et al., 1976), muscular Cr content can be augmented by ∼30% via dietary supplementation with Cr. This increase in Cr allows for greater PCr resynthesis and enhanced performance in high-intensity, repetitive exercise bouts (Harris et al., 1992). Over time, this performance enhancement has been shown to lead to greater gains in muscular strength, lean body mass (LBM), and muscular endurance after a period of resistance training and other high-intensity exercises in both young and older adults (Branch, 2003; Devries & Phillips, 2014).
Indeed, most studies demonstrating the effectiveness of Cr supplementation have used Cr monohydrate (CrM). However, several purported analogs of Cr have been introduced into the consumer market since the late 1990s in an attempt to improve the efficacy of Cr supplementation on skeletal muscle content compared with CrM. Many of these molecules are purported to increase the bioavailability, solubility, or safety of Cr supplementation compared with CrM. Despite previous studies demonstrating that many of these putative forms of Cr fail to improve the ergogenic effects of CrM and augment muscle Cr content (Jagim et al., 2012; Kreider et al., 2022; Spillane et al., 2009), novel supplements continue to be introduced to the market. For example, one such purported analog of Cr, creatyl-L-leucine (CLL), is currently on the market as part of a multi-ingredient blend and is sold under the name “Super Creatine®.” Scientifically, it is not entirely clear what anabolic properties, if any, may manifest from consuming CLL. However, it is important to investigate the potential ergogenic effects of other analogs of Cr within the consumer market given the potential benefits of Cr to enhance muscle mass and strength for all adult ages. While the toxicological effects of CLL have been studied in rodents (Reddeman et al., 2018), to our knowledge, no studies to date have investigated the effects of CLL supplementation on muscle Cr content in humans. Therefore, the purpose of this study was to compare the effects of 2 weeks of dietary supplementation with CrM, CLL, or a placebo (PLA) on muscle Cr content in healthy males and females. We tested the hypothesis that both CrM and CLL would increase muscle Cr content compared with PLA with CrM supplementation yielding enhanced Cr content compared with CLL.
Supplementation Protocol
The supplementation period lasted a total of 14 days and began on the day of participants’ preliminary trial. During this time, participants consumed 5 g of either CrM (Creapure®, Bare Performance Nutrition), CLL, or PLA (maltodextrin, BulkSupplements) in a double-blind manner. CLL was provided by the sponsor (lot no. 201907016). Upon arrival of the supplements to the research facility, the powders were given to a researcher that was not involved in the study for transfer into identical containers labeled A, B, or C. The individual then recorded the contents of each container on a piece of paper, folded the paper, and subsequently sealed the blinding key with a signed and dated piece of tape. The blinding key was then stored in the office of the principal investigator (N.A. Burd) until all analysis was completed. Once blinded, 5 g portions of each condition were weighed to the nearest 0.1 g and transferred to plastic bottles for distribution to participants. Participants were instructed to consume the contents of one of these bottles at the same time daily by filling the bottle with ∼12 oz of warm water, mixing until all the powder was dissolved, and promptly consuming the mixture (within 3 min of preparation). To promote compliance, participants returned empty supplement bottles on exercise days.
Discussion
The identification of more effective Cr supplementation regimens has important implications for performance and clinical nutrition when the goal is to support muscle mass and strength. In this study, we examined the impact of CLL and CrM supplementation (5 g/day for 14 days) on muscle Cr content in healthy males and females against the backdrop of resistance training. The results presented herein demonstrated that 2 weeks of supplementation with CLL did not statistically significantly increase muscle Cr while CrM supplementation significantly increased muscle Cr content. Indeed, a past study evaluated a product (mixed ingredient supplement) containing CLL on strength and LBM outcomes (Schwarz et al., 2019). However, this particular study could not reach any firm conclusions on the potential independent ergogenic effects of CLL. Our study is the first to directly compare CLL versus Cr in isolation on muscle Cr and body composition in humans.
Previous studies on Cr supplementation have reported an increase of 10%–40% in muscle Cr content after a period of supplementation (Greenhaff et al., 1994; Hultman et al., 1996; Jagim et al., 2012). While there is significant heterogeneity in the response to Cr supplementation, this can be explained, in part, by differing supplementation strategies and durations of observation. Most studies use a loading phase (typically 20 g/day for 5–7 days) followed by a lower maintenance dose (∼3–5 g/day). However, CLL, to our knowledge, is currently only available commercially in a multi-ingredient energy drink or pre-workout supplement formulation. Hence, the typical use of CLL as a component of a beverage does not allow for a traditional loading phase. Thus, we elected to forgo the loading phase in our study. While this likely reduced the rate at which muscle Cr accumulated, foundational work in this area demonstrated that this strategy is a valid approach to saturate muscle Cr (Hultman et al., 1996). Along those lines, Hultman et al. (1996) report a ∼12% increase in muscle Cr following 14 days of supplementation with CrM (3 g/day). This is slightly lower than the ∼24% increase we observed in our study for the CrM group, which is to be expected considering participants in the current investigation consumed a higher dose of CrM (5 g/day) compared with Hultman et al. (1996). Moreover, participants in our study completed supervised bouts of resistance exercise 3 days/week throughout the supplementation period. Exercise in combination with Cr supplementation has previously been shown to enhance muscle Cr above supplementation alone (Harris et al., 1992)
The mean increase in muscle Cr was ∼24% for the CrM group with no increase for the CLL group. While no study has characterized the digestive fate of ingested CLL, the absorption of CrM is nearly 100% (Deldicque et al., 2008; Harris et al., 1992), and thus, the potential benefit of consuming a purported creatyl amide, such as CLL, for enhanced solubility and subsequent increase in muscle Cr content is not entirely clear. Future studies could test higher-dose CLL supplementation or a longer supplementation period to enhance muscle Cr. However, based on the results presented from the current study, it seems more straightforward to simply supplement with CrM, especially given we did not observe a statistically significant increase in muscle Cr content in the CLL condition.
We did not observe any changes to LBM as measured by dual-energy X-ray absorptiometry in any groups. While Cr supplementation is known to enhance the training-induced gain in LBM (Branch, 2003; Devries & Phillips, 2014), most demonstrating this effect employ a longer duration of supplementation (i.e., >8 weeks) in combination with a progressive resistance training program. One study reports a significant increase in fat-free mass following 3 days of loading and 7 days of maintenance supplementation at a dose of 20 and 5 g/day, respectively (Safdar et al., 2008). However, this gain in fat-free mass is most likely due to increased fluid retention following Cr supplementation (Bone et al., 2017; Powers et al., 2003) rather than a true increase in myofibrillar protein content. As such, the lack of a significant gain in LBM for our study is in line with what is expected in response to 2 weeks of Cr supplementation
In conclusion, we demonstrated that CrM supplementation significantly increased muscle Cr content by ∼24%, whereas there was no statistically significant change in muscle Cr content in the PLA and/or the CLL conditions. As such, this work does not support the use of CLL as an alternative dietary supplementation strategy to CrM to increase muscle Cr content in healthy males and females.
